EP1231913A2

EP1231913A2 - Treatment of cyclooxygenase-2 mediated disorders using conjugated fatty acid compounds

Info

Publication number: EP1231913A2
Application number: EP00951029A
Authority: EP
Inventors: Zaheer Abbas; Carol Koboldt
Original assignee: Pharmacia LLC
Current assignee: Pharmacia LLC
Priority date: 1999-07-09
Filing date: 2000-06-07
Publication date: 2002-08-21
Also published as: AU6402700A; WO2001003688A2; JP2003506324A; WO2001003688A3

Abstract

This invention is directed to a process for the prevention or treatment of inflammation or other cyclooxygenase-2 mediated disorders. This process comprises administering a conjugated fatty acid to an organism in an amount effective to prevent or treat the cyclooxygenase-2 mediated disorder by inhibiting cyclooxygenase -2. In one embodiment, the process comprises administering a fatty acid compound having formula (I) or a pharmaceutically acceptable salt thereof, wherein R1 is hydrogen, hydrocarbyl, or substituted hydrocarbyl; m is from 6 to 11; and the sum of n and m is from 13 to 16. In another embodiment, the process comprises administering a fatty acid compound having formula (VI) or a pharmaceutically acceptable salt thereof, wherein R6 is hydrogen, hydrocarbyl, or substituted hydrocarbyl; R7 is -OH, -SO¿3?H, -SH, -NH2, -PO3H2, or halogen; v is from 7 to 11; and the sum of v and w is from 11 to 15. This invention is also directed to a process for preparing a fatty acid compound. The process comprises combining a ylide with an aldehyde.

Description

TREATMENT OF

CYCLOOXYGENASE-2 MEDIATED DISORDERS

USING CONJUGATED FATTY ACID COMPOUNDS

FIELD OF THE INVENTION

This invention generally relates to a process for preventing or treating medical disorders by reducing the activity of cyclooxygenase. More specifically, this invention relates to a process for preventing or treating inflammation and other cyclooxygenase-2 ("COX-2") mediated disorders by inhibiting COX-2 using a conjugated fatty acid compound (most preferably, a conjugated eicosadienoic acid compound or a coriolic acid compound). In a particularly preferred embodiment, COX-2 is selectively inhibited in preference to cyclooxygenase- 1 ("COX-1").

This invention further relates to a method for preparing fatty acid compounds, which, in turn, may be used to treat COX-2 mediated disorders.

BACKGROUND OF THE INVENTION

Inflammation is a defense mechanism of organisms caused by a physical or chemical stimulation, such as an injury to tissue. The classic symptoms of inflammation include, for example, pain, heat, redness, swelling, and sometimes loss of tissue function. Histologically, inflammation is characterized by, for example, hyperemia (i. e. , the presence of excess blood in a region of an organism), stasis (i. e. , the stoppage of the flow of blood in a region of an organism), changes in blood composition, changes in the walls of small blood vessels (typically in the form of expansion and perforation), and/or various exudations (i.e., leakages of blood components from the blood vessels and the deposition of such components in tissue or on tissue surfaces).

Many drugs are available for inhibiting inflammatory symptoms and alleviating the tissue destruction caused by inflammation. One general class of anti- inflammatory drugs consists of adrenocortical hormones (i.e., steroidal anti- inflammatory drugs). Although steroidal anti-inflammatory drugs provide strong anti- inflammatory action, they also tend to exhibit strong side-effects, such as hypertension, decreased immunity, hyperglycemia, osteoporosis, myopathy, cataracts, growth arrest, and electrolyte abnormalities. These side-effects are particularly problematic when such drugs are used over a long period of time.

A second general class of anti-inflammatory drugs consists of non-steroidal compositions. Non-steroidal anti-inflammatory drugs typically are directed to inhibiting enzymes in the arachidonic metabolic cascade which forms prostaglandins. This approach is effective for reducing inflammation because prostaglandins (particularly PGG₂, PGH₂, and PGE₂) play a major role in the inflammation process. One enzyme which is often targeted by non-steroidal anti-inflammatory drugs is cyclooxygenase (sometimes referred to as "COX"), which performs the initial reaction in the arachidonic metabolic cascade. See Smith, W.L., "Prostanoid

Biosynthesis and Mechanisms of Action," Am. J. Physiol, 263, F181-F191 (1992). Originally, the COX enzyme was thought to be a single enzyme which produced both pro-inflammatory prostaglandins and homeostatic prostaglandins. More recently, however, it has been discovered that the COX enzyme is actually two enzymes: a constitutive enzyme (COX-1) and an inducible enzyme (COX-2). COX-1 is found in nearly every tissue in the body, including the stomach, kidney, heart, brain, liver, and spleen. It is normally associated with the formation of homeostatic prostaglandins, which are linked to many normal tissue functions, such as gastric and renal functions. COX-2, on the other hand, is typically found in sites of inflammation, and is associated with pro-inflammatory prostaglandin production. Besides causing inflammation, this pro-inflammatory prostaglandin production also has been linked to other diseases, such as cancer. Belury, M.A., "Conjugated Dienoic Linoleate: A Polyunsaturated Fatty Acid with Unique Chemoprotective Properties," Nutrition Reviews, vol. 53, no. 4, 83-89 (1995). Although aspirin and many other conventional non-steroidal anti- inflammatory drugs reduce inflammation (and other COX-2 mediated disorders) by inhibiting of COX-2, they also tend to indiscriminately inhibit COX-1. This is often undesirable because inactivation of COX-1 disrupts normal tissue functions, and can lead to, for example, gastrointestinal injuries, nephrotoxicity (i.e., destruction of kidney tissue), and/or hemopoietic disorders (i.e., disorders in the formation of blood cells). This is particularly true where such drugs are used over a long duration. Given the problems associated with conventional non-steroidal anti- inflammatory drugs indiscriminately inhibiting COX-1, some recent approaches have reportedly been directed to developing non-steroidal anti-inflammatory drugs which selectively inhibit COX-2 in preference to COX-1. For example, in U.S. Patent No. 5,710,140 (and other related patents), Ducharme et al. report selective inhibition of COX-2 in preference to COX-1 by phenyl heterocycle compounds. In U.S. Patent No. 5,643,933, Talley et al. report inhibition of COX-2 in preference to COX-1 by substituted sulfonylphenylheterocycles. In U.S. Patent No. 5,436,265, Black et al. report inhibition of COX-2 in preference to COX-1 by l-aroyl-3-indolyl alkonoic acid compounds. And, Gierse et al. report selective inhibition of COX-2 in preference to COX-1 by N-(2-cyclohexyloxy-4-nitrophenyl)methanesulphonamide and 5-bromo-2- (4-fluorophenyl)-3-(4-methylsulphonylphenyl)-thiophen. See Gierse, J.K., Hauser, S.D., Creely, D.P., Koboldt, C, Rangwala, S.H., Isakson, P.C., and Seibert, K., "Expression and Selective Inhibition of the Constitutive and Inducible Forms of Human Cyclo-Oxygenase," Biochem. J., 305, 479-84 (1995). These approaches, however, remain limited, both in scope and in number. Thus, there still remains a demand for safe, simple, and effective treatments which inhibit COX-2, and, more particularly, for such treatments which selectively inhibit COX-2 in preference to COX-1.

SUMMARY OF THE INVENTION

This invention provides for a safe, simple, and effective process for preventing or treating inflammation and other COX-2 mediated disorders in an organism (human or otherwise) by inhibiting the COX-2 enzyme through the administration of a conjugated fatty acid compound. This invention also provides for a process for preventing or treating inflammation and other COX-2 mediated disorders in an organism by selective inhibition of COX-2 in preference to COX-1, thereby permitting the inhibition of COX-2 with fewer of the adverse side-effects normally associated with aspirin and many other conventional COX-2 inhibitors known in the art. Briefly, therefore, this invention is directed to a process for preventing or treating a cyclooxygenase-2 mediated disorder in an organism having a cyclooxygenase-2 mediated disorder or disposed to having a cyclooxygenase-2 mediated disorder. This process comprises administering a fatty acid compound to the organism in an amount effective to prevent or treat the COX-2 mediated disorder by inhibiting COX-2. In one embodiment, the fatty acid compound has formula (I) or is a pharmaceutically acceptable salt thereof:

O H H H H H H H

R¹ 0 C— (C)_m C=C— C=C— (C)_n C H

(I), wherein R¹ is hydrogen, hydrocarbyl, or substituted hydrocarbyl; m is from 6 to 11; and the sum of n and m is from 13 to 16. In another embodiment, the fatty acid compound has formula (VI) or is a pharmaceutically acceptable salt thereof:

O H H H H H H H

R⁶ O C— (C) _v C-=C— C=C— C (C)_w CH₃

^H R⁷ H (VI),

wherein R⁶ is hydrogen, hydrocarbyl, or substituted hydrocarbyl; R⁷ is -OH, -SO₃H, -SH, -NH₂, -PO₃H₂, or halogen; v is from 7 to 11; and the sum of v and w is from 11 to 15.

