CA2220517A1 - Aryl-imidazolines and aryl-imidazoles useful as alpha-2 adrenergic agonists without cardiovascular side effects - Google Patents

Abstract

Methods are disclosed for treating a mammal with a condition that responds to .alpha.-2 agonist treatment without causing cardiovascular side effects using compounds of formula (II) wherein R1 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.

Description

CA 02220~17 1997-11-07 W 096/35~24 PCT~US96/~6633 Aryl-Imidazolines and Aryl-Imidazoles Useful as Alpha-2 Adrenergic Agonists without Cardiovascular Side Effects Description Field of the Invention 10 The present invention relates to meta-substituted aryl linked imidazolines and imidazoles. More particularly, the invention relates to such compounds which have ~C2 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 20 review of structure activity relationships of this type of compound in relation to adrenergic receptors see R. Ruffolo, Jr. (ed.) in a-Adrenoreceptors: Molecular Biology, Biochemistry and Pharmacology, Prog. Basic Clin. Pharmacol. (Basel, Karger), 8 pp. 75-114 (1991).

r~ r\ r~
HN~NH Cl HN~NH B HN~NH
N~ N ~N ~ N

Cl NH2 N
Para-aminoclonidine Bromonidine Tramazoline SUBSTITUTE SHEET (RULE 26~

CA 02220~17 1997-11-07 W O 96/35~24 PCTrUS96/06633 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 5 imidazoline agents independent of adrenoceptors started in the mid-1980's. Two major subtypes, tentatively designated I, and I2, are recognized. Il sites are labeled with nanomolar affinity by clonidine analogs whereas I2 sites have micromolar affinity for clonidine and are usually labeled by tritiated idazoxan. The 'I' designation (for 0 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-pre~~ g receptors has been published by M.C. Michel and P. Ernsberger in TiPS, 13, pp. 369-379 (Oct.
1992).

NH

O

Cl~J\
Moxonidine Studies to identify the mechanism of the selective antihypertensive action of moxonidine have shown that the effect is mediated mainly by 20 1,-imidazoline receptors in the rostral ventrolateral medulla. [Haxhiu, M.A. et al, J. Cardiovasc. PharInacol. 24 (suppl.1) pp. S1-S8 (1994)].
Similar studies of related compounds have identified rilmenidine as a SUBSTITUTE SHEET (RULE 26~

CA 02220~17 1997-11-07 W 096135~24 PCT~US96/~6633 hypotensive drug, that is more selective for imidazoline receptors than for classical OC2 adrenoceptors [Bousquet, P., 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 5 Ruffolo monograph on page ~9 to reduce or abolish activity at ocz 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 rect:~lors than for o~2-adrenoceptors; at equihypotensive o doses, rilmenidim~ causes less bradycardia and reduction in cardiac output, less sedation, and little or no antinociceptive action compared to clonidine".
A

N </
~/ H O

Rilmendine 15 A few aryl-2-amino-imidazole derivatives are known in the pharmaceutical alts: Jen, et al in J. 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 20 imidazole compo1lnds, the two classes of compounds disclosed are regioisomers of t]ae 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~

CA 02220~17 1997-11-07 W 096/35~2~ PCTAUS96/06633 In addition, several drugs are kno~n 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 5 the nitrogen bridge of clonidine with a methylene bridge has little effect on ~C2 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 ~C2 adrenoceptor activity".

The background of the division of adrenergic receptor system into diffèring categories and subtypes can be briefly described as follows.
Historically, adrenoceptors were first divided in a and ~ types by Ahlquist in 1948. This division was based on pharmacological 5 characteristics. Later, ,~-adrenoceptors were subdivided into ,Bl and ,B2 subtypes, again based on a pharmacological definition by comparison of the relative potencies of 12 agonists. The a-adrenoceptors were also subdivided into o~, and OC2 subtypes, initially based on a presumed localization of ocl receptors postsynaptically and a2 presynaptically. No~, =
2 o however, this physiologic division is no longer used and it is generally accepted that the most useful way to subdivide the oc-adrenoceptors is based on pharmacology using affinities for the antagonists yohimbine and prazosin. At al receptors, prazosin is more potent that yohimbine, whereas the ~C2 receptors, yohimbine is more potent than prazosin.
25 Bylund, et al. first suggested in 1981 that there possible existed subtypes of the a2-adrenoceptors on the basis of radioligand binding studies. This SUBSTITUTE SHEET (RULE 26~

, CA 02220~17 1997-11-07 W 096/3~424 PCTAUS96/06633 initial work was done with various tissues taken from various species.
While receptor heterc~ ~neity among species is considered to be important, the term 'subtype' is usually reserved by pharmacologists for heterogeneity ~hich can be demonstrated within the same species and 5 ideally within a, single tissue. Bylund and coworkers have later demonstrated that some regions of the human and rat brain contain two populations of ~x2-adrenoceptors sites which differ in their affinity for prazosin by 30- to 40- fold.

