Soft Anticholinergic Esters Patent Application (2025)

U.S. patent application number 13/423071 was filed with the patent office on 2012-07-12 for soft anticholinergic esters. Invention is credited to Nicholas S. BODOR.

SOFT ANTICHOLINERGIC ESTERS

Abstract

Soft anticholinergic esters of the formulas: ##STR00001##wherein R.sub.1 and R.sub.2 are both phenyl or one of R.sub.1 andR.sub.2 is phenyl and the other is cyclopentyl; R isC.sub.1-C.sub.8 alkyl, straight or branched chain; and X.sup.- isan anion with a single negative charge; and wherein each asteriskmarks a chiral center; said compound having the R, S or RSstereoisomeric configuration at each chiral center unless specifiedotherwise, or being a mixture thereof.

Related U.S. Patent Documents

Claims

1. A method for eliciting an anticholinergic response in a subjectin need of same, comprising administering to said subject ananticholinergically effective amount of a compound having theformula: ##STR00023## wherein R.sub.1 and R.sub.2 are both phenylor one of R.sub.1 and R.sub.2 is phenyl and the other iscyclopentyl; R is C.sub.1-C.sub.8 alkyl, straight or branchedchain; and X.sup.- is an anion with a single negative charge; andwherein each asterisk marks a chiral center; said compound havingthe R, S or RS stereoisomeric configuration at each chiral centerunless specified otherwise, or being a mixture thereof.

2. A method as claimed in claim 1 for reducing or inhibiting thedevelopment of, or alleviating the symptoms of, a disorder selectedfrom the group consisting of chronic obstructive pulmonary disease,asthma, bronchitis, allergic rhinitis and infectious rhinitis in asubject in need thereof, comprising administering to said subjectan anticholinergically effective amount of a compound having theformula: ##STR00024## wherein R.sub.1 and R.sub.2 are both phenylor one of R.sub.1 and R.sub.2 is phenyl and the other iscyclopentyl; R is C.sub.1-C.sub.8 alkyl, straight or branchedchain; and X.sup.- is an anion with a single negative charge; andwherein each asterisk marks a chiral center; said compound havingthe R, S or RS stereoisomeric configuration at each chiral centerunless specified otherwise, or being a mixture thereof.

3. A method as claimed in claim 1 for reducing or inhibiting thedevelopment of, or alleviating the symptoms of, chronic obstructivepulmonary disease or asthma in a subject in need thereof,comprising administering to said subject an anticholinergicallyeffective amount of a compound having the formula: ##STR00025##wherein R.sub.1 and R.sub.2 are both phenyl or one of R.sub.1 andR.sub.2 is phenyl and the other is cyclopentyl; R isC.sub.1-C.sub.8 alkyl, straight or branched chain; and X.sup.- isan anion with a single negative charge; and wherein each asteriskmarks a chiral center; said compound having the R, S or RSstereoisomeric configuration at each chiral center unless specifiedotherwise, or being a mixture thereof.

4. A method as claimed in claim 1 for inducing mydriasis in theeye(s) of a subject in need thereof, comprising topically applyingto the eye(s) of said subject a mydriatically effective amount of acompound having the formula: ##STR00026## wherein R.sub.1 andR.sub.2 are both phenyl or one of R.sub.1 and R.sub.2 is phenyl andthe other is cyclopentyl; R is C.sub.1-C.sub.8 alkyl, straight orbranched chain; and X.sup.- is an anion with a single negativecharge; and wherein each asterisk marks a chiral center; saidcompound having the R, S or RS stereoisomeric configuration at eachchiral center unless specified otherwise, or being a mixturethereof.

5. A method as claimed in claim 1 for alleviating the symptoms ofoveractive bladder in a subject in need thereof, comprisingadministering to said subject an anticholinergically effectiveamount of a compound having the formula: ##STR00027## whereinR.sub.1 and R.sub.2 are both phenyl or one of R.sub.1 and R.sub.2is phenyl and the other is cyclopentyl; R is C.sub.1-C.sub.8 alkyl,straight or branched chain; and X.sup.- is an anion with a singlenegative charge; and wherein each asterisk marks a chiral center;said compound having the R, S or RS stereoisomeric configuration ateach chiral center unless specified otherwise, or being a mixturethereof.

6. A method as claimed in claim 1 for eliciting an antiperspiranteffect in a subject in need thereof, comprising topically applyingto said subject an antiperspirant effective amount of a compoundhaving the formula: ##STR00028## wherein R.sub.1 and R.sub.2 areboth phenyl or one of R.sub.1 and R.sub.2 is phenyl and the otheris cyclopentyl; R is C.sub.1-C.sub.8 alkyl, straight or branchedchain; and X.sup.- is an anion with a single negative charge; andwherein each asterisk marks a chiral center; said compound havingthe R, S or RS stereoisomeric configuration at each chiral centerunless specified otherwise, or being a mixture thereof.

7. The method as claimed in claim 1, wherein the compound offormula (Ia) is a compound having the formula: ##STR00029## whereinR is methyl or ethyl; and wherein each asterisk marks a chiralcenter; said compound having the R, S or RS stereoisomericconfiguration at each chiral center unless specified otherwise, orbeing a mixture thereof.

8. The method as claimed in claim 1, wherein the compound offormula (Ia) is the compound which is: (a)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-m-ethylpyrrolidinium bromide; (b)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; (c) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-m-ethylpyrrolidinium bromide; (d) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; (e) (2R,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-m-ethylpyrrolidinium bromide; (f) (2R,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-m-ethylpyrrolidinium bromide; (g) (2R,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; (h) (2S,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-m-ethylpyrrolidinium bromide; (i) (2S,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-m-ethylpyrrolidinium bromide; (j) (2R,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; (k) (2R,1'R,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; (l) (2R,1'S,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; (m) (2R,1'R,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; or (n) (2R,1'S,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide.

9. The method as claimed in claim 1, wherein the compound offormula (Ia) is: (a)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmet-hyl)-1-methylpyrrolidinium bromide; (b)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; (c) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-m-ethylpyrrolidinium bromide; or (d) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide.

10. A pharmaceutical combination comprising a compound having theformula: ##STR00030## wherein R1 and R2 are both phenyl or one ofR.sub.1 and R.sub.2 is phenyl and the other is cyclopentyl; R isC.sub.1-C.sub.8 alkyl, straight or branched chain; and X.sup.- isan anion with a single negative charge; and wherein each asteriskmarks a chiral center; said compound having the R, S or RSstereoisomeric configuration at each chiral center unless specifiedotherwise, or being a mixture thereof; and an anti-inflammatorycorticosteroid, betamimetic agent or antiallergic agent, in acombined amount effective for reducing or inhibiting thedevelopment of, or controlling or alleviating the symptoms of,chronic obstructive pulmonary disease, asthma, bronchitis, allergicrhinitis or infectious rhinitis.

11. The combination as claimed in claim 10, wherein the compound offormula (Ia) is a compound having the formula: ##STR00031## whereinR is methyl or ethyl; and wherein each asterisk marks a chiralcenter; said compound having the R, S or RS stereoisomericconfiguration at each chiral center unless specified otherwise, orbeing a mixture thereof.

12. The combination as claimed in claim 10, wherein the compound offormula (Ia) is the compound which is: (a)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-m-ethylpyrrolidinium bromide; (b)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; (c) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-m-ethylpyrrolidinium bromide; (d) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; (e) (2R,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-m-ethylpyrrolidinium bromide; (f) (2R,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-m-ethylpyrrolidinium bromide; (g) (2R,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; (h) (2S,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-m-ethylpyrrolidinium bromide; (i) (2S,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-m-ethylpyrrolidinium bromide; (j) (2R,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; (k) (2R,1'R,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; (l) (2R,1'S,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; (m) (2R,1'R,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; or (n) (2R,1'S,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide.

13. The combination as claimed in claim 10, wherein the compound offormula (Ia) is: (a)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-m-ethylpyrrolidinium bromide; (b)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; (c) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-m-ethylpyrrolidinium bromide; or (d) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide.

14. The combination as claimed in claim 10, wherein theanti-inflammatory corticosteroid is selected from the groupconsisting of budesonide, fluticasone, loteprednol etabonate,etiprednol dichloracetate, mometasone and ciclesonide, or whereinthe betamimetic agent is selected from the group consisting offenoterol, formoterol or salmeterol.

15. A pharmaceutical combination as claimed in claim 14 wherein theanti-inflammatory corticosteroid is loteprednol etabonate, andfurther comprising an enhancing agent for the loteprednol etabonateselected from the group consisting of: (a)11.beta.,17.alpha.-dihydroxyandrost-4-en-3-one-17.beta.-carboxylicacid; (b)11.beta.,17.alpha.-dihydroxyandrosta-1,4-dien-3-one-17.beta.-carboxyl-ic acid; (c) methyl11.beta.,17.alpha.-dihydroxyandrost-4-en-3-one-17.beta.-carboxylate;(d) ethyl11.beta.,17.alpha.-dihydroxyandrost-4-en-3-one-17.beta.-carboxylate-; (e) methyl11.beta.,17.alpha.-dihydroxyandrosta-1,4-dien-3-one-17.beta.-carboxylate;and (f) ethyl11.beta.,17.alpha.-dihydroxyandrosta-1,4-dien-3-one-17.beta.-carboxylate.

16. A method as claimed in claim 2, wherein the compound of formula(Ia) is administered to said subject as a pharmaceuticalcombination comprising said compound of formula (Ia) and ananti-inflammatory corticosteroid, betamimetic agent or antiallergicagent in a combined amount effective for reducing or inhibiting thedevelopment of, or controlling or alleviating the symptoms of, adisorder selected from the group consisting of chronic obstructivepulmonary disease, asthma, bronchitis, allergic rhinitis andinfectious rhinitis.

17. The method as claimed in claim 16, wherein theanti-inflammatory corticosteroid is selected from the groupconsisting of budesonide, fluticasone, loteprednol etabonate,etiprednol dichloracetate, mometasone and ciclesonide, or whereinthe betamimetic agent is selected from the group consisting offenoterol, formoterol or salmeterol.

18. The method as claimed in claim 17, wherein theanti-inflammatory corticosteroid is loteprednol etabonate andwherein the pharmaceutical combination further comprises anenhancing agent for the loteprednol etabonate selected from thegroup consisting of: (a)11.beta.,17.alpha.-dihydroxyandrost-4-en-3-one-17.beta.-carboxylicacid; (b)11.beta.,17.alpha.-dihydroxyandrosta-1,4-dien-3-one-17.beta.-carboxyl-ic acid; (c) methyl11.beta.,17.alpha.-dihydroxyandrost-4-en-3-one-17.beta.-carboxylate;(d) ethyl11.beta.,17.alpha.-dihydroxyandrost-4-en-3-one-17.beta.-carboxylate-; (e) methyl11.beta.,17.alpha.-dihydroxyandrosta-1,4-dien-3-one-17.beta.-carboxylate;and (f) ethyl11.beta.,17.alpha.-dihydroxyandrosta-1,4-dien-3-one-17.beta.-carboxylate.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of copending U.S. patentapplication Ser. No. 12/494,367, filed Jun. 30, 2009, now allowed,which is a divisional of U.S. patent application Ser. No.12/138,013, filed Jun. 12, 2008, now U.S. Pat. No. 7,576,210, whichis a divisional of U.S. patent application Ser. No. 11/598,079,filed Nov. 13, 2006, now U.S. Pat. No. 7,399,861, which claimsbenefit of U.S. Provisional Patent Application No. 60/735,207,filed Nov. 10, 2005, all incorporated by reference herein in theirentireties and relied upon.

[0002] This application is also related to U.S. application Ser.No. 11/598,076 concurrently filed with prior application Ser. No.11/598,079 on Nov. 13, 2006, by the present inventor and claimingbenefit of U.S. Provisional Application No. 60/735,206, filed Nov.10, 2006, now U.S. Pat. No. 7,417,174, as well as application Ser.No. 12/137,896, filed Jun. 12, 2008, as a divisional of Appln. No.11/598,076, now U.S. Pat. No. 7,538,219, and its divisional,application Ser. No. 12/418,939, filed Apr. 6, 2009, now U.S. Pat.No. 8,071,639, as well as the divisional thereof, Application No.13/286,020, filed Oct. 31, 2011, all incorporated by referenceherein in their entireties and relied upon.

BACKGROUND

[0003] Various anticholinergic compounds have been previouslydescribed but are not optimal.

[0004] Muscarinic receptor antagonists are frequently usedtherapeutic agents that inhibit the effects of acetylcholine byblocking its binding to muscarinic cholinergic receptors atneuroeffector sites on smooth muscle, cardiac muscle, and glandcells as well as in peripherial ganglia and in the central nervoussystem (CNS). However, their side effects, which can include drymouth, photophobia, blurred vision, urinary hesitancy andretention, decreased sweating, drowsiness, dizziness, restlessness,irritability, disorientation, hallucinations, tachycardia andcardiac arrhythmias, nausea, constipation, and severe allergicreactions, often limit their clinical use, and even topicalanticholinergics can cause the same unwanted side effects.Glycopyrrolate and triotropium are among the quaternary ammoniumanticholinergics, which have reduced CNS-related side effects asthey cannot cross the blood-brain barrier; however, becauseglycopyrrolate (or, presumably, tiotropium) is eliminated mainly asunchanged drug or active metabolite in the urine, itsadministration is problematic in young or elderly patients andespecially in uraemic patients. To increase the therapeutic indexof anticholinergics, the soft drug approach has been applied in anumber of different designs starting from various lead compoundsover the past 20 years, but there is a need for yet other new softanticholinergics. These novel muscarinic antagonists, just as allother soft drugs, are designed to elicit their intendedpharmacological effect at the site of application, but to bequickly metabolized into their designed-in, inactive metaboliteupon entering the systemic circulation and rapidly eliminated fromthe body, resulting in reduced systemic side effects and increasedtherapeutic index.

SUMMARY

[0005] New soft anticholinergic agents, pharmaceutical compositionscontaining them, processes for their preparation and methods foreliciting an anticholinergic response, especially for treating anobstructive disease of the respiratory tract or for treatingoveractive bladder, are provided.

[0006] In one exemplary embodiment, there is provided a compoundhaving the formula

##STR00002##

wherein R.sub.1 and R.sub.2 are both phenyl or one of R.sub.1 andR.sub.2 is phenyl and the other is cyclopentyl; R isC.sub.1-C.sub.8 alkyl, straight or branched chain; and X.sup.- isan anion with a single negative charge; and wherein each asteriskmarks a chiral center; said compound having the R, S or RSstereoisomeric configuration at each chiral center unless otherwisespecified, or being a mixture thereof.

[0007] In another exemplary embodiment, there is provided acompound having the formula

##STR00003##

wherein R is methyl or ethyl.

[0008] In other exemplary embodiments, processes for preparing thecompounds are provided.

[0009] In other exemplary embodiments, there are providedpharmaceutical compositions comprising one or more of the compoundsof the foregoing formulas and pharmaceutically acceptable carrierstherefor; pharmaceutical combinations comprising one or more of thecompounds of the foregoing formulas and an anti-inflammatorycorticosteroid, a betamimetic agent or an antialleric agent; andmethods of using the subject compositions and combinations.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 depicts the pH profiles of four compounds of theinvention: Compound (a), -.smallcircle.-; Compound (b), -.DELTA.-;Compound (c), -.tangle-solidup.-; and Compound (d), - -.

[0011] FIG. 2 is a representative time-profile of a chemicalhydrolysis with formation of the corresponding acid as hydrolyticproduct [Compound (d), pH 7.3, 37.degree. C.].

[0012] FIG. 3A is a graph of mydriatic activities of glycopyrrolateand soft analogs Compound (c) and Compound (d) at pharmacologicallyequipotent doses (mean.+-.SD shown) showing data for up to 144hours.

[0013] FIG. 3B is a graph of mydriatic activities of glycopyrrolateand soft analogs Compounds (c) and (d) at pharmacologicallyequipotent doses (mean.+-.SD shown) showing data for the first 24hours only.

[0014] FIG. 4 is a graph of mydriatic activities of variouszwitterionic isomers at 0.1% concentrations over a seven hourperiod.

[0015] FIG. 5 is a graph comparing the mydriatic activity of themost active zwitterionic isomers with glycopyrrolate at 0.1%concentrations.

[0016] FIG. 6 is a graph of the heart rate in beats per minuteversus time in minutes showing the protection effect of differentanticholinergics on carbachol-induced bradycardia in anesthetizedrats (mean.+-.SD; n=3-5), including ethyl, n-hexyl and n-octylesters.

[0017] FIG. 7 is a graph of the heart rate in beats per minuteversus time in minutes showing the protective effect of differentanticholinergics, including Compound (w), on carbachol-inducedbradycardia is anesthetized rats (n=3-6).

[0018] FIG. 8 is a graph showing the time course of action ofdifferent anticholinergics, including Compounds (w) and (aa), onelectrically stimulated guinea pig trachea.

[0019] FIG. 9 is a graph showing the time course of the effect ofdifferent anticholingerics including Compounds (w) and (aa), afterwash out of the test drug on electrically stimulated guinea pigtrachea.

[0020] FIG. 10 is a graph of bronchoconstriction (% of baseline)versus time for acetylcholine-induced bronchoconstriction inanesthetized guinea pigs for Compounds (q) and (m) andglycopyrrolate at selected dosages.

DETAILED DESCRIPTION

[0021] Throughout this specification, the following definitions,general statements and illustrations are applicable:

[0022] The patents, published applications, and scientificliterature referred to herein establish the knowledge of those withskill in the art and are hereby incorporated by reference in theirentirety to the same extent as if each was specifically andindividually indicated to be incorporated by reference. Anyconflict between any reference cited herein and the specificteachings of this specification shall be resolved in favor of thelatter. Likewise, any conflict between an art-understood definitionof a word or phrase and a definition of the word or phrase asspecifically taught in this specification shall be resolved infavor of the latter.

[0023] As used herein, whether in a transitional phrase or in thebody of a claim, the terms "comprise(s)" and "comprising" are to beinterpreted as having an open-ended meaning. That is, the terms areto be interpreted synonymously with the phrases "having at least"or "including at least". When used in the context of a process, theterm "comprising" means that the process includes at least therecited steps, but may include additional steps. When used in thecontext of a composition, the term "comprising" means that thecomposition includes at least the recited features or components,but may also include additional features or components.

[0024] The terms "consists essentially of" or "consistingessentially of" have a partially closed meaning, that is, they donot permit inclusion of steps or features or components which wouldsubstantially change the essential characteristics of a process orcomposition; for example, steps or features or components whichwould significantly interfere with the desired properties of thecompounds or compositions described herein, i.e., the process orcomposition is limited to the specified steps or materials andthose which do not materially affect the basic and novelcharacteristics of the invention. The basic and novel featuresherein are the provision of compounds of formula (Ia) and (Ib) andcombinations of those compounds with other drugs, particularly withanti-inflammatory steroids, especially loteprednol etabonate oretiprednol dichloracetate, and most especially in the case ofloteprenol etabonate (LE) further including an inactive metaboliteenhancing agent for the LE as further defined hereinafter.

[0025] The terms "consists of" and "consists" are closedterminology and allow only for the inclusion of the recited stepsor features or components.

[0026] As used herein, the singular forms "a," "an" and "the"specifically also encompass the plural forms of the terms to whichthey refer, unless the content clearly dictates otherwise.

[0027] The term "about" is used herein to mean approximately, inthe region of, roughly, or around. When the term "about" is used inconjunction with a numerical range, it modifies that range byextending the boundaries above and below the numerical values setforth. In general, the term "about" or "approximately" is usedherein to modify a numerical value above and below the stated valueby a variance of 20%.

[0028] As used herein, the recitation of a numerical range for avariable is intended to convey that the variable can be equal toany of the values within that range. Thus, for a variable which isinherently discrete, the variable can be equal to any integer valueof the numerical range, including the end-points of the range.Similarly, for a variable which is inherently continuous, thevariable can be equal to any real value of the numerical range,including the end-points of the range. As an example, a variablewhich is described as having values between 0 and 2, can be 0, 1 or2 for variables which are inherently discrete, and can be 0.0, 0.1,0.01, 0.001, or any other real value for variables which areinherently continuous.

[0029] In the specification and claims, the singular forms includeplural referents unless the context clearly dictates otherwise. Asused herein, unless specifically indicated otherwise, the word "or"is used in the "inclusive" sense of "and/or" and not the"exclusive" sense of "either/or."

[0030] Technical and scientific terms used herein have the meaningcommonly understood by one of skill in the art to which the presentinvention pertains, unless otherwise defined. Reference is madeherein to various methodologies and materials known to those ofskill in the art. Standard reference works setting forth thegeneral principles of pharmacology include Goodman and Gilman's ThePharmacological Basis of Therapeutics, 10.sup.th Ed., McGraw HillCompanies Inc., New York (2001).

[0031] As used herein, "treating" means reducing, preventing,hindering or inhibiting the development of, controlling,alleviating and/or reversing the symptoms in the individual towhich a combination or composition comprising a compound of formula(Ia) or (Ib) has been administered, as compared to the symptoms ofan individual not being so treated. A practitioner will appreciatethat the combinations, compositions, dosage forms and methodsdescribed herein are to be used in concomitance with continuousclinical evaluations by a skilled practitioner (physician orveterinarian) to determine subsequent therapy. Such evaluation willaid and inform in evaluating whether to increase, reduce orcontinue a particular treatment dose, and/or to alter the mode ofadministration.

[0032] The methods described herein are intended for use with anysubject/patient that may experience their benefits. Thus, the terms"subjects" as well as "patients," "individuals" and "warm-bloodedanimals" include humans as well as non-human subjects, particularlydomesticated animals, particularly dogs, cats, horses and cows, aswell as other farm animals, zoo animals and/or endangeredspecies.

[0033] X.sup.- denotes an anion with a single negative charge. Thisanion is an anion of a pharmaceutically acceptable acid.Preferably, X.sup.- is chloride, bromide, iodide, sulfate,phosphate, methanesulfonate, nitrate, maleate, acetate, citrate,fumarate, tartrate, oxalate, succinate, benzoate orp-toluenesulfonate. More preferably, X.sup.- is chloride, bromide,4-toluenesulfonate or methanesulfonate. Most preferably X.sup.- isbromide.

[0034] In formula (Ia), the compounds having the R configurationwith respect to chiral center 2 are of particular interest.

[0035] The moiety R in formulas (Ia) and (Ib) can be methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl or n-octyl or theirbranched chain isomers.

[0036] In the compounds of formulas (Ia) and (Ib), R is preferablyC.sub.1-C.sub.6 straight chain alkyl.

[0037] In the compounds of formula (Ia), compounds wherein one ofR.sub.1 and R.sub.2 is phenyl and the other is cyclopentyl are ofparticular interest.

[0038] Also of particular interest are the compounds of theformula:

##STR00004##

wherein R is methyl or ethyl.

[0039] The following specific compounds of formula (Ia) are ofparticular interest: [0040] (a)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-m-ethylpyrrolidinium bromide; [0041] (b)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; [0042] (c) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-m-ethylpyrrolidinium bromide; [0043] (d) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; [0044] (e) (2R,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-m-ethylpyrrolidinium bromide; [0045] (f) (2R,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-m-ethylpyrrolidinium bromide; [0046] (g) (2R,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; [0047] (h) (2S,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-m-ethylpyrrolidinium bromide; [0048] (i) (2S,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-m-ethylpyrrolidinium bromide; [0049] (j) (2R,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; [0050] (k) (2R,1'R,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; [0051] (l) (2R,1'S,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; [0052] (m) (2R,1'R,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; [0053] (n) (2R,1'S,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide; [0054] (o) (2R,1'R,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(n-hexyloxycarbonylmethyl)--1-methylpyrrolidinium bromide; [0055] (p) (2R,1'S,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(n-hexyloxycarbonylmethyl)--1-methylpyrrolidinium bromide; [0056] (q) (2R,1'R,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(n-hexyloxycarbonylmethyl)--1-methylpyrrolidinium bromide; [0057] (r) (2R,1'S,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(n-hexyloxycarbonylmethyl)--1-methylpyrrolidinium bromide; [0058] (s) (2R,1'R,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-methyl-1-(n-octyloxycarbony-lmethyl)pyrrolidinium bromide; [0059] (t) (2R,1'S,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-methyl-1-(n-octyloxycarbony-lmethyl)pyrrolidinium bromide; [0060] (u) (2R,1'R,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-methyl-1-(n-octyloxycarbony-lmethyl)pyrrolidinium bromide; or [0061] (v) (2R,1'S,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-methyl-1-(n-octyloxycarbony-lmethyl)pyrrolidinium bromide.

[0062] Of these, particular mention may be made of: [0063] (a)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-m-ethylpyrrolidinium bromide; [0064] (b)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyaolidinium bromide; [0065] (c) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-m-ethylpyrrolidinium bromide; or [0066] (d) (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide.

[0067] In formula (Ib), the compound wherein R is ethyl and X.sup.-is Br.sup.- is of special interest. The compounds of formula (Ib)wherein R is methyl, n-hexyl and n-octyl and X.sup.- is Br.sup.-can be made in analogous fashion to the R=ethyl compound and arealso of particular interest.

[0068] Various methods of making the instant compounds areillustrated hereinafter. Generally speaking, the compounds offormula (Ia) can be prepared by reacting a bromoacetate of theformula

BrCH.sub.2COOR

wherein R is as defined above, with a compound of the formula

##STR00005##

wherein R.sub.1 and R.sub.2, the asterisks and the stereoisomericconfigurations are as defined above, and optionally separating theindividual stereoisomers to afford a compound of formula (Ia) and,when desired, exchanging the bromine anion with a different X.sup.-anion wherein X.sup.- is as defined above but other thanBr.sup.-.

[0069] In a particular embodiment, the compound of formula (IIa)has the R configuration with respect to chiral center 2.

[0070] In another particular embodiment, the compound of formula(IIa) has the configuration R or S with respect to chiral center 1'or with respect to chiral center 3'.

[0071] In another embodiment, the process includes separating theindividual stereoisomers of the compound of formula (Ia) aftertheir formation to the extent possible.

[0072] In one particular embodiment, the process comprisesquaternizing a compound of the formula

##STR00006##

with an alkyl bromoacetate of the formula

BrCH.sub.2COOR

wherein R is methyl or ethyl, to afford the desired product.

[0073] In analogous fashion, methods of making the compounds offormula (Ib) are illustrated hereinafter. Generally speaking, theprocess comprises reacting a bromocetate of the formula:

BrCH.sub.2COOR

wherein R is as defined above, with a compound of the formula

##STR00007##

and optionally separating the individual stereoisomers to afford acompound of formula (Ib) and, when desired, exchanging the bromineanion with a different X.sup.- anion wherein X.sup.- is as definedabove but other than Br.sup.-.

[0074] In the case of both the compounds of formula (Ia) and thoseof formula (Ib), use of ICH.sub.2COOR or ClCH.sub.2COOR in place ofBRCH.sub.2COOR can be employed in the above reaction schemes toafford the corresponding compounds in which X.sup.- is I.sup.- orCl.sup.-. Alternatively, ion exchange columns can be used toreplace the Br.sup.- anion in the product of formula (Ia) or (Ib)with a different X.sup.- anion.

[0075] The compounds of formulas (Ia) and (Ib) are of use aspharmaceutical agents because of their anticholinergic activity. Ananticholinergically effective amount of such an agent inhibits theeffect of acetycholine by blocking its binding to muscariniccholinergic receptors at neuroeffector sites. Subjects in need of amethod of eliciting an anticholinergic response are those sufferingfrom conditions which respond to treatment with an anticholinergicagent. Such conditions include obstructive diseases of therespiratory tract, for example asthma and chronic obstructivepulmonary disease, vagally induced sinus bradycardia and heartrhythm disorders, spasms, for example in the gastrointestinal tractor urinary tract (including overactive bladder) and in menstrualdisorders. The compounds of formulas (Ia) and (Ib) can also be usedto induce short-acting mydriasis and thus can be used to dilate thepupils of the eyes in vision testing. Other uses of the compoundsof formulas (Ia) and (Ib) include the treatment of ulcers as wellas topical use as an antiperspirant in the treatment hyperhydrosis(sweating).

[0076] The compounds of formula (Ia) and (Ib) are particularlyuseful in the treatment of obstructive diseases of the respiratorytract. The expression "obstructive disease of the respiratorytract" includes breathing disorders such as asthma, bronchitis,chronic obstructive pulmonary disease (COPD), allergic rhinitis andinfectious rhinitis.

[0077] "Asthma" refers to a chronic lung disease causingbronchoconstriction (narrowing of the airways) due to inflammation(swelling) and tightening of the muscles around the airways. Theinflammation also causes an increase in mucus production, whichcauses coughing that may continue for extended periods. Asthma isgenerally characterized by recurrent episodes of breathlessness,wheezing, coughing, and chest tightness, termed exacerbations. Theseverity of exacerbations can range from mild to life threatening.The exacerbations can be a result of exposure to e.g. respiratoryinfections, dust, mold, pollen, cold air, exercise, stress, tobaccosmoke, and air pollutants.