This invention also provides for a simple process for preparing fatty acid compounds which may be used to prevent or treat COX-2 mediated disorders. One advantage of this process is that it typically does not produce a significant amount of undesirable fatty acid byproducts (e.g., undesirable isomers). The process comprises combining a ylide with an aldehyde. In this embodiment, the fatty acid product has formula (I):

O H H H H H H H

R¹ O C— (C)_m C=-=C— C=C— (C)„ C H

H H H (J). the ylide reagent has formula (II):

R ² H H H H O

R ³ P= C ( C= C ) _χ ( C ) _m C 0 R

R ⁴ (II); the aldehyde reagent has formula (III):

0 H H ^H

H C ( C= C ) _y ( C ) „ C H ₃

(in); R¹, R², R³, and R⁴ are independently hydrocarbyl or substituted hydrocarbyl; m is from 6 to 11; the sum of n and m is from 13 to 16; x is 0 or 1; and the sum of x and y is l.

Other features of this invention will be in part apparent and in part pointed out hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a reaction scheme which may be used to prepare a fatty acid compound containing an unsubstituted fatty acid residue having from 19 to 22 carbons. This scheme comprises forming a ylide, and reacting the ylide with an aldehyde.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In accordance with the present invention, a method has been developed for preventing or treating inflammation and other COX-2 mediated disorders in an organism by inhibiting (i.e., reducing the activity of) the COX-2 enzyme through the administration of a conjugated fatty acid compound. The fatty acid compound preferably has a COX-2 IC₅₀ of less than about 100 μM, more preferably less than about 60 μM, even more preferably less than about 1.5 μM, still even more preferably less than about 1 μM, and most preferably less than 0.5 μM. The fatty acid compound also preferably has a COX-1 IC₅₀ which is greater than about 0.5 μM, more preferably greater than about 1 μM, even more preferably greater than about 1.5 μM, still even more preferably greater than about 60 μM, and most preferably greater than about 100 μM. As used herein, the "IC₅₀" value represents the concentration of the fatty acid compound required to reduce by 50% (as compared to an uninhibited control) the activity of the COX enzyme to produce prostaglandin E₂ ("PGE₂") in the presence of arachidonic acid.

Preferably, the fatty acid compound also selectively inhibits COX-2 in preference to COX-1. In one embodiment, the fatty acid compound which is administered to the organism has a COX-2 IC₅₀ which is less than the COX-1 IC₅₀. In this embodiment, the ratio of the COX-1 IC₅₀ to the COX-2 IC₅₀ preferably is at least about 1.5, more preferably at least about 2, even more preferably at least about 2.5, still even more preferably at least about 3, and most preferably at least about 3.5. In another embodiment, the fatty acid compound which is administered is a pro-drug (i.e., the fatty acid compound reacts following administration to form a different fatty acid compound, which, in turn, acts to inhibit the COX-2 enzyme). For example, if an ester of a fatty acid (e.g., the ethyl ester of cl l,tl3-eicosadienoic acid) is administered orally, it may hydrolyze during digestion to form the corresponding free fatty acid (e.g., cl l,tl3-eicosadienoic acid) and an alcohol (e.g., ethanol); in that instance, the compound which actually acts to inhibit the COX-2 enzyme is the free fatty acid. It is preferred that the fatty acid compound which actually acts to inhibit the COX-2 enzyme has a COX-2 IC₅₀ which is less than the COX-1 IC₅₀. In a particularly preferred embodiment, the fatty acid compound which actually acts to inhibit the COX-2 enzyme has a ratio of the COX-1 IC₅₀ to the COX-2 IC₅₀ which is at least about 1.5, more preferably at least about 2, even more preferably at least about 2.5, still even more preferably at least about 3, and most preferably at least about 3.5.

Two types of fatty acid compounds have been found in accordance with this invention to be particularly suitable for inhibiting COX-2: (1) a conjugated fatty acid compound comprising an unsubstituted fatty acid residue which has from 19 to 22 carbon atoms; and (2) a conjugated fatty acid compound comprising a fatty acid residue which has from 18 to 22 carbon atoms and is substituted with one of the following functional groups: a hydroxyl group (-OH), a sulfo group (-SO₃H), a thio group (-SH), an amino group (-NH₂), a phosphono acid group (-PO₃H₂), or a halogen.

I. The Unsubstituted Conjugated Fatty Acid Compound

A. Structure of the Unsubstituted Fatty Acid Compound The unsubstituted conjugated fatty acid compound generally has the following formula (I) or is a pharmaceutically acceptable salt thereof:

O H H H H H H H

_R 1 _{0 c}— _{( C ) m} C---= C— C= C— ( C ) _n C H

(I), wherein R¹ is hydrogen, hydrocarbyl, or substituted hydrocarbyl; and the sum of n and m is from 13 to 16. Preferably, m is from 6 to 11. In a preferred embodiment, the unsubstituted fatty acid compound is a glyceride, most preferably a triglyceride. In another preferred embodiment, R¹ is hydrogen or a saturated hydrocarbyl (i.e., the hydrocarbyl contains no carbon-carbon double or triple bonds). More preferably, R¹ is hydrogen or a saturated hydrocarbyl containing no greater than 6 carbon atoms (e.g., methyl (-CH₃), ethyl (-CH₂CH₃), propyl (-(CH₂)₂CH₃), isopropyl (-CH(CH₃)₂), butyl (-(CH₂)₃CH₃), pentyl (-

(CH₂)₄CH₃), or hexyl (-(CH₂)₅CH₃)). In an even more preferred embodiment, R¹ is methyl. Most preferably, R¹ is ethyl or hydrogen.

In a particularly preferred embodiment, the fatty acid compound contains an eicosadienoic acid residue (i.e., the sum of n and m is 14). In this embodiment, m preferably is from 7 to 9 (most preferably 9). In one of the more preferred embodiments, the fatty acid compound is the methyl ester of cl l,tl3-eicosadienoic acid ("c" meaning cis, and "t" meaning trans), i.e., the compound having formula (VIII):

In an even more preferred embodiment, the fatty acid compound is the ethyl ester of cl l,t 13 -eicosadienoic acid, i.e., the compound having formula (LX):

In the most preferred embodiment, the fatty acid compound is cl l,tl3-eicosadienoic acid (i.e. , the compound having formula (VII)) or a pharmaceutically acceptable salt thereof:

As shown in Example 4 below, conjugated eicosadienoic acid reduces the activity of COX-2. It also is selective toward inhibiting COX-2 in preference to COX-1. Thus, it may be administered to treat COX-2 mediated disorders, and advantageously causes fewer (if any) of the side effects normally associated with less selective anti-inflammatory drugs (e.g., aspirin). It should further be recognized that although the results in Example 4 suggest that the methyl ester of eicosadienoic acid (i.e., formula (VIII)) does not function as a COX-2 inhibitor, COX-2 inhibition may still be achieved by administering the ester as a pro-drug in a manner such that the ester will form the free fatty acid following administration. For example, if the ester is administered orally to a warm-blooded animal, it will typically hydrolyze during digestion to form the free fatty acid.

B. Preparation of Compounds Having Formula (I) Various methods for preparing mixtures comprising conjugated fatty acid compounds having formula (I) or the pharmaceutically acceptable salts thereof are generally known in the art. For example, in U.S. Patent No. 5,855,917 (incorporated herein by reference), Cook et al. teach preparing a mixture containing conjugated eicosadienoic acids by: (a) the alkaline isomerization of cl l,cl4-eicosadienoic acid; or (b) the alkaline isomerization or enzymatic isomerization of 9, 12-octadecadienoic acid, followed by the enzymatic elongation of the isomerized products.

In a particularly preferred embodiment of this invention, the conjugated fatty acid compound is prepared by forming a ylide, and reacting the ylide with an aldehyde. Figure 1 shows a general reaction scheme for such an embodiment. The benefits of using this reaction scheme include, for example, the fact that it produces a product composition which contains the desired conjugated fatty acid compound, but few (if any) undesirable fatty acid byproducts. This reaction scheme is particularly preferred for forming a carbon-carbon double bond having a cis configuration because the reaction tends to be highly selective toward forming such a configuration, especially at low reaction temperatures (i.e., a reaction temperature which is no greater than about -30°C). Although reactions between a ylide and an aldehyde (or ketone) have been generally disclosed in the art (see, e.g., K.P.C. Vollhardt and N.E.Schore, Organic Chemistry, 657-61 (W.H. Freeman & Co., 2nd ed., 1994) (incorporated herein by reference)), applicants are unaware of such a reaction mechanism being reported for preparing the conjugated fatty acid compounds of formula (I).

