WO1997041852A1

WO1997041852A1 - Angiostatic compounds

Info

Publication number: WO1997041852A1
Application number: PCT/US1997/008066
Authority: WO
Inventors: Rupa Doshi; Jon Nixon; Robert J. Collier, Jr.
Original assignee: Alcon Laboratories, Inc.
Priority date: 1996-05-09
Filing date: 1997-05-08
Publication date: 1997-11-13
Also published as: US5798356A; AU3123697A

Abstract

The present invention is directed to compositions containing chroman-type or benzfuran type compounds and methods for their use in preventing pathological neovascularization.

Description

ANGIOSTATIC COMPOUNDS

Background of the invention

The present invention relates to certain compounds useful in preventing and treating

neovascularization. Specifically, the invention is directed to compositions containing

chroman-type compounds and methods to treat neovascularization.

Angiogenesis is a term used to describe the development of new blood vessels or

neovascularization (L. Diaz-Flores et al., Angiogenesis: an Update, Histology and

Histopathologv. volume 9, pages 807-843 (1994)). Though angiogenesis is a normal

process for the development or maintenance of the vasculature, pathological conditions

(i.e., angiogenesis dependent diseases) arise where blood vessel growth is actually harmful.

Such pathologies include diabetic retinopathies, proliferative vitreoretinopathies, psoriasis,

arthritis and tumor development. The progression of angiogenesis occurs in several phases

which include: elaboration of the angiogenic signal; dissolution of the blood vessel

basement membrane; endothelial cell proliferation; endothelial cell migration; and

formation and differentiation of capillary tubules and loops. Each of these phases is a

potential target for pharmacological intervention.

Tumor growth is dependent on neovascularization. For solid tumors to grow

beyond the size of a pea, they must become vascularized. They do so by secreting their own angiogenic factor(s) which recruit new blood vessels to provide essential nutrients and

oxygen.

Angiogenesis is also associated with important diseases of ocular tissue especially

in older patients and diabetics. Any abnormal growth of blood vessels in the eye can

scatter and block the incident light prior to reaching the retina. Neovascularization can

occur at almost any site in the eye and significantly alter ocular tissue function. Some of

the most threatening ocular neovascular diseases are those which involve the retina. For

example, many diabetic patients develop a retinopathy which is characterized by the

formation of leaky, new blood vessels on the anterior surface of the retina and in the

vitreous causing proliferative vitreoretinopathy. A subset of patients with age related

macular degeneration develop subretinal neovascularization which leads to their eventual blindness.

Another area involving neovascularization and unwanted tissue growth involves the

technique of glaucoma filtration surgery. With this technique, a small fistula or bleb is

surgically formed in the eye to facilitate drainage of aqueous humor, and therefore reduce

the intraocular pressure of the eye. This technique can be frustrated, however, by the

closure of the bleb. The bleb, which is a tissue wound in the eye, has a tendency to close

up due to normal healing processes involving the addition of fibroblasts and/or

neovasculature. Therefore, an agent which inhibits bleb vascularization and fibroblast

proliferation would be useful in maintaining bleb patency.

Current therapy for the treatment of ocular neovascular disease is not very effective.

Retinal neovascularization is often treated with multiple laser burns to the retina to remove

the pathological vasculature. Patients with neovascular diseases of the anterior chamber (e.g. corneal neovascularization, iritis rubeosis) are treated with potent topical ocular

glucocorticoids. These therapies are only partially effective and generally only slow

neovascularization and the progress of the overall disease. In addition, they can cause

severe side effects if used over a relatively long period of time.

Other attempts have been made to provide therapies for the prevention or treatment

of pathological angiogenesis. For example, angiostatic steroids functioning to inhibit

angiogenesis in the presence of heparin or specific heparin fragments are disclosed in

Crum, et al., A New Class of Steroids Inhibits Angiogenesis in the Presence of Heparin or a

Heparin Fragment, Science, volume 230, pages 1375-1378 (1985). Another group of

angiostatic steroids useful in inhibiting angiogenesis is disclosed in commonly assigned

United States Patent No. 5,371 ,078 (Clark et al).

Glucocorticoids, as mentioned above, have also been shown to inhibit angiogenesis.

However, the use of glucocorticoid therapy in general is complicated by the inherent

problems associated with steroid applications. Such problems include elevated intraocular

pressure (Kitazawa, Increased Intraocular Pressure Induced by Corticosteroids, American Journal of Ophthalmology, volume 82, pages 492-493 (1976)).

Still other therapies have included: the use of protamine (S. Taylor, Protamine is an

Inhibitor of Angiogenesis, Nature, volume 297, pages 307-312 (1982)); the use of calcitriol

(European Journal of Pharmacology, volume 178, pages 247-250 (1990)); and the use of

the antibiotic, fumagillin and its analogs, disclosed in EP 354787. The use of a variety of

pharmaceutical proteins has also been proposed for treating angiogenesis. Such therapies

have included: monoclonal antibodies directed to fibroblast growth factor, disclosed in WO 9106668; platelet factor 4, disclosed in WO 9302192; and thrombospondin fragments, disclosed in WO 9316716.

Summary of the Invention

This invention is directed to compositions containing angiostatic compounds and

methods of using these compositions to prevent and/or treat neovascularization in human patients. In particular, the compositions are useful for controlling ocular

neovascularization. Additionally, the compounds are useful in treating glaucoma filtration

bleb failure, pterygium, hyperkeratosis, cheloid formation and polyp formation.

The compositions of the present invention also have the advantage of providing

effective angiostatic therapy which avoids the problems inherent in steroid therapies.

