WO2003053436A1 - Novel benzodifuranimidazoline and benzofuranimidazoline derivatives and their use for the treatment of glaucoma - Google Patents

Abstract

The present invention provides benzodifuran imidazoline derivatives and benzofuran imidazoline derivatives for lowering intraocular pressure and providing ocular neuroprotection.

Description

NOVEL BENZODIFURANIMIDAZOLINE AND BENZOFURANIMIDAZOLINE DERIVATIVES AND THEIR USE FOR THE TREATMENT OF GLAUCOMA

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of glaucoma treatment and ocular

neuroprotection. More particularly, the present invention provides novel compounds,

compositions and methods for treating glaucoma, lowering intraocular pressure and

providing neuroprotection.

2. Description of the Related Art

The disease state referred to as glaucoma is characterized by a permanent loss of

visual function due to irreversible damage to the optic nerve. The several morphologically

or functionally distinct types of glaucoma are typically characterized by elevated IOP,

which is considered to be causally related to the pathological course of the disease. Ocular

hypertension is a condition wherein intraocular pressure is elevated but no apparent loss of

visual function has occurred; such patients are considered to be at high risk for the

eventual development of the visual loss associated with glaucoma. Some patients with

glaucomatous field loss have relatively low intraocular pressures. These so called normal

tension or low tension glaucoma patients can also benefit from agents that lower and

control IOP. If glaucoma or ocular hypertension is detected early and treated promptly

with medications that effectively reduce elevated intraocular pressure, loss of visual

function or its progressive deterioration can generally be ameliorated. Drug therapies that

have proven to be effective for the reduction of intraocular pressure include both agents

that decrease aqueous humor production and agents that increase the outflow facility. Such therapies are in general administered by one of two possible routes, topically (direct

application to the eye) or orally.

There are some individuals who do not respond well when treated with certain

existing glaucoma therapies. There is, therefore, a need for other topical therapeutic

agents that control IOP.

Serotonin (5-hydroxy tryptamine; 5HT) is an endogenous biogenic amine with a

well defined neurotransmitter function in many tissues of the body including the eye [Zifa

and Fillion 1992; Hoyer et al. 1994; Tobin et al. 1988].

5HT is known to interact with at least seven major 5HT receptors (δHT_j - 5HT₇),

and additional subtypes within these families, to initiate intracellular biochemical events

such as stimulation of second messengers (e.g. cAMP, inositol trisphosphate) eventually

leading to the final biological response, for example, tissue contraction or hormone

release, etc. [Hoyer et al. 1994; Martin et al. 1998]. Receptor subtypes within the 5HT_[

family are negatively coupled to adenylyl cyclase (AC) and cause inhibition of cAMP

production, while 5HT₄, 5HT₆, and 5HT₇ receptors are positively coupled to AC and thus

stimulate cAMP production when activated by 5HT [Martin et al. 1998]. The receptors in

the 5HT₂ family are positively coupled to phospholipase C (PLC) and thus generate

inositol phosphates and mobilize intracellular calcium when activated to mediate the

effects of 5HT. The 5HT₃ receptor is unique in that it couples to an ion channel which

gates sodium, potassium, and calcium [Hoyer et al. 1994]. Known compounds exhibiting 5HT₂ agonist activity have typically been designed

to treat numerous central nervous system (CNS)-related conditions, particularly the

treatment of obesity and depression, by activation of 5-HT_2C receptors. Thus, one desired

property of known 5HT₂ agonist compounds is that they easily penetrate the blood brain

barrier. Compounds that readily penetrate the blood-brain-barrier by passive diffusion are

generally lipophilic molecules, which do not contain polar functional groups that might

impede this diffusion.

The utility of 5-HT₂ agonists for controlling IOP in the monkey model of glaucoma

has been established (WO 00/16761). α₂ adrenoceptor agonists are also known for their

use as IOP lowering agents. It is also known that compounds with 5-HT_1A agonist activity

can be useful for the treatment of glaucomatous optic neuropathy (WO 0170223 Al).

Until the present invention, no single compound possessing 5-HT_2A and/or 5-HT_1A agonist

activity along with α₂ adrenoceptor agonist activity has been known.

To treat ocular diseases, it is desirable to administer topically compositions that

will remain in the ocular tissues and not cross the blood brain barrier and enter the CNS.

What are needed are anti-glaucoma drugs with both IOP lowering potency and ocular

neuroprotective activity. It is also desirable that such compounds would not have a

propensity to cross the blood brain barrier. SUMMARY OF THE INVENTION

The present invention overcomes these and other drawbacks of the prior art by

providing benzodifuran imidazoline derivatives and benzofuran imidazoline compounds

for lowering IOP and providing neuiOprotection. More specifically, the present invention

provides compounds of the formula:

wherein A, B and D are independently chosen from either N or C, with the provision that

at least one of A, B or D is N; E is C or N; R is H or C_Malkyl; R² and R³ are independently

H, C_j.3 alkyl, C₂-₃ alkenyl, or R² and R³ taken together can form a 5 or 6 member ring; X is

hydrogen, halogen, C_Malkyl, or CF₃; and the dashed bond may be a single bond or a

double bond; and pharmaceutically acceptable salts and solvates. Preferably the

compound is 2-(8-bromo-benzo-[l,2-b;4,5-b']difuran-4-yl) imidazoline hydrochloride.

