AU715350B2 - Aryl-imidazolines and aryl-imidazoles useful as alpha-2 adrenergic agonists without cardiovascular side effects - Google Patents

Description

WO 96/35424 PCT/US96/06633 1 Aryl-Imidazolines and Aryl-Imidazoles Useful as Alpha-2 Adrenergic Agonists without Cardiovascular Side Effects Description Field of the Invention The present invention relates to meta-substituted aryl linked imidazolines and imidazoles. More particularly, the invention relates to such compounds which have a 2 adrenergic agonist activity.

Background Of the Invention Aryl-2-amino-imidazolines are well-known in the art. Compounds such as moxonidine, para-aminoclonidine, brimonidine and tramazoline are but a few of the compounds which contain this basic structural feature that have also found use as therapeutic agents. For a review of structure activity relationships of this type of compound in relation to adrenergic receptors see R. Ruffolo, Jr. in oc- Adrenoreceptors: Molecular Biology, Biochemistry and Pharmacology, Prog. Basic Clin. Pharmacol. (Basel, Karger), 8 pp. 75-114 (1991).

HN ,,NHN NH HN NH Cl Br N N

N

Cl NH 2 N Para-aminoclonidine Bromonidine Tramazoline SUBSTITUTE SHEET (RULE 26) WO 96/35424 PCT/US96/06633 2 A compound of similar structure is moxonidine. However, moxonidine has been identified pharmacologically as a selective imidazoline receptor agonist, with utility as a centrally-acting antihypertensive agent. The pharmacological investigation of imidazoline agents independent of adrenoceptors started in the mid- 1980's. Two major subtypes, tentatively designated 11 and I2, are recognized. I, sites are labeled with nanomolar affinity by clonidine analogs whereas 12 sites have micromolar affinity for clonidine and are usually labeled by tritiated idazoxan. The designation (for imidazoline) has been intended to encompass not only imidazolines, imidazoles, and imidazolidines, but also such related structures as guanidines and oxazolines, all of which are potential ligands at these sites. A recent review of imidazoline-preferring receptors has been published by M.C. Michel and P. Emsberger in TiPS, 13, pp. 369-379 (Oct.

1992).

NH

H

q 0.

N

N

Cl Moxonidine Studies to identify the mechanism of the selective antihypertensive action of moxonidine have shown that the effect is mediated mainly by 1 1 -imidazoline receptors in the rostral ventrolateral medulla. [Haxhiu, M.A. et al, J. Cardiovasc. Pharmacol. 24 (suppl.1) pp. S1-S8 (1994)].

Similar studies of related compounds have identified rilmenidine as a SUBSTITUTE SHEET (RULE 26) WO 96/35424 PCT/US96/06633 3 hypotensive drug that is more selective for imidazoline receptors than for classical a adrenoceptors [Bousquet, et al., Am. J. Hypertens. 5 pp 47S-50S, 1992]. The rilmenidine structure substitutes an oxazolidine ring for imidazoline. Such heterocyclic ring substitutions are noted in the Ruffolo monograph on page 99 to reduce or abolish activity at a 2 receptors. A study published by Harron, D.W. [Am. J. Hypertens., 5(4, Pt.

2) pp. 91S-98S (Apr. 1992) reported that in experimental studies, "rilmenidine differs from clonidine in that it is more selective for imidazoline receptors than for a 2 -adrenoceptors; at equihypotensive doses, rilmenidine causes less bradycardia and reduction in cardiac output, less sedation, and little or no antinociceptive action compared to clonidine".

N

N

Rilmendine A few aryl-2-amino-imidazole derivatives are known in the pharmaceutical arts: Jen, et al in I. Med. Chem.. 18(1) 90-99 (1975) made and tested a clonidine analog, among a few other related structures for antihypertensive and gastric antisecretory activity. U.S. Patent number 3,459,763 (to Gruenfeld) which discloses a variety of substituted imidazole compounds, the two classes of compounds disclosed are regioisomers of the general structures: phenyl-2-amino-imidazole and N-l-phenyl-2-amino-imidazole. These structures were disclosed as having cardiovascular and anti-inflammatory activities.