Detailed Description of the Invention

The compositions of the present invention contain compounds which have the general formula (I):

wherein:

n is 1 or 2;

R is H, C,-C₆ alkyl or C₃-C₆ cycloalkyl;

Y is H, C,-C₆ alkyl, C₃-C₆ cycloalkyl, O, NR, C(R)₂ , CH(OH) or S(O)_n. ; n' is 0 to 2; R' is H, C(O)R, C(O)N(R)₂, PO₃ ^" , SO₃ ^" , or HO₂C(CH₂)₂(C=O)— ;

R" is H or C,-C₆ alkyl;

R³ is H, C,-C₆ alkyl, (CH₂)_q(OH), — (C=O)O(CH₂)_qCH₃ or

; provided that R' can not be H, when

q is 1 to 10; and

Z, if present, is H, C_rC₆ alkyl, C₃-C₆ cycloalkyl, or selected from the group consisting of:

wherein:

D is O or NR; and

E and E' are independently H, F or Cl.

The compounds of the present invention also include pharmaceutically acceptable salts of the compounds of formula (I).

The following compounds are particularly preferred for use in the compositions and

methods of the present invention:

2-(6-hydroxy-2,5,7,8-tetramethyl-3,4-dihydro-2H-benzo[l,2-b]pyran-2-yl)ethyl 2-(6- methoxy-2-naphthyl)propionate ("Compound A");

2-(5-hydroxy-2,4,6,7-tetramethyl-2,3-dihydro-benzo[l ,2-b]furan-2-yl)methyl 2-(6- methoxy-2-naphthyl)propionate ("Compound B");

N-(2-(6-hydroxy-2,5,7,8-tetramethyl-3,4-dihydro-2H-benzori,2-b]pyran-2-yl)methyl) 2-(6- methoxy-2-naphthyl)propionamide ("Compound C");

2-(5-hydroxy-2,4,6,7-tetramethyl-2,3-dihydro-benzo[l,2-b]furan-2-yl)ethyl 2-(6-methoxy- 2-naphthyl)propionate ("Compound D");

l-[2-(5-hydroxy-2,4,6,7-tetramethyl-2,3-dihydro-benzo[l,2-b]furan-2-yl)2-ethyl]-4-[4,4'- fluorobenzhydryl]piperzine ("Compound E")

2-(6-hydroxy-2,5,7,8-tetramethyl-3,4-dihydro-2H-benzo[l,2-b]pyran-2-yl)ethanol ("Compound F")

Vitamin E succinate (VES) ("Compound G")

2-(6-hydroxy-2,5,7,8-tetramethyl-2,3-dihydro-2H-benzo[l ,2-b]pyran-2-yl)ethyl 2-(3- fluoro-4-phenyl-phenyl)propionate ("Compound H"). The initiation of new blood vessel formation may arise quite differently in various

tissues or as a result of different diseases. Many substances have been found to induce

neovascularization, see, Folkman, et al., Angiogenic Factors, Science, volume 235, pages

442-447 (1987). However, it is believed, that once initiated, the process of

neovascularization is similar in all tissues regardless of the associated disease, Furcht,

Critical Factors Controlling Angiogenesis: Cell Products, Cell Matrix, and Growth

Factors, Laboratory Investigation, volume 55, No. 5, pages 505-509 (1986).

There are many theories associated with the cause of neovascularization, and there

may be different inducers depending on the disease or surgery involved, BenEzra,

Neovasculogenic Ability of Prostaglandins, Growth Factors, and Synthetic

Chemoattractants, American Journal of Ophthalmology, volume 86, No. 4, pages 455-461,

(October, 1978). Regardless of the cause or the associated disease, it is believed that

angiostatic agents work by inhibiting one or more steps in the process of

neovascularization. Therefore, the angiostatic compounds of this invention are useful in

the treatment and prevention of neovascularization associated with a variety of diseases and

surgical complications.

The angiostatic compositions of the present invention are useful in inhibiting

pathological neovascularization in human patients. As used herein, the term "pathological

neovascularization" refers to those conditions where the formation of blood vessels

(neovascularization) is harmful to the patient. Examples of pathological neovascularization

dependent diseases include: head trauma, spinal trauma, systemic or traumatic shock,

stroke, hemorrhagic shock, cancer, arthritis, arteriosclerosis, angiofibroma, arteriovenous

malformations, corneal graft neovascularization, delayed wound healing, diabetic retinopathy, granulations, burns, hemangioma, hemophilic joints, hypertrophic scars, ocular

neovascularization, nonunion fractures, Osier-Weber Syndrome, psoriasis, pyogenic

granuloma, retrolental fibroplasia, pterigium, scleroderma, trachoma, vascular adhesions,

and solid tumor growth.

In particular, the compositions are useful in preventing and treating any ocular

neovascularization, including, but not limited to: retinal diseases (diabetic retinopathy,

chronic glaucoma, retinal detachment, sickle cell retinopathy and subretinal

neovascularization due to senile macular degeneration); rubeosis iritis; proliferative vitreo¬

retinopathy; inflammatory diseases; chronic uveitis; neoplasms (retinoblastoma,

pseudoglioma and melanoma); Fuchs' heterochromic iridocyclitis; neovascular glaucoma;

corneal neovascularization (inflammatory, transplantation and developmental hypoplasia of

the iris); neovascularization following a combined vitrectomy and lensectomy; vascular

diseases (retinal ischemia, choroidal vascular insufficiency, choroidal thrombosis and

carotid artery ischemia); neovascularization of the optic nerve; and neovascularization due

to penetration of the eye or contusive ocular injury.

The use of the compositions of the present invention to ameliorate complications

arising from glaucoma filtration surgery is a particularly important aspect of the invention.

Glaucoma filtration surgery involves the surgical creation of a fistula with a conjuctival

flap which allows the direct drainage of aqueous humor from the anterior chamber into the

conjuctival tissue, thereby lowering the elevated intraocular pressure associated with

glaucoma. However, in many patients, the filtration "bleb" becomes scarred or healed over

so that aqueous drainage can no longer occur. It has been noted that failing filtration blebs

may become vascularized prior to failure. This vascularization may feed the fibroblasts which migrate, proliferate, and block the bleb, or the vascularization itself may also result

in physical blockage of the bleb. It is therefore likely that inhibition of filtration bleb neovascularization may inhibit filtration bleb failure.