In another aspect, the present invention provides compositions containing the

compounds described above. The compositions are most preferably in the form of topical

ophthalmic formulations for delivery to the eye. The compounds of the invention may be

combined with ophthalmologically acceptable preservatives, surfactants, viscosity

enhancers, penetration enhancers, buffers, sodium chloride, and water to form an aqueous,

sterile ophthalmic suspension or solution to form the compositions of the invention. The compositions of the invention are preferably formulated as topical ophthalmic

suspensions or solutions, with a pH of about 5 to 8. The compounds of the invention as

described above will normally be contained in these formulations in an amount .01% to

5% by weight, but preferably in an amount of 0.1% to 2% by weight. Thus, for topical

presentation 1 to 2 drops of these formulations would be delivered to the surface of the eye

1 to 4 times per day according to the routine discretion of a skilled clinician.

The present invention further provides a method of lowering intraocular pressure

and providing ocular neuroprotection in a mammal by administering to a patient in need

thereof a therapeutically effective amount of a composition comprising a compound

having the structure as described above. In preferred embodiments, the composition can

be administered locally to the eye (e.g., topically, intracamerally, or via an implant).

DETAILED DESCRIPTION PREFERRED EMBODIMENTS

Unexpectedly, it has been found that serotonergic compounds which possess

agonist activity at 5HT₂ receptors effectively lower and control elevated IOP and

glaucoma. In addition, the compounds provide neuroprotective activity and are useful for

treating persons suffering from ocular diseases associated with neuronal cell death.

It has been found that serotonergic compounds which possess agonist activity at 5-HT₂

receptors effectively lower and control normal and elevated IOP and are useful for treating

glaucoma, see commonly owned co-pending application, PCT/US99/19888.

Compounds that act as agonists at 5-HT₂ receptors are known and have shown a

variety of utilities, primarily for disorders or conditions associated with the central nervous system (CNS). U.S. Patent 5,494,928 discloses certain 2-(indol-l-yl)-ethylamine

derivatives that are 5-HT_2C agonists for the treatment of obsessive compulsive disorder and

other CNS derived personality disorders. U.S. Patent 5,571,833 discloses tryptamine

derivatives that are 5-HT₂ agonists for the treatment of portal hypertension and migraine.

U.S. Patent 5,874,477 discloses a method for treating malaria using 5-HT_2A/2C agonists.

U.S. Patent 5,902,815 discloses the use of 5-HT_2A agonists to prevent adverse effects of

NMD A receptor hypo-function. WO 98/31354 A2 discloses 5-HT_2B agonists for the

treatment of depression and other CNS conditions. Agonist response at the 5-HT_2A

receptor is reported to be the primary activity responsible for hallucinogenic activity, with

some lesser involvement of the 5-HT_2C receptor possible (Fiorella et al. 1995).

Serotonergic 5-HT_1A agonists have been reported as being neuroprotective in

animal models and many of these agents have been evaluated for the treatment of acute

stroke among other indications. This class of compounds has been disclosed for the

treatment of glaucoma (lowering and controlling IOP), see e.g., WO 98/18458 and EP

0771563 A2. Osborne et al. teach that 8-hydroxydipropylaminotetralin (8-OH-DPAT) (a

5-HT_1A agonist) reduces IOP in rabbits (Osborne et al. 1996). Wang et al. disclose that 5-

methylurapidil, an α_1A antagonist and 5-HT_IA agonist lowers IOP in the monkey, but due

to its α_1A receptor activity (Wang et al. 1997; Wang et al. 1998). Also, 5-HT_1A antagonists

are disclosed as being useful for the treatment of glaucoma (elevated IOP) (e.g. WO

92/0338). Furthermore, DeSai et al. (WO 97/35579) and Macor et al. (U.S. 5,578,612)

disclose the use of 5-HT_! and 5-HT_μlike agonists for the treatment of glaucoma (elevated

IOP). These anti-migraine compounds are 5-HT_{1B D E F} agonists, e.g., sumatriptan and

naratriptan and related compounds. The present invention provides compounds possessing α₂ adrenoceptor agonist

activity along with 5-HT_2A and 5-HT_1A activities having the general structure of Formula I.

Formula I

wherein A, B and D are independently chosen from ether N, C, with the provision that at

least one of A, B or D is N; E is C or N; R is H, C_Malkyl; R² is H, C,.₃ alkyl, or C₂_₃

alkenyl; R³ is H, C₁_₃ alkyl, or C₂.₃ alkenyl; or R² and R³ taken together can form a 5 or 6

member ring; X is chosen from hydrogen, halogen, C_].₄alkyl, CF₃; the dashed bond

indicates that either a single bond or a double bond can exist at this bond location; and

pharmaceutically acceptable salts and solvates. In preferred embodiments, the compound

of the invention is 2-(8-bromo-benzo-[1.2-b;4,5-b']difuran-4-yl) imidazoline

hydrochloride.

ES 323985 discusses that oxymetazoline is currently used for nasal de-congestion

and in an ophthalmic solution to relieve redness of the eye. Although ES 323985 does

discuss IOP lowering activity for oxymetazoline, it does not discuss the use of

oxymetazoline for lowering IOP and ocular neuroprotection. Moreover, oxymetazoline is

not a benzofuran as it lacks the furan substituent(s) and/or the ether substituent (Wang et

al. 1993). Further, none of the claimed compounds are disclosed in ES 323985 or Wang. It is recognized that compounds of Formula I can contain one or more chiral

centers. This invention contemplates all enantiomers, diastereomers and, mixtures thereof.

