CA2222573A1 - The use of alpha-1c-selective adrenoceptor agonists for the treatment of urinary incontinence - Google Patents

Abstract

The present invention provides a method of treating urinary incontinence in a subject which comprises administering to the subject a therapeutically effective amount of an .alpha.1C selective agonist which activates a human .alpha.1C adrenoceptor at least ten-fold more than it activates a human .alpha.1A adrenoceptor and a human .alpha.1B adrenoceptor. The present invention further provides a method of inducing contraction of urethra and bladder neck tissues which comprises contacting the urethra and bladder neck tissues with an effective contraction-inducing amount of an .alpha.1C
selective agonist which activates a human .alpha.1C adrenoceptor at least ten-fold more than it activates a human .alpha.1A adrenoceptor and a human .alpha.1B adrenoceptor. In addition, the invention includes compounds for the treatment of urinary incontinence and for use in inducing contraction of urethra and bladder neck tissues.

Description

CA 02222~73 1997-11-27 WO96~38143 P~CT~S96107979 THE USE OF ALPRA-lC-SELECTIVE ADRENOCEPTOR AGONISTS FOR
THE T~T~F~T OF U~T~Y l~ol~llN~N~

Throughout this application, various references are referred to within parentheses. Disclosures of these ~ publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
Full bibliographic citations for these references may be found immediately preceding the claims.

Bach~lo~.d of the Invention The designation "~" is the appellation recently approved by the IUPHAR Nomenclature Committee for the previously designated ~1C~ cloned subtype as outlined in the 1995 Receptor and Ion ~h~nnel Nomenclature Supplement (Watson and Girdlestone, 1995). However, the designation ~1c;is used throughout this application and the supporting tables and figures to refer to the receptor subtype recently renamed "~". Since in both the old and new nomenclature there has only been one unique receptor subtype which has been designated ~lc (i.e., there is no ~1c under the current nomenclature), ~lC~ iS an unambiguous description of this unique receptor subtype.

Incontinence is a condition characterized by the involuntary loss of urine. It can be divided generally into two types, the first involving an unstable bladder as the underlying cause, and the second involving an insufficiency in bladder outlet closing pressure despite the presence of a stable bladder. The condition may arise from a variety of different pathological, anatomical or neurological factors (Lundberg, 1989).

CA 02222~73 1997-11-27 W O96/38143 PCTrUS96107979 While the prevalence in females is two fold higher, it also affects males (Lundberg, 1989). The greatest incidence is seen in postmenopausal women. It is estimated that at least 10 million Americans suffer from urinary incontinence ~Sand et al., 1990). Incontinence can be treated by surgical and nonsurgical methods.
Conservative approaches include physiotherapy (Kegel exercises) and functional electrical stimulation which aim to strengthen the peri-urethral musculature (Walters et al., 1992). Periurethral injection of polytetraflurorethylene is a more invasive procedure intended to augment the urethral support (Sand et al, 1990). The most radical treatment for stress incontinence is surgery, involving a variety of techniques which seek to improve the alignment of the bladder, urethra, and surrounding structures.

A variety of pharmaceutical agents have been employed with varying success to treat urinary incontinence.
Drugs useful in reducing the contractility of the bladder include anticholinergics, ~-blockers, calcium channel blockers, and tricyclic antidepressants.
Estrogen has been used with some success in increasing bladder outlet resistance, particularly in postmenopausal women. Its actions have been attributed to a "mucosal seal effectn resulting from urethral mucosal cell proliferation (Wein, 1987), although there is now some suggestion that it may also contribute to a restoration of ~-adrenoceptor expression in the urethra (Wein, 1987).

The most commonly employed agents for increasing bladder outlet resistance are the ~-adrenoceptor agonists. These activate ~-adrenoceptors located on the smooth muscle cells of the proximal urethra and bladder neck CA 02222~73 1997-11-27 W O 96138143 PCTrUS96/07979 (Sourander, 1990; Wein, 1987), resulting in contraction and increased closing pressure. The compounds currently employed for this therapy include the non-selective adrenoceptor agents phenylpropanolamine, ephedrine, and phenylephrine (Wein, 1987; Lundberg, 1989). The actions of these drugs are attributable, in part, to direct activation of adrenoceptors and in part to their ability to displace endogenous norepinephrine from sympathetic neurons following uptake into the nerve terminal, a so-called indirect sympathomimetic action (Andersson and Sjogren, 1982). Their lack of selectivity (see Table 3 hereinafter) among the adrenoceptor subtypes and the indirect action of these compounds results in their activating ~ 2-~ and,B-adrenoceptors in the CNS and in the periphery. As a result, any desired therapeutic effect of these agents may be accompani.ed by a constellation of undesirable side effects. One major side effect of their use in incontinence is an increase in blood pressure. This effect is dose-dependent and limits the ability to achieve therapeutically effective circulating concentrations of the drug (Andersson and Sjogren, 1982). In addition, these compounds in some patients produce insomnia, anxiety and dizziness as a result of their stimulant actions in the CNS (Andersson and Sjogren, 1982, Wein, 1987).

Another compound which has been evaluated in urinary incontinence is midodrine, a prodrug which is converted in ~ivo to the active phenylethylamine ST-1059. The clinical efficacy of midodrine has not been demonstrated conclusively (Andersson and Sjogren, 1982). Like the above compounds, its effects may be limited by cross-reactivity with other adrenoceptors (see Table 3) which may limit the maximum achievable dose. A better understanding of the subtypes of ~-adrenoceptors and CA 02222~73 1997-11-27 W O 96/38143 PCTrUS96/07979 their involvement in various physiological processes will facilitate the development of more efficacious drugs for the treatment of incontinence.

The ~-adrenoceptors are specific neuroreceptor proteins located in the peripheral and central nervous systems and on tissues throughout the body. The receptors are important switches for controlling many physiological functions and, thus, represent important targets for drug development. Drugs which interact at these receptors comprise two main classes: agonists, which mimic the endogenous ligands (norepinephrine and epinephrine) in their ability to activate the receptor; and antagonists, which serve to block the actions of the endogenous ligands. Many ~-adrenoceptor drugs of both classes have been developed o~er the past 40 years. Examples in addition to those indicated above, which owe at least part of their action to stimulation of alpha adrenoceptors, include clonidine (agonist; treatment of hypertension), prazosin (antagonist; hypertension), oxymetazoline (agonist, nasal decongestion), and methox~m;ne (treatment of episodes supraventricular tachycardia). While many of these drugs are effective, they also produce undesirable side effects at therapeutic doses (e.g., clonidine produces dry mouth, sedation and orthostatic hypotension in addition to its antihypertensive effect).

During the past 15 years a more precise understanding of ~-adrenoceptors and drugs targeting ~-adrenoceptors has emerged. Prior to 1977, only one ~-adrenoceptor was known to exist. Between 1977 and 1988, it was accepted by the scientific community that at least two ~-adrenoceptors, cx1 and CX2, existed in the central and peripheral nervous systems. Since 1988, new techniques CA 02222~73 1997-11-27 WO 96/38143 P~T~US96107979 in molecular biology have led to the identification of at least six distinct ~-adrenoceptor proteins which are distributed throughout the central and peripheral nervous ~ systems: ~lA~ ~lB ~ ~lC ~ ~2A ~ ~2B and ~2C ( Bylund, 1992). In addition to the cloned ~-adrenoceptors, se~eral putative adrenoceptor subtypes have been recently described based upon functional studies in a variety of mammalian tissues. These receptors, which have not been cloned, are described as ~lH~ ~lL and ~lN (Murmamatsu, 1995) or "atypical ~l'l (Abel, 1995) adrenoceptors. The precise role of each of the subtypes in various physiological responses is only beginning to be understood, but it is clear that distinct subtypes do mediate distinct physiological responses to agonists and antagonists. For example, it has been shown that norepinephrine-induced contractions of the human prostate are mediated by the ~lc-adrenoceptor (Forray et al., 1994). Many adrenoceptor drugs developed before 1992 are not selective for any particular ~-adrenoceptor subtype. It is increasingly evident that this lack of receptor subtype selectivity is an underlying cause of the untoward side-ef~ects of these drugs.

The role of the sympathetic adrenergic nervous system in the storage function of the bladder is well recognized (Wein, 1987; Latifpour et al, 1990). Likew.ise, it is understood in the art that the study of adrenoceptor mechanisms in isolated urethra and bladder tissues is applicable to incontinence therapy (Latifpour et al., 1994; Tsujimoto et al., 1986). Various groups have attempted to identify, through binding and functional studies, ~l receptor subtypes in the urethrae of humans, rabbits, and rats (Yoshida et al., 1991; Testa et al.
1993; Chess-Williams et al., 1994). These efforts have, thus far, failed to provide conclusive evidence for a W 096/38143 CA 02222~73 1997-11-27 PCTÇUS96/07979 particular ~1-adrenoceptor subtype being responsible for the effects of adrenoceptor agonists in the urethra.

This invention relates to the discovery that ~lC-agonists are useful for the treatment of urinary incontinence with the potential for decreased side effects. Data already exists which indicates that the ~1c-adrenoceptor is not involved significantly in the cardiovascular actions of ~-agonists and antagonists (Forray et al., 1994).
Therefore, agonists exhibiting significant binding and functional selectivity for the ~1c-adrenoceptor over other ~1-adrenoceptors, ~2-adrenoceptors, ~-adrenoceptors, as well as histamine receptors and serotonin (5-HT) receptors, are contemplated to be more effective agents, relative to currently available therapies, for the treatment of urinary incontinence.

CA 02222~73 1997-11-27 w096/38143 P~T~S9610797 SummarY o~ the Invention The present invention provides a method o~ treating urinary incontinence in a subject which comprises administering to the subject a therapeutically effective amount of an ~lc selective agonist which activates a human c adrenoceptor at least ten-fold more than it activates a human ~lA adrenoceptor and a human ~lB adrenoceptor.

The present invention further provides a method of inducing contraction of urethra and bladder neck tissues which comprises contacting the urethra and bladder-neck tissues with an effective contraction-inducing amount of an ~lc selective agonist which activates a human ~lc adrenoceptor at least ten-~old more than it activates a human ~lA adrenoceptor and a human ~lB adrenoceptor.

In addition, the invention includes compounds for the treatment of urinary incontinence and for use in inducing contraction of urethra and bladder neck tissues.

CA 02222~73 1997-11-27 W O96138143 PCTrUS96/07979 Brief De~criPtion of the Fiqures Fiqures lA, lB, and lC show correlation of antagonist pKp values determined in functional studies of human urethra versus pKI values measured in binding experiments using cloned human ~1A-adrenoceptors (A), ~1B-adrenoceptors (B), and ~lc-adrenoceptors (C). The slopes and correlation coefficients (r) for the linear regression analysis are presented in each figure.
Fiaures 2A, 2B, and 2C show correlation of antagonist pKB
values determined in functional studies of female rabbit urethra versus pKI values measured in binding experiments using cloned human ~lA-adrenoceptors (A), ~lB-adrenoceptors (B), and ~lc-adrenoceptors (C). The slopes and correlation coefficients (r) for the linear regression analysis are presented in each figure.

Fiqures 3A, 3B, and 3C show correlation of antagonist pKB
values determined in functional studies of male rabbit urethra versus pKI values measured in binding experiments using cloned human ~lA-adrenoceptors (A), ~lB-adrenoceptors (B), and lc-adrenoceptors (C). The slopes and correlation coefficients (r) for the linear regression analysis are presented in each figure.

Fiaures 4A, 4B, and 4C show correlation of antagonist pKB
values determined in functional studies of female dog urethra versus pKI values measured in binding experiments using cloned human ~lA-adrenoceptors (A), alB-adrenoceptors (B), and ~lc-adrenoceptors (C). The slopes and correlation coefficients (r) for the linear regression analysis are presented in each figure.

Fiqures S, 5B, and 5C show correlation of antagonist pKB

W O96138143 PCTnUS96107g79 values determined in functional studies of male dog urethra versus pKI values measured in binding experiments using cloned human a1A-adrenoceptors (A), ~lB-adrenoceptors (B), and ~1c-adrenoceptors (C). The slopes and correlation coefficients (r) for the linear regression analysis are presented in each figure.

Fiqure 6 shows the chemical structures of SK&F 102652, A-61603, SDZ NVI 085, Prazosin, 5-Methyl urapidil, Abanoquil, Compound 1, and ST-1059.

CA 02222~73 1997-11-27 W O96/38143 PCTrUS96107979 Detailed DescriPtion of the Invention The following definitions are presented as an aid in understanding this invention.

Receptor Activation describes the process in which the binding of a compound to the receptor when it is on the surface of a cell leads to a metabolic response within the cell. Such metabolic responses include, but are not limited to, activation of adenylyl cyclase, activation of guanylyl cyclase, hydrolysis of inositol phospholipids, movement of ions across the cell membrane, or contraction in a tissue in the cells of which the receptor is expressed.
Potency means the concentration of an agonist which elicits half of its maximum activation (expressed as ECso or the negative log of the ECso/ i.e., pEC50).

Intrinsic Activity means the magnitude of the maximum activation in a cell or tissue which a particular agonist is capable of eliciting, relative to the maximum activation elicited by a reference full agonist and is expressed as values ranging between unity for full agonists (e.g., norepinephrine in the case of ~-adrenoceptors) and zero for antagonists. Because intrinsic activity as originally defined (Ariens, 1960) is recognized as being dependent upon the receptor system in which it is measured (Kenakin, 1987), intrinsic activity herein is based upon measurements made using the cloned receptor systems described below.