Additionally the angiostatic agents of the present invention are useful in treating

pterygium (primary and recurrent), hyperkeratosis, cheloid formation and polyp formation.

The compounds of formula (I) may be contained in various types of pharmaceutical compositions, in accordance with formulation techniques known to those skilled in the art.

For example, the compounds may be included in tablets, capsules, solutions, suspensions

and other dosage forms adapted for oral administration; solutions and suspensions adapted

for parenteral use; solutions, suspensions and gels adapted for topical ophthalmic use;

solutions and suspensions adapted for intra-vitreal or intra-cameral use; and suppositories

for rectal use. Solutions, suspensions and other dosage forms adapted for topical

application to the involved tissues, such as tissue irrigating solutions, are particularly

preferred for treatment of acute conditions associated with surgery or other forms of

trauma.

The present invention is particularly directed to the provision of compositions

adapted for treatment of ophthalmic tissues. The ophthalmic compositions of the present

invention will include one or more compounds of formula (I) and a pharmaceutically

acceptable vehicle for said compound(s). Various types of vehicles may be used. The

vehicles will generally be aqueous in nature. Aqueous solutions are generally preferred,

based on ease of formulation, as well as a patients' ability to easily administer such

compositions by means of instilling one to two drops of the solutions in the affected eyes.

However, the compounds of formula (I) may also be readily incorporated into other types of compositions, such as suspensions, viscous or semi-viscous gels or other types of solid

or semi-solid compositions. Suspensions may be preferred for compounds of formula (I)

which are relatively insoluble in water. The ophthalmic compositions of the present

invention may also include various other ingredients, such as buffers, preservatives, co- solvents and viscosity building agents.

An appropriate buffer system (e.g., sodium phosphate, sodium acetate or sodium

borate) may be added to prevent pH drift under storage conditions.

Ophthalmic products are typically packaged in multidose form. Preservatives are

thus required to prevent microbial contamination during use. Suitable preservatives

include: benzalkonium chloride, thimerosal, chlorobutanol, methyl paraben, propyl

paraben, phenylethyl alcohol, edetate disodium, sorbic acid, polyquaternium- 1 , or other

agents known to those skilled in the art. Such preservatives are typically employed at a

level of from 0.001 to 1.0 percent by weight, based on the total weight of the composition

(wt.%).

Some of the compounds of formula (I) may have limited solubility in water and

therefore may require a surfactant or other appropriate co-solvent in the composition. Such

co-solvents include: polyethoxylated castor oils, Polysorbate 20, 60 and 80; Pluronic® F-

68, F-84 and P-103 (BASF Corp., Parsippany NJ, USA); cyclodextrin; or other agents

known to those skilled in the art. Such co-solvents are typically employed at a level of

from 0.01 to 2 wt.%.

Viscosity greater than that of simple aqueous solutions may be desirable to increase

ocular absoφtion of the active compound, to decrease variability in dispensing the

formulations, to decrease physical separation of components of a suspension or emulsion of formulation and/or otherwise to improve the ophthalmic formulation. Such viscosity

building agents include, for example, polyvinyl alcohol, polyvinyl pyrrolidone, methyl

cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, carboxymethyl

cellulose, hydroxypropyl cellulose or other agents known to those skilled in the art. Such

agents are typically employed at a level of from 0.01 to 2 wt.%.

The route of administration (e.g., topical, parenteral or oral) and the dosage regimen

will be determined by skilled clinicians, based on factors such as the exact nature of the

condition being treated, the severity of the condition, the age and general physical condition of the patient, and so on.

As indicated above, use of the compounds of formula (I) to prevent or reduce

angiogenesis in ophthalmic tissues is a particularly important aspect of the present

invention. The compounds may also be used as an adjunct to ophthalmic surgery, such as

by vitreal or subconjunctival injection following ophthalmic surgery. The compounds may

be used for acute treatment of temporary conditions, or may be administered chronically,

especially in the case of degenerative disease. The compounds may also be used

prophylactically, especially prior to ocular surgery or noninvasive ophthalmic procedures,

or other types of surgery.

The use of physiologically balanced irrigating solutions as pharmaceutical vehicles

for the compounds of formula (I) is preferred when the compositions are administered

intraocularly. As used herein, the term "physiologically balanced irrigating solution"

means a solution which is adapted to maintain the physical structure and function of tissues

during invasive or noninvasive medical procedures. This type of solution will typically

contain electrolytes, such as sodium, potassium, calcium, magnesium and/or chloride; an W

energy source, such as dextrose; and a buffer to maintain the pH of the solution at or near

physiological levels. Various solutions of this type are known (e.g., Lactated Ringers

Solution). BSS® Sterile Irrigating Solution and BSS Plus® Sterile Intraocular Irrigating

Solution (Alcon Laboratories, Inc., Fort Worth, Texas, USA) are examples of

physiologically balanced intraocular irrigating solutions. The latter type of solution is

described in United States Patent No. 4,550,022 (Garabedian, et al.), the entire contents of

which are hereby incoφorated in the present specification by reference.

The specific type of formulation selected will depend on various factors, such as the

compound or its salt being used, the dosage frequency, and the disease being treated.

Topical aqueous solutions, suspensions, ointments, creams and gels are the preferred

dosage forms for the treatment of pterygium, hyperkeratosis, and cheloid and polyp

formation. Topical ophthalmic formulations are suitable for preventing glaucoma filtration

bleb failure or scar formation associated with ophthalmic surgery.

