FIELD OF THE INVENTION
This present invention generally relates to muscarinic receptor antagonists, which are useful, among other uses, for the treatment of various diseases of the respiratory, urinary and gastrointestinal systems mediated through muscarinic receptors. The invention also relates to the process for the prepration of disclosed compounds, pharmaceutical compositions containing the disclosed compounds, and the methods for treating diseases mediated through muscarinic receptors.
BACKGROUND OF THE INVENTION
Physiological effects elicited by the neurotransmitter acetylcholine are mediated through its interaction with two major classes of acetylcholine receptors - the nicotinic and muscarinic acetylcholine receptors. Muscarinic receptors belong to the superfamily of G-protein coupled receptors and five molecularly distinct subtypes are known to exist (Mi, M2, M3, M4 and M5).
These receptors are widely distributed on multiple organs and tissues and are critical to the maintenance of central and peripheral cholinergic neurotransmission. The regional distribution of these receptor sub-types in the brain and other organs has been documented, (for example, the Mi subtype is located primarily in neuronal tissues such as cereberal cortex and autonomic ganglia, the M2 subtype is present mainly in the heart and bladder smooth miscle, and the M3 subtype is located predominantly on smooth muscle and salivary glands {Nature, 323, p.411 (1986); Science, 237, p.527 (1987)).
A review in Curr. Opin. Chem. Biol, 3, p. 426 (1999), as well as in Trends in Pharmacol. Sci., 22, p. 409 (2001) by Eglen et. al., describes the biological potentials of modulating muscarinic receptor subtypes by ligands in different disease conditions, such as Alzheimer's disease, pain, urinary disease condition, chronic obstructive pulmonary disease, and the like.
The pharmacological and medical aspects of the muscarinic class of acetylcholine agonists and antagonists are presented in a review in Molecules, 6, p. 142 (2001).
Birdsall et. al. in Trends in Pharmacol. Sci., 22, p. 215 (2001) has also summarized the recent developments on the role of different muscarinic receptor subtypes using different muscarinic receptor of knock out mice.
Almost all the smooth muscles express a mixed population of M2 and M3 receptors. Although the M2- receptors are the predominant cholinoreceptors, the smaller population of M3- receptors appears to be the most functionally important as they mediate the direct contraction of these smooth muscles. Muscarinic receptor antagonists are known to be useful for treating various medical conditions associated with improper smooth muscle function, such as overactive bladder syndrome, irritable bowel syndrome and chronic obstructive pulmonary disease. However the therapeutic utility of antimuscarinics has been limited by poor tolerability as a result of treatment related, frequent systemic adverse events such as dry mouth, constipation, blurred vision, headache, somnolence and tachycardia. Thus, there exists a need for novel muscarinic receptor antagonists that demonstrate target organ selectivity.
WO 04/005252 discloses azabicyclo derivatives described as musacrinic receptor antagonists. WO 04/004629, WO 04/052857, WO 04/067510, WO 04/014853, WO 04/014363 discloses 3,6-disubstituted azabicyclo [3.1.0] hexane derivatives described as useful muscarinic receptor antagonists. WO2004/056811 discloses flaxavate derivatives as muscarinic receptor antagonists. WO2004/056810 discloses xanthene derivatives as muscarinic receptor antagonists. WO2004/056767 discloses l-substituted-3-pyrrolidine derivatives as muscarinic receptor antagonists. WO2004/089363, WO2004/089898, WO04069835, WO2004/089900 and WO2004089364 discloses substituted azabicyclohexane derivatives as muscarinic receptor antagonists.
J. Med. Chem., 44, p. 984 (2002), describes cyclohexylmethylpiperidinyl-triphenylpropioamide derivatives as selective M3 antagonist discriminating against the other receptor subtypes. J. Med. Chem., 36, p. 610 (1993), describes the synthesis and antimuscarinic activity of some 1-cycloalkyl-1 -hydroxy- l-phenyl-3-(4-substituted piperazinyl)-2-propanones and related compounds. J. Med. Chem., 34, p.3065 (1991), describes analogues of oxybutynin, synthesis and antimuscarinic activity of some substituted 7-amino-l-hydroxy-5-heptyn-2-ones and related compounds. Bio-Organic Medicinal Chemistry Letters, 15, p.2093 (2005) describes synthesis and activity of analogues of Oxybutynin and Tolterodine.
The present invention fills the need of muscarinic receptor antagonists useful in the treatment of disease states associated with improper smooth muscle function and respiratory disorders
SUMMARY OF THE INVENTION
In one aspect, there are provided muscarinic receptor antagonists, which can be useful as safe and effective therapeutic or prophylactic agents for the treatment of various diseases of the respiratory, urinary and gastrointestinal systems. Also provided are processes for synthesizing such compounds. In another aspect, pharmaceutical compositions containing such compounds are provided together with acceptable carriers, excipients or diluents which can be useful for the treatment of various diseases of the respiratory, urinary and gastrointestinal systems.