The ylide used to prepare the conjugated fatty acid compound preferably has formula (II):

R ²

R ³ P= C ( C= C ) _χ — ( C ) ,

^{R 4} H (II),

wherein m is as defined above for formula (I); R¹, R², R³, and R⁴ are independently hydrocarbyl or substituted hydrocarbyl; and x is 0 or 1. It should be recognized that the ylide actually may resonate between formula (II) and a structure having a positive charge on the phosphorus atom and a negative charge on the adjacent carbon atom of the fatty acid chain: p c — ( c= c ; ⁽ 9 :

The term "ylide" and formula (II) as used herein encompass both structures.

In a particularly preferred embodiment, the ylide has a structure wherein R², R³, and R⁴ are each phenyl (-C₆H₅) or phenyl substituted with at least one hydrocarbyl (most preferably, R², R³, and R⁴ are each unsubstituted phenyl). In another particularly preferred embodiment, m is 9. In a further particularly preferred embodiment, x is 0. Combinations of these embodiments are also particularly preferred.

The ylide preferably is formed by a process comprising first combining a phosphine compound with a haloalkane to form a phosphonium salt having formula (XI):

Here, the phosphine compound has formula (IV):

R - P

(IV); the haloalkane, has formula (V):

H H H H O

R ⁵ C ( C= C ) _χ ( C ) _m C O R ¹

H H (V);

R², R³, and R⁴ are as defined above for the ylide; and R⁵ is halogen (preferably bromine). When preparing the phosphonium salt from the phosphine compound and haloalkane, a slight excess of the phosphine compound preferably is used (i.e., the molar ratio of the phosphine compound to the haloalkane preferably is from about 1.1:1 to about 1.5:1, more preferably from about 1.1 :1 to about 1.3:1, and most preferably about 1.25:1). Large excesses of either the phosphine compound or the haloalkane preferably are avoided due to the extra separation costs to remove such excesses from the product mixture.

The reaction of the phosphine compound with the haloalkane may be conducted in a wide range of solvents which can solubilize the phosphine reagent, the haloalkane, and the phosphonium salt product. Suitable solvents typically include, for example, acetonitrile, toluene, dichloromethane, benzene, acetone, N,N- dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and hexamethylphosphoric triamide (HMPA). Protic solvents (e.g., methanol, ethanol, and water) are typically less preferred because they tend to stabilize the haloalkane compound, making it more difficult for the alkyl group of the haloalkane to dissociate from the halogen so that it may bond with the phosphine compound. In a particularly preferred embodiment, the solvent has a dielectric constant which is from about 2 to about 40 at about 20°C and atmospheric pressure (dielectric constants for many compounds are well known in the art, and may be found in, for example, Handbook of Chemistry and Physics (CRC Press, Inc., Boca Raton, Florida) (incorporated herein by reference)). The solvent also preferably has a boiling point at atmospheric pressure of at least about 50°C, and more preferably at least about 80°C. Preferably, from about 2 to about 100 ml (more preferably from about 5 to about 25 ml, and most preferably from about 5 to about 10 ml) of solvent are used per each mmole of the phosphine compound. The environment in which the phosphine compound and haloalkane are reacted preferably is an inert environment (i.e., an environment that essentially does not react with the reagents or products of the reaction). This environment may contain, for example, nitrogen, a noble gas (e.g., helium, argon, and neon), or a combination thereof. In some embodiments, the more preferred environment contains a noble gas (most preferably argon) rather than nitrogen due to the fact that nitrogen may react with components of the reaction mixture under certain conditions. In other embodiments (particularly large scale embodiments), however, the environment contains nitrogen due to its relatively low cost. In any event, the environment contains essentially no oxygen gas or water, and more preferably contains no oxygen gas or water (oxygen gas tends to oxidize the phosphine compound, and water tends to hydrolyze the resulting ylide). The temperature preferably is sufficient to dissociate the halogen from the rest of the haloalkane compound. Typically, the temperature is at least about 50°C, more preferably from about 50 to about 100°C, and most preferably from about 80 to about 100°C. Such a temperature may often be achieved by refluxing the reaction mixture at about atmospheric pressure. The reaction time varies widely, and depends, for example, on the temperature at which the reaction is conducted (in general, the greater the temperature, the faster the reaction rate) and the concentration of the reagents (in general, the greater the reagent concentrations, the faster the reaction rate). Typical minimum reaction times range from about 6 hours to about 5 days, with the most typical reaction time being about 3 days.

Following the formation of the phosphonium salt, the solvent preferably is evaporated and the excess phosphine compound preferably is removed from the remaining reaction mixture. Removal of the excess phosphine compound may be achieved using, for example, silica gel chromatography by methods well known in the art. Alternatively, for example, the excess phosphine compound may be removed from the reaction mixture by extracting the phosphine compound from the reaction mixture with a non-polar solvent. This extraction is typically performed at a temperature of at least about 20°C. Preferably, the extraction solvent has a dielectric constant such that the phosphonium salt is essentially insoluble in the solvent. It is particularly preferred for the extraction solvent to have a dielectric constant of less than about 2 at about 20°C and atmospheric pressure. Hydrocarbon solvents (e.g., hexane and pentane) are often suitable for this purpose. The amount of extraction solvent may vary widely. In most instances, it is preferred to use from about 50 to about 100 ml of extraction solvent per gram of the phosphine compound initially loaded. In another alternative embodiment, the phosphine compound is removed from the reaction mixture with a solvent containing both a polar component and a non-polar component. This extraction may be conducted in addition to (or as an alternative to) an extraction with an exclusively non-polar solvent. The polar component may be, for example, dichloromethane. Such a polar component typically enhances the amount of phosphine compound extracted from the reaction mixture. It also, however, tends to dissolve phosphonium salt and therefore reduces the yield. If an extraction solvent containing a polar component is used, the polar component preferably makes up no greater than about half the total volume of the extraction solvent. Preferably, from about 50 to about 100 ml of polar/nonpolar extraction solvent is used per gram of the phosphine compound initially loaded.

To form the ylide, the phosphonium salt is combined with a base to deprotonate the phosphonium salt. Various bases are suitable for this reaction, and include, for example, lithium hexamethyldisilazane (LiHMDS), butyllithium, sodium hydride, and alkoxides. The most preferred bases are hindered bases (i.e., a base that essentially will not attack an electrophile, but can accept an electron), such as LiHMDS. The number of moles of base preferably is substantially equal to the number of moles of phosphonium salt. The reaction preferably is conducted in a solvent which has a dielectric constant sufficient to dissolve the phosphonium salt. Often suitable solvents include, for example, N,N-dimethylformamide (DMF), tetrahydrofuran (THF), hexamethylphosphoric triamide (HMPA), and combinations thereof. Preferably, from about 2 to about 100 ml (more preferably from about 5 to about 25 ml, and most preferably from about 5 to about 10 ml) of solvent are used per each mmole of phosphonium salt. During the deprotonation, the reaction mixture preferably is kept under an inert environment. In addition, the temperature preferably is maintained at from about -78 (using, for example, liquid nitrogen as the cooling source) to about -30°C (using, for example, an electronic cooling bath), and more preferably is maintained at from about -78 to about -50°C. Greater temperatures are less preferred because the ylide product at such temperatures tends to decompose to form a phosphonium oxide. The preferred reaction time is dependent on, for example, reaction temperature. At temperatures of from about -78 to about -50°C, the preferred reaction time typically is from about 30 to about 60 minutes. Either during or after the ylide formation (most preferably after the ylide formation), the reaction mixture is combined with an aldehyde to form the desired fatty acid compound. Because the ylide is highly reactive, it typically is not separated from the reaction mixture before being combined with the aldehyde. The aldehyde preferably has formula (III):

H C ( C= C ) _y ( C ) _n C H ₃

H (πi),

wherein n is as defined above for the fatty acid compound (i.e., formula (I)); and y is 0 or 1, such that the sum of x (as defined for formula (II)) and y is 1. The preferred amount of aldehyde is dependent on the amount of phosphonium salt used to form the ylide. Typically, the moles of aldehyde added preferably is slightly less than the number of moles of phosphonium salt used in the deprotonation reaction to form the ylide due to the fact that the aldehyde is generally more expensive. Typically, the molar ratio of aldehyde to phosphonium salt is from about 1:1.1 to about 1:1.5. The reaction preferably is conducted at from about -78 to about -30°C for from about 30 to about 120 minutes under an inert atmosphere. More preferably the temperature ranges from about -50°C to about -78°C, and most preferably is about -78°C. As noted above, use of such low temperatures tends to promote the formation of a cis carbon-carbon double bond.