10 This finding supports the division of the a2 receptor into A and B
subtypes. More recently there have been reports of a third alpha subtype receptor called 2C.

Some examples of alpha2 (a2) adrenergic receptor agonists well known 15 in the art are:
~H

U ~ ~ Dr Clonidine Brimonidine 20 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 a2 adrenergic partial agonist, however, clonidine also has hypotensive cardiovascular effects. It was SUBSTITUTE SHEET (RULE 26~

CA 02220~17 1997-11-07 W O 96/35~24 PCTrUS96106633 reported that clonidine binds to both a2 and imidazoline receptors and that the binding to the imidazoline receptors mediates the blood pressure lowering side effects of clonidine. [See e.g. Codd, E.E., et al. Life Sci., 56 (2) p. 63-74 (2 Dec. 94) and Ernsberger, P., et al., Cardiovasc. Drugs 5 Ther., 8 (Suppl. 1) pp. 27-41 (Mar. 1994)] Brimonodine (UK 14,304) is a newer a2 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.
0 Other activities inferred by I-receptor studies are stimulation of insulin release from pancreatic ~-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, S.A., et al, J. Med. (~hem. 39 (6) 1193-1195(1996).]

A few compounds which have been reported to be a2A selective are dexmedetomidine and oxymetazoline.

N/~ CH

bJ~CH3 (CH3)3C~CH3 Dexmedetomidine Oxymetazoline The identification of subtypes of the a2 receptor has progressed faster than complete pharmacological and physiological characterization of 25 them. Nevertheless, a2A receptors have been identified in the ciliary body of the eye, and so are postulated to have a controlling mechanism SUBSTITUTE SHEET ~RULE 26~

CA 02220~17 1997-11-07 W 09613~2~ PCT~US96/06633 in ocular hypertension and symptomatology of glaucoma (see Jin, Y. et al., J. Ocul. Ph~lrmacol., 10(1) pp. 359-69 (1994). Alpha 2A receptors have also been stud.ied in pain perception, or alternatively, pain alleviation (see Millan, M. J., Eur. J. 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 a2A
10 subtype receptor in preferel~ce to the a2B or a2csubtype receptors is an a2A
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 a2 agonists possess valuable therapeutic utility for treating glaucoma and pain, and for producing sedation.
Summary of the Invention This invention covers methods of using compounds of formula I
selective agonism of a2 adrenoceptors 2 5 HN~
N

Rl~,Rl Formula I
R2~ R2 SUBSTITUTE SHEET(RULE26) CA 02220~17 1997-11-07 W 096/35~24 . PCTrUS96/06633 wherein Q is NH or CH2, a broken line parallel to and adjacent a solid line indicates a single or a double bond, each Rl is independently H, alkyl of 1 to 4 carbon atoms or a halogen atom, each R2 is independently H or an atom or functional group chosen from the group consisting of 5 -N(R4)3, -OR4, F, Cl, Br and -SR4, and wherein each R4 is independently chosen from the group consisting of hydrogen, alkyl of 1 to 4 carbon atoms and alkylcarbonyl of 1 to 5 carbon atoms, and R3 is independently chosen from the group of values for Rl and R2, or R2 and R3 together can form a 5 or 6 membered ring, which can optionally bear methyl, ethyl or 10 oxo substituents, fused to the aryl ring, provided that in either case at least one R2 is -N(R4)3, -OR4, F, Cl, Br, or -SR4 and that the heteroatom bonds directly to the aryl ring.

Preferably the present invention provides a method of treating elevated 15 intraocular pressure, nasal congestion or diarrhea in a ~nammal comprising administering to said mammal a therapeutically effective amount of a compound of the formula II

H~,~j~ A

SUBSTITUTE SHEET (RULE 26 wherein Rl 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.

