Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
  • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

• This invention relates to control of ocular development in general and, more particularly, to the treatment of the eye to prevent and/or arrest the development of myopia (nearsightedness).
• myopia nearsightedness
• myopia i.e., an elongation of the eye along the visual axis.
• myopia afflicts 10% to 75% of the youth of the world, depending upon race, geographic distribution and level of education.
• Myopia is not a trivial maldevelopment of the eye. In its pathologic form, the sclera continues to grow and as result the retina stretches and degenerates resulting in permanent blindness.
• Cycloplegics are topically administered drugs that relax the ciliary muscle of the eye, which is the muscle that focuses the eye by controlling lens dimensions.
• the classic cycloplegic drug is the belladonna alkaloid atropine, available for over a century.
• Atropine is a long-acting non-specific antimuscarinic agent that antagonizes the action of the neurotransmitter acetylcholine (ACh) at autonomic effector cells innervated by postganglionic cholinergic nerves of the parasympathetic nervous system.
• ACh neurotransmitter acetylcholine
• Atropine is impractical in that it causes mydriasis (increase of pupil size) and its action on the ciliary muscle to inhibit ocular focusing impairs near visual work like reading.
• the receptors in the iris and ciliary muscle responsible for the side effects of atropine are of the M3 subtype.
• studies have shown that muscarinic receptors in the retina of a variety of non-human species are comprised of ml , m2 and m4 subtypes. Accordingly, a muscarinic antagonist with low m3 activity would be efficacious in prevention of the development of myopia without the undesirable side effects associated with the use of atropine.
• retinal concentrations of dopamine were found to be reduced during such image deprivation and the ocular administration of a dopamine-related agent, e.g., apomorphine, a dopamine agonist, was found to inhibit or actually prevent the axial enlargement of the eye under conditions ordinarily leading to such enlargement.
• a dopamine-related agent e.g., apomorphine, a dopamine agonist
• Cholinergic receptors are proteins embedded in the wall of a cell that respond to the chemical acetylcholine. Particularly, it is now known that the cholinergic receptors are subdivided into nicotinic and muscarinic receptors and that the muscarinic receptors are not all of the same type. Recent literature indicates that there are at least five types of cholinergic muscarinic receptors (types ml through m5). Receptors of type ml are those present in abundance and thought to be enriched in the brain neural tissue and neural ganglia. The other receptors are concentrated in other tissues such as the heart, smooth muscle tissue or glands.
• pirenzepine (Gastrozepin, LS 519) 5, 11 -Dihydro-l l-[4-methyl-l -piperazinyl) acetyl]-6H-pyrido[2,3-b] benzodiazepin-6-one, and its dihydrochloride are anticholinergic, antimuscarinic, and relatively selective for Ml receptors. See U.S. Pat. No. 5,122.522. It is also known that 4-DAMP (4-diphenylacetoxy-N-methylpiperadine methiodide) is a relatively selective antagonist for smooth muscle (ordinarily called M3 type but variously called type M2 or M3, as the current classification of receptors is in flux).
• Pirenzepine being primarily an Ml antagonist, inhibits axial elongation, but is far less effective at pupil dilation than atropine or another cycloplegic agent. This makes it possible to suppress the development of myopia without dilating the pupil and paralyzing the accommodation activity of the ciliary muscle. Additionally, the administration of a drug topically into the eye of a developing child for a long period of time makes it desirable to have a minimal likelihood of sensitization of the eye. Pirenzepine and atropine test positive in sensitization assays and this is an undesirable side effect.
• This invention is concerned with novel l ,3-dihydro-l - ⁇ 1 - [piperidin-4-yl]piperidin-4-yl ⁇ -2H-benzimidazol-2-ones and 1,3- dihydro- 1 - ⁇ 4-amino- 1 -cyclohexyl ⁇ -2H-benzimidazol-2-ones, their compositions and method of use.
• the novel compounds are selective muscarinic antagonists of the ml . m2, and m4 subtypes with low activity at the m3 subtype.
• the compounds have good ocular penetration (bioavailability) when dosed as a 0.1 - 2% aqueous solution, preferably a 0.5-2% solution.
• the compounds are effective for the treatment and/or prevention of myopia.
• novel compounds of this invention are represented by the structural formula:
• P is phosphorus
• W is O or H2
• B is H, Me, Et, Pr, iPr, CH2OH, C ⁇ 2Me, C ⁇ 2Et,
• Q is phenyl or heterocycle ring unsubstimted or substituted with Me, Et, Pr, Bu, hydroxyl, alkoxy, F, Cl, Br, I. alkylsulfonyl, phenyl or heterocychc;
• heterocycle or heterocychc represents a stable 5- to 7- membered monocyclic heterocychc ring, which is either saturated or unsaturated, and which consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S, and including any bicyclic group in which any of the above defined heterocychc rings is fused to a benzene ring.
• the heterocychc ring may be attached at any heteroatom or carbon atom which results in the creation of a stable strucmre.
• heterocychc rings examples include pyridine, pyrazine, pyrimidine, pyridazine, triazine, imidazole, pyrazole, triazole, quinoline. isoquinoline, quinazoline, quinoxaline, phthalazine, oxazole, isoxazole, thiazole, isothiazole, thiadiazole, oxadiazole, pyrrole, furan. thiophene, hydrogenated derivatives of these heterocyles such as piperidine, pyrrolidine, azetidine, tetrahydrofuran. and N-oxide derivatives of heterocyles containing basic nitrogen.
• alkyl is intended to include both branched and straight chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms (Me is methyl, Et is ethyl, Pr is propyl, Bu is butyl).
• alkoxy represents an alkyl group of indicated carbon atoms attached through an oxygen linkage.
• alkylamino represents an alkyl group of indicated carbon atoms attached through a nitrogen atom linkage.
• dialkylamino represents two alkyl groups of indicated carbon atoms attached through a nitrogen atom linkage.
• small alkyl is intended to indicate those alkyls with Cl to C4 carbon atoms, either branched or linear in connection.
• alkylsulfonyl represents an alkyl group of indicated carbon atoms attached through an sulfonyl (SO2) linkage.
• halo represents fluoro, chloro, bromo or iodo.
• the pharmaceutically acceptable salts of the compounds of formula I include the conventional non-toxic salts or the quarternary ammonium salts of the compounds of formula I formed e.g. from non-toxic inorganic or organic acids.
• such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.
• the pharmaceutically acceptable salts of the present invention can be synthesized from the compounds of formula I which contain a basic or acidic moiety by conventional chemical methods. Generally, the salts are prepared by reacting the free base or acid with stoichiometric amounts or with an excess of the desired salt- forming inorganic or organic acid or base in a suitable solvent or various combinations of solvents.
• the compounds of the present invention may have asymmetric centers and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers, including optical isomers, being included in the present invention.
• novel compounds of this invention are prepared by the following non-limiting procedures:
• the reaction is preferably carried out at room temperature (20- 30°C) at a pH in the range of 2-7 (acidic) by the addition of glacial acetic acid or hydrochloric acid.
• the reaction is preferably carried out at room temperature (20- 30°C) at a pH in the range of 2-7 (acidic) by the addition of glacial acetic acid or hydrochloric acid.