Selectivity of Receptor Activation refers to the ability of an agonist to selectively activate one receptor relative to another receptor. Such selectivity may CA 02222~73 1997-11-27 reflect either (a) the agonist's ability to activate one receptor at a much lower concentration than that required to activate another receptor (i.e., a potency difference) or (b) the agonist's ability to activate one ~eceptor to a much greater degree than another receptor, independent of concentration, (i.e., an intrinsic activity difference) or (c) a combination of both.

Therefore, statements of the form "activates a human ~lc-adrenoceptor at least ten-fold more than it activates any of the following (receptors)" mean and include any such difference whether it is by virtue of a difference in potency, or a difference in intrinsic activity, or-both.

Having due regard to the preceding definitions, the present invention provides a method of treating urinary incontinence in a subject which comprises administering to the subject a therapeutically effective amount of an ~lc selective agonist which activates a human ~lC
adrenoceptor at least ten-fold more than it activates a human ~lA adrenoceptor and a human ~lB adrenoceptor.

The invention further provides a method of treating urinary incontinence in a subject which comprises administering to the subject a therapeutically effective amount of an ~lc selective agonist which activates a human ~lc adrenoceptor at least 50-fold more than it activates a human ~lA adrenoceptor and a human ~lB adrenoceptor.

The invention provides a method of treating urinary incontinence in a subject which comprises administering to the subject a therapeutically effective amount of an c selective agonist which activates a human ~lC
adrenoceptor at least 100-fold more than it activates a human ~lA adrenoceptor and a human ~B adrenoceptor.

CA 02222~73 1997-11-27 W O96/38143 PCTrUS96107979 The invention provides a method of treating urinary incontinence in a subject which comprises administering to the subject a therapeutically effective amount of an ~lC selective agonist which activates a human ~c adrenoceptor at least 200-fold more than it activates a human ~lA adrenoceptor and a human ~lB adrenoceptor.

The ~lc selective agonist used to practice the method of treating urinary incontinence further has the characteristic that it does not antagonize a human ~lA
adrenoceptor and a human ~lB adrenoceptor.

Desirably, the ~1c selective agonist used to practice the method of treating urinary incontinence activates the human ~1c adrenoceptor at least ten-fold more than it activates any human ~2 adrenoceptor and any adrenoceptor. Some examples of a2 adrenoceptors include the ~X2A~ ~Y23~ and a!2C receptorS.

The invention also provides that the ~lc selective agonist used to practice the method of treating urinary incontinence further has the characteristic that it does not antagonize any human ~2 adrenoceptor and any adrenoceptor. Some examples Of ~2 adrenoceptors include the ~2A~ ~2B~ and ~2C receptors.

Desirably, the ~lc selective agonist used to practice the method of treating urinary incontinence activates the human ~1c adrenoceptor at least ten-fold more than it activates a human histamine Hl or H2 receptor.

The invention further provides that the ~lc selective agonist used to practice the method of treating urinary incontinence activates the human ~lc adrenoceptor at least ten-fold more than it activates a human dopamine D1, D2, CA 02222~73 1997-11-27 WO96138143 P~CT~S96J07979 D3, or Ds receptor.

The invention also provides that the ~lc selective agonist used to practice the method of treating urinary incontinence activates the human ~lc adrenoceptor at least ten-fold more than it activates a human serotonin 5-HTlA, 5-HTlD~, 5-HTlD~, 5-HTlE, 5-HTlF, 5-HT2, or 5-HT, receptor.

The present invention further provides a method of inducing contraction of urethra and bladder neck tissues which comprises contacting the urethra and bladder neck tissues with an effective contraction-inducing amount Oc an ~1c selective agonist which activates a human adrenoceptor at least ten-fold more than it activates a human ~lA adrenoceptor and a human ~lB adrenoceptor.

The invention further provides a method o~ inducing contraction of urethra and bladder neck tissues which comprises contacting the urethra and bladder neck tissues with an effective contraction-inducing amount: of an ~lc selective agonist which activates a human ~lc adrenoceptor at least 50-fold more than it activates a human ~lA
adrenoceptor and a human ~lB adrenoceptor.

The invention provides a method of inducing contraction of urethra and bladder neck tissues which comprises contacting the urethra and bladder neck tissues with an effective contraction-inducing amount of an ~lc selective agonist which activates a human ~lc adrenoceptor at least 100-fold more than it activates a human ~lA adrenoceptor and a human ~lB adrenoceptor.

The invention also provides a method of inducing contraction of urethra and bladder neck tissues which comprises contacting the urethra and bladder neck tissues W 096/38143 CA 02222~73 l997-ll-27 PCTrUS96/07979 with an effective contraction-inducing amount of an ~l-selective agonist which activates a human ~lc adrenoceptor at least 200-fold more than it activates a human ~lA
adrenoceptor and a human ~lB adrenoceptor.
The ~lc selective agonist used to practice the method of inducing contraction of urethra and bladder neck tissues further has the characteristic that it does not antagonize a human ~lA adrenoceptor and a human ~lB
adrenoceptor.

Desirably, the ~lc selective agonist used to practice the method of inducing contraction of urethra and bladder neck tissues activates the human ~lc adrenoceptor at least ten-fold more than it activates any human ~2 adrenoceptor and any ~ adrenoceptor. Some examples of ~2 adrenoceptors include the ~2A~ ~2B~ and ~2C receptors The invention also provides that the ~lc selective agonist used to practice the method of inducing contraction of urethra and bladder neck tissues further has the characteristic that it does not antagonize any human ~2 adrenoceptor and any ~ adrenoceptor. Some examples of ~2 adrenoceptors include the ~2A~ ~2B~ and ~2C receptors Desirably, the ~lc selective agonist used to practice the method of inducing contraction of urethra and bladder neck tissues activates the human ~l~ adrenoceptor at least ten-fold more than it activates a human histamine Hl or H2 receptor.

The invention further provides that the ~lc selective agonist used to practice the method of inducing contraction of urethra and bladder neck tissues activates the human ~lc adrenoceptor at least ten-fold more than it CA 02222~73 1997-11-27 w o96r38143 P~C~nUS961~7979 activates a human dopamine Dl, D2, D3, or Ds receptor.

The invention also provides that the ~1c select:Lve agonist used to practice the method of inducing contraction of urethra and bladder neck tissues activates the human ~lc adrenoceptor at least ten-fold more than it activates a ~ human serotonin 5-HTlA, 5-HTlD~, 5-HTlD~, 5-HTlE, 5-HT.~, 5-HT2, or 5-HT7 receptor.

In one embodiment the invention provides a method of treating urinary incontinence which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

. N i.

where n is an integer from 1 to 6; R is H or Cl-C6 alkyli Rl is Cl-C6 alkyl, phenyl, naphthyl, substituted phenyl or naphthyl where the substituent is a halogen, or a Cl-C6 alkyl or alkoxy group; where l :

is an amino group or a heterocyclic group; the heterocyclic group is piperidine, morpholine, piperazine, pyrrolidine, hexamethylene, or thiomorpholine, the CA 02222573 1997-ll-27 W O96/38143 PCTrUS96/07979 heterocyclic group being bonded through the nitrogen atom thereof to the (CH2) n group; the amino group, where R2 is H, Cl-C6 alkyl, benzyl, or benzyhydryl and where R3 is H;
Cl-C10 alkyl; C2-C10 alkenyl; C~-C10 cycloalkyl or cycloalkenyl.

The present invention also provides that the compound has the structure:

0 F~ ~ N~

F', ~ H

The invention further provides that the compound has the structure:
~ N-CH

~N'~

A further embodiment of the invention provides a method of treating urinary incontinence which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

WO96138143 P~-T~S96107979 R~
,~'.'1 ;!~

~7 N N H
R~
!

where m is an integer from 0 to 2; where each of Rl, R2, R3 and R7 is independently H; OH; Cl-C6 alkyl or alkoxy;
halo; amino; acetamido or NHSO2R with R being H or cl- C6 alkyl; where Rl and R2 or R2 and R3 or R3 and R, taken together constitute a methylenedioxy, ethylenedioxy, benzimidazole or indole ring; where each of R4 and Rs are independently H ~r taken together has the foll~owing formula:

where the dashed line represents a single or double bond;
and R6 is H or Cl-C6 alkyl; or a pharmaceutically acceptable salt thereof.

The invention also provides that the compound has the structure:
r ~''' ~
'j CA 02222~73 1997-11-27 W O96/38143 PCTÇUS96/n7979 The invention further provides that the compound has the structure:
~S C~3 HO

Il~N

The invention also provides a method of treating urinary incontinence which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

2~ ~1R~

where each of R1 and R2 is independently H or Cl-C~ alkyl;
where R3 is OH or C1-C4 alkoxy; and R4 is Cl-C4 alkylthio, alkylsulfoxide or alkylsulfone; Cl; Br; I or CF3; where X is O, S, SO, SO2, NH, NR1 or NC(O)R1; in free base or acid addition salt form.

CA 02222~73 l997-ll-27 W 096J38143 P~TAUS96/07979 The invention further provides that the compound has the structure:

~H~
R~

R

The invention specifically provides that the compound has the structure:
~CH3 t~ F~3 ~N~

SC~ ~

This invention is also directed to optical isomers of the compounds described above. The invention also provides for the (-) and (+) enantiomers of all compounds of the subject application described herein. Included in this 25 invention are pharmaceutically acceptable salts and complexes of all of the compounds described herein. The salts include but are not limited to the following acids and bases. The following inorganic acids; hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and boric acid. The organic acids;
acetic acid, trifluoroacetic acid, formic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, maleic acid, citric acid, methanesulfonic acid, trifluoromethanesulfonic acid, benzoic acid, glycolic acid, lactic acid and mandelic acid. The following CA 02222~73 1997-ll-27 W O96/38143 PCTrUS96/07979 inorganic bases; ammonia, hydroxyethylamine and hydrazine. The following organic bases; methylamine, ethylamine, propylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, ethylenediamine, hydroxyethylamine, morpholine, piperazine and guanidine.
This invention further provides for the hydrates, isomorphs and polymorphs of all of the compounds described herein.

The present invention therefore provides a method of treating urinary incontinence, which comprises administering a quantity of any of the ~lc receptor agonists defined herein in a quantity effective against urinary incontinence.
The drug may be administered to a patient afflicted with urinary incontinence by any conventional route of admin-istration, including, but not limited to, intravenous, intramuscular, oral, subcutaneous, intratumoral, intra-dermal, and parenteral. The quantity effective againsturinary incontinence is between 0.001 mg and 10.0 mg per kg of subject body weight. The method of treating urinary incontinence disclosed in the present invention may also be carried out using a pharmaceutical composition comprising any of the ~1c receptor agonists as defined herein and a pharmaceutically acceptable carrier.
The composition may contain between 0. 05 mg and 500 mg of an ~c receptor agonist, and may be constituted into any form suitable for the mode of administration selected.
Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

CA 02222~73 1997-11-27 W 096138~43 P~TnUS96/n79~9 The drug may otherwise be prepared as a sterile solid composition which may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium. Carriers are intended to include necessary and inert binders, suspending agents, lubricants, flavorants, sweeteners, preservatives, dyes, and coatings. The drug may also be formulated as a transdermal patch.

Optimal dosages to be administered may be readily determined by those skilled in the art, and will vary with the particular ~1c receptor agonist in use, the strength of the preparation, the mode of administration, and the advancement of the disease condition. Additional factors depending on the particular patient being treated will result in a need to adjust dosages, including patient age, weight, diet, and time of administration.

The term "therapeutically effective amount" as used herein refers to that amount of pharmaceutical agent that elicits in a tissue, system, animal or human, the biological or medicinal response that is being sought by a researcher, veterinarian, medical doctor or other clinician, which response includes alleviation of the symptoms of the disease being treated. The term "subject," as used herein refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experimenl.

The binding and functional properties of compounds at the different human receptors were determined in vitro using cultured cell lines that selectively express the receptor of interest. These cell lines were prepared by transfecting the cloned cDNA or cloned genomic DNA or constructs containing both genomic DNA and cD~A encoding WO 96138143 CA 0 2 2 2 2 ~ 7 3 19 9 7 - 1 1 - 2 7 PCTtUS96107979 the human ~-adrenoceptors as further described ln detail in Example 10 hereinbelow. In connection with this invention, a number of cloned human receptors discussed herein, either as plasmids or as stably transfected cell lines, have been made pursuant to, and in satisfaction of, the Budapest Treaty on the International Recoynitlon of the Deposit of Microorganisms for the Purpose of Patent Procedure, and are made with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852. Specifically, these deposits have been accorded ATCC Accession Numbers as follows in Table 1:

Table 1 - ATCC Deposits:
Designation Receptor ATCC AccessionDate of No.Deposit Cell lines:
L-~1A human ~lA CRL 11138 09/25/1992 L-~1B human ~lB CRL 11139 09/25/1992 L-~lc human ~lC CRL 11140 09/25/1992 L-~2A human ~2A CRL 11180 11/6/1992 L-NGC-~2B human ~2B CRL 10275 10/25/1989 Y-a2B-2 human ~2B CRL 11888 05/11/1995 L-~2c human ~2C CRL 11181 11/6/1992 Ltk-8-30-84human 5-HTlDlCRL 10421 04/17/1990 Ltk-ll human 5-HT1D2 CRL 10422 04/17/1990 5HTlE-7 human 5-HTlE CRL 10913 11/6/1991 L-5-HTlF human 5-HTlF CRL 10957 12/27/1991 L-5HT-4B human 5-HT4B CRL 11166 10/20/1992 5HTlA-3 human 5-HTlA CRL 11889 05/11/1995 L-NGc-5HT2human 5-HT2 CRL 10287 10/31/1989 Plasmids:
pcEXV-D2 human D2 75344 11/6/1992 pcEXV-H2 human H2 75345 11/6/1992 pcEXV-Hl human H1 75346 11/6/1992 CA 02222~73 1997-11-27 WO96138143 P~S96/07979 Cell transfection~

Transient transfections of COS-7 cells with various plasmids were performed using the DEAE-Dextran method.
which is well-known to those skilled in the art.
Briefly, a plasmid comprising an expression vector for the receptor of interest was added to monolayexs of COS-7 cells bathed in a DEAE-Dextran solution. In order to enhance the efficiency of transfection, dimethyl sulfoxide was typically also added, according to the method of Lopata (Lopata, et al., 1984). Cells were then grown under controlled conditions and used in experiments after about 72 hours.

