Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
  • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
  • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
  • C07D213/79—Acids; Esters
  • C07D213/80—Acids; Esters in position 3
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/06—Anti-spasmodics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/22—Anxiolytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/06—Antiarrhythmics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C233/00—Carboxylic acid amides
  • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
  • C07C233/45—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups
  • C07C233/53—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring
  • C07C233/55—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring having the carbon atom of the carboxamide group bound to a carbon atom of an unsaturated carbon skeleton

Definitions

• the present invention relates to a novel series of anthranilic acid-derived amides (I) having pharmacological activity, to a process for their preparation, to pharmaceutical compositions containing them, and to their use in the treatment of disorders associated with smooth muscle contraction, via potassium channel and chloride channel modulation.
• disorders include, but are not limited to: urinary incontinence, asthma, premature labor, irritable bowel syndrome, congestive heart failure, angina, and cerebral vascular disease.
• Patent No. J6 0097-946-A discloses a series of substituted carboxamide derivatives which exhibit activity as leucotriene antagonists and phospholipase inhibitors.
• Figure 1 is a schematic representation of a single cell recordings of cell current.
• Figure 1 A shows cell outward current of the control.
• Figure IB shows cell outward current following exposure of cells to compound of Example 1.
• Figure 1C shows cell outward current following washout.
• Figure 2 is a schematic representation of the effects of Example 1 on chloride channel current induced by swelling.
• Figure 2A depicts current traces for a cell in isotonic, low osmolarity and after exposure to Example 1.
• Figure 2B depicts voltage dependency of current recorded in the presence of control (C), hypotonic (swelling), Example 1(WY) and washout solutions (washout).
• Figure 2C depicts the time course of the effect on Example 1 on chloride channel current. Description of The Invention
• R,, R 2 , R 3 , R 4 ,R 5 , R fi , R ? and R g are, independently, hydrogen, COOR 15 , halogen, nitro, cyano, C, .10 alkoxy, C, .10 haloalkoxy, sulfonic acid, C,., 0 alkylsulfonyl, C 6. , 2 arylsulfonyl, C 6.12 aralkylsulfonyl, C, .10 alkylsulfinyl,
• R 9 is hydrogen, C, .10 alkyl and C, .10 haloalkyl
• R 10 is hydrogen, C, .10 alkyl, C, . ,,, haloalkyl, or C 2.12 alkylidene;
• R 15 is hydrogen, metal cation, acetylamido, alkoxyacetoyl or a related moiety which delivers the carboxylate in vivo; the dotted line is an optional double bond; with the proviso that when R 10 is an alkylidene moiety, the bond is absent; and
• W is nitrogen or carbon bearing a hydrogen, or R 4 , R 5 or R 6 as hereinbefore defined; or pharmaceutcal salts thereof.
• R , R and R are, independently, hydrogen, cyano, C 0 perhaloalkoxy, sulfonic acid, C, .I0 alkyl sulfonyl, C 6 . 12 arylsulfonyl, C 6.12 aralkylsulfonyl, C,_ 10 alkylsulfinyl, C 6.12 arylsufinyl, C 6 .
• R, R 2 and R 3 may not all simultaneously be hydrogen, and (2) when R, and R 2 are hydrogen, R 3 may not be meta-CF 3 ;
• R 5 , R 6> R 7 and R g are, independently, hydrogen, COOR 15 , halogen, nitro, cyano, C, .10 alkoxy, C, .10 haloalkoxy, sulfonic acid, C, .10 alkylsulfonyl, C 6 _ 12 arylsulfonyl, C 6.12 aralkylsulfonyl, C, .10 alkylsulfinyl, C 6.12 arylsufinyl, C 6 .
• R 4 and R 5 are COOR 15 ;
• R 9 is hydrogen, C,_ 10 alkyl and C, .]0 haloalkyl;
• R 10 is hydrogen, C 0 alkyl, C, .10 haloalkyl, or C 2 _ 12 alkylidene;
• R 15 is hydrogen, metal cation, acetylamido, alkoxyacetoyl or a related moiety which delivers the carboxylate in vivo; the dotted line is an optional double bond; with the proviso that when R 10 is an alkylidene moiety, the bond is absent; and W is nitrogen or carbon bearing a hydrogen, or R 4 , R 5 or R 6 as hereinbefore defined; or pharmaceutical salts thereof.