[0078] "COPD" refers to chronic obstructive pulmonary disease,primarily but not necessarily associated with past and presentcigarette smoking. It involves airflow obstruction, mainlyassociated with emphysema and chronic bronchitis. Emphysema causesirreversible lung damage by weakening and breaking the air sacswithin the lungs. Chronic bronchitis is an inflammatory disease,which increases mucus in the airways and bacterial infections inthe bronchial tubes, resulting in obstructed airflow.

[0079] "Allergic rhinitis" refers to acute rhinitis or nasalrhinitis, including hay fever. It is caused by allergens such aspollen or dust. It may produce sneezing, congestion, runny nose,and itchiness in the nose, throat, eyes, and ears.

[0080] "Infectious rhinitis" refers to acute rhinitis or nasalrhinitis of infectious origin. It is caused by upper respiratorytract infection by infectious rhinoviruses, coronaviruses,influenza viruses, parainfluenza viruses, respiratory syncyticalvirus, adenoviruses, coxsackieviruses, echoviruses, or Group Abeta-hemolytic Streptococci and is generically referred to as thecommon cold. It may produce sneezing, congestion, runny nose, anditchiness in the nose, throat, eyes, and ears.

[0081] The compounds of formula (Ia) and (Ib) are also particularlyuseful in the treatment of overactive bladder (OAB).

[0082] Overactive bladder is a treatable medical condition that isestimated to affect 17 to 20 million people in the United States.Symptoms of overactive bladder can include urinary frequency,urinary urgency, urinary urge incontinence (accidental loss ofurine) due to a sudden and unstoppable need to urinate, nocturia(the disturbance of nighttime sleep because of the need to urinate)or enuresis resulting from overactivity of the detrusor muscle (thesmooth muscle of the bladder which contracts and causes it toempty).

[0083] Neurogenic overactive bladder (or neurogenic bladder) is atype of overactive bladder which occurs as a result of detrusormuscle overactivity referred to as detrusor hyperreflexia,secondary to known neurologic disorders. Patients with neurologicdisorders, such as stroke, Parkinson's disease, diabetes, multiplesclerosis, peripheral neuropathy, or spinal cord lesions oftensuffer from neurogenic overactive bladder. In contrast,non-neurogenic overactive bladder occurs as a result of detrusormuscle overactivity referred to as detrusor muscle instability.Detrusor muscle instability can arise from non-neurologicalabnormalities, such as bladder stones, muscle disease, urinarytract infection or drug side effects or can be idiopathic.

[0084] Due to the enormous complexity of micturition (the act ofurination), an exact mechanism which causes overactive bladder isnot known. Overactive bladder can result from hypersensitivity ofsensory neurons of the urinary bladder, arising from variousfactors including inflammatory conditions, hormonal imbalances, andprostate hypertrophy. Destruction of the sensory nerve fibers,either from a crushing injury to the sacral region of the spinalcord, or from a disease that causes damage to the dorsal rootfibers as they enter the spinal cord can also lead to overactivebladder. In addition, damage to the spinal cord or brain stemcausing interruption of transmitted signals can lead toabnormalities in micturition. Therefore, both peripheral andcentral mechanisms can be involved in mediating the alteredactivity in overactive bladder.

[0085] Current treatments for overactive bladder includemedication, diet modification, programs in bladder training,electrical stimulation, and surgery. Currently, antimuscarinics(which are members of the general class of anticholinergics) arethe primary medication used for the treatment of overactivebladder. The antimuscarinic, oxybutynin, has been the mainstay oftreatment for overactive bladder. However, treatment with knownantimuscarinics suffers from limited efficacy and side effects suchas dry mouth, dry eyes, dry vagina, blurred vision, cardiac sideeffects, such as palpitations and arrhythmia, drowsiness, urinaryretention, weight gain, hypertension and constipation, which haveproven difficult for some individuals to tolerate. Thus, the needfor new anticholinergic agents is evident.

[0086] The compounds of formula (Ia) or (Ib) may be used on theirown or combined with other active substances of formula (Ia) or(Ib) according to the invention.

[0087] The compounds of formula (Ia) or (Ib) may optionally also becombined with other pharmacologically active substances. Theseinclude, in particular, betamimetics, antiallergic agents, andcorticosteroids (also termed "anti-inflammatory steroids","anti-inflammatory corticosteroids" or simply "steroids") andcombinations of these active substances. The combinations withbetamimetics, antiallergics or corticosteroids are of interest inthe treatment of obstructive diseases of the respiratory tract,especially COPD or asthma. Accordingly, they are intended foradministration by oral inhalation, as powders or aerosols.

[0088] Examples of betamimetics which may be used in conjunctionwith the compounds of formula (Ia) or (Ib) include compoundsselected from the group consisting of bambuterol, bitolterol,carbuterol, clenbuterol, fenoterol, formoterol, hexoprenaline,ibuterol, pirbuterol, procaterol, reproterol, salmeterol,sulfphonterol, terbutaline, tulobuterol,4-hydroxy-7-[2-{[2-{[3-(2-phenylethoxy)propyl]sulfonyl}ethyl]amino}ethyl]--2(3H)-benzothiazolone,1-(2-fluoro-4-hydroxyphenyl)-2-[4-(1-benzimidazolyl)-2-methyl-2-butylamin-o]ethanol,1-[3-(4-methoxybenzylamino)-4-hydroxyphenyl]-2-[4-(1-benzimidaz-olyl)-2-methyl-2-butylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-[3-(4-N,N-dimethylaminoph-enyl)-2-methyl-2-propylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-[3-(4-methoxyphenyl)-2-me-thyl-2-propylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-[3-(4-n-butyloxyphenyl)-2--methyl-2-propylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-{4-[3-(4-methoxyphenyl)-1-,2,4-triazol-3-yl]-2-methyl-2-butylamino}ethanol,5-hydroxy-8-(1-hydroxy-2-isopropylaminobutyl)-2H-1,4-benzoxazin-3-(4H)-on-e,1-(4-amino-3-chloro-5-trifluoromethylphenyl)-2-tert.-butylamino)ethanoland1-(4-ethoxycarbonylamino-3-cyano-5-fluorophenyl)-2-(tert.-butylamino)-ethanol, optionally in the form of their racemates, theirenantiomers, their diastereomers, as well as optionally theirpharmacologically acceptable acid addition salts and hydrates. Itis particularly preferable to use, as betamimetics, activesubstances of this kind, combined with the compounds of formula(Ia) or (Ib), selected from among fenoterol, formoterol,salmeterol,1-[3-(4-methoxybenzylamino)-4-hydroxyphenyl]-2-[4-(1benzimidazolyl)-2-met-hyl-2-butylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-[3-(4-N,N-dimethylaminoph-enyl)-2-methyl-2-propylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-[3-(4-methoxyphenyl)-2-me-thyl-2-propylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-[3-(4-n-butyloxyphenyl)-2--methyl-2-propylamino]ethanol,1-[2H-5-hydroxy-3-oxo-4H-1,4-benzoxazin-8-yl]-2-{4-[3-(4-methoxyphenyl)-1-,2,4-triazol-3-yl]-2-methyl-2-butylamino}ethanol, optionally in theform of their racemates, their enantiomers, their diastereomers, aswell as optionally their pharmacologically acceptable acid additionsalts and hydrates. Of the betamimetics mentioned above, thecompounds formoterol and salmeterol, optionally in the form oftheir racemates, their enantiomers, their diastereomers, as well asoptionally their pharmacologically acceptable acid addition saltsand hydrates, are particularly important.

[0089] The acid addition salts of the betamimetics selected fromamong the hydrochloride, hydrobromide, sulfate, phosphate,fumarate, methanesulfonate and xinafoate are preferred according tothe invention. In the case of salmeterol, the salts selected fromamong the hydrochloride, sulfate and xinafoate are particularlypreferred, especially the sulfates and xinafoates. In the case offormoterol, the salts selected from among the hydrochloride,sulfate and fumarate are particularly preferred, especially thehydrochloride and fumarate. Of outstanding importance is formoterolfumarate.

[0090] The corticosteroids which may optionally be used inconjunction with the compounds of formula (Ia) or (Ib), includecompounds selected from among flunisolide, beclomethasone,triamcinolone, budesonide, fluticasone, mometasone, ciclesonide,rofleponide, GW 215864, KSR 592, ST-126, loteprednol etabonate,etiprednol dichloracetate and dexamethasone. The preferredcorticosteroids are those selected from among flunisolide,beclomethasone, triamcinolone, loteprednol etabonate, etiprednoldichloracetate, budesonide, fluticasone, mometasone, ciclesonideand dexamethasone, while budesonide, fluticasone, loteprednoletabonate, etiprednol dichloracetate, mometasone and ciclesonide,especially budesonide, fluticasone, loteprednol etabonate andetiprednol dichloracetate, are of particular importance. Anyreference to steroids herein also includes a reference to salts orderivatives which may be formed from the steroids. Examples ofpossible salts or derivatives include: sodium salts,sulfobenzoates, phosphates, isonicotinates, acetates, propionates,dihydrogen phosphates, palmitates, pivalates or furoates. Thecorticosteroids may optionally also be in the form of theirhydrates.

[0091] When the corticosteroid is loteprednol etabonate, it may beadvantageously combined with an enhancing agent selected from thegroup consisting of: [0092] (a)11.beta.,17.alpha.-dihydroxyandrost-4-en-3-one-17.beta.-carboxylicacid (cortienic acid, or CA); [0093] (b)11.beta.,17.alpha.-dihydroxyandrosta-1,4-dien-3-one-17.beta.-carboxylicacid (.DELTA..sup.1 cortienic acid or .DELTA..sup.1-CA); [0094] (c)methyl11.beta.,17.alpha.-dihydroxyandrost-4-en-3-one-17.beta.-carboxylat-e (cortienic acid methyl ester, or MeCA); [0095] (d) ethyl11.beta.,17.alpha.-dihydroxyandrost-4-en-3-one-17.beta.-carboxylate(cortienic acid ethyl ester, or EtCA); [0096] (e) methyl11.beta.,17.alpha.-dihydroxyandrosta-1,4-dien-3-one-17.beta.-carboxylate((.DELTA..sup.1 cortienic acid methyl ester, or.DELTA..sup.1-MeCA); and [0097] (f) ethyl11.beta.,17.alpha.-dihydroxyandrosta-1,4-dien-3-one-17.beta.-carboxylate(.DELTA..sup.1 cortienic acid ethyl ester, or .DELTA..sup.1-EtCA),wherein the mole ratio of loteprednol etabonate to enhancing agentis from about 5:1 to about 0.5:1. Such combinations with theseinactive metabolites are described in detail in WO 2005/000317 A1,incorporated by reference herein in its entirety and reliedupon.

[0098] Examples of antiallergic agents which may be used as acombination with the compounds of formula (Ia) or (Ib) includeepinastin, cetirizin, azelastin, fexofenadin, levocabastin,loratadine, mizolastin, ketotifen, emedastin, dimetinden,clemastine, bamipin, cexchloropheniramine, pheniramine, doxylamine,chlorophenoxamine, dimenhydrinate, diphenhydramine, promethazine,ebastin, desloratidine and meclizine. Preferred antiallergic agentswhich may be used in combination with the compounds of formula (Ia)or (Ib) are selected from among epinastin, cetirizin, azelastin,fexofenadin, levocabastin, loratadine, ebastin, desloratidine andmizolastin, epinastin and desloratidine being particularlypreferred. Any reference to the abovementioned antiallergic agentsalso includes a reference to any pharmacologically acceptable acidaddition salts thereof which may exist.

[0099] When the compounds of formula (Ia) or (Ib) are used inconjunction with other active substances, the combination withsteroids or betamimetics is particularly preferred of the variouscategories of compounds mentioned above.

[0100] Whether or not the compounds of formula (Ia) or (Ib) areused in conjunction with other active substances as describedabove, they are typically administered in the form of apharmaceutical composition comprising an anticholinergicallyeffective amount of a compound of formula (Ia) or (Ib) and anon-toxic pharmaceutically acceptable carrier therefor.Pharmaceutically acceptable carriers, or diluents, are well-knownin the art. The carriers may be any inert material, organic orinorganic, suitable for administration, such as: water, gelatin,gum arabic, lactose, microcrystalline cellulose, starch, sodiumstarch glycolate, calcium hydrogen phosphate, magnesium stearate,talcum, colloidal silicon dioxide, and the like. Such compositionsmay also contain other pharmaceutically active agents, as notedabove, and/or conventional additives such as stabilizers, wettingagents, emulsifiers, flavoring agents, buffers, binders,disintegrants, lubricants, glidants, antiadherents, propellants,and the like. The carrier, e.g., non-active ingredient, can be just(sterile) water with the pH adjusted to where the activepharmaceutical agent is very soluble. It is preferred that the pHbe at or near 7. Alternatively and preferably, the non-activecarrier agent should be physiological saline with the pH adjustedappropriately.

[0101] The novel compounds of formula (Ia) or (Ib) can beadministered in any suitable way. The compounds can be made up insolid or liquid form, such as tablets, capsules, powders, syrups,elixirs and the like, aerosols, sterile solutions, suspensions oremulsions, and the like.

[0102] The compounds of formula (Ia) or (Ib) can be brought intosuitable dosage forms, such as compositions for administrationthrough the oral, rectal, trandermal, parenteral, nasal, pulmonary(typically via oral inhalation) or topical (including ophthalmic)route in accordance with accepted pharmaceutical procedures. Theroute of administration and thus the dosage form will be chosen inlight of the condition to be treated with the instantanticholinergic agents. By way of illustration only, when thecompound of formula (Ia) or (Ib) is administered to treat COPD orasthma, or other serious obstructive disease of the respiratorytract, the compounds may be advantageously administered viainhalation or insufflation; for such purposes, the compounds areadvantageously in the form of an aerosol or a powder forinhalation. When administered to treat less serious respiratorydisorders such as rhinitis, a nasal spray, mist or gel may beadvantageous. For inducing mydriasis, an ophthalmic formulationsuch as eye drops may be most appropriate. For OAB, a formulationfor oral administration such as tablet or capsules or a transdermalpreparation may be preferred. For treating hyperhydrosis, antopical preparation formulated as an antiperspirant stick, gel,spray, cream or the like would be preferred.

[0103] For purposes of illustration, dosages are expressed based onthe inhalation of an aerosol solution, such as the product AtroventInhalation Aerosol (Boehringer Ingelheim). Adjustments in dosagesfor administration by other modes of inhaled administration arewell known to those skilled in the art.

[0104] In general, a therapeutically effective oranticholinergically effective amount of compound of formula (Ia) or(Ib) is from about 1 .mu.g to about 1,000 .mu.g, e.g., from about10 .mu.g to about 1,000 .mu.g or from about 100 .mu.g to about 1000.mu.g. However, the exact dosage of the specific compound offormula (Ia) or (Ib) will vary depending on its potency, the modeof administration, the age and weight of the subject and theseverity of the condition to be treated. The daily dosage may, forexample, range from about 0.01 .mu.g to about 10 .mu.g per kg ofbody weight, administered singly or multiply in doses e.g. fromabout 1 .mu.g to about 1,000 .mu.g each. The compounds of formula(Ia) or (Ib) can be administered from one to four times daily,e.g., once or twice daily.

[0105] The dosage form for inhalation can be an aerosol. Theminimum amount of an aerosol delivery is about 0.2 ml and themaximum aerosol delivery is about 5 ml. The concentration of thecompounds of formula (Ia) or (Ib) may vary as long as the totalamount of spray delivered is within the about 0.2 to about 5 mlamount and as long as it delivers an anticholinergically effectiveamount of the compound of formula (Ia) or (Ib). It is well known tothose skilled in the art that if the concentration is higher, onegives a smaller dose to deliver the same effective amount.

[0106] The dosage form for inhalation can also be via intranasalspray. The minimum amount of an aerosol delivery is about 0.02 mlper nostril and the maximum aerosol delivery is about 0.2 ml pernostril. The concentration of the compounds of formula (Ia) or (Ib)may vary as long as the total amount of spray delivered is withinabout 0.02 ml per nostril to about 0.2 ml per nostril, e.g.,between about 0.05 ml per nostril and about 0.08 ml per nostril,and it delivers an anticholinergically effective amount of thecompound of formula (Ia) or (Ib).

[0107] Of course, the volume of aerosol or intranasal spray fordelivering an anticholinergically effective amount of the compoundof formula (Ia) or (Ib) depends upon the concentration of thecompound in the aerosol or intranasal spray, i.e., higherconcentrations of the compound of formula (Ia) or (Ib) requiresmaller dosage volumes to deliver a therapeutically effectiveamount and lower concentrations of the compound of formula (Ia) or(Ib) require larger dosage volumes to deliver the sameanticholinergically effective amount.

[0108] Aerosols for inhalation of various pharmaceutical agents arewell known to those skilled in the art, including many aerosols fortreating asthma. Aerosols may be produced with a nebulizer.Typically, the nebulizer is charged with a carrier solution and thecompound of formula (Ia) or (Ib) in an amount sufficient toeffectively deliver an anticholinergically effective amount of thecompound of formula (Ia) or (Ib). For instance, depending upon thenebulizer and its operating conditions, the nebulizer may becharged with several hundred mg of anticholinergic compound inorder to deliver about 1 .mu.g to about 1000 .mu.g, e.g., fromabout 10 .mu.g to about 1000 .mu.g or from about 50 .mu.g to about500 .mu.g, of the compound of formula (Ia) or (Ib).

[0109] The dosage form for inhalation may also be in powder form.Powders for inhalation of various pharmaceutical agents are wellknown to those skilled in the art, including many powders fortreating asthma. When the dosage form is a powder, the compounds offormula (Ia) or (Ib) can be administered in pure form or dilutedwith an inert carrier. When an inert carrier is used, the compoundsare compounded such that the total amount of powder delivereddelivers an "effective amount" of the compounds according to theinvention. The actual concentration of the active compound mayvary. If the concentration is lower, then more powder must bedelivered, if the concentration is higher, less total material mustbe delivered to provide an effective amount of the active compoundaccording to the invention. Any of the foregoing pharmaceuticalcompositions may further comprise one or more additional activesubstances, particularly corticosteroids and/or betamimetics asdiscussed earlier.

[0110] "Pharmaceutically acceptable" refers to those propertiesand/or substances which are acceptable to the patient from apharmacological/toxicological point of view and to themanufacturing pharmaceutical chemist from a physical/chemical pointof view regarding composition, formulation, stability, patientacceptance and bioavailability.

[0111] Suitable preparations for administering the compounds offormula (Ia) or (Ib) include tablets, capsules, suppositories,solutions, etc. Of particular importance (particularly whentreating asthma or COPD or other respiratory disorders) is theadministration of the compounds by inhalation. The proportion ofpharmaceutically active compound or compounds should be in therange from 0.05 to 90% by weight, preferably 0.1 to 50% by weightof the total composition. Suitable tablets may be obtained, forexample, by mixing the active substance(s) with known excipients,for example inert diluents such as calcium carbonate, calciumphosphate or lactose, disintegrants such as corn starch or alginicacid, binders such as starch or gelatin, lubricants such asmagnesium stearate or talc and/or agents for delaying release, suchas carboxymethyl cellulose, cellulose acetate phthalate, orpolyvinyl acetate. The tablets may also comprise several layers.Tablets and other solid oral formulations are of particularinterest in the treatment of OAB or ulcers while opthalmicsolutions, suspensions and gels are of special interest forinducing mydriasis and topical gels, solids and sprays are ofparticular use as antiperspirants.

[0112] Coated tablets may be prepared accordingly by coating coresproduced analogously to the tablets with substances normally usedfor tablet coatings, for example collidone or shellac, gum arabic,talc, titanium dioxide or sugar. To achieve delayed release orprevent incompatibilities the core may also consist of a number oflayers. Similarly the tablet coating may consist of a number orlayers to achieve delayed release, possibly using the excipientsmentioned above for the tablets.

[0113] Syrups or elixirs containing the active substances offormulas (Ia) or (Ib) or combinations thereof as described abovemay additionally contain a sweetener such as saccharin, cyclamate,aspartame, sucralose, glycerol or sugar and a flavor enhancer, e.g.a flavoring such as vanillin or orange extract. They may alsocontain suspension adjuvants or thickeners such as sodiumcarboxymethyl cellulose, wetting agents such as, for example,condensation products of fatty alcohols with ethylene oxide, orpreservatives such as p-hydroxybenzoates.

[0114] Solutions are prepared in the usual way, e.g. with theaddition of isotonic agents, preservatives such asp-hydroxybenzoates, or stabilizers such as alkali metal salts ofethylenediamine tetraacetic acid, optionally using emulsifiersand/or dispersants, while if water is used as the diluent, forexample, organic solvents may optionally be used as solvatingagents or dissolving aids, and transferred into injection vials orampules or infusion bottles.

[0115] Capsules containing one or more active substances orcombinations of active substances may for example be prepared bymixing the active substances with inert carriers such as lactose orsorbitol and packing them into gelatin capsules. Suitablesuppositories may be made for example by mixing with carriersprovided for this purpose, such as neutral fats orpolyethyleneglycol or the derivatives thereof. Excipients which maybe used include, for example, water, pharmaceutically acceptableorganic solvents such as paraffins (e.g. petroleum fractions),vegetable oils (e.g. groundnut or sesame oil), mono- orpolyfunctional alcohols (e.g. ethanol or glycerol), carriers suchas e.g. natural mineral powders (e.g. kaolins, clays, talc, chalk),synthetic mineral powders (e.g. highly dispersed silicic acid andsilicates), sugars (e.g. cane sugar, lactose and glucose),emulsifiers (e.g. lignin, spent sulfite liquors, methylcellulose,starch and polyvinylpyrrolidone) and lubricants (e.g. magnesiumstearate, talc, stearic acid and sodium lauryl sulphate).

[0116] The preparations are administered by the usual methods,preferably by inhalation in the treatment of asthma or COPD orother respiratory disorders. For oral administration the tabletsmay, of course, contain, apart from the above-mentioned carriers,additives such as sodium citrate, calcium carbonate and dicalciumphosphate together with various additives such as starch,preferably potato starch, gelatin and the like. Moreover,lubricants such as magnesium stearate, sodium lauryl sulfate andtalc may be used at the same time for the tabletting process. Inthe case of aqueous suspensions the active substances may becombined with various flavor enhancers or colorings in addition tothe excipients mentioned above.

[0117] The dosage of the compounds of formula (Ia) and (Ib) isnaturally greatly dependent on the route of administration and thecomplaint to be treated. When administered by inhalation thecompounds of formula (Ia) or (Ib) are characterized by highefficacy even at doses in the .mu.g range. The compounds of formula(Ia) or (Ib) can also be used effectively above the .mu.g range.The dosage may then be in the gram range, for example. Particularlywhen administered by a method other than inhalation, the compoundsaccording to the invention may be given in higher doses (in therange from 1 to 1000 mg, for example, although this does not implyany limitation).

[0118] The compounds of formula (Ia) and (Ib), combinations of acompound of formula (Ia) or (Ib) with one or more other activeagents, and compositions comprising a compound of formula (Ia) or(Ib), with or without one or more other active agents, as describedhereinabove are thus useful in a method for eliciting ananticholinergic response in a subject in need of same, comprisingadministering to said subject an anticholinergically effectiveamount of said compound or composition. In particular embodiments,the method is for treating an obstructive disease of therespiratory tract, especially when the disease is chronicobstructive pulmonary disease or asthma, or for treating overactivebladder. In another embodiment, the method comprises inducingmydriasis in the eye(s) of a subject in need of such treatment,comprising topically applying to the eye(s) of said subject amydriatically effective amount of a compound of formula (Ia) or(Ib) or combination or composition comprising it as describedhereinabove. Use of compounds of formula (Ia) or (Ib) in thepreparation of a medicament for treating a condition responsive toan anticholinergic agent (such as any of these conditions disclosedabove) is likewise provided herein.

[0119] In particular embodiments there are provided combinations ofthe compound of formula (Ia) or (Ib) with other active agents,especially one or more antiinflammatory corticosteroids,betamimetic agents or antiallergic agents. In the combinationproducts, the active agents are present in a combined amounteffective to treat the target condition, especially to treat anobstructive disease of the respiratory tract, most especially totreat chronic obstructive pulmonary disease or asthma. In preferredembodiments, the other active agent is a betamimetic agent or anantiinflammatory corticosteroid. Of particular interest arecombinations of a compound of formula (Ia) or (Ib) and acorticosteroid, especially loteprednol etabonate or etiprednoldichloracetate. When loteprednol etabonate is selected as thecorticosteroid, its activity can be enhanced by combination withcortienic acid or .DELTA..sup.1-cortienic acid or a methyl or ethylester of cortienic acid or .DELTA..sup.1-cortienic acid, in a moleratio of from about 5:1 to about 0.5:1. A molar ratio of about 1:1,which can be approximated by a 1:1 ratio by weight, is particularlyconvenient.

Initial Studies

Materials and Methods

Materials

[0120] Glycopyrrolate (glycopyrronium bromide) was kindly providedby Boehringer Ingelheim Chemicals, Inc. Carbamylcholine bromide(carbachol), atropine methylbromide (atropine MeBr), andscopolamine methylbromide (scopolamine MeBr) were obtained fromSigma Chemicals Co. (St. Louis, Mo.).N-[.sup.3H]-Methyl-scopolamine (NMS) was obtained from AmershamBiosciences UK Limited (Buckinghamshire, UK). Cloned humanmuscarinic receptor subtypes M.sub.1-M.sub.4 were obtained fromApplied Cell Science Inc. (Rockville, Md.). Scintiverse BD was fromFisher Scientific Co. (Pittsburgh, Pa.).

[0121] Chemicals used for synthesis were reagent or HPLC grade, andwere obtained from Aldrich (Milwaukee, Wis.) and Fisher ScientificCo. Melting points were taken on Fisher-Johns melting apparatus.NMR spectra were recorded on a Bruker Advance 500 MHz NMRspectrometer and are reported in ppm relative to TMS. Elementalanalyses were performed by Atlantic Microlab Inc (Atlanta,Ga.).

Synthesis

Racemic cyclopentylmandelic acid (1)

[0122] Cyclopentylmagnesium bromide ether solution (100 ml, 2M; 0.2mol) was added drop-wise to benzoylformic acid (15 g, 0.1 mol) in330 ml of anhydrous ethyl ether at 0.degree. C. The mixture wasstirred at 0.degree. C. for 30 min and at room temperature for 24h. The reaction mixture was treated with 1 N HCl, and the aqueoussolution was extracted with ether. The combined ether solution wastreated with K.sub.2CO.sub.3 solution. The potassium carbonatesolution was acidified with HCl and extracted with ether twice. Theether solution was dried with anhydrous sodium sulfate andevaporated to give a crude product. The crude product was washedwith water to get pure racemic cyclopentylmandelic acid 1 (8.0 g,36.4%). Needle-like crystals, m.p.: 153-154.degree. C. .sup.1H NMR(CDCl.sub.3, 500 MHz): 1.28-1.39, 1.42-1.50, 1.51-1.61, 1.63-1.72[8H, m, (CH.sub.2).sub.4], 2.93 [1H, p, CHC(OH)], 7.26-7.30,7.33-7.36, 7.65-7.67 (5H, m, Ph) ppm.

Methyl cyclopentylmandelate (2)

[0123] To a mixture of racemic cyclopentylmandelic acid R/S(.+-.)1(4.47 g, 20 mmol) and potassium carbonate (7.01 g, 50 mmol) in DMF(50 ml), methyl iodide (8.64 g, 60 mmol) was added at roomtemperature. The mixture was stirred at room temperature for 2 h,and then poured into water and extracted with hexanes three times.Evaporation of the dried hexanes extract gave a crude product.Flash chromatography of the crude product on silica gel with 1.5:1hexanes:methylene chloride gave the pure product 2 (3.02 g, 64%)..sup.1H NMR (CDCl.sub.3, 300 MHz): 1.32-1.37, 1.43-1.69 [8H, m,(CH.sub.2).sub.4], 2.90 [1H, p, CHC(OH)], 3.74 (1H, s, OH), 3.77(3H, s, CH.sub.3), 7.25-7.37, 7.63-7.65 (5H, m, Ph) ppm.

N-Methyl-3-pyrrolidinyl cyclopentylmandelate (4)

[0124] A solution of 2 (2.20 g, 9.4 mmol) andN-methyl-3-pyrrolidinol (3, 1.30 g, 13 mmol) in 40 ml of n-heptanewas heated until 20 ml of heptane had been distilled. About 0.003 gof sodium was added, and the solution was stirred and heated for 2h as the distillation was continued. More heptane was added at sucha rate as to keep the reaction volume constant. Additional sodiumwas added at the end of an hour. The solution was then cooled andextracted with 3N HCl. The acid extract was made alkaline withconcentrated NaOH and extracted three times with ether. Removal ofthe dried ether solution gave a crude oil. Flash chromatography ofthe crude product on silica gel with 8:1 EtOAc:EtOH gave pureproduct 4 (2.053 g, 72%). Analysis for C.sub.18H.sub.25NO.sub.3.Calcd: C, 71.26; H, 8.31; N, 4.62. Found: C, 71.55; H, 8.44; N,4.68. .sup.1H NMR (CDCl.sub.3, 500 MHz): 1.27-1.35, 1.40-1.47,1.54-1.60, 1.75-1.90 [8H, m, (CH.sub.2).sub.4], 2.12-2.30,2.52-2.57, 2.64-2.81 (6H, m, CH.sub.2NCH.sub.2CH.sub.2), 2.33, 2.36(3H, 2s, NCH.sub.3), 2.93 [(1H, p, CHC(OH)], 3.83 (1H, bs, OH),5.23 (1H, m, CO.sub.2CH), 7.23-7.36, 7.64-7.67 (5H, m, Ph) ppm.