SUBSTITUTE SHEET (RULE 26) WO 96/35424 PCT/US96/06633 4 In addition, several drugs are known which substitute a methylene group for the bridging amino group in the imidazoline series, compounds such as oxymetazoline, naphazoline and tolazoline are examples. The Ruffolo review indicates at page 95 that "replacement of the nitrogen bridge of clonidine with a methylene bridge has little effect on a 2 adrenoceptor activity..." and elsewhere on p. 95 that the "replacement of the nitrogen atom in clonidine-like imidazolines with either carbon or sulfur produces only a small reduction in a 2 adrenoceptor activity".

The background of the division of adrenergic receptor system into differing categories and subtypes can be briefly described as follows.

Historically, adrenoceptors were first divided in a and 3 types by Ahlquist in 1948. This division was based on pharmacological characteristics. Later, 1-adrenoceptors were subdivided into 3 1 and 3 2 subtypes, again based on a pharmacological definition by comparison of the relative potencies of 12 agonists. The a-adrenoceptors were also subdivided into a, and a 2 subtypes, initially based on a presumed localization of a, receptors postsynaptically and a 2 presynaptically. Now, however, this physiologic division is no longer used and it is generally accepted that the most useful way to subdivide the a-adrenoceptors is based on pharmacology using affinities for the antagonists yohimbine and prazosin. At xa receptors, prazosin is more potent that yohimbine, whereas the a 2 receptors, yohimbine is more potent than prazosin.

Bylund, et al. first suggested in 1981 that there possible existed subtypes of the a 2 -adrenoceptors on the basis of radioligand binding studies. This SUBSTITUTE SHEET (RULE 261 WO 96/35424 PCT/US96/06633 initial work was done with various tissues taken from various species.

While receptor heterogeneity among species is considered to be important, the term 'subtype' is usually reserved by pharmacologists for heterogeneity which can be demonstrated within the same species and ideally within a single tissue. Bylund and coworkers have later demonstrated that some regions of the human and rat brain contain two populations of a 2 -adrenoceptors sites which differ in their affinity for prazosin by 30- to 40- fold.

This finding supports the division of the a 2 receptor into A and B subtypes. More recently there have been reports of a third alpha subtype receptor called 2C.

Some examples of alpha 2 (X2) adrenergic receptor agonists well known in the art are: Cl H Br N H

N

ClH

N

Clonidine Brimonidine Clonidine is clinically useful as a hypotensive agent, and has been studied as a nasal decongestant and as an ocular hypotensive agent and as an anesthetic adjunct. The mechanism of action of clonidine has been described as a centrally acting 2 adrenergic partial agonist, however, clonidine also has hypotensive cardiovascular effects. It was SUBSTITUTE SHEET (RULE 261 WO 96/35424 PCT/US96/06633 6 reported that clonidine binds to both a 2 and imidazoline receptors and that the binding to the imidazoline receptors mediates the blood pressure lowering side effects of clonidine. [See e.g. Codd, et al. Life Sci., 56 p. 63-74 (2 Dec. 94) and Emsberger, et al., Cardiovasc. Drugs Ther., 8 (Suppl. 1) pp. 27-41 (Mar. 1994)] Brimonodine (UK 14,304) is a newer a 2 adrenergic agent which possesses superior therapeutic action as an ocular hypotensive, and has been tested in other a2 agonist responsive conditions. Brimonidine, as is shown by the data in table I at Example 4 also shows significant imidazoline receptor binding affinity.

Other activities inferred by I-receptor studies are stimulation of insulin release from pancreatic P-cells via coupling to ATP-sensitive K+ channels and inhibition of sodium reabsorption in the tubules of the kidneys. It has now been suggested by the present inventors that the imidazoline receptor is a nonfunctional binding site. [See Munk, et al, 1. Med. Chem.. 39 1193-1195(1996).] A few compounds which have been reported to be a2A selective are dexmedetomidine and oxymetazoline.

CH3 H H

CH

3 CH3 HO

CH

3

(CH

3 3 C GCH3 Dexmedetomidine Oxymetazoline The identification of subtypes of the a 2 receptor has progressed faster than complete pharmacological and physiological characterization of them. Nevertheless, a 2 A receptors have been identified in the ciliary body of the eye, and so are postulated to have a controlling mechanism SUBSTITUTE SHEET (RULE 26) WO 96 35424 PCTIUS96/06633 7 in ocular hypertension and symptomatology of glaucoma (see Jin, Y. et al., L. Ocul. Pharmacol. 10(1) pp. 359-69 (1994). Alpha 2A receptors have also been studied in pain perception, or alternatively, pain alleviation (see Millan, M. Eur. T. Pharmacol. 215(2-3) pp. 355-6 (1992).