Following the formation of the fatty acid compound, the reaction mixture preferably is gradually warmed by, for example, placing the reaction mixture in an environment having a temperature of from about 20 to about 25°C. The excess ylide preferably is hydrolyzed, using, for example, an aqueous solution of ammonium acetate or ammonium chloride having a pH of about 7. Afterward, the fatty acid compound may be separated from the reaction mixture using any of the various conventional separation methods known in the art. See, e.g., Example 1 below, which uses extraction with a non-polar organic solvent, followed by chromatographic separation on a silica gel column.

The above reaction scheme is typically used to form esters of fatty acids. In one embodiment of this invention, the ester is further hydrolyzed to form the free fatty acid before being administered as a treatment. Hydrolysis of the ester may be carried out by, for example, acid-catalyzed or base-catalyzed hydrolysis. Many variations of such hydrolysis methods are well known in the art. In one embodiment of this invention, the hydrolysis is a base-catalyzed reaction carried out in an aqueous solution containing a weak base, such as sodium bicarbonate, triethylamine, cesium carbonate, and potassium carbonate, with potassium carbonate being most preferred. If the ester is insoluble in water, an alcohol preferably is added to the solution to solubilize the ester. Preferably, the alcohol has the formula R'-OH, wherein R¹ is as defined in formula (I) (for example, if the ester is a methyl ester, the alcohol is methanol; if the ester is an ethyl ester, the alcohol is ethanol; etc.). Example 2 below further illustrates this method. Regardless of the method for hydrolyzing the ester, the hydrolysis preferably is performed in a non-oxidizing atmosphere (i.e., the atmosphere consists essentially of a non-oxidizing gas(es), such as N₂ and/or a noble gas.

II. The Substituted Conjugated Fatty Acid Compound A. Structure of the Substituted Fatty Acid Compound

The substituted conjugated fatty acid compound generally has the following formula (VI) or is a pharmaceutically acceptable salt thereof:

wherein R⁶ is hydrogen, hydrocarbyl, or substituted hydrocarbyl; R⁷ is an acidic group; and the sum of v and w is from 11 to 15. Preferably, v is from 7 to 11. R⁷ is preferably -OH, -SO₃H, -SH, -NH₂, -PO₃H₂, or halogen, with -OH being most preferred. In a preferred embodiment, the fatty acid compound is a glyceride, and most preferably a triglyceride. In another preferred embodiment, R⁶ is hydrogen or a saturated hydrocarbyl. More preferably, R⁶ is hydrogen or a saturated hydrocarbyl containing no greater than 6 carbon atoms (e.g., methyl (-CH₃), ethyl (-CH₂CH₃), propyl (-(CH₂)₂CH₃), isopropyl (-CH(CH₃)₂), butyl (-(CH₂)₃CH₃), pentyl (-

(CH₂)₄CH₃), or hexyl (-(CH₂)₅CH₃)). In an even more preferred embodiment, R⁶ is methyl. In another even more preferred embodiment, R⁶ is ethyl. In a further even more preferred embodiment, R⁶ is hydrogen.

In a particularly preferred embodiment, the sum of v and w is 11. Even more preferably, the sum of v and w is 11, and R⁷ is -OH. Still even more preferably, the fatty acid compound is coriolic acid (i.e., v is 7, w is 4, R⁶ is hydrogen, and R⁷ is -OH), a salt of coriolic acid, or an ester of coriolic acid. In the most preferred embodiment, the fatty acid compound is 13S-hydroxy-c9, til -coriolic acid (i.e., the compound having formula (X)) or a pharmaceutically acceptable salt thereof:

OH (X).

As shown in Example 4 below, coriolic acid (like eicosadienoic acid) selectively inhibits COX-2 in preference to COX-1. Thus, the side effects normally associated with less selective anti-inflammatory drugs (e.g., aspirin) may be reduced (or altogether avoided) by using coriolic acid. It should be recognized that the fatty acid compound of formula (VI) and salts thereof may be either administered in addition to the fatty acid compound of formula (I) or salts thereof, or as an alternative to the fatty acid compound of formula (I) or salts thereof.

B. Preparation of Compounds Having Formula (VI) Methods for preparing fatty acid compounds of formula (VI) are generally known in the art. For example, hydroxyoctadecadienoic acids occur naturally in vegetable and animal tissues, and can be isolated from them (e.g. , in vegetable extracts of nettle roots, such acids have been reported to be present as both free acids and as ester components of glycerides, ceramides, and phospholipids). Such acids also can be synthesized from oleic acid and from linoleic acid. See, e.g., Streber, U.S. Patent No. 5,102,912 (incorporated herein by reference).

Coriolic acid, in particular, may be prepared from linoleic acid by lipooxygenation under oxygen pressure (2.5 bar), followed by reduction of the hydroperoxide. See Martini, D., Iacazio, G., Ferrand, D., Buono, G., and Triantaphylides, C, "Optimization of Large-Scale Preparation of 13-(S)-hydroperoxy- 9z,l le-octadecadienoic Acid Using Soybean Lipooxygenase. Application to the Chemoenzymatic Synthesis of (+)-Coriolic Acid," Biocatalysis, 11, 47-63 (1994) (incorporated herein by reference). Coriolic acid also may be prepared from an optically active lactol starting material, as described in Bloch, R. and Perfetti, M.T., "An Efficient Synthesis of 13(S)-hydroxy-9z,l le-octadecadienoic (Coriolic) Acid," Tetrahedron Letters, vol. 31, no. 18, pp. 2577-80 (1990) (incorporated herein by reference). In addition, coriolic acid may be prepared, for example, from trilinolein, as described in Gargouri, M. and Legoy, M.D., "Chemoenzymatic Production of (+)- Coriolic Acid from Trilinolein: Coupled Synthesis and Extraction," JAOCS, vol. 74, no. 6, pp. 641-645 (1997) (incorporated herein by reference). Coriolic acid further may be isolated from plants such as, for example, from the oil of Xeranthemum annuum seeds (see Powel, R.C., Smith, C.R., and Wolff, LA., "Geometric Configuration and Etherification Reactions of Some Naturally Occurring 9-Hydroxy- 10,12- and 13-Hydroxy-9,l l-Octadecadienoic Acids," J Org. Chem., 32, 1442-46 (1966) (incorporated herein by reference)). Example 3 below further illustrates the preparation of coriolic acid.

III. Use of the Conjugated Fatty Acid Compounds to Inhibit COX-2 The conjugated fatty acid compounds discussed above may generally be administered (alone or in combination) to prevent or treat all types of inflammation and other COX-2 mediated disorders. For example, these fatty acid compounds may be used to treat swelling, fever, redness of the skin, aches (e.g., tension headaches, migraine headaches, postoperative pain, dental pain, muscular pain, back pain, neck pain, and pain resulting from cancer), arthritis (e.g., rheumatoid arthritis, spondyloarthropathies, gouty arthritis, osteoarthritis, systemic lupus erythematosus, and juvenile arthritis), arthritic symptoms, skin conditions (e.g., dermatitis, psoriasis, eczema, and burns), infections, gastrointestinal conditions (e.g., inflammatory bowel disease, Crohn's disease, gastritis, irritable bowel syndrome, and ulcerative colitis), periarteritis nodosa, thyroiditis, aplastic anemia, Hodgkin's disease, sclerodoma, rheumatic fever, neuromuscular junction disease (e.g., myasthenia gravis), white matter disease (e.g., multiple sclerosis), sarcoidosis, nephrotic syndrome, Behcet's syndrome, polymyositis, gingivitis, nephritis, hypersensitivity, myocardial ischemia, ophthalmic diseases (e.g., retinitis, retinopathies, uveitis, and ocular phtophobia), type I diabetes, chronic lung diseases, asthma, bronchitis, pulmonary inflammation (e.g., inflammation associated with viral infections and cystic fibrosis), allergic reactions (e.g., reactions to food, drugs, insects, and pets), collagen disease, tendinitis, bursitis, rhinitis, post-operative inflammation (e.g., from ophthalmic surgery such as cataract surgery or refractive surgery), vascular diseases, cancer (e.g., colorectal cancer, breast cancer, lung cancer, prostate cancer, bladder cancer, cervic cancer, and skin cancer), central nervous system disorders (e.g., Alzheimer's disease and central nervous system damage resulting from stroke, ischemia, and trauma), menstral cramps, and pre-mature labor. This list is simply for illustration purposes, and is not intended to be exhaustive of the COX-2 mediated disorders which may be treated by the fatty acid compounds described above. As noted previously, the fatty acid compounds described herein which selectively inhibit COX-2 in preference to COX-1 have fewer adverse side effects associated with COX-1 inhibition than do less selective anti-inflammatory drugs. Such fatty acid compounds are therefore particularly valuable for use over long durations, such as in conjunction with preventing or treating allergies and chronic illnesses (e.g., rheumatoid arthritis). For the same reason, the selective fatty acid compounds of this invention are typically well-suited for treating organisms which have preexisting conditions often associated with COX-1 inhibition, such as peptic ulcers, gastritis, regional enteritis, ulcerative colitis, diverticulitis, recurring gastrointestinal lesions, gastrointestinal bleeding, coagulation or other bleeding disorders, and kidney disease (e.g., impaired renal function). The selective fatty acid compounds of this invention are also typically well-suited for treating organisms that are (1) susceptible to non-steroidal anti-inflammatory drug induced asthma, (2) about to have a surgery, and/or (3) taking anticoagulants.