3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide, Compound (a)

[0125] To compound 4 (0.8235 g, 2.71 mmol) in 30 ml of dryacetonitrile, methyl bromoacetate (1.08 g, 7.06 mmol) was added atroom temperature. The mixture was stirred for 2 h. Evaporation ofacetonitrile gave a crude product. The crude product was dissolvedin a small volume of methylene chloride and then poured into 100 mlof dry ethyl ether to precipitate. This procedure was repeatedthree times to obtain Compound (a) as pure product (0.9912 g, 80%).White powder, m.p.: 192-194.degree. C. Analysis forC.sub.21H.sub.30BrNO.sub.5. Calcd: C, 55.27; H, 6.63; N, 3.07.Found: C, 55.11; H, 6.59; N, 3.03. .sup.1H NMR (CDCl.sub.3, 500MHz): 1.23-1.29, 1.31-1.37, 1.41-1.47, 1.53-1.67 [8H, m,(CH.sub.2).sub.4], 2.18-2.23, 2.73-2.80, 4.04-4.16, 4.21-4.25 (6H,m, CH.sub.2NCH.sub.2CH.sub.2), 2.85 [1H, p, CHC(OH)], 3.57 (3H, s,NCH.sub.3), 3.80 (3H, s, CO.sub.2CH.sub.3), 4.66, 4.85 (2H, 2dd,CH.sub.2CO.sub.2), 5.27 (1H, s, OH), 5.52 (1H, m, CO.sub.2CH),7.25-7.28, 7.32-7.35, 7.57-7.59 (5H, m, Ph) ppm.

3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-met-hylpyrrolidinium bromide, Compound (b)

[0126] To compound 4 (0.369 g, 1.22 mmol) in 10 ml of dryacetonitrile, ethyl bromoacetate (0.377 g, 2.25 mmol) was added atroom temperature. The mixture was stirred for 2 h. Evaporation ofacetonitrile gave a crude product. The crude product was dissolvedin a small volume of ethylene chloride and then poured into 50 mlof dry ethyl ether to precipitate. This procedure was repeatedthree times to obtain Compound (b) as pure product (0.45 g, 79%).White powder, m.p.: 192-194.degree. C.

[0127] Analysis for C.sub.22H.sub.32BrNO.sub.5. Calcd: C, 56.17; H,6.86; N, 2.98. Found: C, 56.14; H, 6.89; N, 2.94. .sup.1H NMR(CDCl.sub.3, 500 MHz): 1.35 (3H, t, CH.sub.3CH.sub.2), 1.26-1.33,1.42-1.47, 1.55-1.67 [8H, m, (CH.sub.2).sub.4], 2.14-2.21,2.73-2.79, 4.12-4.17, 4.22-4.29 (6H, m, CH.sub.2NCH.sub.2CH.sub.2),2.86 [1H, p, CHC(OH)], 3.62 (3H, s, NCH.sub.3), 4.25 (2H, q,CH.sub.3CH.sub.2), 4.67, 4.83 (2H, dd, CH.sub.2CO.sub.2), 4.91 (1H,s, OH), 5.53 (1H, m, CO.sub.2CH), 7.25-7.27, 7.32-7.34, 7.57-7.59(5H, m, Ph) ppm.

Resolution of racemic cyclopentylmandelic acid (1)

[0128] (-)-Strychnine (6.10 g) in 50 ml of methanol (suspension)was added to racemic cyclopentylmandelic acid 1, (3.96 g) inmethanol (20 ml) at room temperature. The reaction solution was letto stand for overnight. The crystals were removed by filtration andcrystallized again with hot methanol. The second crop of crystalswas collected by filtration and treated with sodium hydroxidesolution. The basic solution was extracted with methylene chloridetwice (methylene chloride solution discarded), and then acidifiedwith hydrochloric acid to recover the resolved cyclopentylmandelicacid. To this resolved acid (20.6 mg in 0.1 ml of ethyl acetate),13 .mu.L of (+)-.alpha.-phenylethylamine was added. The precipitatewhich formed was washed with hexane three times and dried undervacuum. The precipitate was identified by NMR as optically purecyclopentylmandelic acid, R(-)1, (1.49 g, 37.6%). M.p.:121-122.degree. C. [.alpha.].sup.25.degree..sub.D=-22.5.degree.(c=1 g/100 ml, CHCl.sub.3). .sup.1H NMR (CDCl.sub.3, 500 MHz):1.28-1.39, 1.42-1.50, 1.51-1.61, 1.64-1.73 [8H, m,(CH.sub.2).sub.4], 2.93 [1H, p, CHC(OH)], 7.25-7.28, 7.32-7.35,7.64-7.65 (5H, m, Ph) ppm.

Methyl (-)-cyclopentylmandelate, R(-)2

[0129] To a mixture of (-)-cyclopentylmandelic acid, R(-)1, (1.83g, 8.3 mmol) and potassium carbonate (2.87 g, 21 mmol) in DMF (21ml), methyl iodide (3.53 g, 25 mmol) was added at room temperature.The mixture was stirred at room temperature for 2 h, and thenpoured into water and extracted with hexanes three times.Evaporation of the dried hexanes extract gave a crude product.Flash chromatography of the crude product on silica gel with 1.5:1hexanes:methylene chloride gave pure product R(-).sub.2 (1.95 g,100%). Analysis for C.sub.18H.sub.18O.sub.3. Calcd: C, 71.77; H,7.74. Found: C, 71.88; H, 7.80. .sup.1H NMR (CDCl.sub.3, 500 MHz):1.32-1.36, 1.43-1.61 [8H, m, (CH.sub.2).sub.4], 2.90 [1H, p,CHC(OH)], 3.71 (1H, s, OH), 3.79 (3H, s, CH.sub.3), 7.25-7.28,7.31-7.35, 7.63-7.65 (5H, m, Ph) ppm.

N-Methyl-3-pyrrolidinyl (-)-cyclopentylmandelate, 2R-4

[0130] A solution of R(-).sub.2 (1.85 g, 7.9 mmol) andN-methyl-3-pyrrolidinol (3, 1.05 g, 10.4 mmol) in 40 ml ofn-heptane was heated until 20 ml of heptane had distilled.Approximately 0.003 g of sodium was added, and the solution wasstirred and heated for 2 h as the distillation was continued. Moreheptane was added at such a rate as to keep the reaction volumeconstant. Additional sodium was added at the end of an hour. Thesolution was then cooled and extracted with 3N HCl. The acidextract was made alkaline with concentrated NaOH and extractedthree times with ether. Removal of dried ether solution gave acrude oil. Flash chromatography of the crude product on silica gelwith 8:1 EtOAc:EtOH gave 2R-4 as a mixture of two diastereoisomersin an NMR-estimated ratio of 1:1, (1.68 g, 70%). Analysis forC.sub.18H.sub.25NO.sub.3.0.2H.sub.2O. Calcd: C, 70.42; H, 8.34; N,4.5. Found: C, 70.60; H, 8.26; N, 4.63. .sup.1H NMR (CDCl.sub.3,500 MHz): 1.28-1.37, 1.40-1.47, 1.51-1.70, 1.73-1.80, 1.83-1.90[8H, m, (CH.sub.2).sub.4], 2.14-2.21, 2.27-2.35, 2.36-2.42,2.52-2.55, 2.64-2.81 (6H, m, CH.sub.2NCH.sub.2CH.sub.2), 2.33, 2.37(3H, 2s, NCH.sub.3), 2.93 [1H, p, CHC(OH)], 3.78 (1H, bs, OH), 5.22(1H, m CO.sub.2CH), 7.24-7.27, 7.31-7.35, 7.64-7.66 (5H, m, Ph)ppm.

(2R)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmethyl)--1-methylpyrrolidinium bromide, Compound (c)

[0131] To compound 2R-4 (0.15 g, 0.49 mmol) in 6 ml of dryacetonitrile, methyl bromoacetate (0.194 g, 1.27 mmol) was added atroom temperature. The mixture was stirred for 6 h. Evaporation ofacetonitrile gave a crude product. The crude product was dissolvedin a small volume of methylene chloride and then poured into 50 mlof dry ethyl ether to precipitate. This procedure was repeatedthree times to obtain the product, Compound (c) (0.1879 g, 83%), asa mixture of four diastereoisomers in an NMR-estimated ratio of1:1:2:2. White powder, m.p.: 153-155.degree. C.[.alpha.].sup.25.degree..sub.D=+0.5.degree. (c=1 g/100 mlCHCl.sub.3). Analysis for C.sub.21H.sub.30BrNO.sub.5.0.2H.sub.2O.Calcd: C, 54.86; H, 6.62; N, 3.05. Found: C, 54.75; H, 6.66; N,3.01. .sup.1H NMR (CDCl.sub.3, 500 MHz): 1.30-1.37, 1.41-1.50,1.55-1.73 [8H, m, (CH.sub.2).sub.4], 1.93-2.00, 2.12-2.26,2.75-2.95, 3.00-3.03, 4.30-4.50, 4.57-4.61 [7H, m, CHC(OH) andCH.sub.2NCH.sub.2CH.sub.2], 3.09, 3.30 (1H, 2s, OH), 3.64, 3.66,3.84, 3.95, 3.97 (3H, 5s, NCH.sub.3), 3.74, 3.77, 3.79, 3.81 (3H,4s, CO.sub.2CH.sub.3), 4.78, 4.83; 4.90, 4.97; 5.30, 5.35; 5.37,5.41 (2H, 4 groups of 2dd, CH.sub.2CO.sub.2), 5.53 (1H, m,CO.sub.2CH), 7.23-7.29, 7.31-7.38, 7.56-7.60 (5H, m, Ph) ppm.

(2R)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)--1-methylpyrrolidinium bromide, Compound (d)

[0132] To compound 2R-4 (0.22 g, 0.73 mmol) in 10 ml of dryacetonitrile, ethyl bromoacetate (0.21 ml, 0.316 g, 1.89 mmol) wasadded at room temperature. The mixture was stirred for 22 hours.Removal of acetonitrile gave a crude product. The crude product wasdissolved in small volume of ethylene chloride and then poured into50 ml of dry ethyl ether to precipitate. This procedure repeatedthree times to obtain the product, Compound (d) (0.3085 g, 90%) asa mixture of four diastereoisomers in an NMR-estimated ratio of1:1:2:2. White powder, m.p.: 143-145.degree. C.[.alpha.].sup.25.degree..sub.D=+5.6.degree. (c=1 g/100 mlCHCl.sub.3). Analysis for C.sub.22H.sub.32BrNO.sub.5.0.3H.sub.2O.Calcd: C, 55.53; H, 6.91; N, 2.94. Found: C, 55.46; H, 6.85; N,2.97. .sup.1H NMR (CDCl.sub.3, 500 MHz): 1.26, 1.28, 1.32, 1.35(3H, 4t, CH.sub.3CH.sub.2), 1.44-1.50, 1.53-1.63, 1.65-1.70 [8H, m,(CH.sub.2).sub.4], 1.93-2.00, 2.04-2.11, 2.18-2.25, 2.76-2.96,3.01-3.04, 4.09-4.26 [7H, m, CHC(OH) andCH.sub.2NCH.sub.2CH.sub.2], 3.06, 3.28 (1H, 2s, OH), 3.66, 3.69,3.81, 3.82, 3.94, 3.96 (3H, 6s, NCH.sub.3), 4.61, 4.69; 4.76, 4.85;5.17, 5.22; 5.26, 5.30 (2H, 4 set of dd, CH.sub.2CO.sub.2),4.26-4.52 (2H, m, CH.sub.3CH.sub.2), 5.53 (1H, m, CO.sub.2CH),7.24-7.29, 7.31-7.38, 7.56-7.60 (5H, m, Ph) ppm.

pH Profile

[0133] The stabilities of the soft glycopyrrolates in standardphosphate buffers (0.05 M) of various pH (pH 6.00-8.40) wereinvestigated at 37.degree. C. Aliquots of 4.4 mM of the compoundsin water solution were added to the buffer solutions to give afinal concentration of 0.44 mM. At appropriate time intervals,samples were taken and analyzed by HPLC to monitor thedisappearance of the soft analogs and the formation of itshydrolysis products. The pseudo-first-order rate constant (k,min.sup.-1) and half-life (t.sub.1/2, min) of the disappearance ofthe compound in the buffer were calculated.

In Vitro Studies

[0134] The stability of soft glycopyrrolates in biological media invitro was determined by measuring the pseudo-first-order rateconstants (k, min.sup.-1) and half-lives (t.sub.1/2, min) of thedisappearance of the compound in rat blood and plasma. Aliquots of22 mM were added to the biological medium at 37.degree. C. to yielda final concentration of 0.7 mM. At appropriate time intervals,samples (0.15 ml) were withdrawn and mixed with 0.3 ml of 5%dimethylsulfoxide in acetonitrile solution. The mixtures werecentrifuged, and the supernatants were analyzed by HPLC.Experiments were performed in triplicates.

Analytical Method

[0135] The HPLC system used for the analysis of the compounds offormula (I) and their hydrolysis products was as follows: ASupelcosil LC-8 column (25 cm.times.4.6 mm) was used with a mobilephase of acetonitrile (42%) and aqueous solution (58%) containingsodium phosphate (10 mM), acetic acid (0.1%), and triethylamine(0.1%). At a flow rate of 1 ml/min, the retention times were 6.02min for Compounds (a) and (c), 7.27 min for Compounds (b) and (d)and 4.14 min (hydrolysis product), respectively. With an injectionvolume of 10 .mu.l, the detection limit was 1 .mu.g/ml.

Receptor Binding Affinity

[0136] Receptor binding studies were performed withN-[.sup.3H]-methylscopolamine (NMS) in assay buffer(phosphate-buffered saline, PBS, without Ca.sup.++ or Mg.sup.++, pH7.4) following the protocol obtained from Applied Cell Science Inc.(Rockville, Md.). A 10 mM NaF solution was added to the buffer asan esterase inhibitor. The assay mixture (0.2 ml) contained 20.mu.l diluted membranes (receptor proteins, final concentration:M.sub.1, 38 .mu.g/ml; M.sub.2, 55 .mu.g/ml; M.sub.3, 27 .mu.g/ml;and M.sub.4, 84 .mu.g/ml). The final concentration of NMS for thebinding studies was 0.5 nM. Specific binding was defined as thedifference in [.sup.3H]NMS binding in the absence and presence of 5.mu.M atropine for M.sub.1 and M.sub.2 or 1 .mu.M atropine forM.sub.3 and M.sub.4. Incubation was carried out at room temperaturefor 120 min. The assay was terminated by filtration through aWhatman GF/C filter (presoaked with 0.5% polyethyleneimine). Thefilter was then washed six times with 1 ml ice cold buffer (50 mMTris-HCl, pH 7.8, 0.9% NaCl), transferred to vials, and 5 ml ofScintiverse was added. Final detection was performed on a Packard31800 liquid scintillation analyzer (Packard Instrument Inc.,Downer Grove, Ill.). Data obtained from the binding experimentswere fitted to the %[.sup.3H] NMSbound=100-[100x.sup.n/k/(1+x.sup.n/k)] equation, to obtain the Hillcoefficient n, and then to %[.sup.3H] NMSbound=100-[100x.sup.n/IC.sub.50/(1+x.sup.n/IC.sub.50)], to obtainIC.sub.50s (x being the concentration of the tested compound).Based on the method of Cheng and Prusoff (Cheng & Prusoff1973), K.sub.i was derived from the equationK.sub.i=IC.sub.50/(1+L/K.sub.d), where L is the concentration ofthe radioligand. IC.sub.50 represents the concentration of the drugcausing 50% inhibition of specific radioligand binding, and K.sub.drepresents the dissociation constant of the radioligand receptorcomplex. Experiments were performed in triplicates. Data wereanalyzed by a non-linear least-square curve-fitting procedure usingScientist software (MicroMath Inc., Salt Lake City, Utah).

pA.sub.2 Values

[0137] Male guinea pigs obtained from Harlan Sprague Dawley Inc.(Indianapolis, Ind.) and weighing about 400 g were used afterovernight fasting. Animals were sacrificed by decapitation, and theileum (the region of 5 cm upward of the cecum) was isolated andremoved. The ileum was cut into 2.5 cm pieces and suspended in anorgan bath containing 30 ml of mixture of Tyrode's solution and 0.1mM hexamethonium bromide. The organ bath was constantly aeratedwith oxygen and kept at 37.degree. C. One end of the ileum stripwas attached to a fixed support at the bottom of the organ bath,and the other end to an isometric force transducer (Model TRN001,Kent Scientific Corp., Conn.) operated at 2-10 g range. The ileumstrip was kept at a 2 g tension, and carbachol was used asantagonist. The ileum contracted cumulatively upon the addition ofconsecutive doses of carbachol (10-20 .mu.l of2.times.10.sup.-4-2.times.10.sup.-3 M in water solution).Contractions were recorded on a physiograph (Kipp & ZonenFlarbed Recorder, Holland). After the maximum response wasachieved, the ileum was washed three times, and a fresh Tyrode'ssolution containing appropriate concentration of the antagonist[Compound (a), (b), (c) or (d), glycopyrrolate, or scopolamine] wasreplaced. An equilibration time of 10 min was allowed for theantagonists before the addition of carbachol. Four to six trialswere performed for each antagonist.

Pharmacological Activities of Soft Glycopyrrolates

[0138] The mydriatic effects of the soft drugs (a), (b), (c) and(d) in rabbit eyes have been compared with that of glycopyrrolate.Four healthy male New-Zealand white rabbits weighting about 3.5 kgwere used. To investigate the dose-mydriatic-responserelationships, 100 .mu.l of various concentrations of the compounds(0, 0.5, and 1% for the soft drugs and 0, 0.05, 0.1, and 0.2% forglycopyrrolate) were administered in the eyes to determine thepharmacodynamically equivalent doses, the lowest doses that inducethe maximum pupil dilations. Drug solutions were applied to oneeye; only water was applied to the other eye that served ascontrol. Experiments were carried out in a light- andtemperature-controlled room. At appropriate time intervals, thepupil diameters of both eyes were recorded. Difference in pupildiameters between each time-point and zero time-point werecalculated for both treated and control eyes and reported asmydriatic responses [(treated-control)/control in %]. Control eyedilations were monitored to determine whether systemic absorptionhad occurred or not. For each compound, four trials have beenconducted. Animal studies were performed in accordance with theGuide for the Care and Use of Laboratory Animals adopted by theNational Institute of Health, USA. Institutional animal care anduse committee (IACUC) approval was obtained prior to the initiationof this research and during its execution.

Statistical Analysis

[0139] Stability, receptor binding, and pA.sub.2 activities werecompared using both t-tests and nonparametric Mann-Whitney U testsfor the compound-pairs of interest. Pharmacological activities(maximum response R.sub.max% and area under the effect curvesAUC.sup.eff) were compared using ANOVA followed by Tukey-Kramermultiple comparison tests as a parametric post hoc test (Jones2002). A significance level of p <0.05 was used in all cases.All statistical analyses were performed using NCSS (Number CruncherStatistical Systems, Kaysville, Utah, USA).

Results and Discussion

Synthesis

[0140] The new soft glycopyrrolate derivatives, compounds (a) and(b)

[0141][3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(alkoxycarbonylmethy-l)-1-methylpyrrolidinium bromide; alkoxy being methoxy and ethoxyfor (a) and (b), respectively], have been synthesized as shown inScheme 1 except for the second, resolution step. This involvedGrignard reaction of cyclopentylmagnesium bromide withbenzoylformic acid in anhydrous ether to give the racemiccyclopentylmandelic acid 1; (ii) methylation of 1 with methyliodide and potassium carbonate in DMF at room temperature to yieldmethyl cyclopentylmandelate 2; (iii) transesterification of 2 with1-methyl-3-pyrrolidinol 3 in heptane to giveN-methyl-3-pyrrolidinyl cyclopentylmandelate 4; and (iv)quaternization of 4 with alkyl bromoacetate in acetonitrile to givethe final product 5 [Compound (a) or Compound (b)]. These areracemic soft glycopyrrolate derivatives, and they have beencharacterized by NMR and elemental analysis.

[0142] Because stereospecificity is known to be important atmuscarinic receptors, improved anticholinergic activity beingobtained with the 2R configuration of glycopyrrolate-typesubstances, these soft drug candidates have also been preparedstarting with optically pure cyclopentylmandelic acid, R(-)1.Racemic 1 was resolved by repeated crystallization of the saltsproduced between acid 1 and (-)-strychnine. Optically pure freeacid was recovered by basification of the salts with sodiumhydroxide solution followed by acidification with hydrochloricacid. The obtained left rotatory)(-22.5.degree. optically pureR(-)1 was characterized by NMR. Grover and coworkers reported thehighly stereoselective synthesis of(S)-cyclopentyl-phenylglycoxilic acid using (S)-mandelic acid in2000, and they found (S)-cyclopentyl-phenylglycoxilic acid to havepositive optical rotation. Accordingly, R(-)1, which was found tohave an optical rotation of [.alpha.]=-22.5.degree., is the R form.The NMR of the salt formed by the resolved cyclopentylmandelic acidR(-)1 and (+)-.alpha.-phenylethylamine gave a single pentaplet forthe CHC(OH) group; whereas the salt of the unresolved 1 and(+)-.alpha.-phenylethylamine gave two pentaplets for CHC(OH).

[0143] The soft glycopyrrolate Compounds (c) and (d) having 2Rconfigurations have been synthesized from R(-)-cyclopentylmandelicacid R(-)1 by the route shown in Scheme 1, and they were alsocharacterized by NMR and elemental analysis. The optical rotationsof Compound (c) and Compound (d) were +0.5.degree. and+5.6.degree., respectively.

##STR00008##

[0144] The racemic Compound (a) and Compound (b) had much simplerNMR spectra than the corresponding resolved compounds, Compound (c)and Compound (d). These molecules have a total of three chiralcenters as shown in Scheme 1. In Compound (c) and Compound (d), oneof the chiral centers was resolved, but two others remained; hence,they both are mixture of four diastereoisomers complicating theirNMR spectra. For example, the CH.sub.3CH.sub.2 methyl group showedonly one triplet at 1.35 ppm in Compound (b), where it is notsubject to unequal chemical environments; however, it showed fourtriplets at 1.26, 1.28, 1.32, and 1.35 ppm, respectively inCompound (d), which has one resolved and two unresolved chiralcenters and is a mixture of four diastereoisomers (RRR, RSR, RRS,RSS). Also, the AB system of Compound (b)'s CH.sub.2CO.sub.2 groupshowed one set of double-doublet signals at 4.67 and 4.83 ppm, butthe same system in Compound (d) showed four sets of double-doubletsignals at 4.61, 4.69; 4.76, 4.85; 5.17, 5.22; and 5.26, 5.30ppm.

pH Profile

[0145] In the pH range of 6.00-8.40 and at 37.degree. C., thechemical hydrolysis of the present soft glycopyrrolate compoundswas significantly pH-dependent. As shown in FIG. 1, they are morestable under acidic condition, and the ethyl derivatives are morestable than the corresponding methyl derivatives. The half-lives ofCompounds (a), (c), (b) and (d) in aqueous solutions at pH 6.0 were91, 77, 155, and 134 h, respectively. However, at pH 8.4, thecorresponding half-lives decreased to 8, 7, 16, and 12 min,respectively. The pH profiles are displayed in FIG. 1, and theresults indicate a base-catalyzed hydrolysis of the compounds witha correlation coefficient of 0.997-0.998. For illustrativepurposes, the time-profile of the disappearance of Compound (c) andthe concurrent formation of the corresponding acid at pH 7.4 isshown in FIG. 2.

In Vitro Stability

[0146] In vitro stability studies have been performed using ratblood and plasma by measuring the pseudo-first-order rate constant(k, min.sup.-1) and half-life (t.sub.1/2, min) of the disappearanceof the parent compounds (Table 1). At 37.degree. C. and pH 7.4, thehydrolysis of soft glycopyrrolate analogs was relatively fast inplasma with half-lives of 19.5, 20, 44, and 34 min for Compounds(a), (c), (b) and (d), respectively, and significantly slower inblood (57, 57, 97, and 86 min, respectively; p<0.05 for allcompounds, t-test or nonparametric Mann-Whitney U test), indicatingthat blood cell binding is significant enough to slow thehydrolytic degradation of these esters. The ethyl esters were morestable than the methyl derivatives (p<0.05, t-test ornonparametric Mann-Whitney U test).

TABLE-US-00001 TABLE 1 Pseudo-first-order rate constant (k,min.sup.-1) and half-life (t.sub.1/2, min) for the disappearance ofsoft analogs in rat plasma and blood. Data represent mean .+-. SDof three experiments. Compound Medium k .times. 10.sup.-3,min.sup.-1 t.sub.1/2, min r.sup.2 (a) plasma 36.4 .+-. 5.0 19.5.+-. 2.7 0.998 blood 12.3 .+-. 1.3 57.0 .+-. 5.8 0.998 (b) plasma15.8 .+-. 0.2 44.0 .+-. 0.4 0.997 blood 7.2 .+-. 0.2 96.6 .+-. 2.90.996 (c) plasma 34.5 .+-. 3.2 20.0 .+-. 2.1 0.993 blood 12.4 .+-.1.4 56.7 .+-. 6.1 0.997 (d) plasma 20.9 .+-. 3.0 33.8 .+-. 4.80.998 blood 8.0 .+-. 0.1 86.4 .+-. 1.2 0.998

In Vitro Pharmacodynamic Evaluation

[0147] To evaluate the relative potency of the newly synthesizedsoft analogs, receptor binding affinities, pK.sub.i, and guinea pigileum contraction ability, pA.sub.2, were determined

Receptor Binding Studies

[0148] The receptor binding affinities of the compounds determinedby radioligand binding assays using human cloned muscarinicreceptor subtypes, M.sub.1-M.sub.4. are presented in Table 2. The2R isomers, Compounds (c) and (d), had pK.sub.i values that are inthe 8.7-9.5 range; somewhat less, but close to those observed forthe known highly active antagonists that served as lead for thepresent design, N-methylscopolamine and glycopyrrolate (9.2-9.9 and8.7-9.9, respectively). As expected, the racemic forms, Compounds(a) and (b), showed lower receptor binding affinities than theircorresponding 2R isomers (differences significant at p<0.05level for M.sub.3, t-test or nonparametric Mann-Whitney U test),confirming that stereospecificity is important at thesereceptors.

TABLE-US-00002 TABLE 2 Receptor binding affinities and pA.sub.2values. Subtypes of cloned muscarinic receptors .sup.a CompoundM.sub.1 M.sub.2 M.sub.3 M.sub.4 pA.sub.2 .sup.b (c) 8.89 .+-. 0.048.87 .+-. 0.05 9.00 .+-. 0.06 9.52 .+-. 0.01 8.31 .+-. 0.05 (0.83.+-. 0.11) (1.10 .+-. 0.11) (0.83 .+-. 0.01) (0.83 .+-. 0.01) (a)7.91 .+-. 0.05 7.79 .+-. 0.11 7.80 .+-. 0.10 8.29 .+-. 0.19 7.90.+-. 0.13 (1.02 .+-. 0.12) (1.25 .+-. 0.01) (1.17 .+-. 0.18) (1.12.+-. 0.05) (d) 8.67 .+-. 0.16 8.84 .+-. 0.34 8.74 .+-. 0.02 8.85.+-. 0.13 8.55 .+-. 0.16 (0.86 .+-. 0.08) (0.92 .+-. 0.01) (1.09.+-. 0.15) (0.89 .+-. 0.02) (b) 7.51 .+-. 0.17 7.32 .+-. 0.07 7.54.+-. 0.15 7.94 .+-. 0.09 7.36 .+-. 0.34 (0.91 .+-. 0.09) (1.23 .+-.0.06) (1.18 .+-. 0.08) (1.18 .+-. 0.09) glycopyrrolate 9.76 .+-.0.05 9.19 .+-. 0.18 8.73 .+-. 0.05 9.90 .+-. 0.08 8.57 .+-. 0.12(1.37 .+-. 0.20) (0.99 .+-. 0.11) (1.14 .+-. 0.25) (1.02 .+-. 0.01)scopolamine 9.69 .+-. 0.01 9.18 .+-. 0.21 9.29 .+-. 0.12 9.92 .+-.0.21 9.16 .+-. 0.19 methyl bromide (0.92 .+-. 0.10) (1.02 .+-.0.02) (1.07 .+-. 0.01) (0.90 .+-. 0.04) .sup.a Data of the receptorbinding experiments represent mean .+-. S.D. of 3 experiments. Thenumbers in parentheses denote Hill slopes. .sup.b pA.sub.2 valueswere determined on 4-6 ileum strips obtained from differentanimals. Data represent mean .+-. SD.

pA.sub.2 Studies

[0149] The pA.sub.2 values determined from guinea pig ileumcontraction assays, which represent the negative logarithm of themolar concentration of the antagonist that produces a two-foldshift to the right in an agonist's concentration-response curve,are a classical functional study of anticholinergic affinity (atM.sub.3 muscarinic receptors). For the soft anticholinergics of thepresent study, the pA.sub.2 values obtained from ileum longitudinalcontractions by using carbachol as agonists with the method of vanRossum (Table 2) were, in general, comparable to the pK.sub.ivalues obtained in the M.sub.3 receptor binding studies. The 2Risomers were again significantly more active than the correspondingracemates, and the most active soft analog [Compound (d),pA.sub.2=8.55.sub..+-.0.16] showed activity similar toglycopyrrolate (pA.sub.2=8.57.sub..+-.0.12).