The enantiomers, diastereomers, N-oxides, polymorphs, pharmaceutically acceptable salts and pharmaceutically acceptable solvates of these compounds as well as metabolites having the same type of activity are also provided, as well as pharmaceutical compositions comprising the compounds, their metabolites, enantiomers, diastereomers, N-oxides, polymorphs, solvates or pharmaceutically acceptable salts thereof, in combination with a pharmaceutically acceptable carrier and optionally included excipients.
Other aspects will be set forth in the description which follows, and in part will be apparent from the description or may be learnt by the practice of the invention.
In accordance with one aspect, there are provided compounds having the structure of Formula I
(Formula Removed)
and its pharmaceutically accepted salts, pharmaceutically acceptable solvates, enantiomers,
diastereomers, polymorphs or N-oxides wherein
R1 and R2 can be independently selected from alkyl, alkenyl, alkynyl, cycloalkyl or aryl.
R3 can represent hydrogen, lower alkyl, hydroxy, amino, alkoxy.
X can represent oxygen or NR7 wherein R7 can represent hydrogen, lower alkyl or aralkyl
R4 can represent hydrogen, alkyl, -COR8 (wherein R8 can represent alkyl, cycloalkyl, aryl, aralkyl
or heteroaryl) or -CHR9R10 (wherein R9 and R10 can be independently selected from hydrogen,
alkyl, aryl)
In accordance with a second aspect, there is provided a method for treatment or prophylaxis of an animal or a human suffering from a disease or disorder of the respiratory, urinary and gastrointestinal systems, wherein the disease or disorder is mediated through muscarinic receptors. The method includes administration of at least one compound having the structure of Formula I.
In accordance with a third aspect, there is provided a method for treatment or prophylaxis of an animal or a human suffering from a disease or disorder of the respiratory system such as bronchial asthma, chronic obstructive pulmonary disorders (COPD), pulmonary fibrosis, and the like; urinary system which induce such urinary disorders as urinary incontinence, lower urinary tract symptoms (LUTS), etc.; and gastrointestinal system such as irritable bowel syndrome, obesity, diabetes and gastrointestinal hyperkinesis with compounds as described above, wherein the disease or disorder is associated with muscarinic receptors.
In accordance with a fourth aspect, there are provided processes for preparing the compounds as described above.
The compounds described herein exhibit significant potency in terms of their activity, as determined by in vitro receptor binding Pharmaceutical compositions for the possible treatment for the disease or disorders associated with muscarinic receptors are provided. In addition, the compounds can be administered orally or parenterally. The following definitions apply to terms as used herein:
As used herein the term "alkyl" refers to a monoradical branched or unbranched saturated hydrocarbon chain having from 1 to 20 carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, t-butyl, n-hexyl, n-decyl, tetradecyl, and the like.
It may further be substituted with one or more substituents selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, cycloalkenyl, acyl, acylamino, acyloxy, amino, aminocarbonyl, alkoxycarbonylamino, azido, aralkyloxy, cyano, halogen, hydroxy, keto, thiocarbonyl, carboxy, carboxyalkyl, arylthio, thiol, alkylthio, aryl, aryloxy, aminosulfonyl, aminocarbonylamino, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, SO2-aryl and -SO2-heteroaryl. Unless otherwise constrained by the definition, all substituents may optionally be further substituted by 1-3 substituents chosen from alkyl, carboxy,
carboxyalkyl, aminocarbonyl, hydroxy, alkoxy, halogen, CF3, amino, substituted amino, cyano, and -S(O)nR.9, where R9 is alkyl, aryl, or heteroaryl and n is 0, 1 or 2.
It may further be substituted with one or more substituents selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, cycloalkenyl, acyl, acylamino, acyloxy, amino, aminocarbonyl, alkoxycarbonylamino, azido, cyano, halogen, hydroxy, keto, thiocarbonyl, carboxy, carboxyalkyl, arylthio, thiol, alkylthio, aryl, aryloxy, aminosulfonyl, aminocarbonylamino, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, SO2-aryl and -SO2-heteroaryl. Unless otherwise constrained by the definition, all substituents may optionally be further substituted by 1-3 substituents chosen from alkyl, carboxy, carboxyalkyl, aminocarbonyl, hydroxy, alkoxy, halogen, CF3, amino, substituted amino, cyano, and -S(O)nR9, where R9 is alkyl, aryl, or heteroaryl and n is 0, 1 or 2.
As used herein the term "cycloalkyl" refers to (un)saturated cyclic alkyl groups of from 3 to 20 carbon atoms having a single cyclic ring or multiple condensed rings. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, cyclopropylene, cyclobutylene and the like, or multiple ring structures such as adamantanyl, and bicyclo [2.2.1]heptane.
It may further be substituted with one or more substituents selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, cycloalkyl, cycloalkenyl, acyl, acylamino, acyloxy, amino, aminocarbonyl, alkoxycarbonylamino, azido, cyano, halogen, hydroxy, keto, thiocarbonyl, carboxy, carboxyalkyl, arylthio, thiol, alkylthio, aryl, aryloxy, aminosulfonyl,
aminocarbonylamino, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl and -SO2-heteroaryl. Unless otherwise constrained by the definition, all substituents may optionally be further substituted by 1-3 substituents chosen from alkyl, carboxy, carboxyalkyl, aminocarbonyl, hydroxy, alkoxy, halogen, CF3, amino, substituted amino, cyano, and -S(O)nRg, where R9 is alkyl, aryl or heteroaryl and n is 0, 1 or 2.