Stable cell lines were obtained using means which are well-known in the art. For example, a suitable host cell was typically cotransfected, using the calcium phosphate technique, with a plasmid comprising an expression vector for the receptor of interest and a plasmid cGmprising a gene which allows selection of successfully transfected cells. Cells were then grown in a controlled environment, and selected for expression of the receptor in interest. By continuing to grow and select cells, stable cell lines were obtained expressing the receptors described and used herein.

B;ndi~ assaYs The binding of a test compound to a receptor of interest was generally evaluated by competitive binding assays using membrane preparations derived from cells which expressed the receptor. First, conditions were determined which allowed measurement of the specific binding of a compound known to bind to the receptor.
Then, the binding of the known compound to the receptor 3 CA 0 2 2 2 2 ~ 7 3 19 9 7 - 1 1 - 2 7 PCT/US96/07979 in membrane preparations was evaluated in the presence of several different concentrations of the test compound.
Binding of the test compound to the receptor resulted in a reduction in the amount of the known compound which was abound to the receptor. A test compound having a high affinity for the receptor of interest would displace a given fraction of the bound known compound at a concentration lower than the concentration which would be required if the test compound had a low affinity for the receptor of interest.

The data shown in the Table 2 indicate that it is the ~,_-adrenoceptor which is responsible for mediating the contractile response to adrenoceptor agonists in the urethra of mAmm~ls, particularly humans. This in vitro property is recoghized in the art as correlating with efficacy in treating urinary incontinence in vivo.

This invention will be better understood from the 20 Experimental Details which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative o~
the invention as described more fully in the claims which follow thereafter.
Ex~rimental Details Phenylephrine, prazosin, 5-methylurapidil, and BMY 7378 were obtained from Research Biochemicals, Inc. Other compounds were prepared according to the examples which follow.

Synthesis of (+)-N-t5-(4,5-Dihydro-lH-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetra-hydronaphthalen-1-yllmethAne~ulfonamide (A-61603) 5-Nitro-6-:methoxy-1-tetralone. To a solution of 100 ml of 7096 HNO3 was added 6-methoxytetralone (Aldrich Chemical Co., Milwaukee, Wisconsin, 4.0 g, 23 mmol) over 1 h period at 0 ~C The 5 resulting solution was stirred for 24 h at 25 ~C. The reaction mixture was then poured into water t:o yield a yellow precipitate, which was subj ected to column chromatography (40~ EtOAc-Hexane) to yield 2.2 g (43~) o~
the desired product. lH NMR (300 MHz, CDCl3): ~ 2.12 (qt, 2H, J=6.9 Hz), 2.61 (t, 2H, J=6.9 Hz), 2.83 (t, 2H, J=6.9 Hz), 3.93 (s, 3H), 6.98 (d, lH, J=8.9 Hz), 8.14 (d, lH, J=8.9 Hz) .

Synthesis of (1)-6-Methoxy-5-nitro-1-trimethy.lsilyloxy-1,2,3,4-tetrahydronaph-thalene-1-carbonitrile. To a solution of 5-nitro-6-methoxy-1-tetralone (0.93 g, 4.2 mmol) in 20 ml of CH2Cl2 was added ZnI2 (100 mg, 0.31 mmol) and TMSCN (0.84 ml, 6.3 mmol) and the resulting solution was stirred for 2 h at 25 ~C. The reaction mixture was concentrated in vacuo to provide the desired product as a colorless oil, which was used in the next step without purification. lH NMR (300 MHz, CDCl3):
0.028 (s, 9H), 1.88-2.35 (m, 4H), 2.67 (t, 2H, J=6.1 Hz), 3.88 (s, 3H), 6.97 (d, lH, J=8.9 Hz), 7.71 (d, lH, J=8.9 Hz) .

Synthesi~3 of (~) - 6 -Methoxy- 5 -ni tro- 3, 4 -dihydronaphthalene-1-carbonitrile. A soluti.on of 6-methoxy- 5 -nitro- 1 - trimethylsilyloxy- 1, 2, 3, 4 -tetrahydronaphthalene-1-carbonitrile (1.3 g, 4.0 mmol) and AcCl (1.0 ml) in 20 ml AcOH was stirred for 2 h at 80-100 ~C. The resulting reaction mixture was concentrated in vacuo to yield the desired product as a colorless oil (0.86 g, 4.0 mmol, 96~ for two steps), which was subjected to the following s'cep without any CA 02222~73 1997-11-27 W 096/38143 PCTrUS96/07979 further purification. lH NMR (300 MHz, CDCl3):~ 2.48 (dt, 2H, J=2.3, 6.7 Hz), 2.78 (t, 2H, J=8.9 Hz), 3.89 (s, 3H), 6.82 (t, lH, J=2.3 Hz), 6.92 (d, lH, J=8.9 Hz), 7.53 (d, lH, J=8.9 Hz).

Synthesis of (i)-6-Methoxy-5-nitro-l~2~3~4-tetrahydronaphthalene-1-carbonitrile. To a solution of 6-methoxy-5-nitro-3,4-dihydronaphthalene-1-carbonitrile (0.41 g, 1.8 mmol) in 10 ml of EtOH was added NaBH4 (0.20 g, 5.3 mmol) and the resulting reaction mixture was stirred for 30 min at 25 ~C. The solvent was removed in vacuo to yield an oily residue which was dissolved in EtOAc and washed with brine. The organic layer was dried over Na2SO4 and concentrated in vacuo to yield the desired product as a colorless oil (0.42 g, >95~) which was subjected to a following reaction without purification.
H NMR (300 MHz, CDCl3):~ 1.86 (m ,2H), 2.04 (m, 2H), 2.62 (q, 2H, J=6.3 Hz), 3.83 (s, 3H), 4.17 (t, lH, J=6.3 Hz), 7.10 (d, lH, J=8.9 Hz), 7.45 (d, 2H, J=8.9 Hz).
Synthesis of (i)-6-Methoxy-5-amino-1,2,3,4-tetrahydronaphthalen-l-carbonitrile. A solution of 6-methoxy-5-nitro-1,2,3,4-tetrahydronaphthalene-1-carbonitrile (0.42 g, 1.8 mmol) and catalytic amount of 10~ Pd/C in 100 ml of MeOH was stirred under H2 for 12 h at 25 ~C. The reaction mixture was filtered and concentrated in vacuo to yield the desired product as a colorless oil (0.36 g, >95~). lH NMR (300 MHz, CDCl3):
1.93 (m, 4H), 2.48 (qt, 2H, J=5.7Hz), 3.79 (s, 3H), 3.99 (t, lH, J=6.3 Hz), 6.68 (dd, 2H, J=8.0, 8.9 Hz).

Synthesis of (i)-N-(5-cyano-2-methoxy-5~6~7~8-tetrahydronaphthalen-1-yl)methane-sulfonamide. To a solution of 6-methoxy-5-amino-1,2,3,4-tetrahydronaphthalene-l-carbonitrile (1.7 g, 8.3 mmol) in CA 02222~73 1997-11-27 W096J38143 P~T~S96/07979 20 ml of anhydrous pyridine was added methanesulfonyl chloride (;.9 ml, 1.2 mmol). The resulting solution was stirred for 2 h at 25 ~C. The reaction mixture was concentrated in vacuo to yield an oily residue which was redissolved in EtOAc and washed with aqueous NaHCO~. The organic layer was dried over Na2SO4 and concentrated in vacuo to provide an oily residue which was purified by column chromatography (EtOAc, neat) to yield 1.5 g (65~) of the desired product. lH NMR (300 MHz, CDCl3):~ 1.82 (m,lH), 1.94 (m, lH), 2.14 (m, 2H), 2.96 (s, 3H), 3 03 (q, 2H, J=8.8 Hz), 3.84 (s, 3H), 3.96 (t, lH, J=6.3 Hz), 6.85 (d, lH, J=8.9 Hz), 7.39 (d, lH, J=8.9 Hz).

Synthesis of ($)-N-t5-(4,5-Dihydro-lH-imidazol-2-yl)-2-methoxy-5,6,7,8-tetra-hydronaphthalen-1-yl]me~h~ns~ulfonamide. N-(5-Cyano-2-methoxy-5,6~,7,8-tetrahydronaphthalen-1-yl)methane-sulfonamide (1.5 g, 5.4 mmol) was dissolved in 200 ml of MeOH and cooled to O ~C.
The solution was then treated with dry HCl gas for 2 h, sealed tightly and stored for 12 h at 25 ~C. The solvent was removed and the residue was redissolved in 100 ml of MeOH, followed by addition of ethylenediamine (0.67 ml, 10 mmol). The resulting solution was stirred at reflux for 12 h. The reaction mixture was concentrated in vacuo, yielding an oily residue which was subjected to column chromatography (25~ NH3 sat'd MeOH-CHCl3) to provide 1.3 g (76~) of the desired product. lH NMR (300 MHz, CD30D): ~ 1.78 (m, 2H), 1.96 (m, lH), 2.12 (m, lH), 2.95 (broad t, 2H), 2.98 (s, 3H), 3.82 (s, 3H), 3.90 (broad s, 4H), 4.11 (t, lH, J=6.3 Hz), 6.96 (t, lH, J=8.9 Hz), 7.05 (t, lH, J=8.9 Hz).

Synthesis of (+)-N-t5-(4,5-Dihydro-lH-i~idazo1-2-yi)-2-hydroxy-5,6,7,8-tetra-hydronaphthalen-1-yl~meth~ne~ulfonamide (A-61603). To a solution of N-[5-CA 02222~73 1997-ll-27 W O96/38143 PCT~US96/n7979 (4,5-dihydro-lX-imidazol-2-yl)-2-methoxy-5,6,7,8-tetra-hydronaphthalen-l-yl]methanesulfonamide (0.3 g, 0.9 mmol) in 100 ml of CHCll was added BBr3 (2.0 ml, 2.0 mmol) at -78 ~C. The resulting reaction mixture was stirred for 12 h at 25 ~C. The reaction mixture was then recooled to -78 ~C and 2 ml of MeOH was added. The reaction mixture was warmed to 25 ~C and stirred for another 3 h. It was then concentrated in vacuo to provide a light yellow solid (0.35 g, ~95~) which was identified as the HBr salt of the desired product, mp 263-265 ~C. lH NMR (300 MHz, CD30D): ~ 1.78 (m, 2H), 1.90 (m, lH), 2.04 (m, lH), 2.93 (broad t, 2H), 3.05 (s, 3H), 3.86 (s, 4H), 4.06 (t, lH, J=6.3 Hz), 6.75 (d, lH, J=8.9 Hz), 6.92 (d, lH, J=8.9 Hz), 9.58 (s, lH); Anal. Cal. For Cl~HlgN3O3S.1.0~3r requires C, 43.7; H, 5.12; N, 10.7. Found: C, 42.8; H, 4.95; N, 10.3.

SynthesisofN-t2-Hydroxy-5-t2-methylamino)ethyl]phenyl~-methanesulfonamide (SR&F 102652)4-Hydroxy-N-methyl-3-nitrobenzeneacetamide. A mixture of 5 g (25.38 mmol) of 3-nitro-4-hydroxyphenylacetic acid (from Aldrich Chemical Co., Milwaukee, Wisconsin) in 20 mL of thionyl chloride was heated at reflux for 45 min. The reaction mixture was cooled and poured into 80 mL of hexane. The resulting precipitate was collected by filtration, washed with hexane, and air-dried to yield a yellow solid. A
solution of this acid chloride in 100 mL of dichloromethane was cooled in an ice bath and stirred while excess methylamine was distilled in dropwise. The mixture was stirred at room temperature overnight. The precipitated solid was collected by filtration and dissolved in water. It was acidified to pH 2 with 3 N HCl and extracted with dichloromethane to yield a yellow CA 02222~73 1997-11-27 W ~96138143 PCTnUS96107979 -29- ~
solid 4.2 g(80~). The title compound was used in the next step without further purification. lH NMR (300 MHz, CDCl3) ~ 10.50 (s, lH), 7.97 (s, lH), 7.50 (d, lH, J =
9.0 Hz), 7.11 (d, lH, J = 9.0 Hz), 5.4 (brs, lH), 3.49 5 (s, 2H), 2.78 (d, 3H, J = 6.0 Hz).