The fatty acid compounds described herein generally may be used to treat a wide variety of organisms, particularly warm-blooded organisms. For example, these fatty acid compounds may be used to treat humans, compamon animals (e.g., dogs and cats), farm animals (e.g., horses, cattle, goats, pigs, rabbits, and sheep), mice, rats, and wild animals. Most preferably, the fatty acid compounds are used to treat warmblooded animals having a cyclooxygenase-2 mediated disorder or disposed to having a cyclooxygenase-2 mediated disorder. The mode of administration may vary widely. Generally, the fatty acid compounds of this invention may be administered by any acceptable means which results in the prevention, reduction, or elimination of the targeted inflammation or other COX-2 mediated disorder. For example, such fatty acid compounds may be administered orally (e.g., in the form of a tablet, capsule, syrup, solution, emulsion, food supplement, or drink supplement); parenterally (e.g., via subcutaneous injection, intramuscular injection, intrasternal injection, intravenous injection, or infusion techniques); topically (e.g., in the form of an ointment or eye drops), particularly when the inflammation is localized near the surface of the skin; via inhalation (e.g., in the form of an inhalant); via nasal spray; or rectally (e.g., in the form of a suppository). For most types of COX-2 mediated disorders, it is typically preferred to administer the fatty acid compound orally.

Before being administered, the fatty acid compound may be combined with a conventionally used pharmaceutically acceptable compound(s) which is compatible with the fatty acid compound. This pharmaceutically acceptable compound may be, for example, another drug, such as, for example, a lipooxygenase. The pharmaceutically acceptable compound may also be, for example, an adjuvant, excipient, corrigent (e.g., a flavoring agent), coloring agent, preserving agent, or other additive. The excipient may be, for example, an inert diluent (e.g., calcium carbonate, sodium carbonate, lactose, calcium phosphate, sodium phosphate, peanut oil, liquid paraffin, or olive oil), a binder (e.g., starch, gelatin, or acacia), a granulating or disintegrating agent (e.g., corn starch or alginic acid), or a lubricant (e.g., magnesium stearate, stearic acid, or talc). The fatty acid compound may also be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby sustaining action over a longer period. Such a coating may contain, for example, glyceryl monostearate or glyceryl distearate. If administered rectally, it is often preferable to mix the fatty acid compound with a non-irritating excipient (e.g., cocoa butter or a polyethylene glycol) which is solid at ordinary temperatures, but liquid at the rectal temperature such that it will melt in the rectum to release the drug.

In general, the fatty acid compound may be administered with one or more other fatty acid compounds, and therefore does not have to necessarily be isolated from other fatty acid compounds before being administered. Further, when administering the fatty acid compound orally, the fatty acid compound typically may be administered in the presence of other compounds that are ordinarily found in foods and beverages (e.g., proteins, sugars, fats, vitamins, etc.).

The preferred dosage will vary, depending generally on factors such as the sex, age, weight, diet, rate of excretion, and physical condition of the recipient; the form of the fatty acid compound; the time and route of administration; the severity, type, stage, and location of the inflammation or other COX-2 mediated disorder to be prevented or treated; and the existence of any concurrent treatments. In general, the fatty acid compound is administered to the organism in an amount which is effective to prevent or treat the targeted cyclooxygenase-2 mediated disorder by reducing the activity of COX-2. If the fatty acid compound is administered orally, the preferred daily dose typically is from about 0.0001 to about 2 g/Kg (i.e., grams of fatty acid compound per kilograms of recipient), more preferably from about 0.001 to about 2 g/Kg, and most preferably from about 0.001 to about 1 g/Kg. In general, it is preferred that the fatty acid compound make up from about 1 to about 10,000 ppm by weight (more preferably from about 100 to about 10,000 ppm by weight, and most preferably from about 1,000 to about 10,000 ppm by weight) of the recipient's diet when administered orally. It should be recognized that in most instances, the foregoing upper limits are not particularly critical because the fatty acid compounds used herein are generally relatively non-toxic. Oral dosages typically are administered from 1 to 6 times per day, and more typically from 1 to 3 times per day. DEFINITIONS

Unless otherwise stated, the following definitions should be used: The term "hydrocarbyl" is defined as a group consisting exclusively of carbon and hydrogen. The hydrocarbyl may be branched or unbranched, may be saturated or unsaturated, and may comprise one or more rings. Suitable hydrocarbyl moieties include alkyl, alkenyl, alkynyl, and aryl moieties. They also include alkyl, alkenyl, alkynyl, and aryl moieties substituted with other aliphatic or cyclic hydrocarbyl groups, such as alkaryl, alkenaryl and alkynaryl.

The term "substituted hydrocarbyl" is defined as a hydrocarbyl wherein at least one hydrogen atom has been substituted with (1) an atom other than hydrogen and carbon, or (2) a group of atoms which contains at least one atom other than hydrogen and carbon. For example, the hydrogen atom may be replaced by a halogen atom, such as a chlorine or fluorine atom. The hydrogen atom alternatively may be substituted by an oxygen atom to form, for example, a hydroxy group, an ether, an ester, an anhydride, an aldehyde, a ketone, or a carboxylic acid. The hydrogen atom also may be replaced by a nitrogen atom to form, for example, an amide or a nitro functionality. In addition, the hydrogen atom may be replaced with a sulfur atom to form, for example, a thio group or a sulfo group.

The term "pharmaceutically acceptable salt" embraces alkali metal salts and addition salts of free acids or free bases. The nature of the salt is not critical, provided that it is pharmaceutically acceptable. Mixtures of two or more pharmaceutically acceptable salts may also be used. Suitable pharmaceutically acceptable acid addition salts of the therapeutic compounds discussed herein may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acid. Examples of suitable organic acids include aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclyl, carboxylic, and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, stearic, salicylic, ?-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, toluenesulfonic, 2-hydroxyethanesulfonic, sulfanilic, cyclohexylaminosulfonic, algenic, b-hydroxybutyric, galactaric, and galacturonic acid. Suitable pharmaceutically acceptable base addition salts of the therapeutic compounds discussed herein include metallic salts, organic salts, and ammonium salts. More preferred metallic salts include, but are not limited to, alkali metal (group la) salts, alkaline earth metal (group Ila) salts, and salts made from other physiological acceptable metals. Such salts include, for example, aluminum, calcium, lithium, magnesium, potassium, cesium, sodium, copper, iron, silver, and zinc salts. Preferred organic salts can be made from tertiary amines and quaternary ammonium salts, including in part, tromethamine, diethylamine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N- methylglucamine), and procaine. Typically, it is more preferred to use a sodium, potassium, calcium, or magnesium salt. Still, in some embodiments, an ammonium salt is more preferred. In other embodiments, a lithium salt is more preferred. And, in yet other embodiments, a zinc salt is more preferred. All of the foregoing salts may be prepared by conventional means from the corresponding therapeutic compounds discussed herein by reacting, for example, the appropriate acid or base with the compounds.

EXAMPLES

The following examples are intended to further illustrate and explain the process of the present invention.

Example 1 : Preparation of the methyl ester of cll.tl3-eicosadienoic acid

This example demonstrates one embodiment of the method for preparing an eicosadienoic acid compound, namely the methyl ester of cl 1, tl3-eicosadienoic acid having formula (VIII):

First a phosphonium salt having the formula [(C₆H₅)₃P⁺-(CH₂)₁₀CO₂CH₃]Bf was prepared. Approximately 3.51 g (13.39 mmol) of triphenylphosphine (i.e., (C₆H₅)₃P, Cat. No. T8,440-9, Aldrich Chemical Co., Milwaukee, WI) and 3.0 g (10.74 mmol) of methyl 11-bromo undecanoate (i.e., Br(CH₂)₁₀CO₂CH₃, Cat. No. 44,746-3, Aldrich) were combined in the presence of 80 ml of acetonitrile (CH₃CN). This mixture was refluxed under a dry nitrogen atmosphere for 5 days. Afterward, the solvent was evaporated until a dry mass remained. The triphenylphosphine was extracted from the dry mass with 500 ml of hexane and then with 500 ml of a solvent containing dichloromethane and hexane (the volumetric ratio of dichloromethane to hexane was 1 :2). Approximately 6.2 g of a thick residual oil remained after the extraction. This product was analyzed using gas chromatography/mass spectroscopy, which verified the molecular weight and carbon-carbon double bond positions of the resulting phosphonium salt compound.