As used herein the term "alkoxy" refers to the group O-alkyl wherein alkyl is the same as defined above.
As used herein the term "haloalkyl" refers to alkyl substituted with halogen.
As used herein the term "halogen" refers to fluoro, bromo, chloro or iodo.
As used herein the term "aryl" refers to phenyl or naphthyl ring and the like optionally substituted with 1 to 3 substituents selected from the group consisting of halogen (F, CI, Br, I), hydroxy, alkyl, cyano, nitro and carboxy.
The term "aralkyl" refers to aryl linked through alkyl (wherein alkyl is the same as defined above) portion and the said alkyl portion contains carbon atoms from 1 -6 and aryl is as defined above.
As used herein the term "carboxy" refers to -C(=O)O-R12 wherein R12 is selected from the group consisting of hydrogen, alkyl and cycloalkyl.
As used herein the term "heteroaryl" refers to an aromatic ring structure containing 5 or 6 carbon atoms, or a bicyclic aromatic group having 8 to 10 carbon atoms, with one or more heteroatom(s) independently selected from the group consisting of N, O and S optionally substituted with 1 to 3 substituent(s) selected from the group consisting of halogen (F, Cl, Br, I), hydroxy alkoxy, cyano, nitro, optionally substituted amino.
Examples of heteroaryl groups are pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, pyrrolyl, oxazolyl, thiazolyl, thienyl, isoxazolyl, triazinyl, furanyl, benzofuranyl, indolyl, benzothiazolyl, benzoxazolyl, benzo[l,3]dioxol-5-yl-ethyl, and the like.
The term "pharmaceutically acceptable solvates" refers to solvates with waters (i.e hydrates) or pharmaceutical ly acceptable organic solvents. Such solvates are also encompassed within the scope of this invention.
The phrase "pharmaceutically acceptable salts" of the compounds of Formula I include acid addition salts such as hydrochloride, hydrobromide, hydrofluoric, sulphate, bisulfate, phosphate, hydrogen phosphate, acetate, brosylate, citrate, fumarate, glyconate, lactate, maleate, mesylate, succinate, and tartarate. Quaternary ammonium salts such as alkyl salts, aralkyl salts, and the like, of the organic bases may be readily formed by treatment of the organic bases with the appropriate quaternary salts forming substances, which include, for example methyl chloride, methyl bromide, methyl iodide, methyl sulphate, methyl benzene sulphonate, methyl p-toluene sulphonate, ethyl chloride, ethyl bromide, ethyl iodide, n-propyl chloride, n-propyl bromide, n-propyl iodide,
isopropyl bromide, n-butyl chloride, n-butyl bromide, isobutyl bromide, seobutylbromide, n-amyl bromide, n-hexyl chloride, benzyl chloride, benzyl bromide, and ethyl sulphate.
The present invention also includes within its scope prodrugs of these agents. In general, such "prodrugs" will be functional derivatives of these compounds, which are readily convertible in vivo into the required compound. Conventional procedure for the selection and preparation of suitable prodrug derivatives are described, for example, in "design of prodrugs", ed. H Bundgaard and, Elsevier, 1985.
The crystalline or amorphous forms of compounds disclosed herein may exist as polymorphs and as such are intended to be included in the present invention.
The compounds of present invention include stereoisomers. The term "stereoisomer" refers to compounds, which have identical chemical composition, but differ with regard to arrangement of the atoms and the groups in space. These include, diastereomers, geometrical isomers, atropisomer and comformational isomers. Geometric isomers may occur when a compound contains a double bond or some other feature that gives the molecule a certain amount
of structural rigidity. An enantiomer is a stereoisomer of a reference molecule that is the nonsuperimposable mirror image of the reference molecule. A diastereomer is a stereoisomer of a reference molecule that has a shape that is not the mirror image of the reference molecule. An atropisomer is a conformational of a reference compound that converts to the reference compound only slowly on the NMR or laboratory time scale. Conformational isomers (or conformers or rotational isomers or rotamers) are stereoisomers produced by rotation about o bonds, and are often rapidly interconverting at room temperature. Racemic mixtures are also encompassed within the scope of this invention.
DETAILED DESCRIPTION OF THE INVENTION
The compounds disclosed herein may be prepared by techniques well known in the art and familiar to the average synthetic organic chemist. In addition, the compounds of the present invention may be prepared by the following reaction sequences as depicted in SO2emes I
(Scheme Removed)
The compounds of Formula IV, V, VII and VIII can be prepared by following the procedure
described in SO2eme I. Thus the preparation comprises condensing a compound of Formula II
(wherein X is the same as defined earlier) with a compound of Formula III (wherein R1, R2 and R3
are the same as defined earlier) to give a compound of Formula IV, which undergoes deprotection
to give a compound of Formula V,
Path a: which is reacted with a compound of Formula VI (wherein hal is CI, Br or I and R8 is the
same as defined earlier) to give a compound of Formula VII
Path b: which undergoes reductive amination with a compound of Formula R9CHO (wherein R9 is
the same as defined earlier) to give a compound of Formula VIII
The condensation of a compound of Formula II with a compound of Formula III to give a compound of Formula IV can be carried out in an organic solvent (for example, dimethylformamide, tetrahydrofuran, diethyl ether dioxane or chloroform) in the presence of a base (for example, N-methylmorpholine, triethylamine, diisopropylethylamine or pyridine) with a condensing agent for example, l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HC1) or dicyclohexylcarbodiimide.