Synthesis of 4-Methoxy-N-methyl-3-nitrobenzeneacetamide.
To a solution of 4.2 g (19.8 mmol) of 4-hydroxy-N-methyl-3-nitrobenzeneacetamide in 50 mL of DMF containing 5.5 g of anhydrous potassium carbonate was added 5.6 mL
of dimethyl sulfate. The mixture was heated at 60-70 ~C
for 45 min, treated with an additional 3.0 mL of methyl sulfate, and heated for another 30 min. The mixture was cooled, poured into 200 mL water, and extracted with dichloromethane. The extracts were washed with water, dried and evaporated to give a solid which was recrystallized from ethanol-water to afford 3.7 g of a yellowish solid (82~).lH NMR (300 MHz, CDC].3) ~ 7.71 (s, lH), 7.47 (d, lH, J = 9.0 Hz), 7.25 (d, lH, J - 8.4 Hz), 5.71 (brs, lH),3.90 (s, 3H) 3.47 (s, 2H), 2.75 (d, 3H, J = 4.8 Hz).

S y n t h e~3i 8 o f 4 - M e t h ox y - N - m e t h y 1 -3 [(methylsulfonyl)amino]-benzeneacetamide. A solution of 3.6 g (16.3 mmol) of 4-methoxy-N-methyl-3-nitrobenzeneacetamide was hydrogenated using hydrogen gas at 50 psi in 40 mL of ethanol over 200 mg of Pd/C (10~) for 6 h. The catalyst was removed by filt:ration and solvent was evaporated to give 2.9 g of white solid. This solid was dissolved in 30 mL of pyridine and treated dropwise with 1.5 mL ~19.4 mmol) of methanesul~onyl chloride in 5 mL of pyridine. The reaction mixture was warmed to 65 ~C for 30 min and then stirred at room temperature overnight. The pyridine was evaporated and the residue taken up in 40 mL of water, adjusted to pH

W096/38143 CA 02222~73 Igs7-ll-27 PCT~S96/07979 6.7, and cooled in an ice bath. The resulting precipitate was removed by filtration and dried to give 1.7 g of an off-white solid(42~). Recrystallization from methanol gave white crystals.lH NMR (300 MHz, CDC13) ~ 7.35 (s, lH), 7.04 (d, lH, J = 9.0 Hz), 6.85 (d, lH, J = 8.4 Hz),6.78 (brs, lH),5.41 (brs, lH), 3.85 (s, 3H) 3.46 (s, 2H),2.94 (s, 3H), 2.73 (d, 3H, J = 4.8 Hz).

SyntheRi Q ofN-t2-Methoxy-5-~2-methylamino)ethyl]phenyl]-methanesulfonamide. A solution of 0.8 g (2.89 mmol) of 4-methoxy-N-methyl-3-[(methylsulfonyl)amino]-benzeneacetamide in 20 ml of dry THF was stirred and cooled in ice as a 1 M solution of borane in THF (15 mL) was added dropwise. After the addition was complete, the mixture was warmed to 65 ~C for 6 h. It was cooled and treated with 25 mL of methanol, followed by 1 mL of 6 N
HCl . The mixture was evaporated to yield a white residue which was dissolved in a minimum amount of hot methanol, filtered, and treated with ethyl acetate until cloudy and allowed to crystallize. The solid was removed by filtration and dried to give 0.54 g of white crystals (72~ H NMR (300 MHz, CD30D) ~ 7.28 (s, lH), 7.07 (d, lH, J = 9.0 Hz), 7.00 (d, lH, J = 8.4 Hz), 3.83 (s, 3H) 3.19-3.14 (m, 2H),2.90-2.85(m, 2H), 2.86 (s, 3H) 2.66 (s, 3H,).

Synthe~isofN-t2-Hydroxy-5-[2-methylamino)ethyl]phenyl]-methanesulfonamide ~SR&F 102652). A suspension of 0.2 g of N-[2-methoxy-5-[2-methylamino)ethyl]phenyl]-methanesulfonamide in 10 mL of dichloromethane in a dryice-2-propanol bath was treated with 4 mL of 1 M BBr3in dichloromethane. It was allowed to warm to room temperature and stirred overnight. The mixture was treated with 50 mL of methanol, stirred for 1 h, evaporated , and treated again with methanol, and W O96J38143 P'CTAUS96~0797g evaporated to dryness. This residue was taken up in minimum volume of hot methanol, treated with ethyl acetate, and allowed to crystallize to afford 0.17 g (67~) of tan crystals, mp 188-189 ~C.lH NMR (300 MHz, CD30D) ~ 7.23 (s, lH), 6.97 (d, lH, J = 9.0 Hz), 6 . 85 (d, lH, J = 8.4 Hz), 3.21-3.16 (m, 2H), 2~92 (s, 3H) 2.90-2.84(m, 2H), 2.68 (s, 3H,). Anal. Calcd for ClOHl7BrN203S . O . 05 CH2Cl2: C, 36. 69; H, 5.23; N, 8 . 50 .
Found: C, 36.57; H, 5.14; N, 8.26.
EXAMP~E 3 Synthesis of (i)-4-Methyl-6-methoxy-9-thiomethoxy-3,4,4a,5,10,10a-heY~hydro-2H-naphtho[2,3-b~1,4]-oxazine (SDZ NVI 085)1,4 Dihydro-5-methoxynaphthale!ne. To a refluxing solution of 1-methoxynaphthalene (Aldrich Chemical Co., Milwaukee, Wisconsin, 5.5 g, 34 mmol) in 80 ml of EtOH was added sodium (5.7 g, 250 mmol) in pieces under Ar. When all of the sodium was consumed, the reaction was cooled to 25 ~C and stirred for additional 3 h. The reaction mixture was carefully quenched by adding 100 ml of water and extracted with EtOAc. The organic layer was dried over Na2SO4 and concentrated in vacuo to yield an oily residue which was purified by column chromatography (30~ EtOAc-Hexane) to yield 2.8 g (52~) of the desired product as a colorless oil. lH NMR
(300 MHz, CDCl3): ~ 3.27(m, 2H), 3.39 (m, 2H), 3.82 (s, 3H), 5.85 (m, 2H), 6.67-6.75 (m, 2H), 7.13 (t, lH, J=7.9 Hz).
~ynthesis of (+)-6-Methoxy-la,2,7,7a-tetrahydro-1-oxa-cyclopropan~b]naphthalene. To a solution of 1,4 dihydro-5-methoxynaphthalene (2.8 g, 17.5 mmol) in 50 ml of CH2Cl2 was added MCPBA (8.5 g, 50 mmol) in one portion. The resulting solution was stirred for 3 h at 0 ~C . The CA 02222~73 1997-ll-27 W O96/38143 PCTrUS96/07979 reaction mixture was diluted with CH2Cl2 and poured in~o a mixture of ice (50 g) and NaHCO3 sat'd aqueous solution (150 ml). The organic layer was separated and the aqueous layer was extracted with CH2Cl2. The comDined organic layers were washed with aqueous NaHCO3, dried over MgSO4 and concentrated in vacuo to yield an oil which was subjected to column chromatography (CH2Cl2, neat) to yield 1.7 g (59~) of the desired product as a colorless oil.
lH NMR (300 MHz, CDCl3): ~ 2.78 (d, lH, J=18.2 Hz), 3.21(AB q, 2H, J=18.3 Hz), 3.47 (m, 3H), 3.77 (s, 3H), 6.66(m, 2H), 7.08 (t, lH, J=7.8 Hz).

Synthe~3is of ( + ) - 3 -Azido- 5 -methoxy- 1, 2, 3, 4 -tetrahydronaphthalen-2-ol. To a solution of 6-methoxy-la~2~7~7a-tetrahydro-l-oxa-cyclo-propan[b]naphthalene (1.7 g, 9.7 mmol) in 20 ml of DMSO was added sodium azide (5.6 g, 86 mmol) and H2SO4 (0.2 ml). The resulting suspension was stirred for 17 h at 25 ~C. The reaction mixture was diluted with EtOAc and washed with brine.
The organic layer was dried over MgSO4 and concentrated in vacuo, yielding an oil which was subjected to column chromatography (EtOAc, neat) to provide 1.9 g (89~) of a mixture of two regioisomers in a 1:1 ratio. Two isomers were separated by fractional recrystallization in hexane to provide 0.6 g of the desired product, mp 83-84 ~C. lH
NMR (300 MHz, CDCl3): ~ 1.61 (s, lH), 2.60 (dd, lH, J=10.6, 17.0 Hz), 2.84 (dd, lH, J=10.1, 15.9 Hz), 3.19 (dd, lH, J=5.3, 15.9 Hz), 3.37 (dd, lH, J=5.8, 17.0 Hz), 3.70 (m, lH), 3.84 (s, 3H), 3.88 (m, lH), 6.70(m, 2H), 7.17(t, lH, J=7.8 Hz).

Synthesis of ( + ) - 3 -Amino - 5 -methoxy- 1, 2, 3, 4 -tetrahydl o.,aphthalen- 2 - ol . A solution of 3-azido-5-methoxy-1,2,3,4-tetrahydronaphthalen-2-ol (1.8 g, 8~0 mmol) in 150 ml of MeOH was stirred with 10~ Pd/C (20 mg) CA 02222~73 l997-ll-27 W O96J38143 PCTAUSg~07g7~

under H2 (18 psi) for 4 h. The reaction mixture was filtered and concentrated in vaCuo to provide 1.4 g (91~) of the desired product as a colorless oil which was used in the next reaction without purification. lH NMR ~300 MHz, CDCl3~: ~ 2.0 (broad s, 2H), 2.24 (dd, lH, J=10.4, 16.8 Hz), 2.84 (m, 2H), 3.16 (m, 2H), 3.59 (m, lH), 3.78 (s, 3H), 6.66 (m, 2H), 7.09 (t, lH, J=7.9 Hz).

Synthesis of (+)-6-Methoxy-4a,5,10,10a-tetrahydro-4H-naphthol2,3-b]tl,4]oxazin-3-one. To a solution of 3-amino-5-methoxy-1,2,3,4-tetrahydronaphthalen-2-ol(1.7g, 8.8 mmol) and triethylamine (1.5 ml, 11 mmol~ in 100 ml of CH2Cl2 was added chloroacetyl chloride (1.0 g, 8.9 mmol) in 10 ml of CH2Cl2 dropwise at 0 ~C. The resulting solution was stirred for 1.5 h at 25 ~C. The reaction mixture was ther. diluted with EtOAc and washed with lN
aqueous HCl. Organic layer was dried over Na2SO4 and concentrated in vacuo, yielding an oil which corresponds to the amide. The oily residue was redissolved in 20 ml of THF, and NaH (0.35 g, 8.8 mmol) and tetrabutylammonium iodide (0.25 g, 0.67 mmol) were added into the solution at 0 ~C. The reaction mixture was stirred for 12 h at 25 ~C. It was diluted with EtOAc and washed with brine. The organic layer was dried over Na2SO4 and concentrated in vacuo, yielding an oily residue which was purified by column chromatography (50~ CH2Cl2-EtOAc) to provide 1.4 g (68~) of the desired product. 1H NMR (300 MHz, CDCl3):
2.45 (dd, lH, J=10.5, 16.7 Hz), 2.85 (m, lH), 3.11-3.34 (m, 2H), 3.65(m, 2H), 3.79 (s, 3H), 4.28 (AB q, 2H, 16.7 Hz), 6.68 (m, 2H), 7.07 (t, lH, J=7.9 Hz).

Synthesis of (+)-6-Methoxy-3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3-b]tl,4]oxazine. To a solution of 6-methoxy-4a~5~lo~loa-tetrahydro-4H-naphto[2~3-b][l~4]oxazin-3-one (1.4 g, 6.0 mmol) in 100 ml of THF was added 10 ml o~

W 096/38143 CA 02222~73 1997-11-27 PCTrUS96/07979 LiAlH4 solution in THF (10 mmol). The resulting solution was stirred for 2 h at reflux. The reaction was quenched with ice and the reaction mixture was then diluted with EtOAc. Filtration of the reaction mixture provided a clean organic layer which was concentrated in vacuo to yield 1.2 g (92~) of the desired product as a colorless oil. lH NMR (300 MHz, CDCl3): ~ 1.85 (s, lH), 2.34(dd, lH, J=12.0, 17.0 Hz), 2.78-3.16 (m, 6H), 3.50 (ddd, lH, J=5.2, 6.8, 10.5 Hz), 3.74 (m, lH), 3.81 (s, 3H), 3.94 (m, lH), 6.70 (m, 2H), 7.16 (t, lH, J=7.6 Hz).

Synthesis of (I)-6-Methoxy-3,4,4a,5,10,10a-hexahydro-2H-naphtho12,3-b]~1,4]oxazine-4-carboxylic acid benzyl ester. To a solution of 6-methoxy-3,4,4a,5,10,10a-hexahydro-2H-naphtho-[2,3-b][1,4]oxazine (0.62 g, 2.7 mmol) in 10 ml of CH2Cl2 was added triethylamine (1.0 ml, 7.2 mmol) and benzyl chloroformate (0.6 ml, 4.1 mmol).
The resulting mixture was stirred at 25 ~C for 3 h. It was then diluted with 100 ml of EtOAc and washed with brine. The organic layer was dried over Na2SO4 and concentrated in vacuo, yielding an oily residue which was subjected to column chromatography (20~ EtOAc-CH2Cl2) to provide 0.46 g (48~) of the desired product. lH NMR (300 MHz, CDCl3): ~ 2.45 (dd, lH, J=10.6, 16.6 Hz), 2.83 (dd, lH, J=10.3, 16.0 Hz), 3.04 (dd, lH, J=5.0, 16.0 Hz), 3.68-3.88 (m, 6H), 3.76 (s, 3H), 4.02(m, lH), 5.16 (A~3 q, 2H, J=17.6 Hz), 6.66 (m, 2H), 7.09 (t, lH,J=7.8 Hz).