Next, the phosphonium salt was reacted with a base to form a mixture containing a ylide having the formula (C₆H₅)₃PCH(CH₂)₉CO₂CH₃. Approximately 1.62 g (3 mmol) of the phosphonium salt was placed into a mixture containing 15 ml of dry tetrahydrofuran (THF) and 5 ml of dry hexamethylphosphoramide (HMPA, i. e. , [N(CH₃)₂]₃PO, Cat. No. HI, 160-2, Aldrich). The resulting mixture was cooled to -78°C, and then 3 ml (502 mg, 3 mmol) of lithium hexamethyldisilazane (LiHMDS, i.e., LiN[Si(CH₃)₃]₂, Cat. No. 22,577-0, Aldrich) was added. This mixture was stirred at -78°C for 45 minutes to yield a reaction mixture containing the ylide. This ylide-containing mixture was then reacted with tra«s-2-nonanal

(CH₃(CH₂)₅(CH)₂CHO, Cat. No. 25,565-3, Aldrich) to form the methyl ester of cl 1, tl3-eicosadienoic acid. More specifically, about 280 mg (2 mmol) of traw.s-2-nonanal in 4 ml of dry THF was added to the ylide-containing mixture. This mixture was then stirred at -78°C for 1 hr and then allowed to naturally warm to room temperature.

To separate the methyl ester fatty acid product, 50 ml of 25% (by weight) aqueous ammonium acetate having a pH of 7 was first added to the reaction mixture to hydrolyze the excess ylide. Afterward, the methyl ester fatty acid product was extracted twice from the reaction mixture with hexane (between about 175 ml and 350 ml). The hexane mixture was then dried over anhydrous sodium sulfate (about 10 g per 100 ml of total mixture, including the hexane), and filtered. The hexane was evaporated from the mixture under a slight vacuum to form a residual liquid containing the methyl ester fatty acid product. The residual liquid was then separated on a homemade silica gel gravity chromatography column using a hexane/dichloromethane eluant (the volumetric ratio of hexane to dichloromethane in the solvent was 7:3). Approximately 220 mg of the methyl ester fatty of cl 1, tl3- eicosadienoic acid was obtained. The composition was verified using gas chromatography/mass spectroscopy.

Example 2: Preparation of cll,tl3-eicosadienoic acid

In this example, the methyl ester of cl 1, tl3-eicosadienoic acid prepared in Example 1 was hydrolyzed to form cl l,tl3-eicosadienoic acid. Approximately 200 mg (0.62 mmol) of the methyl ester was combined with 856 mg (6.2 mmol) of K₂CO₃ in 15 ml of an aqueous solution containing 20% (by volume) methanol. This mixture was stirred at room temperature for 48 hours, and neutralized with an aqueous solution containing 10% (by weight) HCl. The free fatty acid was extracted with 150 ml ethyl acetate, and then the ethyl acetate was evaporated. This yielded approximately 130 mg of cl l,t 13 -eicosadienoic acid.

Example 3: Preparation of coriolic acid

Approximately 5 g of linoleic acid (i.e., t9,tl2-octadecadienoic acid, Cat. No. L-1376, Sigma Chemical Co., St. Louis, Missouri) was combined with 20 ml of octane, 160 ml of 0.1 M borate buffer (pH = 9.6), and 100 mg soybean lipooxygenase (Cat. No. L-8383, Sigma Chemical) at 20°C. For two hours, the bi-phasic mixture was stirred with a magnetic stirrer (800 rpm) while pure oxygen was bubbled through the mixture at a flow rate of 30 ml/min. Afterward, the mixture was centrifuged and the octane layer was separated and diluted with 100 ml of ethanol and 500 mg of NaBH₄. This mixture was then stirred for 1 hour. Subsequently, the liquid was evaporated and the fatty acid product was extracted from the remaining residue with 250 ml ethyl acetate, and washed with a plentiful amount of water and brine. The ethyl acetate was evaporated, and the resulting residue was separated on a homemade silica gel gravity chromatography column using a eluant containing equal volumes of hexane and ethyl acetate. This yielded about 11 mg of coriolic acid. Example 4: Use of fatty acid compounds to inhibit cyclooxygenase

Several fatty acid compounds were analyzed in vitro to determine the extent to which they reduce the activity of COX-1 and COX-2. This analysis was conducted using the procedure set forth in Gierse, J.K., Hauser, S.D., Creely, D.P., Koboldt, C, Rangwala, S.H., Isakson, P.C, and Seibert, K., "Expression and Selective Inhibition of the Constitutive and Inducible Forms of Human Cyclo-Oxygenase," B iochem. J., 305, 479-84 (1995) (incorporated herein by reference). Briefly, Sf21 insect cells expressing COX-1 or COX-2 were homogenized. To analyze each fatty acid compound, several samples were prepared which contained the crude homogenate suspended in a buffer solution containing (1) 25 mM Tris, (2) sufficient HCl to impart a pH of 8.1, (3) 0.25 M sucrose, and (4) 1% (weight/volume) of 3-[3- cholamidopropyl)dimethylammonio]-l-propane-sulfonate (also known as "CHAPS," Sigma, St. Louis, Missouri). Each sample contained about 2-10 μg of protein from the homogenized cells. The fatty acid compound was added to the samples in varying concentrations of from 0.001 tolOO μM. The samples were then incubated at room temperature for 10 minutes. Afterward, a sufficient amount of arachidonic acid was added to each sample to impart a 10 μM arachidonic acid concentration, and the samples were again incubated at room temperature for another 10 minutes. The COX activity was determined by measuring the amount of prostaglandin E₂ produced (PGE₂ concentrations were measured using e.l.i.s.a. (Caymen)).

Table 1 shows the results. The cl l,tl3-eicosadienoic acid inhibited COX-2, and was selective to COX-2 in preference to COX-1 (the ratio of the IC₅₀'s for COX-1 to COX-2 was at least about 3.8). Although the methyl ester of cl l,tl3-eicosadienoic acid was inactive toward inhibiting COX-2, it was also inactive toward inhibiting COX- 1 (unlike the methyl ester of c9,tl 1 -CLA, which did have a measurable inhibiting effect toward COX-1).

Table 1 also shows that the 13(S)hydroxy-c9,tl l-(+)coriolic acid had an inhibiting effect on COX-2, and was inactive toward inhibiting COX-1. Table 1 Comparison of the Extent to which Various Fatty Acid Compounds Inhibit COX-1 and COX-2 Fatty Acid Compound IC₅₀ (μM)¹

COX-1 COX-2 c9,tl l-CLA² (97% pure) 0.74 <0.137 c9,tl l-CLA² (97% pure) 0.42 0.29 c9,tl l-CLA (75% pure) 0.9 0.43 c9,cl l-CLA (98% pure) 28 >100 t9,tl l-CLA (98% pure) 0.339 <0.137 t9,tl l-CLA (98% pure) 0.45 0.14 methyl ester of c9,tl 1-CLA 44 >100 cl l,tl3-eicosadienoic acid 1.87 0.49 cl l,tl3-eicosadienoic acid 2.63 <0.137 methyl ester ofcl l,tl3 -eicosadienoic acid >100 >100 13(S)hydroxy-c9,tl l-(+)coriolic acid >100 56

1. "IC₅₀" is the concentration of fatty acid compound necessary to inhibit 50% of the enzyme's (i.e., COX-1 or COX-2) activity. 2. "CLA" is conjugated linoleic acid.

* * * * * * * * *

The above description of the preferred embodiment is intended only to acquaint others skilled in the art with the invention, its principles, and its practical application, so that others skilled in the art may adapt and apply the invention in its numerous forms, as may be best suited to the requirements of a particular use. The present invention, therefore, is not limited to the above embodiments, and may be variously modified.

Claims

We Claim:

1. A process for the treatment or prevention of a cyclooxygenase-2 mediated disorder in an organism having a cyclooxygenase-2 mediated disorder or disposed to having a cyclooxygenase-2 mediated disorder, the process comprising administering a fatty acid compound to the organism in an amount effective to prevent or treat the cyclooxygenase-2 mediated disorder by inhibiting cyclooxygenase-2, wherein the fatty acid compound has formula (I) or is a pharmaceutically acceptable salt thereof:

H H H H H H H

R ¹ O C— ( C ) _m C = C— C= C— ( C ) _n C H

H H H _(I);

R¹ is hydrogen, hydrocarbyl, or substituted hydrocarbyl; m is from 6 tol 1; and n and m have a sum which is from 13 to 16.