The deprotection of a compound of Formula IV to give a compound of Formula V can be carried out in an organic solvent (for example, methanol, ethanol, propanol or isopropylalcohol) in the presence of a deprotecting agent (for example, palladium on carbon in presence of hydrogen gas or palladium on carbon with a source of hydrogen gas (for example, ammonium formate solution, cyclohexene or formic acid)).
The reaction of a compound of Formula V with a compound of Formula VI (Path a) to give a compound of Formula VII can be carried out in an organic solvent (for example, dichloromethane, dichloroethane, carbon tetrachloride or chloroform) in the presence of a base (for example, triethylamine, pyridine, N-methylmorpholine or diisopropylethylamine) and catalyst (for example, dimethylaminopyridine or 4-(pyrrolidino)pyridine.
The reductive amination of a compound of Formula V with a compound of Formula R9CHO (path b) to give a compound of Formula VIII can be carried out in an organic solvent (for example, dichloromethane, dichloroethane, chloroform or carbon tetrachloride) with reducing agent (for example, sodium triacetoxyborohydride or sodium cyanoborohydride).
Compounds prepared following SO2eme I are:
Hydroxy-diphenyl-acetic acid l-(2-benzyloxy-acetyl)-pyrrolidin-3-ylmethyl ester (Compound No.
1),
2-Hydroxy-2-phenyl-pent-4-ynoic acid l-benzyl-pyrrolidin-3-ylmethyl ester (Compound No. 2),
N-(l-Benzyl-pyrrolidin-3-ylmethyl)-2-cyclopentyl-2-hydroxy-N-methyl-2-phenyl-acetamide (Compound No. 3), 2-Cyclopentyl-2-hydroxy-N-methyl-2-phenyl-N-pyrrolidin-3-ylmethyl-acetamide (Compound No.
4), 2-Cyclopentyl-2-hydroxy-N-methyl-N-(l-methyl-pyrrolidin-3-ylmethyl)-2-phenyl-acetamide
(Compound No. 5),
N-(l-Benzyl-pyrrolidin-3-ylmethyl)-2-cyclohexyl-2-hydroxy-N-methyl-2-phenyl-acetamide
(Compound No. 6),
N-( 1 -Benzyl-pyrrolidin-3-ylmethyl)-2-hydroxy-N-methyl-2,2-diphenyl-acetamide (Compound No.
7),
2-Cyclohexyl-2-hydroxy-N-methyl-2-phenyl-N-pyrrolidin-3-ylmethyl-acetamide (Compound No.
8),
N-[l-(2-Benzyloxy-acetyl)-pyrrolidin-3-ylmethyl]-2-cyclopentyl-2-hydroxy-N-methyl-2-phenyl-acetamide (Compound No. 9),
and its pharmaceutically accepted salts, pharmaceutically acceptable solvates, enantiomers, diastereomers, polymorphs or N-oxides.
In the above SO2emes, where specific bases, condensing agents, protecting groups, deprotecting agents, solvents, catalysts, temperatures, etc. are mentioned, it is to be understood that other bases, condensing agents, protecting groups, deprotecting agents, solvents, catalysts, temperatures, etc. known to those skilled in the art may be used. Similarly, the reaction temperature and duration may be adjusted according to the desired needs.
Suitable salts of the compounds represented by the Formula I were prepared so as to solubilize the compound in aqueous medium for biological evaluations, as well as to be compatible with various dosage formulations and also to aid in the bioavailability of the compounds. Examples of such salts include pharmacologically acceptable salts such as inorganic acid salts (for example, hydrochloride, hydrobromide, sulphate, nitrate and phosphate), organic acid salts (for example, acetate, tartarate, citrate, fumarate, maleate, tolounesulphonate and methanesulphonate). When carboxyl groups are included in the Formula I as substituents,
they may be present in the form of an alkaline or alkali metal salt (for example, sodium, potassium, calcium, magnesium, and the like). These salts may be prepared by various techniques, such as treating the compound with an equivalent amount of inorganic or organic, acid or base in a suitable solvent.
Particular compounds are shown in Table below:

(Table Removed)
Because of their valuable pharmacological properties, the compounds described herein may be administered to an animal for treatment orally, or by a parenteral route. The pharmaceutical compositions described herein can be produced and administered in dosage units, each unit containing a certain amount of at least one compound described herein and/or at least one physiologically acceptable addition salt thereof. The dosage may be varied over extremely wide limits as the compounds are effective at low dosage levels and relatively free of toxicity. The compounds may be administered in the low micromolar concentration, which is therapeutically effective, and the dosage may be increased as desired up to the maximum dosage tolerated by the patient.