A selective or subtype selective agonist as the term is used in this invention indicates a compound that binds to, and activates, a specific receptor subtype in preference to other receptors of related but different subtype(s). For example, a compound that binds to and activates the oa subtype receptor in preference to the og or 2c subtype receptors is an X2A selective agonist. Activation means that the receptor is induced to initiate a biochemical event that is controlled or operated by that particular receptor. Activation can further be thought of in terms of a signal transduction process which mediates the signal triggered by 15 receptor activation to intracellular effector structures.

From this summary of the state of the art it is apparent that compounds which are selective a 2 agonists possess valuable therapeutic utility for *2 treating glaucoma and pain, and for producing sedation.

*o P:kWPDOCS\CRNSPECL\65921 l.SPE. 8 Summary of the Invention This invention provides a method of treating nasal congestion in a mammal comprising administering to said mammal a therapeutically effective amount of a compound of the formula II

N

*4 4 4 4 4.

a 4 0 4.

4 a..

4 .4 4. 4 4 4404 44 a 4 4 444 4 4 4 4.

a 4 .4 4449 4a44.

4 4. 4 44 *4 a.

4a 4 WO 96/35424 PCT/US96/06633 9 wherein R, is H, alkyl of 1 to 4 carbon atoms or a halogen atom, X is O or NH and A is H or an oxo group and wherein said compound does not cause a concomitant reduction in blood pressure of said mammal.

More preferably in the method of the present invention the compound of formula II has the structure

/N

H

R,

H

N N

A

H

H

wherein R 1 represents methyl or bromine and A is hydrogen or an oxo group.

SUBSTITUTE SHEET (RULE 26) WO 96/35424 PCT/US96/06633 Most preferably in the method of the present invention the compound of formula II has the structure

/N

N Me H N

N

H

N

Pharmaceutically acceptable salts of the compounds of formula I are also within the scope of the present invention. Pharmaceutically acceptable acid addition salts of the compounds of the invention are those formed from acids which form non-toxic addition salts containing pharmaceutically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, phosphate or acid phosphate, acetate, maleate, fumarate, oxalate, lactate, tartrate, citrate, gluconate, saccharate, orp-toluenesulfonate salts. A pharmaceutically acceptable salt may be any salt which retains the activity of the parent compound and does not impart any deleterious or untoward effect on the subject to which it is administered and in the context in which it is administered.

Organic amine salts may be made with amines, particularly ammonium salts such as mono-, di- and trialkyl amines or ethanol amines. Salts may also be formed with caffeine, tromethamine, and similar SUBSTITUTE SHEET (RULE 26) WO 96/35424 PCT/US96/06633 11 molecules. Where there is a nitrogen sufficiently basic as to be capable of forming acid addition salts such may be formed with any inorganic or organic acids or alkylating agent such as methyl iodide. Any of a number of simple organic acids such as mono-, di-, or tri-acid may also be used. A pharmaceutically acceptable salt may be prepared for any compound of the invention having a functionality capable of forming such a salt, an acid salt of an amine group.

General Embodiments Definitions The terms "ester" and "amide" as used here refer to and cover any compound falling within the definition of those terms as classically used in organic chemistry.

Some compounds of the present invention contain the (2-imidazolyl) amino structure which is represented as: K NH

N

N..j

H

This group attaches via the exocyclic nitrogen to the aryl ring. Other compounds of the present invention have the (2-imidazolinyl) amino group represented in structure by: rKNH rKNH

KN

N HN

HN

N N sr N

H

H

SUBSTITUTE SHEET (RULE 261 WO 96/35424 PCT/US96/06633 12 These compounds exist as tautomers (formally equivalent isomers capable of interchanging double bond positions and a proton between the heteroatoms) wherein the double bond can shift from one nitrogen to another either within or without the ring but always terminating at the 2-carbon of the ring. The chemical nomenclature of these compounds is: 2 -amino-imidazolines for the forms where the double bond is positioned within the ring and 2 -imino-imidazolidines when the double bond is positioned outside the ring. No tautomeric form of these compounds places a double bond at the 4-5 position of the imidazole ring.