• Deprotection by the usual methods hydrogenation or acidic hydrolysis followed by basification
• the preferred compounds can be obtained after isolation and purification.
• the more preferred trans -isomer is either formed selectively by control of the reaction conditions, or separated by chromatography.
• novel starting materials of the formula (IV) can be obtained by conventional procedures such as those described in the Examples section.
• the starting materials of the formula (V) are either commercially available or can be obtained by conventional procedures such as those described in the Examples section.
• the reaction is preferably carried out at room temperature (20- 30°C) at a pH in the range of 2-7 (acidic) by the addition of glacial acetic acid or hydrochloric acid.
• Deprotection by the usual methods hydrogenation or acidic hydrolysis followed by basification
• the more preferred trans -isomer is either formed selectively by control of the reaction conditions, or separated by chromatography.
• novel starting materials of the formula (IV) can be obtained by conventional procedures such as those described in the Examples section.
• the starting materials of the formula (VI) are either commercially available or can be obtained by conventional procedures such as those described in the Examples section.
• the reaction is preferably carried out at room temperature (20-
• Deprotection by the usual methods hydrogenation or acidic hydrolysis followed by basification
• the more preferred trans- isomer is either formed selectively by control of the reaction conditions, or separated by chromatography.
• novel starting materials of the formula (VII) can be obtained by conventional procedures such as those described in the Examples section.
• the starting materials of the formula (ID) are either commercially available or can be obtained by conventional procedures such as those described in the Examples section.
• the compounds of the present invention include, but are not limited by the following Tables (1-4).
• the selectivity of the compounds can be measured by radioligand displacement from ml-m5 receptors expressed in Chinese hamster ovary cells (CKO) as described in the Examples section.
• the functional activity of the compounds can be assessed by measuring the agonist induced contractile response on muscle tissue from rabbit vas deferens (M l ), the guinea pig left atria (M2), or the guinea pig ileum (M3) as described in the Examples section.
• the functional activity at the human muscarinic receptors can be assessed by measuring agonist induced phosphoinositide hydrolysis in CHO cells expressing the human ml and m3 receptors or agonist inhibition of foskolin-stimulated adenylate cyclase activity in CHO cells expressing the human m2 receptor as described in the Examples section.
• the instant compounds of this invention are useful in treating and/or preventing the development of myopia.
• Therapy to inhibit axial-elongation myopia during maturation can be administered by the use of the agent in eye drops. Indeed, in the vast majority of cases, treatment agents are administered to human eyes by the application of eye drops. Eye drops are typically made up at a concentration of active agent between about 0.1 and 2% in the ophthalmic medium. A 0.5%-2% solution of the instant muscarinic antagonist in water would be a likely concentration for clinical use. A pH of about 4.5 to about 7.5 is expected to be acceptable as an ophthalmic drop and practical in terms of known solubility and stability of piperidines. Phosphate buffering is also common for eye drops and is compatible with the instant muscarinic antagonist.
• a common regimen for application of eye drops is one to three times a day spaced evenly throughout waking hours. More effective agents may require fewer applications or enable the use of more dilute solutions. Alternatively, ointments and solid inserts are now coming into increased use in clinical practice. These aid the ocular penetration of the drug. It is, of course, also possible to administer the above-described active agents in therapeutically effective amounts and dosages in pills, capsules, or other preparations of systemic administration.
• amblyopia is evidenced by poor visual acuity in the eye resulting in poor visual performance. Normally, visual acuity improves during maturation. It is known that amblyopia may occur in humans from unknown causes or as part of strabismus. It is possible that administration of therapeutically effective amounts and dosages of the instant muscarinic antagonist might prevent or inhibit the development of permanent or persistent amblyopia in maturing humans with decreased likelihood of sensitization of the eye. It is also possible that humans who have already developed amblyopia from other or even unknown causes might be aided by similar therapeutic treatment with the aforementioned agents.
• Step 1 A mixture of 100 g 4-piperidone hydrochloride hydrate, 1 L ether, 300 mL of water, 500 mL of saturated aqueous
• Step 2 A mixture of 20.6 g N-t-butyloxycarbonyl-4-piperidone, 15 g of 4-(2-oxo-l-benzimidazolinyl)piperidine, 300 mL of 1 ,2- dichloroethane, 4.2 mL of glacial acetic acid and 24 g of sodium triacetoxyborohydride was stirred at room temperature for 48 h. The reaction mixture was poured into 500 mL chloroform and 500 mL saturated aqueous Na 2 C ⁇ 3 and the layers separated. The aqueous layer was extracted with 2 X 250 mL of chloroform and the combined organic layers dried over MgS ⁇ 4 and concentrated under reduced pressure.
• Step 3 A stirred solution of 4 g of 1 ,3-dihydro- 1 - ⁇ 1 -[ 1 -(t- butyloxycarbonyl)piperidin-4-yl]piperidin-4-yl ⁇ -2H-benzimidazol-2-one in 150 mL of 1 N HC1 was heated to reflux for 4 h, cooled and concentrated to dryness. After drying overnight under vacuum, there was obtained 4.0 g of 1 ,3-dihydro- 1 - ⁇ 1 -[ 1 -piperidin-4-y l]piperidin-4- yl ⁇ -2H-benzimidazol-2-one dihydrochloride salt as a white solid.
• Step 4 To a stirred solution of 6 g of 1 ,3-dihydro- 1 - ⁇ 1 -[ 1 - piperidin-4-yl]piperidin-4-yl ⁇ -2H-benzimidazol-2-one dihydrochloride salt and 20 mL of triethylamine in 500 mL of dichloromethane was added 3 g of pyrimidine-5-carboxylic acid chloride. After 2 h, 50 mL of dilute aqueous ammonia was added and the mixture stirred for an additional 30 min. The organic layer was separated, the aqueous layer extracted with two additional 200 mL portions of chloroform and the combined organic extracts dried over MgSC and concentrated under reduced pressure.
• Step 1 A mixture of 69 g of 4-chloro-3-nitro-toluene, 50 g of ethyl 4-amino-l -piperidinecarboxylate, 24 g of sodium carbonate, 0.1 g of sodium iodide and 120 mL of cyclohexanol was heated to 150°C for 72 h. After cooling the cyclohexanol was distilled off under reduced pressure and the residue partitioned between 1 L of ethyl acetate and 1 L of water. The organic extract was dried over MgSC ⁇ i and concentrated under reduced pressure.
• Step 2 A mixture of 8.23 g of ethyl 4-(4-methyl-2-nitroanilino)- 1 -piperidinecarboxylate, 200 mL of tetrahydrofuran , 225 mL of ethanol and 2 g of 5% platinum on carbon was stirred under an atmosphere of hydrogen for 7 h. The catalyst was filtered off and the filtrate concentrated to to a thick oil.
• Step 3 A mixture of 5 g of ethyl 4-(5-methyl-2-oxo-l - benzimidazolinyl)piperidine-l -carboxylate and 20 mL of 2N NaOH was heated under reflux for 12 h. The resulting solution is cooled and stirred for for 30 minutes with 5 g of ammonium chloride and extracted with three 200 mL portions of chloroform. The combined organic extracts were dried over MgS ⁇ 4, concentrated under reduced pressure and triturated with ether. The solid product 4-(5-methyl-2-oxo-l- benzimidazoliny piperidine weighed 3.5 g after drying.
