Disclosed are compounds for treating or preventing a disease or disorder responsive to antagonism of a P2Y14R receptor agonist in a mammal in need thereof, for example, compounds of formulas (I) and (II), wherein R1-R8, X, Y, Z, X′, Y′, Z′, and A are as defined herein, that are useful in treating an inflammatory such as asthma, cystic fibrosis, and sterile inflammation of the kidney.

BACKGROUND OF THE INVENTION

Extracellular nucleotides released by tissue and organs during stress or injury activate a class of cell-surface receptors (P2Rs) to boost the innate and adaptive immune responses (1-3). This mechanism acts as a time-dependent component of the signaling purinome, along with the anti-inflammatory adenosine receptors (ARs, also termed P1 receptors), to protect the organism in various challenged circumstances. The P2Y14receptor (P2Y14R) responds to endogenous agonists uridine-5′-diphosphoglucose and uridine-5′-diphosphate to mediate inflammatory activity, in part by activating neutrophil motility (4-6). Structurally, the P2Y14R belongs to the δ-branch of rhodoposin-like G protein-coupled receptors (GPCRs). Three subtypes of the P2YRs are preferentially coupled to inhibition of adenylate cyclase through guanine nucleotide inhibitory (Gi) protein: P2Y12R, P2Y13R and P2Y14R. Selective P2Y14R antagonists are sought as potential agents for treating asthma, sterile inflammation of the kidney, diabetes and neurodegeneration (7-12). However, only a few antagonists are known, so there is a clear need for more competitive P2Y14R antagonists. Other subtypes of the P2YR family in general, e.g., P2Y2R and P2Y6R, are also associated with proinflammatory effects, and their antagonists are desired for providing anti-inflammatory activity (13, 14).

Antagonists of the P2Y14R were first reported by Black and colleagues (58), and of the two classes of antagonists reported, naphthoic acids and pyrido[4,3-d]pyrimidines, only the former appeared to be competitive antagonists. Thus, there is an unmet need for diverse competitive P2Y14R antagonists.

BRIEF SUMMARY OF THE INVENTION

The invention provides a compound of formula (I) or formula (II).

R3is selected from the group consisting of NHR4, COOR5, (C≡C)n(CH2)mR6, CONH(CH2)qNH2,

m, o, and q are independently integers of 1 to about 10, and

n is zero or an integer of 1 to about 10,

or a pharmaceutically acceptable salt thereof, as well as stereoisomers thereof.

The present invention further provides a compound of formula (III)

wherein R1is CF3, R2is COOH, and R3is a 4-8 membered heterocyclic ring having at least one nitrogen atom.

The present invention further provides a compound of formula (IV) or (V):

The invention also provides a method of antagonizing a P2Y14R receptor in a mammal in need thereof, comprising administering to the mammal an effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof.

The invention further provides a method of treating or preventing an inflammatory condition in a mammal in need thereof, comprising administering to the mammal an effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof.

The invention additionally provides a method of treating pain in a mammal in need thereof, comprising administering to the mammal an effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof.

DETAILED DESCRIPTION OF THE INVENTION

In an aspect, the invention provides a compound of formula (I) or formula (II):

wherein X, Y, Z are CH or N,

R3is selected from the group consisting of NHR4, COOR5, (C≡C)n(CH2)mR6, CONH(CH2)qNH2, COCF3,

m, o, and q are independently integers of 1 to about 10, and

n is zero or an integer of 1 to about 10,

or a pharmaceutically acceptable salt thereof, as well as stereoisomers thereof.

In certain aspects, the compound is of formula (I).

In certain aspects of the compound of formula (I), R3is selected from NH2, NHCOCH3, NHCOC6H5, NHCOC(CH3)3,

In certain of these aspects, R3is selected from CONH(CH2)3NH2, CH2CONH2, (CH2)3NH2, and C≡CCH2NH2.

In certain aspects, the compound is of formula (II).

In certain aspects of formula (II), R3is selected from Br, (CH2)2—CN,

In certain particular aspects, R3is

In a particular aspect, the compound is:

In certain aspects, R3is selected from

In certain particular aspects, R3is

and wherein the configuration of the three chiral centers is (S,S,S).

In certain aspects, R3is selected from

In a particular aspect, the compound is:

wherein the configuration about the three chiral centers is (S,S,S).

The present invention further provides a compound of formula (III)

wherein R1is CF3, R2is COOH, and R3is a 4-8 membered heterocyclic ring having at least one nitrogen atom, wherein the heterocyclic ring comprises 1, 2, or 3 rings, fused or linked at one or more atoms, and wherein the heterocyclic ring is saturated or unsaturated, and wherein the heterocyclic ring is optionally substituted with one or more of substituents selected from the group consisting of alkyl, alkoxy, and hydroxy. Examples of compounds of formula (III) those wherein R3is:

The present invention further provides a compound of formula (IV) or (V):

Referring now to terminology used generically herein, the term “alkyl” means a straight-chain or branched alkyl substituent containing from, for example, 1 to about 6 carbon atoms, preferably from 1 to about 4 carbon atoms, more preferably from 1 to 2 carbon atoms. Examples of such substituents include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isoamyl, hexyl, and the like.

