WO2003047515A2

WO2003047515A2 - P2x7 receptor antagonists

Info

Publication number: WO2003047515A2
Application number: PCT/US2002/038126
Authority: WO
Inventors: Kenneth A. Jacobson
Original assignee: The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services
Priority date: 2001-11-30
Filing date: 2002-11-27
Publication date: 2003-06-12
Also published as: AU2002359524A1; AU2002359524A8; WO2003047515A3

Abstract

Disclosed are antagonists of the P2X7 receptor in an animal, for example, an antagonist of the formula (I) which may be monomeric or dimeric; wherein R1-R3, for example, are aryl sulfonyl or heteroaryl sulfonyl; and R4 is H or alkyl. Also disclosed are pharmaceutical compositions comprising one or more of these antagonists and methods of using these antagonists, including blocking an ATP-induced toxic process in the blood cell of an animal, for example, in the treatment or prevention of septic shock, inflammation, stroke or neurodegenerative disease.

Description

P2X₇ RECEPTOR ANTAGONISTS

CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of U.S. provisional patent application No. 60/334,130, filed on November 30, 2001, the disclosure of which is incorporated by reference.

FIELD OF THE INVENTION The present invention relates to compounds which block the P2X₇ receptor in an animal, pharmaceutical compositions comprising one or more of these compounds, and uses of these compounds in the treatment or prevention of a disease, state, or condition in an animal.

BACKGROUND OF THE INVENTION P2 receptors, which are activated by ATP (adenosine 5'-triphosphate) and other purine/pyrimidine nucleotides, consist of two families: G protein-coupled receptors termed P2Y, of which five mammalian subtypes have been cloned, and ligand-gated cation channels (receptors) termed P2X, of which seven mammalian subtypes have been cloned. Fredholm et al., Trends Pharm. Set, 75:79-82 (1997); North et al., Current Opinion in NeuroBiology, 7:346-357 (1997). The nomenclature of P2 receptors and their various ligand specificities have been reviewed. Jacobson et al., Handbook of Experimental Pharmacology, 51:1, 129-175 (2001); Jacobson et al., The P2 Nucleotide Receptors, 81-108 (1997); Bhagwat et al., Eur. J. Med. Chem., 32:183-193 (1997); Fischer, Exp. Opin. Ther. Patents, .9:385-399 (1999). The P2X- receptor (formerly the P2Z receptor) is expressed primarily in blood cells (monocytes, macrophages, and lymphocytes), in the brain (on microglial cells Ferrari et al., J. Biol. Chem., 274:13205-13210 (1999)), and in the salivary gland. A characteristic of the P2X₇ receptor is that at high μM concentrations of agonists, it forms or activates a large pore in addition to a cation channel. This pore increases permeability indiscriminately to molecules having a molecular weight of <900. 2'- and 3'-O-(4-benzoylbenzoyl)-ATP ("BzATP") is among the most potent agonists at P2X₇ receptors, but also has nanomolar potency at V2 _X receptors. Bianchi et al., Eur. J. Pharmacol, 37<5:127-138 (1999). Affinity labeling of the P2X₇ receptor in mast cells has been carried out using [³H]-BzATP. Erb et al., J. Biol Chem., 265:7424-7431 (1990).

In macrophages, activation of the P2X. receptor triggers the processing and release of interleukin lβ (IL-lβ). In the immune system, activation of the P2X₇ receptor activation leads to apoptosis or programmed cell death, Ferrari et al., Neuropharmacology, 36: 1295-1301 (1997); Coutinho-Silva et al., Am. J. Physiol., 276:Cl 139-1147 (1999); Humphreys et al., J. Biol. Chem., 275:26792-26798 (2000). BzATP (5 mM) caused apoptosis in dendritic cells, which play a significant role in T- cell activation. Coutinho-Silva et al., supra. BzATP (1 mM) was very effective in activating the transcription factor NFAT in N9 microglial cells, suggesting purinergic modulation of early inflammatory gene expression in the nervous and immune systems. Ferrari et al., J. Biol Chem., supra. Activation of the P2X₇ receptor in rat microglia triggers the release of TNF-α. Hide et al., J. Neurochem., 75:965-972 (2000). The foregoing shows that there exists a need for compounds which can deactivate or block the P2X₇ receptor. The advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION The present invention provides antagonists of the P2X₇ receptor in an animal. The antagonists are substituted tyrosine derivatives, e.g., L-tyrosine derivatives, of the general structure R₁-Tyr(OR₂)-piperazinyl-R₃. A large hydrophobic group is present as R] on the alpha N of Tyr. R₂ and R₃ are, for example, aryl or heteroaryl groups. The present invention further provides a pharmaceutical composition comprising one or more of the antagonists. The present invention also provides methods of treating an animal, e.g., a mammal, afflicted with a disease, or preventing a disease, state, or condition, comprising administering to the mammal at least one of the antagonists. The present invention further provides a method of blocking an ATP-induced toxic process in the blood cell of an animal. The present invention also provides a method of blocking a P2X₇ receptor in an animal. The present invention also provides methods of inhibiting the binding of a ligand to a P2X₇ receptor.