Synthes~s of (l)-4-Methyl-6-methoxy-3,4,4a,5,10,1Oa-hexahydro-2H-naphtho~2,3-b][1,4]oxazine. To a solution of 6-methoxy-3,4,4a,5,10,10a-hexahydro-2H-naphtho-[2,3-b][l,4]oxazine-4-carboxylic acid benzyl ester (0.40 g, 1.1 mmol) in THF was added 2.8 ml of LiAlH4 solution(1.0 M)in THF. The resulting solution was stirred at reflux ~or 3 h. The reaction mixture was quenched with ice, CA 02222~73 1997-11-27 W 096/38143 P~TrUS96/07979 diluted with EtOAc and filtered through Celite. The organic layer was dried over NazSO4 and concentrated in vacuo, yielding an oily residue which was subjected to column chromatography (5~ MeOH-EtOAc) to yield 0.19 g (75%) of the desired product. lH NMR (300 MHz, CDCl,~:
2.07 (m, lH), 2.28 (m, lH), 2.37 (s, 3H), 2.49 (m, lH!, 2.77 (m, 2H), 2.97 (dd, lH, J=5.5, 16.1 Hz), 3.28 (dd, lH, J=5.5, 17.0 HZ), 3.55 (m, lH), 3.79 (s, 3H), 3.86 (m, 2H), 6.67 (m, 2H), 7.09 (t, lH, J=7.8 Hz).
Synthesis of (+)-4-Methyl-6-methoxy-9-iodo-3,4,4a,5,10,10a-h~Y~hydro-2H-naphto-t2~3-~]tl~l4]oxazine~
A solution of 4-methyl-6-methoxy-3,4,4a,5,10,10a-hexahydro-2H-naphtho[2,3-b][1,4]oxazine (0.37 g, 1.6 mmol) was dissolved in 7 ml of AcOH and heated to 50 ~C.
To the solution of the amine was added a solution of Hg(OAc)2 (0.62 g, 19 mmol) and I2 (1.0 g, 3.8 mmol) in 30 ml of AcOH. The resulting solution was stirred for 1 h at 50 ~C and 1.5 h at 25 ~C. The reaction mixture was filtered to remove mercury salts and concentrated in vacuo, yielding an oily residue which was subjected to column chromatography (5~ NH3 sat'd MeOH-EtOAc) to yield O.25 g (44~) of the desired product. lH NMR (300 MHz, CDCl3): ~ 2.04 (m, lH), 2.32 (dd, lH, J=11.0, 18.0 Hz), 2.41 (s, 3H), 2.49 (m, lH), 2.62 (dd, lH, J=11.0, 17.0 Hz), 2.76 (m, lH), 3.12 (dd, lH, J=6.0, 17.0 Hz), 3.33 (dd, lH, J=5.6, 17.0 Hz), 3.56 (m, lH), 3,81 (s, 3H), 3.88 (m, 2H), 6.47 (d, lH, J=7.8 Hz), 7.66 (d, lH, J=7.8 Hz).
Synthesis of (+)-trans-4-Methyl-6-methoxy-9-thiomethoxy-3,4,4a,5,10,10a-h~Y~hydro-2H-naphtho[2,3-b]tl,4]oxazine (SDZ NVI 085). To a suspension of CH3SLi (0.30 g, 5.5 mmol) in 6 ml of DMSO was added 4-methyl-6-methoxy-9-iodo-3,4,4a,5,10,10a-hexahydro-2H-naphtho-[2,3-CA 02222~73 1997-11-27 b] [1,4]oxazine (0.25 g, 0.7 mmol) and Cu2O (1.3 g, 9.
mmol). The reaction mixture was stirred for 5 h at 80 "C.
It was diluted with EtOAc and washed with 4N NH40H several times. The organic layer was dried over Na2SO and 5 concentrated in vacuo to yield an oily residue which was subjected to column chromatography (S~ MeOH-CH2Cl2) to yield 0.15 g (7996) of the desired product. lH NMR (300 MHz, CDCl3): ~ 2.09 (m, lH), 2.32 (dd, lH, J=10.5, 16.5 Hz), 2.39 (s, 3H), 2.41 (s, 3H), 2.45-2.80 (m, 3H), 3.27-3.40 (m, 2H), 3.60 (m, lH), 3.82 (s, 3H), 3.90 (m, 2H), 6.72 (d, lH, J=7.8 Hz), 7.14 (d, lH, J=7.8 Hz). The product obtained was converted to the HCl salt and recrystallized from EtOAc-Et2O to obtain 0.17 g of the product as a white solid: mp 215-217 ~C; Anal. Cal. For C1sH2lNO2S.l.OHCl requires C, 56.9; H, 6.69; N, 4.43.
Found: C, 56.5; H, 6.77; N, 4.38.

Synthesisof 1-(6,7-Dimethoxy-3,4-dihydro-lN-isoquinolin-2-yl)eth~nr~n~. To a stirred solution of 6,7-dimethoxy-1~2~3~4-tetrahydroisoquinoline (3.00 g, 15.4 mmol, 1.00 equiv) in anhydrous pyridine (100 mL) under argon at room temperature was added acetic anhydride (14.5 mL, 154 mmol, 10.0 equiv) over 15 min. The resulting mixture was stirred at room temperature for 2 h, and then at reflux for 6 h. The volatiles were removed by rotary evaporation at 80 ~C under high vacuum. The residue was flash chromatographed on silica gel (MeOH-CH2Cl2 8:92) to afford 3.21 g (89~) of viscous brown oil. The lH NMR
spectrum reflected the presence of two slowly interconverting conformers in a ratio of 1.2 :1 at room temperature. lH NMR (300 MHz, CDC13) for conformer 1: ~
2.18 (s, 3 H), 2.83 (t, J = 5.9 Hz, 2 H), 3.67 (t, ~ =
5.9 Hz, 2 H), 3.86 (s, 3 H), 3.87 (s, 3 H), 4.66 (s, 2 CA 02222~73 1997-11-27 WO g6138~43 P~T/US96~07~7g H), 6.63 (s, 1 H), 6.65 (s, 1 H) . For conformer 2: ~
2.19 (s, 3 H), 2.77 (t, J = 5.9 Hz, 2 H), 3.81 (t, J =
5.9 Hz, 2 H), 3.86 (s, 3 H), 3.87 (s, 3 H), 4.56 (s, 2 H), 6.59 (s, 1 H), 6.63 (s, 1 H) .
- Synthe sis o f 2-tl-(6,7-Dimethoxy-3,4-dihydro-lH-isoquinolin-2-yl)-ethylidineamino]-4,5-dimethoxybenzonitrile. To a stirred solution of 1- (6,7-dimethoxy- 3,4 -dihydro- lH- isoquinolin- 2 -yl ) ethanone ( l .00 g, 4.25 mmol, 1.00 equiv) in CHCl3 at room temperature under argon was added POCl3 (143 ~lL, 1.53 mmol, 0.36 equiv) . After 10 min, 2-amino-4,5-dimethoxybenzonitrile (763 mg, 4.28 mmol, 1.01 equiv) was added and the mixture was heated at ref lux overnight . The mixture was cooled to room temperature and poured into 1 M aq. NaOH solution (50 mL), and the aqueous phase was extracted with CH2Cl2 (3 x 50 TnL) . The combined organic solutions were dried over MgSO4 and concentrated . The residue was f lash chromatographed on silica gel (MeOH-CH2Cl2 5:95) to afford 482 mg (28~) of yellow solid: lH NMR (300 MHz, CDCl3) ~
2.02 (s, 3 H), 2.87 (t, LJ = 6.0 Hz, 2 ~I), 3.78 (t, J =
6.0 Hz, 2 H), 3.85 (s, 6 H), 3.87 (s, 6 H), 4.70 (s, 2 H), 6.35 (s, 1 H), 6.65 (s, 2 H), 6.92 (s, 1 H); CIMS
(CH4) 424 (M + C2Hs) ', 396 (M + H) + .
Synthesisof2-(6,7-Dimethoxy-3,4-dihydro-lH-i~30quinolin-2-yl)-6,7-dimethoxyquinolin-4-ylamine hemifumarate hydrate (abanoquil). To a stirred solution of 2- [1- (6,7-dimethoxy-3, 4 -dihydro- 1~- isoquinoli.n- 2 -yl ) -ethylitli n~mi no] -4,5-dimethoxybenzonitrile (471 mg, 1.19 mmol, 1.00 equiv) in refluxing anhydrous N,N-dimethylacetamide (24 mL) under argon was added ZnCl2 (339 mg, 2.49 mmol, 2.10 equiv) in three portions over 1 h .
The solvent was removed by distillation at 70 ~ C under 35 high vacuum. Ether (40 mL) was added to the residue, W 096/38143 CA 02222~73 1997-11-27 PCTrUS96/07979 which was broken up with a stirring rod, and the mixture was stirred at 0~C to precipitate the product. The supernatant was discardea, and the precipitate was washed twice more at 0~C with ether. The solid residue was stirred with 1 M aq. NaOH (25 mL) and CH2Cl2 (25 m~) for 10 min, and the aqueous phase was extracted with CH2C1 (2 x 25 mL). The combined organic solutions were dried over MgSO4 and concentrated to give 493 mg of brown oil, which was flash chromatographed on silica gel (MeOH-CH2Cl2 12:88 followed by 2-propylamine-CH2Cl2 5:95) to afford 151 mg (38~) of 2-(6,7-dimethoxy-3,4-dihydro-lH-isoquinolin-2-yl)-6,7-dimethoxyquinolin-4-ylamine as a tan solid: lH
NMR (300 MHz, CDCl3) ~ 2.90 (t, ~ = 5.7 Hz, 2 H), 3.81 (t, ~ = 5.7 Hz, 2 H), 3.86 (s, 3 H), 3.88 (s, 3 H), 3.93 (s, 3 H), 3.97 (s, 3 H), 4.64 (s, 2 H), 6.05 (s, 1 H), 6.66 (s, 1 H), 6.75 (s, 1 H), 7.02 (s, 1 H), 7.23 (s, 1 H);
CIMS (CH4) 424 (M + C2Hs)', 396 (M + H)+. To a solution of 2-(6,7-dimethoxy-3,4-dihydro-lH-isoquinolin-2-yl)-6,7-dimethoxyquinolin-4-ylamine (150 mg) in hot CH2Cl2 (4.5 mL) and MeOH (1.5 mL) was added a solution of fumaric acid (22.8 mg, 0.196 mmol, 0.50 equiv) in hot MeOH (3.0 mL). - The resulting mixture was concentrated and the product was recrystallized from MeOH with hot filtration to afford, after filtration, 85 mg of light brown solid:
m.p. 239-240 ~C. Calcd. for C22H2sN3O4 0.5 C4 H4O4 ~ 0.75 H 2~ C, 61.73; H, 6.15; N, 9.00. Found: C,61.77; H, 6.17; N, 8.91.

Synthesi~ of (+)-2,6-Dimethyl-4-(4-nitrophenyl) -1,4-dihydro-pyridine-3,5-dicarboylicacidt3-(4,4-diphenyl piperidin-1-yl)propyl] ester methyl ester. A solution of methyl 3-aminocrotonate (265 mg, 2.3 mmol, 1.0 equiv), 4-nitrobenzaldehyde (348 mg, 2.3 mmol, 01.0 equiv), and CA 02222~73 1997-11-27 W O96138143 PCTrUS96M7979 acetoacetic acid 3-[4,4-diphenylpiperidin-1-yl)propyl]
ester (872 mg, 2.3 mmol, 1.0 equiv; Flockerzi, D.; Ulrich, W.-R. U.S. Patent 4,975,440, 1990) in isopropanol was ~ refluxed under argon with stirring for 68 hours. Cooling and removal of solvent gave a residue, which was puri~ied by flash chromatography (SiO~, EtOAc-hexane 1:1 and 2:1 followed by EtOAc) to afford 717 mg (51~) of yellow solid: lH NMR ~300 MHz, CDCl3) ~ 1.73 (m, 2 H), 2.22 (m, 2 H), 2.30-2.51 (m, 8 H), 2.34 (s, 3 H), 2.35 (s,~3 H), 3.63 (s, 3 H), 4.05 (dt, ~ = 2.1, 7.9 Hz, 2 H), 5.06 ~s, 1 H), 5.73 (br s, 1 H), 7.14 (m, 2 H), 7.27 (m, 8 H), 7.42 (dm, J = 8.8 Hz, 2 H), 8.06 (dm, J = 8.~ Hz, 2 H!;
13C NMR (75 MHz, CDCl3) ~ 15.30, 19.65, 26.32, 36.11, 39.88, 44.60, 50.60, 51.12, 55.34, 62.66, 102.99, 107.55, 123.39, 125.67, 127.12, 128.33, 128.65, 144.~30, 144.93, 146.36, 147.50, 154.78, 166.91, 167.43; IR (neat) 1698.0, 1684.7, 1517.5, 1345.7 cm~1; CIMS (NH3) 610 (M + 1)+, 553, 338.