In general, the doses utilized for the above described puφoses will vary, but will be

in an effective amount to inhibit or reduce neovascularization. As used herein, the term

"pharmaceutically effective amount" to inhibit or reduce neovascularization, is that amount

which inhibits formation of new blood vessels or reduces the number of blood vessels

which are involved in the pathological condition. The angiostatic compounds will

normally be contained in these formulations in an amount from about 0.01 to about 10.0

weight/percent. Preferable concentrations range from about 0.1 to about 5.0

weight/percent. Thus, for topical administration, these formulations are delivered to the

disease site one to six times a day, depending on the routine discretion of the skilled

clinician. Systemic administration, for example, in the form of tablets or suppositories is useful for the treatment of polyp formation. Tablets containing 10-1000 mg of a compound

can be taken 2-3 times per day depending on the discretion of the skilled clinician.

The compositions of the present invention are further illustrated by the following

examples.

Example 1

Topical compositions useful for controlling ocular neovascularization:

Example 2

A preferred topical composition useful for controlling neovascularization:

The above formulation is prepared by first placing a portion of the purified water into a beaker and heating to 90°C. The hydroxypropylmethylcellulose (HPMC) is then added to the heated water and mixed by means of vigorous vortex stirring until all of the HPMC is dispersed. The resulting mixture is then allowed to cool while undergoing mixing in order to hydrate the HPMC. The resulting solution is then sterilized by means of autoclaving in a vessel having a liquid inlet and a hydrophobic, sterile air vent filter.

The sodium chloride and the edetate disodium are then added to a second portion of the purified water and dissolved. The benzalkonium chloride is then added to the solution, and the pH of the solution is adjusted to 7.4 with 0.1M NaOH/HCl. The solution is then sterilized by means of filtration.

Compound A is sterilized by either dry heat or ethylene oxide. If ethylene oxide sterilization is selected, aeration for at least 72 hours at 50°C. is necessary. The sterilized angiogenic compound is weighed aseptically and placed into a pressurized ballmill container. The tyloxapol, in sterilized aqueous solution form, is then added to the ballmill container. Sterilized glass balls are then added to the container and the contents of the container are milled aseptically at 225 φm for 16 hours, or until all particles are in the range of approximately 5 microns.

Under aseptic conditions, the micronized drug suspension formed by means of the preceding step is then poured into the HPMC solution with mixing. The ballmill container and balls contained therein are then rinsed with a portion of the solution containing the sodium chloride, the edetate disodium and benzalkonium chloride. The rinse is then added aseptically to the HPMC solution. The final volume of the solution is then adjusted with purified water and, if necessary, the pH of the solution is adjusted to pH 7.4 with NaOH/HCl.

Example 3

Formulation for oral administration:

Tablet:

10-1000 mg of an angiostatic compound with inactive ingredients such as starch, lactose and magnesium stearate can be formulated according to procedures known to those skilled in the art of tablet formulation.

Example 4

Formulation for sterile intraocular injection:

Example 5

Preferred formulation for a topical ocular solution:

Example 6

A preferred formulation for oral administration:

Tablet:

5-100 mg of Compound A with inactive ingredients such as starch, lactose and magnesium stearate can be formulated according to procedures known to those skilled in the art of tablet formulation. Example 7

Formulations for topical dermatological use:

Cream: 1 mg/g of an angiostatic compound in cream base of purifed water, emulsifying wax, propylene glycol, stearic acid, isopropyl palmitate, synthetic beeswax, polysorbate 60, potassium sorbate, sorbic acid, propyl gallate, citric acid, and sodium hydroxide.

Ointment: 1 mg/g of an angiostatic compound in base of mineral oil and polyethylene.

Example 8

Formulation for suppository:

10-500 mg of an angiostatic compound with the following inactive ingredients: glycerin, butylateal hydroxytoluene, butylated hydroxyanisole, edetic acid, polyethylene glycol, and sodium chloride.

The compositions and methods of the present invention are further illustrated by the

following in vitro and in vivo biological activity examples of the compounds of formula

(I):

Example 9

Representative compounds of the present invention were assayed for their efficacy in inhibiting endothelial cell proliferation. Briefly, human umbilical vein endothelial cells (HUVEC) were seeded in 6-well plates at a density of 2500/cm . At mid-log phase the cells were treated with a test compound or vehicle control. Forty-eight (48) hours later the cells were detached from the plate and counted using a coulter counter. Viability tests were conducted using trypan blue. The data is expressed as percent inhibition based on the control number of endothelial cells counted. The results are illustrated in Table 1 below:

TABLE 1

The data of Table 2 demonstrates the endothelial cell anti-proliferative efficacy of representative compounds of the present invention, as compared to naproxen and vitamin E.

Example IQ

Representative compounds of the present invention were assayed for their efficacy in inhibiting ³H-thymidine uptake in DNA as a measure of DNA synthesis (R.I. Freshney, Culture of Animal Cells. 3rd Edition, John Wiley and Sons, N.Y., N.Y., pages 277-278 (1994)). Briefly, human lung microvascular endothelial cells in mid-log phase were exposed to either a representative compound of the present invention or the vehicle (1.0% DMSO) for 18 hours. Cells were then incubated with H-thymidine for another 6 hours. DNA synthesis was stopped by the addition of cold 5% TCA solution and the relative synthesis assessed by measuring H-thymidine incoφorated into the acid-insoluble product. The data is expressed as percent inhibition of uptake based on tritium counts in the vehicle control reactions. The results are presented in Table 2 below: TABLE 2

The data of Table 2 demonstrates the efficacy of representative compounds of the present invention in inhibiting endothelial cell proliferation based on the inhibition of DNA synthesis.