While the invention has been described and disclosed below in connection with certain preferred embodiments and procedures, it is not intended to limit the invention to those specific embodiments. Rather it is intended to cover all such alternative embodiments and modifications as fall within the spirit and scope, of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts the formulas of known compounds 1 and 2, reported to be P2X₇ receptor antagonists. Figure 2 depicts reaction schemes useful in the preparation of some P2X₇ receptor antagonists in accordance with an embodiment of the invention. The reaction schemes illustrate, for example, the introduction of quinoline- and isoquinoline- sulfonyl groups at the R group. Figure 3 depicts reaction schemes useful in preparing some other P2X₇ receptor antagonists in accordance with an embodiment of the invention. The reaction schemes illustrate, for example, a method of varying the R₂ and R₃ groups.

Figure 4 depicts reaction schemes useful in preparing certain other P2X₇ receptor antagonists in accordance with an embodiment of the invention. The reaction schemes illustrate, for example, a method of varying the substituent on the N^α-atom and the D- configuration.

Figure 5 depicts an effect of certain P2X₇ receptor antagonists in hP2X₇-HEK cells. The adherent cells are pre-incubated with antagonists for 15 min prior to stimulation for 10 min with 3 mM ATP (final concentration). K⁺ content in these nitric acid extracts is assayed by atomic absorbance spectrophotometry. Duplicate or triplicate wells are run for all test conditions in each separate experiment. To obtain the results depicted in Fig. 5 A, hP2X_?-HEK cells are pre-incubated with or without 3 μM 1 prior to stimulation with 3 mM ATP. Data points represent the mean (± SD) K⁺ content from 9 separate experiments. To obtain the results depicted in Fig. 5B, hP2X₇-HEK cells are pre-incubated with or without the indicated concentrations of selected antagonists prior to stimulation with 3 mM ATP. Data points represent the mean (± SD) K⁺ contents from triplicate wells in a single experiment. IC₅₀ values discussed in Example 2 are rough estimates from visual inspection of the concentration-response relationships. Figure 6 depicts reaction scheme 1 useful in preparing certain P2X₇ receptor antagonists in accordance with another embodiment of the invention.

Figure 7 depicts reaction scheme 2 useful in preparing certain other P2X₇ receptor antagonists in accordance with another embodiment of the invention.

Figure 8 depicts reaction scheme 3 useful in preparing some P2X₇ receptor antagonists in accordance with another embodiment of the invention.

Figure 9 depicts reaction scheme 4 useful in preparing some other P2X₇ receptor antagonists in accordance with another embodiment of the invention.

Figure 10 depicts the formulas of certain dimeric P2X₇ receptor antagonists in accordance with an embodiment of the invention. Figures 11-18 depict reaction schemes to prepare embodiments of the antagonists. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a compound of the formula:

5. which may be monomeric or dimeric; wherein Rι-R₃, each independently is selected from the group consisting of H, alkyl sulfonyl, alkoxy sulfonyl, aryl sulfonyl, heteroaryl sulfonyl, arylalkenyl sulfonyl, arylalkoxy sulfonyl, alkyl carbonyl, alkoxy carbonyl, alkoxyalkoxy carbonyl, arylalkoxy carbonyl, aryloxy carbonyl, aryl carbonyl, arylalkyl carbonyl, arylalkyl, and arylalkenyl carbonyl; wherein the aryl or heteroaryl portions of 0 Rι-R may have 1-4 aromatic rings, preferably 1-3 aromatic rings, and each of the aromatic rings may be optionally substituted with one or more substituents selected from the group consisting of alkyl, alkoxy, halo, haloalkyl, nitro, amino, isothiocyanato, acetylamino, thioureido, and aminoalkylthioureido; the alkyl and alkoxy portions of Ri— R₃ may have 1-20 carbon atoms, preferably 1-6 carbon atoms; and the heteroaryl 5 includes one or more of N, O, and S atoms, preferably N; and R₄ is H or alkyl having 1- 6 carbon atoms, typically 1-3 carbon atoms.

Examples of aryl groups include phenyl, naphthyl, anthracenyl, fluorenyl, biphenyl, and phenanthrenyl, preferably phenyl, naphthyl, and fluorenyl. Examples of heteroaryl groups include quinolinyl, isoquinolinyl, indolyl, furyl, thienyl, imidazolyl, 0 pyridyl, pyrimidinyl, and preferably quinolinyl and isoquinolinyl.