The compounds described herein can be produced and formulated as their enantiomers, diastereomers, N-Oxides, polymorphs, solvates and pharmaceutically acceptable salts, as well as metabolites having the same type of activity. Pharmaceutical compositions comprising the molecules of Formula I or metabolites, enantiomers, diastereomers, N-oxides, polymorphs, solvates or pharmaceutical ly acceptable salts thereof, in combination with pharmaceutical ly acceptable carrier and optionally included excipient can also be produced.
The examples mentioned below demonstrate general synthetic procedures, as well as specific preparations of particular compounds. The examples are provided to illustrate the details of the invention and should not be constrained to limit the scope of the present invention.
Examples
Various solvents, such as acetone, methanol, pyridine, ether, tetrahydrofuran, hexanes, and dichloromethane, were dried using various drying reagents according to procedures described in the literature. IR spectra were recorded as nujol mulls or a thin neat film on a Perkin Elmer Paragon instrument, Nuclear Magnetic Resonance (NMR) were recorded on a Varian XL-300 MHz or Bruker 400 MHz instrument using tetramethylsilane as an internal standard.
Synthesis of 2-hvdroxv-2-cvclopentvl-2-phenyl acetic acid
The title compound was prepared following the procedure described in J. Amer. Chem. Soc. 75, 2654 (1953); J. Org. Chem. 2000, 65, 6283-6287.
Synthesis of (l-benzyl-pyrrolidin-3-ylmethyl)-methyl-amine
Step a: Synthesis of l-(benzyl-pyrrolidin-3-yl)-methanol
A solution of the compound l-benzyl-5-oxo-pyrrolidine-3-carboxylic acid methyl ester (1.0 eq.) (commercially available) in toluene was cooled to 0°C under inert atmosphere. To the mixture was added solution of borane (3.75 eq.) in dimethyl sulphide and refluxed the mixture for 16 hours at 100°-110°C. The resulting reaction mixture was cooled to room temperature and subsequently to -5° to -10°C followed by the addition of sodium bicarbonate solution dropwise. The mixture was slowly brought to room temperature and subsequently refluxed the reaction mixture for 2 hours. The mixture was cooled and organic layer was separated. Aqueous layer was extracted with toluene. The combined toluene layers were washed with water and brine solution. The organic solvent was evaporated under reduced pressure to furnish the title compound. Yield: 99.14% Step b: Synthesis of l-benzyl-3-methanesulphonyl-pyrrolidine
To a solution of the compound obtained from step a above (1.0 eq.) in dichloromethane (10ml) was added triethylamine (2 eq.) and dimethylaminopyridine (catalytic amount). The mixture was cooled to 0°C followed by the addition of methane sulphonyl chloride (1.5 eq.) dropwise and stirred the mixture for 14 hours at room temperature. The mixture was dilute with dichloromethane, washed with saturated sodium bicarbonate solution, water and brine, dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure. The residue thus obtained was purified by column chromatography to furnish the title compound. Yield: 90.53%
Step c: Synthesis of l-benzyl-pyrrolidin-3-ylmethyl)-methyl-amine
To a solution of the compound obtained from step b above (4.0 g) in methanol (40 ml) was added aqueous methylamine (4%, 40 ml) and heated the mixture for 16 hours at 85-90°C in autoclave. The solvent was evaporated under reduced pressure and the residue thus obtained was diluted with water and acidified with hydrochloric acid (1:1) and washed with dichloromethane. The aqueous layer was basified with sodium hydroxide solution (20%). The mixture was extracted with ethyl acetate, washed with water and brine, dried over anhydrous sodium sulphate and concentrated under reduced pressure to furnish the title compound. Yield: 84.72%
Example 1: Synthesis of N-(1-benzyl-pyrrolidin-3-ylmethyl)-2-cyclopentyl-2-hvdroxy-N-methyl-2-phenyl-acetamide (Compound No. 3)
To a solution of the compound 2-cyclopentyl-2-hydroxy-2-phenyl acetic acid (1 eq.) in dimethylformamide was added hydroxybenzotriazole (1.5 eq.), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1 eq.) and dimethylaminopyridine (catalytic amount). The reaction mixture was stirred at 15-20°C for 2 hours followed by the addition of N-methylmorpholine (2 eq.) and a solution of the compound (1-benzyl-pyrrolidin-3-ylmethyl)-methyl-amine (leq.) in dimethylformamide (10 ml). The resulting reaction mixture was stirred at 15-20°C for 1 hour and subsequently at room temperature for 14 hours. To the mixture was added water and stirred for 15 minutes. The aqueous layer was extracted with ethylacetate. The organic layer was washed with sodium bicarbonate solution, water and brine, dried over anhydrous sodium sulphate and concentrated under reduced pressure. The residue thus obtained was purified by column chromatography to furnish the title compound. Yield: 75.34% Mass(m/z): 407.0 (M++l). IR: 1623.8 cm-1, 2951.2 cm-1, 3357.5 cm-1.