Example 11

Representative compounds of the present invention were assayed for their efficacy in inhibiting angiogenesis in a chorioallantoic membrane (CAM) model (See, McNatt, et al., Angiostatic Activity and Metabolism ofCortisol in The Chorioallantoic Membrane (CAM) of the Chick Embryo, Journal of Steriod Biochemistry and Molecular Biology, volume 42, No. 7, pages 687-693 (1992)). Briefly, 10 μg of the test compound or reference compound (tetrahydrocortisol) were suspended in a liposome/agarose bead and placed on the CAM of a 5-6 day chicken embryo. Following 2 days of incubation, the CAMs were scored for relative angiostatic activity by comparing percent responding embryos per nanomole test compound to percent responding embryos for the reference compound. The results are illustrated in Table 3 below:

TABLE 3

e at ve ng ostat c actor

As Table 3 demonstrates, representative compounds of the present invention show angiostatic efficacy in the CAM model.

Example 12

Representative compounds of the present invention were assayed for their efficacy in inhibiting angiogenesis in vivo, in a rabbit model of lipopolysaccharide (LPS) induced corneal neovascularization. Briefly, the test compound and LPS (a stimulus for initiating neovascularization) are incoφorated into separate Elvax-40 pellets as described in D.

BenEzra, The Rabbit Cornea. A Model for the Study of Angiogenic Factors, in "Ocular

Circulation and Neovascularization, Documenta Opthalmalogia. Proceedings Series 50, pages 335-340 (1987) and D. BenEzra, Thrombospondin and In Vivo Angiogenesis Induced by Basic Fibroblast Growth Factor or Lipopolysaccharide, Investigative Opthalmology and Visual Sciences, volume 34, No. 13, pages 3601-3608 (1993), which are surgically implanted into rabbit cornea. The area covered by new vessel formation in response to LPS is measured and suppression of neovascularization by test compounds is estimated at the end of the test period. The data is expressed as a relative area units in relation to the Elvax control (100). The results are illustrated in Table 4 below: TABLE 4

The data of Table 4 demonstrate the efficacy of representative compounds of the present

invention in in vivo inhibition of neovascularization.

Some of the compounds of the present invention may contain a nonsteroidal anti-

inflammatory agent (NSAIA) component, and others may contain a calcium channel

blocker (flunarizine) component. These individual moieties may add additional pharmaceutical benefit to the angiostatic efficacy of the compounds of the present

invention.

The compounds of the present invention are synthesized by known methods in the

art. Compounds containing a non-steroidal anti-inflammatory agent (flurbiprofen or

naproxen) can be made by methods illustrated in Scheme 1 and 2, and Examples 13-16.

Compounds containing a flunarizine moiety may be made by methods disclosed in

commonly assigned PCT Patent Publication No. WO/9515958, the entire contents of which

are hereby incoφorated by reference. Other compounds of the present invention are

commercially available from: Sigma Chemical Co. (St. Louis, Missouri) and Aldrich

Chemical Co. (Milwaukee, Wisconsin). Scheme 1

A-OH + H-X-(CH₂)_n-Y-(CH₂)_m-Z — > A-X-(CH₂)_n-Y-(CH₂)_πι-Z (eq. l)

11 III I

A-OH — > A-Cl + H-X-(CH₂)_n-Y-(CH₂)_m-Z — > A-X-(CH₂)_n-Y-(CH₂)_m-Z (eq.2) II IV III I

A-OH — > A-0^"M⁺ + L-(CH₂)_n-Y-(CH₂)_π,-Z— >A-0-(CH₂)_n-Y-(CH₂)_m-Z (eq. 3)

II V L=C1, BR, I, OMs, OTs I

VI

A-OH — ^ A-O M⁺ + Br-CH₂-C(0)OEt — > A-0-CH₂-C(0)OEt + II V VII

H-NR-(CH₂)_n-Y-(CH₂)_m-Z — > A-NR-(CH₂)_n-Y-(CH₂)_m-Z (eq. 4)

VIII I

The conversion of the carboxylic acid containing nonsteroidal anti-inflammatory agents (II) to esters or amides (I) may be carried out by the following methods:

(i) As illustrated in equation 1 above, carboxylic acids (II) may be reacted with the appropriate amine or alcohol derivative (III) in the presence of a coupling reagent, such as dicyclohexylcarbodiimide or l-(3-dimethylaminopropyl)-3-ethyl carbodiimide HCI, and 4-dimethylamine pyridine or 1-hydroxybenzotriazole, in an inert organic solvent, such as acetonitrile or tetrahydrofuran, and at a temperature from 0°C to 50°C.

(ii) As illustrated in equation 2 above, carboxylic acids (II) may be converted to acid chlorides (IV) by reacting them with a reagent such as thionyl chloride or oxalyl chloride, in the presence of an inert solid or neat, at a temperature from 0°C to 80°C. The resulting acid chloride (IV) may be reacted with the desired amine or alcohol (III) in an inert solvent such as tetrahydrofuran, in the presence of pyridine or a tertiary amine, such as triethylamine. W

(iii) As illustrated in equation 3 above, esters (I) may be formed by reacting carboxylate anions (V), formed by reacting the carboxylic acid (II) with a base such as sodium hydride, with a halide (iodide, bromide, chloride) or sulfonate (mesylate, tosylate) (VI), in a solvent such as acetonitrile or dimethylformamide, at a temperature from 0°C to 100°C.

(iv) As illustrated in equation 4 above, amides (I) may be prepared by reacting carboxylate anions (V), formed by reacting carboxylic acid (II) with a base such as sodium hydride, with ethyl bromoacetate. The resulting ester (VII) is reacted with the desired amine (VIII), neat or in an inert solvent, such as acetonitrile or dimethylformamide, at a temperature from 0°C to 100°C.

The intermediate compounds (X) of Scheme 2 below, which can be used as compounds (III) and (VIII), were prepared using the general methods described in Journal of Organic Chemistry, volume 54, pages 3282-3292, (1989). The nitrile (IX) can be reduced using a reagent such as lithium aluminum hydride to afford the amine (X), which may be isolated as the hydrochloride salt.

The use of certain protecting groups and deprotection steps may be necessary, as will be appreciated by those skilled in the art.