A further group of the present invention involves compounds which have in imidazoline ring but not an exocyclic amino group, but rather a methylene group in its place. Compounds of this type can tautomerize to give different double bond position within the ring only as shown below.

HN N N NH CH CH2 Hydrogen-bond acceptors are well-defined in the art [see for example, Wilson and Gisvold's Textbook of Organic Medicinal and Pharmaceutical Chemistry., R.F. Doerge J.B. Lipincott Co. 1982, pp.

41-43 or Physical Organic Chemistry, N.S. Isaacs Universities Press (Belfast) Ltd., 1987 pp. 62-67]. A hydrogen bond is a bond in which a hydrogen atom serves to hold two other atoms together. These "other atoms" must themselves be capable of forming hydrogen bonds. Atoms SUBSTITUTE SHEET (RULE 26) WO 96/35424 PCT/US96/06633 13 with hydrogen bond forming capability have at least one unshared electron pair together with a complete octet of electrons. A list of atoms contemplated by the invention includes: F, 0, N, C1, Br and S. The Hbond consists of an attractive force which exists between a hydrogen atom covalently bound to an atom from the list given above a hydroxy group, and a second atom, not necessarily the same, from the list. The lone pair atom which has the covalent bond to hydrogen is called the hydrogen bond donor. The other atom which hydrogen bonds with this donated hydrogen is called the hydrogen bond acceptor.

Some groups such as hydroxy or primary or secondary amine can act as both hydrogen bond donors and hydrogen bond acceptors. Groups such as ethers or tertiary amines are hydrogen bond acceptors only. The valence of quaternary amines which have no free lone pair are incapable of being hydrogen bond acceptors. Hydrogen bond acceptor groups specifically contemplated by the present invention are primary, secondary, and tertiary amines, hydroxyl and ethers functions, amides and esters, fluoro, chloro and bromo groups and thiols and thioethers.

The term "alkyl" as used here refers to and includes normal and branch chained alkyl groups. The term "lower alkyl", unless specifically stated otherwise, includes normal alkyl of 1 to 4 carbons, branch chained alkyl of 3 or 4 carbons. Similarly, the terms "alkenyl" and "alkynyl" include normal and branch chained groups having 2 to 4 carbons when the chains are normal, and 3 or 4 carbons when the chains are branched.

SUBSTITUTE SHEET (RULE 26) WO 96/35424 PCT/US96/06633 14 Brief Description of the Drawing Figures Figure I compares the effect of clonidine and a representative compound of this invention for lowering intraocular pressure (IOP).

Figure II compares the effect of clonidine and a representative compound of this invention for lowering blood pressure.

Detailed Description of the Invention The compounds of Formula I as described above including all stereoisomers, tautomers as previously defined and mixtures thereof which comply with the constraints of the present compounds are included within the scope of the present invention.

The present compounds may be prepared in a manner analogous to the procedures in: Commonly assigned PCT application 95/US/01150 filed on 25 January, 1995 by Munk, et al. discloses certain phenyl-2-aminoimidazoles which have subtype selected a 2 A activity and methods for making them. The contents of this PCT application are hereby incorporated by reference in their entirety. U.S. Patent No. 5,091,528 by Gluchowski and commonly assigned with the present application discloses methods of making 2 -amino-imidazolinyl)-benzoxazine compounds encompassed by the methods of the present invention. The content of US 5,091,528 is hereby incorporated by reference in its entirety.

Similarly, U.S. Patent No. 5,112,822 by Gluchowski and also commonly assigned discloses methods of making 2 -amino-imidazolinyl)tetrahydroquinoxaline compounds and is hereby incorporated by reference in its entirety. Other compounds, such as oxymetazoline, are well-known compounds in the art and are commercially available.

SUBSTITUTE SHEET (RULE 26) WO 96/35424 PCT/US96/06633 The present meta-substituted aryl linked imidazolines and imidazoles are useful to provide one or more desired therapeutic effects in a mammal. Among the desired therapeutic effects are an alteration, preferably a decrease in the rate of fluid transport in the gastrointestinal tract of a mammal, a reduction in or maintenance of the intraocular pressure in at least one eye of a mammal; and an increase in the renal fluid flow in at least one kidney of a mammal, or a decrease in nasal congestion in the air passages of a mammal. Thus, for example, the present compounds may be used as a anti-diarrhea agent, a medication for use in the treatment or management of glaucoma, a medication for use in the treatment or management of kidney disease and/or a treatment for congested nasal passages. One important feature of many of the present compounds is that the desired therapeutic effect is achieved with reduced or absent side effects, in particular, lacking effects on the blood pressure or the mammal to which the present compound or compounds is/are administered.