• R l5 is selected from the group consisting of hydrogen, a metal cation, a moiety selected from:
• R n , R 12 , R 13 , and R 14 are, independently, hydrogen, C 0 alkyl, C 6 ., 2 aryl, or C 6.12 aralkyl.
• W is nitrogen, or a carbon bearing one of hydrogen, halogen, nitro, cyano, or C, .10 haloalkyl. More preferably W is carbon bearing a hydrogen.
• R,, R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R g are, independently, selected from hydrogen, halogen, COOR 15 , nitro, cyano, C, .6 alkoxy, C,_ 6 alkanoyl, C, .6 haloalkoxy, C 6 _ 10 aryloyl, C, .6 alkylsulfonyl, C,.
• R, R 2 and R 3 may not simultaneously be hydrogen, (2) when R, and R 2 are hydrogen, R 3 is not meta-CF ⁇ , and (3) at least one of R 4 and R 5 is COOR ]5 .
• R,, R 2 and R 3 are selected from hydrogen, C,_ 6 alkyl, C, .6 haloalkyl, C, .6 alkoxy, nitro, C,_ 6 dialkylamino and halogen with the provisos that (1) R,, R 2 and R 3 may not simultaneously be hydrogen and (2) when R, and R 2 are hydrogen, R 3 is not met ⁇ -CF3. With the foregoing provisos, it is further preferred that at least one of R,, R 2 and R 3 is perhaloalkyl more preferably trifluoromethyl. Where one of R,, R 2 and R 3 is perhaloalkyl, it is preferred that said substituent is 4-CF 3 .
• one of R 6 , R 7 and R g is a halogen, more preferably chloro and most preferably 4- chloro.
• R 4 is COOR 15 , and more preferably COOH.
• R 10 is alkylidene and more preferably methylidene.
• R 9 is C,_ 6 alkyl and most preferably methyl. The double bond is present in preferred embodiments of the invention.
• Preferred compounds of the present invention are: (E)-2-[2-Methyl-3-(4-trifluoromethyl-phenyl)-acryloylamino]-benzoic acid lithium salt; 2- [3-(4-Trifluoromethyl-phenyl)propionyl-amino]-benzoic acid;
• the pharmaceutically acceptable salts of the basic compounds of this invention are those derived from such organic and inorganic acids as: lactic, citric, acetic, tartaric, fumaric, succinic, maleic, malonic, hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, and similarly known acceptable acids.
• salts of the compounds in this invention may be formed with bases such as alkali metals (Na, K,
• Li Li or alkaline earth metals (Ca or Mg).
• Alkyl as used herein means a branched or straight chain having from 1 to 12 carbon atoms and more preferably from 1 to 6 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl and hexyl.
• Alkenyl as used herein means a branched or straight chain having from 2 to 12 carbon atoms and more preferably from 1 to 6 carbon atoms, the chain containing at least one carbon-carbon double bond. Alkenyl, may be used synonymously with the term olefin and includes alkylidenes.
• alkenyl groups include ethylene, propylene and isobutylene.
• Alkoxy as used herein means an alkyl-O group in which the alkyl group is as previously described.
• alkoxy groups include methoxy, ethoxy, n-propoxy, i- propoxy, n-butoxy, and t-butoxy.
• Haloalkyl refers to an alkyl group, as defined above, in which one or more hydrogen atoms are replaced with a halogen.
• Perhaloalkyl refers to alkyl groups in which each of the hydrogens are replaced with halogen atoms.
• Exemplary haloalkyl groups include chloromethyl, dibromomethyl, and the perhaloalkyl, trifluoromethyl.
• Aryl as used herein means mono or bicyclic aromatic ring having from 6 to 12 carbon atoms. Monocyclic rings preferably have 6 or 7 members and bicyclic rings preferably have 8, 9 or 10 membered ring structures. Exemplary aryl groups include phenyl and naphthyl. The aryl group may be substituted with one or more substituents.
• Substituted aryl groups preferably have one to three substituents which may include alkyl, alkoxy, perhaloalkyl, halogen, nitro, amino, carboxy, carboxyalkyl, alkylamino, and dialkylamino.
• “Aralkyl” as used herein means an aryl-alkyl group in which the aryl and alkyl group are previously defined. Exemplary aralkyl groups include benzyl and phenethyl.
• Alkanoyl refers to -C(O)R where R is alkyl as previously defined.
• Aryloyl refers to -C(O)R where R is aryl as previously defined.