Scheme 2

Compounds of formula (I) may exist as mixtures of stereoisomers. The preparation of the individual stereoisomers may be effected by preparing and resolving the acids (II), by known methods, and then using a single stereoisomer as starting material. Compounds (III), (VI) and (VIII) may be prepared as single stereoisomers from compounds of formula (XI_a-b), shown in Table 5 below, using known methods:

Table 5

XL Xi,

wherein:

W is (CH₂)_p-Q; p is 0-1 ; Q is CH₂OH or CO₂H;

R' is H, C(O)R, C(O)NR₂, PO₃ ^' , or SO₃ ^" ; and R" is H or C,-C₆ alkyl.

The alcohols (XI_a-b) may be resolved by forming esters with optically active carboxylic acids, separating the diastereomers, and then hydrolyzing the resolved diastereomers. The corresponding carboxylic acids (XI_a-b) may be resolved by forming an ester with an optically active alcohol, separating the diastereomers, and then hydrolyzing the resolved diastereomers. Or, the carboxylic acids (XI_a-b) may be resolved by forming an amine salt with an optically active amine. Separation by recrystallization and neutralization of the resolved carboxylic acid salt may be utilized to provide the resolved carboxylic acid. Resolution of the esters and amides (I) may also be effected using chromatographic techniques known to those skilled in the art.

The amines of formula (I), where Y is NR, may be converted to amine salts by reacting the amine with acids of sufficient strength to produce an organic or inorganic salt. The pharmaceutically acceptable anions include: acetate, bromide, chloride, citrate, maleate, fumarate, mesylate, phosphate, sulfate and tartrate.

Methods of synthesizing the compounds formula (I) are further illustrated by the following examples:

Example 13

S^ynthesis of N-[⁽6-hvdroxv-2.5.7.8-tetramethvl-3.4-dihvdro-2H-l -benzo[l ,2-h]pyran-2- vnmethvl] 2-(6-methoxy-2-naphthvnpropionamide

The intermediate, (6-hydroxy-2,5,7,8-tetramethyl-3,4-dihydro-2H-l -benzo[l,2-b]pyran-2- yl)methylamine, was first synthesized:

A 1 molar (M) ethereal solution of lithium aluminum hydride (Aldrich, 32.4 mL, 32.43 mmol) was added slowly over a 5 minute period to a chilled, (4-6°C) stirring solution of (2- cyano-6-hydroxy-2,5,7,8-tetramethyl-3,4-dihydro-2H- 1 -benzof 1 ,2-b]pyran in tetrahydrofuran (50 mL). After 2 hours, the reaction mixture was quenched by the slow sequential addition of 10% aqueous tetrahydrofuran (30 mL), 15% sodium hydroxide (10 mL) and then water (20 mL), while stirring. The resulting suspension was filtered through celite, and the celite pad was washed with ethyl ether (400 mL). The organic layer was separated, dried (Na₂SO₄), and concentrated in vacuo. resulting in a residue. A 1 M ethereal solution of hydrochloride was then added to a solution of the residue in ethyl ether (100 mL), a solid formed, and the solid was then collected by filtration and washed with ethyl ether to give 2.31 g (65.4% yield) of a white solid. The product was used crude in the next reaction.

1 H-NMR (DMSO-d₆/TMS): 1.15 (s, 3H), 1.75 (t, 2H), 1.99 (s, 6H), 2.01 (s, 3H), 2.54 (t, 2H), 2.98 (s, 2H).

MS (Cl): 236 (m+1). The hydrochloride salt of (6-hydroxy-2,5,7,8-tetramethyl-3,4-dihydro-2H-l-benzo[l,2- b]pyran-2-yl)methylamine (0.30 g, 1.10 mmole) and 6-methoxy-α-methyl naphthaleneacetic acid (Aldrich, 0.28 g, 1.21 mmole) were stirred in the presence of dimethylaminopyridine (Aldrich, 0.26 g, 2.20 mmole) and l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (Janssen Chimica-Spectrum, 0.21 g, 1.10 mmole), in tetrahydrofuran (4.0 mL) under an atmosphere of nitrogen. After stirring 17 hours at ambient temperature, the reaction mixture was diluted with ethyl acetate (70 mL), washed with water (2x 15 mL), followed by brine (15 mL) and then dried (sodium sulfate). The mixture was concentrated in vacuo and the residue subjected to flash chromatography (silica gel, 100-50:0-50, v:v, hexanes:ethyl acetate). The appropriate fractions were concentrated in vacuo, and the resulting crystalline foam suspension was then washed in hexanes to give 0.28 g (58.3% yield) of N-[(5-hydroxy-2,5,7,8-tetramethyl-3,4-dihydro- 2H-l-benzopyran-2-yl)methyl]-2-(6-methoxy-2-naphthyl)propionamide as a white amoφhous solid.

Η-NMR (CDCl₃) d 1.03-1.08 (d,3H), 1.57-1.64 (m, 6H), 1.70 (t, 2H,). 2.04-2.05 (m, 6H,), 2.48-2.51 (m, 2H), 3.16-3.58 (m, 2H), 3.74 (q, IH), 3.91 (s, 3H), 4.91 (br s, IH), 5.751 (t, IH), 7.01-7.19 (m, 2H), 7.29-7.40 (t, IH), 7.52-7.81 (m, 3H).

Elemental Analysis: Calculated for C₂₈H₃₃NO₄ Calculated: C, 75.14; H, 7.43; N, 3.13. Found: C, 75.04; H, 7.50; N, 2.97. Melting point: 67-70°C.