The term “cycloalkyl,” as used herein, means a cyclic alkyl substituent containing from, for example, about 3 to about 8 carbon atoms, preferably from about 4 to about 7 carbon atoms, and more preferably from about 4 to about 6 carbon atoms. Examples of such substituents include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. The cyclic alkyl groups may be unsubstituted or further substituted with alkyl groups such as methyl groups, ethyl groups, and the like.

The term “alkylcarbonyl,” as used herein, refers to an alkyl group linked to a carbonyl group and further linked to a molecule via the carbonyl group, e.g., alkyl-C(═O)—. The term “alkoxycarbonyl,” as used herein, refers to an alkoxy group linked to a carbonyl group and further linked to a molecule via the carbonyl group, e.g., alkyl-O—C(═O)—.

The term “halo” or “halogen,” as used herein, means a substituent selected from Group VIIA, such as, for example, fluorine, bromine, chlorine, and iodine.

The term “tetrazolyl”, as used herein, refers to a group of the formula:

In any of the above aspects, the compound or salt of formula (I) or formula (II) can have at least one asymmetric carbon atom. When the compound or salt has at least one asymmetric carbon atom, the compound or salt can exist in the racemic form, in the form of its pure optical isomers, or in the form of a mixture wherein one isomer is enriched relative to the other. In particular, in accordance with the present invention, when the inventive compounds have a single asymmetric carbon atom, the inventive compounds may exist as racemates, i.e., as mixtures of equal amounts of optical isomers, i.e., equal amounts of two enantiomers, or in the form of a single enantiomer. As used herein, “single enantiomer” is intended to include a compound that comprises more than 50% of a single enantiomer (i.e., enantiomeric excess more than 60%, more than 70%, more than 80%, more than 90%, or up to 100% pure enantiomer).

When the compound or salt has more than one chiral center, the compound or salt can therefore exist as a mixture of diastereomers or in the form of a single diastereomer. As used herein, “single diastereomer” is intended to mean a compound that comprises more than 50% of a single diastereomer (i.e., diastereomeric excess more than 60%, more than 70%, more than 80%, more than 90%, or up to 100% pure diastereomer).

The phrase “pharmaceutically acceptable salt” is intended to include nontoxic salts synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. Generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found inRemington's Pharmaceutical Sciences,18th ed., Mack Publishing Company, Easton, P A, 1990, p. 1445, and Berge, S. M., at al.,Journal of Pharmaceutical Science,66, 1-19 (1977).

Suitable bases include inorganic bases such as alkali and alkaline earth metal bases, e.g., those containing metallic cations such as sodium, potassium, magnesium, calcium and the like. Non-limiting examples of suitable bases include sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate. Suitable acids include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic, methanesulfonic acid, benzenesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, maleic acid, tartaric acid, fatty acids, long chain fatty acids, and the like. Preferred pharmaceutically acceptable salts of inventive compounds having an acidic moiety include sodium and potassium salts. Preferred pharmaceutically acceptable salts of inventive compounds having a basic moiety (e.g., a dimethylaminoalkyl group) include hydrochloride and hydrobromide salts. The compounds of the present invention containing an acidic or basic moiety are useful in the form of the free base or acid or in the form of a pharmaceutically acceptable salt thereof.

It should be recognized that the particular counterion forming a part of any salt of this invention is usually not of a critical nature, so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole.

It is further understood that the above compounds and salts may form solvates, or exist in a substantially uncomplexed form, such as the anhydrous form. As used herein, the term “solvate” refers to a molecular complex wherein the solvent molecule, such as the crystallizing solvent, is incorporated into the crystal lattice. When the solvent incorporated in the solvate is water, the molecular complex is called a hydrate. Pharmaceutically acceptable solvates include hydrates, alcoholates such as methanolates and ethanolates, acetonitrilates and the like. These compounds can also exist in polymorphic forms.

The present invention further provides a pharmaceutical composition comprising a compound as described above and a pharmaceutically acceptable carrier. The present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount, e.g., a therapeutically effective amount, including a prophylactically effective amount, of one or more of the aforesaid compounds, or salts thereof, of the present invention.

The pharmaceutically acceptable carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the compound, and by the route of administration. It will be appreciated by one of skill in the art that, in addition to the following described pharmaceutical compositions; the compounds of the present invention can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.

The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, or diluents, are well known to those who are skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active compounds and one which has no detrimental side effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particular active agent, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention. The following formulations for oral, aerosol, parenteral, subcutaneous, intravenous, intraarterial, intramuscular, interperitoneal, intrathecal, rectal, and vaginal administration are merely exemplary and are in no way limiting.

The compounds of the present invention, alone or in combination with other suitable components, can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer.

Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The compound can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, glycerol ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

Oils, which can be used in parenteral formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters. Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene-polypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-beta-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (3) mixtures thereof.

The compounds of the present invention may be made into injectable formulations. The requirements for effective pharmaceutical carriers for injectable compositions are well known to those of ordinary skill in the art. See Pharmaceutics and Pharmacy Practice, J. B. Lippincott Co., Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986).

Topical formulations, including those that are useful for transdermal drug release, are well-known to those of skill in the art and are suitable in the context of the invention for application to skin. Topically applied compositions are generally in the form of liquids, creams, pastes, lotions and gels. Topical administration includes application to the oral mucosa, which includes the oral cavity, oral epithelium, palate, gingival, and the nasal mucosa. In some aspects, the composition contains at least one active component and a suitable vehicle or carrier. It may also contain other components, such as an anti-irritant. The carrier can be a liquid, solid or semi-solid. In aspects, the composition is an aqueous solution. Alternatively, the composition can be a dispersion, emulsion, gel, lotion or cream vehicle for the various components. In one aspect, the primary vehicle is water or a biocompatible solvent that is substantially neutral or that has been rendered substantially neutral. The liquid vehicle can include other materials, such as buffers, alcohols, glycerin, and mineral oils with various emulsifiers or dispersing agents as known in the art to obtain the desired pH, consistency and viscosity. It is possible that the compositions can be produced as solids, such as powders or granules. The solids can be applied directly or dissolved in water or a biocompatible solvent prior to use to form a solution that is substantially neutral or that has been rendered substantially neutral and that can then be applied to the target site. In aspects of the invention, the vehicle for topical application to the skin can include water, buffered solutions, various alcohols, glycols such as glycerin, lipid materials such as fatty acids, mineral oils, phosphoglycerides, collagen, gelatin and silicone based materials.

Additionally, the compounds of the present invention may be made into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.

The dose administered to a mammal, particularly, a human, in accordance with the present invention should be sufficient to effect the desired response. Such responses include reversal or prevention of the adverse effects of the disease for which treatment is desired or to elicit the desired benefit. One skilled in the art will recognize that dosage will depend upon a variety of factors, including the age, condition, and body weight of the human, as well as the source, particular type of the disease, and extent of the disease in the human. The size of the dose will also be determined by the route, timing and frequency of administration as well as the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular compound and the desired physiological effect. It will be appreciated by one of skill in the art that various conditions or disease states may require prolonged treatment involving multiple administrations.

Suitable doses and dosage regimens can be determined by conventional range-finding techniques known to those of ordinary skill in the art. Generally, treatment is initiated with smaller dosages that are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. The present inventive method typically will involve the administration of about 0.1 to about 300 mg of one or more of the compounds described above per kg body weight of the animal or mammal.

The therapeutically effective amount of the compound or compounds administered can vary depending upon the desired effects and the factors noted above. Typically, dosages will be between 0.01 mg/kg and 250 mg/kg of the subject's body weight, and more typically between about 0.05 mg/kg and 100 mg/kg, such as from about 0.2 to about 80 mg/kg, from about 5 to about 40 mg/kg or from about 10 to about 30 mg/kg of the subject's body weight. Thus, unit dosage forms can be formulated based upon the suitable ranges recited above and the subject's body weight. The term “unit dosage form” as used herein refers to a physically discrete unit of therapeutic agent appropriate for the subject to be treated.

Alternatively, dosages are calculated based on body surface area and from about 1 mg/m2to about 200 mg/m2, such as from about 5 mg/m2to about 100 mg/m2will be administered to the subject per day. In particular aspects, administration of the therapeutically effective amount of the compound or compounds involves administering to the subject from about 5 mg/m2to about 50 mg/m2, such as from about 10 mg/m2to about 40 mg/m2per day. It is currently believed that a single dosage of the compound or compounds is suitable, however a therapeutically effective dosage can be supplied over an extended period of time or in multiple doses per day. Thus, unit dosage forms also can be calculated using a subject's body surface area based on the suitable ranges recited above and the desired dosing schedule.

In certain aspects, the invention provides a method of antagonizing a P2Y14R receptor in a mammal in need thereof, comprising administering to the mammal an effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof.

In certain aspects, the invention provides a method of treating or preventing an inflammatory condition in a mammal in need thereof, comprising administering to the mammal an effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof.

In these aspects, the inflammatory condition is selected from the group consisting of asthma, cystic fibrosis, and sterile inflammation of the kidney.

In certain aspects, the invention provides a method of treating pain in a mammal in need thereof, comprising administering to the mammal an effective amount a compound of the invention or a pharmaceutically acceptable salt thereof.

In certain aspects, the invention provides a compound of the invention or a pharmaceutically acceptable salt thereof, for use in antagonizing a P2Y14R receptor in a mammal in need thereof.

In certain aspects, the invention provides a compound of the invention or a pharmaceutically acceptable salt thereof, for use in treating or preventing an inflammatory condition in a mammal in need thereof.