In the above definitions, the total number of carbon atoms in a substituent group is

indicated by the ._j prefix where the numbers i and j define the number of carbon atoms;

this definition includes straight chain, branched chain, and cyclic alkyl or (cyclic

alkyl)alkyl groups.

It is important to recognize that a substituent may be present either singly or

multiply when incorporated into the indicated structural unit. For example, the substituent

halogen, which means fluorine, chlorine, bromine, or iodine, would indicate that the unit

to which it is attached may be substituted with one or more halogen atoms, which may be

the same or different.

The compounds of the invention can be administered systemically or locally to the

eye (e.g., topically, intracamerally, or via an implant). The compounds are preferrably

incorporated into topical ophthalmic formulations for delivery to the eye. The compounds

may be combined with ophthalmologically acceptable preservatives, surfactants, viscosity

enhancers, penetration enhancers, buffers, sodium chloride, and water to form an aqueous,

sterile ophthalmic suspension or solution. Ophthalmic solution formulations may be

prepared by dissolving a compound in a physiologically acceptable isotonic aqueous

buffer. Further, the ophthalmic solution may include an ophthalmologically acceptable

surfactant to assist in dissolving the compound. Additionally, the ophthalmic solution

may contain an agent ' to increase viscosity, such as, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, methylcellulose,

polyvinylpyrrolidone, or the like, to improve the retention of the formulation in the

conjunctival sac. Gelling agents can also be used, including, but not limited to, gellan and

xanthan gum. In order to prepare sterile ophthalmic ointment formulations, the active

ingredient is combined with a preservative in an appropriate vehicle, such as, mineral oil,

liquid lanolin, or white petrolatum. Sterile ophthalmic gel formulations may be prepared

by suspending the active ingredient in a hydrophilic base prepared from the combination

of, for example, carbopol-940, or the like, according to the published formulations for

analogous ophthalmic preparations; preservatives and tonicity agents can be incorporated.

The compounds of the invention are preferably formulated as topical ophthalmic

suspensions or solutions, with a pH of about 5 to 8. The compounds will normally be

contained in these formulations in an amount .01% to 5% by weight, but preferably in an

amount of 0.1 % to 2% by weight. Thus, for topical presentation 1 to 2 drops of these

formulations would be delivered to the surface of the eye 1 to 4 times per day according to

the routine discretion of a skilled clinician.

The compounds can also be used in combination with other IOP lowering agents,

such as, but not limited to, β-blockers, prostaglandins, carbonic anhydrase inhibitors, and

miotics. The compounds can also be used in combination with other agents useful for

treating glaucoma, such as, but not limited to, calcium channel blockers and NMDA

antagonists. These agents may be administered topically, but usually systemically. The following examples are included to demonstrate preferred embodiments of the

invention. It should be appreciated by those of skill in the art that the techniques disclosed

in the examples which follow represent techniques discovered by the inventor to function

well in the practice of the invention, and thus can be considered to constitute preferred

modes for its practice. However, those of skill in the art should, in light of the present

disclosure, appreciate that many changes can be made in the specific embodiments which

are disclosed and still obtain a like or similar result without departing from the spirit and

scope of the invention.

Example 1 Synthetic Scheme for 2-(8-bromo-benzo-[l,2-b;4,5-b'1difuran-4-yl) imidazoline hydrochloride

Examples of the compounds of this invention may be prepared by the synthetic

route describe by Scheme 1. Briefly , the commercially available bis ethanol ether is

treated with thionyl chloride in the presence of a organic base preferably pyridine in a

solvent such as methylene chloride to form 2. The halogenated ether 2 is brominated

using bromine in the presence of a Lewis acid such as zinc chloride in a solvent such as

acetic acid to give compound 3. The di-bromide is cyclized to 4 with n-butyl lithium in a

solvent such as dioxane or tetrahydrofuran maintained at a temperature of -40 to 0° C.

Formylation with dichloromethyl methyl ether in the presence of stannic chloride in an

inert solvent such as methylene chloride provides 5. Reduction of the aldehyde with

sodium borohyride in a solvent such as ethanol or isopropyl alcohol yields the alcohol 6.

The alcohol is converted to the chloride 7 by treatment with thionyl chloride in the

presence of pyridine in a solvent such as methylene chloride. The nitrile 8 is formed by

reacting 7 with sodium cyanide in a solvent such as DMSO at a temperature of 40-80° C.

Bromination of the nitrile with a mixture of bromine and acetic acid at temperatures 0 to 20° C yields compound 9. Reduction of the bis dihydrofuran with 2,3-dichloro-5,6-

dicyano-l,4-benzoquinone (DDQ) in a solvent such as dioxane at temperatures between 80

to 130° C yields compound 10. Treatment of the nitrile 10 with hydrogen chloride gas in a

solution of ethanol and ether provides the imino ester, 11. Cyclization of the imino ester

with ethylenediamine in ethanol and conversion of the product to the hydrochloride salt

using a solution of hydrogen chloride in ethanol yields imidazoline benzodifuran 12.

Scheme 1

^■ ₂ / acetic acid

Example 2 2-(8-bromo-benzo-[l,2-b;4,5-b'ldifuran-4-yl) imidazoline hydrochloride

2-(8-Bromo-benzo-[l,2-b;4,5-b']difuran-4-yl) imidazoline hydrochloride was

prepared by the multi-step procedure described below.