Example 14

Synthesis of 2-⁽6-hvdroxv-2.5.7.8-tetramethvl-3.4-dihvdro-2H-benzo[1.2-b]pyran-2ynethyl 2-(6-methQxy-2-n,aphthyl)prQpiQhate

A solution of 1 ,3-dicyclohexylcarbodiimide (Aldrich, 0.89 g, 4.31 mmol) in acetonitrile (25 mL), was added dropwise to a stirring slurry of (+)-6-methoxy-a-methyl-2- naphthaleneacetic acid (Aldrich, 0.90 g, 3.91 mmol), 2-hydroxy-2,5,7,8-tetramethyl-3,4- dihydro-2H-benzo[l ,2-b]pyran-2yl)ethanol (0.98 g, 3.91 mmol, USP 5,266,709 column 45) and 1-hydroxybenzotriazole hydrate (Aldrich, 0.59 g, 4.31 mmol), in acetonitrile (50 mL). After stirring for 18 hours, the reaction mixture was concentrated in vacuo. The residue was partitioned between water (30 mL) and methylene chloride (30 mL). The layers were separated, and the aqueous layer was extracted with methylene chloride (2 x 20 mL). The combined organic extracts were washed with water (20 mL), then dried (magnesium sulfate) and concentrated in vacuo. Flash chromatography (silica gel, 2:8, v:v, ethyl acetate :hexanes) of the residue afforded a white solid upon the concentration of the appropriate fractions. The white solid was recrystallized from an ethyl acetate-hexanes mixture to give 0.60 g (33.1% yield) of 2-(6-hydroxy-2,5,7,8-tetramethyl-3.4-dihydro-2H- benzo[l,2-b]pyran-2yl)ethyl 2-(6-methoxy-2-naphthyl)propionate, a mixture of diastereomers, as a white solid.

Η NMR (CDC1₃) d 1.1 (d, 3H), 1.6-1.5 (m, 3H), 1.6 (m, 2H), 1.9 (m,2H). 2.0 (s, 6H), 2.1 (s, 3H), 2.4 (t, 2H), 3.8 (q, 2H), 3.9 (s, 3H), 4.2 (s. IH), 4.1-4.4 (m, 2H), 7.1 -7.7 (m,6H).

Elemental Analysis: Calculated for C₂₉H₃₄O₅ Calculated: C, 75.30; H, 7.41. Found: C, 75.24; H. 7.46. Melting Point: 99.5-101.5°C.

Example 15

S^ynthesis of 2-r5-hvdroxy-2.4.6.7-tetramethyl-3.4-dihvdro-hen7.o[L2-blfuran-2ynethyl 2- r6-methoxy-2-naphthvnpropionate

A solution of 2-(5-hydroxy-2,4,6,7-tetramethyl-2,3-dihydrobenzo[l ,2-b]furan-2-yl)ethanol (1.30 g, 5.51 mmol) and 6-methoxy-α-methyl naphthaleneacetic acid (Aldrich, 1.39 g, 6.06 mmol) was stirred in the presence of dimethylaminopyridine (0.67 g, 5.51 mmol) and l-(3- dimethylaminopropyI)-3-ethyl-carbodiimide hydrochloride (1.06 g, 5.51 mmol), in tetrahydrofuran (25 mL). The reaction mixture was stirred at ambient temperature under nitrogen for 24 hours, diluted with ethyl acetate (150 mL), washed with water (2x 40 mL) and then brine (30 mL). The organic extract was dried (sodium sulfate) and concentrated in vacuo. The residue was subjected to flash chromatography (silica gel, 100-50:0-50, v:v, hexanesxthyl acetate), and the appropriate fractions were combined to give 1.84 g (74.5% yield) of a foam residue. Fractional crystallization and recrystallization from methylene chloride-hexanes gave 0.40 g (13.0% yield) of white solid.

Η-NMR (CDC1₃): 1.34 (s, 3H), 1.54-1.57 (d, 3), 1.99 (t, 2H), 2.01 (s, 3H), 2.05 (s, 3H), 2.10 (s, 3), 2.73-2.81 (d, 1), 2.90-2.97 (d, 1), 3.77-3.89 (q, IH), 3.91 (s, 3H), 4.102 (s, IH, 4.165-4.29 (m, 2H), 7.10-7.16 (m, 2H), 7.35-7.40 (m, IH), 7.64-7.70 (m, 2H).

Elemental Analysis: Calculated for C₂₈H₃₂O₅ 0.1 mole CH₂C1₂. Calculated: C, 73.84; H, 7.10. Found: C, 73.85, 73.83; H, 7.12. Melting point: 129.5-131°C.

Example W

Synthesis of 2-(6-hvdroxy-2.5.7.8-tetramethvl-3.4-dihvdro-2H-henzo [1.2-b]pyran- 7.y1)ethyl 2-(3-fluoro-4-phenyl-ρhenvnpropionate

The intermediate, 2-(6-benzyloxy-2,5,7,8-tetramethyl-3,4-dihydro-2H-benzo[l,2-b]pyran-

2yl)ethyl 2-(3-fluoro-4-phenyl-phenyl)propionate, was first synthesized:

A solution of flubiprofen (Sigma, 2.0 g, 8.2 mmol), 2-(6-benzyloxy-2,5,7,8-tetramethyl- 3,4-dihydro-2H-benzo[l,2-b]pyran-2-yl)ethanol (2.4 g, 8.2 mmol) 1-hydroxybenzotriazole hydrate (Aldrich, 2.4 g, 13.9 mmol) and l-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (Aldrich, 2.8 g, 12.3 mmol), in acetonitrile (40 ml), was stirred at ambient temperature. After 72 hours, the reaction mixture was concentrated in vacuo and the residue partitioned between water and methylene chloride. A solid formed which was removed by filtration and discarded. The layers were separated and the aqueous layer was extracted with methylene chloride (2 x 25 ml). The combined organic extracts were then dried (magnesium sulfate) and concentrated in vacuo. The residue was chromatographed (silica gel, 2:8, v:v, ethyl acetate:hexane). Concentration of the appropriate fractions afforded 3.0 g (64% yield, mixture of stereoisomers) of the product as a clear oil.