• Aralkanoyl refers to -C(O)R where R is aralkyl as previously defined.
• Carboxamido refers to -CONH 2 .
• alkyl-, aryl, and aralkylsulfamido refer to groups in which the R is alkyl, aryl or aralkyl as previously defined.
• Sulfmyl refers to the radical -SOR.
• alkyl-, aryl-, and aralkylsulfinyl refer to groups in which the R is alkyl, aryl or aralkyl, as previously defined.
• alkyl-, aryl-, and aralkylsulfonyl refer to groups in which the R is alkyl, aryl or aralkyl, as previously defined.
• Halogen as used herein means chloro, fluoro, bromo and iodo.
• Carbon number refers to the number of carbons in the carbon backbone and does not include carbon atoms occurring in substituents such as an alkyl or alkoxy substituents.
• perhaloalkoxy refers to an alkoxy group, as defined above, in which each hydrogen atoms of the alkyl group has been replaced by a halogen.
• the present invention also provides a process for the preparation of compounds of formula (I). Methods of preparation are shown in Schemes 1 and 2. Starting materials (II) and (III) are allowed to react (Scheme 1) in the presence of an appropriate base such as, but not limited to, n-butyllithium, .sec-butyllithium, tert- butyllithium, lithium diisopropylamimide, lithium, potassium or sodium hexamethyldisilazide, or lithium, potassium or sodium tetramethylpiperadide, Scheme 1
• an appropriate base such as, but not limited to, n-butyllithium, .sec-butyllithium, tert- butyllithium, lithium diisopropylamimide, lithium, potassium or sodium hexamethyldisilazide, or lithium, potassium or sodium tetramethylpiperadide, Scheme 1
• condensation product (IV) wherein R,, R 2 , R 3 , R 9 , and R10 9 , respectively, are as defined hereinbefore, or a group of atoms convertible thereto. Saponification of the ester provides the intermediate carboxylic acid (V).
• Carboxylic acid intermediate (V) can subsequently be coupled (Scheme 2) to the amine of an appropriately derivatized anthranilic acid of the formula (VI) or (VII) utilizing one of the following established coupling procedures (Method A: (COCl) 2 , cat. DMF, CH 9 C1 2 , then add the neat acid chloride to a solution of anthranilic acid in sodium hydroxide; Method B: diisopropylcarbodiimide, DMAP, CH 2 C1 2 , then add methyl anthranilate; or Method C: (COCl) 2 , cat.
• the reactions mentioned above may be carried out in aprotic solvents such as diethyl ether, dichloroethane, dioxane or THF at low to ambient temperatures.
• aprotic solvents such as diethyl ether, dichloroethane, dioxane or THF at low to ambient temperatures.
• sodium hydroxide is used as a base
• other inorganic bases which may also suffice are lithium hydroxide or potassium hydroxide, etc.
• triethylamine may be optionally substituted with any trialkylamine.
• Compounds of Formula (I) may also be prepared using solid phase synthesis.
• the compounds of formula (I), and their pharmaceutically acceptable salts have been found to relax smooth muscle. They are therefore useful in the treatment of disorders associated with smooth muscle contraction, disorders involving excessive smooth muscle contraction of the urinary tract (such as incontinence), or of the gastro-intestinal tract (such as irritable bowel syndrome), asthma, and hair loss.
• the compounds of formula (I) are active as potassium channel activators which render them useful for treatment of peripheral vascular disease, congestive heart failure, stroke, anxiety, cerebral anoxia and other neurodegenerative disorders.
• compounds of formula (I) may also be active as chloride channel blockers, which again renders them useful for treatment of the above stated disorders.
• Compounds of the present invention are characterized by their potent smooth muscle relaxing properties in vitro.
• the compounds of this invention exert their smooth muscle relaxatory activity via activation of potassium channels and/or blocking of chloride channels.
• Members of this series are expected to be active in vivo as evidenced by in vivo activity shown in a model of rat bladder hypertrophy (Table II).
• the compounds of the present invention are unique in that they possess intrinsic selectivity for bladder tissue over vascular tissue as demonstrated by bladder/aorta IC 50 ratios (Table 1). Comparative compound, Tranilast ® was shown not to be a potent or bladder selective smooth muscle relaxant.
• the present invention accordingly provides a pharmaceutical composition which comprises a compound of this invention in combination or association with a pharmaceutically acceptable carrier.