Syn~thesiR of (~)-2,6-Dimethyl-4-(4-nitrophenyl) -1,4-dihydro-pyridine-3,5-dicarboxylicacid [3-(4,4-diphenylpiperidin-1-yl)propyl] estermLethyl ester hydrochloride (Compound 1). To a solution of= 2,6-dimethyl-4-(4-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylicacid[3-(4,4-diphenyl-piperidin-1-yl)propyl]
ester methyl ester (710 mg, 1.16 mmol, 1.0 equiv) in EtOH
(5 mL) was added a solution of HCl in ether (1.0 M, 1.5 mL, 1.5 mmol, 1.3 equiv). The solvents were removed and the residue was dissolved in CH2Cl2. This solution was added dropwise to 25 mL of ether to afford, after filtration, 500 mg of yellow crystalline solid: m.p.
152-153 ~C. Calcd. for C36H39N3O6 HCl: C, 66.92; H, 6.24;
N, 6.50. Found: C, 66.70; H, 5.99; N, 6.27.

WO 96138143 CA O 2 2 2 2 ~ 7 3 19 9 7 - 1 1 - 2 7 PCT/US96/07979 Synthesis of (+)-2-Amino-1-(2, 5 dimethoxyphenyl) ethanol (ST-1059)(2, 5, Dimethoxyphenyl)-hydroxy-acetonitrile.
To a solution of 4.0 g ~24 mmol) of 2.5-dimethoxybenzaldehyde in 40 mL of dichloromethane containing 0.078 g(5~ mmol) of KCN and 0.31 g tS~ mmol) of 18-crown-6, was added trimethylsilylcyanide 2.62 g(26.4 mmol) dropwise. The reaction mixture was stirred at room temperature for 24 h. The reaction mixture was concentrated, dissolved in chloroform and washed with water, dried (sodium sulfate), concentrated in vacuG and purified by flash chromatography (silica gel;
hexane:ethyl acetate, 8:2) to afford 2.85 g (66~) of the desired compound as a yellow oil. lH NMR (300MHz, CDCl~) ~ 6.95 (d, lH, J = 2.7 Hz), 6.85-6.84 (m, 2H), 5.52 (d, lH, J = 7.5 Hz), 3.93 (d, lH, J = 7.5 Hz), 3.83 (s, 3H), 3.73 (s, 3H) Synthesis o~(+)-2-Amino-1-(2, 5 dimethoxyphenyl) ethanol (ST-1059). A solution of 2.84 g (14.7 mmol) of (2, 5 dimethoxyphenyl)-hydroxy acetonitrile in 10 ml of dry THF was stirred and cooled using ice bath as a l M
solution of borane in THF (90 mL) was added dropwise.
After the addition was complete, the mixture was heated at reflux for 20 h. It was cooled and treated with 40 mL
of 6 N hydrochloric acid and washed with ethyl acetate.The aqueous layer was neutralized with 1 N sodium hydroxide and extracted with ethyl acetate and concentrated to afford the desired compound as a white solid 1.5 g (52~ H NMR (300MHz, CDCl3) ~ 6.97 (d, lH, J = 2.5 Hz), 6.73-6.69(m, 2H), 4.83-4.81(t, lH, J = 4.1 Hz), 3.72(s, 3H), 3.71(s, 3H), 2.89-2.65(m, 2H), 2.16(brs, 2H) CA 02222~73 1997-11-27 W096J38~43 PCT~S96S07979 ~XAMPLE 7 The binding and functional properties of compounds at the ~ different human receptors were determined in ~vitro using cultured cell lines that selectively express the receptor of interest. These cell lines were prepared by transfecting the cloned cDNA or cloned genomic DNA or constructs containing both genomic DNA and cD~A encoding the human ~-adrenoceptors as follows:
Human ~l~ Adrenoce~tor. The entire coding region of the alA receptor (1719 bp), including 150 basepairs of 5' untranslated sequence (5' UT) and 300 bp of 3' untranslated sequence (3' UT), was cloned into the~BamHI
and ClaI sites of the polylinker-modified eukaryotic expression vector pCEXV-3, called EXJ.HR. The construct involved the ligation of partial overlapping human lymphocyte geno~nic and hippocampal cDNA clones: 5' sequences were contained on a 1.2 kb SmaI-XhoI genomic fragment (the vector-derived BamHI site was used for subcloning instead of the internal insert-derived SmaI
site) and 3' sequences were contained on an 1 3 kb XhoI-ClaI cDNA fragment (the ClaI site was from the vector polylinker). Stable cell lines were obtained by cotransfection with the plasmid ~lA/EXJ ~expression vector containing the ~lA receptor gene) and the plasmid pGCcos3neo (plasmid containing the aminoglycoside transferase gene) into LM(tk-), CHO, and NIH3T3 cells, using calcium phosphate technique. The cells were grown, in a controlled environment ~37~C , 5~ C~2), as monolayers in Dulbecco's modified Eagle's Medium (GIBCO, Grand Island, NY) containing 25mM glucose and supplemented with lO~ bovine calf serum, 100 units/ml penicillin g, and 100 ~g/ml streptomycin sulfate. Stable clones were then selected for resistance to the antibiotic G-418 (1 CA 02222~73 1997-11-27 W O96/38143 PCTrUS96/07979 mg/ml), and membranes were harvested and assayed for their ability to bind [3H]prazosin as described below (see "Radioligand Binding assays").

~uma~ ~,p AdrenoceDtor. The entire coding region of the ~lB receptor (1563 bp), including 200 basepairs and 5~
untranslated sequence (5' UT) and 600 bp of 3' untranslated sequence t3' UT), was cloned into the EcoRI
site of pCEXV-3 eukaryotic expression vector. The construct involved ligating the full-length containing EcoRI brainstem cDNA fragment from ~ ZapII into the expression vector. Stable cell lines were obtained as described above.

Human ~l~ AdrenocePtor. The entire coding region of the cYlC receptor (1401 bp), including 400 basepairs of 5' untranslated sequence (5' UT) and 200 bp of 3~
untranslated sequence (3' UT), was cloned into the KpnI
site of the polylinker-modified pCEXV-3-derived eukaryotic expression vector, EXJ.RH. The construct involved ligating three partial overlapping fragments: a 5' 0.6kb HincII genomic clone, a central 1.8 EcoRI
hippocampal cDNA clone, and a 3' 0.6Kb PstI genomic clone. The hippocampal cDNA fragment overlaps with the 5' and 3' genomic clones so that the HincII and PstI
sites at the 5' and 3' ends of the cDNA clone, respectively, were utilized for ligation. This full-length clone was cloned into the KpnI site of the expression vector, using the 5' and 3' KpnI sites of the fragment, derived from vector (i.e., pBluescript) and 3'-untranslated sequences, respectively. Stable cell lines were obtained as described above.

CA 02222~73 1997-11-27 W O ~6138143 PCTrUS96/07979 Radioliqand Bindinq Assavs.

Human ~,-AdrenocePtors. Transfected cells from culture flasks were scraped into 5ml of 5mM Tris-HCl, 5mM EDTA, pH 7.5, and lysed by sonication. The cell lysates were centrifuged at 1000 rpm for 5 min at 4~C, and the supernatant was centrifuged at 30,000 x g for 20 min a.
4~C. The pellet was suspended in 50mM Tris-HCl, lmM
MgCl2, and 0.1~ ascorbic acid at pH 7.5. Binding of the ~1 antagonist [3H]prazosin (0.5 nM, specific activity:
about 76.2 Ci/mmol) to membrane preparations of LM(tk-) cells was done in a final volume of 0 25 ml and incubated at 37~C for 20 min. Nonspecific binding was defined as that binding which remained in the presence of 10 ~M
phentolamine(a concentration at least 100-fold greater than the affinity of phentolamine at any human ~-adrenoceptors). The reaction was stopped by filtration through GF/B filters using a cell harvester. Equilibrium competition binding assays, routinely consisting of 7 different concentrations of the tested compounds, were analyzed using a non-linear regression curve-fitting computer program to obtain IC50 values. The ICso values were converted to affinity constants (pKI) by the method of Cheng and Prusoff (1973).
Human ~2-Adrenoce~tors. To determine the affinity of compounds at the ~2 receptors, LM(tk-) cell li.nes stably transfected with the genes encoding the ~ZA~ ~2B/ and ~.
receptors were used. Cell lysates were prepared as described above (see Radioligand Binding Assays), and suspended in 25mM glycylglycine buffer (pH 7.6 at room temperature). Equilibrium competition binding assays were performed using [3H]rauwolscine (0.5nM), and nonspecific binding was determined by incubation with lO~M phentolamine. The bound radioligand was separated W O 96/38143 CA 02222~73 1997-11-27 PCTrUS96107979 by filtration through GF/B filters using a cell harvester.

Human Histamine H1 RecePtor. The coding sequence of the human histamine H1 receptor, homologous to the bovine Hl receptor, was obtained from a human hippocampal cDNA
library, and was cloned into the eukaryotic expression vector pCEXV-3. The plasmid DNA for the Hl receptor is designated pcEXV-Hl, and was deposited on November 6, 1992 under ATCC Accession No. 75346. This construct was transfected into COS-7 cells by the DEAE-dextran method.
Cells were harvested after 72 hours and lysed by sonication in 5mM Tris-HCl, 5mM EDTA, pH 7.5. The cell lysates were centrifuged at 1000 rpm for 5 min at 4~C, and the supernatant was centrifuged at 30,000 x g for 20 min. at 4~C. The pellet was suspended in 37.8 mM NaHPO4, 12.2 mM KH2PO4, pH 7.5. The binding of the histamine H~
antagonist [3H]mepyramine (lnM, specific activity: about 24.8 Ci/mM) was done in a final volume of 0.25 ml and incubated at room temperature for 60 min. Nonspecific binding was determined in the presence of 10 ~M
mepyramine. The bound radioligand was separated by filtration through GF/B filters using a cell harvester.

Human Histamine H2 Receptor. The coding sequence of the human H2 receptor was obtained from a human placenta genomic library, and cloned into the cloning site of PCEXV-3 eukaryotic expression vector. The plasmid DNA
for the H2 receptor is designated pcEXV-H2, and was deposited on November 6, 1992 under ATCC Accession No.
75345. This construct was transfected into COS-7 cells by the DEAE-dextran method. Cells were harvested after 72 hours and lysed by sonication in 5mM Tris-HCl, 5mM
EDTA, pH 7.5. The cell lysates were centrifuged at 1000 rpm for 5 min at 4~C, and the supernatant was centrifuged CA 02222~73 1997-11-27 WO g6~38143 P~r/USg61~7g7g at 30,000 x g for 20 min at 4 ~C. The pellet was suspended in 37.8 mM NaHPO4, 12.2 mM K2PO4, pH 7.5. The blnding of the histamine H2 antagonist [3H]tiotidine (5nM, specific activity: about 70 Ci/mM) was done in a final volume of 0.25 ml and incubated at room temperature for min. Nonspecific binding was determi~ed in the presence of 10 ~M histamine. The bound radioligand was separated by filtration through GF/B ~ilters using a cell harvester.

Human Serotonin RecePtors. 5-HTlDa, 5-HTlD~, 5-HTlE, 5-HTlF, and 5-HT,Receptors: The cell lysates of LM(t:k-) clonal cell line stably transfected with the genes encoding each of these 5-HT receptor-subtypes were prepared as described above. The cell line for the 5-HT1D~ receptor, designated as Ltk-8-30-84, was deposited on April 17, 1990, and accorded ATCC Accession No. CRL 10421. The cell for the 5-HTlD~ receptor, designated as Ltk-ll, was deposited on April 17, 1990, and accorded ATCC Accession No. CRL 10422. The cell line for the 5-HTlE receptor, designated 5-HTlE-7, was deposited on November 6, 1991, and accorded ATCC Accession No. CRL 10913. The cell line for the 5-HT1F receptor, designated L-5-HT1F, was deposited on December 27, 1991, and accorded ATCC Accession No. CRL
10957. The cell line for the 5-HT7 receptor, designated as L-5-HT-4B, was deposited on October 20, 1992, and accorded ATCC Accession No. CRL 11166. These preparations were suspended in 50mM Tris-HCl buffér (pH
7.4 at 37~C) containing 10 mM MgCl2, 0.2 mM EDTA, 10~M
pargyline, and 0.1~ ascorbate. The affinities of compounds were determined in equilibrium competition binding assays by incubation for 30 minutes at 37~C in the presence of 5nM [3H]serotonin. Nonspecific binding was determined in the presence of 10~M serotonin. The bound radioligand waC separated by filtration through CA 02222~73 1997-ll-27 W O96/38143 PCTrUS96/07979 GF/B filters using a cell harvester.

Human 5-HT2 RecePtor. The coding sequence of the human 5-HT2 recept~r was obtained from a human brain cortex cDNA
library, and cloned into the cloning site o~ pCEXV-3 eukaryotic expression vector. This construct was transfected into COS-7 cells by the DEAE-dextran method.
Cells were harvested after 72 hours and lysed by sonication in 5mM Tris-HCl, 5mM EDTA, pH 7.5. This cell line was deposited with the ATCC on October 31, 1989, designated as L-NGC-5-HT2, and was accorded ATCC Accession No. CRL 10287. The cell lysates were centrifuged at 1000 rpm for 5 minutes at 4~C, and the supernatant was centrifuged at 30,000 x g for 20 minutes at 4~C. The pellet was suspended in 50mM Tris-HCl buffer (pH 7.7 at room temperature) containing 10 mM MgSO4, 0.5mM EDTA, and 0.1~ ascorbate. The affinity of compounds at 5-HT2 receptors were determined in equilibrium competition binding assays using [3H]ketanserin (lnM). Nonspeci~ic binding was defined by the addition of 10~M mianserin.
The bound radioligand was separated by filtration through GF/B filters using a cell harvester.