• Step 4 A mixture of 4.5 g N-t-butyloxycarbonyl-4-piperidone, 4.7 g of 4-(5-methyl-2-oxo-l -benzimidazolinyl)piperidine, 65 mL of 1 ,2-dichloroethane, 1.3 mL of glacial acetic acid and 6.4 g of sodium triacetoxyborohydride was stirred at room temperature for 48 h. The reaction mixture was poured into 500 mL chloroform and 500 mL saturated aqueous Na 2 CO ? and the layers separated. The aqueous layer was extracted with 2 X 250 mL of chloroform and the combined organic layers dried over MgS04 and concentrated under reduced pressure.
• Step 5 A stirred solution of 6.18 g of 5-methyl- 1 ,3-dihydro-l - ⁇ 1 -[ 1 -(t-butyloxycarbonyl)piperidin-4-yl]piperidin-4-yl ⁇ -2H- benzimidazol-2-one in 200 mL of IN HCl was heated to reflux for 4 h, cooled and concentrated to dryness. After drying overnight under vacuum, there was obtained 6 g of 5-methyl- l ,3-dihydro-l - ⁇ 1-[ 1 - piperidin-4-y llpiperidin-4-y 1 ⁇ -2H-benzimidazol-2-one dihydrochloride salt as a white solid.
• Step 6 To a stirred solution of 6 g of 5-methyl- 1 ,3-dihydro-l -
• reaction mixture was diluted with 100 mL of chloroform and the resulting solution was washed with 10 mL of saturated Na2C03, dried over MgSC and concentrated under reduced pressure.
• Preparative thin layer chromatography on silica gel eluting with 90:10:5 CHCl3:CH3 ⁇ H: cone.
• the dihydrochloride salt was recystaUized from ethanol: Analysis calculated for C23H27N5O2-2HC1-0.5 C2H-7O C: 57.32, H: 6.43, N: 13.97 found C: 57.32, H: 6.57, N: 13.77.
• Step 1 A mixture of 10 g of 1 ,4-cyclohexaned ⁇ one mono-ethyleneketal, 13.8 g of 1,2-phenylenediamine, 180 mL of 1 ,2-dichloroethane, 4 mL of glacial acetic acid and 19 g of sodium triacetoxyborohydride was stirred at room temperature for 48 h. The reaction mixture was poured into 200 mL chloroform and 200 mL saturated IN NaOH and the layers separated. The aqueous layer was extracted with 2 X 50 mL of chloroform and the combined organic layers dried over MgS ⁇ 4 and concentrated to dryness under reduced pressure.
• Step 2 A mixture of 7 g of the ethylene ketal of l ,3-dihydro- l -(4- oxocyclohexyl)-2H-benzimidazol-2-one, 100 mL of glacial acetic acid, 50 mL of water and 50 mL of cone. HCl was heated under reflux for 12 h. The mixture was concentrated under reduced pressure, diluted with 100 mL of water and extracted into 3 X 200 mL of CHC . The combined organic extracts were washed with 100 mL of water, 100 mL of saturated Na2C03. dried over MgS04 and concentrated under reduced pressure.
• Step 3 A mixture of 1.5 g of l,3-dihydro-l -(4-oxocyclohexyl)-2H- benzimidazol-2-one, 1.21 g of tert-butyl-1 -piperazinecarboxylate, 20 mL of 1,2-dichloroethane, 0.4 mL of acetic acid and 1.8 g of sodium triacetoxyborohydride was stirred at room temperature for 48 h. The reaction mixture was poured into 500 mL chloroform and 500 mL saturated aqueous Na2C03 and the layers separated.
• Step 4 A stirred solution of 0.52 g of trans -l ,3-dihydro-l - ⁇ 4-[4-(rert -butyloxycarbonyl)piperazin- 1 -yl]- 1 -cyclohexyl ⁇ -2H-benzimidazol-2- one in 15 mL of IN HCl was heated to reflux for 1 h, cooled, basified to pH 10 with 6N NaOH and extracted 2 x 50 mL of CHCI3. The combined extracts were dried over MgS04 and concentrated under reduced pressure.
• Step 5 To a stirred solution of 0.044 g of trans - 1 ,3-dihydro- 1 - ⁇ 4-( 1 - piperazinyl)- 1 -cyclohexyl ⁇ -2H-benzimidazol-2-one and 20 mL of triethylamine in 3 mL of dichloromethane was added 0.03 g of pyrimidine-5-carboxylic acid chloride. After 2 h, 5 mL of dilute aqueous ammonia was added and the mixture stirred for an additional 30 min. The organic layer was separated, the aqueous layer extracted with two addtional 20 mL portions of chloroform and the combined organic extracts dried over MgS ⁇ 4 and concentrated under reduced pressure.
• Step 1 To a stirred solution of 2.0 g of ethyl 4-amino- 1 - piperidinecarboxylate and 2 mL of triethylamine in 50 mL of chloroform was added 2.07 g of nicotinoyl chloride hydrochloride.
• Step 2 A mixture of 1.2 g of the ethylene ketal of 4-(3- pyridinecarbonylamino)- 1 -piperidinecarboxylate and 20 mL of 6N HCl was heated under reflux for 6 h. The mixture was cooled, extracted with 20 mL of dichloromethane, then basified with 6N NaOH and extracted with 3 X 50 mL of chloroform.
• Step 3 A mixture of 0.13 g of l ,3-dihydro-l -(4- oxocyclohexyl)-2H-benzimidazol-2-one, 0.12 g of 4-(3- pyridinecarbonylamino)piperidine, 5 mL of 1 ,2-dichloroethane, 0.04 mL of glacial acetic acid and 0.161 g of sodium triacetoxyborohydride was stirred at room temperature for 48 h. The reaction mixture was poured into 50 mL chloroform and 50 mL saturated aqueous Na 2 C ⁇ and the layers separated. The aqueous layer was extracted with 2 X 25 mL of chloroform and the combined organic layers dried over MgSC.
• the reaction mixture was poured into 10 mL dichloromethane and 10 mL saturated aqueous NaHCC> 3 and the layers separated.
• the aqueous layer was extracted with 2 X 10 mL of dichloromethane and the combined organic layers dried over MgSC and concentrated under reduced pressure.
• Step 1 To a stirred solution of 0.108 g of 3- aminomethylpyridine and 0.280 g of di-2-pyridylcarbonate in 5 mL of dichloromethane was added 0.209 mL of triethylamine. After 12 h, the mixture was diluted with 50 mL of dichloromethane and washed with 50 mL of satutrated sodium bicarbonate, dried over MgSC> 4 and concentrated under reduced pressure. The residue was filtered through a short column of silica gel , eluting with ethyl acetate ( 100 mL) and the eluate concentrated to dryness.
• Step 2 A mixture of 0.108 g of l ,3-dihydro- l- ⁇ l-[ l-piperidin-4- yl]piperidin-4-yl ⁇ -2H-benzimidazol-2-one, 0.070 g of N-(3- pyridylmethyl)-0-2-pyridylurethane and 0.062 mL of triethylamine in 5 mL of dichloromethane was added 2.07 g of nicotinoyl chloride hydrochloride.