5 More preferably in the method of the present invention the compound of formula II has the structure H['~
Rl H
N ~ N~i A

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

SUBSTITUTE SHEET (RULE 26 CA 02220~17 1997-11-07 W 096/35424 PCT~US96/n6633 Most preferably in the method of the present invention the compound of formula II has the structure ~N
H~
Me H
N ~, 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 10 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, or~-toluenesulfonate salts. A pharmaceutically 15 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.

20 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~

CA 02220~l7 l997-ll-07 W O 96l3~121 PCT/US96/06633 molecules. ~There there is a nitrogen sufficiently basic as to be capable of forming acicl addition salts such may be formed with any inorganic or organic acids or alkylating agent such as methyl iodide. Any of a number of sirnple organic acids such as mono-, di-, or tri-acid may also 5 be used. A pharmaceutically acceptable salt may be prepared for any compound of the invention having a functionality capable of forming such a salt, e.g., an acid salt of an amine group.

General Embodiments Definitions The terms "eClter" and "arnide" as used here refer to and cover any compound faLling within the d~ nition of those terms as classically used 15 in organic chemistry.

Some compolmds of the present invention contain the (2-imidazolyl) amino structL:Lre which is represented as:
~\NH
N~<
H

This group attaches via the exocyclic nitrogen to the aryl ring. Other compounds of the present invention have the (2-imidazolinyl) amino 25 group represented in structure by:
\NH 1~\NH l \N
N~< ~ ~ HN~ HN~
N ~f ! HN--r SUBSTITUTE SHEET (RULE 26~

CA 02220~l7 l997-ll-07 W 096/35~2~ PCTAUS96/06633 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 5 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 0 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 5 methylene group in its place. Compounds of this type can tautomerize to give different double bond position within the ring only as shown below.
r~ r~
HN~,N N~NH
2 o CH2 CH2 Hydrogen-bond acceptors are well-defined in the art [see for example, Wilson and Gisvold's Textbook of Organic Medicinal and 25 Pharmaceutical Chemistry., R.F. Doerge (ed.) 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 SU~STITUTE SHEET (RULE 26~

CA 02220~l7 l997-ll-07 W 09613~424 PCTAUS~6~065~3 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, O, N, Cl, Br and S. The H-bond consists of an attractive force which exists between a hydrogen 5 atom covalently bound to an atom from the list given above (e.g. a hydroxy group, -O-H) and a second atom, not ~ecP~s~rily 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 amme can act as both hydrogen bond donors and hydrogen bond acc~Lol~. Groups such as ethers or terl:iary amines are hydrogen bond acceptors only. The valence of quaternary amines which have no free lone pair are incapable 15 of being hydrogen bond accel~tors. Hydrogen bond acceptor groups specifically conL~ lated 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.

20 The term "alkyl" as used here refers to and includes normal and branch chained alkyl groups. The term "lower alkyl", unless specifi~ Ally 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 bran,ch chained groups having 2 to 4 carbons when the 25 chains are normal, and 3 or 4 carbons when the chains are branched.

SUBSTITUTE SHEET (RULE 26~

CA 02220~l7 l997-ll-07 W 096/35124 PCT~US96/06633 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-amino-imidazoles which have subtype selected a2A activity and methods for 2 o making them. The contents of this PCT application are hereby incorporated by rerer~llce 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) =

CA 02220~l7 l997-ll-07 W 096135~2~ PCTAUS96/06633 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 5 tract of a mammal, a reducf:ion 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 0 for use in the treatment or management of glaucoma, a medication for use in the trea~nent or management of kidney disease and/or a treatment for cclngested nasal passages. One irnportant feature of many of the present compounds is that the desired therapeutic effect is achieved with reduced or a~sent side effects, in particular, lacking effects 15 on the blood pressure or the mammal to which the present compound or compounds is/are administered.

Pler~lled compounds of the invention with reference to the Examples 1-24 in Table 1 are those compounds which show high affinity for a2 20 rece~Lol~ (low Ki values) in the binding assays. Particularly preferred are those compouncls in which the Ki (binding affinity) value for the ~~2 receptors is from 0.0001 to 10. With respect to the structural features which are pref,erred in the present invention, the elements which are preferred in providing the desired binding respect to the structural 25 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 R2 and R3, more preferably with a nitrogen atom bonding to the aryl ring at R2 and even more preferably with a nitrogen atom bonding to the ary ring at R2, and even more ~le~ldbly SUBSTITUTE SHEET (RU~E 26~

CA 02220~l7 l997-ll-07 W O 96/35424 PCTrUS96/06633 with a nitrogen or oxygen atom bonding to the aryl ring at R3. Other preferred embodiments include compounds of formula 1 wherein both Rls are methyl, one R2 is hydroxy or methoxy, the other R2 is hydrogen, and R3 is_-butyl.