1H NMR: 1.256-1.663 (m, 10H), 2.014-2.559 (m, 6H), 2.713-2.951 (m, 4H), 3.399-3.461 (m, 2H), 3.801 (s, 2H), 7.105-7.334 (m, 10H).
Following compounds were prepared similarily by coupling an appropriate acid with an appropriate amine or alcohol,
2-Hydroxy-2-phenyl-pent-4-vnoic acid l-benzyl-pyrrolidin-3-ylmethyl ester (Compound No. 2)
Mass(m/z): 364.0 (M++l)
IR: 1745.2 cm-1, 2923.7 cm-1, 3412.7 cm-1
1H NMR: 1.86-1.96 (m, 4H), 2.03-2.59 (m, 6H), 2.79-2.91 (m, 3H), 4.04-4.09 (m, 2H), 7.31-7.74 (m, 10H).
N-(l-benzyl-pyrrolidin-3-ylmethyl)-2-cyclohexyl-2-hydroxy-N-methyl-2-phenyl-acetamide
(Compound No. 6)
Mass(m/z): 421.0 (M++l)
IR: 1623.0 cm-1, 2925.3 cm-1, 3383.8 cm-1
1H NMR (CDC13): 1.17-1.37 (m, 6H), 1.41-1.48 (m, 2H), 1.72-2.61 (m, 12H), 2.75-3.62 (m, 6H),
7.20-7.39 (m, 10H).
N-(l-benzyl-pyrrolidin-3-ylmethyl)-2-hydroxv-N-methyl-2,2-diphenyl-acetamide (Compound No.
2)
Mass(m/z): 415.0 (M++l)
IR: 1747.5 cm-1, 2925.1 cm-1, 3421.4 cm-1
1H NMR (CDC13): 1.20-2.01 (m, 3H), 2.12-2.43 (m, 4H), 2.87-2.91 (m, 2H), 2.95-3.27 (m, 4H),
3.77 (s,2H), 7.01-7.32 (m, 15H).
Example 2: Synthesis of 2-cvclopentyl-2-hvdroxv-N-methyl-2-phenvI-N-pyrrolidin-3-ylmethyl-acetamide (compound No. 4)
To a solution of the Compound No. 3 (leq.) in methanol (20 times) was added palladium on carbon
(10% dry) and ammonium formate. The mixture was refluxed for 25 minutes. The mixture was
cooled and filtered through celite bed. The bed was washed with dichloromethane and basified the
aqueous layer with sodium hydroxide (2N) to a pH 14. The mixture was extracted with ethylacetate.
The organic layer was washed with water and brine, dried over anhydrous sodium sulphate, filtered
and concentrated under reduced pressure to furnish the title compound. Yield: 96.36%
Mass(m/z): 317.0 (M++l)
IR: 1625.7 cm-1, 2958.0 cm-1, 3375.7 cm-1
1H NMR: 1.25-1.32 (m, 2H), 1.60-1.72 (m, 6H), 1.95-2.05 (m, 5H), 2.77-3.04 (m, 8H), 3.17-3.48
(m, 2H), 7.28-7.42 (m, 5H).
Following compounds were prepared similarily,
2-Cyclohexyl-2-hydroxy-N-methyl-2-phenyl-N-pyrrolidin-3-ylmethyl-acetamide (Compound No.
8)
Mass(m/z): 331.0 (M++l)
IR: 1620.0 cm-1, 2926.7 cm-1, 3385.4%
1H NMR (CDC13): 1.10-1.32 (m, 10H), 1.66-1.70 (m, 4H), 1.98-2.04 (m, 4H), 2.40-2.87 (m, 5H),
3.35 (s,2H).
Example 3: Synthesis of 2-cvclopentyl-2-hvdroxv-N-methyl-N-(l-methyl-pyrroIidin-3-ylmethyl)-2-phenyl-acetamide (Compound No. 5)
To a solution of the Compound No. 4 (0.2 g) in acetonitrile (10-15 ml) was added 37% aqueous
formaldehyde (1.7 ml) and sodium triacetoxyborohydride (0.16 g) and stirred at room temperature
for one hour. Added acetic acid till the pH of reaction mixture is neutral. The reaction mixture was
stirred for 2 hours at room temperature. Evaporated acetonitrile completely and added water.
Basified to pH 14 with aqueous sodium hydroxide solution (10%). Extracted with ethyl acetate. The
organic layer was separated, washed with water and brine, dried over anhydrous sodium sulphate,
filtered and concentrated under reduced pressure to furnish the title compound. Yield: 52.67%
Mass(m/z): 331.0 (M++l)
IR: 1623.6 cm'1, 2925.6 cm'1, 3383.7 cm-1
1H NMR: 1.256-1.334 (m, 4H), 1.450-1.513 (m, 7H), 1.587-1.764 (m, 8H), 2.449-2.754 (m, 2H),
2.970-3.409 (m, 4H), 7.30-7.415 (m, 5H).