• Step 1 A mixture of 1.5 g of l ,3-dihydro-l -(4-oxocyclohexyl)-
• Step 2 A stirred solution of 0.52 g of l ,3-dihydro-l - ⁇ fr ⁇ /w -4-[4- (tert -butylcarbonyOpiperazin- 1 -yl]-l -cyclohexyl ⁇ -2H -benzimidazol-2- one in 15 mL of IN HCl was heated to reflux for 1 h, cooled and basified with 6N NaOH. The basic mixture was extracted with 2 X 50 mL portions of chloroform. The combined organic extracts were dried over MgS04 and concentrated under reduced pressure.
• Step 3 To a stirred solution of 0.044 g of 1,3-dihydro-l- [ trans -4-[ l-piperazinyl]-l -cyclohexyl ⁇ -2H-benzimidazol-2-one and 0.2 mL of triethylamine in 3 mL of dichloromethane was added 0.030 g of pyrimidine-5-carboxylic acid chloride. After 2 h, 5 mL of dilute aqueous ammonia was added and the mixture stirred for an additional 30 min. The organic layer was separated, the aqueous layer extracted with two addtional 20 mL portions of chloroform and the combined organic extracts dried over MgSC ⁇ t and concentrated under reduced pressure.
• Step 1 A mixture of 1.5 g of l,3-dihydro- l -(4-oxocyclohexyl)- 2H-benzimidazol-2-one, 1.12 g of ethyl 4-amino-l - piperidinecarboxylate, 20 mL of 1 ,2-dichloroethane, 0.40 mL of glacial acetic acid and 1.79 g of sodium triacetoxyborohydride was stirred at room temperature for 48 h. The reaction mixture was poured into 50 mL chloroform and 50 mL saturated aqueous Na 2 C ⁇ and the layers separated.
• Step 2 A stirred solution of 1.2 g of l,3-dihydro-l - ⁇ trat « -4-[ l- ethoxycarbonyl-4-piperidinylamino]- 1 -cyclohexyl ⁇ -2H-benzimidazol-2- one in 20 mL of 6N HCl was heated to reflux for 12 h, cooled and basified with 6N NaOH. The basic mixture was extracted with 2 X 50 mL portions of chloroform. The combined organic extracts were dried over MgS ⁇ 4 and concentrated under reduced pressure.
• Step 3 To a stirred solution of 0.050 g of l,3-dihydro-l- ⁇ fr ⁇ ns -4-[4-piperidinylamino]-l -cyclohexyl ⁇ -2H-benzimidazol-2-one and 0.023 mL of triethylamine in 1.5mL of dichloromethane was added 0.024 g of nicotinoyl chloride hydrochloride. After 12 h, 20 mL of saturated sodium carbonate was added, the organic layer was separated, and the aqueous layer extracted with two addtional 20 mL portions of chloroform. The combined organic extracts dried over MgS ⁇ 4 and concentrated under reduced pressure.
• Step 2 To a stirred solution of oxalyl chloride (0.83 g) in dry dichloromethane (15 mL) at -60 °C was added dimethylsulfoxide (1.0 mL) in dichloromethane (3 mL) dropwise. The reaction mixture was stirred at -60 °C for 10 min, then N-tert -butyloxycarbonyl-tratzs -4- aminocyclohexanol (1.28 g) in dichloromethane (70 mL) was added over 15 min. The resulting mixture was stirred at -60 °C for a further 15 min, then triethylamine (4.2 mL) was added and the solution was allowed to warm to room temperature.
• Step 3 A mixture of N-tert -butyloxycarbonyl-4- aminocyclohexanone (1.32 g), 4-(2-keto-l-benzimidazolinyl)piperidine ( 1.48 g), sodium triacetoxyborohydride (1.97 g), acetic acid (0.35 mL), 1 ,2-dichloroethane (50 mL) and tetrahydrofuran (60 mL) was stirred at room temperature for 3 days. Saturated aqueous Na2C03 (30 mL) and H2O (30 mL) were added and the mixture was extracted with dichloromethane (2 x 100 mL). The combined organic extracts were concentrated to dryness under reduced pressure.
• Step 4 The mixture of cis and trans isomers isolated in Step 3 (0.85 g) was stirred in ethyl acetate (100 mL) at 0 °C and gaseous HCl was bubbled in until the mixture was saturated with the acid. Stirring was continued for 20 min, then the reaction mixture was concentrated in vacuo. The residue was dissolved in chloroform, washed with saturated aqueous Na2C ⁇ 3, and the organic layer was dried over MgS ⁇ 4 and evaporated under reduced pressure.
• Step 5 To the mixture of cis and trans isomers isolated in Step 4 (90 mg) was added tetrahydrofuran (3 mL), dimethylformamide (2 mL), triethylamine (0.12 mL) and nicotinoyl chloride hydrochloride (76 mg). The reaction mixture was stirred at room temperature for 18 h then concentrated to dryness under reduced pressure.
• Step 1 To 1 mL of benzyl alcohol cooled in an ice bath was added 0.1 g of 60% sodium hydride oil dispersion. After 15 min., 0.1 g of 6-chloro-2-pyrazinoic acid was added. The mixture was stirred at room temperature forl h, then acidified with 2 mL of 1 N HCl. The mixture was cooled in an ice bath and the white precipitate collected by filtration.
• Step 2 From 1 ,3-dihydro- 1 - ⁇ 1 -[ 1 -piperidin-4-yl]piperidin-4- yl ⁇ -2H-benzimidazol-2-one dihydrochloride salt and 6-benzyloxy-2- pyrazinoic acid using the procedure described for Example 3, there was obtained l ,3-dihydro-l- ⁇ l-[ l-(6-benzyloxy-2- pyrazinecarbonyl)piperidin-4-yl]piperidin-4-yl ⁇ -2H-benzimidazol-2-one as a white solid: ⁇ H NMR (400 MHz, CDCI 3 ) 9.98 (s, IH), 8.46 (m, IH), 8.36 (m, IH), 7.47-7.05 (m, 9H), 5.43 (m, 2H), 4.81 (br s, IH), 4.39 (br s, IH), 3.9 (br s, IH), 2.85 (m, I
• Step 1 A mixture of 0.15 g of l,3-dihydro-l- ⁇ l-[l-(6-chloro-2- pyrazinecarbonyl)piperidin-4-yl]piperidin-4-yl ⁇ -2H-benzimidazol-2- one, 0.26 g of sodium azide and 5 mL of anhydrous N,N- dimethylformamide was stirred for 24 h, then concentrated to dryness under reduced pressure. The residue was partitioned between 50 mL of chloroform and 5 mL of saturated sodium carbonate and the organic extracts dried over MgSC .
• Step 2 The crude l ,3-dihydro-l- ⁇ l -[l-(6-azido-2- pyrazinecarbonyl)piperidin-4-yl]piperidin-4-yl ⁇ -2H-benzimidazol-2-one was hydrogenated under 1 atm. of hydrogen in 20 mL of ethanol over 0.05 g of 5% palladium on carbon. The catalyst was removed by filtration and the filtrate concentrated to dryness under reduced pressure. Preparative thin layer chromatography using 20% methanol/10% cone.