Any suitable method of admi.~istering 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 10 in an effective manner, e.g., 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 ~~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 96135.S2~ 17 PCT~US96/06633 T~ble 1 K~
Struct;ure ¦ al azA a~s a2c Il ~A I~ ¦
E~ample HN NH
6,034 8.9 ~9 70 121,200 N~[~

nple 2 513 3.8 8.3 30 8.9 10,000 12,452 CN
Tnple 3 181 2.9 4.8 21 19 64,790 38,642 N ~

C I~NH2 E~ Tnple4 H N~N H 1,433 2 17 27 48 5,199155 N ~N ~l F.~nple 5 1,850 2.7 52 44 17 7,193614 N~T~N~, b~N

SUBSTITUTE SHEET (RULE 26~

CA 02220~l7 lgg7-ll-07 w 096/35l24 pcTrus96lo6633 Table 1 (con't.) K~
S ~ ~Ul~ ¦ a1 a2~ a2B a2c Il I2~ B

F.~Tnple 6 HN NH ~ 38,573 147 1,029 2,012 56 100,000 100,000 N~N

C~J~ N~
E~a m ple 7 H N N H 5,100 35 262 463 1,988 100,000 10,000 I

~N

rnple 8 H N N H 8,715 9.6 139 124 100,000 100,000 10,000 I I
N~[~XN~

H
F.~rnJple 9 H N~N H 1,117 6.1 47 120 4,575 7,561 4,588 N ~N ~
N

H
rn~ple 10 H N N H 6,606 24 194 2g3 3,952 24,827 3,2~6 ~ 7 N ~N 1 N
H
SUBSTITUTE SHEET (RULE 26~

W O96135~21 PCTAUS96/06633 Table 1 (con't.) K~
st~ructure I ~1 ~~ azB a2C Il I2A I2B I

E~ample 11 / \' H N~N H 2,403 3.1 28 20 1,835 1,084 2,032 N~O~
~OJ
F.~rnple 1~
H N~N H 1,452 5.2196 93 340 100,000 607 N~o E~aInple 13 H N~N H 129 0.25 4 3.5 831 100,000 1,223 N~O~
~oJ

~ample 14 /~
HN~NH H 8,987 52 842 11,600 4,500 6,517 N~N~O

E~rnple 1~
/~
HN~NH 2,262 17 241 134100,000 N ~3~N ~0 SUBSTITUTE SHEET (RULE 2~

W 096/35~24 PCTrUS96/06633 Table 1 (con't.) K~
Sbructure ¦ a1 a~ azs a2c I

rnple 16 H N~N H 473 1.2 30 8.9 7,112 100,000 12,135 N ~N ~
o E~ample 17 H ~ 14,784 6.3 436.~4 5,30027,035 9,283 HN~

F~Tn~le 18 H N~N 5,214 ~ 295 180 19 141 13 H N~3,~

Example 19 H N~N 7,613 10 434512 13 6,180 179 HN~

E~aI~ple 20 /=\ . =
H N~N 11,316 30 8954~7 412 H N~[~3~ O H

SUBSTITUTE SHEET (RULE 2~

CA 022205l7 l997-ll-07 W 096135~2~ PCTAUS96~06633 Table 1 (con't.) K~
Struch~ al a~A a~s a2c Il I2A
e_ F\
HN N
2,14? L7 82 19 444 7,708 686 H N ~,~0 ~
oJ
F~nple 22 ~=~
HN, N
y 1 4,17~ 4.9 221 88 4,320100,000 870 H N~O ~
~<
F~rr~ple 23 r~
H N~ ~N
199 0.7,7 97 13 7,41712,873 2,692 ~1~

E~ample 24 HN N
~ 1 30,550 3~ 1,156 212 227 6,367 ~6 H N~N~

SUBSTITUTE SHEET (RULE 26~

CA 02220~17 1997-11-07 W O 96/3512~ 22 PCT~US96/06633 Fxperimental Assays: Binding Affinities and Receptor Activation Receptor Binding Assays 5 Example 25:
Tissue preparation: Membrane suspensions were prepared from human cerebral cortex (HDD, for al receptors) obtained from the UCI Organ and Tissue Bank. Briefly, tissues (lg) were homogenized in 25 mL of ice-cold 5 mM Tris, pH 7.4 with a Polytron homogenizer for 30 sec at setting #7, 10 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-HCl buffer, pH 7.4, then centrifuged at 49,000 x g for 20 minutes. The pellet fraction was washed 3 15 times (resuspended in Tris-HCl buffer and centrifuged for 20 minutes at 49,000 x g). The pellet was then stored at -80~ C until the binding assay.