Example 4: Synthesis of N-[1-(2-benzyloxy-acetyl)-pyrrolidin-3-ylmethyll-2-cyclopentyl-2-hydroxv-N-methyl-2-phenyl-acetamide (compound No. 9)
To a solution of the Compound No. 4 (leq.) in dichloromethane (10 ml) was added triethylamine (2 eq.) and dimethylaminopyridine (catalytic amount). The mixture was cooled to 0-5°C and added benzyloxy acetyl chloride (1.5 eq.). The reaction mixture was stirred at 0-5°C for 30 minutes followed by stirring at room temperature for 16 hours. The mixture was quenched with saturated sodium bicarbonate solution. The organic layer was separated and aqueous layer was extracted with dichloromethane. The combined organic layers were washed with water and brine, dried over anhydrous sodium sulphate and concentrated under reduced pressure. The residue thus obtained was purified by column chromatography to furnish the title compound. Yield: 88.53% Mass (m/z): 465 (M++l), 487 (M++Na).
IR: 1630 cm-1, 2927.1 cm-1, 3391.9 cm-1
1H NMR: 1.12-1.26 (m, 4H), 1.61-1.75 (m, 6H), 2.32-2.37 (m, 6H), 2.88-3.38 (m, 4H), 4.13-4.63
(m,6H), 7.23-7.41 (m, 10H).
Following compound was prepared similarily,
Hydroxy-diphenyl-acetic acid l-(2-benzyloxy-acetyl)-pyrrolidin-3-ylmethyl ester (Compound No.
1)
Mass(m/z): 460.0 (M++l).
IR: 1707.9 cm-1, 2926.8 cm-1, 3031.6 cm-1
1H NMR: 1.255 (s, 2H), 3.30-3.88 (m, 2H), 3.966-4.250 (m, 4H), 4.485-4.87 (m, 5H), 5.183-5.285
(m, 1H), 7.225-7.406 (m, 15H).
Biological Activity
Radioligand Binding Assays:
The affinity of test compounds for M2 and M3 muscarinic receptor subtypes were determined by [3H]-N-Methylscopolamine (NMS) binding studies using rat heart and submandibular gland respectively as described by Moriya et al., (Life Sci, 1999,64(25): 2351-2358) with minor modifications. Specific binding of [3H]-NMS was also determined using membranes from Chinese hamster ovary (CHO) cells expressing cloned human muscarinic receptor subtypes.
Membrane preparation:
(a) Rat tissues
Submandibular glands and heart were isolated and placed in ice-cold homogenising buffer (HEPES 20mM, 10mM EDTA, pH 7.4) immediately after sacrifice. The tissues were homogenised in ten volumes of homogenising buffer and the homogenate was filtered through two layers of wet gauze and filtrate was centrifuged at 500g for 10min. The supernatant was subsequently centrifuged at 40,000g for 20 min. The pellet thus obtained was resuspended in homogenising buffer (HEPES 20
mM, EDTA 10mM, pH 7.4) and were stored at -70°C until the time of assay.
(b) CHO cells expressing human recombinant receptors
The cell pellets were homogenised for 30sec at 12,000 to 14,000 rpm, with intermittent gaps of 10-15 sec in ice-cold homogenising buffer (20mM HEPES, 10mM EDTA, pH 7.4). The homogenate was then centrifuged at 40,000g for 20 min at 4°C. The pellet thus obtained was resuspended in homogenising buffer containing 10% sucrose and was stored at -70°C until the time of assay.
Ligand binding assay:
The compounds were dissolved and diluted in dimethyl sulphoxide. The membrane homogenates (5-10 µg protein) were incubated in 250 uL of assay buffer (20mM HEPES, pH 7.4) at 24-25 °C for 3hrs. Non-specific binding was determined in the presence of 1 µM Atropine. The incubation was terminated by vacuum filtration over GF/B fiber filter mats (Wallac) using Skatron cell harvester. The filters were then washed with ice-cold 50mM Tris HC1 buffer (pH 7.4). The filter mats were dried and transferred to 24 well plates (PET A No Cross Talk) followed by addition of 500 µl of scintillation cocktail. Radioactivity retained on filters was counted in Microbeta scintillation counter. The IC50 & Kd were estimated by using the non-linear curve-fitting program using GraphPad Prism software. The value of inhibition constant, Ki was calculated from competitive binding studies by using Cheng & Prusoff s equation {Biochem Pharmacol, 1973,22: 3099-3108), Ki = IC50 / (1+[L]/Kd), where [L] is the concentration of ligand [3H]-N-methyl scopolamine used in the particular experiment and Kd is the estimate of affinity of receptors to the ligand. The final result is expressed as the pKi value - the negative logarithm of Ki.
Compound Nos. 1-9 exhibited pKi in the range of 5-10 at rat M2 and M3 muscarinic receptors.

WE CLAIM:
1. A compound having the structure of Formula I
(Formula Removed)
and its pharmaceutical ly accepted salts, pharmaceutically acceptable solvates, enantiomers,
diastereomers, polymorphs or N-oxides wherein
R1 and R2 can be independently selected from alkyl, alkenyl, alkynyl, cycloalkyl or aryl.
R3 can represent hydrogen, lower alkyl, hydroxy, amino, alkoxy.