In these aspects, the inflammatory condition is selected from the group consisting of asthma, cystic fibrosis, and sterile inflammation of the kidney.

In certain aspects, the invention provides a compound of the invention or a pharmaceutically acceptable salt thereof, for use in treating pain in a mammal in need thereof.

Chemistry

Schemes 1-14 depict exemplary syntheses of compounds of the invention.

Schemes 1A and 1B show synthesis of triazole-containing derivatives of the compound of formula (I) with acyl or acylamino substituents for R3.

Scheme 2 shows synthesis of triazole-containing derivatives of the compound of formula (I) with acyl or acylamino substituents for R3.

Scheme 3 shows synthesis of triazole-containing derivatives of the compound of formula (I) with cyclic groups for R3.

Scheme 4 shows a synthesis of naphthalene-containing isoxazole derivative 32.

Scheme 5 shows a synthesis of amide-containing isoxazole derivative 33.

Scheme 6 shows a synthesis of triazole-containing derivatives 34-36.

Scheme 7 shows a synthesis of compounds 37a-37c.

Scheme 8 shows the preparation of 4-bromophenyl intermediates containing modified piperidine rings for Suzuki coupling.

Scheme 9 shows a general scheme for preparation of derivatives of the lead P2Y14R antagonist, piperidine 1, via Suzuki coupling followed by ester hydrolysis. Compound 63 is 3-(4-bromophenyl)pyrrolidine hydrochloride, starting material for 75.

Scheme 10 shows a preparation of bridged piperidine analogues 114 and 116 containing a 2-azanorbornane moiety, 119 and 121 containing a lactam; and ring-opened 134 and 136 containing 1-amino-3-hydroxymethylcyclopentane, and 138 and 140 containing 1-amino-3-carboxylcyclopentane. The synthesis maintained absolute stereochemistry originating from chiral bicyclic precursor 176.

Scheme 11 shows a preparation of analogues 115 and 117 with 2-azanorbornane, 120 and 122 with lactam, 135 and 137 with 1-amino-3-hydroxymethylcyclopentane, and 139 and 141 with 1-amino-3-carboxylcyclopentane with absolute stereochemistry from 192.a

Scheme 12 shows a preparation of N-acetyl 2-azanorbornane derivatives.

Scheme 13 shows a preparation of nortropane derivatives from hydroxylated intermediate 127s.

Scheme 15 shows the preparation of isoquinuclidine derivatives from hydroxylated intermediate 206.

Scheme 16 shows a preparation of compounds 1c and 1d.

Scheme 17 shows a preparation of phosphate 3b.

Synthesis of Compounds of Formula (IV) or (V):

PPTN analogues with β-D-glucose bridged with triazole were prepared via click reaction (Scheme 18). The azide 523 was obtained from substitution of bromo starting material 522 with sodium azide. PPTN analogues 525-527 modified with alkynes tethered at the N of piperidine were prepared by reacting PPTN with iodo-substituted alkynes. The click reaction between alkynes 525-527 and azide 523 was catalyzed by CuSO4/sodium ascorbate at 90° C. overnight and provided acetyl protected ligands 504, 506, and 507, which were further hydrolyzed to yield PPTN analogues 508, 510, and 511 with β-D-glucose bridged by various-length alkyltriazolyl linker, respectively.

EXAMPLES

Pharmacological Characterization

The inhibition by the compounds in accordance with aspects of the invention of the binding of fluorescent tracer 2 was measured in hP2Y14R-expressing CHO (references 1 and 2).

Therefore, several of the compounds (11, 19, 29, 32) were compared in affinity at hP2Y14R and mP2Y14R (FIG.2) using the fluorescent binding procedures.2

The solubility (using the pION method)2,25,26and lipophilicity (based on the HPLC retention time)2,27were determined for the 5-(hydroxymethyl)isoxazol-3-yl compounds 29 and 35.

Absorption, distribution, metabolism, excretion and toxicology (ADMET) properties were determined for compound 32 (Tables 4 and 5), by the same methods as in Jung et al.2

The potent 5-(hydroxymethyl)isoxazol-3-yl derivative 32 was first tested in a well-established mouse model of neuropathic pain caused by chronic constriction of the sciatic nerve (CCI)32at a dose of 10 μmol/kg (4.9 mg/kg, i.p.,FIG.3). Previously, it was demonstrated that 1a, 1b, 3a and other P2Y14R antagonists of this series were effective in reducing chronic pain in this model and reached full reversal in some cases (Table S3, Supporting information).2,7A maximal 71.0±17.4% reversal of CCI-induced mechano-allodynia was observed 1 h post-injection of 32, and the degree of protection declined during the following 2 h and was not statistically significant at 3 or 5 h. No side effects were evident at this dose.