Mydriatic Activities of Soft Analogs

[0150] The mydriatic effects of the new soft derivatives, Compounds(a) and (b), were compared to those of glycopyrrolate in rabbits.Mydriatic responses were recorded at appropriate time-intervalsafter the administration of the drugs as % changes in pupil size.Maximum response (R.sub.max, % change in pupil size at 1 h afteradministration) and area under the response-time curve(AUC.sup.eff) are shown in Table 3. Whereas, there are nosignificant differences among the R.sub.max maximum responses amongall treatments considered (compounds and concentrations of Table3), there clearly are among the AUC.sup.effs (p<0.05, ANOVAfollowed by Tukey-Kramer multiple comparison test). Glycopyrrolate(0.1%, 0.2%) gave significantly longer-lasting effects (largerAUC.sup.effs) than any of the soft drugs. The soft ethyl compoundsseem somewhat more potent than corresponding methyl analogs, andthe 2R isomers seem more potent than the isomeric mixtures. Inagreement with soft drug design principles, their duration ofaction is much shorter than that of the "hard" glycopyrrolate asillustrated in FIG. 3A and FIG. 3B for pharmacodynamicallyequipotent doses. The mydriatic activity of Compound (c), Compound(d), and glycopyrrolate lasted for 24, <48, and 144 h,respectively, indicating that the soft drugs are easily hydrolyzedand rapidly eliminated from the body after the desiredpharmacological effect is achieved. In agreement with this andunlike other traditional anticholinergics, these soft drugs did notinduce dilation of the pupil in the contralateral (water-treated)eye, indicating no or just low systemic side-effects. Therefore,these compounds are safe, promising short acting anticholinergicswith the possibility of largely reduced unwanted side effects.

TABLE-US-00003 TABLE 3 Maximum response (R.sub.max, % change inpupil size at 1 h after administration) and area under theresponse-time curve (AUC.sup.eff). Data indicate mean .+-. SD offour trials. Compound Conc. (%) R.sub.max (%)AUC.sup.eff.sub.(0-144 h) (a) 0.5 45.83 .+-. 4.81 185 .+-. 35 159.58 .+-. 15.72 467 .+-. 114 (b) 0.5 44.65 .+-. 13.99 596 .+-. 2741 58.33 .+-. 12.27 645 .+-. 409 (c) 0.5 52.92 .+-. 13.41 752 .+-.342 1 57.08 .+-. 11.66 875 .+-. 197 (d) 0.5 53.96 .+-. 13.27 1170.+-. 308 1 56.04 .+-. 11.69 1532 .+-. 526 glycopyrrolate 0.05 51.46.+-. 7.71 2779 .+-. 443 0.1 55.83 .+-. 6.42 4074 .+-. 459 0.2 56.04.+-. 10.10 5047 .+-. 1631

[0151] In conclusion, a set of new glycopyrrolate-based softanticholinergics has been designed, synthesized, and tested. Theywere found to have receptor binding affinities comparable to thoseof glycopyrrolate or N-methylscopolamine, and good, butshort-lasting mydriatic activity with no or just minimal systemiceffects due to their soft nature that allows easy, one-stepmetabolism into a designed-in metabolite after exerting theirdesired pharmacological activity.

Further Studies

[0152] Purpose. Because stereospecificity is known to be importantat muscarinic receptors, isomers of both N-substituted softanticholinergics based on glycopyrrolate, (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(alkoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide methyl and ethyl esters, Compounds (c)and (d), and their zwitterionic metabolite, (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-methyl-1-carboxymethylpyrro-lidinium inner salt, were synthesized and their pharmacologicalactivities were evaluated in vitro and in vivo.

[0153] Methods. The isomers of Compounds (c) and (d) weresynthesized with both optically pure methyl-cyclopentylmandelateand 3-hydroxy-N-methylpyrrolidine. Trans-esterification followed byquarternization with alkyl bromoacetate gave four isomers of themethyl or ethyl ester with the nitrogen chiral center unresolved.The hydrolysis of these four isomers followed by HPLC separationresulted in eight fully resolved isomers of the corresponding acid.The pharmacological activities were assessed using the in vitroreceptor-binding assay, guinea pig ileum pA.sub.2-assay, and invivo rabbit mydriatic effect. The results were compared with thatof conventional anticholinergic agents such as glycopyrrolate,N-meythylscopolamine, and tropicamide as well as that of previouslyprepared racemates and 2R isomers.

[0154] Results. The receptor binding at cloned human muscarinicreceptors (M.sub.1-M.sub.4 subtypes), pK.sub.i values, of thesenewly synthesized methyl and ethyl ester isomers were in the6.0-9.5 range, and zwitterion isomers in 5.0-8.6 range. In bothcases, 2R isomers were found significantly more active than 2Sisomers (27-447 times for methyl ester isomers, and 6 to 4467 timesfor zwitterion isomers). Among four isomers of the methyl esterCompound (c) (with chiral center 1' unresolved), the 3'R isomerswere more active than the corresponding 3'S isomers (1.5-12.9times). However, in the case of zwitterion isomers, the 3'S isomerswere not always more active than the corresponding 3'R isomers,indicating that activity determined based on chiral center 3' issignificantly affected by the configuration of other two chiralcenters, 2 and 1'. In the completely resolved 8 zwitterion isomers(all the three chiral centers resolved), it was found that 1'Sisomers were more active than the corresponding 1R isomers in allcases (1.8-22.4 times). The results also indicate that some isomersshowed good M.sub.3/M.sub.2 muscarinic-receptorsubtype-selectivities (about 3-5 times), and 2R and 3'S were thedetermining configurations for this property. Guinea pig ileumassays and rabbit mydriasis test on zwitterion isomers doubleconfirmed the stereospecificity. In rabbit eyes, some 2R-zwitterionisomers showed mydriatic potencies similar to glycopyrrolate andexceeded tropicamide, but their mydriatic effects lastedconsiderably less time, and they did not induce dilation of thepupil in the contralateral, water-treated eyes. These resultsindicate that, in agreement with their soft nature, they arelocally active, but safe and have a low potential to cause systemicside effects. The pharmacological potency of eight zwitterionisomers was concluded to be (2R1'S3'S,2R1'S3'R and2R1'R3'S)>2R1'R3'R>2S1S3'R>(2S1'S3'S and2S1'R3'R)>2S1'R3'S.

[0155] Conclusions. The stereospecificity and M.sub.3/M.sub.2muscarinic-receptor subtype-selectivity of soft anti-cholinergics,Compounds (c) and (d) and their zwitterionic metabolite, have beendemonstrated. Adding to the previous results, safe use of thesesoft drugs has been confirmed.

Introduction

[0156] Stereospecificity of anticholinergics is important atmuscarinic receptors, Compounds (c) and (d), (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(alkoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide, and their common zwitterionicmetabolite, (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-methyl-1-carboxymethylpyrro-lidinium inner salt, have shown promising activity and safety inanimal studies. These compounds indeed exhibited stereospecificitytoward muscarinic receptors, and the anticholinergic activity hasbeen improved with the 2R configuration. In addition, thezwitterionic metabolite also showed a moderate M.sub.3/M.sub.2muscarinic-receptor subtype-selectivity that indicates a reducedsystemic cardiac side effect. However, the molecules of this typeof soft analogs possess three chiral centers, so that each racemiccompound may contains up to eight different isomers, that is2R1'R3'R,2R1'R3'S,2R1'S3'R,2R1'S3'S,2S1'R3'R, 2S1R3'S,2S1'S3'R, and2S1'S3'S as displayed below:

##STR00009##

[0157] Thus, the above-described investigations in the softglycopyrrolate isomers based on one resolved chiral center (2R or2S) only expressed that 2R enantiomers (a mixture of fourdiastereoisomers 2R1'3'R, 2R1R3'S, 2R1'S3'R, 2R1'S3'S) were moreactive than 2S enantiomers (a mixture of 2S1'R3'R, 2S1'R3'S,2S1'S3'R, 2S1'S3'S). In this section, further investigations in thestereospecificity of these soft glycopyrrolates are reported usingfive partially-resolved soft anticholinergics isomers and eightfully resolved zwitterion metabolite, isomers (as described for 2Rand 2S enantiomers). The compounds were systematically synthesizedand isomers were separated. The relative pharmacological activitiesand M.sub.3/M.sub.2 muscarinic-receptor subtype-selectivities wereinvestigated by in vitro receptor-binding assay, in vitro guineapig ileum pA.sub.2-assay, and in vivo mydriatic effect inrabbits.

Materials and Methods

Materials

[0158] Glycopyrrolate (glycopyrronium bromide) was kindly providedby Boehringer Ingelheim Chemicals, Inc. Carbamylcholine bromide(carbachol), atropine methylbromide (atropine MeBr), andscopolamine methylbromide (scopolamine MeBr) were obtained fromSigma Chemicals Co. (St. Louis, Mo.), and tropicamide (1%) wasobtained from Bausch & Lomb Pharmaceutical (Tampa, Fla.).N-[.sup.3H]-Methyl-scopolamine (NMS) was obtained from AmershamBiosciences UK Limited (Buckinghamshire, UK). Cloned humanmuscarinic receptor subtypes M.sub.1-M.sub.4 were obtained fromApplied Cell Science Inc. (Rockville, Md.). Scintiverse BD was fromFisher Scientific Co. (Pittsburgh, Pa.). (R)-3-hydroxy pyrrolidinehydrochloride and (S)-3-hydroxy pyrrolidine hydrochloride were fromAstatech Inc. (Monmouth Junction, N.J.).N-[.sup.3H]-Methyl-scopolamine (NMS) was from Amersham BiosciencesUK Limited (Buckinghamshire, UK). Cloned human muscarinic receptorsubtypes M.sub.1-M.sub.4 were from Applied Cell Science Inc.(Rockville, Md.). Scintiverse BD was from Fisher Scientific Co.(Pittsburgh, Pa.). Other chemicals used for synthesis were reagentor HPLC grade, and were obtained from Aldrich (Milwaukee, Wis.) andFisher Scientific Co. Melting points were taken on Fisher-Johnsmelting apparatus. NMR spectra were recorded on Bruker Advance 300,400 and 500 MHz NMR spectrometers, and are reported in ppm relativeto TMS. NOESY was performed using 2D NMR spectrometer,Mercury-300BB Animal studies were performed in accordance with theGuide for the Care and Use of Laboratory Animals adopted by theNational Institute of Health, USA. Institutional animal care anduse committee (IACUC) approval was obtained prior to the initiationof this research and during its execution.

Synthesis of 2R-isomers

Racemic cyclopentylmandelic acid, 1

[0159] Cyclopentylmagnesium bromide ether solution (100 ml, 2M; 0.2mol) was added drop-wise to benzoylformic acid (15 g, 0.1 mol) in330 ml of anhydrous ethyl ether at 0.degree. C. The mixture wasstirred at 0.degree. C. for 30 min and at room temperature for 24h. The reaction mixture was treated with 1 N HCl, and the aqueoussolution was extracted with ether. The combined ether solution wastreated with K.sub.2CO.sub.3 solution. The potassium carbonatesolution was acidified with HCl and extracted with ether twice. Theether solution was dried with anhydrous sodium sulfate andevaporated to give a crude product. The crude product was washedwith water to get pure racemic cyclopentylmandelic acid 1 (8.0 g,36.4%). Needle-like crystal, m.p.: 153-154.degree. C. .sup.1H NMR(CDCl.sub.3, 300 MHz): 1.28-1.39, 1.42-1.50, 1.51-1.61, 1.63-1.72[8H, m, (CH.sub.2).sub.4], 2.93 [1H, p, CHC(OH)], 7.26-7.30,7.33-7.36, 7.65-7.67 (5H, m, Ph) ppm.

Resolution of racemic cyclopentylmandelic acid, R(-)1

[0160] (-)-Strychnine (11.4 g) in 100 ml of methanol (suspension)was added to racemic cyclopentylmandelic acid 1 (7.5 g) in methanol(20 ml) at room temperature. The reaction solution was allowed tostand overnight. The crystals were filtered and crystallized againwith hot methanol. The second crop of crystals was collected byfiltration and treated with sodium hydroxide solution. The basicsolution was extracted with methylene chloride twice (methylenechloride solution discarded), and then acidified with hydrochloricacid to recover the resolved cyclopentylmandelic acid. To thisresolved acid (20.6 mg in 0.1 ml of ethyl acetate), 13 .mu.L of(+)-.alpha.-phenylethylamine was added. The precipitate whichformed was washed with hexane three times and dried under vacuum.The precipitate was identified by NMR as optically purecyclopentylmandelic acid, R(-)1, (2.5 g, 33.3%). M.p.:121-122.degree. C. [.alpha.].sup.25.degree..sub.D=-22.5.degree.(c=1 g/100 ml, CHCl.sub.3). .sup.1H NMR (CDCl.sub.3, 300 MHz):1.28-1.39, 1.42-1.50, 1.51-1.61, 1.64-1.73 [8H, m,(CH.sub.2).sub.4], 2.93 [1H, p, CHC(OH)], 7.25-7.28, 7.32-7.35,7.64-7.65 (5H, m, Ph) ppm.

Methyl (-)-cyclopentylmandelate, R(-)2

[0161] To a mixture of (-)-cyclopentylmandelic acid, R(-)1, (1.83g, 8.3 mmol) and potassium carbonate (2.87 g, 21 mmol) in DMF (21ml), methyl iodide (3.53 g, 25 mmol) was added at room temperature.The mixture was stirred at room temperature for 2 h, and thenpoured into water and extracted with hexane three times.Evaporation of dried hexane extract gave a crude product. Flashchromatography of the crude product on silica gel with 1.5:1hexane:methylene chloride gave pure product R(-)2 (1.90 g, 95%)..sup.1H NMR (CDCl.sub.3, 300 MHz): 1.32-1.36, 1.43-1.61 [8H, m,(CH.sub.2).sub.4], 2.90 [1H, p, CHC(OH)], 3.71 (1H, s, OH), 3.79(3H, s, CH.sub.3), 7.25-7.28, 7.31-7.35, 7.63-7.65 (5H, m, Ph)ppm.

(R)-3-Hydroxy-N-Methyl pyrrolidine, (R)3

[0162] In a 100 ml flask, 2 g (R)-3-Hydroxy pyrrolidine, 25 ml THF,0.49 g paraformaldehyde and 1.5 g formic acid (90%) were added. Themixture was stirred under reflux for 5 hours (until all soliddisappeared), then cooled at 0.degree. C. and added with 10 ml ofNaOH solution (10 N) to adjust the pH to about 10. The organiclayer was separated and dried over MgSO.sub.4. After filtering thedried solution and removing the solvent (THF), an oily product (1.5g, 92%) of (R).sub.3 was obtained. .sup.1H NMR (CDCl.sub.3, 300MHz): 1.50-1.60 (m, 1H), 1.98-2.10 (m, 1H), 2.25 (s, 3H), 2.25-2.40(m, 2H), 2.50-2.60 (m, 1H), 2.61-2.70 (m, 1H), 3.80 (brs, 1H),4.20-4.30 (m, 1H).

(S)-3-Hydroxy-N-Methyl pyrrolidine, (S)3

[0163] Synthesis of (S).sub.3 was the same as for (R).sub.3, exceptthat the starting material was (S)-3-Hydroxy pyrrolidine. Theresultant product (S).sub.3 (1.5 g, 92%) was also an oil. .sup.1HNMR (DMSO-D6 300 MHz): 1.50-1.60 (m, 1H), 1.98-2.05 (m, 1H), 2.15(s, 3H), 2.15-2.35 (m, 2H), 2.45-2.52 (m, 1H), 2.61-2.70 (m, 1H),4.20 (brs, 1H), 4.60-4.70 (m, 1H).

3(R)-N-Methyl-3-pyrrolidinyl-2(R)-cyclopentylmandelate, 4

[0164] A solution of R(-).sub.2 (0.7 g, 3 mmol) and (R).sub.3 (0.7g, 7 mmol) in 40 ml of toluene was heated until 20 ml of toluenehad distilled. Approximately 0.003 g of sodium was added, and thesolution was stirred and heated for 2 h as the distillation wascontinued. More toluene was added at such a rate as to keep thereaction volume constant. Additional sodium was added at the end ofan hour. The solution was then cooled and extracted with 3N HCl.The acid extract was made alkaline with concentrated NaOH andextracted three times with ether. Removal of dried ether solutiongave a crude oil. Flash chromatography of the crude product onsilica gel with 8:1 of EtOAc and EtOH gave an oil product of 4 (0.4g, 44%). .sup.1H NMR (CDCl.sub.3, 300 MHz): 1.28-1.37, 1.51-1.70,1.83-1.90 [8H, m, (CH.sub.2).sub.4], 2.27-2.40 (m, 3H), 2.52-2.55(m, 1H), 2.64-2.72 (m, 1H), 2.74-2.81 (m, 1H), 2.33 (3H, s,NCH.sub.3), 2.93 [1H, p, CHC(OH)], 3.85 (1H, bs, OH), 5.22 (m, 1H),7.24-7.27, 7.31-7.35, 7.64-7.66 (5H, m, Ph)ppm.

3(S)-N-Methyl-3-pyrrolidinyl-2(R)-cyclopentylmandelate, 5

[0165] Synthesis of 5 was the same as for 4, except (S).sub.3 wasused instead of (R).sub.3. The resultant product 5 (0.35 g, 39%)was also an oil. .sup.1H NMR (CDCl.sub.3, 400 MHz): 1.28-1.37,1.51-1.70, 1.75-1.82 [8H, m, (CH.sub.2).sub.4], 2.15-2.22 (m, 1H),2.30-2.40 (m, 2H), 2.65-2.70 (m, 1H), 2.70-2.82 (m, 2H), 2.35 (3H,s, NCH.sub.3), 2.95 [1H, p, CHC(OH)], 3.82 (1H, bs, OH), 5.22 (m,1H), 7.24-7.27, 7.31-7.35, 7.64-7.66 (5H, m, Ph)ppm.

(2R,3'R)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmet-hyl)-1-methylpyrrolidinium bromide, 6 [Compound (e)]

[0166] To Compound 4 (0.3 g, 0.98 mmol) in 12 ml of dryacetonitrile, methyl bromoacetate (0.5 g, 3.2 mmol) was added atroom temperature. The mixture was stirred for 6 h. Evaporation ofacetonitrile gave a crude product. The crude product was dissolvedin a small volume of methylene chloride and then poured into 50 mlof dry ethyl ether to precipitate. This step was repeated threetimes to obtain the pure product 6, or Compound (e), (0.3 g, 70%)as a white powder that was a mixture of two diastereoisomers in aNMR-estimated ratio of 2:1. .sup.1H NMR (CDCl.sub.3, 400 MHz):1.30-1.37, 1.41-1.50, 1.55-1.70 [8H, m, (CH.sub.2).sub.4],2.10-2.27 (m, 1H), 2.79-2.95 (m, 2H), 3.05, 3.60 (2s, total 3H,N--CH.sub.3), 3.75, 3.79 (2s, total 3H, O-Me), 3.95-4.40 (m, 4H),4.68, 5.16 (2AB, total 2H, N--CH2-COOMe), 5.52-5.58 (m, 1H),7.23-7.29, 7.31-7.38, 7.56-7.60 (5H, m, Ph)ppm.

(2R,3'S)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmet-hyl)-1-methylpyrrolidinium bromide, 7a [Compound (f)]

[0167] To Compound 5 (0.16 g, 0.52 mmol) in 8 ml of dryacetonitrile, methyl bromoacetate (0.3 g, 1.9 mmol) was added atroom temperature. Following the same procedure for 6 [Compound (e)]the pure product 7a [Compound (f)] (0.16 g, 80%) was obtained.Compound (f) was also a white powder and a mixture of twodiastereoisomers in a NMR-estimated ratio of 2:1. .sup.1H NMR(CDCl.sub.3, 400 MHz): 1.30-1.70 [8H, m, (CH.sub.2).sub.4],1.95-2.00, 2.10-2.20 (m, 1H), 2.75-2.95 (m, 2H), 3.30, 3.70 (2s,total 3H, N--CH3), 3.78, 3.82 (2s, total 3H, O-Me), 4.00-4.42 (m,4H), 4.90, 5.38 (2AB, total 2H, N--CH2-COOMe), 5.52-5.58 (m, 1H),7.23-7.29, 7.31-7.38, 7.56-7.60 (5H, m, Ph)ppm.

(2R,3'S)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmeth-yl)-1-methylpyrrolidinium bromide, 7b [Compound (g)]

[0168] To Compound 5 (0.16 g, 0.52 mmol) in 10 ml of dryacetonitrile, ethyl bromoacetate (0.32 g, 1.9 mmol) was added atroom temperature. The mixture was stirred for 22 hours, and theremoval of acetonitrile gave a crude product. The crude product wasdissolved in small volume of ethylene chloride, and then pouredinto a 50 ml of dry ethyl ether to afford a precipitate. Thisprocedure was repeated three times, and the pure product 7b, orCompound (g) (0.16 g, 80%) was obtained. Compound (g) was also awhite powder and a mixture of two diastereoisomers in aNMR-estimated ratio of 2:1. .sup.1H NMR (CDCl.sub.3, 400 MHz):1.32, 1.35 (2t, 3H, CH.sub.3CH.sub.2), 1.40-1.50, 1.53-1.63,1.65-1.80 [8H, m, (CH.sub.2).sub.4], 1.93-2.11 (m 2H), 2.80-2.96 M,2H), 3.30, 3.70 (2s, 3H, N--CH3), 4.10-4.60 (m, 6H), 4.79, 5.30(2H, 2set of dd, CH.sub.2CO.sub.2), 5.53 (1H, m), 7.24-7.29,7.31-7.38, 7.56-7.60 (5H, m, Ph)ppm.

Hydrolysis of Esters

[0169] Compounds (e) and (f) were combined with equimolar ratios of0.1 N NaOH. The mixtures were stirred at room temperature for 3hours to obtain the corresponding racemic zwitterionic products, 8and 9 in aqueous solution (colorless, pH about 6.5). Compound 8 is(2R,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyr-rolidinium inner salt. Compound 9 is (2R,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyr-rolidinium inner salt.

HPLC separations for 8a, 8b, and 9a, 9b

[0170] The solutions of 8 and 9 each contained two isomers, 8a, 8band 9a, 9b, at a ratio of 2:1 that could be separated by HPLC. TheHPLC system consisted of a Spectra Physics (San Jose, Calif.) SP8810 isocratic pump, a SP 8450 UV/Vis detector (wavelength set to230 nm), a SP 4290 integrator, and a Supelco Discovery RP Amide C16column. The mobile phase consisted of acetonitrile and water at aratio of 30:70. With 100 .mu.L injection at a flow rate of 1mL/min, the retention times were 7.2 min for 8a and 9a, and 8.5 minfor 8b and 9b. The effluence corresponding to each isomer wascollected, and the solvent was evaporated to obtain the finalzwitterionic isomers, 8a, 8b, and 9a, 9b as following:

[0171] (2R,1'R,3R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyr-rolidinium inner salt, 8a: white powder .sup.1H NMR (CDCl.sub.3,300 MHz): 1.30-1.65 (m, 8H), 2.02-2.12 (m, 1H), 2.20-2.60 (brs,1H), 2.60-2.80 (m, 1H), 2.82-2.92 (m, 1H), 3.30 (s, 3H), 3.55-3.65(m, 1H), 3.72-3.82 (m, 1H), 3.90-4.05 (m, 2H), 4.10-4.15 (m, 1H),5.38-5.45 (m, 1H), 7.15-7.20 (m, 1H), 7.32-7.38 (m, 2H), 7.55-7.62(m, 2H).

[0172] (2R,1'S,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyr-rolidinium inner salt, 8b: .sup.1H NMR (CDCl.sub.3, 300 MHz):1.30-1.75 (m, 8H), 2.02-2.10 (m, 1H), 2.10-2.40 (brs, 2H),2.70-2.80 (m, 1H), 2.80-2.90 (m, 1H), 2.95 (s, 3H), 3.55-3.65 (m,2H), 3.85-4.0 (m, 3H), 4.05-4.10 (m, 1H), 5.38-5.45 (m, 1H),7.15-7.20 (m, 1H), 7.25-7.30 (m, 2H), 7.50-7.60 (m, 2H).

[0173] (2R,1'R,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyr-rolidinium inner salt, 9a: white powder, .sup.1H NMR (CDCl.sub.3,500 MHz): 1.30-1.65 (m, 8H), 2.02-2.12 (m, 1H), 2.50-2.60 (m, 1H),2.78-2.88 (m, 1H), 3.25 (s, 3H), 3.65-4.05 (m, 4H), 4.15-4.30 (brs,2H), 5.30-5.40 (m, 1H), 7.13-7.23 (m, 1H), 7.26-7.32 (m, 2H),7.55-7.60 (m, 2H).

[0174] (2R,1'S,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyr-rolidinium inner salt, 9b: white powder, .sup.1H NMR (CDCl.sub.3,500 MHz): 1.30-1.70 (m, 8H), 1.90-1.98 (m, 1H), 2.65-2.70 (m, 1H),2.85-2.90 (m, 1H), 3.15 (s, 3H), 3.65-3.90 (m, 4H), 4.05-4.10 (M,1H), 4.15-4.22 (brs, 1H), 5.35-5.42 (m, 1H), 7.18-7.23 (m, 1H),7.27-7.32 (m, 2H), 7.53-7.58 (m, 2H).

Determination of Absolute Configurations

[0175] Nuclear overhauser effect (NOE) has been used to identifythe absolute configuration of the product 8b. Compound wasdissolved in CDCl.sub.3, and the 2D .sup.1H-.sup.1H NOESY spectrumwas taken by Mercury-300BB.

Synthesis of 2S-isomers

Cis-(2S,5S)-2-(tert-butyl)-5-phenyl-1,3-dioxolan-4-one, 10

[0176] S(+)-mandelic acid in hexane suspension (50 g, 328 mmol) wasadded with pivaldehyde (42.7 ml, 396 mmol) thentrifluoromethanesulfonic acid (1.23 ml, 14 mmol) at roomtemperature. The mixture was warmed to 36.degree. C., and thereaction was followed by TLC for 5 hr until no starting materialcould be detected. The mixture was then cooled to room temperatureand added with 8% aqueous NaHCO.sub.3. The water layer was removedand the organic layer was dried over Na.sub.2SO.sub.4. Afterfiltration and removal of the solvent, 62.17 g of the crude productwas obtained. Recrystallization of the crude product gave 44.71 gof pure cis-(2S,5S)-2-(tert-butyl)-5-phenyl-1,3-dioxolan-4-one in88% yield as a needle-like crystal. .sup.1H NMR (CDCl.sub.3, 300MHz): 1.08 (s, 9H), 5.24 (s, 1H), 5.33 (s, 1H), 7.40-7.46 (m,5H)ppm. .sup.13C NMR (CDCl.sub.3, 300 MHz): 23.6, 34.4, 77.0,109.3, 127.0, 128.7, 129.2, 133.4, 147.2.

Cis-(2S,5S)-2-(tert-butyl)-5-phenyl-5-cyclopentyl-1,3-dioxolan-4-one,11

[0177] At -78.degree. C., a lithium bis-(trimethylsilyl)amide inhexane solution (120 ml, 120 mmol, 1.0M in hexane) was added tocompound 10 (25 g, 113.5 mmol, dissolved in 100 ml of dried THF),stirred for 1 hr, followed by addition of cyclopentyl bromide (25g, 168 mmol). This reaction was kept at -78.degree. C. for 4 hr,then slowly warmed up to room temperature and continued forovernight. The completion of the reaction was followed by TLC. Withstirring, a solution of 10% of NH.sub.4Cl (25 ml) was added in themixture. Then, the mixture was poured into a separation funnelcontaining 10% NH.sub.4Cl solution (200 ml). The aqueous layer wasdiscarded, and the organic layer was dried over Na.sub.2SO.sub.4.The solvent was removed to give a crude product, which was thenre-crystallized in hexane to give a pure product, 11 (20.36 g,yield 63%, white crystal). .sup.1H NMR (CDCl.sub.3, 300 MHz): 1.15(s, 9H), 1.55-1.95 (m, 8H), 2.74 (m, 1H), 5.62 (s, 1H), 7.44-7.56(m, 3H), 7.88-7.91 (m, 2H) ppm. .sup.13C NMR (CDCl.sub.3, 300 MHz):23.5, 24.5, 25.3, 26.6, 35.6, 50.9, 83.2, 110.6, 124.9, 127.5,127.9, 138.9, 173.7.