2. The process of claim 1 wherein m is from 7 to 9, and m and n have a sum of 14.

3. The process of claim 2 wherein m is 7.

4. The process of claim 2 wherein m is 8.

5. The process of claim 2 wherein m is 9.

6. The process of claim 1 wherein the fatty acid compound is a triglyceride.

7. The process of claim 1 wherein the fatty acid compound is admimstered to treat a cyclooxygenase-2 mediated disorder in an organism having the cyclooxygenase-2 mediated disorder.

8. The process of claim 1 wherein the fatty acid compound is admimstered to prevent a cyclooxygenase-2 mediated disorder in an organism disposed to having the cyclooxygenase-2 mediated disorder.

9. The process of claim 1 wherein the fatty acid compound is administered to prevent or treat swelling, fever, skin redness, an ache, arthritis, a skin disease, an infection, a gastrointestinal condition, periarteritis nodosa, thyroiditis, aplastic anemia, Hodgkin's disease, sclerodoma, rheumatic fever, neuromuscular junction disease, white matter disease, sarcoidosis, nephrotic syndrome, Behcet's syndrome, polymyositis, gingivitis, nephritis, hypersensitivity, myocardial ischemia, an ophthalmic disease, type I diabetes, a chronic lung disease, asthma, bronchitis, pulmonary inflammation, an allergic reaction, collagen disease, tendinitis, bursitis, rhinitis, post-operative inflammation, a vascular disease, a central nervous system disorder, cancer, menstral cramps, or pre-mature labor.

10. The process of claim 1 wherein the fatty acid compound is administered to prevent inflammation.

11. The process of claim 1 wherein the fatty acid compound is administered to treat inflammation.

12. The process of claim 1 wherein the fatty acid compound is admimstered to prevent cancer.

13. The process of claim 1 wherein the fatty acid compound is administered to treat cancer.

14. The process of claim 1 wherein the fatty acid compound is administered to prevent or treat breast cancer, prostate cancer, colorectal cancer, lung cancer, bladder cancer, cervic cancer, or skin cancer.

15. The process of claim 1 wherein the fatty acid compound is administered to prevent or treat Alzheimer's disease or central nervous system damage resulting from stroke, ischemia, or trauma.

16. The process of claim 1 wherein the fatty acid compound is administered to prevent or treat a tension headache, a migraine headache, postoperative pain, dental pain, muscular pain, back pain, neck pain, or pain resulting from cancer.

17. The process of claim 1 wherein the fatty acid compound is admimstered to prevent or treat rheumatoid arthritis, spondyloarthropathies, gouty arthritis, osteoarthritis, systemic lupus erythematosus, or juvenile arthritis.

18. The process of claim 1 wherein the fatty acid compound is administered to prevent or treat dermatitis, psoriasis, eczema, or a burn.

19. The process of claim 1 wherein the fatty acid compound is administered to prevent or treat inflammatory bowel disease, Crohn's disease, gastritis, irritable bowel syndrome, or ulcerative colitis.

20. The process of claim 1 wherein the fatty acid compound is administered to prevent or treat myasthenia gravis.

21. The process of claim 1 wherein the fatty acid compound is administered to prevent or treat multiple sclerosis.

22. The process of claim 1 wherein the fatty acid compound is admimstered to prevent or treat retinitis, retinopathies, uveitis, or ocular phtophobia.

23. The process of claim 1 wherein R¹ is a hydrocarbyl containing no greater than 6 carbon atoms.

24. The process of claim 1 wherein R¹ is methyl.

25. The process of claim 1 wherein R is ethyl

26. The process of claim 1 wherein R¹ is hydrogen.

27. The process of claim 1 wherein the fatty acid compound comprises a sodium, potassium, cesium, lithium, zinc, calcium, magnesium, copper, iron, silver, or ammonium ion.

28. The process of claim 1 wherein the fatty acid compound has formula (VII) or is a pharmaceutically acceptable salt thereof:

29. The process of claim 1 wherein the fatty acid compound has formula (VIII):

30. The process of claim 1 wherein the fatty acid compound has formula (IX):

31. The process of claim 1 wherein the fatty acid compound is selective toward inhibiting cyclooxygenase-2 in preference to inhibiting cyclooxygenase- 1.

32. The process of claim 1 wherein the fatty acid compound has a cyclooxygenase-2 IC₅₀ of less than about 0.5 μM, and a cyclooxygenase- 1 IC₅₀ of greater than about 1.5 μM.

33. The process of claim 1 wherein the fatty acid compound has a cyclooxygenase-2 IC₅₀ and a cyclooxygenase- 1 IC₅₀ such that the ratio of the cyclooxygenase- 1 IC₅₀ to the cyclooxygenase-2 IC₅₀ is at least about 3.5.

34. The process of claim 1 wherein the fatty acid compound reacts after administration to form a compound which is selective toward inhibiting cyclooxygenase-2 in preference to inhibiting cyclooxygenase- 1.

35. The process of claim 1 wherein the fatty acid compound reacts after administration to form a compound which has a cyclooxygenase-2 IC₅₀ of less than about 0.5 μM, and a cyclooxygenase- 1 IC₅₀ of greater than about 1.5 μM.

36. The process of claim 1 wherein the fatty acid compound reacts after administration to form a compound which has a cyclooxygenase-2 IC₅₀ and a cyclooxygenase- 1 IC₅₀ such that the ratio of the cyclooxygenase- 1 IC₅₀ to the cyclooxygenase-2 IC₅₀ is at least about 3.5.

37. The process of claim 1 wherein the fatty acid compound is admimstered orally.

38. The process of claim 1 wherein the fatty acid compound is admimstered through a subcutaneous injection, intramuscular injection, intrasternal injection, intravenous injection, or infusion.

39. The process of claim 1 wherein the fatty acid compound is administered topically.

40. The process of claim 1 wherein the fatty acid compound is administered via inhalation.

41. The process of claim 1 wherein the fatty acid compound is administered via nasal spray.

42. The process of claim 1 wherein the fatty acid compound is administered rectally.

43. The process of claim 1 wherein the amount of fatty acid compound administered per day equals from about 0.0001 to about 2 grams per kilogram of the organism's total weight.

44. The process of claim 1 wherein the amount of fatty acid compound administered per day equals from about 0.001 to about 2 grams per kilogram of the organism's total weight.

45. The process of claim 1 wherein the amount of fatty acid compound admimstered per day equals from about 0.001 to about 1 grams per kilogram of the organism's total weight.

46. The process of claim 1 wherein the amount of fatty acid compound administered per day equals from about 1 to about 10,000 ppm by weight of the organism's diet.

47. The process of claim 1 wherein the amount of fatty acid compound administered per day equals from about 100 to about 10,000 ppm by weight of the organism's diet.

48. The process of claim 1 wherein the amount of fatty acid compound administered per day equals from about 1,000 to about 10,000 ppm by weight of the organism's diet.

49. The process of claim 1 wherein the fatty acid compound is contained in a pharmaceutical composition which also comprises lipooxygenase.

50. The process of claim 1 wherein the process further comprises administering a second fatty acid compound having formula (VI) or a pharmaceutically acceptable salt thereof:

° H H H H H H H

R ⁶ O C— ( C ) _v C= c— C= C— C ( C ) _w C H ₃

H R ⁷ H (VI),

wherein R⁶ is hydrogen, hydrocarbyl, or substituted hydrocarbyl; R⁷ is -OH, -SO₃H, -SH, -NH₂, -PO₃H₂, or halogen; v is from 7 to 11; and v and w have a sum which is from 11 to 15.

51. The process of claim 50 wherein R⁷ is -OH.

52. The process of claim 50 wherein v is 7, w is 4, and R⁷ is -OH.

53. The process of claim 50 wherein the second fatty acid compound is a triglyceride.

54. A process for the prevention or treatment of a cyclooxygenase-2 mediated disorder in an organism having a cyclooxygenase-2 mediated disorder or disposed to having a cyclooxygenase-2 mediated disorder, the process comprising administering a fatty acid compound to the organism in an amount effective to prevent or treat the cyclooxygenase-2 mediated disorder by inhibiting cyclooxygenase-2, wherein the fatty acid compound has formula (VI) or is a pharmaceutically acceptable salt thereof:

° H H H H H H H

R ⁶ O C— ⁽ C ) „ C-= C— C= C— C ( C ) _w C H ₃

H R ⁷ H (VI);

R⁶ is hydrogen, hydrocarbyl, or substituted hydrocarbyl; R⁷ is -OH, -SO₃H, -SH, -NH₂, -PO₃H₂, or halogen; v is from 7 to 11 ; and v and w have a sum which is from 11 to 15.