Selected compounds were examined for their ability to reduce eosinophils in the bronchoalveolar lavage fluid (BALF) in an ovalbumin/Aspergillusmouse asthma model.2,17,33Compounds 1a, 1b, 3a and 32 (10 mg/kg) were administered prior to allergen challenge at a dose of 20 μmol/kg, i.p. Airway inflammation was determined two days post-challenge (FIG.4). Also, the corresponding prodrug derivatives 37a-37c of triazole 3a, 5-(hydroxymethyl)isoxazole 32 and N-acetyl-piperidine 1b derivatives, respectively, were administered at the same dose. Eosinophil counts in the BALF were reduced following administration of reference antagonist 1a or several of the prodrugs. In fact, 37b and 37c significantly reduced eosinophils to a greater extent than the parent antagonists, which showed no significant reduction. Other immune cells showed no significant effect, except the lymphocyte count following administration of prodrug 37a trended higher than in vehicle control.

This example demonstrates synthesis of compounds, in accordance with aspects of the invention.

General Procedures

Method A: A mixture of compound (1 eq) and potassium hydroxide (5 eq) in methanol:water (2:1) was stirred at 50° C. This mixture was neutralized with 1N HCl until pH was 5-6. The slightly acidic mixture was evaporated under reduced pressure and purified by silica gel column chromatography (dichloromethane:methanol:acetic acid=95:5:0.1) or semipreparative HPLC (10 mM triethylammonium acetate buffer:acetonitrile=80:20 to 20:80 in 40 min) to afford the compound as a white solid.

Method B: A solution of compound in trifluoroacetic acid:tetrahydrofuran (2:1) was stirred at room temperature. The solvent was evaporated with toluene under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol=95:5) or semipreparative HPLC (10 mM triethylammonium acetate buffer:acetonitrile=80:20 to 20:80 in 40 min) to afford the compound as a white solid.

General procedure: Suzuki reaction

Method C: A mixture of compound 38 (1 eq), Pd(PPh3)4(0.06 eq) and potassium carbonate (3 eq) in N,N-dimethylformamide was purged with nitrogen gas for 15 min, and then various aryl halides (1.2 eq) was added to the mixture. The mixture was stirred at 85° C. for 5 h, and then allowed to be cooled at room temperature. This mixture was partitioned ethyl acetate (5 mL) and water (10 mL). The aqueous layer was extracted with ethyl acetate (5 mL×2), and the combined organic layer was washed with brine (3 mL), dried (MgSO4or Na2SO4), filtered and evaporated under reduced pressure. The residue was purified by silica gel column chromatography to afford the compound as a white solid.

Method D: The mixture of compound 38 (1 eq) and aryl halide (1.2 eq) in dimethoxyethane/2M Na2CO3aqueous solution (10:1) was purged with nitrogen gas for 30 min, and then PdCl2(dppf) (0.1 eq) was added to the mixture. The mixture was stirred at 60° C. for 4 h. After cooling at room temperature, this mixture was partitioned ethyl acetate (5 mL) and water (10 mL). The aqueous layer was extracted with ethyl acetate (5 mL×2), and then the combined organic layer was washed with brine (3 mL), dried (MgSO4or Na2SO4), filtered and evaporated under reduced pressure. The residue was purified by silica gel column chromatography to afford the compound as a white solid.

Method E: A mixture of compound 38 (1 eq) and aryl bromide (1.2 eq) in DMF/H2O (10:1; 20 mM) was purged with nitrogen gas for 30 min, and then Pd(PPh3)4(0.05 eq) and Na2CO3(3 eq) were added to the mixture, which was stirred for 1 h at 40-120° C. The aqueous layer was extracted with ethyl acetate twice, and the combined organic layer was washed with brine, dried (MgSO4or Na2SO4), filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography to afford the compound as white solids.

Method F: The mixture of compound 38 (1 eq), Pd(PPh3)4(0.06 eq), potassium carbonate (3 eq) and various aryl halides (1.2 eq) in N,N-dimethylformamide was purged with nitrogen gas for 15 min. The mixture was stirred at 100° C. for 30-90 min in microwave. After microwave irradiation, and then allowed to be cooled at room temperature. This mixture was partitioned ethyl acetate (5 mL) and water (10 mL). The aqueous layer was extracted with ethyl acetate (5 mL×2), and the combined organic layer was washed with brine (3 mL), dried (Na2SO4), filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography to afford the compound as a white solid.

Compound 71 (121 mg, 0.523 mmol) was dissolved in acetonitrile (4 mL) and cooled to 0° C. in an ice bathe. tert-Butyl nitrite (124 μL, 1.047 mmol) was added to the reaction mixture and stirred at 0° C. for 30 min. After that, azidotrimethylsilane (206 μL, 1.57 mmol) was added to the reaction mixture at 0° C. by dropwise. The resulting solution was stirred at room temperature for 12 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:ethyl acetate=3:1) to afford compound 74 (50 mg, 37%) as a white solid;1H NMR (400 MHz, CDCl3) δ 7.98 (s, 1H), 7.26 (s, 1H), 3.99 (s, 3H).