• the present invention provides a pharmaceutical composition which comprises an effective amount of a compound of this invention and a pharmaceutically acceptable carrier.
• compositions are preferably adapted for oral administration. However, they may also be adapted for other modes of administration, for example, parenteral administration for patients suffering from heart failure or intransally for patients suffering from asthma.
• a composition of the invention is in the form of a unit dose.
• Suitable unit dose forms include tablets, capsules and powders in sachets or vials.
• Such unit dose forms may contain from 0.1 to 100 mg of a compound of the invention and preferably from 2 to 50 mg.
• Still further preferred unit dosage forms contain 5 to 25 mg of a compound of the present invention.
• the compounds of the present invention can be administered orally at a dose range of about 0.01 to 100 mg/kg or preferably at a dose range of 0.1 to 10 mg/kg.
• Such compositions may be administered from 1 to 6 times a day, more usually from 1 to 4 times a day.
• compositions of the invention may be formulated with conventional excipients, such as a filler, a disintegrating agent, a binder, a lubricant, a flavoring agent and the like. They are formulated in conventional manner, for example, in a manner similar to that used for known antihypertensive agents, diuretics and ⁇ - blocking agents.
• the present invention further provides a compound of the invention for use as an active therapeutic substance.
• Compounds of formula (I) are of particular use in the induction of smooth muscle relaxation.
• the present invention further provides a method of treating smooth muscle disorders in mammals including man, which comprises administering to the afflicted mammal an effective amount of a compound or a pharmaceutical composition of the invention.
• the acid chloride was then added to a homogeneous solution of anthranilic acid (1.19 g, 8.69 mmol) in 2.5 N aqueous NaOH (6.95 mL, 17.4 mmol) at 5°C, resulting in the instantaneous formation of a white precipitate.
• the reaction mixture was then warmed to RT, whereupon it was diluted with a minimal amount of water to facilitate stirring, which was continued for an additional 1.5 h.
• the mixture was acidified to pH 2 by addition of concentrated HCl (1.63 mL), diluted with 2.0 N HCl, and stirred for 1.5 h.
• Step 2 was prepared the title compound (781 mg, 52%): mp 215-216°C; 1H NMR DMSO-d 6 ) ⁇ 13.52 (br s, 1 H), 11.33 (s, 1 H), 8.59 (dd, 1 H), 8.01 (dd, 1 H), 7.58-7.72 (m, 6H), 7.18 (ddd, 1 H), 6.94 (d, 1 H); IR (KBr) 3331, 3123, 3072, 1705, 1670, 1626, 1606, 1583, 1526, 1487, 1449, 1413, 1402, 1389, 1261, 1224, 1146, 1071, 966, 882, 816, 750, 656 cm 1 ; MS (m/z) 345/347 [M + ].
• the aqueous phase was extracted again with EtOAc (50 mL); the combined organic phases were washed with 2% NaHCO 3 (2 x 25 mL), then diluted with hexanes (25 mL), and washed consecutively with water (3 x 25 mL) followed by brine (25 mL).
• the smooth muscle relaxing activity of the compounds of this invention was established in accordance with standard pharmaceutically accepted test procedures with representative compounds as follows: Sprague-Dawley rats (150-200 g) are rendered unconscious by CO2 asphyxiation and then euthanized by cervical dislocation. The bladder is removed into warm (37 deg.C) physiological salt solution (PSS) of the following composition (mM): NaCl, 118.4; KC1, 4.7; CaCl 2 , 2.5; MgSO 4 , 4.7; H 2 O, 1.2; NaHCO 3 , 24.9; KH 2 PO 4 , 1.2; glucose, 11.1; EDTA, 0.023; gassed with 95% O 2 ; 2/5% CO 2 ; pH 7.4.
• PES physiological salt solution
• the bladder is opened and then cut into strips 1-2 mm in width and 7-10 mm in length.
• the strips are subsequently suspended in a 10 mL tissue bath under an initial resting tension of 1.5 g.
• the strips are held in place by two surgical clips one of which is attached to a fixed hook while the other is attached to an isometric force transducer.
• the preparations which usually exhibit small spontaneous contractions, are allowed to recover for a period of 1 hour prior to a challenge with 0.1 ⁇ M carbachol.
• the carbachol is then washed out and the tissue allowed to relax to its resting level of activity. Following a further 30 min period of recovery an additional 15 mM KC1 are introduced into the tissue bath.