• the dihydrochloride salt was precipitated from ethanol/toluene: Analysis calculated for C22H26N6 ⁇ 2 -0.4 CH3CH 2 OH « 0.5 CH3C6H5: C: 58.08, H: 6.38, N: 15.45 found C: 58.07, H: 6.51, N: 15.49.
• Step 1 A mixture of 45 g of 2-aminomethylaniline, 60 g of di- tert -butyldicarbonate, 1000 mL of dichloromethane was stirred for 18 h and washed with 500 mL of 2N NaOH. The organic extract was dried over MgS ⁇ 4 and concentrated under reduced pressure. Drying under vacuum gave 47 g of 2-(tert -butoxycarbonylaminomethyl)aniline as a white crystalline solid.
• iH NMR 400 MHz, CDC1 ) 7.1 (t, IH), 7.05 (d, IH), 6.65 (dd, 2H), 4.8 (br s, IH), 4.2 (br m, 4H), 1.44 (s. 9H).
• Step 2 A mixture of 15.5 g o ⁇ l- ⁇ tert -butoxycarbonyl aminomethyl)aniline, 15 g of N-t-butyloxycarbonyl-4-piperidone, 250 mL of 1 ,2-dichloroethane, 4.2 mL of glacial acetic acid and 25 g of sodium triacetoxyborohydride was stirred at room temperature for 48 h. The reaction mixture was poured into 500 mL chloroform and 500 mL saturated aqueous Na 2 CCh and the layers separated. The aqueous laver was extracted with 2 X 250 mL of chloroform and the combined organic layers dried over MgS ⁇ 4 and concentrated under reduced pressure.
• Step 3 To a stirred solution of 27.1 g of ten -butyl 4-(2-tert -butoxycarbonylaminomethylanilino)-l -piperidinecarboxylate and 30 mL of triethylamine in 400 mL of dichloromethane was added dropwise 60 mL of a 1.93 M solution of phosgene in toluene. After stirring for 12 h, 200 mL of IN NaOH was added. The mixture was shaken, and the organic layer separated, dried over MgS ⁇ 4 and concentrated under reduced pressure.
• Step 4 A stirred solution of 25 g of 1 ,3 -dihydro- ⁇ -[ ⁇ -ten -butoxycarbonylpiperidin-4-yl]-3-te/ t -butoxycarbonyl- 1H-3, 4- dihydroquinazolin-2-one carboxylate in 1 L of ethyl acetate cooled to -50°C was saturated with hydrogen chloride gas for 15 min. The resulting mixture was allowed to warm to room temperature and stir for 4 h. The white solid precipitate was collected by filtration.
• Step 5 A mixture of 0.52 g of l ,3-dihydro-l-[piperidin-4-yl]- lH-3,4-dihydroquinazolin-2-one, 0.65 g of N-t-butyloxycarbonyl-4- piperidone, 10 mL of 1 ,2-dichloroethane, 0.3 mL of glacial acetic acid and 1 g of sodium triacetoxyborohydride was stirred at room temperature for 48 h. The reaction mixture was poured into 50 mL chloroform and 50 mL saturated aqueous Na 2 C0 3 and the layers separated.
• Step 6 A stirred solution of 0.9 g of 1 ,3 -dihydro- 1 - ⁇ ⁇ -[ ⁇ -(tert -buty loxycarbony l)piperidin-4-y l]piperidin-4-y 1 ⁇ - 1 H-3 ,4- dihydroquinazolin-2-one in 40 mL of IN HCl was heated to reflux for 6 h, cooled and concentrated to dryness.
• Step 7 To a stirred solution of 0.58 g of l,3-dihydro-l - ⁇ 1-
• Step 1 To a stirred mixture of 1.1 g of 5-(l -aminoethyl)pyrimidine (O. Cervinska and P. Malon, Coll. Czechoslov. Chem. Commun. 1977, 42 , 3464-72.), 17 mL of ethanol and 1.36 g of K2CO3 heated to reflux was added dropwise over 30 min, a solution of 4 g of 1 ,1 -dimethyl- 4oxopiperidinium iodide in 70 mL of water. When the addition was complete, the mixture was heated under reflux for an additional 2 h, cooled, basified to pH 9 with K2CO3 and extracted with 5 times with 50 mL portions of methylene chloride.
• 5-(l -aminoethyl)pyrimidine O. Cervinska and P. Malon, Coll. Czechoslov. Chem. Commun. 1977, 42 , 3464-72.
• Step 2 A mixture of 0.24 g of l -( l-(5-pyrimidinyl)-ethyl)-4- oxopiperidine, 0.24 g of l-(4-piperidinyl) benzimidazol-2H-one, 4 mL of 1 ,2-dichloroethane, 0.12 mL of glacial acetic acid and 0.45 g of sodium triacetoxyborohydride was stirred at room temperature for 48 h. The reaction mixture was poured into 100 mL dichloromethane and 25 mL saturated aqueous Na2C ⁇ 3 and the layers separated.
• the bis-maleate salt Analysis calculated for C24H32N6 ⁇ »2 C4H4O4O.5 H2 ⁇ : C58.08, H: 6.25, N: 12.70; found C: 58.28, H: 6.13, N: 12.47.
• Step 1 A mixture of 5 g of 1 ,4-cyclohexanedione mono-ethyleneketal, 4.3 g of l ,3-dihydro-l -(4-piperidinyl)-benzimidazol-2H-one, 75 mL of 1 ,2-dichloroethane, 1.2 mL of acetic acid and 5.45 g of sodium triacetoxyborohydride was stirred at room temperature for 48 h. The reaction mixture was poured into 500 mL chloroform and 500 mL 0 saturated aqueous Na2C ⁇ 3 and the layers separated.
• Step 2 A mixture of 7.0 g of the ethylene ketal of l ,3-dihydro-l - ⁇ l -[4- 0 oxocyclohex-l-yl]piperidin-4-yl ⁇ -2H-benzimidazol-2-one, 80 mL of glacial acetic acid, 80 mL of water and 20 mL of cone. HCl was heated under reflux for 2 h, then allowed to cool ovemight. The mixture was concentrated under reduced pressure, diluted with 100 mL of saturated Na2C ⁇ 3 and extracted into 3 X 200 mL of CHCI3. The combined organic extracts were dried over MgS ⁇ 4 and concentrated under reduced pressure.
• CDCI3 + 5% CD3OD 8.44 (s, I H), 8.12 (m, 2H), 7.36 (m, IH), 7.09 (m. 3H), 4.3 (br m, IH), 3.65 (br m, 2H), 3.2 (br m, 2H), 2.45 (m, 5H), 2.0 (m, 2H), 1.85 (br m, 4H), 1.65 (m, 4H): Analysis calculated for C22H28N6O-1.05 H2 ⁇ »0.25 CHCI3: C: 60.56, H: 7.17, N: 18.68: found C: 60.81. H: 7.17, N: 18.68.
• Step 1 A mixture of 0.10 g of trans -l ,3-dihydro-l - ⁇ 4-(l - piperaziny 1)- 1 -cyclohexyl ⁇ -2H-benzimidazol-2-one, 0.037 mL of 3- pyridinecarboxaldehyde, 15 mL of 1 ,2-dichloroethane, 0.10 mL of glacial acetic acid and 0.1 1 g of sodium triacetoxyborohydride was stirred at room temperature for 24 h. The reaction mixture was poured into 10 mL dichloromethane and 10 mL saturated aqueous NaHC ⁇ 3 and the layers separated.