Step A: l,4-Bis(2-chloroethoxy)benzene

Bis(2-hydroxyethyl)hydroquinone (50g, 0.25mol) was dissolved in 500ml of

CH₂C1₂ and cooled to 0° C, pyridine (48ml, 0.6mol) and thionyl chloride (41ml, 0.58ml)

were added drop wise such that the temperature did not exceed 5 °C. The mixture was

allowed to warm to room temperature and was stirred over night. The solvent volume was

reduced to 150ml. Aqueous 2N HCl (150ml) was added slowly and the layers were

separated. The aqueous layer was extracted with CH₂C1₂ (3x100ml). The combined organic

layer was washed with 2N HCl (150ml), saturated NaCl solution (150ml), dried over

anhydrous MgSO filtered and evaporated to a white solid. Recrystallization from

ethanol afforded a white solid (73g). CIMS m/z 236 (M+H)⁺.

Step B: l,4-Bis(2-chloroethoxy)-2,5-dibromobenzene

l,4-Bis(2-chloroethoxy)benzene (40g, 0.17mol) was suspended in acetic acid

(400ml) and zinc chloride (56g, 0.41mol) was added. Bromine (57, 0.36mol) dissolved in

acetic acid (80ml) was added drop wise to the suspension over 1.5h. The reaction was

stirred at room temperature over night, during which time a precipitate formed. The solids

were filtered, washed with acetic acid and ethanol and dried. A crystalline white product

was obtained (45g). CIMS m/z 393 (M+H)⁺. Step C: 2,3,6,7-Tetrahydrobenzol[l,2-b;4,5-b']difuran

A solution of l,4-Bis(2-chloroethoxy)-2,5-dibromobenzene (15g, 0.036mol) in dry THF (300ml) was cooled to 0 °C under nitrogen. A solution of 2.5 M n-butyl lithium in

hexane (30ml, 0.075mol) was added through a syringe very quickly to the well stirred

solution. The reaction mixture was stirred at 0 °C for 10 min, and the solvent was

removed in vacuo. The residue was partitioned between ether (300ml) and water (200ml).

The organic layer was washed with water (200 ml), dried over MgSO₄ and filtered. The

solution was evaporated on a rotary evaporator until solids formed. The solids were

filtered and dried to afford 4.3g of 2,3,6,7-tetr-ιhydrobenzol[l,2-b;4,5-b']difuran. CIMS

m/z 163 (M+H)⁺.

Step D: 4-Formyl-2,3,6,7-tetrahydrobenzol[l,2-b;4,5-b']difuran

Tin(IN) chloride (11.7 ml, O.lmol) was added through a syringe to a solution of

2,3,6,7-tetrahydrobenzol[l,2-b;4,5-b']difuran (12.6 g, 0.078 mol) in 300 ml of dry CH₂C1₂

at 0 °C under Ν₂, and the mixture was stirred for 5 min. Dichloromethyl methyl ether (7

ml, 0.078 mol) in 20 ml of CH,C1₂ was added into the mixture dropwise over a 10 min

period. After the mixture was stirred for 30 min, the reaction was quenched by the

addition of 100 ml of ice water. The aqueous layer was extracted with CH₂C1₂ (2x100ml).

The organic layers were combined and the resulting solution was washed with 3N HCl

(3x150ml), H₂O (200 ml), and a saturated NaCl solution (200ml), dried over anhydrous

MgSO4, filtered and evaporated to a white solid. Recrystallization from CH₂Cl₂-hexane

yielded 12.2 g of the product as a yellow solid. CIMS m/z 191 (M+H)⁺. Step E: 4-Hydroxymethyl-2,3,6,7-tetrahydrobenzol[l,2-b;4,5-b']difuran

A solution of NaBH₄ (2g, 0.053 mol) in 40 ml of 90% EtOH was added dropwise

to a solution of 4-Formyl-2,3,6,7-tetrahydrobenzol[l,2-b;4,5-b']difuran (10 g, 0.053 mol)

in 200 ml of EtOH. The solution was stirred at room temperature for 30 min and at 60° C

for 10 min. After cooling to 0° C, 5 ml of IN HCl was added and the solvent was

evaporated. Ethyl acetate (80 ml) was added to the residue, and the resulting mixture was

washed with H₂O (50 ml), saturated NaCl solution (50ml), dried over anhydrous MgSO4,

filtered and evaporated to a residue. Chromatography of the residue on silica gel, eluting

with 30 % ethyl acetate in hexane, gave 7.5 g of 4-hydroxymethyl-2,3,6,7-

tetrahydrobenzolfl ,2-b;4,5-b']difuran as a white solid. CIMS m/z 193 (M+H)\

Step F: 4-Chloromethyl-2,3,6,7-tetrahrdrobenzol[l,2-b;4,5-b']difuran

Pyridine (4 ml, 0.05 mol) was added to a solution of 4-hydroxymethyl-2,3,6,7-

tetrahrdrobenzol[l,2-b;4,5-b']difuran (4 g, 0.021 mol) in 50 ml of CH₂C1₂ and the mixture

was cooled to 0 °C. Thionyl chloride (3.5 ml, 0.048 mol) was added dropwise. The

resulting mixture was allowed to warm to room temperature and stirred for 6 h. After

cooling, the mixture was washed with 1 N NaOH (2x50 ml), saturated NaCl solution

(100ml), dried over anhydrous MgSO_ι, filtered and evaporated to a residue.