Preferred compounds of the invention with reference to the Examples 1- 24 in Table 1 are those compounds which show high affinity for a 2 receptors (low Ki values) in the binding assays. Particularly preferred are those compounds in which the Ki (binding affinity) value for the a 2 receptors is from 0.0001 to 10. With respect to the structural features which are preferred in the present invention, the elements which are preferred in providing the desired binding respect to the structural features which are preferred in the present invention, the elements which are preferred in providing the desired binding characteristics are the presence of a fused ring at R 2 and R 3 more preferably with a nitrogen atom bonding to the aryl ring at R 2 and even more preferably with a nitrogen atom bonding to the ary ring at R 2 and even more preferably SUBSTITUTE SHEET (RULE 26) WO 96/35424 PCT/US96/06633 16 with a nitrogen or oxygen atom bonding to the aryl ring at R 3 Other preferred embodiments include compounds of formula 1 wherein both Ris are methyl, one R 2 is hydroxy or methoxy, the other R 2 is hydrogen, and R 3 is t-butyl.

Any suitable method of admi:aistering the present compound or compounds to me mammal to be treated may be used. The particular method of administration chosen is preferably one which allows the present compound or compounds to have the desired therapeutic effect in an effective manner, low medication concentration and low incidence of side effects. In many applications, the present compound or compounds are administered to a mammal in a manner substantially similar to the used to administer c 2 agonists, to obtain the same or similar therapeutic effect.

The invention is further illustrated by the following non-limiting examples which are illustrative of specific modes of practicing the invention and are not intended as limiting the scope of the appended claims.

SUBSTITUTE SHEET (RULE 26) WO 96/35424 PCT/US96/06633 Table 1 Kt Structure Example1

HNYNH

N

Example 2 HN NH Nrc

CI

acl (x2A 5,034 8.9 513 3.8 0C2B a2C Ii I2A 12BI 49 70 12 1,200 30 8.9 10,000 12,452 Example 3 HN NH

N

CI

NH

2 Example 4

HNYNH

NN

Example HN NH

NN

21 19 64,790 38,642 1,433 2 17 27 48 5,199 155 1,850 2.7 52 44 17 7,193 614 SUBSTITUTE SHEET (RULE 261 WO 96/35424 PCT/US96/06633 18 Table 1 (con't.) Structure cxl IX2A cX2B ac Ii 12A I2B Example 6 HN.<N H N c I

N

38,573 147 1,029 2,012 56 100,000 100,000 Exam:Ide 7

HNYNH

5,100 35 262 .463 1,988 100,000 10,000 Example 8 8,715 9.6 124 100,000 100,000 10,000 Example 9 HN NH 'Br H

XN

H

Example HN NH E' r H N N

H

1,117 6.1 47 120 4,575 7,561 4,588 6,606 24 194 293 3,952 24,827 3,256 SUBSTITUTE SHEET (RULE 261 WO 96/35424 PCT[US9S/06633 Structure Exainple 11 HNyNH1 0 Example .12 HNyNH N0 0 Example 13 Hfl

NH

N J0 Table 1 (con't.) K1 cX2A CX2B (X2 C Ii I2A

IMB

2,403 3.1 1,452 5.2 28 2D 1,835 1,084 2,032 93 340 100,000 507 129 0.25 4 3.5 831 100,000 1,223 Example 14 HNyNH1 N. 8,987 52 842 11,600 4,500 6,517 -o Example H N N H 0 2,262 17 241 134 100,000 SUBSTITUTE SHEET (RULE 26) WO 96/35424 PCT/US96/06633 Table 1 (con't.) Kt Structure Example 16 al X2A CX2B 2 1i 2A I2BI 473 1.2 30 8.9 7,112 100,000 12,135 Examjle17 14,784 6.3 436 229 5,300 27,035 9,283 Example 18 H N N

HN

Examp~le 19 H N N H N Example 5,214 52 295 180 19 141 7,613 10 434 512 13 6,180 179 11,316 30 895 SUBSTITUTE SHEET (RULE 26 WO 96/35424 WO 9635424PCT/US96/06633 21 Table 1 (con't.) Structure Example 21 HN YN