tert-Butyl (1S,4S,5S)-5-(4-(3-(hydroxycarbamoyl)-6-(4-(trifluoromethyl)phenyl)naphthalen-1-yl)phenyl)-2-azabicyclo[2.2.1]heptane-2-carboxylate (215). Carbonyldiimidazole (32 mg, 0.2 mmol) was added to a solution of 213 (5.9 mg, 0.01 mmol) in THF (1 mL). The resulting solution was stirred at rt overnight to give intermediate 214 (MS m/z [M+H]+for C38H35N3O3F3calculated 638.2, found 638.2). Into the reaction residue was added hydroxylamine hydrochloride (14 mg, 0.2 mmol) and the reaction mixture was stirred at rt for 2 h to give 215 (HRMS m/z [M−t-butyl+2H]+for C31H26N2O4F3calculated 547.1845, found 547.1840) as single product. 215 was acid-labile and decomposed during column chromatography.

This example demonstrates inhibition of fluorescent antagonist (2) binding in hP2Y14R-expressing CHO cells (X, Y, Z=CH, unless noted). The results are set forth in Tables 1 and 2.

This Example illustrates a method of preparing compounds of formula (III). The synthesis includes a Grignard reaction of ketone starting materials for the generation of bromophenyl intermediates, Suzuki coupling of bromophenyl intermediates with boronic acid pinacol ester to obtain the phenylnaphthalenecarboxylate core structure, hydrolysis of esters to free carboxylic acid, Boc/benzyl deprotection to free amine, and conversion of analogues to their related ligands, as illustrated herein. Compound 405 with azetidine was prepared via Suzuki coupling between commercially available 3-(4-bromophenyl)azetidine 421 and naphthalenyl boronic acid pinacol ester 422 followed by ester hydrolysis.

This Example illustrates a method of preparation of PPTN analogues with 2-azaspiro[3.3]heptane, octahydrocyclopenta[c]pyrrole (B), 3-azabicyclo[3.1.1]heptane (C), 1-azacycloheptane (D), and azocane (E) were summarized in Scheme 2. In a one-pot reaction, treatment of ketones 424, 428, 432, 436, and 440 with in-situ generated Grignard reagent 4-bromophenylmagnesium bromide provided bromophenyl intermediates 425, 429, 433, 437, and 441, respectively. Derived from a racemic starting material 428, intermediate 429 is a racemic mixture of left-handed and right-handed helix enantiomers. Stereoselective Grignard reaction of 432 yielded enantiomer 433 with hydroxyl group and one-carbon bridge identified as trans to each other.

The Suzuki coupling of naphthalenyl boronic acid pinacol ester 422 with the 4-(4-bromo-phenyl) intermediates 425, 429, 433, 437, and 441 gave their corresponding esters of analogues 426, 430, 434, 438, and 442. The ester hydrolysis of 426, 430, 434, 438, and 442 provided the derivatives 427, 431, 435, 439, and 443. Boc deprotection of 427 with TFA in DCM offered product 408 in addition to unexpected dehydrated product 407. Compound 408 was unstable in the reaction residue in the presence of methanol and partially reacted with methanol and methanol-d4to give O-methylated ligand 409. Thus, methanol should be avoided if methylated by-product is not needed. Similarly, boc deprotection of 443 gave product 420 and dehydrated compound 419. Nevertheless, boc deprotection of 439 meanwhile dehydrated the hydroxyl group to yield alkene mixture 417. Boc deprotection of compound 435 simply gave ligand 415. Pd-catalyzed debenzylation of 431 with H2afforded ligand 411, which was further dehydrated by refluxing in TFA to give 412. Reduction of 407, 412, 417, and 419 by Pd-catalyzed hydrogenation offered PPTN analogues 406 with 2-azaspiro[3.3]heptane, 413 with octahydrocyclopenta[c]pyrrole (B), 416 with 1-azacycloheptane (D), and 418 with azocane (E), respectively. Stereoselective deoxygenation of 415 with sodium borohydride in sulfuric acid afforded 414. Thus, hygroscopic sulfuric acid was employed to readily generate tertiary benzylic carbocation, which was further reduced by sodium borohydride to yield deoxygenated product.

Chemical Synthesis

General Information. Materials and Methods. All chemicals and anhydrous solvents were obtained directly from commercial sources. All reactions were carried out under argon atmosphere using anhydrous solvents unless specified otherwise. Room temperature (rt) refers to 25±5° C. Silica-gel precoated with F254 on aluminum plates were used for TLC. The spots were examined under ultraviolet light at 254 nm and further visualized by anisaldehyde or cerium ammonium molybdate stain solution. Column chromatography was performed on silica gel (40-63 μm, 60 Å). NMR spectra were recorded on a Bruker 400 MHz spectrometer. Chemical shifts are given in ppm (δ), calibrated to the residual solvent signal peaks of CDCl3(7.26 ppm), CD3OD (3.31 ppm), or DMSO-d6(2.50 ppm) for1H NMR with coupling constant (J) values reported in Hz. High resolution mass (HRMS) measurements were performed on a proteomics optimized Q-TOF-2 (Micromass-Waters). The RP-HPLC was performed using Phenomenex Luna 5 μm C18(2)100A, AXIA, 21.2 mm×250 mm column. Antagonist purity was determined as >95% using Agilent ZORBAX SB-Aq, 5 μm, 4.6 mm×150 mm column attached to Agilent 1100 HPLC system. The HPLC traces for compounds tested in vivo (xxx) were included in Supporting Information.

This Example illustrates the affinity of compounds of formula (III). The affinity was determined using a whole cell fluorescent binding assay, which was shown to underestimate the actual affinity.

This example demonstrates the species dependence of binding of various P2Y14R antagonists, as determined in a fluorescent antagonist binding assay in whole cells (N=3-4). As is apparent from the results set forth in Table 2, compound 29 had slightly enhanced affinity (IC50184 nM) at the mP2Y14R vs the human homologue, while 11, 19 and 32 had nearly the same affinity in the two species. Desirably, receptor affinity is maintained across species, facilitating performance in in vivo preclinical testing in small animals, with the mouse being the most commonly used species in initial evaluation.

This example demonstrates some properties of amidomethyl ester prodrugs 37a-c.aThe results are set forth in Table 3. The triazole-containing compound 37a was the most rapidly cleaved, while the naphthalene-containing compounds 37b and 37c did not reach 50% cleavage after 72 h at 37° C. All of the compounds were stable for 24 h upon aqueous incubation in the absence of PLE.

This examples demonstrates some in vitro and in vivo ADMET data for compound 32 compared to known P2Y14R antagonist 5.aThe compound was stable in simulated gastric and intestinal fluids, in plasma of three species, and in human and mouse liver microsomes.

This example demonstrates some pharmacokinetic parametersafor compound 32 administered by intravenous and intraperitoneal routes. The results are set forth in Table 5.

This example demonstrates a binding comparison of various P2Y14R antagonists at mP2Y14R and hP2Y14R. The results are set forth in Table 6.

This example demonstrates ADMET values for two P2Y14R antagonists in this study, compared to reference quinuclidine 4.

This example demonstrates IC50values of hERG ion channel block by selected P2Y14R antagonists and positive control (cisapride) determined in a patch clamp assay. Value in parentheses indicates EC50value for stimulation of hERG channel activity. None of the compounds inhibited hERG, but one antagonist 20 stimulated hERG activity with an EC50of 18 μM.

This example illustrates the preparation of prodrugs and the Plasma Protein Binding (PPB) of selected derivatives in three Species, including three active drugs and two prodrugs.

The results obtained are set forth in Table 9.

This example illustrates the evaluation of compound 114 in Chronic Constriction Injury Mouse Model of Neuropathic Pain.

Compound 114 was evaluated in a validated model of chronic neuropathic pain in the mouse, i.e. following chronic constriction of the sciatic nerve (CCI). This compound was administered by the i.p. route (10 μmol/kg). The reversal of CCI-induced mechano-allodynia by the compound was nearly complete (89.3±10.1%) at 1 h post-injection, and significant partial protection was maintained through 3 h but was absent at 5 h (FIG.6). No obvious side effects were observed in the mice.

This example illustrates the antagonist potency of prodrugs 142 and 143.

The carbamate prodrug 142 and the double prodrug 143 having carbamate and ester moieties were derived from the most potent antagonist 114. Prodrugs 142 and 143 were tested in the mouse asthma model in comparison to the HCl salt form of the parent drug 114. The N,N-dimethylamidomethyl ester prodrug 141 and the carbamate prodrug 142 reduced allergic inflammation more effectively than the parent drug 15. Intriguingly, the double prodrug 143 displayed an even stronger potency, dramatically reducing the recruitment of neutrophils, eosinophils, and lymphocytes into the airway. The results are depicted inFIG.7. These promising results suggest that double prodrug 143 could be a candidate for further drug development and therapeutic treatment of asthma and other inflammatory diseases.

Experimental Procedures

Chemical Synthesis

General Information. Materials and Methods. All chemicals and anhydrous solvents were obtained directly from commercial sources. All reactions were carried out under argon atmosphere using anhydrous solvents unless specified otherwise. Room temperature (rt) refers to 25±5° C. Silica-gel precoated with F254 on aluminum plates were used for TLC. The spots were examined under ultraviolet light at 254 nm and further visualized by anisaldehyde or cerium ammonium molybdate stain solution. Column chromatography was performed on silica gel (40-63 μm, 60 Å). NMR spectra were recorded on a Bruker 400 MHz spectrometer. Chemical shifts are given in ppm (δ), calibrated to the residual solvent signal peaks of CDCl3(7.26 ppm), CD3OD (3.31 ppm), or DMSO-d6(2.50 ppm) for1H NMR with coupling constant (J) values reported in Hz. High resolution mass (HRMS) measurements were performed on a proteomics optimized Q-TOF-2 (Micromass-Waters). The RP-HPLC was performed using Phenomenex Luna 5 μm C18(2)100A, AXIA, 21.2 mm×250 mm column. Antagonist purity was determined as ≥95% using Agilent ZORBAX SB-Aq, 5 μm, 4.6 mm×150 mm column attached to Agilent 1100 HPLC system. The HPLC traces for compounds tested in vivo (xxxx) were included in Supporting Information.