Η NMR (CDC1₃) d: 1.23-1.27 (m, 3H), 1.53-1.57 (m, 3H), 1.75 (m, 2H), 1.95 (m, 2H), 2.08 (s, 3H), 2.14 (s, 3H), 2.21 (s, 3H), 2.55 (t, 3H), 3.75 (m, 2H), 4.3 (m, IH), 4.65 (s, 2H), 7.1-7.7 (m, 13H).

A solution of 2-(6-benzyloxy-2,5,7,8-tetramethyl-3,4-dihydro-2H-benzo

[l,2-b]pyran-2yl)ethyl 2-(3-fluoro-4-phenyl-phenyl)propionate in ethyl acetate was treated with 10% palladium on charcoal (Aldrich, 0.5 g). The resulting mixture was hydrogenated on a Parr Apparatus [initial pressure 60 pounds/inch² (psi)]. After 18 hours, the reaction mixture was filtered, and the resulting solution concentrated in vacuo. The residue was subjected to flash chromatography (silica gel, 2:8, v:v, ethyl acetate:hexane). Concentration of the appropriate fractions afforded a clear oil. Hexane was added to the oil and a white solid formed upon standing. The white solid was collected by filtration to afford 0.91 g (36% yield) of 2-6-hydroxy-2,5,7,8-tetra methyl-,4-dihydro-H-benzo[I ,2-b]pyran-yl)ethyl 2-(3-fluoro-4-phenyl-phenyl) propionate as a mixture of stereoisomers.

Η NMR (CDC1₃) d: 1.22- 1.23 (m, 3H), 1.51 -1.55 (m, 3H), 1.65-1.8 (m, 2H), 1.85-2.00 (m, 2H), 2.08 (s, 6H), 2.14 (s, 3H), 2.57 (t, 2H), 3.75 (q, IH), 4.1-4.5 (m, 2H), 7.10-7.65 (m, 8H).

Elemental Analysis: Calculated for C₃₀H₃₃FO₄. Calculated: C,75.60; H, 6.98. Found: C/75.69; H,7.01. Melting point: 85-87°C. The invention in its broader aspects is not limited to the specific details shown and described above. Departures may be made from such details within the scope of the

accompanying claims without departing from the principles of the invention and without

sacrificing its advantages.

Claims

What is claimed is:

1. A composition for the treatment of pathological neovascularization

comprising an effective amount of a compound according to formula (I):

wherein: n is 1 or 2;

R is H, C,-C₆ alkyl or C₃-C₆ cycloalkyl;

Y is H, C,-C₆ alkyl, C₃-C₆ cycloalkyl, O, NR, C(R)₂ , CH(OH) or S(O)„. ; n" is 0 to 2;

R' is H, C(O)R, C(O)N(R)₂, PO₃ ^" , SO₃ ^" or HO₂C(CH₂)₂(C=O)— ;

R" is H or C,-C₆ alkyl;

R³ is H, C,-C₆ alkyl, (CH₂)_q(OH), — (C=O)O(CH₂)_qCH₃ or

; provided that R' can not be H, when R³ is ³ , q is 1 to 10; and

wherein:

D is O or NR; and

E and E' are independently H, F or Cl; and pharmaceutically acceptable salts thereof.

2. The composition according to Claim 1, wherein: R is H, R' is H; R" is CH₃;

R ,3^JisCH₃;andYisC,-C₂alkyl.

3. The composition according to Claim 1, wherein Z is:

The composition according to Claim 1 , wherein Z is:

W

5. The composition according to Claim 1, wherein Z is:

6. The composition according to Claim 2, wherein Z is:

The composition according to Claim 2, wherein Z is:

8. The composition according to Claim 2, wherein Z is:

9. The composition according to Claim 1 , wherein the compound is selected from the group consisting of:

10. The composition according to Claim 1 , wherein the composition comprises a physiologically balanced irrigating solution.

1 1. The composition according to Claim 1 , wherein the composition is used to treat glaucoma filtration surgery bleb failure, pterygium, hyperkeratosis, cheloid formation and polyp formation.

12. A method for treating pathological neovascularization which comprises administering to a human patient an effective amount of a pharmaceutical composition comprising a compound according to formula (I):

wherein: n is 1 or 2;

R is H, C,-C₆ alkyl or C₃-C₆ cycloalkyl;

Y is H, C,-C₆ alkyl, C₃-C₆ cycloalkyl, O, NR, C(R)₂ , CH(OH) or S(O)_n. ; n' is 0 to 2;

R' is H, C(O)R, C(O)N(R)₂, PO₃\ SO₃ ^" or HO₂C(CH₂)₂(C=O)— ;

R" is H or C,-C₆ alkyl;

R³ is H, C,-C₆ alkyl, (CH₂)_q(OH), — (C=O)O(CH₂)_qCH₃ or

provided that R' can not be H, when R³ is q is 1 to 10; and

Z, if present, is H, C,-C₆ alkyl, C₃-C₆ cycloalkyl, or selected from the group consisting of:

wherein:

D is O or NR; and

13. The method according to Claim 12, wherein: R is H, R' is H; R" is CH₃; R³ is CH₃; and Y is C_rC₂ alkyl.

14. The method according to Claim 12, wherein Z is:

15. The method according to Claim 12, wherein Z is:

16. The method according to Claim 12, wherein Z is:

17. The method according to Claim 13 , wherein Z is:

18. The method according to Claim 13, wherein Z is:

19. The method according to Claim 13, wherein Z is:

20. The method according to Claim 12, wherein the compound is selected from the group consisting of:

21. The method according to Claim 12, wherein the composition comprises a physiologically balanced irrigating solution.

22. The method according to Claim 12, which comprises administering the composition to a human patient to treat glaucoma filtration surgery bleb failure, pterygium, hyperkeratosis, cheloid formation and polyp formation.