5-HTl~ rece~tor. The cell line for the 5-HTlA receptor, designated 5-HTlA-3, was deposited on May 11, 1995, and accorded ATCC Accession No. CRL 11889. The cDNA
corresponding to the 5-HTlA receptor open reading frames and variable non-coding 5'- and 3'-regions, was cloned into the eukaryotic expression vector pCEXV-3. These constructs were transfected transiently into COS-7 cells by the DEAE-dextran method, and harvested after 72 hours.
Radioligand binding assays were performed as described above for the 5-HT2 receptor, except that [3H]-8-oH-DPAT
was used as the radioligand and nonspecific binding was CA 02222~73 1997-11-27 WO96~38143 P~S9610797 determined by the addition of 10~M mianserin.

Human Dopamine D7 Rece~tors. The affinity of compounds at the D2 receptor were determined using membrane preparations from COS-7 cells transfected with the gene encoding the human D2 receptor. The coding region for the human D2 receptor was obtained from a human striatum cDNA library, and cloned into the cloning site of PCDNA
1 eukariotic expression vector. The plasmid DNA ~or the D2 receptor is designated pcEXV-D2, and was deposited on November 6, 1992 under ATCC Accession No. 75344. This construct was transfected into COS-7 cells by the DEAE-dextran method. Cells were harvested after 7~ hours and lysed by sonication in 5mM Tris-HCl, 5mM EDT~, pH 7.5.
The cell lysates were centrifuged at 1000 rpm for 5 minutes at 4~C, and the supernatant was centrifuged at 30,000 x g for 20 minutes at 4~C. The pellet was suspended in 50 mM Tris-HCl (pH 7.4) containing lmM EDTA, 5mM KCl, 1.5mM CaCl2, 4mM MgCl2, and 0.1~ ascorbic acid.
The cell lysates were incubated with ~3Hjspiperone (2nM), using 10~M (+)Butaclamol to determine nonspeci~ic binding.

Other Dopamine receptors were prepared by known methods.
(Dl: Dearry et al., Nature, 347, 72, (1990), deposited with the European Molecular Biological Laboratory (EMBL) Genbank as X55760; D3: Sokoloff, P. et al., Nature, 347, 146 (1990), deposited with the European Molecular Biological Laboratory (EMBL) Genbank as X53944; DL:
Slln~h~a, R.R., et al., Nature, 350, 614 (1991), deposited with EMBL Genbank as X58454-HU HD 5DR).
-CA 02222~73 1997-11-27 W 096138~43 PCTrUS96107979 Functional Assays.

~,-AdrenocePtor-Mediated Phos~hoinositide Accumulation in Cultured Cell Lines. The agonist activity of test compounds was assayed by measuring their ability to generate phosphoinositide production in cells stably transfected with each of the three cloned human ~ -adrenoceptor subtypes. Cells were plated in 96-well plates and grown to confluence. The day before the assay the growth medium was changed to 100 ~1 of medium containing 1~ serum and 0.5 ~Ci [3H]myo-inositol, and the plates were incubated overnight in a CO2 incubator (5~ CO2 at 37oc)~Immediately before the assay, the medium was removed and replaced by 200 ~1 of PBS containing 10 mM
LiCl, and the cells were equilibrated with the new medium for 20 min. During this interval cells were also equilibrated with the antagonist, added as 10 ~1 aliquot of a 20-fold concentrated solution in PBS.

The [3H]inositol-phosphate (IP) accumulation was started by adding 10 ~1 of a solution containing the agonist. To the first well 10 ~1 were added to measure basal accumulation, and 11 different concentrations of agonist were assayed in the following 11 wells of each plate row.
All assays were performed in duplicate by repeating the same additions in two consecutive plate rows. The plates were incubated in a CO2 incubator for 1 hr. The reaction was terminated by adding 15 ~1 of 50~ (v/v) trichloroacetic acid (TCA), followed by a 40 min incubation at 4~C.

After neutralizing TCA with 40~1 of lM Tris, the content of the wells was transferred to a Multiscreen HV filter plate (Millipore) containing Dowex AG1-X8 (200-400 mesh, formate form). The filter plates were prepared adding 200 CA 02222~73 1997-11-27 W O96/38143 P~T~US96107979 ~l of Dowex AG1-X8 suspension (50~ v/v, water:resin) to each well. The filter plates were placed on a vacuum manifold to wash or elute the resin bed. Each well was washed 2 times with 200 ~l of water, followed by 2 x 200 ~l of 5mM sodium tetraborate/60 mM ammonium formate. The [3H]IPs were eluted into empty 96-well plates with 200 ~l of 1.2 M ammonium formate/0.1 formic acid. The content of the wells was added to 3 mls of scintillation cocktail, and the radioactivity was determined by liquid scintillation counting.

-Adrenoce~tor-Mediated Inhibition of Forskolin-Stimulated Adenyl~l Cyclase. The agonist activity of test compounds was assayed by measuring their ability to inhibit adenylyl cyclase in cells stably transfected with each of the three cloned human ~2-adrenoceptors. LM(tk-) cells expressing the ~2A- or the ~2C- ~ as well as Y1 cells expressing the ~29-adrenoceptor were used. The cell line for the ~2~-adrenoceptor, designated as Ya2B-2, was deposited on May 11, 1995, and accorded ATCC Accession No. CRL 11888. The formation of cyclic AMP was measured in cultures incubated with DMEM containing 1 mM
theophylline. Twelve concentrations of the test compounds (from 10 pM to lOO~M) were added to the incubation medium and incubated at 37~C for 20 min. Following this incubation step, 10 ~M forskolin was added to stimulate the formation of cyclic AMP, and the cultures were incubated for another 10 min. The reaction was stopped by replacing the incubation medium with 100 mM HCl. The intracellular levels of cyclic AMP were measured by radioimmunoassay. The data from concentration-response curves was fitted to a four-parameter logistic equation, by non-linear regression analysis, to determine the pECso and intrinsic activity.

W 096/38143 CA 02222~73 1997-11-27 PCTrUS96/07979 Isolated Tissue Assays.

Protocol for the Identification of ~l-Adrenoceptors in Mammalian Urethra from Functional Studies.

Using a battery of agonists and antagonists which exhibit selectivity among the ~1-adrenoceptor subtypes, a pharmacological profile of the receptor which mediates the contractile response to ~-agonists in the urethra of male hl~mAn.~, and male and female dogs and rabbits was determined. In addition, similar studies were done using bladder neck tissue from female dogs. In order to identify the specific receptor subtype in each tissue, the pharmacological profile was compared to the profiles for these same drugs at the cloned human ~lA~ ~lBt or ~lC
subtypes.

Methods.

Tissue samples from the proximal urethra of male humans and male and female rabbits, as well as tissue samples from both the proximal urethra and bladder neck of male and female dogs were cut into transverse strips (3xlOmm) and suspended under 0.5g tension in Krebs' physiological buffer at 37~C. To determine agonist potency (pECsO)and antagonist affinity constants (pKB), concentration-effect curves to the non-selective agonist phenylepherine were constructed in the absence and in the presence of increasing concentrations of the antagonist. Up to four sequential curves were constructed in each tissue.
Antagonists were allowed to equilibrate for lh before each concentration-effect curve, and the drugs were completely washed out in between successive curves. In each experiment, one tissue served as a "time control", in which no antagonist was added so that changes in .
CA 02222~73 1997-11-27 W O96J38143 P~CTnUS96107979 tissue sensitivity could be assessed as a function of time. In most instances, antagonist pK~ values were determined by Schild analysis (Arunlakshana and Schild,1959). In instances in which a single concentration of antagonist was used, pKB values were determined by the equation: pK8 = log ((CR-1)/[Antagonist]), where CR is defined as the ratio of the agonist ECso in the presence of the antagonist to that in the absence of the antagonist. In addit:Lon to the antagonist studies, the ~1c-selective agonists A-61603 and SK&F 102652 were used to characterize the receptor subtype in the female dog urethra, and to compare the receptor profiles in the urethra with that in the bladder neck. In these experiments, two concentration effect curves were constructed on each tissue, one in the absence and the second in the presence of prazosin. The pKB values derived for prazosin using A-61603 and SK&F
102652 as the agonists were compared to the pKB obtained for prazosin using phenylephrine as the agonist, to verify that each agonist interacted with a common ~1-adrenoceptor site.

Determination of ~-Adrenoce~tor Activitv in the Isolated Rat Riaht Atrium. R ght atria were removed from rats and placed immediately into oxygenated Krebs solution at 37~C.
The Krebs solution was replaced three times at 5 min intervals and the tissues were tensioned three times to 0.5g. In spontaneously beating atria, a control concentration effect curve to isoprenaline was generated.
~-Adrenoceptor-mediated increase in atrial rate were measured as the response. After complete wash out of the isoprenaline, a concentration effect curve was performed using the agonists A-61603, SK&F 102652, and SDZ NVI 085, __~p to a concentration of lOO~M. If no response was 35 ~bserved, the drug was left in the bath while another F~ .
_ ~

L
3~ .

WO96t38143 CA 02222~73 1997-ll-27 PCT~S96/07979 concentration-effect curve to isoprenaline was generated.
pEC50 values for agonists were calculated by logistic curve fitting. The antagonist effects of the test compounds were measured using the dose-ratio method, by comparing the shift in pEC50 for lsoprenaline.

Results.

Table 2 shows the pKI values determined from binding assays for various antagonists at the cloned human ~i-adrenoceptor subtypes and the corresponding pK~ values determined from contractile studies in urethral and bladder neck tissues obtained from human, dog, and rabbit.
Figures 1-5 illustrate the data from Table 2 in a graphical format. For each of the mammalian tissues, the pK3 value for each antagonist (abcissa) is plotted against the pKI values determined for each of the three cloned human ~-adrenoceptor subtypes (ordinate). The slopes and correlation coefficients (r) for the linear regression analysis are presented in each figure. In each case, the antagonist data derived from the functional experiments correlates best with the ~1c-subtype.
Table 3 shows the pKI, pEC50, and intrinsic activity at the cloned ~l- and ~2-subtypes for the various agonists.
In particular, A-61603 and SK&F 102652 each fully stimulate inositol phosphate production in cells transfected with the human ~1c-adrenoceptor, but are virtually inactive at the ~lA- and ~1~-subtypes. Table 3 also indicates that while both A-61603 and SK&F 102652 are selective among the ~1-adrenoceptors, these compounds also possess significant activity at ~2-adrenoceptors. The cross-reactivity binding profiles of these drugs and CA 02222~73 1997-11-27 W 096~38143 P/CTnUS96M797 other agonists are shown in Table 4.

Because o~ the ability of A-61603 and SK&F 102652 to fully stimulate ~lc-adrenoceptors, but not ~lA- or ~lB-subtypes, these compounds were used in the female dogtissues to compliment the antagonist-based pharmacological characterization. Additionally, these two compounds were used to establish that the ~l-subtype in the urethra is identical to the ~l-subtype in the bladder neck. The potency of these agonists and the pKB
value for prazosin in antagonizing their effects in each tissue are as follows:
Bladder Neck pEC50 Prazosin PKB
A-616036.8 8.3 15 SK&F 102652 5.7 7.8 Urethra pEC~o Prazosin PKB
A-616036.7 8.3 SK&F 102652 6.0 8.5 In each tissue, the magnitude of the contractions produced by A-61603 and SK&F 102652 was similar to the magnitude of the contraction produced by phenylephrine.
In addition, the contractions produced by A-61603 and SK&F 102652 were highly sensitive to prazosin, confirming their action at an ~l-adrenoceptor site. The high degree of selectivity of these compounds for the alc-subtype over the ~lA and ~lB subtypes, indicates that it is the ~lc-subtype which mediates contraction of the urethra as well as the bladder neck.

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CA 02222~73 1997-11-27 WO96/38143 P~CT~S96~07979 Re f erences Abel, P.W. et al. (1995) The atypical alpha-1-adrenoceptor. Pharmacol. Comm., 6, 29-38.

Andersson, K.-E. and Sjogren, C. (1982) Aspects on the physiology and pharmacology of the bladder and urethra.
Progress in Neurobiology, 19, 71-89.

Ariens, E.J. et al., (1960) Receptor reserve and threshold phenomena. Arch I~t Pharmacdyn Ther, 127, 459-478.

Arunlakshana,O. and Schild, H.O. (1959) Some quantitative uses of drug antagonists. Br J .Pharacol Chemother, 14, 48-58.

Chess-Williams, R. et al. (1994) Alpha-lA-adrenoceptor subtype mediates contraction of the rat urethra.
Auton Pharmacol, 14, 375-381.
Bylund, D.B. (1992) FASEB J, 6, 832.

Cheng, Y.-C. and Prusoff, W.H. (1973) Biochem Pharmacol, 22, 3099-3108.
Forray, C. et al. (19 94) The alpha-1-adrenocept:or that mediates smooth muscle contraction in human prostate has the pharmacological properties of the cloned human alpha-1C subtype. Mol Pharmacol, 45, 703-708.
Hatano, A. et al. (1994) Pharmacological eviclence of distinct alpha-1-adrenoceptor subtypes mediating the contraction of human prostatic urethra and peripheral artery. Brit J Pharmacol, 113, 723-728.

CA 02222~73 1997-11-27 W 096/38143 PCTrUS96/07979 Johnson, D.A. (1991) Pharmacology and safety of phenylpropanolamine. Drug Development Research 22, 197-207.