• Step 1 A mixture of ethyl 4-oxocyclohexanecarboxylate (1.86 g), 4-(2- oxo-l -benzimidazolinyl)piperidine ( 1.98 g), 1 ,2-dichloroethane (40 mL), glacial acetic acid (0.52 mL) and sodium triacetoxyborohydride (3.47 g) was stirred at room temperature for 72 h. The reaction mixture was poured into dichloromethane (50 mL) and saturated Na2C03 (50 mL) and the layers separated.
• the aqueous layer was extracted with 2 X 50 mL of dichloromethane and the combined organic layers dried over MgS ⁇ 4 and concentrated to dryness under reduced pressure.
• the crude product was purified by flash column chromatography on silica, eluting with a gradient of ethyl acetate; 0-8% methanol to yield the cis and trans isomers of ethyl l -[4-(2-keto- l - benzimidazolinyl)piperidin-l-yl]cyclohexane-4-carboxylate as a colorless solid (2.23 g).
• Step 2 To a stirred solution of the cis and trans isomers of ethyl 1 -[4- (2-keto- 1 -benzimidazoliny l)piperidin- 1 -yl]cyclohexane-4-carboxylate (2.16 g) in dry toluene (150 mL) at -90 °C under argon, was added diisobutylaluminum hydride (1 1.6 mL of a 1.5 M solution in toluene). The reaction mixture was stirred at -90 °C for lh, then quenched with methanol (1 mL) followed by saturated sodium potassium tartrate (50 mL).
• Step 3 To a stirred solution of 5-bromopyrimidine (3.98 g) in 1 : 1 diethyl ether; tetrahydrofuran (120 mL) at -1 10 °C under argon, was added n-butyllithium (10.4 mL of a 1.6 M solution in hexanes) at such a rate that the temperature was maintained at -1 10 °C.
• Step 1 To a stirred solution of oxalyl chloride (68 mg) in dry dichloromethane (4 mL) at -60 °C, under argon, was added dry dimethyl sulfoxide (0.107 mL) dropwise. The resulting mixture was stirred for 5 min, then added to a solution of ( ⁇ )- i-l ,3-dihydro-l -( l - ⁇ 4-[(5-pyrimidinyl)hydroxymethyl]cyclohex-l -yl ⁇ p ⁇ peridin-4-yl)-2H- benzimidazol-2-one (1 12 mg) in dichloromethane (5 mL) at -60 °C.
• Step 1 A mixture of l ,3-dihydro-l - ⁇ l -[4-oxocyclohex-l -yl]piperidin- 4-yl ⁇ -2H-benzimidazol-2-one (200 mg), 3-aminopyridine (29 mg), 1 ,2- dichloroethane (1.5 mL), acetic acid (0.087 mL) and sodium triacetoxyborohydride (180 mg) was stirred at room temperature for 20 h. The reaction mixture was poured into saturated aqueous Na2C ⁇ (10 mL) and extracted with dichloromethane (3 X 30 mL). The combined organic extracts were dried over MgS04 and concentrated under reduced pressure.
• CDCI3) 9.28 (br s, IH), 8.01 (d, IH), 7.93 (d, IH), 7.32 (m, IH). 7.09-7.03 (m, 4H), 6.86 (ddd, IH), 4.36 (tt, IH), 3.22 (tt, IH), 3.09 (d. 2H), 2.50-2.39 (m. 5H), 2.23 (d, 2H), 2.00 (d, 2H), 1.86 (m, 2H), 1.47 (q, 2H), 1.21 (q, 2H).
• hydrochloride salt was precipitated from methanol/diethyl ether: analysis calculated for C23H29 503 HC1-0.65 CH3OH C: 54.45, H: 6.68, N: 13.42; found C: 54.44, H: 6.60, N: 13.29.
• Step 1 Sodium borohydride (0.69 g) was added in portions to a stirred solution of acetylpyrazine (1.49 g) in ethanol (300 mL) at 0 °C. The mixture was allowed to warm to room temperature and stirred for 20 min, then water (100 mL) was added and the mixture adjusted to pH 7 with 1 N hydrochloric acid. The neutralized solution was concentrated in vacua to a volume of 200 mL, saturated with sodium chloride, and extracted with ethyl acetate (3 X 500 mL). The organic extracts were dried (Na2S ⁇ 4) and concentrated to give a solid which was washed with dichloromethane (4 X 100 mL). The dichloromethane washings were concentrated to give 2-( 1 -hydroxyethyl)pyrazine (1.38 g) as a colorless oil.
• Step 2 To a stirred solution of 2-( l -hydroxyethyl)pyrazine (0.859 g) and diphenylphosphoryl azide (2.48 g) in dry toluene (12 mL) at 0 °C, under argon, was added l ,8-diazabicyclo[5.4.0]undec-7-ene ( 1.35 mL) dropwise. The resulting mixture was allowed to warm slowly to room temperature and stirred for 22 h then quenched with water (8 mL). The organic layer was removed and the aqueous phase extracted with 10 mL ethyl acetate.
• Step 3 2-(l-Azidoethyl)pyrazine (0.77 g) and 10% palladium on carbon (0.075 g) were stirred in ethanol (40 mL) under an atmosphere of hydrogen at room temperature for 2 h.
• Step 4 To a stirred mixture of 2-( 1 -aminoethy l)pyrazine (225 mg), ethanol (10 mL) and K2CO3 (72 mg) heated to reflux was added dropwise over 30 min, a solution of 4 g of l ,l -dimethyl-4- oxopiperidinium iodide (2.1 g) in water (36 mL).
• Step 5 A mixture of l -(l-(2-pyrazinyl)-ethyl)-4-oxopiperidine (37 mg), 4-(2-oxo-l -benzimidazolinyl)piperidine (41 mg), 1 ,2- dichloroethane (0.75 mL), glacial acetic acid (0.01 1 mL) and sodium triacetoxyborohydride (60 mg) was stirred at room temperature for 48 h. The reaction mixture was poured into dichloromethane (5 mL) and saturated aqueous Na2C ⁇ 3 (3 mL) and the layers separated.
• Step 1 Sodium borohydride (1.75 g) was added in portions to a stirred solution of ace tylpyri dine (12.1 g) in ethanol (100 mL) at 0 °C. The mixture was allowed to warm to room temperature and stirred for 20 min, then concentrated under reduced pressure to remove ethanol. The thick residue was diluted with 300 mL of dichloromethane, 25 mL of water and 5 mL of 20% NaOH. Thed aqueous layer was saturated with sodium chloride,and extracted with ethyl acetate (4 X 150 mL). The combined organic extracts were dried (MgS04) and concentrated. Evaporative distillation of the crude oily product (14.0 g) at 1mm, oven temperature 85-95°C gave 1 1.4 g of 3-(l -hydroxyethyl)pyridine as a colorless oil.
• Step 2 A mixture of 5 g of 3-( l-hydroxyethyl)pyridine, 80 mL of t- butylmethylether, 0.85 g of lipase from Pseudom ⁇ nas fluorescens on Celite® (30 wt%, prepared as described by D. Bianchi, P. Cesti and E. Battistel, J. Org. Chem. 1988, 53 , 5531 -34) and 2.0 mL of acetic anhydride was allowed to stir in a stoppered flask for 24 h. The mixture was filtered and concentrated under reduced pressure.