S(+)-Cyclopentylmandelic Acid, 12

[0178] To a solution ofcis-(2S,5S)-2-(tert-butyl)-5-cyclopentyl-5-phenyl-1,3-dioxolan-4-one(14.35 g, 50 mmol) in 100 ml methanol and 50 ml water, 15 g of KOHwas added slowly. The mixture was stirred and heated (65.degree.C.) to reflux for 3-4 hr, then cooled down to the room temperature,and methanol was removed. To the aqueous solution, 100 ml of ethylacetate was added, then acidified to pH 1 with 3N HCl. The mixturewas poured into a separation funnel, and the organic layer wasseparated. The aqueous layer was extracted two times with ethylacetate (50 ml). The combined organic layers were dried overNa.sub.2SO.sub.4, filtered, and the solvent was removed to provide13.44 g of yellowish crude product, which was re-crystallized togive a pure product of S(+)-cyclopentylmandelic acid, 12 (6.89 g,yield 62%, white crystal). .sup.1H NMR (CDCl.sub.3, 300 MHz):1.28-1.75 (m, 8H), 2.94 (m, 1H), 7.24-7.34 (m, 3H), 7.62-7.68 (m,2H). .sup.13C NMR (CDCl.sub.3, 300 MHz): 25.9, 26.3, 26.4, 26.9,47.1, 79.2, 125.8, 127.7, 128.2, 140.8, 180.9.

Methyl S(+)-cyclopentylmandelate, 13

[0179] S(+)-cyclopentylmandelic acid, 12 (5.5 g, 25 mmol), andpotassium carbonate (8.61 g, 63 mmol) in DMF (60 ml) solution wasadded with methyl iodide (10.6 g, 75 mmol). The mixture was stirredat room temperature for 3 hr, poured into water, and extracted withhexane for three times. Evaporation of dried hexane extract gave apure product of S(+)-cyclopentylmandelate, 13 (5.85 g, 100%, clearoil). .sup.1H NMR (CDCl.sub.3, 300 MHz): 1.32-1.61 [8H, m,(CH.sub.2).sub.4], 2.90 [1H, p, CHC(OH)], 3.76 (s, 3H), 3.78 (s,1H), 7.25-7.35 (m, 3H), 7.63-7.65 (m, 2H). .sup.13C NMR(CDCl.sub.3, 300 MHz): 25.9, 26.2, 26.3, 26.8, 47.1, 53.2, 79.1,125.8, 127.3, 128.0, 141.6, 176.0.

(R)-3-Hydroxy-N-Methylpyrrolidine, (R)3

[0180] In a 100 ml flask, 4 g (R)-3-Hydroxy pyrrolidinehydrochloride salt, 50 ml THF and 1.3 g NaOH were added and stirredfor 20 min. Then, 1.1 g paraforaldehyde and 4.8 g formic acid (90%)were added. The mixture was heated (60.degree. C.) and stirred atreflux for 2 hr until all solid disappeared. The mixture was cooledto 0.degree. C., combined with 6.5 ml of 10 N NaOH solution (pHabout 10), and extracted twice by ethyl ether (50 ml). The combinedorganic layer was dried over Na.sub.2SO.sub.4. Evaporation of thedried organic layer gave a yellowish, oily product of (R).sub.3(3.0 g, 92%). .sup.1H NMR (CDCl.sub.3, 300 MHz): 1.65-1.75 (m, 1H),2.15-2.36 (m, 2H), 2.33 (s, 3H), 2.55-2.59 (m, 2H), 2.76-2.85 (m,1H), 4.30-4.40 (m, 1H), 4.8-5.10 (brs, 1H). .sup.13C NMR(CDCl.sub.3, 300 MHz): 35.4, 41.9, 54.7, 64.9, 70.9.

(S)-3-Hydroxy-N-Methylpyrrolidine, (S)3

[0181] Synthesis of (S).sub.3 was the same as for (R).sub.3, exceptthe starting material was (S)-3-Hydroxypyrrolidine hydrochloridesalt. The resultant product (S).sub.3 (3.10 g, 95%) was also anoil. .sup.1H NMR (CDCl.sub.3, 300 MHz): 1.50-1.60 (m, 1H),2.05-2.30 (m, 2H), 2.28 (s, 3H), 2.40-2.50 (m, 2H), 2.70-2.80 (m,1H), 4.25-4.30 (m, 1H), 4.80 (brs, 1H). .sup.13C NMR (CDCl.sub.3,300 MHz): 35.4, 41.9, 54.7, 64.9, 70.9.

(3R) N-Methyl-3-pyrrolidinyl-(S)-cyclopentylmandelate, 14

[0182] In a 250 ml 3-neck flask equipped with Dean-Stark condenser,a mixture of methyl S(+)-cyclopentylmandelate, 13 (2 g, 8.8 mmol),(R)-3-hydroxy-N-methylpyrrolidine, (R)-3 (2 g, 20 mmol), and 100 mlof heptane was stirred and heated (110.degree. C.) until 20 ml ofheptane had been distilled. The temperature was reduced to25.degree. C., and approximately 0.003 g of sodium was added. Themixture was stirred and heated to 110.degree. C. again for 3 hr asthe distillation was continued. An additional piece of sodium(0.002 g) was added at the 1 hr point. More heptane was added atsuch a rate as to keep the reaction volume constant. The mixturewas cooled to 0.degree. C., mixed with 5 ml of water, and theorganic layer was separated. The organic layer was extracted with3N HCl. The acid extract was made alkaline (pH 10) with 5N NaOH andextracted three times with ether. Removal of dried ether solution(over Na.sub.2SO.sub.4) gave a clear, oily product 14 (1.6 g,61.5%). .sup.1H NMR (CDCl.sub.3, 300 MHz): 1.28-1.80 [m, 9H],2.15-2.25 (m, 1H), 2.30-2.40 (m, 1H), 2.37 (s, 3H), 2.65-2.80 (m,3H), 2.90-3.00 (m, 1H), 3.85 (1H, brs, OH), 5.22 (m, 1H), 7.20-7.35(m, 3H), 7.64-7.70 (m, 2H). .sup.13C NMR (CDCl.sub.3, 300 MHz):26.0, 26.4, 26.5, 26.7, 32.1, 42.0, 47.1, 54.8, 62.0, 76.5, 79.1,125.8, 127.3, 128.0, 141.7, 175.3.

(3S) N-Methyl-3-pyrrolidinyl-(S)-cyclopentylmandelate, 15

[0183] Following the same procedure as for 14, except (S)-3 wasused instead of (R)-3, a clear, oily product of 15 (2.33 g, 89.6%)was obtained. .sup.1H NMR (CDCl.sub.3, 300 MHz): 1.24-1.70 (m, 9H),1.80-1.88 (m, 1H), 2.25-2.40 (m, 2H), 2.35 (s, 3H), 2.55-2.70 (m,2H), 2.75-2.82 (m, 1H), 2.90-3.00 (m, 1H), 3.95 (1H, bs, OH), 5.22(m, 1H), 7.24-7.40 (m, 2H), 7.64-7.69 (m, 5H). .sup.13C NMR(CDCl.sub.3, 300 MHz): 26.0, 26.3, 26.4, 26.7, 32.6, 42.0, 47.1,54.9, 61.6, 76.4, 79.2, 125.8, 127.3, 128.0, 141.7, 175.2.

(2S,3'R)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmet-hyl)-1-methylpyrrolidinium bromide, 16 [Compound (h)]

[0184] Compound 14 (0.6 g, 1.96 mmol) in 30 ml of dry acetonitrilewas combined with methyl bromoacetate (1.0 g, 6.4 mmol) at roomtemperature. The mixture was stirred for 3 hr. Evaporation ofacetonitrile gave a crude product. The crude product was dissolvedin a small volume of methylene chloride and poured into a 100 ml ofdry ethyl ether to obtain a precipitate. This procedure wasrepeated three times and gave compound (h) as the product (0.81 g,89%, white powder). .sup.1H NMR (CDCl.sub.3, 300 MHz): 1.30-1.70(m, 8H), 1.82-1.95 (brs, 1H), 2.10-2.20 (m, 1H), 2.75-2.90 (m, 2H),3.25, 3.60 (2s, total 3H, N--CH3), 3.75, 3.79 (2s, total 3H, O-Me),4.10-4.60 (m, 4H), 4.92, 5.35 (2AB, total 2H, N--CH2-COOMe),5.52-5.58 (m, 1H), 7.23-7.38 (m, 3H), 7.56-7.60 (m, 2H). .sup.13CNMR (CDCl.sub.3, 300 MHz): 25.8, 25.9; 26.3, 26.4; 26.4, 26.5;27.0, 27.0; 29.8, 30.1; 45.9, 46.8; 50.2, 51.4; 53.2, 53.2; 62.2,63.2; 64.6, 64.7; 69.6, 69.7; 72.8, 73.1; 79.4, 79.6; 125.7, 125.7;127.6, 127.9; 128.2, 128.4; 141.0, 141.2; 165.3, 165.5; 173.9,174.2.

(2S,3'S)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(methoxycarbonylmet-hyl-1-methylpyrrolidinium bromide, 17 [Compound (i)]

[0185] Following the same procedure as for Compound (h), exceptcompound 15 was used instead of compound 14, the product Compound(i) (0.8 g, 88%, white powder) was obtained. .sup.1H NMR(CDCl.sub.3, 300 MHz): 1.30-1.75 (m, 8H), 1.80-1.90 (brs, 1H),2.15-2.30 (m, 1H), 2.78-2.95 (m, 2H), 3.10, 3.65 (2s, total 3H,N--CH3), 3.75, 3.78 (2s, total 3H, O-Me), 4.15-4.52 (m, 4H), 4.85,5.38 (2AB, total 2H, N--CH2-COOMe), 5.50-5.58 (m, 1H), 7.23-7.38(m, 3H), 7.56-7.66 (m, 2H). .sup.13C NMR (CDCl.sub.3, 300 MHz):25.8, 25.9; 26.2, 26.3; 26.3, 26.4; 26.8, 26.9; 29.4, 29.6; 45.6,46.9; 50.1, 51.4; 53.1, 53.1; 62.2, 63.3; 64.8, 64.8; 69.5, 69.8;72.8, 73.2; 79.4, 79.6; 125.6, 125.9; 127.6, 127.9; 128.2, 128.4;140.7, 141.1; 165.2, 165.5; 173.9, 174.2.

Hydrolysis of Esters & HPLC Separations

[0186] The procedures used for obtaining the 2S-isomers 18a, 18b,19a and 19b (white powder) were the same as for 2R-isomers 8a, 8b,9a and 9b.

[0187] (2S,1'R,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyr-rolidinium inner salt, 18a: .sup.1H NMR (CDCl.sub.3, 300 MHz):1.30-1.65 (m, 8H), 2.02-2.45 (m, 2H), 2.82-2.90 (m, 1H), 3.10-3.18(m, 1H), 3.25 (s, 3H), 3.50-4.05 (m, 6H), 5.34-5.40 (m, 1H),7.23-7.38 (m, 3H), 7.50-7.68 (m, 2H).

[0188] (2S,1'S,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyr-rolidinium inner salt, 18b: .sup.1H NMR (CDCl.sub.3, 300 MHz):1.45-1.85 (m, 9H), 2.05-2.15 (m, 1H), 2.80-2.90 (m, 1H), 3.00-3.10(m, 1H), 3.35 (s, 3H), 3.70-3.80 (m, 1H), 3.90-4.10 (m, 4H),4.22-4.35 (m, 1H), 5.50-5.60 (m, 1H), 7.36-7.55 (m, 3H), 7.72-7.80(m, 2H).

[0189] (2S,1'R,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyr-rolidinium inner salt, 19a: .sup.1H NMR (CDCl.sub.3, 300 MHz):1.20-1.65 (m, 8H), 1.95-2.10 (m, 1H), 2.20 (brs, 1H), 2.40-2.50 (m,1H), 2.78-2.90 (m, 1H), 3.15 (s, 3H), 3.70-3.90 (m, 2H), 3.96-4.20(m, 4H), 5.38-5.50 (m, 1H), 7.20-7.38 (m, 3H), 7.55-7.65 (m,2H).

[0190] (2S,1'S,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyr-rolidinium inner salt, 19b: .sup.1H NMR (CDCl.sub.3, 300 MHz):1.35-1.70 (m, 8H), 2.00-2.15 (m, 1H), 2.70-2.90 (m, 2H), 3.00 (s,3H), 3.42 (brs, 1H), 3.58-3.68 (m, 2H), 3.80-3.95 (m, 3H),4.08-4.18 (m, 1H), 5.38-5.48 (m, 1H), 7.20-7.40 (m, 3H), 7.55-7.62(m, 2H).

Receptor Binding Affinity

[0191] Receptor binding studies on soft anticholinergics isomersand their zwitterionic metabolite isomers, as well asglycopyrrolate, and N-methylscopolamine were performed withN-[.sup.3H]-methyl-scopolamine (NMS) in assay buffer(phosphate-buffered saline, PBS, without Ca.sup.++ or Mg.sup.++, pH7.4), following the protocol from Applied Cell Science Inc.(Rockville, Md.). A 10 mM NaF solution was added to the buffer asan esterase inhibitor. The assay mixture (0.2 mL) contained 20.mu.L diluted receptor membranes (receptor proteins: M.sub.1, 38.mu.g/mL; M.sub.2, 55 .mu.g/mL; M.sub.3, 27 .mu.g/mL; M.sub.4, 84.mu.g/mL). The final concentration of NMS for the binding studieswas 0.5 nM. Specific binding was defined as the difference in[.sup.3H]NMS binding in the absence and presence of 5 .mu.Matropine for M.sub.1 and M.sub.2 or 1 .mu.M atropine for M.sub.3and M.sub.4. Incubation was carried out at room temperature for 2hr. The assay was terminated by filtration through a Whatman GF/Cfilter (presoaked overnight with 0.5% polyethyleneimine) The filterwas then washed six times with 1 mL ice cold buffer (50 mMTris-HCl, pH 7.8, 0.9% NaCl), transferred to vials, and 5 mL ofScintiverse was added. Detection was performed on a Packard 31800liquid scintillation analyzer (Packard Instrument Inc., DownerGrove, Ill.). Data obtained from the binding experiments werefitted to the equation %[.sup.3H] NMSbound=100-[100x.sup.n/k/(1+x.sup.n/k)], to obtain the Hillcoefficient n, and then to the equation %[.sup.3H] NMSbound=100-[100x.sup.n/IC.sub.50/(1+x.sup.n/IC.sub.50)], to obtainthe IC.sub.50 values (x being the concentration of the testedcompound). Based on the method of Cheng and Prusoff K, was derivedfrom the equation K.sub.i=IC.sub.50/(1+L/K.sub.d), where L is theconcentration of the radioligand. IC.sub.50 represents theconcentration of the drug causing 50% inhibition of specificradioligand binding, and K.sub.d represents the dissociationconstant of the radioligand receptor complex. Data were analyzed bya non-linear least-square curve-fitting procedure using Scientistsoftware (MicroMath Inc., Salt Lake City, Utah).

Determination of pA.sub.2 Values

[0192] Male guinea pigs weighing about 400 g were obtained fromHarlan Inc. (Indianapolis, Ind.) and fasted overnight. Animals weresacrificed by decapitation, and the ileum (the region of 5 cmupward of the cecum) was isolated and removed. The ileum was cutinto 2.5 cm pieces and suspended in an organ bath containing 30 mLof mixture of Tyrode's solution and 0.1 mM hexamethonium bromide.The organ bath was constantly aerated with oxygen and kept at37.degree. C. One end of the ileum strip was attached to a fixedsupport at the bottom of the organ bath, and the other end to anisometric force transducer (Model TRN001, Kent Scientific Corp.,Conn.) operated at 2-10 g range. The ileum strip was kept at a 2 gtension, and carbachol was used as antagonist. The ileum contractedcumulatively upon the addition of consecutive doses of carbachol(10-20 .mu.L of 2.times.10.sup.-4-2.times.10.sup.-3M in watersolution). Contractions were recorded on a physiograph (Kipp &Zonen Flarbed Recorder, Holland). After the maximum response wasachieved, the ileum was washed three times, and a fresh Tyrode'ssolution containing appropriate concentration of the antagonist(anticholinergic compound tested) was replaced. An equilibrationtime of 10 min was allowed for the antagonists before the additionof carbachol. In each experiment, 5 to 6 different concentrationswere used, and a Schild plot was used to obtain the pA.sub.2values. Four trials were performed for each antagonist.

In Vivo Mydriatic Studies

[0193] The mydriatic effects of eight completely resolvedzwitterionic isomers were compared to those of glycopyrrolate,tropicamide, (.+-.)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyr-rolidinium inner salt [(.+-.)-GA] and (2R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyr-rolidinium inner salt [(2R)-GA] in rabbit eyes. Four healthy, maleNew-Zealand white rabbits weighting about 3.5 kg were used. 100.mu.L of compound in water solution (pH 6.5) at variousconcentrations were administered in the eyes. Compound solutionswere applied to one eye, and water was applied to the other eyethat served as control. Experiments were carried out in a light-and temperature-controlled room. At appropriate time intervals, thepupil diameters of both eyes were recorded. Percent difference inpupil diameters between each time-point and zero time-point werecalculated for both treated and control eyes and reported asmydriatic responses. Control eye dilations were monitored todetermine whether systemic absorption had occurred or not. The areaunder the mydriatic response-time curve (AUC.sup.eff) wascalculated by the trapezoidal rule, and it was used to compare theactivity and duration of action of the tested compounds.

Statistical Analysis

[0194] Receptor binding affinities and pA.sub.2 values werecompared using student t-tests. Mydriatic activities (maximumresponse Rmax % and area under the effect curves AUC.sub.eff) werecompared using ANOVA. A significance level of P<0.05 was used inall cases.

Results and Discussion

Synthesis

[0195] Five soft anticholinergic ester isomers and eightzwitterionic metabolite acid isomers were newly synthesized. The 2Rdiastereoisomers [Compounds (e), (f), (g), 8a, 8b, 9a and 9b] wereobtained by the synthetic pathways described below.

##STR00010## ##STR00011##

[0196] As shown in Scheme 2, first the racemic cyclopentylmandelicacid 1 was synthesized with cyclopentylmagnesium bromide andbenzoylformic acid. This racemic acid was resolved by repeatedcrystallization of the salts produced between this acid and(-)-strychnine. The left rotatory)(-22.5.degree. optically purefree acid R(-)1 was recovered by basification of the salts withsodium hydroxide solution followed by acidification withhydrochloric acid. Methylation of R(-)1 with methyl iodide andpotassium carbonate in DMF at room temperature yields methyl 2R(-)cyclopentylmandelate, R(-).sub.2. Transesterfication of R(-).sub.2with R-3-hydroxy-N-methylpyrrolidine, (R)-3 (made fromR-3-hydroxypyrrolidine with paraformaldehyde and formic acid), gave(3R)-N-methyl-3-pyrrolidinyl-2R-cyclopentylmandelate 4; or withS-3-hydroxy-N-methylpyrrolidine (S)-3 (made fromS-3-hydroxypyrrolidine with paraformaldehyde and formic acid), gave(3S)-N-methyl-3-pyrrolidinyl-2R-cyclopentyl mandelate 5.Quarternization of 4 and 5 with methyl or ethyl bromoacetate inacetonitrile gave 6 [Compound (e)], 7a [Compound (f)], and 7b[Compound (g)]. Each of these has two diastereoisomers, due to thenitrogen chiral center, with a ratio of 2 to 1 (R:S=2:1) that wasshown in .sup.1H NMR spectra. Hydrolysis of 6 [Compound (e)] and 7a[Compound (f)] gave their zwitterionic inner salts 8 and 9. Eachzwitterionic salt also possesses two diastereoisomers with a ratioof 2 to 1 that could be separated by HPLC to give zwitterionicisomers 8a, 8b & 9a and 9b. From .sup.1H NMR,8a, 8b, and 9a, 9bwere evidenced to be pairs of diastereoisomers based on chiralnitrogen. To identify the absolute configuration of these isomers,8b was chosen and dissolved in CDCl.sub.3 for the investigation ofnuclear overhauser effect (NOE). The 2D .sup.1H-.sup.1H NOESYspectrum showed that the methyl group on the nitrogen was at thesame side as the hydrogen at the 3-position of pyrrolidinium ring.Accordingly, the configuration of the nitrogen should be the Sform, and the absolute stereochemistry of 8b was proved to be(2R,1'R,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyr-rolidinium inner salt. Therefore, 8a was (2R,1'R,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyr-rolidinium inner salt; 9a was (2R,1'R,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyr-rolidinium inner salt; and 9b was (2R,1'S,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyr-rolidinium inner salt.

[0197] Grover and coworkers previously reported [J. Org. Chem. 65:6283-6287 (2000)] the highly stereoselective synthesis of(S)-cyclopentylmandelic acid in five steps starting with(S)-mandelic acid. Modification of their procedure afforded pureS(+)-cyclopentylmandelic acid in three steps with good yield. Asdepicted in Scheme 3, reaction of S(+)-mandelic acid withpivaldehyde in the presence of the catalysttrifluoromethanesulfonic acid gave the product ofcis-(2S,55)-2-(tert-butyl)-5-phenyl-1,3-dioxolan-4-one, 10, inabout 90% yield. At -78.degree. C., deprotonation of 10 withlithium bis(trimethylsilyl)amide followed by adding cyclopentylbromide generatedcis-(2S,5S)-2-(tert-butyl)-5-cyclopentyl-5-phenyl-1,3-dioxolan-4-one,11. Base hydrolysis of 11 with potassium hydroxide, followed byacidification with hydrochloric acid provided the expected(S)-(+)-cyclopentylmandelic acid 12. After this step, the sameprocedures as for 8a, 8b, 9a and 9b including methylation,esterification, quaternization and hydrolyses were followed to givethe final four zwitterionic isomers, (2S,1'R,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyr-rolidinium inner salt 18a [2S1'R3'R-GA]; (2S,1'S,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyr-rolidinium inner salt 18b [2S1'S3'R-GA]; (2S,1'R,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyr-rolidinium inner salt 19a [2S1'R3'S-GA]; and (2S,1'S,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(carboxymethyl)-1-methylpyr-rolidinium inner salt 19b [2S1'S3'S-GA]. They were alsocharacterized by NMR.

##STR00012## ##STR00013##

Receptor Binding Studies

[0198] The receptor binding affinities of soft analogs, pK.sub.i,determined by radioligand binding assays using human clonedmuscarinic receptor subtypes, M M.sub.4, are presented in Table4.

TABLE-US-00004 TABLE 4 Receptor binding affinities, M.sub.3/M.sub.2selectivities, and pA.sub.2 values. Subtypes of cloned muscarinicreceptors .sup.a Selectivity .sup.b Compound M.sub.1 M.sub.2M.sub.3 M.sub.4 M.sub.3/M.sub.2 pA.sub.2 .sup.c (a) .sup.d 7.91.+-. 0.05 7.79 .+-. 0.11 7.80 .+-. 0.10 8.29 .+-. 0.19 1.0 .+-. 0.07.90 .+-. 0.13 (1.02 .+-. 0.12) (1.25 .+-. 0.08) (1.17 .+-. 0.18)(1.12 .+-. 0.05) (b) .sup.d 7.51 .+-. 0.17 7.32 .+-. 0.07 7.54 .+-.0.15 7.94 .+-. 0.09 1.8 .+-. 0.7 7.36 .+-. 0.34 (0.91 .+-. 0.09)(1.23 .+-. 0.06) (1.18 .+-. 0.08) (1.18 .+-. 0.09) (.+-.)-GA .sup.d6.19 .+-. 0.06 5.48 .+-. 0.13 5.84 .+-. 0.07 6.44 .+-. 0.06 2.4.+-. 0.7 6.42 .+-. 0.30 (1.11 .+-. 0.06) (1.02 .+-. 0.20) (1.01.+-. 0.07) (0.84 .+-. 0.06) (c) .sup.e 8.89 .+-. 0.04 8.87 .+-.0.05 9.00 .+-. 0.06 9.52 .+-. 0.01 1.4 .+-. 0.2 8.31 .+-. 0.05(0.83 .+-. 0.11) (1.10 .+-. 0.11) (0.83 .+-. 0.01) (0.83 .+-. 0.01)(d) .sup.e 8.67 .+-. 0.16 8.84 .+-. 0.34 8.74 .+-. 0.02 8.85 .+-.0.13 1.1 .+-. 1.1 8.55 .+-. 0.16 (0.86 .+-. 0.08) (0.92 .+-. 0.01)(1.09 .+-. 0.15) (0.89 .+-. 0.02) 2R-GA .sup.e 8.11 .+-. 0.16 7.48.+-. 0.12 8.12 .+-. 0.10 8.23 .+-. 0.12 4.4 .+-. 0.3 7.20 .+-. 0.19(1.12 .+-. 0.25) (0.95 .+-. 0.11) (0.80 .+-. 0.01) (1.02 .+-. 0.10)(e) .sup.f 8.99 .+-. 0.04 9.01 .+-. 0.06 9.06 .+-. 0.14 9.45 .+-.0.01 1.1 .+-. 0.1 -- (1.19 .+-. 0.12) (1.03 .+-. 0.09) (1.03 .+-.0.18) (1.52 .+-. 0.66) (f) .sup.f 8.50 .+-. 0.03 7.90 .+-. 0.048.60 .+-. 0.09 8.87 .+-. 0.09 5.0 .+-. 1.1 .sup. -- .sup.h (1.30.+-. 0.20) (1.07 .+-. 0.17) (1.04 .+-. 0.27) (1.08 .+-. 0.01) (h).sup.f 7.23 .+-. 0.01 7.22 .+-. 0.03 6.99 .+-. 0.08 7.57 .+-. 0.010.6 .+-. 0.1 -- (0.98 .+-. 0.06) (1.09 .+-. 0.18) (1.15 .+-. 0.13)(1.11 .+-. 0.03) (i) .sup.f 6.40 .+-. 0.05 6.47 .+-. 0.08 5.95 .+-.0.02 6.39 .+-. 0.01 0.3 .+-. 0.0 -- (0.92 .+-. 0.09) (0.99 .+-.0.16) (1.06 .+-. 0.03) (1.44 .+-. 0.75) (j) .sup.f 8.68 .+-. 0.118.21 .+-. 0.10 8.64 .+-. 0.07 8.71 .+-. 0.38 2.8 .+-. 0.8 -- (1.21.+-. 0.33) (1.27 .+-. 0.11) (1.33 .+-. 0.16) (1.15 .+-. 0.03) 8a.sup.g 7.04 .+-. 0.09 6.43 .+-. 0.07 6.95 .+-. 0.04 7.00 .+-. 0.053.5 .+-. 0.2 6.32 .+-. 0.23 (0.97 .+-. 0.13) (0.85 .+-. 0.21) (1.06.+-. 0.04) (0.93 .+-. 0.01) 8b .sup.g 8.13 .+-. 0.06 7.63 .+-. 0.028.15 .+-. 0.02 8.33 .+-. 0.04 3.3 .+-. 0.0 7.45 .+-. 0.21 (1.25.+-. 0.01) (0.82 .+-. 0.15) (0.84 .+-. 0.17) (1.00 .+-. 0.06) 9a.sup.g 7.98 .+-. 0.01 7.39 .+-. 0.09 8.04 .+-. 0.01 8.15 .+-. 0.065.2 .+-. 0.7 7.33 .+-. 0.28 (1.02 .+-. 0.03) (0.80 .+-. 0.22) (0.96.+-. 0.03) (1.01 .+-. 0.06) 9b .sup.g 8.32 .+-. 0.04 7.64 .+-. 0.018.46 .+-. 0.12 8.56 .+-. 0.07 5.5 .+-. 1.1 7.15 .+-. 0.12 (1.01.+-. 0.01) (1.00 .+-. 0.04) (0.80 .+-. 0.21) (0.86 .+-. 0.06) 18a.sup.g 5.87 .+-. 0.04 5.65 .+-. 0.06 5.54 .+-. 0.16 5.79 .+-. 0.120.8 .+-. 0.1 5.14 .+-. 0.38 (1.06 .+-. 0.05) (1.24 .+-. 0.07) (1.02.+-. 0.12) (0.88 .+-. 0.04) 18b .sup.g 6.67 .+-. 0.06 6.35 .+-.0.01 6.22 .+-. 0.05 6.47 .+-. 0.01 0.7 .+-. 0.0 5.69 .+-. 0.13(1.08 .+-. 0.03) (1.01 .+-. 0.01) (1.04 .+-. 0.08) (1.30 .+-. 0.28)19a .sup.g <4.5 <4.5 <4.5 <4.5 -- <4 -- -- -- -- 19b.sup.g 5.84 .+-. 0.06 5.61 .+-. 0.00 5.61 .+-. 0.09 5.85 .+-. 0.051.0 .+-. 0.2 5.03 .+-. 0.26 (1.13 .+-. 0.10) (1.20 .+-. 0.02) (1.03.+-. 0.01) (0.95 .+-. 0.18) glycopyrrolate 9.76 .+-. 0.05 9.19 .+-.0.18 8.73 .+-. 0.05 9.90 .+-. 0.08 0.4 .+-. 0.2 8.57 .+-. 0.12(1.37 .+-. 0.20) (0.99 .+-. 0.11) (1.14 .+-. 0.25) (1.02 .+-. 0.01)scopolamine 9.69 .+-. 0.01 9.18 .+-. 0.21 9.29 .+-. 0.12 9.92 .+-.0.21 1.3 .+-. 0.4 9.16 .+-. 0.19 methyl bromide (0.92 .+-. 0.10)(1.02 .+-. 0.02) (1.07 .+-. 0.01) (0.90 .+-. 0.04) .sup.a Receptorbinding at cloned human muscarinic receptors (M.sub.1-M.sub.4subtypes); pK.sub.i data represent mean .+-. SD of 3 experiments,and the numbers in parentheses denote Hill slopes. .sup.bM.sub.3/M.sub.2 affinity ratio (times) .sup.c pA.sub.2 values weredetermined on 4-6 ileum strips obtained from different animals, anddata represent mean .+-. SD. .sup.d Racemic forms. .sup.e Isomersbased on the chiral center 2. .sup.f Isomers based on the chiralcenters 2 & 3'. .sup.g Isomers based on the chiral centers 2,3', & 1'. .sup.h Data not available or not detectable.