• the isometric force developed by the bladder strips is measured using a concentration required to elicit 50% inhibition of pre-drug contractile activity (IC50 concentration) and is calculated from this concentration-response curve.
• IC50 concentration concentration required to elicit 50% inhibition of pre-drug contractile activity
• the maximum percentage inhibition of contractile activity evoked by a test compound is also recorded for concentrations of test compound less than or equal to 30 ⁇ M. The results of this study are shown in Table I.
• Tranilast ® ⁇ 2 14.4 ⁇ 4.5 5 15.59 ⁇ 8.96 1.08
• Tranilast is (E)-2-[3-(3,4-Dimethoxy-phenyl)-acryloylamino]-benzoic acid. *Percent inhibition at 30 ⁇ M
• Compounds are dissolved in PEG-200 and administered by gastric gavage or intraveneously in a volume of 5 mL/kg.
• All drugs are administered at the arbitrary dose of 10 mg/kg p.o. to groups of 4 rats.
• the animals are anesthetized with halothane.
• halothane Through a midline incision the bladder and urethra are exposed and a ligature of 4-0 silk is tied around the proximal urethra in the presence of a stainless steel rod (1 mm diameter) to produce a partial occlusion. The rod is then removed. The abdominal region is closed using surgical staples and each rat receives 150,000 units of bicillin C-R. The animals are allowed six weeks to develop sufficient bladder hypertrophy. After six weeks, the ligature is removed under halothane anesthesia and a catheter (PE 60) with a cuff is placed in the dome of the bladder and secured with a purse string suture.
• PE 60 catheter
• the catheter is tunneled under the skin and exteriorized through an opening in the back of the neck.
• the abdominal incision is sutured and the free end of the catheter sealed.
• the rats receive an injection of bicillin C-R (150000 units/rat).
• Two days later the animals are used in cystometrical evaluations.
• the animals are placed in the metabolic cages and the catheter is attached (using a "T" connector) to a Statham pressure transducer (Model P23Db) and to a Harvard infusion pump.
• a plastic beaker attached to a force displacement transducer (Grass FTO3) is placed under the rat's cage to collect and record urine volume.
• Basal bladder pressure the lowest bladder pressure during cystometry
• Threshold pressure bladder pressure immediately prior to micturition
• the mean value of each variable is calculated before and after compound administration. For each compound the changes in the variables measured are compared to the values obtained before treatment and expressed as percent inhibition. The data are also subjected to 2-way analysis of variance to determine significant (p ⁇ 0.05) changes in the variable measured.
• the most characteristic finding in this rat model is spontaneous bladder contractions which develop during filling.
• the compounds which inhibit spontaneous contractions by at least 50% at 10 mg/kg p.o. or i.v. (arbitrary chosen dose) are considered active.
• Rat detrusor cells were isolated in a manner previously described for guinea-pig detrusor (Sheldon and Argentieri, 1995). Male Sprage-Dawley rats (200-400 grams) were euthanized by CO 2 inhalation and exsanguination. Their urinary bladders were rapidly removed and placed in 37°C physiological solution with the following composition (mM): Na glutamate (80.0), NaCl (54.7), KC1 (5.0), NaHCO 3 (25.0), MgCl 2 » 2H 2 O (2.5), D-glucose (11.8) and CaCl 2 (0.2) gassed with CO 2 -O 2 , 95%/5% for a final pH of 7.4.
• the dome of the bladder was isolated from the trigon region and the mucosa was removed. This tissue was then cut into 2-3 mm wide strips and placed in fresh buffer for 1 hour. Tissues were then transferred into 10.0 ml of an isolation buffer containing the above composition plus collagenase type VIII (1.0 mg/ml) and pronase (0.25 mg/ml). After 10 minutes the isolation buffer was replaced with fresh isolation buffer for an additional 10 min. The tissue was then washed 3 times in fresh collagenase and pronase free solution and stored at room temperature until studied.
• Cells for study were prepared by triturating 1-2 pieces of detrusor tissue in 2 mL of fresh isolation buffer for 5 minutes with a polished Pasteur pipette, (tip diameter -1.5 ml) attached to a modified Harvard Respirator pump (Harvard Apparatus, Southnatic, MA) at a rate of 20x/min. with an approximate volume of 5 mL.