The invention provides the following aspects:

1. A compound of formula (I) or formula (II):

wherein X, Y, Z are CH or N,

m, o, and q are independently integers of 1 to about 10, and

n is zero or an integer of 1 to about 10, or a pharmaceutically acceptable salt thereof, or stereoisomers thereof.

2. The compound or salt or stereoisomers thereof of aspect 1, wherein R1is CF3.

3. The compound or salt or stereoisomers thereof of aspect 1 or 2, wherein the compound is of formula (I).

4. The compound or salt or stereoisomers thereof of any one of aspects 1-3, wherein R3is selected from NH2, NHCOCH3, NHCOC6H5, NHCOC(CH3)3,

5. The compound or salt or stereoisomers thereof of any one of aspects 1-3, wherein R3is COOR5.

6. The compound or salt or stereoisomers thereof of any one of aspects 1-3, wherein R3is (C≡C)m(CH2)mR6or CONH(CH2)qNH2.

7. The compound or salt or stereoisomers thereof of aspect 6, wherein R3is selected from CONH(CH2)3NH2, CH2CONH2, (CH2)3NH2, and C≡CCH2NH2.

8. The compound or salt or stereoisomers thereof of any one of aspects 1-3, wherein R3is selected from Br, (CH2)2—CN,

9. The compound or salt or stereoisomers thereof of aspect 1 or 2, wherein the compound is of formula (II).

10. The compound or salt or stereoisomers thereof of aspect 9, wherein R3is selected from Br, (CH2)2—CN,

11. The compound or salt or stereoisomers thereof of aspect 9, wherein R3is

11. The compound or salt or stereoisomers thereof of any one of aspects 1-10, wherein R2is H.

12. The compound or salt or stereoisomers thereof of any one of aspects 1-10, wherein R2is COOR12, CH2CON(R11)2, CHR7OCOR8, (CH2)oNR9, tetrazolyl, CH2OPO(OH)2, or

13. The compound or salt or stereoisomers thereof of aspect 1, wherein the compound is:

14. The compound or salt or stereoisomers thereof of aspect 9, wherein R3is selected from

15. The compound or salt or stereoisomers thereof of aspect 14, wherein R3is

and wherein the configuration of the three chiral centers is (S,S,S).

16. The compound or salt or stereoisomers thereof of aspect 9, wherein R3is selected from

17. The compound or salt or stereoisomers thereof of any one of aspects 14-16, wherein R2is H.

18. The compound or salt or stereoisomers thereof of any one of aspects 14-16, wherein R2is COOR12, CH2CON(R11)2, CHR7OCOR8, (CH2)oNR9, tetrazolyl, CH2OPO(OH)2, or

19. The compound or salt or stereoisomers thereof of aspect 1, wherein the compound is:

wherein the configuration about the three chiral centers is (S,S,S).

20. A pharmaceutical composition comprising a compound or salt or stereoisomers thereof of any one of aspects 1-19 and a pharmaceutically acceptable carrier.

21. A compound or salt or stereoisomers thereof of any one of aspects 1-19, for use in antagonizing a P2Y14R receptor in a mammal in need thereof.

22. A compound or salt or stereoisomers thereof of any one of aspects 1-19, for use in treating or preventing an inflammatory condition in a mammal in need thereof.

23. The compound for use according to aspect 22 wherein the inflammatory condition is selected from the group consisting of asthma, cystic fibrosis, and sterile inflammation of the kidney.

24. A compound or salt or stereoisomers thereof of any one of aspects 1-19, for use in treating pain in a mammal in need thereof

25. A method of antagonizing a P2Y14R receptor in a mammal in need thereof, comprising administering to the mammal an effective amount of a compound or salt or stereoisomers thereof of any one of aspects 1-19.

26. A method of treating or preventing an inflammatory condition in a mammal in need thereof, comprising administering to the mammal an effective amount of a compound or salt or stereoisomers thereof of any one of aspects 1-19.

27. The method according to aspect 26, wherein the inflammatory condition is selected from the group consisting of asthma, cystic fibrosis, and sterile inflammation of the kidney.

28. A method of treating pain in a mammal in need thereof, comprising administering to the mammal an effective amount of a compound or salt or stereoisomers thereof of any one of aspects 1-19.

28. The method according to aspect 27, wherein the inflammatory condition is selected from the group consisting of asthma, cystic fibrosis, and sterile inflammation of the kidney.

29. A method of treating pain in a mammal in need thereof, comprising administering to the mammal an effective amount of a compound or salt or stereoisomers thereof of any one of aspects 1-20.

REFERENCES