Chromatography of the residue on silica gel, eluting with 10 % ethyl acetate in hexane,

gave 2.5 g of the product as a white solid. CIMS m/z 211 (M+H)⁺. Step G: 4-Acetonitrile-2,3,6,7-tetrahrdrobenzol[l,2-b;4,5-b']difuran

4-Chloromethyl-2,3,6,7-tetrahydrobenzol[l,2-b;4,5-b']difuran (2 g, 0.01 mol) in 20

ml of DMSO was added dropwise to a solution of sodium cyanide (0.75 g, 0.015 mol) in

20 ml of DMSO at 70 °C. The mixture was stirred at 70 °C for 40 min. After cooling, 50

ml of ice-water was added. The precipitate formed was filtered, washed with water and

dried giving white solid 8 (1.4g). CIMS m/z 202 (M+H)⁺.

Step H: 4-Acetonitrile-8-bromo-2,3_.6,7-tetrahrdrobenzol[l,2-b;4,5-b'] difuran

Bromine (1.1 g, 0.007 mol) in 10 ml of acetic acid was added dropwise to a

suspension of 4-acetonitrile-2,3,6,7-tetrahydrobenzol[l,2-b;4,5-b']difuran (1.4 g, 0.007

mol) in 20 ml of acetic acid at 15° C. The mixture was stirred at 15° C for 15 min. The

precipitate formed was filtered, washed with acetic acid and ethanol and dried to yield 1.4

g of the product as a white solid. CIMS m/z 281 (M+H)⁺.

Step I: 4-Acetonitrile-8-bromo-[l,2-b;4,5-b']difuran

A solution of DDQ in 70 ml of dioxane was added dropwise to a solution of 4-

acetonitrile-8-bromo-2,3,6,7-tetrahrdrobenzol[l,2-b;4,5-b']difuran (1.4g, 0.005 mol) in 70

ml of dioxane. The mixture was stirred at reflux for 24 h. After cooling, the precipitate

that formed was filtered and washed with dioxane. The filtrate was evaporated to a

residue, which was subjected to chromatography on silica gel, eluting with 10 % ethyl

acetate in hexane, to yield 0.61 g of 10 as a white solid. CIMS m/z 277 (M+H)⁺, mp 169-

170° C. Step J: Ethyl (8-bromo-[l,2-b;4,5-b']difuran- 4-yl)acetimidate

hydrochloride

An excess of dry HCl gas was passed through a solution of 4-acetonitrile-8-bromo-

[l,2-b;4,5-b']difuran (0.6 g, 0.0022 mol) in 50 ml of anhydrous ether and 3 ml of absolute

ethanol at 0 °C. The resulting mixture was allowed to stirred at 0 °C for 1 h and at room

temperature over night. The white solid formed was collected by filtration, washed with

ether and dried to give white crystal of the product (0.6 g). ESMS m/z 323 (M+H)⁺, mp

239-240 °C (dec).

Step K: 2-(8-Bromo-benzo-[l,2-b;4,5-b']difuran- 4-yl)imidazoline

hydrochloride

A solution of ethylenediamine (0.8 ml, 0.012 mol) in absolute ethanol (5 ml) was

added dropwise to a suspension of ethyl (8-bromo-[l,2-b;4,5-b']difuι-an-4-yl)acetimidate

hydrochloride (0.54 g, 0.0015 mol) in absolute ethanol (50 ml) at 0° C. The resulting

mixture was stirred at 0° C for 1 h and then refluxed for 20 min. The solvent was

evaporated and the residue was dissolved in 20 ml of ethanol. A solution of IN HCl in

ether was added to the solution above to reach a pH of 3 and the mixture was stirred at

room temperature overnight. The white solid that formed (0.4 g) was filtered, dried and

recrystallized from MeOH/ether to afford the product (0.32 g). APCIMS m/z 320 (M+H)⁺,

mp 264-265° C (dec). *H NMR (CDC1₃ ) D 8.21-8.19 ( s, 2H ), 7.43 (s, 1H ), 7.08 ( s, 1H

), 4.47 (s, 2H ), 3.83 ( s, 4H ), 3.32 ( s, 2H), 13c NMR ( CDCI3) D 168.10 ( C ), 149.45 (

C ), 148.49 ( C), 147.99 ( CH ), 147.63 ( CH ), 126.26 ( C ), 126.13 ( C ), 106.64 ( CH ),

106.53 ( CH ), 106.24 ( C ), 93.40 ( C ), 44.24 ( CH₂ ), 24.15 ( CH₂ ). Anal. (C₁₄H_πBrN₂O₂ HCl), Cal: C, 47.29%; H, 3.40%; N, 7.87%; found: C, 47.05%; H, 3.56%;

N, 7.98%.

Example 3 5-HT₂ Receptor Binding Assay

In order to determine the relative affinities of serotonergic compounds at the 5-HT₂

receptors, their ability to compete for the binding of the agonist radioligand [^I25I]DOI to

brain 5-HT₂ receptors is determined as described below with minor modification of the

literature procedure (Johnson et al. 1987). Aliquots of post mortem rat cerebral cortex

homogenates (400 μl) dispersed in 50 mM TrisHCl buffer (pH 7.4) are incubated with

[^{1 5}I]DOI (80 pM final) in the absence or presence of methiothepin (10 μM final) to define

total and non-specific binding, respectively, in a total volume of 0.5 ml. The assay

mixture is incubated for 1 hour at 23 °C in polypropylene tubes and the assays terminated

by rapid vacuum filtration over Whatman GF/B glass fiber filters previously soaked in

0.3% polyethyleneimme using ice-cold buffer. Test compounds (at different

concentrations) are substituted for methiothepin. Filter-bound radioactivity is determined

by scintillation spectrometry on a beta counter. The data are analyzed using a non-linear,

iterative curve-fitting computer program (Bowen et al. 1995) to determine the compound

affinity parameter. The concentration of the compound needed to inhibit the [¹²⁵I]DOI

binding by 50% of the maximum is termed the IC₅₀ or K, value. Compounds are

considered to possess high affinity for the 5-HT₂ receptor if their IC₅₀ or Kj values are <50

nM.