55. The process of claim 54 wherein R⁷ is -OH.

56. The process of claim 54 wherein v is 7, w is 4, and R⁷ is -OH.

57. The process of claim 54 wherein the fatty acid compound is a triglyceride.

58. The process of claim 54 wherein the fatty acid compound is administered to treat a cyclooxygenase-2 mediated disorder in an organism having the cyclooxygenase-2 mediated disorder.

59. The process of claim 54 wherein the fatty acid compound is admimstered to prevent a cyclooxygenase-2 mediated disorder in an organism disposed to having the cyclooxygenase-2 mediated disorder.

60. The process of claim 54 wherein the fatty acid compound is administered to prevent or treat swelling, fever, skin redness, an ache, arthritis, a skin disease, an infection, a gastrointestinal condition, periarteritis nodosa, thyroiditis, aplastic anemia, Hodgkin's disease, sclerodoma, rheumatic fever, neuromuscular junction disease, white matter disease, sarcoidosis, nephrotic syndrome, Behcet's syndrome, polymyositis, gingivitis, nephritis, hypersensitivity, myocardial ischemia, an ophthalmic disease, type I diabetes, a chronic lung disease, asthma, bronchitis, pulmonary inflammation, an allergic reaction, collagen disease, tendinitis, bursitis, rhinitis, post-operative inflammation, a vascular disease, a central nervous system disorder, menstral cramps, or pre-mature labor.

61. The process of claim 54 wherein the fatty acid compound is administered to prevent inflammation.

62. The process of claim 54 wherein the fatty acid compound is administered to treat inflammation.

63. The process of claim 54 wherein the fatty acid compound is administered to prevent or treat a tension headache, a migraine headache, postoperative pain, dental pain, muscular pain, back pain, or neck pain.

64. The process of claim 54 wherein the fatty acid compound is administered to prevent or treat rheumatoid arthritis, spondyloarthropathies, gouty arthritis, osteoarthritis, systemic lupus erythematosus, or juvenile arthritis.

65. The process of claim 54 wherein the fatty acid compound is admimstered to prevent or treat dermatitis, psoriasis, eczema, or a burn.

66. The process of claim 54 wherein the fatty acid compound is admimstered to prevent or treat inflammatory bowel disease, Crohn's disease, gastritis, irritable bowel syndrome, or ulcerative colitis.

67. The process of claim 54 wherein the fatty acid compound is administered to prevent or treat myasthenia gravis.

68. The process of claim 54 wherein the fatty acid compound is administered to prevent or treat multiple sclerosis.

69. The process of claim 54 wherein the fatty acid compound is administered to prevent or treat retinitis, retinopathies, uveitis, or ocular phtophobia.

70. The process of claim 54 wherein the fatty acid compound is administered to prevent or treat colorectal cancer, lung cancer, bladder cancer, cervic cancer, or skin cancer.

71. The process of claim 54 wherein the fatty acid compound is administered to prevent or treat Alzheimer's disease or central nervous system damage resulting from stroke, ischemia, or trauma.

72. The process of claim 54 wherein R⁶ is a hydrocarbyl containing no greater than 6 carbon atoms.

73. The process of claim 54 wherein R⁶ is methyl.

74. The process of claim 54 wherein R⁶ is ethyl.

75. The process of claim 54 wherein R⁶ is hydrogen.

76. The process of claim 54 wherein the fatty acid compound comprises a sodium, potassium, cesium, lithium, zinc, calcium, magnesium, copper, iron, silver, or ammonium ion.

77. The process of claim 54 wherein the fatty acid compound has formula (X) or is a pharmaceutically acceptable salt thereof:

OH (X).

78. The process of claim 54 wherein the fatty acid compound is admimstered orally.

79. The process of claim 54 wherein the fatty acid compound is administered through a subcutaneous injection, intramuscular injection, infrastemal injection, intravenous injection, or infusion.

80. The process of claim 54 wherein the fatty acid compound is administered topically.

81. The process of claim 54 wherein the fatty acid compound is administered via inhalation.

82. The process of claim 54 wherein the fatty acid compound is administered rectally.

83. The process of claim 54 wherein the fatty acid compound is contained in a pharmaceutical composition which also comprises lipooxygenase.

84. The process of claim 54 wherein the fatty acid compound is selective toward inhibiting cyclooxygenase-2 in preference to inhibiting cyclooxygenase- 1.

85. The process of claim 54 wherein the fatty acid compound has a cyclooxygenase-2 IC₅₀ of less than about 100 μM, and a cyclooxygenase- 1 IC₅₀ of greater than about 100 μM.

86. The process of claim 54 wherein the fatty acid compound has a cyclooxygenase-2 IC₅₀ of less than about 60 μM, and a cyclooxygenase- 1 IC₅₀ of greater than about 100 μM.

87. The process of claim 54 wherein the fatty acid compound has a cyclooxygenase-2 IC₅₀ and a cyclooxygenase- 1 IC₅₀ such that the ratio of the cyclooxygenase- 1 IC₅₀ to the cyclooxygenase-2 IC₅₀ is at least about 1.5.

88. The process of claim 54 wherein the fatty acid compound reacts after administration to form a compound which is selective toward inhibiting cyclooxygenase-2 in preference to inhibiting cyclooxygenase- 1.

89. The process of claim 54 wherein the fatty acid compound reacts after being admimstered to form a compound which has a cyclooxygenase-2 IC₅₀ of less than about 100 μM, and a cyclooxygenase- 1 IC₅₀ of greater than about 100 μM.

90. The process of claim 54 wherein the fatty acid compound reacts after being admimstered to form a compound which has a cyclooxygenase-2 IC₅₀ of less than about 60 μM, and a cyclooxygenase- 1 IC₅₀ of greater than about 100 μM.

91. The process of claim 54 wherein the fatty acid compound reacts after being admimstered to form a compound which has a cyclooxygenase-2 IC₅₀ and a cyclooxygenase- 1 IC₅₀ such that the ratio of the cyclooxygenase- 1 IC₅₀ to the cyclooxygenase-2 IC₅₀ is at least about 1.5.

92. The process of claim 54 wherein the amount of fatty acid compound administered per day equals from about 0.0001 to about 2 grams per kilogram of the organism's total weight.

93. The process of claim 54 wherein the amount of fatty acid compound administered per day equals from about 0.001 to about 2 grams per kilogram of the organism's total weight.

94. The process of claim 54 wherein the amount of fatty acid compound admimstered per day equals from about 0.001 to about 1 grams per kilogram of the organism's total weight.

95. The process of claim 54 wherein the amount of fatty acid compound administered per day equals from about 1 to about 10,000 ppm by weight of the organism's diet.

96. The process of claim 54 wherein the amount of fatty acid compound administered per day equals from about 100 to about 10,000 ppm by weight of the organism's diet.

97. The process of claim 54 wherein the amount of fatty acid compound administered per day equals from about 1,000 to about 10,000 ppm by weight of the organism's diet.

98. A process for preparing a fatty acid compound, the process comprising combining a ylide with an aldehyde, wherein the fatty acid compound has formula (I):

H H H H H H H

R¹ 0 C— (C)_m C=C— C=C— (C)_n C H

(i); the ylide has formula (II):

R² H H H H O

R³ — P=C — (cr=c)_x — (C)_m c o — R¹

^{R4 H} (ii); the aldehyde has formula (LTI): O H H ^H

H C ( C=-= C ) _y ( C ) „ C H ₃

(HI);

R¹, R², R³, and R⁴ are independently hydrocarbyl or substituted hydrocarbyl; m is from 6 to 11 ; n and m have a sum which is from 13 to 16; x is 0 or 1 ; and x and y have a sum of 1.

99. The process of claim 98 wherein m is from 7 to 9, and m and n have a sum of 14.

100. The process of claim 98 wherein m is 9.

101. The process of claim 98 wherein x is 0.

102. The process of claim 98 wherein R², R³, and R⁴ are each -C₆H₅.

103. The process of claim 98 wherein R¹ is a hydrocarbyl group having no greater than 6 carbon atoms.

104. The process of claim 98 wherein R¹ is -CH₃.

105. The process of claim 98 wherein R¹ is -CH₂CH₃.

106. The process of claim 98 wherein the fatty acid compound has formula (VIII):

107. The process of claim 98 wherein the fatty acid compound has formula (IX):

108. The process of claim 98 wherein the ylide is formed by a process comprising combining a phosphine compound with a haloalkane to form a phosphonium salt, and reacting the phosphonium salt with a base, wherein the phosphonium salt has formula (XI):

the phosphine compound has formula (IV):

R " P

^R (IV),

the haloalkane has formula (V):

H H H H O

R ⁵ C ( C= C ) _X ( C ) _m C O R

(V), and and R⁵ is halogen.

109. The process of claim 108 wherein R⁵ is bromine.

110. The process of claim 108 wherein the base comprises butyllithium, lithium hexamethyldisilazane, sodium hydride, or an alkoxide.