HNI

cI a OC2A& am2 awlC hi I2A I2B 686 2,147 1.7 82 19 444 7,708 Example 22 4,175 4.9 88 4,320 100,000 870 .0

N

199 0.27 30,550 31 97 13 7,417 12,873 2,692 ExamTule 24 HN N 1,156 212 227 6,367 SUBSTITUTE SKEET (RULE 261 WO 96/35424 PCT/US96/06633 22 Experimental Assays: Binding Affinities and Receptor Activation Receptor Binding Assays Example Tissue preparation: Membrane suspensions were prepared from human cerebral cortex (HDD, for cal receptors) obtained from the UCI Organ and Tissue Bank. Briefly, tissues (Ig) were homogenized in 25 mL of ice-cold mM Tris, pH 7.4 with a Polytron homogenizer for 30 sec at setting #7, and centrifuged for 10-12 minutes at 300 x g at 4° C. The supernatant was filtered through 2 layers of gauze and diluted 1:2 with 50 mM Tris-HC1 buffer, pH 7.4, then centrifuged at 49,000 x g for 20 minutes. The pellet fraction was washed 3 times (resuspended in Tris-HC1 buffer and centrifuged for 20 minutes at 49,000 x The pellet was then stored at -80' C until the binding assay.

Cell preparation: Chinese hamster ovary (CHO) cells expressing the human x 2 A and human a 2 C (CHO-C10 and CHO-C4 respectively) receptors and CHO cells (CHO-RNG) expressing the rat a 2 B adrenoceptor were grown to near confluence in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum using standard cell culture methods. Cells were harvested by scraping and placed into cold buffer of the following composition: 50 mM Tris-HC1, 5 mM EDTA, pH 7.4).

Cells were then homogenized with a Polytron homogenizer for 2 x sec at setting and centrifuged for 20 minutes at 49,000 x g. The pellet fraction was washed (resuspended in Tris-HC1, pH 8 buffer and SUBSTITUTE SHEET (RULE 26) WO 96/35424 PCT/US96/06633 23 centrifuged for 15-20 minutes at 49,000 x g) 2 times and stored at -100 until binding assay.

Binding Studies: The radioligands 3 H]rauwolscine (specific activity Ci/mmol) and 3 H)prazosin (specific activity 76 Ci/mmol) were obtained from New England Nuclear, Boxton, MA. Frozen membrane pellet was resuspended in 25 mM glycine/glycine, pH 7.5 and incubated with radioligand under the following conditions: CHO-C10,

CHO-RNG,

CHO-C4-[ 3 H]rauwolscine, 22C, 30 minutes; RKC-[ 3 H]rauwolscine,

OC,

120 minutes; and, HCC-[ 3 H]prazosin, 22'C, 30 minutes in a final volume of 500 ul. At the end of the incubation period, the samples were filtered through glass filters (Whatman GF/B) in a 96 well cell harvester and rapidly washed four times with 4 mL of iced-cold 50 mM Tris-HC1 buffer.

The filters were then oven dried and transferred to scintillation vials containing 10 mL of Beckman's Ready Protein® scintillation cocktail for counting. Specific binding defined by 10 uM phentolamine for competition studies were as follows: 2.4 nM 3 H]brimonidine-RbICB 62%; 2.4 nM 3 H]rauwolscine-RbICB 75%; 2 nM 3 H]rauwolscine-RbKc 88%; 0.3 nM 3 H] rauwolscine-CHO-C10 99%; 0.4 nM 3 H]rauwolscine- CHO-RNG 99%, 0.3 NM 3 H]prazosin 87%; and 1 nM 3 H]rauwolscine- CHO-C4 90%. Protein concentrations were determined with a protein assay kit from Bio Rad. Binding isotherms, equilibrium dissociation and affinity constants were analyzed and determined by the non-linear least squares curve fitting programs EBDA (BioSoft) or AccuFit Competition/Saturation by Beckman.

SUBSTITUTE SHEET (RULE 26) WO 96/35424 PCT/US96/06633 24 Example 26: Cell Preparation: Chinese hamster ovary (CHO) cells expressing the human a 2 A (CHO-C10) and the rate a2B (CHO-RNG) human a 2

A

adrenoceptors were grown to near confluence in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum using standard cell culture methods. Cells were harvested by scraping and placed into cold buffer of the following composition: 50 mM Tris-HC1, mM EDTA, pH Cells were then homogenized with a Polytyron homogenizer for 2 x 10 sec at setting and centrifuged for 20 minutes at 49,000 x g. The pellet fraction was washed (resuspended in Tris-HCl, pH 8 buffer and centrifuged for 15-20 minutes at 49,000 x g) 2 times and stored at 100 C until binding assay.