X can represent oxygen or NR7 wherein R7 can represent hydrogen, lower alkyl or aralkyi
R4 can represent hydrogen, alkyl, -COR8 (wherein Rg can represent alkyl, cycloalkyl, aryl, aralkyi
or heteroaryl) or -CHR9R10 (wherein R9 and R10 can be independently selected from hydrogen,
alkyl, aryl).
2. A compound which is:
Hydroxy-diphenyl-acetic acid l-(2-benzyloxy-acetyl)-pyrrolidin-3-ylmethyl ester (Compound No.
1),
2-Hydroxy-2-phenyl-pent-4-ynoic acid l-benzyl-pyrrolidin-3-ylmethyl ester (Compound No. 2),
N-(l-Benzyl-pyrrolidin-3-ylmethyl)-2-cyclopentyl-2-hydroxy-N-methyl-2-phenyl-acetamide
(Compound No. 3),
2-Cyclopentyl-2-hydroxy-N-methyl-2-phenyl-N-pyrrolidin-3-ylmethyl-acetamide (Compound No.
4),
2-Cyclopentyl-2-hydroxy-N-methyl-N-(l-methyl-pyrrolidin-3-ylmethyl)-2-phenyl-acetamide
(Compound No. 5),
N-(l-Benzyl-pyrrolidin-3-ylmethyl)-2-cyclohexyl-2-hydroxy-N-methyl-2-phenyl-acetamide
(Compound No. 6),
N-( 1 -Benzyl-pyrrolidin-3-ylmethyl)-2-hydroxy-N-methyl-2,2-diphenyl-acetamide (Compound No.
7),
2-Cyclohexyl-2-hydroxy-N-methyI-2-phenyl-N-pyrrolidin-3-ylmethyl-acetamide (Compound No.
8),
N-[l-(2-Benzyloxy-acetyl)-pyrrolidin-3-ylmethyl]-2-cyclopentyl-2-hydroxy-N-methyl-2-phenyl-acetamide (Compound No. 9),
3. A pharmaceutical composition comprising a therapeutically effective amount of a compound as
defined in claim 1 and 2 optionally together with pharmaceutically acceptable carriers, excipients or
diluents.
4. A method for treatment or prophylaxis of an animal or a human suffering from a disease or disorder of the respiratory, urinary and gastrointestinal systems, wherein the disease or disorder is mediated through muscarinic receptors, comprising administering to said animal or human, a therapeutically effective amount of a compound according to claim 1 and 2.
5. A method according to claim 4 wherein the disease or disorder is urinary incontinence, lower urinary tract symptoms (LUTS), bronchial asthma, chronic obstructive pulmonary
disorders (COPD), pulmonary fibrosis, irritable bowel syndrome, obesity, diabetes or gastrointestinal hyperkinesis.
6. A method for treatment or prophylaxis of an animal or a human suffering from a disease or disorder of the respiratory, urinary and gastrointestinal systems, where the disease or disorder is mediated through muscarinic receptors, comprising administering to said animal or human, a therapeutically effective amount of the pharmaceutical composition according to the claim 3.
7. A method according to claim 6 wherein the disease or disorder is urinary incontinence, lower urinary tract symptoms (LUTS), bronchial asthma, chronic obstructive pulmonary disorders (COPD), pulmonary fibrosis, irritable bowel syndrome, obesity, diabetes and gastrointestinal hyperkinesis.
8. A process of preparing a compound of Formula IV, V, VII and VIII and its pharmaceutically acceptable salts, pharmaceutically acceptable solvates, esters, stereoisomers, N-oxides, polymorphs, prodrugs, or metabolites
(Formula Removed)
wherein
R1, R2, R.3, R4, Rs, R9 and X are the same as defied in claim 1.
9. The process according to claim 8, wherein condensation of a compound of Formula II with a
compound of Formula II to give a compound of Formula IV is carried out in an organic solvent
selected from dimethylformamide, tetrahydrofuran, diethyl ether and dioxane in the presence of a
base selected from N-methylmorpholine, triethylamine, diisopropylethylamine and
pyridine with a condensing agent selected from l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and dicyclohexylcarbodiimide.
10. The process according to claim 8, wherein deprotection of a compound of Formula IV to give a compound of Formula V is carried out in an organic solvent selected from methanol, ethanol, propanol and isopropylalcohol in the presence of a deprotecting agent selected from palladium on carbon in presence of hydrogen gas and palladium on carbon with a source of hydrogen gas selected from ammonium formate solution, cyclohexene and formic acid.
11. The process according to claim 8, wherein reaction of a compound of Formula V with a compound of Formula VI to give a compound of Formula VII is carried out in an organic solvent selected from dichloromethane, dichloroethane, carbon tetrachloride and chloroform in the presence of a base selected from triethylamine, pyridine, N-methylmorpholine and diisopropylethylamine and catalyst selected from dimethylaminopyridine and 4-(pyrrolidino)pyridine
12. The process according to claim 8, wherein reductive amination of a compound of Formula V with a compound of Formula R9CHO to give a compound of Formula VIII is carried out in an organic solvent selected from dichloromethane, dichloroethane, chloroform and carbon tetrachloride with reducing agent selected from sodium triacetoxyborohydride and sodium cyanoborohydride.