Example 4 5-HT₂ Functional Assay: Phosphoinositide (PI) turnover assay

The relative agonist activity of serotonergic compounds at the 5-HT₂ receptor can

be determined in vitro using the ability of the compounds to stimulate the production of [³H]inositol phosphates in [³H]myo-inositol-labeled A7r5 rat vascular smooth muscle cells

by their ability to activate the enzyme phospholipase C. These cells are grown in culture

plates, maintained in a humidified atmosphere of 5% CO₂ and 95% air and fed semi-

weekly with Dulbecco's modified Eagle medium (DMEM) containing 4.5 g/1 glucose and

supplemented with 2mM glutamine, 10 μg/ml gentamicin, and 10% fetal bovine serum.

For the purpose of conducting the phosphoinositide (PI) turnover experiments, the A7r5

cells are cultured in 24-well plates as previously described (Griffin et al. 1998). Confluent

cells are exposed for 24-30 hrs to 1.5 μCi [³H]-myo-inositol (18.3 Ci/mmol) in 0.5 ml of

serum-free medium. Cells are then rinsed once with DMEM/F-12 containing 10 mM LiCl

prior to incubation with the test agent (or solvent as the control) in 1.0 ml of the same

medium for 1 hr at 37°C, after which the medium is aspirated and 1 ml of cold 0.1 M

formic acid added to stop the reaction. The chromatographic separation of [³H]-inositol

phosphates ([³H]-IPs) on an AG- 1-X8 column is performed as previously described

(Griffin et al. 1998) with sequential washes with H₂O and 50 mM ammonium formate,

followed by elution of the total [³H]-IPs fraction with 1.2 M ammonium formate

containing 0.1 M formic acid. The eluate (4 ml) is collected, mixed with 15 ml

scintillation fluid, and the total [³H]-IPs determined by scintillation counting on a beta-

counter. Concentration-response data are analyzed by the sigmoidal fit function of the

Origin Scientific Graphics software (Microcal Software, Northampton, MA) to determine

agonist potency (EC₅₀ value) and efficacy (E_max). Serotonin (5-HT) is used as a positive

control (standard) agonist compound and the efficacy of test compounds is compared to

that of 5-HT (set at 100%). The concentration of the compound needed to stimulate the

production of [³H]-IPs by 50% of the maximum response is termed the EC₅₀ value. Compounds are considered potent agonists if their EC₅₀ values in this functional assay are

< 1 μM and are considered full agonists if their efficacy is > 80% of that of 5-HT.

The above procedures were used to generate the data shown in Table 1.

Table 1. 5-HT2 Receptor Binding and Functional Data.

Example 5 Acute IOP Response in Lasered (Hypertensive) Eyes of Conscious

Cynomolgus Monkeys

Intraocular pressure (IOP) can be determined with an Alcon Pneumatonometer

after light corneal anesthesia with 0.1%) proparacaine. Eyes are washed with saline after

each measurement. After a baseline IOP measurement, test compound is instilled in one

30 μL aliquot to the right eyes only of nine cynomolgus monkeys. Nehicle is instilled in

the right eyes of six additional animals. Subsequent IOP measurements are taken at 1, 3,

and 6 hours.

Example 6 5-HT_]A Receptor Binding Assay

5-HT_ΪA binding studies were performed with human cloned receptors expressed in

Chinese hamster ovary (CHO) cells using (³H)8-OH DP AT as the ligand. -Membranes

from Chinese hamster ovary cells (CHO) expressing cloned 5-HT_1A receptors

(manufactured for ΝEΝ by Biosignal, Inc., Montreal, Canada) were homogenized in

approximately 40 volumes of 50 mM Tris pH 7.4 for 5 sec. Drug dilutions were made

using a Beckman Biomek 2000 robot (Beckman Instruments, Fullerton, CA). Incubations were conducted with membrane prep, test compounds, and 0.25 nM [³H]8-OH-DPAT

(NEN, Boston, MA) in the same buffer at 27°C for 1 h. Assays were terminated by rapid

vacuum filtration over Whatman GF/B glass fiber filters pre-soaked in 0.3%

polyethyleneimme. Bound radioactivity was measured using liquid scintillation

spectrometry. Data were analyzed using non-linear curve fitting programs (Sharif et al.

1999).

Ligand binding studies can also be run using membrane preparations from calf and

rat brain (local source) and human cortex membranes. Specific brain regions were

dissected out, homogenized in 10 volumes of 0.32 M sucrose and centrifuged for 10 min at

700 x g. The resulting supernatant was centrifuged at 43,500 x g for 10 min and the pellet

re-suspended in 50 mM Tris-HCl (pH 7.7, 25°C) using a 10 sec polytron treatment.

Aliquots were stored at -140° C. To remove endogenous serotonin, the preps were

incubated at 37° C for 10 min prior to the experiment. Assay incubations were terminated

by rapid filtration over Whatman GF/C filters using a Brandel cell harvester. K_; values

were calculated using the Cheng-Prusoff equation (De Vry et al. 1998).