• a polished Pasteur pipette (tip diameter -1.5 ml) attached to a modified Harvard Respirator pump (Harvard Apparatus, Southnatic, MA) at a rate of 20x/min. with an approximate volume of 5 mL.
• PES physiological salt solution
• Example 1 All cells exposed to Example 1 (dissolved in DMSO and further diluted in PSS) responded with an increase in outward current that was reversible upon washout (Figure 1). Exposure to the sulfonyl urea glyburide had no effect (data not shown). Previous studies have shown that this current is iberiotoxin sensitive which is a hallmark of the maxi K + channel. It is concluded therefore, that the compound of Example 1 is capable of activating the maxi K + current in isolated rat bladder cells consistent with its ability to inhibit KCl-induced contractions of intact tissue in vitro.
• NHBE Normal human bronchial epithelial cells
• the normal external solution contained (in mM): N-methyl d-Glucamine chloride (NMDGC1). 140, MgCl 2 2, CaCl 2 2 and 4-(2-hydroxyethyl)-l-piperazine ethanesulfonic acid (HEPES) 10.
• the normal pipette solution contained (in mM): NMDGC1. 125, HEPES 10, MgCl 2 2, ethylenebis(oxonitrilo)tetraacetate (EGTA) 2, and ATP Mg 5, the pH adjusted to 7.3 with TRIS.
• glutamic acid E C1 approximately -31 mV according Nernst equation).
• the osmolarity for standard external solution was about 280 mOsm. Hypotonic solutions were obtained by diluting the normal external solution with distilled water. The whole cell voltage clamp recording techniques were utilized to record membrane ionic currents. The volume of the recording chamber was 1 ml and the rate of superfusion was 4 ml/min. Membrane currents were recorded using an Axopatch 200A Patch Clamp Amplifier (Axon Instruments, Inc., Foster City, CA). Under voltage clamp conditions, the cells were stimulated at 0.2 Hz to be sure channels were completely recovered between two voltage steps.
• FIG. 2 shows that Example 1 inhibited swelling induced chloride channel current on guinea pig bladder cell.
• A current traces from -100 to 60 mV were recorded in the isotonic, low osmolarity (210 mOsm) and after application of 20 ⁇ M Example 1 in same cell.
• Example 1 significantly reduced swelling induced chloride current.
• Panel B is four superimposed ramp test traces recorded in the presence of control, hypotonic, Example 1 and washout solutions.
• the C panel depicts the time course of the effect of Example 1 on I ci swelhng at 60 mV monitored from the same cell. The figure shows that I cl swellmg reached a steady state within 15 min following the addition of 210 mOs solution and with a 67 % block in presence of 20 ⁇ M Example 1.
• the compounds of this invention have a pronounced effect on smooth muscle contractility and are useful in the treatment of gastrointestinal, cardiovascular, metabolic and central nervous system disorders such as urinary incontinence, irritable bladder and bowel disease, asthma, hypertension, stroke, cystic fibrosis, cardiac arrhythmias, peripheral vascular disease, congestive heart failure, anxiety neurodegenerative disease, and similar diseases as mentioned above, which are amenable to treatment with potassium channel activating and/or chloride channel blocking compounds by administration, orally, parenterally, or by aspiration to a patient in need thereof.
• gastrointestinal, cardiovascular, metabolic and central nervous system disorders such as urinary incontinence, irritable bladder and bowel disease, asthma, hypertension, stroke, cystic fibrosis, cardiac arrhythmias, peripheral vascular disease, congestive heart failure, anxiety neurodegenerative disease, and similar diseases as mentioned above, which are amenable to treatment with potassium channel activating and/or chloride channel blocking compounds by administration, orally, parenterally, or by aspiration to a

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• Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
• Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
• Pyridine Compounds (AREA)

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• 1998
  • 1998-08-03 WO PCT/US1998/016099 patent/WO1999007669A1/en not_active Application Discontinuation
  • 1998-08-03 BR BR9811845-5A patent/BR9811845A/pt not_active Application Discontinuation
  • 1998-08-03 CA CA002297412A patent/CA2297412A1/en not_active Abandoned
  • 1998-08-03 EP EP98938288A patent/EP1003712A1/en not_active Withdrawn
  • 1998-08-03 AU AU86845/98A patent/AU8684598A/en not_active Abandoned
  • 1998-08-03 JP JP2000507205A patent/JP2001513526A/ja active Pending
  • 1998-08-03 CN CN98809866A patent/CN1273579A/zh active Pending

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