Kenakin, T.P. (1987) In: Pharmacological Analysis of Drug-Receptor Interaction. pp 190-192, Raven Press, New York.

Latifpour, J. (1990) Autonomic receptors in urinary tract: sex and age differences. J Pharmacol Exp Ther, 253, 661-667.

Lopata et al. (1984) High-level expression of a chloramphenicol acetyltransferase gene by DEAE-dextran-mediated DNA transfection coupled with a dimethylsulfoxide or glycerol shock treatment. Nucl. Acids ~es., 12, 5707-5717.

Muramatsu, I. et al. (1995) Functional subclassification of vascular alpha-1-adrenoceptor.
Pharmacol. Comm., 6, 23-28.

Receptor and Ion Channel Nomenclature Supplement (1995) Trends Pharmacol Sci., p 9.
Sand, P.K. et al.,(1990) Advances in nonoperative treatment of genuine stress incontinence. Current Opinion in Obstetrics and Gynecology, 2, 599-604.

Sourander, L.B. (1990) Treatment of urinary incontinence: The place of drugs. Gerontology 36, 19-26.

Testa, R. et al. (1993) Characterization of alpha-1 adrenoceptor subtypes in prostate and prostatic urethra of rat, rabbit, dog, and man. Eur J Pharmacol 249, 307-315.

CA 02222~73 1997-11-27 W O96138143 PC~nUS96S07979 Tsujimoto, G. et al., (1986) Alpha adrenoceptors in the rabbit bladder base smooth muscle: alpha-l adrenoceptors mediate contractile responses. J
Pharmacol Exp Ther, 228, 384-389.
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261, No. 18, 2685-2690.

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1995 Receptor and Ion Channel Nomenclature Supplement, 9-12.

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Claims (65)

What is claimed is:

1. A method of treating urinary incontinence in a subject which comprises administering to the subject a therapeutically effective amount of an .alpha.1C selective agonist which activates a human .alpha.1C.
adrenoceptor at least ten-fold more than: it activates a human .alpha.1A adrenoceptor and a human .alpha.1B
adrenoceptor.

2. The method of claim 1, wherein the .alpha.1C selective agonist further has the characteristic that it does not antagonize a human .alpha.1A adrenoceptor and a human .alpha.1B adrenoceptor.

3. The method of claim 1, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates any human .alpha.2 adrenoceptor and any .beta. adrenoceptor.

4. The method of claim 1, wherein the .alpha.1C selective agonist further has the characteristic that it does not antagonize any human .alpha.2 adrenoceptor and any .beta. adrenoceptor.

5. The method of claim 1, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates a human histamine H1 or H2 receptor.

6. The method of claim 1, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates a human dopamine D1, D2, D3, or D5 receptor.

7. The method of claim 1, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates a human serotonin 5-HT1A, 5-HT1D.alpha., 5-HT1D.beta., 5-HT1E, 5-HT1F, 5-HT2, or 5-HT7 receptor.

8. A method of treating urinary incontinence in a subject which comprises administering to the subject a therapeutically effective amount of an .alpha.1C selective agonist which activates a human adrenoceptor at least 50-fold more than it activates a human .alpha.1A adrenoceptor and a human .alpha.1B
adrenoceptor.

9. The method of claim 8, wherein the .alpha.1C selective agonist further has the characteristic that it does not antagonize a human .alpha.1A adrenoceptor and a human .alpha.1B adrenoceptor.

10. The method of claim 8, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates any human .alpha.2 adrenoceptor and any .beta. adrenoceptor.

11. The method of claim 8, wherein the .alpha.1C selective agonist further has the characteristic that it does not antagonize any human .alpha.2 adrenoceptor and any .beta. adrenoceptor.

12. The method of claim 8, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates a human histamine H1 or H2 receptor.

13. The method of claim 8, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates a human dopamine D1, D2, D3, or D5 receptor.

14. The method of claim 8, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates a human serotonin 5-HT1A, 5-HT1D.alpha., 5-HT1D.beta., 5-HT1E, 5-HT1F, 5-HT2, or 5-HT7 receptor.

15. A method of treating urinary incontinence in a subject which comprises administering to the subject a therapeutically effective amount of an .alpha.1C selective agonist which activates a human .alpha.1C
adrenoceptor at least 100-fold more than it activates a human .alpha.1A adrenoceptor and a human .alpha.1B
adrenoceptor.

16. The method of claim 15, wherein the .alpha.1C selective agonist further has the characteristic that it does not antagonize a human .alpha.1A adrenoceptor and a human .alpha.1B adrenoceptor.

17. The method of claim 15, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates any human .alpha.2 adrenoceptor and any .beta. adrenoceptor.

18. The method of claim 15, wherein the .alpha.1C selective agonist further has the characteristic that it does not antagonize any human .alpha.2 adrenoceptor and any .beta. adrenoceptor.

19. The method of claim 15, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates a human histamine H1 or H2 receptor.

20. The method of claim 15, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates a human dopamine D1, D2, D3, or D5 receptor.

21. The method of claim 15, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates a human serotonin 5-HT1A, 5-HT1D.alpha., 5-HT1D.beta., 5-HT1E, 5-HT1F, 5-HT2, or 5-HT7 receptor.

22. A method of treating urinary incontinence in a subject which comprises administering to the subject a therapeutically effective amount of an .alpha.1C selective agonist which activates a human .alpha.1C
adrenoceptor at least 200-fold more than it activates a human .alpha.1A adrenoceptor and a human .alpha.1B
adrenoceptor.

23. The method of claim 22, wherein the .alpha.1C selective agonist further has the characteristic that it does not antagonize a human .alpha.1A adrenoceptor and a human .alpha.1B adrenoceptor.

24. The method of claim 22, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates any human .alpha.2 adrenoceptor and any .beta. adrenoceptor.

25. The method of claim 22, wherein the .alpha.1C selective agonist further has the characteristic that it does not antagonize any human .alpha.2 adrenoceptor and any .beta. adrenoceptor.

26. The method of claim 22, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates a human histamine H1 or H2 receptor.

27. The method of claim 22, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates a human dopamine D1, D2, D3, or D5 receptor.

28. The method of claim 22, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates a human serotonin 5-HT1A, 5-HT1D.alpha., 5-HT1D.beta., 5-HT1E, 5-HT1F, 5-HT2, or 5-HT7 receptor.

29. A method of inducing contraction of urethra and bladder neck tissues which comprises contacting the urethra and bladder neck tissues with an effective contraction-inducing amount of an .alpha.1C
selective agonist which activates a human .alpha.1C
adrenoceptor at least ten-fold more than it activates a human .alpha.1A adrenoceptor and a human .alpha.1B
adrenoceptor.

30. The method of claim 29, wherein the .alpha.1C selective agonist further has the characteristic that it does not antagonize a human .alpha.1A adrenoceptor and a human .alpha.1B adrenoceptor.

31. The method of claim 29, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates any human .alpha.2 adrenoceptor and any .beta. adrenoceptor.

32. The method of claim 29, wherein the .alpha.1C selective agonist further has the characteristic that it does not antagonize any human .alpha.2 adrenoceptor and any .beta. adrenoceptor.

33. The method of claim 29, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates a human histamine H1 or H2 receptor.

34. The method of claim 29, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates a human dopamine D1, D2, D3, or D5 receptor.

35. The method of claim 29, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates a human serotonin 5-HT1A, 5-HT1D.alpha., 5-HT1D.beta., 5-HT1E, 5-HT1F, 5-HT2, or 5-HT7 receptor.

36. A method of inducing contraction of urethra and bladder neck tissues which comprises contacting the urethra and bladder neck tissues with an effective contraction-inducing amount of an .alpha.1C
selective agonist which activates a human .alpha.1C
adrenoceptor at least 50-fold more than it activates a human .alpha.1A adrenoceptor and a human .alpha.1B
adrenoceptor.

37. A method of claim 36, wherein the .alpha.1C selective agonist further has the characteristic that it does not antagonize a human .alpha.1A adrenoceptor and a human .alpha.1B adrenoceptor.

38. The method of claim 36, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates any human .alpha.2 adrenoceptor and any .beta. adrenoceptor.

39. The method of claim 36, wherein the .alpha.1C selective agonist further has the characteristic that it does not antagonize any human .alpha.2 adrenoceptor and any .beta. adrenoceptor.

40. The method of claim 36, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates a human histamine H1 or H2 receptor.

41. The method of claim 36, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates a human dopamine D1, D2, D3, or D5 receptor.

42. The method of claim 36, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates a human serotonin 5-HT1A, 5-HT1D.alpha., 5-HT1D.beta., 5-HT1E, 5-HT1F, 5-HT2, or 5-HT7 receptor.

43. A method of inducing contraction of urethra and bladder neck tissues which comprises contacting the urethra and bladder neck tissues with an effective contraction-inducing amount of an .alpha.1C
selective agonist which activates a human .alpha.1C
adrenoceptor at least 100-fold more than it activates a human .alpha.1A adrenoceptor and a human .alpha.1B
adrenoceptor.

44. The method of claim 43, wherein the .alpha.1C selective agonist further has the characteristic that it does not antagonize a human .alpha.1A adrenoceptor and a human .alpha.1B adrenoceptor.

45. The method of claim 43, wherein the .alpha.1C selective agonist activates the human a1C adrenoceptor at least ten-fold more than it activates any human .alpha.2 adrenoceptor and any .beta. adrenoceptor.

46. The method of claim 43, wherein the .alpha.1C selective agonist further has the characteristic that it does not antagonize any human .alpha.2 adrenoceptor and any .beta. adrenoceptor.

47. The method of claim 43, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates a human histamine H1 or H2 receptor.

48. The method of claim 43, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates a human dopamine D1, D2, D3, or D5 receptor.

49. The method of claim 43, wherein the .alpha.1C selective agonist activates the human a1C adrenoceptor at least ten-fold more than it activates a human serotonin 5-HT1A, 5-HT1D.alpha., 5-HT1D.beta., 5-HT1E, 5-HT1F, 5-HT2, or 5-HT7 receptor.

50. A method of inducing contraction of urethra and bladder neck tissues which comprises contacting the urethra and bladder neck tissues with an effective contraction-inducing amount of an .alpha.1C
selective agonist which activates a human .alpha.1C
adrenoceptor at least 200-fold more than it activates a human .alpha.1A adrenoceptor and a human .alpha.1B
adrenoceptor.

51. The method of claim 50, wherein the .alpha.1C selective agonist further has the characteristic that it does not antagonize a human .alpha.1A adrenoceptor and a human .alpha.1B adrenoceptor.

52. The method of claim 50, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates any human .alpha.2 adrenoceptor and any .beta. adrenoceptor.

53. The method of claim 50, wherein the .alpha.1C selective agonist further has the characteristic that it does not antagonize any human .alpha.2 adrenoceptor and any .beta. adrenoceptor.

54. The method of claim 50, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates a human histamine H1 or H2 receptor.

55. The method of claim 50, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates a human dopamine D1, D2, D3, or D5 receptor.

56. The method of claim 50, wherein the .alpha.1C selective agonist activates the human .alpha.1C adrenoceptor at least ten-fold more than it activates a human serotonin 5-HT1A, 5-HT1D.alpha., 5-HT1D.beta., 5-HT1E, 5-HT1F, 5-HT2, or 5-HT7 receptor.

57. A method of treating urinary incontinence in a subject which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

wherein n is an integer from 1 to 6; R is H or C1-C6 alkyl; R1 is C1-C6 alkyl, phenyl, naphthyl, substituted phenyl or naphthyl wherein the substituent is a halogen, or a C1-C6 alkyl or alkoxy group; wherein is an amino group or a heterocyclic group; the heterocyclic group is piperidine, morpholine, piperazine, pyrrolidine, hexamethylene, or thiomorpholine, the heterocyclic group being bonded through the nitrogen atom thereof to the (CH2)n group; the amino group, wherein R2 is H, C1-C6 alkyl, benzyl, or benzyhydryl and wherein R3 is H; C1-C10 alkyl; C2-C10 alkenyl; C3-C10 cycloalkyl or cycloalkenyl.

58. The method of claim 57, wherein the compound has the structure:

59. The method of claim 58, wherein the compound has the structure:

60. A method of treating urinary incontinence in a subject which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

wherein m is an integer from 0 to 2; wherein each of R1, R2, R3 and R7 is independently H; OH; C1-C6 alkyl or alkoxy; halo; amino; acetamido or NHSO2R
with R being H or C1-C6 alkyl; wherein R1 and R2 or R2 and R3 or R3 and R7 taken together constitute a methylenedioxy, ethylenedioxy, benzimidazole or indole ring; wherein each of R4 and R5 are independently H or taken together has the following formula:

wherein the dashed line represents a single or double bond; and R6 is H or C1-C6 alkyl; or a pharmaceutically acceptable salt thereof.

61. The method of claim 60, wherein the compound has the structure:

62. The method of claim 61, wherein the compound has the structure:

63. A method of treating urinary incontinence in a subject which comprises administering to the subject a therapeutically effective amount of a compound having the structure:

wherein each of R1 and R2 is independently H or C1-C4 alkyl; wherein R3 is OH or C1-C4 alkoxy; and R4 is C1-C4 alkylthio, alkylsulfoxide or alkylsulfone; C1; Br; I or CF3; wherein X is O, S, SO, SO2, NH, NR1 or NC(O)R1; in free base or acid addition salt form.

64. The method of claim 63, wherein the compound has the structure:

65. The method of claim 64, wherein the compound has the structure: