Prostaglandin E synthase inhibitors and methods for utilizing the same

Compounds and compositions are provided that can inhibit microsomal prostaglandin E synthase-1 (mPGES-1). The compounds and compositions can reduce inflammation in a subject, such as inflammation caused by an inflammation disorder or symptoms thereof. Pharmaceutical compositions comprising the compound are also provided. Furthermore, methods are provided for reducing inflammation and/or inhibiting mPGES-1. The methods can comprise administering an effective amount of the composition to a subject.

TECHNICAL FIELD

The presently-disclosed subject matter relates to prostaglandin E synthase (PGES) inhibitors, and in particular, microsomal PGES-1 (mPGES-1) inhibitors. Embodiments of the presently-disclosed subject matter also relate to methods of utilizing mPGES-1 inhibitors to treat inflammatory disorders in a subject in need thereof.

BACKGROUND

Prostaglandin E2(PGE2) is one of the most important prostanoids with diverse biological activity.1The biosynthetic pathway of PGE2has been well characterized and involves three sequential enzymatic actions.2The first step in this pathway, involves the release of arachidonic acid (AA) from the membrane, by the action of phospholipase A2(PLA2).2This is followed by the conversion of AA to prostaglandin H2(PGH2) by the action of cyclooxygenase COX-1 or COX-2.2Finally, PGH2is converted to PGE2by the action of terminal prostaglandin E synthase (PGES) enzymes,3particularly microsomal PGES-1 (mPGES-1).4It has been known that mPGES-1 couples with COX-25-6and plays a key role in a number of disease conditions, including inflammation, arthritis, fever, pain, cancer, stroke, and bone disorders.7-13Human mPGES-1 has been recognized as a promising target of next-generation therapeutics for the above diseases.14

Currently available non-steroidal anti-inflammatory drugs (NSAIDs) inhibit either cyclooxygenase (COX)-1 or COX-2 or both.15These inhibitors have several deleterious side effects including ulcers, bleeding within the gastrointestinal tract, or increased risk of cardiovascular events.16The withdrawal of rofecoxib (Vioxx) due to side effects further highlights the need to develop improved, safer anti-inflammatory drugs.15The COX inhibitors prevent the production of all prostaglandins downstream of PGH2, which results in a lot of problems. For example, blocking the production of prostaglandin-I2(PGI2) has been reported to play a role in cardiovascular events.17Unlike COX inhibition, inhibition of terminal mPGES-1 will only block the production of PGE2without affecting the normal production of other prostaglandins including PGI2. Reported knock-out studies identified mPGES-1 as a central switch in pyresis.18The mPGES-1 knock-out studies also revealed a decrease in inflammatory response in a collagen-induced arthritis model.19In contrast to COX-2, mPGES-1-deficient mice were reported to be viable, fertile and have normal phenotype.19Ischemic stroke induced in mPGES-1 null mice was reported to show significant reduction in the infarct size and volume.10, 14Thus, mPGES-1 inhibitors are expected to retain the anti-inflammatory effect as COX inhibitors without the side effects of COX inhibitors.

Although mPGES-1 inhibitors are expected to be potentially valuable therapeutic agents, few inhibitors of mPGES-1 were identified in experimental screening efforts. The COX-2 inhibitor NS-398, 5-Lipoxygenase activating protein (FLAP) inhibitor MK-886, and the active metabolite of another NSAID sulindac, were found to inhibit mPGES-1 with an IC50of 20, 1.6, and 80 μM, respectively.20-21,22Leukotriene C4 was reported to inhibit mPGES-1 with micromolar IC50, probably by competing with glutathione (GSH).20In addition to small molecules,23several polyunsaturated fatty acids and stable analogs of PGE2were reported to inhibit mPGES-1.24Riendeau22recently reported a series of mPGES-1 inhibitors synthesized based on the scaffold of MK-886 (FLAP inhibitor). Unfortunately, all of these inhibitors are not sufficiently potent against mPGES-1 in the tested living cells.

Thus, there remains a need for novel compounds that more potently inhibit mPGES-1. There also remains a need for methods of treating inflammatory disorders that do not have the problems discussed above. However, known mPGES-1 inhibitors are not sufficiently potent, and known anti-inflammatory agents are associated with many adverse side effects, such as ulcers and gastrointestinal bleeding. Hence, novel compounds that more potently inhibit mPGES-1 activity and are thereby able to treat inflammatory disorders are highly desired.

SUMMARY

In some embodiments, the presently-disclosed subject matter includes a compound of the formula:

or pharmaceutically acceptable salts thereof; wherein R1is selected from the group consisting of H, halide, Me, OMe, OEt, NO2, OH, and, together with the ring to which it is attached, a bicyclic ring system; wherein R2is alkyl; wherein R3is selected from the group consisting of H and Me; and wherein X is selected from the group consisting of O or S. In one embodiment, R1is selected from the group consisting of: H, Cl, Br, I, Me, OMe, OEt, NO2, OH, and, taken together with the ring to which it is attached, a bicyclic ring system. In another embodiment, R2is selected from the group consisting of:

In a further embodiment, the compound includes the formula selected from the group consisting of:

In some embodiments, the presently-disclosed subject matter includes a compound of the formula:

or pharmaceutically acceptable salts thereof, wherein R1is selected from the group consisting of H, an alkyl, an alkyl halide, an ether, and a carboxylic acid; wherein R2is selected from the group consisting of H, a halide, an alkyne, and an aromatic; and wherein R3is selected from the group consisting of H, a carboxyl, a carboxylic acid, and an alkyl. In one embodiment, R1is selected from the group consisting of:
H,

In another embodiment, R2is selected from the group consisting of:
H,

In a further embodiment, R3is selected from the group consisting of:
H,

In some embodiments, the compound includes the formula selected from the group consisting of:

In some embodiments, the presently-disclosed subject matter includes a compound of the formula:

or pharmaceutically acceptable salts thereof, wherein R is selected from the group consisting of an alkyl and an alkoxy; and wherein n is from 1 to 6. In one embodiment, R is selected from the group consisting of:

In another embodiment, the compound has the formula selected from the group consisting of:

In some embodiments, the presently-disclosed subject matter includes a compound of the formula:

or pharmaceutically acceptable salts thereof, wherein R is a substituted phenyl; and wherein X is O. In one embodiment, the compound is of the formula:

In some embodiments, the presently-disclosed subject matter includes a compound of the formula:

or pharmaceutically acceptable salts thereof, wherein R is selected from the group consisting of an aliphatic side chain and an alkyl; wherein X is selected from the group consisting of H, NO2, Br, and OMe; and wherein R′ and R″ are independently selected from the group consisting of CN, COOH, COOEt, CONH2, and NO2. In one embodiment, the compound is of the formula:

In some embodiments, the presently-disclosed subject matter includes a compound of the formula:

or pharmaceutically acceptable salts thereof, wherein R1is an alkyl; wherein each R2is independently selected from the group consisting of H and an alkyl; wherein X is selected from the group consisting of H and a halogen; and wherein Y is selected from the group consisting of S and O. In one embodiment, the compound is of the formula:

In some embodiments, the presently-disclosed subject matter includes a compound of the formula:

or pharmaceutically acceptable salts thereof, wherein R is selected from the group consisting of an aliphatic side chain and an alkyl; wherein X is selected from the group consisting of H and Cl; wherein Y is CN; wherein Z is selected from the group consisting of CN, COOH, and, together with Y, a heterocyclic group of the formula:

wherein R1is selected from the group consisting of O and S; and wherein R2is selected from the group consisting of H and CH2COOH. In one embodiment, the compound is of the formula:

In some embodiments, the presently-disclosed subject matter includes a pharmaceutical composition comprising one of the compounds disclosed herein and a pharmaceutically-acceptable carrier. In one embodiment, the pharmaceutical composition further comprises a second compound or composition having mPGES-1 inhibition activity, having anti-inflammatory activity, being useful for treatment of an inflammation disorder, being useful for treatment of symptoms associated inflammation and/or an inflammation disorder, or combinations thereof.

In some embodiments, the presently-disclosed subject matter includes a kit comprising one of the compounds disclosed herein and a device useful for administration of the compound. In one embodiment, the kit further comprises a second compound or composition, or a treatment device having mPGES-1 inhibition activity, anti-inflammatory activity, being useful for treatment of an inflammation disorder, and/or being useful for treatment of symptoms associated inflammation and/or an inflammation disorder.

In some embodiments, the presently-disclosed subject matter includes a method of reducing inflammation in a subject, comprising administering to the subject an effective amount of one of the compound disclosed herein. In one embodiment, the subject includes an inflammation disorder or symptoms thereof. In another embodiment, the inflammation disorder is selected from the group consisting of inflammation, arthritis, fever, pain, cancer, stroke, bone disorders, and combinations thereof. In a further embodiment, the compound inhibits microsomal prostaglandin E synthase-1 (mPGES-1).

In some embodiments, the presently-disclosed subject matter includes a method of reducing inflammation in a subject, comprising administering to the subject an effective amount of a compound selected from the group consisting of:

In one embodiment, the subject includes an inflammation disorder or symptoms thereof. In another embodiment, the inflammation disorder is selected from the group consisting of inflammation, arthritis, fever, pain, cancer, stroke, bone disorders, and combinations thereof. In a further embodiment, the compound inhibits microsomal prostaglandin E synthase-1 (mPGES-1).

Further features and advantages of the presently-disclosed subject matter will become evident to those of ordinary skill in the art after a study of the description, figures, and non-limiting examples in this document.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The presently-disclosed subject matter meets some or all of the above-identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document. To avoid excessive repetition, this Description does not list or suggest all possible combinations of such features.

The presently-disclosed subject matter includes a compound having a structure represented by the formula I:

or pharmaceutically acceptable salts thereof, wherein R1includes H, halide, Me, OMe, OEt, NO2, OH, or taken together with the ring to which it is attached, a bicyclic ring system as in the formula:

R2includes an alkyl; R3includes H or Me; and X includes O or S.

In some embodiments of the compound having the structure of formula I, R1includes H, Cl, Br, I, Me, OMe, OEt, NO2, OH, or taken together with the ring to which it is attached, a bicyclic ring system.

In some embodiments of the compound having the structure of formula I, R2includes:

In some embodiments of the compound of formula I, the compound has the structure selected from the group consisting of:

In some embodiments of the compound of formula I, the compound has the structure of:

The presently-disclosed subject matter includes a compound having a structure represented by the formula:

or pharmaceutically acceptable salts thereof, wherein R1includes H, an alkyl, an alkyl halide, an ether, or a carboxylic acid; R2includes H, a halide, an alkyne, or an aromatic; and R3includes H, a carboxyl (e.g., CO2H), a carboxylic acid (e.g., CH2CO2H), or an alkyl.

In some embodiments of the compound having the structure of formula II, R1is selected from the group consisting of:

In some embodiments of the compound having the structure of formula II, R2is selected from the group consisting of:

In some embodiments of the compound having the structure of formula II, R3is selected from the group consisting of:

In some embodiments of the compound having the structure of formula II, the compound has the structure selected from the group consisting of:

The presently-disclosed subject matter includes a compound having a structure represented by the formula:

or pharmaceutically acceptable salts thereof, wherein R is an alkyl or alkoxy; and n is 1, 2, 3, 4, 5, or 6.

In some embodiments of the compound having the structure of formula III, R is selected from the group consisting of:

In some embodiments of the compound having the structure of formula III, the compound has the structure selected from the group consisting of:

The presently-disclosed subject matter includes a compound having a structure represented by the formula:

or pharmaceutically acceptable salts thereof, wherein R is selected from the group consisting of substituted phenyl; and X is O.

In some embodiments, the compound of formula IV has the structure of

The presently-disclosed subject matter includes a compound having a structure represented by the formula:

or pharmaceutically acceptable salts thereof.

In some embodiments of the compound having the structure of formula V, R includes an aliphatic side chain or an alkyl; X is selected from the group consisting of H, NO2, Br, or OMe; and R′ and R″ are independently selected from the group consisting of CN, COOH, COOEt, CONH2, and NO2.

In some embodiments of the compound having the structure of formula V, the compound is selected from the group consisting of:

The presently-disclosed subject matter includes a compound having a structure represented by the formula:

or pharmaceutically acceptable salts thereof.

In some embodiments of the compound having the structure of formula VI, X includes H or a halogen such as Cl; R1includes an alkyl; each R2independently includes H or an alkyl; and Y includes S or O.

In some embodiments of the compound having the structure of formula VI, the compound has the structure selected from the group consisting of:

The presently-disclosed subject matter further includes a compound having a structure represented by the formula:

or pharmaceutically acceptable salts thereof.

In some embodiments of the compound having the structure of formula VII, X includes H or Cl; Y includes CN; Z includes CN or COOH; and R includes an aliphatic side chain or an alkyl. In some embodiments of the compound having the structure of formula VII, Y and Z together form a heterocyclic group, such as, but not limited to, a five membered heterocyclic group. In one embodiment, for example, Y and Z together form a thiazolidine group having the structure:

wherein R1includes O or S; and R2includes H or CH2COOH.

In some embodiments of the compound having the structure of formula VII, the compound has the structure selected from the group consisting of:

The term “alkyl” refers to alkyl groups with the general formula CnH2n+1, where n=about 1 to about 18 or more. The groups can be straight-chained or branched. Alkyl, when used herein, also comprise “lower alkyls,” which refer to alkyl groups with the general formula CnH2n+1, where n=1 to about 6. In some embodiments, n=1 to about 3. Examples include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, t-butyl, isobutyl, n-pentyl, isopentyl, neopentyl, n-hexyl, and the like. The alkyl group can be substituted or unsubstituted. For example, the alkyl group can be substituted with one or more groups including, but not limited to, optionally substituted alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein.

In this regard, the term alkyl is inclusive of “cycloalkyl,” which refers to a non-aromatic carbon-based rings composed of at least three carbon atoms, such as cyclopropyl, cyclohexyl, and the like. Like other alkyls, cycloalkyls can be substituted or unsubstituted. The substituted moieties can be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as an “alkylcycloalkyl.” Again, the practice of using a general term, such as “cycloalkyl,” and a specific term, such as “alkylcycloalkyl,” is not meant to imply that the general term does not also include the specific term.

The term “fluorocarbon” refers to compounds that comprise carbon and fluoride bonded together. Fluorocarbons can comprise any type of bond and may be fluoroalkyl, fluoroalkene, or the like. Examples of fluorocarbons include CF4, C2F6, C2F4, and the like.

The term “aryl,” refers to an aromatic group containing ring carbon atoms and having about 5 to about 14 ring carbon atoms and up to a total of about 18 ring or pendant carbon atoms. Examples include, but are not limited to, phenyl, biphenyl, naphthalene, α-naphthyl, β-naphthyl, tolyl, xylyl, benzene, phenoxybenzene, and the like. The term “aryl” also includes “heteroaryl,” which is defined as a group that contains an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Likewise, the term “non-heteroaryl,” which is also included in the term “aryl,” defines a group that contains an aromatic group that does not contain a heteroatom. The aryl group can be substituted or unsubstituted. The term “biaryl” is a specific type of aryl group and is included in the definition of “aryl.” Biaryl refers to two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.

As described above, each of the groups mentioned herein, including the groups defined above, could be substituted or unsubstituted. For example, “alkyl” can include substituted alkyl, substituted with hydroxyl, heteroatoms, or lower alkyl groups. As a further example, “aryl” can include substituted aryl, substituted with alkyl, cycloalkyl, amino, nitro, thiol, or the like.

Suitable formulations include aqueous and non-aqueous sterile injection solutions that can contain antioxidants, buffers, bacteriostats, bactericidal antibiotics and solutes that render the formulation isotonic with the bodily fluids of the intended recipient; and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents.

The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The formulations can be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and can be stored in a frozen or freeze-dried (lyophilized) condition requiring only the addition of sterile liquid carrier immediately prior to use.

For oral administration, the compositions can take the form of, for example, tablets or capsules prepared by a conventional technique with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycollate); or wetting agents (e.g., sodium lauryl sulphate). The tablets can be coated by methods known in the art.

Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional techniques with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g. lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration can be suitably formulated to give controlled release of the active compound. For buccal administration the compositions can take the form of tablets or lozenges formulated in conventional manner.

The compounds can also be formulated as a preparation for implantation or injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).

The compounds can also be formulated in rectal compositions (e.g., suppositories or retention enemas containing conventional suppository bases such as cocoa butter or other glycerides), creams or lotions, or transdermal patches.

The compounds disclosed herein have utility as PGES inhibitors, and in particular, mPGES-1. In this regard, the compounds and pharmaceutical compositions of the presently disclosed subject matter have anti-inflammatory utilities. In this regard, in some embodiments, the pharmaceutical compositions of the presently-disclosed subject matter further include a second compound having PGES inhibition activity, having anti-inflammatory activity, being useful for treatment of an inflammation disorder, and/or being useful for treatment of symptoms associated inflammation and/or an inflammation disorder.

The presently-disclosed subject matter further includes kits. In some embodiments, a kit can include a compound or pharmaceutical composition as described herein, packaged together with a second compound, composition, or treatment device having PGES inhibition activity, having anti-inflammatory activity, being useful for treatment of an inflammation disorder, and/or being useful for treatment of symptoms associated inflammation and/or an inflammation disorder. By way of providing non-limiting examples of treatment devices that could be included in a kit of the presently-disclosed subject matter, inflammation can be treated in some cases with application of a device that changes temperature at a site of interest, e.g., a cooling pack or a heating pack.

In some embodiments, a kit can include a compound or pharmaceutical composition as described herein, packaged together with a device useful for administration of the compound or composition. As will be recognized by those or ordinary skill in the art, the appropriate administration aiding device will depend on the formulation of the compound or composition that is selected and/or the desired administration site. For example, if the formulation of the compound or composition is appropriate for injection in a subject, the device could be a syringe. For another example, if the desired administration site is cell culture media, the device could be a sterile pipette.

The presently-disclosed subject matter further includes methods of inhibiting mPGES. In some embodiments, the method can include contacting any of the compounds or compositions described herein with mPGES-1, thereby forming a complex with the compound and mPGES-1. In some embodiments, the method can include administering an effective amount of a compound or pharmaceutical composition, as described herein, including but not limited to the compounds set forth herein, and compositions thereof.

As will be recognized by one of ordinary skill in the art, the term “inhibiting” or “inhibition” does not refer to the ability to completely inactivate all target biological activity in all cases. Rather, the skilled artisan will understand that the term “inhibiting” refers to decreasing biological activity of a target, such as a prostaglandin E synthase, such as can occur with a ligand binding site of the target is blocked, or when a non-native complex with the target is formed. Such decrease in biological activity can be determined relative to a control, wherein an inhibitor is not administered and/or placed in contact with the target. For example, in some embodiments, a decrease in activity relative to a control can be about a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% decrease. The term “inhibitor” refers to a compound of composition that inactivates or decreases the biological activity of a target, such as a prostaglandin E synthase.

Without being bound by theory or mechanism, in some embodiments the compounds disclosed herein inhibit mPGES-1 by blocking its interaction with the PGH2, COX-2, or other substrates. Thus, the presently-disclosed subject matter also includes methods that find utility from the blocking mPGES-1 interaction with PGH2, COX-2, or other substrates. In this regard, the presently-disclosed subject matter includes methods of reducing and/or inhibiting inflammation, and methods of treating an inflammation disorder, and/or symptoms associated inflammation and/or an inflammation disorder. Such methods can include administering an effective amount of a compound of pharmaceutical composition as described herein to a subject. Non-limiting examples of inflammation disorders include inflammation, arthritis, fever, pain, cancer, stroke, and bone disorders

In some embodiments of a method of treating an inflammation disorder or symptoms thereof in a subject in need thereof, the method includes administering to the subject an effective amount of a compound, including any of the compounds described above. In some embodiments the compound inhibits prostaglandin E synthase (PGES), and in particular, some embodiments inhibit microsomal PGES-1 (mPGES-1). Thus, some embodiments include a method for inhibiting mPGES-1, comprising administering to a subject an effect amount of a compound, including any of the compounds described above.

In some embodiments, compounds disclosed herein that are mPGES-1 inhibitors are potent against both human and mouse mPGES-1 enzymes.

The presently-disclosed subject matter further includes methods for selecting and synthesizing embodiments of the present invention can utilize structure-based virtual screening to identify small-molecule inhibitors from a large drug-like database. In some embodiments a large database of lead compounds can be virtually screened to retrieve putative mPGES-1 inhibitors. From that screening, essential amino acids involved in antagonist recognition can be identified and a primary topographical interaction model can be made to guide subsequent virtual screening processes. Without being bound by theory or mechanism, an inhibitor's binding pocket of the mPGES-1 protein can overlap with both the binding site of the PGH2substrate and GSH cofactor in mPGES-1 protein.

EXAMPLES

Example 1 Synthetic Protocol Compounds of Formula I

The synthesis of BAR series (compounds of Formula I) can be generally described by the schemes illustrated inFIG. 1andFIG. 2of this Example. (for BAR042-044).

The substituted hydroxybenzaldehyde or hydroxy naphthaldehyde was treated with alcohol tosylate or alkyl bromide in the presence of potassium carbonate as acid capturer.25,26The forming aldehyde intermediate was usually pure enough after aqueous work-up and removal of solvents which could be used for the subsequent step without further purification. However, analytical samples could be obtained by flash chromatography using a mixture of hexanes and ethyl acetate as eluent. The final product, substituted benzylidenebarbituric acid derivatives were obtained by the condensation of the aldehyde intermediate and barbituric acid (or 1,3-dimethylbarbituric acid, 2-thiobarbituric acid) in reflux ethanol/water (4:1, v/v).27,28The precipitate formed was washed with hot water and ethanol, and dried under vacuum to form the analytical pure sample.

I-42˜I-44 were synthesized by the reduction of benzylidene double bond of I-02, I-03, and I-08, using sodium borohydride in methanol as reducing agent solution.29

Example 2 Synthetic Protocol of the Compounds of Formula II

Commercially available isatin (or 5-iodoisatin) and 2,4-thiazolidinedione were used as starting materials to construct the building blocks of substituted isatin and 2,4-thiazolidinedione N-acetic acid, respectively. After treatment with potassium hydroxide in hot ethanol, the potassium salt of 2,4-thiazolidinedione was precipitated out for the N-substitution by tert-butyl bromoacetate. The removal of tert-butyl ester in TFA/DCM (1:1, v/v) at room temperature led to the formation of important building block 2,4-thiazolidinedione N-acetic acid.32N-substituted isation was prepared by potassium carbonate promoted reaction between isatin and alkyl bromide (or alcohol tosylate if the bromide was not commercially available).33While for 1,5-disubstituted isatin, N-substitution on 5-iodoisatin by 4-chlorobenzyl bromide in the presence of potassium carbonate was followed by the Suzuki cross-coupling reaction with aryl boronic acid (ArB(OH)2) using [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) complex with dichloromethane (0) as catalyst and sodium bicarbonate as base activator in refluxing dimethoxyethane (DME) and distilled water (DME/H2O 4:1) under the protection of nitrogen gas.34,35The final product was obtained as red to brown powders by the Knovenagal-type condensation of the isatin-based building block with 2,4-thiazolidinedione N-acetic acid in the presence of ammonium acetate in refluxing glacial acetic acid, as described inFIG. 3.

Some of the compounds in this series were designed by switching the positions of hydrophilic and hydrophobic groups, as Cy4TZISA, whose acetic acid group occupied N-position of isatin and aliphatic group linked to 2,4-thiazolidinedione moiety. These compounds were readily synthesized following similar protocol as described previously, as shown inFIG. 4.

Example 3 Synthetic Protocol of Compounds of Formulae III and IV (Maleimide Derivatives and Substituted Dinitrobenzene Derivatives)

Maleimide derivatives were synthesized via the condensation of 4-maleimidobutyric acid or 6-maleimidohexanoic acid36with aryl amines,37as shown inFIG. 5.

In the synthesis of dinitrobenzene derivatized potent inhibitors, the oxygen on phenyl derivative nucleophilically substituted one of the fluorine atom on 1,2-difluoro-4,5-dinitrobenzene using potassium carbonate as acid capturer, as shown inFIG. 6.38

Example 4 Synthetic Protocol of Compounds of Formula V

The compounds of Formula V were prepared following a two-step protocol.39-42O-Substitution of substituted 4-hydroxybenzaldehyde afforded the aldehyde intermediate and the latter was coupled with malononitrile, 2-cyanoacetic acid or 2-cyanoacetamide. An example of the synthesis of V-04 is illustrated inFIG. 7.

Example 5 Synthetic Protocol of Compounds of Formulae VI and VII

The compounds of Formula VI were synthesized according to a multi-step protocol. 4-Alkyloxyacetophenone, obtained from the reaction of 4-hydroxyacetophenone and alkyl bromide, or acetophenone was condensed with 4-chlorophenylhydrazine in reflux ethanol containing 5% glacial acetic acid. The ethylidene hydrazine was formed as precipitate at room temperature and filtered off. The next step was Vilsmeier-Haack-Arnold ring closing formylation, by treating with POCl3/DMF. The produced 1H-pyrazole-4-carbaldehyde intermediate was coupled with barbituric acid or 2-thiobarbituric acid in refluxing EtOH/H2O (4:1) to afford the final product. The synthetic protocol for compounds with Formula VII followed similar strategy as those with formula VI, except the final step which was the coupling with 2,4-thiazolidinedione derivatives. An example of the synthesis of VI-01 is depicted inFIG. 8.

Example 6 Characterization of Inhibition In Vitro

Studies were conducted to characterize the inhibitory activity against recombinant mPGES-1 of the compounds synthesized in accordance with Examples 1-4, and disclosed herein.

Briefly, FreeStyle 293-F cells were cultured following manufacturer's manual in FreeStyle 293 expression medium on orbit rotate shaker in 8% CO2incubator at 37° C. Cells were transfected with 1.5 μg/mL of mPGES-1/pcDNA3 construct using FreeStyle Max reagent at a cell density of 1×106for 2 days. Transfected cells were collected, washed, and sonicated in TSES buffer (15 mM Tris-HCl, pH 8.0 plus 0.25 M sucrose, 0.1 mM EDTA and 1 mM DTT) on ice. The broken cells were first centrifuged at 12,500×g for 10 min. The supernatant was further centrifuged at 105,000×g for 1 hr at 4° C. The pellet was washed and homogenized in PBS buffer. The crude microsomal mPGES-1 preparation was aliquoted and stored at −80° C. The crude protein concentration was 8 mg/mL.

The enzyme activity assays were performed on ice in 1.5 ml microfuge tubes by using the expressed mPGES-1. The reaction mixture contained: 0.2 M Na2HPO4/NaH2PO4, pH 7.2, 10 μL; 0.1 M GSH, 2.5 μL; diluted microsomal enzyme (80 μg/mL), 1 μL; PGH2(0.31 mM in DMF), 5 μL; 1 μL, inhibitor; and H2O in a final reaction volume of 100 μL. PGH2was stored in dry ice and used to initiate the reaction.

Compounds were incubated with the enzyme for 15 min at room temperature before the addition of cold PGH2(1 μM final) to initiate the enzyme reaction. After 30 s, 10 μL of SnCl2(40 mg/mL) in ethanol was added to stop the reaction. The nonenzymatic conversion of PGH2to PGE2was performed in the same buffer devoid of enzyme. The reaction mixture was placed on ice until PGE2production was determined by the PGE2enzyme immunoassay as described earlier. IC50values of the inhibitors were calculated by using the GraphPad Prism 4.0 program. The results are set forth in the tables provided in Examples 7-10.

Example 7 Characterization of Inhibition of Compounds of Formula I

Example 8 Characterization of Inhibition of Compounds of Formula II

Example 9 Characterization of Inhibition of Compounds of Formulae III and IV

TABLE 3IC50aagainst mPGES-1 (nM)CompoundStructureHuman enzymeMouse enzymeIII-011920 ± 300n.d. (48)III-02613 ± 1032880 ± 496III-03n.d.b(63)cn.d. (55)IV-01_296 ± 48n.d. (91)aData are expressed as means ± SD of single determinations obtained in triplicate.bn.d. = not detected.cThe % inhibition of the compound at a concentration of 10 μM against mPGES-1 (IC50values were determined if the compounds resulted in 70 % or higher inhibition).

Example 10 Characterization of Inhibition of Compounds of Formula V

Example 11 Characterization of Inhibition of Compounds of Formulae VI and VII

While the terms used herein are believed to be well understood by one of ordinary skill in the art, the definitions set forth herein are provided to facilitate explanation of the presently-disclosed subject matter.

Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “an inhibitor” includes a plurality of such inhibitors, and so forth.

Throughout this document, various references are mentioned. All such references, including those listed below, are incorporated herein by reference.

REFERENCES