[0199] The pK.sub.i of newly synthesized isomers were compared withthat of the racemic and 2R isomeric parent soft drugs [the methylester Compound (c) and the ethyl ester Compound (d)], racemic and2R isomeric GA (the zwitterionic metabolite) as well as those ofglycopyrrolate and N-methylscopolamine pK.sub.i of the racemicforms, Compound (a) and Compound (b), showed lower receptor bindingaffinities than their corresponding 2R isomers (7.8-8.3 vs.8.7-9.5), confirming that stereospecificity is important at thesereceptors. The potencies of these 2R isomers are similar to thoseof glycopyrrolate (8.7-9.9) and N-methylscopolamine (9.2-9.9).Resolution of 2 and 3' chiral centers of racemic Compound (a)resulted in four stereoisomers, Compounds (e), (f), (h) and (i)with pK.sub.i values of 9.0-9.5, 7.9-8.9, 7.0-7.6 and 6.0-6.5,respectively. These numbers indicate that among the methyl esterisomers, not only 2R isomers are more potent than the corresponding2S isomers, but also that 3'R isomers are more potent than 3'Sisomers. The 2R3'S isomer of the ethyl ester, Compound (j), showeda pK.sub.i value of 8.2-8.7, the same as the 2R3'S isomer of themethyl ester. In the same table, the M.sub.3/M.sub.2muscarinic-receptor subtype-selectivities were also calculated.Contrary to the previously reported 2R isomer of the methyl andethyl esters, Compounds (c) and (d), that show no M.sub.3/M.sub.2subtype selectivity, the 2R3'S isomers of the methyl and ethylesters, Compounds (e) and (j), show significantly increasedM.sub.3/M.sub.2 muscarinic-receptor subtype-selectivity (p<0.01,t-test assuming equal variances). The M.sub.3 affinity was5.0.+-.1.1 times of M.sub.2 affinity in the case of Compound (e),and 2.8.+-.0.8 times in the case of Compound (j). This indicatesthat the configuration of chiral center 3' may play an importantrole in the safety profile of this type of softanticholinergics.

[0200] The receptor-binding pK.sub.i of racemic (.+-.) GA andisomeric 2R-GA obtained earlier are also shown in Table 4. Inagreement with soft drug design principles that the acidic moietyformed by hydrolysis of the parent soft drug ester inactivates thedrug, the zwitterions were found considerably less active thantheir corresponding parent esters, e.g. pK.sub.i of (.+-.)GA,5.5-6.4, vs. Compound (a), 7.8-8.3, and Compound (b), 7.3-7.9; andpK.sub.a of 2R-GA, 7.5-8.2, vs. Compound (c), 8.9-9.5, and Compound(d), 8.7-8.9 (3-4). As discussed previously, the zwitterionicmetabolite retains some activity because the electronicdistribution in its structures somewhat resembles those of theneutral, active anticholinergics. In this study, to obtain a betterpicture of the stereospecificity/stereoselectivity of this type ofanticholinergic, the zwitterionic form was chosen as a modelcompound for the investigation, since the zwitterion GA, either inits racemic or its 2R isomeric form, was very soluble and stable inaqueous solutions (buffer or biological media, pH 6-8). Inaddition, 2R-GA has been found active at topical sites (e.g. inrabbit eyes), and could be excreted unchanged, rapidly throughurine (t.sub.1/2 10-15 min after i.v. in rats). In Table 4, thepK.sub.i of the completely resolved eight isomers of .+-.GA,2R1'R3'R-GA, 2R1'S3'R-GA, 2R1R3'S-GA, 2R1'S3'S-GA, 2S1'R3'R-GA,2S1'S3'R-GA, 2S1'R3'S-GA, 2S1'S3'S-GA was in a wide range of4.5-8.6. In all cases, the 2R isomers are more potent than the 2Sisomers, and the 1'S isomers are more potent than the 1R isomers.The comparative potencies for 3'R and 3'S isomers varied dependingon the configuration of chiral center 2, e.g. 2R1'R3'S>2R1'R3'Rand 2R1'S3'S>2R1'S3'R; but 2S1'R3'R>2S1R3'S and2S1'S3'R>2S1'S3'S. Also, the same as previous methyl esterisomers, among 2R isomers of the acid, the 2R3'S isomers (2R1R3'Sand 2R1'S3'S) showed highest M.sub.3/M.sub.2 muscarinic-receptorsubtype-selectivities (5.2-5.5 times) followed by the 2R3'R isomers(2R1'R3'R and 2R1'S3'R,3.3-3.5 times). The 2S isomers did not showany M.sub.3/M.sub.2 selectivity. Thus, the importance of the chiralcenter 2 and 3' configuration (2R3'S) on the M.sub.3/M.sub.2selectivity of this type of anticholinergics has beendemonstrated.

[0201] In order to show the comparative stereoselectivity (times)based on each chiral center, the ratio of binding activities ofeach corresponding paired isomers was calculated, and the resultsare shown in Table 5. The results displayed are comparativepotencies (times) calculated from the receptor binding affinities,pK.sub.i, in Table 4. The difference in receptor binding affinitiesbetween 2R and 2S isomers is significant (27 to 447 times for themethyl ester isomers, and 6 to 4467 times for zwitterion isomers).The 3'R isomers of the methyl ester (with chiral center 1unresolved, 2R3'R & 2S3R methyl esters) are more active (1.5 to12.9 times) than their corresponding 3'S isomers (2R3'S & 2S3'Smethyl esters). However, in the acid, the 3'S isomers were notalways more active than the corresponding 3R isomers, e.g. in 2Risomers, 3'S>3'R (2R1'R3'S>2R1'R3'R and2R1'S3'S>2R1'S3'R); but in 2S isomers, 3'R>3'S(2S1'R3'R>2S1'R3'S and 2S1'S3'R>2S1'S3'S). Also, there aremore significant differences between 2R1'R3'S and 2R1'R3'R thanbetween 2R1'S3'S and 2R1'S3'R (8.7 to 14.1 times vs. 1.0 to 2.0times), and between 2S1'R3'R and 2S1'R3'S than between 2S1'S3'R and2S1'S3'S (11.0 to 23.4 times vs. 4.1 to 6.8 times). These resultsindicate that the activity based on chiral center 3' can beaffected by the configuration of the other two chiral centers, 2and 1'. When comparing all eight zwitterion isomers (with all threechiral centers resolved), it clearly shows that 1'S isomers weremore active than the corresponding 1'R isomers in all cases(1.8-22.4 times).

TABLE-US-00005 TABLE 5 Comparative stereoselectivities.sup.aSubtypes of cloned muscarinic receptors.sup.b Descrip- CompoundM.sub.1 M.sub.2 M.sub.3 M.sub.4 tion.sup.f Methyl Esters 2R3'S/125.9 26.9 446.7 302.0 2R > 2S 2S3'S.sup.c 2R3'R/ 57.5 61.7117.5 75.9 2S3'R.sup.c 2R3'R/ 3.1 12.9 2.9 3.8 3R > 3S2R3'S.sup.d 2S3'R/ 6.8 5.6 11.0 1.5 2S3'S.sup.d Zwitterions2R1'R3'R/ 14.8 6.0 25.7 16.2 2R >> 2S 2S1'R3'R.sup.c2R1'S3'R/ 28.8 19.1 85.1 72.4 2S1'S3'R.sup.c 2R1'R3'S/ 3020.0 776.23467.4 4466.8 2S1'R3'S.sup.c 2R1'S3'S/ 302.0 107.2 707.9 512.92S1'S3'S.sup.c 2R1'R3'S/ 8.7 9.1 12.3 14.1 3S > 3R2R1'R3'R.sup.d 2R1'S3'S/ 1.5 1.0 2.0 1.7 2R1'S3'R.sup.d 2S1'R3'R/23.4 14.1 11.0 19.5 3R > 3S 2S1'R3'S.sup.d 2S1'S3'R/ 6.8 5.5 4.14.2 2S1'S3'S.sup.d 2R1'S3'R/ 12.3 15.8 15.8 21.4 1R < 1S2R1'R3'R.sup.e 2R1'S3'S/ 2.2 1.8 2.6 2.6 2R1'R3'S.sup.e 2S1'S3'R/6.3 5.0 5.2 4.8 2S1'R3'R.sup.e 2S1'S3'S/ 21.9 12.9 12.9 22.42S1'R3'S.sup.e .sup.aAffinity ratio (times) between each twoisomers based on each of the three different chiral centers..sup.bReceptor binding at cloned human muscarinic receptors(M.sub.1-M.sub.4 subtypes) .sup.cAffinity ratio based on the chiralcenter 2. .sup.dAffinity ratio based on the chiral center 3..sup.eAffinity ratio based on the chiral center 1. .sup.fConcludedstereoselectivities

[0202] In all cases, the Hill coefficients (n) were not verydifferent from unity indicating that, in general, drug-receptorinteractions obeyed the law of action and binding took place atonly one site.

pA.sub.2 Studies

[0203] The pA.sub.2 values determined from guinea pig ileumcontraction assays, which represent the negative logarithm of themolar concentration of the antagonist that produces a two-foldshift to the right in an agonist's concentration-response curve,are a classical functional study of anticholinergic affinity (atM.sub.3 muscarinic receptors). For the soft anticholinergics of thepresent study, the pA.sub.2 values obtained from ileum longitudinalcontractions by using carbachol as agonist with the method of vanRossum [Arch. Int. Pharcodyn. 143: 299-330 (1963)] are presented inTable 4. The pA.sub.2 values are in general, comparable to thepK.sub.a values obtained in the M.sub.3 receptor binding studies.The pA.sub.2 values of newly developed zwitterionic isomerssignificantly differed between 2R and 2S configurations (6.32 to7.45 and <4 to 5.69, respectively, p<0.01, t-test assumingequal variances). Similar to the above reported 2R isomer (2R-GA),the pA.sub.2 values of completely resolved 2R isomers (2R1'R3'R-GA,2R1'S3'R-GA, 2R1'R3'S-GA, and 2R1'S3'S-GA) are 1 to 2 less thanthose of the corresponding 2R ethyl and methyl parent ester softdrugs, indicating a one to two order of magnitude less activity ofthese zwitterionic compounds. The retained moderate activity ofsome zwitterionic metabolite isomers is probably due to aspatially-close structures that resembles those of the neutral,active anticholinergics. In the active 2R isomers, while2R1'R3'R-GA showed a lower value (6.32), all others showed asimilar moderate contraction activity (about 7.15 to 7.45).

Mydriatic Activities

[0204] The mydriatic effects of the fully resolved eightzwitterionic isomers were compared to those of (.+-.)GA, 2R-GA,glycopyrrolate and tropicamide in vivo in rabbits. Following a 100.mu.l topical administration, the mydriatic responses were recordedat appropriate time-intervals as % changes in pupil size. Themaximum response (R.sub.max, % change in pupil size at 30 min to 1h after administration) and area under the response-time curve(AUC.sup.eff.sub.0-168h) are shown in Table 6.

TABLE-US-00006 TABLE 6 Maximum response (R.sub.max, maximum %change in pupil size) and area under the response-time curve(AUC.sub.eff) after topical administration (0.1 mL)..sup.a CompoundConc. (%) R.sub.max (%) AUC.sup.eff.sub.0-168 h (.+-.)GA.sup.b 0.011.85 .+-. 2.14 0.7 .+-. 0.9 1 45.37 .+-. 8.19 119 .+-. 342R-GA.sup.b 0.01 31.00 .+-. 7.14 73 .+-. 24 0.1 50.34 .+-. 7.92 182.+-. 40 2R1'R3'R-GA (8a) 0.1 24.40 .+-. 8.33 89 .+-. 50 2R1'S3'R-GA(8b) 0.1 51.79 .+-. 16.62 308 .+-. 106 2R1'R3'S-GA (9a) 0.1 43.90.+-. 7.63 216 .+-. 29 2R1'S3'S-GA (9b) 0.1 47.32 .+-. 19.64 274.+-. 134 2S1'R3'R-GA (18a) 0.1 0.00 .+-. 0.00 0 .+-. 0 0.4 7.44.+-. 0.60 11 .+-. 1 2S1'S3'R-GA 18(b) 0.1 3.87 .+-. 4.49 13 .+-. 150.4 14.88 .+-. 1.19 37 .+-. 3 2S1'R3'S-GA 19(a) 0.1 0.00 .+-. 0.000 .+-. 0 0.4 0.00 .+-. 0.00 0 .+-. 0 2S1'S3'S-GA 19(b) 0.1 3.87.+-. 4.49 13 .+-. 15 0.4 11.01 .+-. 3.81 28 .+-. 2glycopyrrolate.sup.b 0.05 48.73 .+-. 12.66 2476 .+-. 847 0.1 52.95.+-. 10.93 3732 .+-. 866 tropicamide.sup.b 0.5 44.64 .+-. 11.17 451.+-. 121 .sup.aData represent mean .+-. SD of four trials..sup.bData adapted from other testing.

[0205] The results indicate that, as in the in vitro studies, the2R isomers are much more potent than the 2S isomers (even when the2S dose was increased to 0.4%); and 2R1'R3'R-GA is less potent thanthe other three 2R isomers. In FIG. 4, the activity-time profilesof four 2R and one 1'S isomers (the most active S isomer) at 0.1%are displayed. The pupil-dilating potency of the most potent three2R isomers at a dose of 0.1% is similar to that of 0.05 to 0.1% ofglycopyrrolate and 0.5% of tropicamide, however, their duration ofactions was much shorter than that of the "hard" glycopyrrolate(AUC 200-300 vs. 2500, respectively), and somewhat shorter thanthat of tropicamide, in agreement with soft drug design principles.The activities of 2R1'S3'R-GA (the most active zwitterionic isomer)and glycopyrrolate lasted for 10 h and 144 h, respectively, asdisplayed in FIG. 5. These results indicate that a goodpharmacological effect can be achieved by some 2R zwitterionicisomers, and these isomers can be rapidly eliminated from the body.Furthermore, the active 2R zwitterionic isomers did not cause anyobservable irritation reactions, such as eye-closing, lacrimation,mucous discharge as well as change in the intraocular pressureduring the topical applications; and unlike other conventionalanticholinergics, these 2R zwitterionic isomers did not inducedilation of the pupil in the contralateral (water-treated) eyes,indicating no or low systemic side-effects. Therefore, these softdrugs are safe, promising short acting anticholinergics with thepossibility of largely reduced unwanted side effects.

CONCLUSION

[0206] Isomers of N-substituted soft anticholinergics based onglycopyrrolate, the methyl and ethyl esters, and their zwitterionicmetabolite were synthesized and separated. Their pharmacologicalactivities were evaluated in vitro and in vivo. The receptorbinding (pK.sub.i) results indicate that stereo-specificity andstereo-selectivity are very important in these softanticholinergics. There were three chiral centers presented in thestructure of these compounds. The most significant improvement ofthe receptor binding activity was observed in 2R configuration,followed by 1'S. The activities of 3'R and 3'S could be affected bythe configurations of the other two chiral centers. The improvementof M.sub.3/M.sub.2 muscarinic-receptor subtype-selectivity wasfound most significant in 2R3'S configurations followed by 2R3'R.The configuration of chiral center 1' showed no effect onM.sub.3/M.sub.2 muscarinic-receptor subtype-selectivity. Comparableresults obtained from guinea pig ileum assays (pA.sub.2), andrabbit mydriasis test on zwitterionic isomers further confirmed thestereo-specificity of these anticholinergics. The pharmacologicalpotency of eight zwitterionic isomers was determined to be2R1'S3'S=2R1'S3'R=2R1'R3'S>2R1R3'R>2S1'S3R>2S1'S3'S=2S1R3'R&g-t;2S1R3'S (student t-test, p<0.05). When topically administered(0.1%) in rabbit eyes, some 2R-zwitterion isomers (2R1'S3'S,2R1'S3'R and 2R1R3'S) showed similar mydriatic potencies to that ofglycopyrrolate and tropicamide, however, their mydriatic effectswere of considerably shorter duration, and they did not inducedilation of the pupil in the contralateral, water-treated eyes,indicating that, in agreement with their soft nature, they arelocally active, but safe and have a low potential to cause systemicside effects. The usefulness and safety of theseglycopyrrolate-based soft anticholinergics have been thereforefurther proved.

Further Synthesis and Biological Testing

[0207] A series of pure stereoisomeric soft glycopyrrolateanalogues 3, 4 and 5 below was synthesized by using chiralintermediates and by careful separation of the stereoisomers formedduring the last quaternization step of the synthesis. Thestereochemistry of the products was elucidated by using various 1Dand 2D NMR techniques. Anticholinergic activity of the newcompounds was determined by receptor binding studies and further byperforming tests on isolated organs and by in vivo tests. Receptorbinding revealed that in the higher alkyl ester series the(2R,1'R,3'R) and the (2R,1'S,3'S) isomers were the compoundsshowing the highest receptor affinity; furthermore, it demonstratedthe confines of the length of the alkyl chain. In vitro isolatedorgan experiments correlated well with the receptor bindingresults, and in vivo investigations indicated the soft character ofthe compounds.

[0208] One of the most effective anticholinergic compounds isglycopyrrolate[3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1,1-dimethylpyrrolidiniumbromide, 1 below] containing a quaternary N-atom the charge ofwhich prevents its crossing through lipid membranes and therefore,compared to e.g atropine, glycopyrrolate has reduced CNS-relatedside effects. The molecule contains two chiral centers: at position2 of the acyl group and at position 3' of the pyrrolidinyloxymoiety and hence this compound can exist in the form of the fourstereoisomers (2R,3'R), (2R,3'S), (2S,3'R), (2S,3'S). The marketeddrug is a mixture of stereoisomers while the (2R,3'R) form is knownas Ritropirronium bromide.

##STR00014##

[0209] Soft analogues of glycopyrrolate such as compounds offormulas 2, 3 and 6 below containing three chiral centers (inaddition to the two centers in the parent molecule the quaternarynitrogen bearing two different substituents is also asymmetric)have also been described above and have been shown to possess theexpected soft character. This has been reflected by the relativelyshort duration of action and low systemic side effects. Inaddition, it has been shown above that enzymatic hydrolysis of theesters 2 and 3 yielded the corresponding zwitterionic acid 6 whichis much less active in rats and is rapidly eliminated. Theinitially prepared soft analogues were either mixtures of allpossible eight stereoisomers or mixtures of four stereoisomerscontaining the (R)-form of the cyclopentylmandeloyl unit while theremaining two chiral centers were in racemic forms.

Soft Analogues of Glycopyrrolate

TABLE-US-00007 ##STR00015## [0210] R 2 methyl 3 ethyl 4 n-hexyl 5n-octyl 6 H

[0211] Considering the favorable biological test results ofcompounds 2 and 3 the question emerged whether any of the purestereoisomers could have any advantage over the others or not.Therefore, the aim of the present work was to synthesize and testpure stereoisomers of the soft glycopyrrolate analogues 3, 4 and 5and at the same time to study the influence of higher alkyl groupsas R upon the extent and time course of anticholinergic activity.Thus, the primary target molecules were the pure stereoisomers ofthe hexyl esters 4 and octyl esters 5 with the proviso that theconfiguration of C-2 in the cyclopentylmandeloyl unit was fixed as(R) since literature data showed (R)-cyclopentylmandeloylderivatives to be more active anticholinergics than their(S)-counterparts. This meant that only two chiral centers (N-1' andC-3' in the pyrrolidinyloxy moiety) and consequently only fourstereoisomers had to be taken into consideration. In addition, forcomparison, analogous pure stereoisomers of the ethyl esters 3 werealso synthesized.

Synthesis of the Pure Stereoisomers

[0212] The target compounds were prepared by quaternization of thekey intermediates 10 (see Scheme 4) with the bromoacetates 11, 12and 13, respectively, wherein 10 was used either as a mixture ofthe (2R,3'S) and (2R,3'R) diastereomers (Method A) or as theindividual diastereomers (Method B).

[0213] The diastereomeric mixture of compound 10 was prepared firstby the known transesterification of (R)-methyl cyclopentylmandelate(8) with racemic 1-methyl-3-pyrrolidinol [(R,S)-(9)]. Later it wasfound that much higher yield could be reached by direct coupling of(R)-cyclopentylmandelic acid (7) with (R,S)-(9) under Mitsunobuconditions. On the other hand, the individual diastereomers of 10were obtained via two different routes. Thus, transesterificationof (R)-methyl cyclopentylmandelate (8) with(S)-1-methyl-3-pyrrolidinol [(S)-(9)] as above proceeded withretention of configuration at C-3' and yielded (2R,3'S)-10. On theother hand, direct coupling of (R)-cyclopentylmandelic acid (7)with the same (S)-(9) under Mitsunobu conditions (with inversion ofconfiguration at C-3') led to (2R,3'R)-10.

##STR00016##

[0214] Next, in Method A, quaternization of the mixture of(2R,3'S)-10 and (2R,3'R)-10 with the bromoacetates 12 and 13,respectively, giving rise to the formation of a new chiral centerat N-1', led to mixtures of the four stereoisomeric targetcompounds 4a-d and 5a-d, respectively. The individual stereoisomerscould be separated only partially by a combination of variouschromatographic and crystallization methods (see Experimentalbelow).

[0215] Isolation of the pure stereoisomers was simpler in the otherapproach: in Method B, quaternization of (2R,3'S)-10 with thebromoacetates 11 and 12, respectively, as above afforded only twostereoisomers, i.e. the (2R,1'R,3'S) and the (2R,1'S,3'S) versionsboth of compounds 3 and 4, respectively, and separation of thesecomponents was achieved with less difficulty. On the other hand, infull analogy with the abovesaid, the final quaternization startingwith (2R,3'R)-10 resulted in the formation of the other pair ofisomers, i.e. the (2R,1'R,3'R) and the (2R,1'S,3'R) versions of thefinal products 3 and 4, respectively. Finally, from among thetwelve target compounds (3a-d, 4a-d and 5a-d) two pairs ofcompounds, i.e. 3a+3b and also further 5a+5d were obtained asinseparable mixtures while all other stereoisomers could beisolated in pure state. Note that 3a-d are also referred to hereinas Compounds (k), (l), (m) and (n), respectively; 4a-d are alsoreferred to herein as Compounds (o), (p), (q) and (r),respectively; and 5a-d are also referred to herein as Compounds(s), (t), (u) and (v), respectively.

Structure Elucidation and Assignment of Stereochemistry

[0216] The structures and stereochemistry of the new targetcompounds were elucidated by detailed NMR studies and the purity ofthe samples was confirmed by HPLC. The assignment of the individualstereoisomers is illustrated below using the example of the fourisomeric hexyl esters 4a-d. A complete .sup.1H and .sup.13C NMRsignal assignment was achieved by applying .sup.1H, .sup.13C, DEPT,and two-dimensional .sup.1H, .sup.1H-COSY, .sup.1H, .sup.1H-TOCSYand .sup.1H, .sup.13C-HSQC correlation experiments. Thecharacteristic .sup.1H and .sup.13C chemicals shifts are compiledin Table 7. Due to the high similarity of the chemical shifts ofthe isomers 4a-d, a simple differentation of the structures was notpossible, only the .sup.+NCH.sub.3 and .sup.+NCH.sub.2COOR chemicalshifts exhibited characteristic differences. To reveal thestereochemistry, one-dimensional selective NOESY, two-dimensionalROESY and NOESY spectra were run, affording evidences ofinterprotonic distances less than 5 .ANG.. The double arrows inScheme 5 denote the detected relevant NOE .sup.1H/.sup.1H stericproximities.

TABLE-US-00008 TABLE 7 Characteristic .sup.1H and .sup.13C chemicalshifts of isomers 4a-d in CDCl.sub.3. 4a [(o)] 4b [(p)] 4c [(q)] 4d[(r)] 2R, 1'R, 3'S 2R, 1'S, 3'S 2R, 1'R, 3'R 2R, 1'S, 3'R .sup.1H.sup.13C .sup.1H .sup.13C .sup.1H .sup.13C .sup.1H .sup.13C 1 --174.5 -- 174.6 -- 174.6 -- 174.5 2 -- 79.8 -- 79.7 -- 79.4 -- 79.82'.sub.cis 4.36 70.3 4.16 68.9 3.91 70.0 4.20 70.5 2'.sub.trans4.46 4.68 4.55 4.38 3' 5.52 73.1 5.57 73.4 5.55 73.3 5.53 73.14'.sub.cis 2.06 31.5 1.96 30.1 2.24 29.6 2.25 30.1 4'.sub.trans2.79 2.93 3.05 2.88 5'.sub.cis 4.08 65.0 4.03 65.1 4.17 65.1 4.2065.3 5'.sub.trans 4.18 4.39 4.44 4.34 NCH.sub.3 cis -- 3.24 50.73.03 50.6 -- NCH.sub.3 trans 3.69 51.9 -- -- 3.69 51.9 NCH.sub.2cis 4.74; 62.8 -- -- 4.52; 62.9 4.86 4.69 NCH.sub.2 trans -- 5.16;63.8 5.08; 63.7 -- 5.20 5.19 Ph.sub.ipso 141.4 141.1 140.8 141.2Ph.sub.ortho 7.59 126.1 7.57 126.0 7.58 126.0 7.59 126.0Ph.sub.meta 7.34 128.5 7.36 128.6 7.37 128.6 7.36 128.6 Ph.sub.para7.27 128.2 7.30 128.2 7.27 128.0 7.27 128.0 HC--C-2 2.87 46.8 2.9646.1 2.93 45.6 2.89 46.8

##STR00017##

[0217] Selective irradiation of the .sup.+NCH.sub.3 signal in 4bresulted in NOE intensity enhancement at the H.sub.cis-4' and atthe ortho hydrogen signals, which unambiguously proved the 1'Sconfiguration and at the same time, the depicted preferredconformation of the O-acyl moiety. Irradiation of the NCH.sub.3signal in 4a marked out only the hydrogen atom H.sub.trans-2',located on the same side of the pyrrolidine ring. In the case ofcompound 4d, the appearance of a strong .sup.+NCH.sub.3/H-3' crosspeak in the ROESY spectrum gave evidence of the 1'S configuration,whereas the H-3'/H.sub.ortho response revealed the conformation ofthe O-acyl group.

[0218] In compound 4c due to the unfavourable signal overlapping(e.g. .sup.+NCH.sub.3 and H-4'.sub.trans), the two-dimensionalmeasurement does not work. Here, the one-dimensional selectiveNOESY was utilized again. Irradiating the H.sub.ortho hydrogenatoms a small, but significant NOE was observed at the.sup.+NCH.sub.3 signal, which is in accordance with the depictedconfiguration and conformation.

[0219] Due to the pseudorotation of the pyrrolidine ring and thehigh flexibility of the compounds 4a-d, conformational averagedstructures are expected. Despite this, the anomalous upheld shiftof the .sup.+NH.sub.3 signals (3.24 and 3.03 ppm) can be explainedby the well known anisotropic shielding effect of the aromaticring, wherein the hydrogen atoms located above the plane of thearomatic ring show smaller chemical shifts. The smaller chemicalshifts of the NCH.sub.2 (cis) hydrogens in 4a (4.74; 4.86) and 4d(4.52; 4.69) are in accord with the relative steric arrangement.Preference of the conformations of compounds 4a-d, where thearomatic ring is oriented towards the nitrogen atom, is in accordwith the stabilization of the positive charge on the nitrogen bythe .pi.-system of the phenyl group.