Binding studies: Determination of Ki The radioligands 3 H] rauwolscine (specific activity 80 Ci/mmol) and 3

H]

prazosin (specific activity 76 Ci/mmol) were obtained from New England Nuclear, Boston, MA. Frozen membrane pellet was resuspended in 25 mM glycine/glycine, pH 7.4 and incubated with radioligand under the following conditions: CHO-C10,

CHO-RNG,

CHO-C4-[ 3 H]rauwolscine, 22' C, 30 min.; and, HCC-[ 3 H]prazowsin, 22'C, minutes in a final volume of 500 ul. At the end of the incubation period, the samples were filtered through glass fiber filters (Whatman GF/B) in a 96 well cell harvester and rapidly washed four times with 4 mL of iced-cold 50 mM Tris-CH1 buffer. The filters were then oven dried and transferred to scintillation vials containing 5 mL of Beckman's Ready Protein® scintillation cocktail for counting. Specific binding defined by 10 uM phentolamine for competition studies were as follows: SUBSTITUTE SHEET (RULE 26) WO 96/35424 PCT/US96/06633 0.3 nM[ 3 H]rauwolscine-CHO-C10 99%; 0.4 nM[ 3 H]rauwolscine-CHO-RNG 99%, and 0.3 nM 3 H]prazosin-HCC 87%. Protein concentrations were determined with a protein assay kit from Bio Rad. Binding isotherms, equilibrium dissociation and affinity constants were analyzed and determined by the non-linear least squares curve fitting programs AccuFit Competition/Saturation by Beckman.

Preparation of Bovine ventrolateral medulla (BVLM) membranes Example 27: Fresh bovine brain stems were obtained from a local slaughter house.

After removal of pia-arachnoid, the medulla was isolated by dissecting the brain stem about 1 cm posterior and 1 cm caudal to the obex. The ventral quadrants of the medulla excluding the pyramids were used as the VLM. For each preparation, 30 to 40 VLM were used. Initial homogenization was performed in 20 volumes of 5 mM HEPES buffer (pH 7.4 at 4"C) containing 250 mM sucrose, 50 uM Calpain I inhibitor (Boehginer Mannheim, Indianapolis, IN), 100 uM 1,10-phenanthroline (Sigma, St. Louis, MO) and 50 uM Pefabloc (Boehginer Mannheim, Indianapolis, IN), using Virtis homogenizer at setting 7 with three second pulses, followed by three passes in a teflon-glass tissue homogenizer. The inhibitors were added to prevent degradation by proteases and peptidases. The homogenates were then centrifuged at 1000xg for 10 min and the resulting pellets were re-homogenized and centrifuged. Supernatants resulted from both runs were combined and centrifuged for 20 min at 4 8000xg. The pellets obtained were resuspended in a teflon-glass homogenizer in 50 mM Tris-HCl with mM EDTA (pH 7.7 at centrifuged, and resuspended in 50 mM Tris- SUBSTITUTE SHEET (RULE 26) WO 96/35424 PCT/US96/06633 26 HC1 with 25 mM NaC1, pH 7.7 at room temp. To remove endogenous ligands, the homogenate was incubated 30 min at room temp before it was centrifuged for 20 min at 4 8 0 0 0xg. The pellets were then washed with 50 mM Tris-HCl buffer, (pH 7.4 at 4°C) and loaded on top of 5 mM HEPES/0.85 M sucrose (pH 7.4 at Pellets obtained after centrifugation at 4 8 0 0 0xg for 20 min were saved. The fatty connective tissue on the top layer was discarded. The partially purified

VLM

membrane pellets were then washed twice with 50 mM Tris-HCl, pH 7.7 at 4"C, flash frozen in dry ice/acetone slush, and stored at -100°C until use. Receptor binding experiments were performed within days after the membrane preparation.