• Step 4 A mixture of 1.7 g of 3-( l 'R-azidoethyl)pyridine, 0.25 g of 5% palladium on carbon and 150 mL of ethanol was stirred under an atmosphere of hydrogen at room temperature for 4 h. The mixture was filtered through a pad of celite and the filtrate concentrated under reduced pressure to yield 1.5 g of 3-( l'R-aminoethyl)pyridine as a pale oil.
• Step 6 A mixture of l -(rR-(3"-pyridinyl)-ethyl)-4-oxopiperidine (250 mg), 4-(2-oxo-l -benzimidazolinyl)piperidine (530 mg), 1 ,2- dichloroethane (7 mL), glacial acetic acid (0.2 mL) and sodium 5 triacetoxyborohydride (450 mg) was stirred at room temperature for 48 h. The reaction mixture was partitioned between chloroform (3 x 50 mL) and saturated aqueous Na2C ⁇ 3 (5 mL) and the combined organic layers dried over MgS04 and concentrated under reduced pressure.
• the citrate salt Analysis calculated for C25*H33N5 ⁇ « 1.0 C6H8 ⁇ 7 «1.0 CH3CO2CH2CH3O.5 H 2 0: C: 56.00, H: 6.50, N: 9.60 found C: 55.97, H: 6.42, N: 9.61.
• citrate salt Analysis calculated for C25H33N5 ⁇ » 1.0 C6H8 ⁇ vl .O H2O: C: 59.12, H: 6.88, N: 1 1.12 found C: 59.47, H: 6.78, N: 10.78.
• Step 1 To a stirred solution of 4.06 g of 5-pyrimidineacetic acid (F. Zymalkowski and E. Reimann, Arch. Phar . 1966 299 362-7) and triethylamine (6.55 g) in methylene chloride (150 mL) at 0 °C was added ethyl chloroformate (6.38 g) followed by 4- dimethylaminopyridine (0.72 g). The mixture was stirred at 0 °C for 1 h, then the solution was diluted with methylene chloride (200 mL) and saturated ammonium chloride ( 150 mL) was added.
• Step 2 A solution of ethyl 5-pyrimidinylacetate (3.98 g) and iodomethane (17.0 g) in dry THF (200 mL) was added to a solution of lithium bis(trimethylsilyl)amide (60 mmol) in dry THF (200 mL) at -70 °C at such a rate that the temperature did not rise above -65 °C.
• the reaction mixture was stirred under argon at -70 °C for 2 h, then allowed to warm to room temperature overnight and quenched with sulfuric acid (2.94 g) in water (100 mL).
• Step 3 A mixture of ethyl 2-methyl-2-(5-pyrimidinyl)propionate (4.64 g), 1.0 N lithium hydroxide (26 mL) and THF ( 100 mL) was heated to reflux under argon for 26 h. The mixture was allowed to cool, adjusted to pH 7 with acetic acid, and concentrated to dryness to give 4.1 1 g of lithium 2-methyl-2-(5-pyrimidinyl)propionate as a pale solid.
• Step 4 A mixture of lithium 2-methyl-2-(5-pyrimidinyl)propionate (4.1 1 g), diphenylphosphoryl azide (13.2 g), triethylamine (4.86 g) and anhydrous tert-butyl alcohol (100- mL) was heated to reflux for 6 h, then allowed to cool and concentrated in vacuo. The residue was partitioned between chloroform (250 mL) and water ( 100 mL), and the chloroform was extracted, dried (MgS04) and evaporated under reduced pressure.
• Step 5 The crude 2-(5-pyrimidinyl)-2-propylisocyanate was treated with a mixture of 1.0 N sodium hydroxide (26 mL), water (20 mL) and THF ( 100 mL) at 0 °C for 30 min, then the mixture was poured into ethyl acetate (200 mL) and water (100 mL).
• the aqueous layer was acidified to pH 5 with 10% citric acid, extracted twice with ethyl acetate, and then adjusted to pH 1 1 , saturated with sodium chloride, and extracted with methylene chloride (3 X 250 mL).
• the combined methylene chloride extracts were dried (Na2S ⁇ 4) and concentrated in vacuo to give 2-(5-pyrimidinyl)-2-propylamine (950 mg) as a colorless oil.
• Step 6 To a stirred mixture of 2-(5-pyrimidinyl)-2-propylamine (660 mg), ethanol (9 mL) and K2CO3 (665 mg) heated to reflux was added dropwise over 30 min, a solution of l ,l-dimethyl-4-oxopiperidinium iodide (1.9 g) in water (36 mL). When the addition was complete, the mixture was heated under reflux for an additional 2 h, cooled, basified to pH 10 with Na2C ⁇ 3 and extracted with ethyl acetate (3 ⁇ 100 mL). The combined organic extracts were dried over MgS04 and concentrated under reduced pressure.
• Step 7 A mixture of l -(2-(5-pyrimidinyl)-prop-2-yl)-4-oxopiperidine ( 1 17 mg), 4-(2-oxo-l -benzimidazolinyl)piperidine ( 128 mg), 1 ,2- dichloroethane (2.5 mL), glacial acetic acid (0.031 mL) and sodium triacetoxyborohydride ( 170 mg) was stirred at room temperature for 48 h. The reaction mixture was poured into dichloromethane (15 mL) and saturated aqueous Na2C ⁇ 3 (8 mL) and the layers separated.
• Step 1 A mixture of 0.65 g of ethyl 2-amino-5-pyrimidinecarboxylate (P. Schenone, L. Sansebastiano and L. Mosti, J. Heterocyclic Chem., 1990, 27 , 295-305) , 1.7 g of di-tert-butyldicarbonate and 50 mL of dichloromethane was allowed to stir overnight then concentrated under reduced pressure.
• ethyl 2-amino-5-pyrimidinecarboxylate P. Schenone, L. Sansebastiano and L. Mosti, J. Heterocyclic Chem., 1990, 27 , 295-305
• 1.7 g of di-tert-butyldicarbonate and 50 mL of dichloromethane was allowed to stir overnight then concentrated under reduced pressure.
• Step 3 From 1 ,3-dihydro- 1 - ⁇ 1 -[ 1 -piperidin-4-yl]piperidin-4-y 1 ⁇ -2H- benzimidazol-2-one dihydrochloride salt and 2-tert- butoxycarbonylamino-5-pyrimidinecarboxylic acid using the procedure described for Example 3, there was obtained l ,3-dihydro- l - ⁇ l -[ l -(2- tert-butoxycarbonylamino-5-pyrimidinecarbonyl)piperidin-4- yl]piperidin-4-yl ⁇ -2H-benzimidazol-2-one as a solid: ] H NMR (400 MHz, CDCI3) 9.95 (m, IH), 8.81 (s, 2H), 7.28 (m, 2H), 7.08 (m, 2H), 4.77 (s, IH), 4.35 (m, IH), 3.80 (s, IH), 3.12 (m, 4H).