Example 7 5-HT_{1 A} Functional Assays

The function of Compounds of the present invention can be determined using a

variety of methods to assess the functional activity of 5-HT_1A agonists. One such assay is

performed using hippocampal slices from male Sprague-Dawley rats, measuring the

inhibition of forskolin-stimated adenylate cyclase (Lopez-Rodriguez et al. 1999; Morin et

al. 1991 ; De Vry et al. 1998). Rat hippocampal membranes were homogenized in 25

volumes of 0.3 M sucrose containing ImM EGTA, 5 mM EDTA, 5 mM dithiothreitol, and 20 mM Tris-HCl, pH 7.4 at 25°C. The homogenate was centrifuged for 10 m in at 1,000 x

g. The supernatant subsequently was centrifuged at 39,000 x g for 10 min. The resulting

pellet was re-suspended in homogenization buffer at a protein concentration of

approximately 1 mg/ml and aliquots were stored at -140°C. Prior to use, the membranes

were rehomogenized in a Potter-Elvehjem homogenizer. Fifty μl of the membrane

suspension (50 μg protein) were added to an incubation buffer containing 100 mM NaCl, 2

mM magnesium acetate, 0.2 mM ATP, 1 mM cAMP, 0.01 mM GTP, 0.01 mM forskolin,

80 mM Tris-HCl, 5 mM creatine phosphate, 0.8 U/μl creatine phosphokinase, 0.1 mM

IBMX, 1-2 μCi α-[³²P]ATP. Incubations with test compounds (10 min at 30°C) were

initiated by the addition of the membrane solution to the incubation mixture (prewarmed 5

min at 30°C). [³²P]cAMP was measured according to the method of Salomon (Salomon

1979). Protein was measure using the Bradford assay (Bradford 1976).

Functional activity can also be determined in recombinant human receptors

according to the method of Schoeffter et al. (1997). HeLa cells transfected with

recombinant human 5-HT_1A receptors were grown to confluence in 24-well plates. The

cells were rinsed with 1 ml of Hepes-buffered saline (in mM) NaCl 130, KC1 5.4, CaCl₂,

1.8, MgSO₄ 0.8, NaH₂PO₄ 0.9, glucose 25, Hepes 20, pH 7.4, and phenol red 5 mg/1. The

cells were labelled with 6 μCi/ml of [³H] adenine (23 Ci/mmol, Amersham, Rahn AG,

Zurich, Switzerland) in 0.5 ml of saline at 37 °C for 2 hr. The plates were subsequently

rinsed twice with 1 ml of buffered saline containing ImM isobutylmethylxanthine. The

cells were incubated for 15 min in 1 ml of this solution (37 °C) in the presence or absence

of 10 μM forskolin and the test compound. The buffer was then removed and 1 ml of 5%

trichloroacetic acid (TCA) containing 0.1 mM cAMP and 0.1 mM ATP was added to extract the samples. After 30 min at 4°C, the TCA extracts were subjected to

chromatographic separation on Dowex AG 50W-X4 and alumina columns (Salomon

1991). Cyclic AMP production was calculated as the ratio [³H]cAMP/([³H]cAMP +

[³H]ATP).

Table 2. 5-HT1A Receptor Binding and Functional Data.

Example 8 Alpha-2 Adrenergic Receptor Assay Methods Cell culture. For the alpha-2A assays, HT29 human clonic adenocarcinoma cells

were grown in McCoy's 5A Medium Modified supplemented with 10%) (v/v) heat-

inactivated fetal bovine serum in a humidified atmosphere of 5% CO₂/95% air. Cells were

sub-cultured with 0.5%> Trypsin/5.3 mM EDTA in 48 wells plates with confluence being

reached in approximately 4 days. Growth medium was replaced with fresh medium, 24

hours before assay of confluent cells in order to avoid the nutrient exhaustion.

Cyclic AMP functional assays. Confluent cultures of HT29 cells were washed

twice with 0.5 ml of 15mM Hepes-buffered DMEM (DMEM/F12), then incubated with

0.5 ml DMEM/F12 containing 0.25mM 3-Isobutyl-l-methyl-xanthine (IBMX) for 20

minutes. At the end of this period the appropriate serially diluted α2-adrenergic agonists

was added and the cells were further incubated for 10 minutes. Then the appropriate

concentration of forskolin (for HT29 cells 4μM) was added and the cells were incubated for an additional 10 minutes. At the end of the incubation period the media was aspirated

and 150 μl of 0.1 M acetic acid, pH 3.5 was added. The plates were incubated at 4o C for

20 minutes. Then 220 μl of 0.1 M sodium acetate, pH 11.5-12 was added. The plates

were stored at -20° C. After this, a commercially available cAMP ELISA kit was used to

quantify the amount of cAMP generated in the receptor assay. In all these alpha-2 receptor

assays, an inhibition of cAMP production reflected a receptor-mediated process.

Table 3. Alpha2A Receptor Binding and Functional Data.

All of the compositions and/or methods disclosed and claimed herein can be made

and executed without undue experimentation in light of the present disclosure. While the

compositions and methods of this invention have been described in terms of preferred

embodiments, it will be apparent to those of skill in the art that variations may be applied

to the compositions and/or methods and in the steps or in the sequence of steps of the

method described herein without departing from the concept, spirit and scope of the

invention. More specifically, it will be apparent that certain agents which are both

chemically and structurally related may be substituted for the agents described herein to

achieve similar results. All such substitutions and modifications apparent to those skilled

in the art are deemed to be within the spirit, scope and concept of the invention as defined

by the appended claims. References

The following references, to the extent that they provide exemplary procedural or

other details supplementary to those set forth herein, are specifically incorporated herein

by reference.