Biology: Evaluation of the Anticholinergic Activity

Receptor Binding

[0220] Evaluation of the affinity of the soft glycopyrrolateanalogues 3a-d, 4a-d and 5a-d above for muscarinic receptors wascarried out using [.sup.3H]QNB as ligand and rat cortical membranepreparation as a source of the receptor. The affinity (summarizedin Table 8) of these compounds for the muscarinic receptors (mainlyM.sub.1 in this preparation) was found one or two orders ofmagnitude lower than those of the reference compoundsglycopyrrolate (Ki=0.8 nM) and atropine (Ki=1.9 nM) but the instantcompounds were still strong antagonists of the muscarinic receptor.The nature of the interaction was characterized by the steep Hillslope, the value of which was close to unity indicating theantagonistic action. The difference between the effects of the purestereoisomers was seen most clearly within the hexyl series as inthis case all the four possible stereoisomers were isolated in purestate. The compounds 4b (2R,1'S,3'S) and 4c (2R,1'R,3'R) wereequally, approximately four-fold, more active than 4a (2R,1'R,3'S)or 4d (2R,1'S,3'R). The same tendency was clear in case of the lessactive octyl series (5b, 5c), even though the other two isomers(5a+5d) were tested as a mixture. The above (2R,1'S,3'S) and(2R,1'R,3'R) compounds contain the larger quaternizing group(CH.sub.2COOR) in trans position to the cyclopentylmandeloyloxymoiety and this fact suggests that the sterically less crowdednature of these isomers may contribute to the higher receptoraffinity, in contrast with the sterically more crowded cisisomers.

TABLE-US-00009 TABLE 8 Receptor binding strength of theglycopyrrolate analogues Compound Ki (nM), Average .+-. SD Hillslope 3a + 3b [(k) + (l)] 65 .+-. 8 -0.96 .+-. 0.09 3c [(m)] 16.+-. 1 -0.93 .+-. 0.08 3d [(n)] 16 .+-. 1 -1.07 .+-. 0.02 4a [(o)]67 .+-. 9 -1.22 .+-. 0.08 4b [(p)] 13 .+-. 1 -1.19 .+-. 0.05 4c[(q)] 15 .+-. 1 -1.24 .+-. 0.04 4d [(r)] 58 .+-. 5 -1.15 .+-. 0.055a + 5d [(s) + (v)] 303 .+-. 7 -1.20 .+-. 0.08 5b [(t)] 60 .+-. 5-1.22 .+-. 0.11 5c [(u)] 68 .+-. 6 -1.26 .+-. 0.06

[0221] On the other hand, in the ethyl ester series the isomers 3c(2R,1'R,3'R) and 3d (2R,1'S,3'R), i.e. the compounds wherein thecyclopentylmandeloyloxy moiety is attached to the pyrrolidine ringin the .alpha.-position, have higher affinity indicating that inthis case the steric position of the smaller CH.sub.2COOEt grouphas less influence upon receptor affinity.

[0222] The effect of the length of the alkyl chain in the estergroup upon the receptor binding seemed to be negligible up to 6carbon moiety but when the longer chain was used this alreadyaffected receptor binding (compare the whole hexyl and octylseries).

Ex Vivo Experiments with Isolated Organs

[0223] Determination of the pA.sub.2 values in guinea pig tracheaand ileum assay resulted in the expected results. In line with thereceptor binding experiments in both of the isolated organpreparations, atropine and glycopyrrolate were more active (Table9) than the instant selected glycopyrrolate analogues chosen torepresent compounds with markedly different receptor affinities.The ethyl and hexyl side chain containing compounds (3c and 4b)were practically equally effective while the octyl chain containingesters showed weaker activity.

TABLE-US-00010 TABLE 9 pA.sub.2 values in two types of isolatedorgan experiments* Trachea Ileum Antagonist pA.sub.2 Slope .+-.S.E. pA.sub.2 Slope .+-. S.E. Atropine 8.85 0.98 .+-. 0.02.sup.a8.52 0.96 .+-. 0.30.sup.a Glycopyrrolate 9.43 1.53 .+-. 0.07.sup.a9.66 1.03 .+-. 0.37.sup.a 3c [(m)] 8.12 1.54 .+-. 0.18.sup.a 8.480.76 .+-. 0.08.sup.a 4b [(p)] 8.21 1.14 .+-. 0.10.sup.a 8.14 1.31.+-. 0.67.sup.a 5a + 5d [(s) + (v)] 7.23 0.79 .+-. 0.07.sup. 6.641.03 .+-. 0.37.sup.a *data are presented of mean estimates intissue samples from four animals .sup.adeviation from unity is notsignificant (P > 0.05)

In Vivo Experiments

Carbachol Induced Bradycardia in the Rat

[0224] The bradycardia protective effect of the selected newcompounds was comparable both to their receptor binding affinityand their activity in the isolated organ experiments. In line withthis, their in vivo activity (FIG. 6) was lower than that ofglycopyrrolate (GP) but more importantly their duration of actionwas notably shorter than that of the parent compound, indicatingtheir potential soft character.

Experimental

Chemistry

[0225] Melting points were determined on a Boetius microscope andare uncorrected. Purity of the compounds was tested on TLC plates(silica gel, Merck). The spots were visualized under UV lightand/or by exposure to iodine vapours. NMR spectra were recorded inCDCl.sub.3, DMSO-d.sub.6 or CD.sub.3OD solutions using a BrukerAvance 500 spectrometer, operating at 500/125 MHz(.sup.1H/.sup.13C). Chemical shifts are given on the .delta.-scaleand were referenced to TMS. Pulse programs for the 1D and 2D NMRexperiments were taken from the Bruker software library. Forstructure elucidation and NMR signal assignment .sup.1H, .sup.13C,DEPT-135, selective 1D-NOESY, .sup.1H, .sup.1H-COSY, .sup.1H,.sup.1H-TOCSY, .sup.1H, .sup.13C-HSQC, .sup.1H, .sup.13C-HMBC,.sup.1H, .sup.1H-ROESY and .sup.1H, .sup.1H-NOESY spectra wererecorded.

[0226] Analytical HPLC of compounds 3, 4 and 5 was performed usinga Waters (Milford, Mass.) HPLC system consisting of a model 510isocratic pump working at 1 ml/min flow rate, a WISP programmableautoinjector with 10 .mu.l injection volume and a model 486 singlechannel variable wavelength UV detector with 220 nm presetwavelength. The applied HPLC stationary phase was a Prontosil 120C18 AQ 5 .mu.m column with 150*4 mm geometry. Column temperature:40.degree. C. The optimal mobile phase was a mixture of 30 mMammonium acetate/MeOH/acetonitrile, in a ratio of 55/17.5/27.5(v/v/v) for compounds 3, in a ratio of 34/16/55 (v/v/v) forcompounds 4 and in a ratio of 18/20/60 (v/v/v) for compounds 5. Theenantiomeric purity of 2-cyclopentylmandelic acid (7) wasdetermined by chiral ligand exchange chromatography on a NucleosilChiral-1 5 .mu.m, 250*4 mm chiral HPLC column. The mobile phase was0.5 mM CuSO.sub.4/acetonitrile 97/3 (v/v), flow rate: 1 ml/min,column temperature: 60.degree. C., detection wavelength: 220 nm.The retention time of the individual enantiomers was 13.5 min (R)and 15.5 min (S), respectively. The observed selectivity was1.18.

[0227] (R)-2-Cyclopentylmandelic acid [(R)-(7)] was obtained byresolution of the racemic acid with (-)-cinchonidine, (R)-methyl2-cyclopentylmandelate [(R)-(8)] was prepared as described in theart while (R,S)- and (S)-1-methyl-3-pyrrolidinol, respectively[(R,S)- and (S)-(9), respectively], were synthesized in two stepsstarting with (R,S)- and (S)-malic acid, respectively, aspreviously described. Ethyl bromoacetate (11) was purchased fromAldrich while the homologous n-hexyl (12) and n-octyl bromoacetates(13) were prepared by reaction of the corresponding alcohol withbromoacetyl bromide. Found values of elemental analyses agreed withcalculated vaues within the range of .+-.1%.

Preparation of the Quaternized Target Compounds 3, 4 and 5

Method A

[0228] A mixture of (2R,3'R)-10 and (2R,3'S)-10 (1.0 mM), togetherwith the alkylating agent 12 or 13 (2.0 mM) in acetonitrile (12 ml)was stirred for 2 hours at room temperature. After completion ofthe reaction, the solvent was evaporated and the products wereisolated as given below in the description of the individualcompounds.

Method B

[0229] A mixture of (2R,3'R)-10 or (2R,3'S)-10 (0.3 mM) and thealkylating agent 11 or 12 (0.6 mM) in acetonitrile (5 ml) wasallowed to react and the crude product was isolated as describedunder Method A above.

(2R,1'R,3'S)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide (3a) [Compound (k)] and (2R,1'S,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonyloxymethyl)-1--methylpyrrolidinium bromide (3b) [Compound (l)]

[0230] Method B above was followed starting with (2R,3'S)-10. Thecrude product was purified by silica gel chromatography elutingwith chloroform-methanol 9:1 and 3a and 3b were isolated as aninseparable mixture. Yield: 54%, mp. 163.degree. C., ratio 3a/3b(.sup.1H-NMR): 4:1.

[0231] 3a .sup.1H NMR (CDCl.sub.3) .delta. 3.68 (3H, s, NCH.sub.3),4.78 (1H, d, NCH.sub.2), 4.89 (1H, d, NCH.sub.2), 5.55 (1H, m,H-3'); 3b .sup.1H NMR (CDCl.sub.3) .delta. 3.27 (3H, s, NCH.sub.3),5.26 (1H, d, NCH.sub.2), 5.30 (1H, d, NCH.sub.2), 5.51 (1H, m,H-3').

(2R,1'R,3'R)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide (3c) [Compound (m)] and (2R,1'S,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(ethoxycarbonylmethyl)-1-me-thylpyrrolidinium bromide (3d) [Compound (n)]

[0232] Method B above was followed starting with (2R,3'R)-10 andthe products 3c and 3d were separated by silica gel chromatographyof the crude product eluting with chloroform-methanol 9:1.

[0233] 3c: yield: 19%, mp. 98.degree. C., purity (HPLC): 93%.

[0234] .sup.1H NMR (CDCl.sub.3) .delta. 1.29 (3H, t,CH.sub.3CH.sub.2O), 2.24 (1H, m, H.sub.c-4'), 2.94 (1H, m,HC--C-2), 3.02 (3H, s, NCH.sub.3), 3.07 (1H, m, H.sub.t-4'), 3.88(1H, m, H.sub.c-2'), 4.13 (1H, m, H.sub.c-5'), 4.23 (2H, q,CH.sub.3CH.sub.2O), 4.46 (1H, m, H.sub.t-5'), 4.56 (1H, m,H.sub.t-2'), 5.10 (1H, d, NCH.sub.2), 5.22 (1H, d, NCH.sub.2), 5.56(1H, m, H-3'), 7.28 (1H, t, Ph.sub.p), 7.37 (2H, t, Ph.sub.m), 7.58(2H, d, Ph.sub.o).

[0235] 3d: yield: 28%, mp. 70.degree. C., purity (HPLC): 96%.

[0236] .sup.1H NMR (CDCl.sub.3) .delta. 1.33 (3H, t,CH.sub.3CH.sub.2O), 2.24 (1H, m, H.sub.c-4'), 3.66 (3H, s,NCH.sub.3), 4.65 (1H, d, NCH.sub.2), 4.74 (1H, d, NCH.sub.2), 5.54(1H, m, H-3'), 7.27 (1H, t, Ph.sub.p), 7.35 (2H, t, Ph.sub.m), 7.59(2H, d, Ph.sub.o).

(2R,1'R,3'S)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(n-hexyloxycarbonylmethyl)--1-methylpyrrolidinium bromide (4a) [Compound (o)] and (2R,1'S,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(n-hexyloxycarbonylmethyl)--1-methylpyrrolidinium bromide (4b) [Compound (p)]

[0237] Method B above was followed starting with (2R,3'S)-10 andthe products 4a and 4b were separated by silica gel chromatographyof the crude product eluting with chloroform-methanol 9:1.

[0238] 4a: yield: 18%, mp. 146.degree. C., purity (HPLC): 93%.

[0239] 4b: yield: 23%, mp. 125-128.degree. C., purity (HPLC):96%.

For NMR data of 4a-b see above.

(2R,1'R,3'R)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(n-hexyloxycarbonylmethyl)--1-methylpyrrolidinium bromide (4c) [Compound (q)] and (2R,1'S,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(n-hexyloxycarbonylmethyl)--1-methylpyrrolidinium bromide (4d) [Compound (r)]

[0240] Method B above was followed starting with (2R,3'R)-10 andthe products 4c and 4d were separated by silica gel chromatographyof the crude product eluting with chloroform-methanol 9:1.

[0241] 4c: yield: 20%, mp. 138.degree. C., purity (HPLC): 98%.

[0242] 4d: yield: 55%, mp. 116.degree. C., purity (HPLC): 95%.

For NMR data of 4c-d see above.

[0243] As an alternative, upon preparing compounds 4a-d byfollowing Method A above, the products 4b and 4c could be isolatedin pure state as described below while 4a and 4d were obtained inthe form of an inseparable mixture. Thus, the crude product wastriturated with ethyl acetate to give the mixture 4a+4d [Compounds(O) and (r)] as a solid, yield: 42%. The mother liquor wasconcentrated to dryness and the residue was purified by columnchromatography on silica gel eluting with chloroform-methanol 9:1.Subsequently compounds 4b (yield: 12%) [Compound (p)]; and 4c(yield: 6%) [Compound (q)] were separated by preparative thin layerchromatography developing with chloroform-methanol 9:1.

(2R,1'R,3'S)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(n-octyloxycarbonylmethyl)--1-methylpyrrolidinium bromide (5a) [Compound (s)], (2R,1'S,3'S)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(n-octyloxycarbonylmethyl)--1-methylpyrrolidinium bromide (5b) [Compound (t)], (2R,1'R,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(n-octyloxycarbonylmethyl)--1-methylpyrrolidinium bromide (5c) [Compound (u)] and (2R,1'S,3'R)3-(2-cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-(n-octyloxycarbonylmethyl)--1-methylpyrrolidinium bromide (5d) [Compound (v)]

[0244] By following Method A above and purification of the crudeproduct by silica gel column chromatography eluting withchloroform-methanol 9:1 the compounds 5a and 5d [Compound (s) and(v)] were obtained in the form of a mixture inseparable by TLC andHPLC. On the other hand, 5b [Compound (t)] and 5c [Compound (u)]could be separated by a final preparative thin layerchromatography, developing with chloroform-methanol 9:1.

[0245] Mixture of 5a and 5d: yield: 47%, ratio 5a/5d (.sup.1H-NMR):1:1.

[0246] 5a .sup.1H NMR (CDCl.sub.3) .delta. 2.10 (1H, m,H.sub.c-4'), 3.64 or 3.67 (3H, s, NCH.sub.3), 4.71 (1H, d,NCH.sub.2), 4.81 (1H, d, NCH.sub.2); 5d .sup.1H NMR (CDCl.sub.3).delta. 2.28 (1H, m, H.sub.c-4'), 3.64 or 3.67 (3H, s, NCH.sub.3),4.54 (1H, d, NCH.sub.2), 4.67 (1H, d, NCH.sub.2).

[0247] 5b: yield: 10%, mp. 30.degree. C., purity (HPLC): 86%.

[0248] .sup.1H NMR (CDCl.sub.3) .delta. 0.90 (3H, t,CH.sub.3CH.sub.2), 2.00 (1H, m, H.sub.c-4'), 2.93 (1H, m, HC--C-2and 1H, m, H.sub.t-4'), 3.27 (3H, s, NCH.sub.3), 4.10 (1H, m,H.sub.c-5'), 4.18 (2H, t, CH.sub.3CH.sub.2), 4.18 (1H, m,H.sub.c-2'), 4.29 (1H, m, H.sub.t-5'), 4.58 (1H, m, H.sub.t-2'),5.16 (2H, s, br, NCH.sub.2), 5.56 (1H, m, H-3'), 7.27 (1H, t,Ph.sub.p), 7.35 (2H, t, Ph.sub.m), 7.57 (2H, d, Ph.sub.o).

[0249] 5c: yield: 7.5%, purity (HPLC): 93%.

[0250] 5c .sup.1H NMR (CDCl.sub.3) .delta. 0.89 (3H, t,CH.sub.3CH.sub.2), 2.28 (1H, m, H.sub.c-4'), 2.93 (1H, m, HC--C-2),2.53 (3H, s, NCH.sub.3), 3.05 (1H, m, H.sub.t-4'), 3.86 (1H, m,H.sub.c-2'), 4.12 (1H, m, H.sub.c-5') 4.16 (2H, t,CH.sub.3CH.sub.2), 4.30 (1H, m, H.sub.t-5'), 4.50 (1H, m,H.sub.t-2'), 4.97 (1H, d, NCH.sub.2), 5.10 (1H, d, NCH.sub.2), 5.58(1H, m, H-3'), 7.27 (1H, t, Ph.sub.p), 7.38 (2H, t, Ph.sub.m), 7.59(2H, d, Ph.sub.o).

Mixture of (2R,3'R) and (2R,3'S)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-methylpyrrolidine[(2R,3'R)-10 and (2R,3'S)-10]

[0251] A solution of diisopropyl azodicarboxylate (1.5 mM) intetrahydrofuran (1 ml) was added dropwise to a mixture of (R)-7(1.5 mM), (R,S)-9 (1.64 mM) and triphenylphosphine (1.5 mM) intetrahydrofuran (4 ml) at room temperature. The reaction mixturewas stirred at room temperature for 2 hours and the solvent wasremoved in vacuo. The residue was suspended in ethyl acetate andextracted with 1N hydrochloric acid. The aqueous solution was madealkaline with 5N aqueous sodium hydroxide, followed by extractionwith ether. The organic layer was dried over magnesium sulfate andconcentrated in vacuo giving the title compound as a colorless oil.Yield: 87%, purity (HPLC): 97% (total area of two unresolvedpeaks). 1:1 mixture of (2R,3'R)-10 and (2R,3'S)-10:

[0252] .sup.1H NMR (CDCl.sub.3) .delta. 2.35 and 2.39 (3H, s,NCH.sub.3), 2.56 and 2.68 (1H, m, H.sub.c-2'), 2.94 (1H, m,HC--C-2), 3.75 (1H, s, HO--C-2), 5.24 (1H, m, H-3'), 7.27 (1H, t,Ph.sub.p), 7.36 (2H, t, Ph.sub.m), 7.67 (2H, d, Ph.sub.o).

(2R,3'S)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-methylpyaolidine(2R,3'S)-10

[0253] The methyl ester (R)-(8) was submitted totransesterification with (S)-9 by following the known method ofFranko and Lunsford. Yield: 28%, purity (HPLC): 97%.

[0254] .sup.1H NMR (CDCl.sub.3) .delta. 2.39 (3H, s, NCH.sub.3),2.68 (1H, m, H.sub.c-2'), 2.94 (1H, m, HC--C-2), 3.79 (1H, s, br,HO--C-2), 5.24 (1H, m, H-3'), 7.27 (1H, t, Ph.sub.p), 7.35 (2H, t,Ph.sub.m), 7.67 (2H, d, Ph.sub.o).

(2R,3'R)3-(2-Cyclopentyl-2-phenyl-2-hydroxyacetoxy)-1-methylpyrrolidine(2R,3'R)-10

[0255] (R)-(7) was coupled with (S)-9 under Mitsunobu conditions asdescribed above under 3.1.1.7. Yield: 49%, purity (HPLC): 95%.

[0256] .sup.1H NMR (CDCl.sub.3) .delta. 2.34 (3H, s, NCH.sub.3),2.93 (1H, m, HC--C-2), 3.78 (1H, m, HO--C-2), 5.23 (1H, m, H-3'),7.27 (1H, t, Ph.sub.p), 7.34 (2H, t, Ph.sub.m), 7.67 (2H, d,Ph.sub.o).

Biology: Test Methods

Receptor Binding Assay

[0257] The binding of [.sup.3H]quinuclidinyl benzylate([.sup.3H]QNB; Amersham, 42.0 Ci/mmol) to muscarinic receptors wasmeasured according to the method of Yamamura and Snyder (1974),with some modifications, as described previously by Barlocco et al.(1997). Briefly, male Sprague-Dawley rats (180-220 g) weredecapitated and cerebral cortices removed, discarding the whitematter. Pooled tissue was homogenized in 10 volumes of ice-cold 50mM Tris-HCl buffer (pH 7.4) by using a motor driven glasshomogenizer. The homogenate was centrifuged at 4.degree. C. and30,000 g for 10 min. The pellet was washed twice with the samebuffer by resuspension, followed by centrifugation at 4.degree. C.and 30,000 g for 10 min. The final pellet was resuspended in 10volumes of Tris-HCl buffer and stored at -20.degree. C. before use.Protein content was determined by the method of Bradford (1976)using bovine serum albumin as the standard.

[0258] In all binding experiments, membranes (0.25 mg/ml protein),radioligand and competing drugs were incubated in a final volume of1 ml of 50 mM Tris-HCl buffer (pH 7.4) for 60 min at 25.degree. C.For saturation studies, membranes were incubated with 0.01-2 nM[.sup.3H]QNB. In competition experiments, the final concentrationof the radioligand was 0.2 nM and competing drugs were given in 8concentrations. Non-specific binding was determined with 1 .mu.Matropine. The incubation was terminated by rapid vacuum filtrationover Whatman GF/B filters using a Brandel Cell Harvester. Sampleswere washed immediately with 3.times.4 ml ice-cold Tris-HCl bufferand placed in 6 ml scintillation fluid. Radioactivity was estimatedby liquid scintillation counting.

[0259] Data are the mean.+-.S.E.M. of at least three experimentsrun in duplicate. GraphPad Prism 3.0 (GraphPad Software Inc., SanDiego, Calif., USA) was used to perform linear and non-linearregression analysis of the data. Saturation binding parameters(K.sub.d and B.sub.max) were determined by linear regressionanalysis of the transformed saturation binding data. Competitionbinding isotherms were analyzed by non-linear regression to deriveestimates of the IC.sub.50 values and Hills slopes. IC.sub.50values were converted to K.sub.i values according to the equationof Cheng and Prusoff (1973).

Guinea Pig Trachea and Ileum Assay (pA.sub.2 ValueDetermination)

[0260] Tracheal preparations were made as described previously indetails by Preuss and Goldie (1999). Briefly: tracheas wereisolated from male Dunkin-Hartley guinea pigs (280-300 g) and thering preparations (2-3 mm in width) were suspended under 500 mgresting tension in an organ bath containing Krebs' bicarbonatebuffer aerated with 95% O.sub.2/5% CO.sub.2. Changes in isometrictension were measured by a force displacement transducer(Experimetria, Budapest, Hungary) coupled to a Watanabe recorder.Cumulative concentration-effect curves were constructed tocarbachol in the absence or presence of the antagonists. In eachanimal two preparations were used as time control (i.e., repeatedcarbachol curves in the absence of any antagonist) the remainingtwo preparations were used to test responses in the presence of twodifferent concentrations of the antagonist.

[0261] Antagonists were added to the organ bath 30 min prior tocommencement of the agonist concentration versus effect curves.Schild plots were constructed for the antagonists against carbacholand pA.sub.2 values as well as slope estimates were obtained.

[0262] The ileum longitudinal muscle strips with adhering myentericplexus were also prepared from male guinea-pigs (200-400 g). Asegment of small intestine (8-10 cm) 10 cm proximal to theileo-coecal valve was dissected. The longitudinal muscle strip wasobtained by mounting segments of the whole ilea on a 1 ml pipetteand gently tearing away the outer longitudinal muscle layer with acotton swab. Longitudinal muscle strips were cut into 3-4 cmpieces. The strips were mounted in an organ bath containing Tyrodesolution at 37.degree. C. under a resting tension of 500 mg.Contractions were recorded isometrically with the same strain gaugesystem as above and registered on the Watanabe type polygraph. Thetissues were left to equilibrate for 30 min. Dose-response curvesto the agonist were constructed by addition of acetylcholine inincreasing concentrations. The doses were given at 10 min intervalswith 1 min contact time. After a 40 min equilibration period, thepreparations were incubated with the antagonist for 20 min, and asecond concentration-response curve to acetylcholine wasconstructed. The agonist (Ach) was non-cumulatively added at 10 minintervals (concentration range: 10.sup.-10 to 10.sup.-5 M).Antagonists were applied in the concentration range of 10.sup.-8 to10.sup.-5 M, depending on the individual test compound.

[0263] Responses were measured as changes in isometric tension andcalculated as a percentage of the maximum response attained in theinitial concentration-response curve. Determination of antagonistpotencies was done by constructing Schild double logarithmic plotof log (DR-1) versus -log M concentrations of the antagonist, andthe slope of the plot was computed. If the slopes of the plots werenot significantly different from unity, the interaction wasaccepted as competitive in nature, and antagonist potencies wereexpressed as pA.sub.2 values (Arunlakshana & Schild, 1959). Ifthe slopes of the plots were significantly different from unity,the method of Ariens & Van Rossum (1957) was used to determinepD' values for characterization of non-competitive antagonism.Statistical significance was assessed by ANOVA followed by Dunnetttest.

Antagonistic Effect on Carbachol Induced Bracycardia

[0264] The experimental procedure described previously in detail byJuhasz et al. (1998) was followed. Male Sprague-Dawley rats,weighing 300-350 g were anesthetized with sodium pentobarbital (50mg/kg i.p.). Baseline electrocardiography (ECG) recordings wereperformed after 15 min stabilization periods. Needle electrodeswere inserted subcutaneously into the limbs of the anesthetizedrats and were joined to a Watanabe recorder. Recording of the heartrate (1/min) was taken before, during and after the administrationof any of the compounds until basic ECG parameters returned tobaseline at a paper speed of 25 mm/sec. All drugs were administeredby direct injection into the jugular vein. Anticholinergic drugswere administered in the approximate pharmacodynamic equivalentdoses (0.2, 2.0 umol/kg) at 0 time, while carbachol (5-8 ug/kg) wasinjected at -5, 5, 10, 15, 20, 30, 45, 60 min.

Synthesis and Biological Testing of Tiotropium Derivatives

Structure of the Compounds

##STR00018##

[0265] b) Synthesis of a New Tiotropium Analog

[0266] By following the inactive metabolite approach, ametabolically sensitive ester function was introduced into thetiotropium molecule resulting in a new soft anticholinergic analog.Intermediate XIII was prepared by the known Grignard reaction ofdimethyl oxalate with 2-thienylmagnesium bromide (XII, see Scheme 6below). Compound XIII was then submitted to knowntransesterification with scopine (XIV) catalyzed by sodium metalgiving the corresponding ester XV. Finally, quaternization withethyl bromoacetate gave the target compound II (R=Et).

[0267] 2-Thienylmagnesium bromide was prepared in the usual mannerfrom magnesium and 2-bromothiophene in ether. Scopine (XIV) wasobtained from scopolamine hydrobromide by treatment with sodiumborohydride in ethanol in moderate yield as described in GB1,469,781 (1974).

##STR00019##

[0268] The above synthesis of the soft tiotropium bromide analogleads to a single isomer in a yield of 70%. Tlc indicated nofurther new component in the reaction mixture of the finalquaternization step and it could be shown by NMR (two dimensionalROESY technique) that the ethoxycarbonylmethyl group is inproximity to the oxirane ring. This finding is somewhat surprisingas the other isomer with N-methyl pointing toward the oxirane ringwould be sterically less crowded.

Stereochemistry of Compound (w) Obtained by Two-Dimensional RoesyExperiment:

##STR00020##

[0269] The numbers denote .sup.1H chemical shifts and couplingconstants, the double arrows indicate steric proximities.

Preparation of6.beta.,7.beta.-epoxy-3.beta.-hydroxy-8-ethoxycarbonylmethyl-8-methyl-1.a-lpha.H,5.alpha.H-tropanium bromide, di-2-thienylglycolate, Compound(w)

[0270] The scopine ester, represented by formula XV in Scheme 6above, was prepared as described above, or by conventional methodsas described in EP418716 (equivalent to U.S. Pat. No. 5,610,163).Then the scopine ester (70 mg, 0.18 ml) is dissolved in 2 ml ofacetone. Ethyl bromocetate (150 microliters, 0.45 mM) is added andthe mixture is allowed to react at 20.degree. C. for eight days.The solvent is evaporated in vacuo, 8 ml of water is added and theorganic material is extracted with chloroform. The desiredquaternary salt is in the aqueous phase and is obtained bylyophilization. Yield 70 mg (70%). Melting point: 115.degree. C.Thin layer chromotography on Al.sub.2O.sub.3: R.sub.f=0.3(CHCl.sub.3--CH.sub.3OH, 4:1) (3 times 4 ml). The product, Compound(w), has the structural formula II shown in Scheme 6 above.