• Step 4 The l ,3-dihydro-l- ⁇ l -[ l -(2-tert-butoxycarbonylamino-5- pyrimidinylcarbonyl)piperidin-4-yl]piperidin-4-yl ⁇ -2H-benzimidazol-2- one was dissolved in ethvl acetate, cooled to -50°C and treated with a stream of HCl gas for 2 min. The reaction mixture was allowed to warm to room temperature and stir for several hours, and then concentrated to dryness under reduced pressure.
• Step 1 To a stirred solution of l ,3-dihydro-l - ⁇ l-[4-oxocyclohex-l - ° yl]piperidin-4-yl ⁇ -2H-benzimidazol-2-one (100 mg) in methanol (4 mL) at 0 °C, under nitrogen, was added rerf-butylamine-borane (28 mg). The reaction mixture was stirred at 0 °C for 1 h, then quenched with water (2 mL) and concentrated to remove the methanol.
• Step 1 To a stirred solution of 1 ,3 -dihydro- 1- ⁇ l -[4-oxocyclohex-l - yl]piperidin-4-yl ⁇ -2H-benzimidazol-2-one (100 mg) in dry tetrahydrofuran (25 mL) at -78 °C, under nitrogen, was added L- Selectride (0.385 mL of a 1.0 M solution in tetrahydrofuran). The reaction mixture was stirred at -78 °C for 30 min, then quenched with water (3 mL). The solution was allowed to warm to room temperature and ethyl acetate (50 mL) was added.
• hydrochloride salt was precipitated from diethyl ether/methylene chloride: analysis calculated for Cl RH25N3O2-HC1-0.70 H2O C: 59.48, H: 7.32, N: 1 1.56; found C: 59.46, H: 7.28, N: 1 1.23.
• muscarinic antagonists for ml-m5 receptors expressed in Chinese hamster ovary cells were determined using the technique described by Dorje et al., J. Pharmacol. Exp. Ther. 256: 727-733 (1991).
• CHO cells When 80- 100% confluent, CHO cells were harvested, and transferred to centrifuge tubes containing CHO buffer (20 mM HEPES at pH 7.4 containing 5mM MgCl2). The cells were homogenized using a Brinkman Polytron homogenizer for 30 seconds at a setting of 5, on ice. The homogenate was centrifuged at 40,000 x g for 15 minutes at 4°C in a Beckman J2-21 M centrifuge. The supernatant was discarded and the homogenization/centrifugation step repeated once.
• Pelleted membranes were resuspended in CHO buffer to a concentration of one flask harvested (75 cm 2 ) per mL of buffer, mixed well and aliquoted in cryovials ( lmL/vial). The vials were stored at -70°C until used in the assay.
• the binding incubation was done in polypropylene macrowell tube strips in a final volume of 0.5 mL of HEPES buffer (20 mM; pH 7.4 containing 5 mM MgCl2) containing 0.1 mL of cell membrane suspension, 3H-N-methylscopolamine (NEN Corporation, NET-636, 70-87 Q/mmole) at a final concentration of approximately 0.2 nM and the competing drug in a varying range of concentrations or vehicle. After the addition of the cell homogenate the tubes were agitated on a vortex mixer and then placed in a water bath at 32°C.
• tissue segments are attached to platinum electrodes with 4-0 surgical silk and placed in a 10 mL jacketed tissue bath containing Krebs buffer at 30°C, bubbled with 5% CO2 / 95% O2.
• the tissues are connected to a Statham-Gould force transducer; 0.75 gram of tension is applied and the tissues are electrically stimulated.
• EFS parameters are 0.05 Hz; 0.5 ms duration; voltage is set to 30% of 50 V at 25 ohms and increased until a supramaximal voltage is achieved.
• the contractions are recorded on a Gould strip chart recorder. The tissues are washed every 20 minutes and allowed to equilibrate.
• a concentration response curve to the selective ml receptor agonist McN-A-343 is determined. Tissues are washed every 20 minutes for 60 minutes. The vehicle or compound is added to the bath and the tissues are incubated for 30 minutes, then the McN-A-343 concentration response is repeated. EC50 values are determined for both vehicle and tissues treated with the compound before and after treatment. Antagonist dissociation constants (K b ) are calculated by the dose-ratio method. Compounds displayed Kb values at m l generally consistent with the radioligand binding assay described in Example 30 in the range of 5 to 100 nM.
• Each atria is attached to platinum electrodes with 4-0 surgical silk and placed in a 10 mL jacketed tissue bath containing Krebs buffer at 37°C, bubbled with 5% CO2 / 95% O2.
• the tissues are connected to a Statham-Gould force transducer; 0.75 gram of tension is applied and the tissues are electrically stimulated. [EFS parameters are 3 Hz; 4 ms duration; voltage is set to 5 V.]
• the contractions are recorded on a Gould strip chart recorder.
• the tissues are washed every 20 minutes and allowed to equilibrate.
• a concentration response curve to the agonist carbachol is determined. Tissues are washed every 20 minutes for 60 minutes.
• K b Antagonist dissociation constants
• the lumen of the remainder is flushed with oxygenated Krebs solution [NaCl, 1 18 mM; KCl, 4.7 mM; CaCl 2 , 2.5 mM: KH2PO4, 1.2 mM; MgS0 4 , 1.2 mM; NaHC03, 25 mM; dextrose, 1 1 mM] warmed to 30°C.
• the ileum is cut into 2.5 cm segments and each segment is mounted on a glass pipette.
• a scalpel is used to lightly cut the surface of the tissue and a cotton swab used to tease the longitudinal muscle free from the underlying circular muscle.
• Preconfluent CHO cells were labeled for 24 hours with 4 ⁇ Cj/mL of [ 3 H] myo-inositol (specific activity 15-20 Ci /mmole).
• the cells were detached from flasks using 1 mM EDTA in phosphate buffer saline, centrifuged for 5 minutes at 200x g, and resuspended in assay buffer (1 16 mM NaCl; 10 mM LiCl; 4.7 mM KCl; 1.2 mM MgS0 4 ; 2.5 mM CaCb; 1.2 mM KH2PO4; 5 mM NaHC ⁇ 3; 1 1 mM dextrose, 20 mM HEPES; pH 7.4 at 37°C) to the desired volume.
• Kb values were generated by performing carbachol concentration-response curves in the absence and presence of a fixed concentration of compound.
• Compounds displayed Kb values at ml and m3 generally consistent with the radioligand binding assay described in Example 30 in the range of 1 to 100 nM at m l and 4,000 to 20,000 at m3.
• Preconfluent CHO cells were harvested using 1 mM
• EDTA in phosphate buffer saline and washed one time by centrifugation in a HEPES buffered physiological salt solution.
• the cell concentration was adjusted to 3.3 X 106 cells / mL in the HEPES buffer containing 1.3 micromolar isobutylmethylxanthine.
• Muscarinic agonist 50 microliters of carbachol; 1 micromolar final concentration

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  • 1995-10-24 WO PCT/US1995/013710 patent/WO1996013262A1/en not_active Application Discontinuation
  • 1995-10-24 CA CA002200468A patent/CA2200468A1/en not_active Abandoned
  • 1995-10-24 EP EP95937615A patent/EP0786997A4/de not_active Withdrawn
  • 1995-10-24 JP JP51469196A patent/JP2002515008A/ja active Pending
  • 1995-10-24 AU AU39674/95A patent/AU701127B2/en not_active Ceased

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