Cell preparation: Chinese hamster ovary (CHO) cells expressing the human a2A and human a2C (CHO-C10 and CHO-C4 respectively) 2 0 receptors and CHO cells (CHO-RNG) expressing the rat a2B 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-HCl, 5 mM EDTA, pH 7.4).
25 Cells were then homogenized with a Polytron homogenizer for 2 x 10 sec at setting #7, and centrifuged for 20 minutes at 49,000 x g. The pellet fraction was washed (resuspended in Tris-HCl, pH 8 buffer and SUBSTITUTE SHEET (RIJLE 26~

CA 02220~17 1997-11-07 W 09613~24 PCT/US96/06633 centrifuged for 15-20 minut~s at 49,000 x g) 2 times and stored at -100 ~
until ~mding assay.

Binding Studies: The radioligands [3H]rauwolscine (specific activity 80 Ci/mmol) and [3H)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-[3H]rallwolscine, 22~C, 30 minutes; RKC-[3H]rauwolscine, 0~C, 0 120 minutes; amd, HCC-[3H]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 fi]ters (WhatmLan GF/B) in a 96 well cell harvester and rapidly washedL four times with 4 mL of iced-cold 50 mM Tris-HCl buffer.
The filters were then oven dried and transferred to scintillation vials containing 10 rnL of Beckma,n's Ready Protein~) scintillation cocktail for counting. Speci:~ic binding defined by 10 uM phentolamine for competition stuclies were as follows: 2.4 nM [3H]brimonidine-RbICB
62%; 2.4 nM [3H]rauwolscine-RbICB 75%; 2 nM [3H]rauwolscine-RbKc 88%; 0.3 nM [3H] rauwolscine-CHO-C10 99%; 0.4 nM [3H]rauwolscine-2 0 CHO-RNG 99%, 0.3 NM [3H]prazosin 87%; and 1 nM [3H]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/Sal:uration by Beckman.

SUBSTITUTE SHEET (RULE 26 CA 02220~17 1997-11-07 Example 26:

Cell Preparation: Chinese hamster ovary (CHO) cells expressing the human CC;~A (CHO-C10) and the rate OC2B (CHO-RNG) human a2A
5 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-HCl, 5 mM EDTA, pH 7.4). Cells were then homogenized with a PolyLy,oll homogenizer for 2 x 10 sec at setting #7, 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.

15 Binding studies: Determination of Ki The radioligands [3H] rauwolscine (specific activity 80 Ci/mmol) and [3H]
prazosin (specific activity 76 Ci/mmol) were obtained from New England Nuclear, Boston, MA. Frozen membrane pellet was 2 0 resuspended in 25 mM glycine/glycine, pH 7.4 and incubated with radioligand under the following conditions: CHO-C10, CHO-RNG, CHO-C4-[3H]rauwolscine, 22~ C, 30 min.; and, HCC-[3H]prazowsin, 22~C, 30 minutes in a final volume of 500 ul. At the end of the incubation period, the samples were filtered through glass fiber filters (Whatman 25 GF/B) in a 96 well cell harvester and rapidly washed four times with 4 mL of iced-cold 50 mM Tris-CHl buffer. The filters were then oven dried and transferred to scintillation vials containing 5 mL of Beckman's Ready Protein(É~) scintillation cocktail for counting. Specific binding ~ _ defined by 10 uM phentolamine for competition studies were as follows:

SUBSTITUTE SHEET (RULE 2B) CA 02220~17 1997-11-07 W ~96)3~424 PCT~US96/06633 0.3 nM[3H]rauw~lscine-CHO-C10 99%; 0.4 nM[3H]rauwolscine-CHO-RNG
99%, and 0.3 n~ [3H]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 E~ovine ventrolateral medulla (BVLM) membranes 0 Example 27:

Fresh bovine bra,in 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 pcsterior 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) cl~ntaining 250 mM sucrose, 50 uM Calpain I inhibitor (Boehginer Mam~heim, IndiaLnapolis, IN), 100 uM 1,10-phenanthroline (Sigma, St. Louis, MO) and 50 uM Pefabloc (Boehginer Mannheim, Indianapolis, I~J), using Virtis homogenizer at setting 7 with three 10 second pulses, f~llowed by three passes in a teflon-glass tissue homogenizer. 1~-e 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 2() min at 48000xg. The pellets obtained were resuspended in a teflon-glass homogenizer in 50 mM Tris-HCl with 5 mM EDTA (pH 7.7 at 4~C), centrifuged, and resuspended in 50 mM Tris-SUBSTITUTE SHEET (RULE 26~

CA 02220~17 1997-11-07 W 096/35121 PCTrUS96/06633 HCl with 25 mM NaCl, 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 48000xg. 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 4~C). Pellets obtained after centrifugation at 48000xg 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.

Il imidazoline receptor binding assay F~mple 28:
I, imidazoline receptor binding affinity was determined from radioligand binding of 3H-clonidine (NEN, Boston, MA) to bovine VLM
membranes. Specific activity of 3H-clonidine was 43 Ci/mmol. Kd of 3H-clonidine binding to the Il imidazoline receptor was determined by saturation experiments and Ki of other ligands studied were determined 2 o by competition experiments. The radioligand binding assay was performed in Teflon 96-wells with the Biomek-1000 robotics (Beckman Instruments, Fullerton, CA). Each well contained 4 mN 3H-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 MgCl2, pH 7.4 (0.1 mM ascorbic acid was added just before the protein). After 50 min of incubation at 25~C, 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-HCl, pH 7.4 at 4~C in a Brandel Harvester (Brandel, Gaithersburg, M.D.). Both 'hot' and 'cold' saturation SUBSTITUTE SHEET (RULE 26~

CA 02220~17 1997-11-07 W 096135~24 PCTA~S96~06633 experiments were performed. In 'hot' saturation experiments, studies were performed with 3H-clonidine ranging from 0.1 to 50 nM. In 'cold' saturation experiments, studies were performed with 2 nM 3H-clonidine with 20 different concentrations of the unlabeled clonidine, ranging 5 from 0.1 nM to 1 uM urLlabeled clonidine. Non specific binding was defined by paral]el incubatiorLs containing 10-5 M phentolamine or naphazolirLe. In-Lidazoline binding was determined by parallel incubations in which the alpha-adrenergic sites were masked with 10-5 M
norepinephrine. During competition experiments, ligands of 20 0 concentrations ranged from 10-ll to 10 l were used. Radioactivity was counted in a Becl~man 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 15 were repeated at ]east twice.

Representative compourLds 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 2 o studies and Kl a;re measures of the affinity of a compound for a particular receptor.

Example 29 25 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 3 0 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~

CA 02220~17 1997-11-07 W O 96/35424 PCTrUS96/06633 16 showed a greater effect in lowering IOP than clonidine without lowering IOP in the untreated eye.

Fxample 30 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 10 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 15 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.

Sl lBsTlTuTE SHEET (RULE 26

Claims (12)

1) A use, in the manufacture of a medicament, of a compound of the formula II

wherein R1 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 as an active ingredient in a composition which treats elevated intraocular pressure without causing a concomitant reduction in blood pressure of said mammal in admixture with an inert carrier.

2) The use of claim 1, wherein the compound of formula II
has the structure wherein R1 represents methyl or bromine and A is hydrogen or an oxo group.

3) The use of claim 1, wherein the compound of formula II
has the structure

4) A use, in the manufacture of a medicament, of a compound of the formula II

wherein R1 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 as an active ingredient in a composition which treats nasal congestion without causing a concomitant reduction in blood pressure of said mammal in admixture with an inert carrier.

5) The use of claim 4, wherein the compound of formula II
has the structure wherein R1 represents methyl or bromine and A is hydrogen or an oxo group.

6) The use of claim 4, wherein the compound of formula II
has the structure

7) A use, in the manufacture of a medicament, of a compound of the formula II

wherein R1 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 as an active ingredient in a composition which treats diarrhea without causing a concomitant reduction in blood pressure of said mammal in admixture with an inert carrier.

8) The use of claim 7, wherein the compound of formula II
has the structure wherein R1 represents methyl or bromine and A is hydrogen or an oxo group.

9) The use of claim 7, wherein the compound of formula II
has the structure

10) Canceled

11) Canceled

12) Canceled