Preparation of6.beta.,7.beta.-epoxy-3.beta.-hydroxy-8-methyl-8-(2,2,2-trichloroethoxyca-rbonylmethyl)-1.alpha.H,5.alpha.H-tropanium bromide,di-2-thienylglycolate, Compound (x)

[0271] To the scopine ester XV (0.5 mM) in 3 ml of anhydrousacetonitrile, 1.5 mM of trichloroethyl bromoacetate was added. Themixture was stirred under argon for three days and the acetonitrilewas removed under reduced pressure. To the oily residue, 15 ml ofwater was added and extracted with chloroform (3 times 5 ml). Theaqueous solution was lyophilized to give the product as a whitesolid. Yield: 257 mg (79%). Melting point 105.degree. C.,R.sub.f=0.65 (CHCl.sub.3--CH.sub.3OH, 4:1). The product has thestructural formula:

##STR00021##

Preparation of6.beta.,7.beta.-epoxy-3.beta.-hydroxy-8-carboxymethyl-8-methyl-1.alpha.H,-5.alpha.H-tropanuim, di-2-thienylglycolate inner salt, Compound(aa)

[0272] A suspension of 0.35 mM Compound (x) and Zn dust (0.6 mM) inacetic acid (1.5 ml) was stirred for 3 hours. To that mixture,water (2 ml) and chloroform (2 ml) were added and filtered. Thesolvents were evaporated in vacuo, 3 ml of water was added and thesolution was lyophilized. The crude product was dissolved inmethanol (2 ml) and purified by chromatography on Sephadex LH-20.The resulting oil was dissolved in methanol (2 ml) and precipitatedwith ethyl acetate (1 ml) to give the solid product, Compound (aa).Yield 67 mg (38%), melting point 158-165.degree. C. (decomp).R.sub.f=0.45 (CHCl.sub.3--CH.sub.3OH 4:1) on aluminum oxide. Theproduct has the structural formula:

##STR00022##

Pharmacology

1: Receptor Binding Assay

[0273] Evaluation of the affinity of the compound was made using[.sup.3H]QNB as ligand and rat cortical membrane preparation as asource of the receptor. The ethyl ester Compound (w) bound to themuscarinic receptors (mainly M.sub.1 in this preparation) with highaffinity (Table 10) although this affinity was several-fold lowerthan those of the reference compounds. The steep Hill slope closeto unity indicates the antagonistic nature of its action.

TABLE-US-00011 TABLE 10 Affinities of tiotropium ethyl esterderivative [Compound (w)] and reference compounds for muscarinicreceptors Compounds K.sub.i (nM) Hill Slope Number of exps.Atropine 1.9 .+-. 0.2 -1.10 .+-. 0.04 4 Glycopyrrolate 0.8 .+-.0.10 -1.07 .+-. 0.03 4 Compound (w) 7.2 .+-. 0.5 -1.00 .+-. 0.04 4The K.sub.i values are for inhibition of [.sup.3H]QNB binding torat brain cortex membranes. Values are the mean .+-. S.E.M. of atleast three experiments run in duplicate.

2: Experiments with Isolated Organs

[0274] In isolated organ experiments the measurement ofantimuscarinic effect of Compound (w) was carried out using guineapig tracheal ring preparations, where smooth muscle contraction ismediated primarily by muscarinic M.sub.3 cholinoceptors althoughactivation of M.sub.2-receptors also plays a role in the developingcontraction. Compound (w) showed excellent activity in this test;it was more active than atropine and only slightly less effectivethan ipratropium bromide (Table 11).

TABLE-US-00012 TABLE 11 Schild-plot analysis of the antagonismagainst carbachol in isolated tracheal rings of guinea pigs.Antagonist pA.sub.2 Slope .+-. S.E. atropine 8.85 0.98 .+-.0.02.sup.a ipratropium Br 9.18 1.11 .+-. 0.14.sup.a Compound (w)8.82 1.03 .+-. 0.08.sup.a Data are presented of mean estimates intissue from four animals pA.sub.2: the abscissa intercept of theSchild-plot drawn .sup.aIndicates slope estimates not significantlydifferent (P > 0.05) from unity.

3. Determination of the Antagonistic Effect of AnticholinergicAgents on Charbachol Induced Bradycardia in Anesthetized Rats

[0275] Intravenous administration of the cholinomimetic carbacholcauses sinus bradycardia (increasing the PP cycle and RR cyclelength of the ECG) in anesthetized rats. This effect, which ismediated mainly by muscarinic M.sub.2 receptors, can be preventedby prior administration of anticholinergic agent. The bradycardiaprotective effect of Compound (w) was compared to those of atropineand ipratropium bromide in this system. See FIG. 7. Compound (w)was less active than an equimolar dose of atropine, and wasslightly less active than a 10-fold lower dose of ipratropiumbromide indicating that Compound (w) may have lower affinity forM.sub.2 than M.sub.1 or M.sub.3 muscarinic receptors. See FIG.7.

Examination of the Time Course of the Anticholinergic Effect ofCompound (w) and Compound (aa) in Electrically Stimulated GuineaPig Trachea

Experimental Procedures:

[0276] The procedure described by Takahashi T. et al., (Am J RespirCrit. Care Med, 150:1640-1645, 1994) was used with slightmodifications.

[0277] Male Dunkin-Hartley guinea pigs (300-500 g) wereexterminated; the tracheas were rapidly removed, and placed inoxygenated normal Krebs buffer solution. The epithelium was removedand the trachea was spirally cut into 15 mm long strips. Two stripsfrom one animal were prepared and suspended between parallelstainless steel wire field electrodes in 10-ml organ bathscontaining buffer solution, which was continually gassed by a 95%O.sub.2 and 5% CO.sub.2 mixture. The tissues were allowed toequilibrate for 1 h with frequent washing, under a resting tensionof 1.0 g.

[0278] Indomethacin 10.sup.-5(M) was present throughout the studiesto block the formation of endogenous prostaglandins. Before theexperiment, capsaicin (10.sup.-5M) was added and washed out 30 minafter the pre-treatment to deplete endogenous tachykinins. Tissueswere also pretreated with propranolol (10.sup.-6M) 10 min beforethe experiment to inhibit the effects of endogenouscatecholamines

[0279] Isometric contractile responses were measured usingforce-displacement transducers (Experimetria, Hungary) connected toa Watanabe polygraph. A stimulator (CRS-ST-01-04, Experimetria)provided biphasic square-wave impulses with a supramaximal voltageof 40 V at source and 0.5 ms duration. Stimulations were applied ata frequency of 4 Hz for 15 sec followed by a 100 sec restinginterval. After at least four stable responses of equal magnitudewere obtained, the antagonist (submaximal dose) was introduced andwas left in the system until the maximal effect of the drug wasobserved. Thereafter the test drug was washed out. Furtherstimulations were delivered for at least 6 additional hours oruntil the responses returned to about 50% of the originalresponses. Appropriate time controls were run in parallel for allstudies.

Statistical Analysis

[0280] Contractile responses were expressed as the percentage ofthe own maximal contraction. The time for offset t.sub.1/5 ort.sub.1/2 of action was defined as the time from washout of thetest antagonist to attainment of 20 or 50% recovery of cholinergicresponses.

Results:

[0281] Typical tracings were obtained during the experiments.Continuous, stable, long-lasting contraction is achieved withelectrical stimulation. Upon the addition of anticholinergic agentsthe inhibition develops with varying speed, and the inhibitoryeffect of the compounds last for very different periods after thewashout. In FIG. 8, the time course of action of the differentanticholinergic compounds is shown; calculated results aresummarized in Table 12. In FIG. 9, the time course of theinhibition of the examined compounds are displayed; the resultscalculated from this data are summarized in Table 13.

[0282] The differences between the on and off rates of theCompounds (w) and (aa) are very notable.

TABLE-US-00013 TABLE 12 Time course of action of differentanticholinergics in electrically stimulated guinea pig trachealstrips Compound t.sub.1/5 onset (min) t.sub.1/2 onset (min)Atropine 2.5 4.6 Ipratropium Br 3.0 7.0 Tiotropium 8.0 12 Cpd (w)0.6 1.2 Cpd (aa) 0.9 2.0

TABLE-US-00014 TABLE 13 Time course of recovery from inhibitionafter washing out of different anticholinergics in electricallystimulated guinea pig tracheal strips Compound t.sub.1/5 offset(min) t.sub.1/2 offset (min) Atropine 10 31 Ipratropium Br 6.5 19Tiotropium >360 >360 Cpd (w) 60 130 Cpd (aa) 27 140

Investigation of the Anticholinergic Action of Compounds inAcetylcholine Induced Bronchoconstriction in Anesthetized GuineaPigs

Experimental Procedure

[0283] Male Hartley guinea pigs (320.+-.120 g) (Charles River) werehoused under standard conditions. Guinea pigs were anesthetizedwith urethane (2 g/kg, intraperitoneally), the trachea wascannulated and the animal was respired using a small animalrespiratory pump (Harvard Apparatus LTD, Kent UK). Respiratory backpressure was measured and recorded using a rodent lung functionrecording system (MUMED, London UK). For drug administration theright jugular vein was cannulated. Following the surgicalpreparation guinea pigs were allowed to stabilize for 20 minutes.Ten minutes before acetylcholine administration the animals weredisconnected from the ventilator and either the vehicle (10 mglactose) or different amounts of the drug (suspended in the sameamount of vehicle) were administered intratracheally. The tracheawas reconnected to the ventilator and changes in pulmonarymechanics were followed. Acetylcholine (10 .mu.g/kg) wasadministered intravenously in every 10 minutes six times.

Results

[0284] Compounds (q) and (m) of the invention and glycopyrrolateall exhibited a protective effect on the acetylcholine-inducedbronchoconstriction provoked in this test. Glycopyrrolate wasadministered at a dose of 0.01 mg/kg, while Compounds (q) and (m)were administered at a dose of 0.1 mg/kg. See FIG. 10.

[0285] This test is a model for asthma, chronic obstructivepulmonary disorder and other obstructive respiratory tractdisorders in which the effectiveness of the compounds of formulas(Ia) and (Ib) can be evaluated.

Test for Bronchodilatory Effect of Inhaled Test Compounds in Balb/cMice

[0286] Female BALB/c mice, weight range 19-22 g, are obtained, forexample from Charles River Laboratories (Kingston, N.C.). Theyreceive food and water ad libitum.

[0287] Compounds for aerosol administration are prepared in sterileDulbecco's Phosphate Buffered Saline. Mice are placed in acarousel-style, nose only, exposure chamber and allowed to inhaleaerosols for five minutes, using an ICN SPAG-2 nebulizer. Thisnebulizer generates a mean aerosol particle size of 1.3 microns ata rate of approximately 0.25 ml/minute.

[0288] Ten minutes and 36 hours later, the mice are moved to wholebody plethysmograph chambers. Bronchoconstriction is induced in themice by administration of an 80 mg/ml methacholine (MC) aerosolinto the plethysmograph chambers for 5 minutes. The mice areallowed to inhale an aerosol containing 80 mg/ml methacholinefollowing inhalation treatment with DPBS vehicle (Dulbecco'sPhosphate Buffered Saline), or 80 mg/ml methacholine followinginhalation treatment with test compound. The average enhanced pause(Penh, lung resistance), corresponding to airflow resistance, isdetermined and statistically analyzed using Kruskal-Wallis one wayANOVA. In order to determine the baseline, saline aerosol (withoutmethacholine) is also separately administered to the mice.

[0289] This procedure is a model for inhalation treatment ofasthma, chronic obstructive pulmonary disorder and otherobstructive respiratory tract disorders in which the effectivenessof the compounds of formulas (Ia) or (Ib) can be tested.

Test for Frequency of Micturition in Female Sprague-Dawley Rats

[0290] Ten female Sprague-Dawley rats having a mean weight of about245-285 g are anesthetized with urethane (1.2 g/k, sc.). A midlineincision is performed to expose the bladder and a 23G catheter isinserted into the bladder dome for the measurement of intravesicalpressure. A non-stop transvesical cystometrogram, as described inJ. Pharmacological. Methods, 15, pp. 157-167 (1986), is used, at afilling rate of 0.216 ml/min. of saline, to access the filling andvoiding characteristics of the bladder. Through the continuouscystometry method thus afforded, consecutive micturition can berecorded. Test compound is given at intravenous doses after theinitial baseline micturition sequence is reliably measured forapproximately 12 min. From these recordings, the absolute values inmaximum pressure obtained and the frequency of micturition ismeasured. A dose response curve illustrating the effect of testcompound on the absolute micturition pressures in the range of 1-50mg/kg can be obtained. This procedure is a model for overactivebladder (OAB) in which the compounds of the formulas (Ia) and (Ib)can be tested.

[0291] The following Examples illustrate numerous formulationssuitable for administering the compounds of formula (Ia) and (Ib)to treat various conditions responsive to treatment with ananticholinergic agent.

[0292] In these Examples, percentages are by weight unlessotherwise indicated.

EXAMPLES OF PHARMACEUTICAL FORMULATIONS

Example 1

TABLE-US-00015 [0293] Tablets per tablet Compound of formula (Ia)or (Ib), e.g. 100 mg Compound (d) or (m) or (w) lactose 140 mg cornstarch 240 mg polyvinylpyrrolidone 15 mg magnesium stearate 5 mg500 mg

[0294] The finely ground active substance, lactose and some of thecorn starch are mixed together. The mixture is screened, thenmoistened with a solution of polyvinylpyrrolidone in water,kneaded, wet-granulated and dried. The granules, the remaining cornstarch and the magnesium stearate are screened and mixed together.The mixture is compressed to produce tablets of suitable shape andsize.

Example 2

TABLE-US-00016 [0295] Tablets per tablet Compound of formula (Ia)or (Ib), e.g. 80 mg Compound (d) or (m) or (w) lactose 55 mg cornstarch 190 mg microcrystalline cellulose 35 mg polyvinylpyrrolidone15 mg sodium-caroxymethyl starch 23 mg magnesium stearate 2 mg 400mg

[0296] The finely ground active substance, some of the corn starch,lactose, microcrystalline cellulose and polyvinylpyrrolidone aremixed together, the mixture is screened and worked with theremaining corn starch and water to form a granulate which is driedand screened. The sodium carboxymethyl starch and the magnesiumstearate are added and mixed in and the mixture is compressed toform tablets of a suitable size.

Example 3

TABLE-US-00017 [0297] Ampule solution Compound of formula (Ia) or(Ib), e.g. 50 mg Compound (d) or (m) or (w) sodium chloride 50 mgwater for inj. 5 ml

[0298] The active substance is dissolved in water at its own pH oroptionally at pH 5.5 to 6.5 and sodium chloride is added to make itisotonic. The solution obtained is filtered free from pyrogens andthe filtrate is transferred under aseptic conditions into ampuleswhich are then sterilized and sealed by fusion. The ampules contain5 mg, 25 mg and 50 mg of active substance.

Example 4

TABLE-US-00018 [0299] Metering aerosol Compound of formula (Ia) or(Ib), e.g. 0.005 Compound (d) or (m) or (w) Sorbitan trioleate 0.1Monofluorotrichloromethane and 2:3 ad 100difluorodichioromethane

[0300] The suspension is transferred into a conventional aerosolcontainer with a metering valve. Preferably, 50 .mu.l of suspensionare delivered per spray. The active substance may also be meteredin higher doses if desired (e.g. 0.02% by weight).

Example 5

TABLE-US-00019 [0301] Solutions (in mg/100 ml) Compound of formula(Ia) or (Ib), e.g. 333.3 mg Compound (d) or (m) or (w) Formoterolfumarate 333.3 mg Benzalkonium chloride 10.0 mg EDTA 50.0 mgHCl(ln) ad pH 3.4

[0302] This solution may be prepared in the usual manner.

Example 6

TABLE-US-00020 [0303] Powder for inhalation Compound of formula(Ia) or (Ib), e.g. 6 .mu.g Compound (d) or (m) or (w) Formoterolfumarate 6 .mu.g Lactose monohydrate ad 25 mg

[0304] The powder for inhalation is produced in the usual way bymixing the individual ingredients together.

Example 7

TABLE-US-00021 [0305] Powder for inhalation Compound of formula(Ia) or (Ib), e.g. 10 .mu.g Compound (d) or (m) or (w) Lactosemonohydrate ad 5 mg

[0306] The powder for inhalation is produced in the usual way bymixing the individual ingredients together.

Further Formulations Obtained Analogously to Methods Known in theArt

A: Inhalable Powders

Example 8

TABLE-US-00022 [0307] Ingredients .mu.g per capsule Compound offormula (Ia) or (Ib), e.g. 100 Compound (d) or (m) or (w)budesonide 200 lactose 4700 Total 5000

Example 9

TABLE-US-00023 [0308] Ingredients .mu.g per capsule Compound offormula (Ia) or (Ib), e.g. 100 Compound (d) or (m) or (w)fluticasone propionate 125 lactose 4775 Total 5000

Example 10

TABLE-US-00024 [0309] Ingredients .mu.g per capsule Compound offormula (Ia) or (Ib), e.g. 100 Compound (d) or (m) or (w)mometasone furoate .times. H.sub.2O 250 lactose 4650 Total 5000

Example 11

TABLE-US-00025 [0310] Ingredients .mu.g per capsule Compound offormula (Ia) or (Ib), e.g. 100 Compound (d) or (m) or (w)ciclesonide 250 lactose 4650 Total 5000

Example 12

TABLE-US-00026 [0311] Ingredients .mu.g per capsule Compound offormula (Ia) or (Ib), e.g. 50 Compound (d) or (m) or (w) budesonide125 lactose 4825 Total 5000

Example 13

TABLE-US-00027 [0312] Ingredients .mu.g per capsule Compound offormula (Ia) or (Ib), e.g. 50 Compound (d) or (m) or (w)fluticasone propionate 200 lactose 4750 Total 5000

Example 14

TABLE-US-00028 [0313] Ingredients .mu.g per capsule Compound offormula (Ia) or (Ib), e.g. 75 Compound (d) or (m) or (w) mometasonefuroate .times. H.sub.2O 250 lactose 4675 Total 5000

Example 15

TABLE-US-00029 [0314] Ingredients .mu.g per capsule Compound offormula (Ia) or (Ib), e.g. 75 Compound (d) or (m) or (w)ciclesonide 250 lactose 4675 Total 5000

Example 16

TABLE-US-00030 [0315] Ingredients .mu.g per capsule Compound offormula (Ia) or (Ib), e.g. 100 Compound (d) or (m) or (w) ST-126250 lactose 4650 Total 5000

Example 17

TABLE-US-00031 [0316] Ingredients .mu.g per capsule Compound offormula (Ia) or (Ib), e.g. 50 Compound (d) or (m) or (w) ST-126 125lactose 4825 Total 5000

Example 18

TABLE-US-00032 [0317] Ingredients .mu.g per capsule Compound offormula (Ia) or (Ib), e.g. 100 Compound (d) or (m) or (w)loteprednol etabonate 200 lactose 4700 Total 5000

Example 19

TABLE-US-00033 [0318] Ingredients .mu.g per capsule Compound offormula (Ia) or (Ib), e.g. 100 Compound (d) or (m) or (w)etiprednol dichloroacetate 200 lactose 4700 Total 5000

Example 20

TABLE-US-00034 [0319] Ingredients .mu.g per capsule Compound offormula (Ia) or (Ib), e.g. 100 Compound (d) or (m) or (w)loteprednol etabonate 125 lactose 4775 Total 5000

Example 21

TABLE-US-00035 [0320] Ingredients .mu.g per capsule Compound offormula (Ia) or (Ib), e.g. 50 Compound (d) or (m) or (w) etiprednoldichloroacetate 125 lactose 4825 Total 5000

Example 22

TABLE-US-00036 [0321] Ingredients .mu.g per capsule Compound offormula (Ia) or (Ib), e.g. 100 Compound (d) or (m) or (w)loteprednol etabonate 200 .DELTA..sup.1 - cortienic acid methylester 200 lactose 4500 Total 5000

Example 23

TABLE-US-00037 [0322] Ingredients .mu.g per capsule Compound offormula (Ia) or (Ib), e.g. 100 Compound (d) or (m) or (w)loteprednol etabonate 200 .DELTA..sup.1 - cortienic acid 200lactose 4500 Total 5000

Example 24

TABLE-US-00038 [0323] Ingredients .mu.g per capsule Compound offormula (Ia) or (Ib), e.g. 100 Compound (d) or (m) or (w)loteprednol etabonate 125 .DELTA..sup.1 - cortienic acid or 125.DELTA..sup.1 - cortienic acid methyl ester lactose 4650 Total5000

Example 25

TABLE-US-00039 [0324] Ingredients .mu.g per capsule Compound offormula (Ia) or (Ib), e.g. 50 Compound (d) or (m) or (w)loteprednol etabonate 125 .DELTA..sup.1 - cortienic acid or 125.DELTA..sup.1 - cortienic acid methyl ester lactose 4700 Total5000

B. Propellant-Containing Aerosols for Inhalation (wherein TG 134ais 1,1,1,2-Tetrafluoroethane and TG 227 is1,1,1,2,3,3,3-Heptafluoropropane)

Example 26

Suspension Aerosol

TABLE-US-00040 [0325] Ingredients % by weight Compound of formula(Ia) or (Ib), e.g. 0.050 Compound (d) or (m) or (w) budesonide 0.4soya lecithin 0.2 TG 134a:TG227 (2:3) to 100

Example 27

Suspension Aerosol

TABLE-US-00041 [0326] Ingredients % by weight Compound of formula(Ia) or (Ib), e.g. 0.020 Compound (d) or (m) or (w) fluticasonepropionate 0.3 isopropyl myristate 0.1 TG 227 to 100

Example 28

Suspension Aerosol

TABLE-US-00042 [0327] Ingredients % by weight Compound of formula(Ia) or (Ib), e.g. 0.020 Compound (d) or (m) or (w) mometasonefuroate .times. H.sub.2O 0.6 isopropyl myristate 0.1 TG 227 to100

Example 29

Suspension Aerosol

TABLE-US-00043 [0328] Ingredients % by weight Compound of formula(Ia) or (Ib), e.g. 0.020 Compound (d) or (m) or (w) ciclesonide 0.4isopropyl myristate 0.1 TG 134a:TG227 (2:3) to 100

Example 30

Suspension Aerosol

TABLE-US-00044 [0329] Ingredients % by weight Compound of formula(Ia) or (Ib), e.g. 0.039 Compound (d) or (m) or (w) ciclesonide 0.4absolute ethanol 0.5 isopropyl myristate 0.1 TG 134a:TG227 (2:3) to100

Example 31

Solution Aerosol

TABLE-US-00045 [0330] Ingredients % by weight Compound of formula(Ia) or (Ib), e.g. 0.039 Compound (d) or (m) or (w) fluticasonepropionate 0.2 absolute ethanol 0.5 isopropyl myristate 0.1 TG134a:TG227 (2:3) to 100

Example 32

Solution Aerosol

TABLE-US-00046 [0331] Ingredients % by weight Compound of formula(Ia) or (Ib), e.g. 0.039 Compound (d) or (m) or (w) mometasonefuroate .times. H.sub.2O 0.6 absolute ethanol 0.5 isopropylmyristate 0.1 TG 134a:TG227 (2:3) to 100

Example 33

Solution Aerosol

TABLE-US-00047 [0332] Ingredients % by weight Compound of formula(Ia) or (Ib), e.g. 0.039 Compound (d) or (m) or (w) ciclesonide 0.4absolute ethanol 0.5 isopropyl myristate 0.1 TG 134a:TG227 (2:3) to100

Example 34

Solution Aerosol

TABLE-US-00048 [0333] Ingredients % by weight Compound of formula(Ia) or (Ib), e.g. 0.039 Compound (d) or (m) or (w) ST-126 0.6absolute ethanol 0.5 isopropyl myristate 0.1 TG 134a:TG227 (2:3) to100

Example 35

Solution Aerosol

TABLE-US-00049 [0334] Ingredients % by weight Compound of formula(Ia) or (Ib), e.g. 0.039 Compound (d) or (m) or (w) ST-126 0.4absolute ethanol 0.5 isopropyl myristate 0.1 TG 134a:TG227 (2:3) to100

Example 36

TABLE-US-00050 [0335] Ingredients % by weight Compound of formula(Ia) or (Ib), e.g. 0.05 Compound (d) or (m) or (w) loteprednoletabonate 0.4 soya lecithin 0.2 TG 134a:TG227 (2:3) to 100

Example 37

TABLE-US-00051 [0336] Ingredients % by weight Compound of formula(Ia) or (Ib), e.g. 0.020 Compound (d) or (m) or (w) loteprednoletabonate 0.3 isopropyl myristate 0.1 TG 227 to 100

Example 38

TABLE-US-00052 [0337] Ingredients % by weight Compound of formula(Ia) or (Ib), e.g. 0.020 Compound (d) or (m) or (w) etiprednoldichloracetate 0.4 isopropyl myristate 0.1 TG 227 to 100

Example 39

TABLE-US-00053 [0338] Ingredients % by weight Compound of formula(Ia) or (Ib), e.g. 0.020 Compound (d) or (m) or (w) loteprednoletabonate 0.4 isopropyl myristate 0.1 TG 134a:TG227 (2:3) to100

Example 40

TABLE-US-00054 [0339] Ingredients % by weight Compound of formula(Ia) or (Ib), e.g. 0.039 Compound (d) or (m) or (w) loteprednoletabonate 0.4 absolute ethanol 0.5 isopropyl myristate 0.1 TG134a:TG227 (2:3) to 100

Example 41

TABLE-US-00055 [0340] Ingredients % by weight Compound of formula(Ia) or (Ib), e.g. 0.05 Compound (d) or (m) or (w) loteprednoletabonate 0.4 .DELTA..sup.1- cortienic acid or .DELTA..sup.1-cortienic acid 0.4 methyl ester soya lecithin 0.2 TG134a:TG227(2:3) to 100

Example 42

TABLE-US-00056 [0341] Ingredients % by weight Compound of formula(Ia) or (Ib), e.g. 0.02 Compound (d) or (m) or (w) loteprednoletabonate 0.3 .DELTA..sup.1- cortienic acid or .DELTA..sup.1-cortienic acid 0.3 methyl ester isopropyl myristate 0.1 TG227 to100

Example 43

TABLE-US-00057 [0342] Ingredients % by weight Compound of formula(Ia) or (Ib), e.g. 0.04 Compound (d) or (m) or (w) loteprednoletabonate 0.4 .DELTA..sup.1- cortienic acid or .DELTA..sup.1-cortienic acid 0.4 methyl ester isopropyl myristate 0.1 TG 227 to100

Example 44

TABLE-US-00058 [0343] Ingredients % by weight Compound of formula(Ia) or (Ib), e.g. 0.02 Compound (d) or (m) or (w) loteprednoletabonate 0.4 .DELTA..sup.1- cortienic acid or .DELTA..sup.1-cortienic acid 0.4 methyl ester isopropyl myristate 0.1TG134a:TG227 (2:3) to 100

Example 45

TABLE-US-00059 [0344] Ingredients % by weight Compound of formula(Ia) or (Ib), e.g. 0.039 Compound (d) or (m) or (w) loteprednoletabonate 0.4 .DELTA..sup.1- cortienic acid or .DELTA..sup.1-cortienic acid 0.4 methyl ester absolute ethanol 0.5 isopropylmyristate 0.1 TG134a:TG227 (2:3) to 100

C. Ophthalmic Formulations

Example 46

Eye Drops

TABLE-US-00060 [0345] Compound of formula (Ia) or (Ib), e.g. 0.05%w/v Compound (d) or (m) or (w) Tween 80 2.5% w/v Ethanol 0.75% w/vBenzalkonium chloride 0.02% w/v Phenyl ethanol 0.25% w/v Sodiumchloride 0.60% w/v Water for injection q.s. 100 volumes

Example 47

Eye Drops

TABLE-US-00061 [0346] Compound of formula (Ia) or (Ib), e.g. 0.04%w/v Compound (d) or (m) or (w) Tween 80 2.5% w/v Ethanol 0.75% w/vBenzalkonium chloride 0.02% w/v Phenyl ethanol 0.25% w/v Sodiumchloride 0.60% w/v Water for injection q.s. 100 volumes

Example 48

Eye Drops

TABLE-US-00062 [0347] Compound of formula (Ia) or (Ib), e.g. 0.035%w/v Compound (d) or (m) or (w) Povidone 0.6% w/v Benzalkoniumchloride 0.02% w/v Sodium edetate U.S.P. 0.10% w/v Glycerin U.S.P.2.5% w/v Tyloxapol U.S.P. 3.0% w/v Sodium chloride 0.3% w/v Sodium.gamma.-aminobutyrate 1.0% w/v Sterile distilled water q.s. 100volumes

[0348] The ingredients listed above are combined, then the pH ischecked and, if necessary, adjusted to 5.0-5.5 by basifying withsodium hydroxide or acidifying with hydrochloric acid.

[0349] Yet other compositions of the invention can be convenientlyformulated using known techniques.

[0350] While this description has been couched in terms of variouspreferred or exemplary embodiments, the skilled artisan willappreciate that various modifications, substitutions, omissions andchanges may be made without departing from the spirit thereof.Accordingly, it is intended that the scope of the foregoing belimited only by the broadest statements herein and by the scope ofthe following claims, including equivalents thereof.

* * * * *