I imidazoline receptor binding assay Example 28: I, imidazoline receptor binding affinity was determined from radioligand binding of 3 H-clonidine (NEN, Boston, MA) to bovine VLM membranes. Specific activity of 3 H-clonidine was 43 Ci/mmol. Kd of 3 H-clonidine binding to the I, imidazoline receptor was determined by saturation experiments and Ki of other ligands studied were determined by competition experiments. The radioligand binding assay was performed in Teflon 9 6-wells with the Biomek-1000 robotics (Beckman Instruments, Fullerton, CA). Each well contained 4 mN 3 H-Clonidine and 0.3 to 0.5 mg of bovine VLM protein in 5 mM HEPES buffer containing 0.5 mM EGTA and 0.5 mM MgCl 2 pH 7.4 (0.1 mM ascorbic acid was added just before the protein). After 50 min of incubation at the reaction was terminated by rapid filtration over Whatman GF/B filters treated with 0.1% polyethyleneimine and washed with 12 ml ice cold 50 mM Tris-HC1, pH 7.4 at 4°C in a Brandel Harvester (Brandel, Gaithersburg, Both 'hot' and 'cold' saturation SUBSTITUTE SHEET (RULE 26) WO 96/35424 PCT/US96/06633 27 experiments were performed. In 'hot' saturation experiments, studies were performed with 3 H-clonidine ranging from 0.1 to 50 nM. In 'cold' saturation experiments, studies were performed with 2 nM 3 H-clonidine with 20 different concentrations of the unlabeled clonidine, ranging from 0.1 nM to 1 uM unlabeled clonidine. Non specific binding was defined by parallel incubations containing 10- M phentolamine or naphazoline. Imidazoline binding was determined by parallel incubations in which the alpha-adrenergic sites were masked with 10- M norepinephrine. During competition experiments, ligands of concentrations ranged from 10-1 to 10' were used. Radioactivity was counted in a Beckman LS-3801 scintillation counter. Data were captured and analyzed with Accufit saturation and competition softwares modeled both for one-site and two-site fits (Beckman Instruments, Fullerton, CA) using an IBM compatible computer. All experiments were repeated at least twice.

Representative compounds of the present invention were tested according to the procedures given above. Results of these tests are tabulated in Table 1 above as specific examples. The receptor binding studies and K 1 are measures of the affinity of a compound for a particular receptor.

Example 29 The compound of Example 16 was compared to clonidine for lowering intraocular pressure (IOP) by topical administration of a single drop of 0.001% of the compound in an ophthalmically acceptable vehicle to one eye of a rabbit. The untreated eye was used as the control. The results are reported in Figure 1. As shown, clonidine shows a systemic effect, in that the IOP of the untreated eye is lowered to the same extent as the treated eye. In contrast, the eye treated with the compound of Example SUBSTITUTE SHEET (RULE 26) WO 96/35424 PCT/US96/06633 28 16 showed a greater effect in lowering IOP than clonidine without lowering IOP in the untreated eye.

Example The compound of Example 16 and clonidine were tested for systemic effect by injecting 10 ug/kg of each compound into a rabbit. In comparison to a saline control, the clonidine lowered the mean arterial blood pressure, substantially, while the compound of Example 16 did not effectively lower the mean arterial blood pressure. These results are reported in Figure 2.

While the invention has been described in terms of certain preferred embodiments and specific examples, they are not intended as limiting the scope of the present invention which should be determined solely on the basis of the appended claims, as such claims are read in light of the disclosure.

SUBSTITUTE SHEET (RULE 26)

Claims (1)

1. 9. 9 9 S :0 *5.l* 9@ 9 0 0 S 900S *509 SO S S. SSfrSSO S 005955 *9 SO *9 *5 9 Q S S S wherein RI represents methyl. or bromine and A is hydrogen or an oxo group- 3) The use of claim 1 wherein the compound of formulaRI has the structure H Me N H H Substitute Sheet P:\WPDOCS\CRN\SPECI\659211.SPE 19/11/99 -31- 4) A method for the treatment of nasal congestion without causing a concomitant reduction in blood pressure which comprises administering to a mammal in need of such treatment a therapeutically effective amount of a composition set forth in claim 1. Methods for the treatment of nasal congestion without causing a concomitant reduction in blood pressure involving/containing compounds of the formula II, substantially as hereinbefore described. DATED this 19th day of November, 1999 0 ALLERGAN SALES INC 00 O By its Patent Attorneys S DAVIES COLLISON CAVE 0 *e 0 0 0