United States Patents

5,494,928

5,571,833

5,578,612

5,874,477

5,902,815

Foreign Patents and Published Applications

EP 0771563A2.

PCT/US99/19888

WO 92/0338

WO 97/35579

WO 98/18458

WO 98/31354 A2

WO 00/16761

WO 01/70223 Al

Other Publications

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Fiorella et al., PSYCHOPHARM. 121(3):347-356 (1995). Griffin et al., J. PHARMACOL. EXPT. THER. 286(1):411-418 (1998).

Hoyer et al., PHARMACOL. REV. 46:157-203 (1994).

Johnson et al., NEUROPHARMACOLOGY, 26(12):1803-1806 (1987).

Lopez-Rodriguez et al, J. MED. CHEM. 42(l):36-49 (1999).

Martin et al., TRENDS PHARMACOL. SCI. 19:2-4 (1998).

Morin et al, J. NEUROCHEM. 56(4):1114-1120 (1991).

Osborne, et al, OPHTHALMOLOGICA, 210:308-314 (1996).

Salomon, ADV. CYCLIC NUCLEOTIDE RES. 10:35-55 (1979).

Salomon, METHODS IN ENZYMOLOGY 195: 22-28 (1991).

Schoeffter et al. NEUROPHARM. 36:429-437 (1997).

Sharif et al, J. PHARMAC. PHARMACOL. 51: 685-694 (1999).

Tobin et α/., J. NEUROSCI. 8:3713-3721 (1988).

Wang et al, ARCH. OPHTHALMOL. 111:535-538 (1993).

Wang, et al, CURRENT EYE RESEARCH, 16(8):769-775 (1997).

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Zifa and Fillion, PHARMACOL. REV. 44:401-458 (1992).

Claims

We Claim:

1. A compound of the formula:

wherein A, B and D are independently chosen from either N or C, with the provision that

at least one of A, B or D is N;

E is C or N;

R is H or C_Malkyl;

R² and R³ are independently H, C^ alkyl, C₂-₃ alkenyl, or R² and R³ taken together can form

a 5 or 6 member ring;

X is hydrogen, halogen, C₁ alkyl, or CF₃; and

the dashed bond may be a single bond or a double bond;

and pharmaceutically acceptable salts and solvates.

2. The compound of claim 1, wherein the compound is 2-(8-bromo-benzo-

[l,2-b;4,5-b']difuran-4-yl) imidazoline hydrochloride.

3. A method for lowering inraocular pressure and providing neuroprotection

comprising administering to a patient in need thereof a therapeutically effective amount of

a composition comprising a compound of the formula:

wherein A, B and D are independently chosen from either N or C, with the provision that

at least one of A, B or D is N;

E is C orN;

R is H or C_Malkyl;

R² and R³ are independently H, C ₃ alkyl, C₂.₃ alkenyl, or R² and R³ taken together can form

a 5 or 6 member ring;

X is hydrogen, halogen, C_Malkyl, or CF₃; and

the dashed bond may be a single bond or a double bond;

and pharmaceutically acceptable salts and solvates.

4. The method of claim 3, wherein the compound is 2-(8-bromo-benzo-[l,2-

b;4,5-b"]difuran-4-yl) imidazoline hydrochloride.

5. A composition for lowering and controlling normal or elevated intraocular

pressure and providing ocular neuroprotection, comprising a compound of the formula:

wherein A, B and D are independently chosen from either N or C, with the provision that

at least one of A, B or D is N;

E is C orN;

R is H or C_Malkyl;

R² and R³ are independently H, C,.₃ alkyl, C₂.₃ alkenyl, or R² and R³ taken together can form

a 5 or 6 member ring;

X is hydrogen, halogen, C_Malkyl, or CF₃; and

the dashed bond may be a single bond or a double bond;

and pharmaceutically acceptable salts and solvates.

6. The composition of claim 5, wherein the compound is 2-(8-bromo-benzo-

[l,2-b;4,5-b"]difuran-4-yl) imidazoline hydrochloride.

7. The composition of claim 6, further comprising ophthalmologically

acceptable preservatives.

8. The composition of claim 6, further comprising ophthalmologically

acceptable surfactants.

The composition of claim 6, further comprising an agent to increase

viscosity.

10. The composition of claim 9, wherein the agent is selected from the group

consisting of hydroxymethylcellulose, hydroxyethylcellulose,

hydroxypropylmethylcellulose, methylcellulose, and polyvinylpyrrolidone.

11. The composition of claim 6, further comprising ophthalmologically

acceptable preservatives, ophthalmologically acceptable surfactants and at least one agent

to increase viscosity.

12. The composition of claim 6, further defined as a topical ophthalmic

suspension or solution having a pH of about 5 to about 8.

13. The composition of claim 12, wherein the concentration of the compound is

from .01% to 5%> by weight.

14. The composition of claim 13, wherein the composition of the compound is

from .25% to 2% by weight.

15. The composition of claim 6, further comprising at least one agent selected

from the group consisting of β-blockers, prostaglandins, carbonic anhydrase inliibitors,

and miotics.

16. The composition of claim 6, further comprising at least one agent selected

from the group consisting of calcium channel blockers and NMDA antagonists.