According to an embodiment of the invention, Ri is selected from the group consisting of heteroaryl sulfonyl, aryl sulfonyl, aryl carbonyl, arylalkoxy carbonyl, alkoxy carbonyl; and arylalkenyl carbonyl; R₂ is selected from the group consisting of aryl sulfonyl and arylalkyl; and R₃ is selected from the group consisting of H, alkoxy 5 carbonyl, and arylalkoxy carbonyl. As a specific example, the heteroaryl sulfonyl can be quinoline sulfonyl or isoquinoline sulfonyl; the aryl sulfonyl can be benzene sulfonyl, -toluene sulfonyl, or naphthalene sulfonyl; the aryl carbonyl can be benzoyl or toluoyl; arylalkoxy carbonyl can be benzyloxy carbonyl, or fluorenylmethoxy carbonyl; the alkoxy carbonyl can be t-butoxy carbonyl or ethoxy carbonyl; the arylalkyl can be 0 benzyl; and the arylalkenyl carbonyl can be phenylethenyl carbonyl. In preferred embodiments of the invention, R₄ is H and R R₃ are as follows: Ri is isoquinoline 5-sulfonyl; R₂ is isoquinoline 5-sulfonyl or quinoline 8-sulfonyl; R₃ is H or t-butoxy carbonyl; and; Ri and R₂ are quinoline 8-sulfonyl and R₃ is H or t-butoxy carbonyl; Ri is ethoxy carbonyl; R₂ is quinoline 8-sulfonyl; and R₃ is t-butoxy carbonyl; Ri is benzyloxy carbonyl or benzoyl; R₂ is quinoline 8-sulfonyl; and R₃ is t-butoxy carbonyl; Ri is benzene sulfonyl, -toluene sulfonyl, -methoxybenzene sulfonyl, or 1- naphthalene sulfonyl; R is quinoline 8-sulfonyl; and R₃ is t-butoxy carbonyl; Ri is benzyloxy carbonyl; R₂ is benzyl, benzene sulfonyl, -toluene sulfonyl, -methoxy benzene sulfonyl, naphthalene 1 -sulfonyl, or naphthalene 2-sulfonyl; and R₃ is t-butoxy ^• carbonyl; Ri is benzyloxy carbonyl; R₂ is benzene sulfonyl; and R₃ is t-butoxy carbonyl, ethoxy carbonyl, or benzoyl; R_t is benzyloxy carbonyl; R₂ is isoquinoline 5-sulfonyl; and R₃ is benzoyl; Ri is benzyloxy carbonyl; R₂ is benzyl; and R₃ is t-butoxy carbonyl; Ri is trαπs-phenylethenyl carbonyl; R₂ is benzene sulfonyl orp-nitrobenzene sulfonyl; and R₃ is benzoyl or 4-nitrobenzoyl; Ri is trørøs-4-nitrophenylethenyl carbonyl; R₂ is benzene sulfonyl or 4-nitrobenzene sulfonyl; and R₃ is benzoyl or 4-nitrobenzoyl; Ri is trαns-2-methoxyphenylethenyl carbonyl or trøns-3-chlorophenylethenyl carbonyl; R₂ is -toluene sulfonyl; and R₃ is benzoyl; Ri is benzyloxy carbonyl; R₂ is p-toluene sulfonyl, 4-nitrobenzene sulfonyl, 4-aminobenzene sulfonyl, or 4-isothiocyanatobenzene sulfonyl; and R₃ is benzoyl, t-butoxy carbonyl, 4-aminobenzoyl, 4-acetylamino benzoyl, or 4-isothiocyanato benzoyl; Ri is 3-chlorobenzyloxy carbonyl or 4-nitro-benzyloxy carbonyl; R₂ is p-toluene sulfonyl; and R₃ is benzoyl.

The compounds of the present invention can be monomeric or dimeric. The dimeric compound includes two monomeric compounds such that R_l3 R₂, or R₃ of one of the two monomeric compounds is linked to R_ls R₂, or R₃ of the other of the two monomeric compounds through a linker. Any suitable linker can be employed, for example, a linker comprising a thiourea group can be employed. In embodiments, R₂ of one of the monomeric compounds is linked to R₂ of the other of the two monomeric compounds; R₂ or R₃ of one of the two monomeric compounds is linked to the R₃ of the two monomeric compounds. A preferred dimeric compound has the formula shown below.

Examples of dimeric compounds are shown in Fig. 10. In an embodiment, a preferred dimer is 222.

The compounds of the present invention can be prepared by any suitable method, for example, by the methods outlined in Figures 2-4 and 6-9 and described in Example 1. Illustratively, compounds in accordance with an embodiment of the invention can be prepared by methods in which the i-R₃ positions are systematically varied in structure through facile acylation reactions. Each of the three positions can be optimized in sequence, for example, through parallel synthesis alternating with biological evaluation, consisting of screening at a single concentration (e.g., initially 3 μM), leading to the identification and optimization of potent P2X₇ antagonists.

For example, as shown in Figures 2-4, it is preferable to replace groups at two positions on the structure of the lead compound, 1, the tyrosyl N -methyl group, and the N-phenyl-piperazinyl group, which could be replaced conveniently with a N-t- butyloxycarbonyl- (Boc-) -piperazinyl group. Boc is a group favorable for biological screening, and also as a preferred protecting group for synthetic intermediates. Amides are formed readily using bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-C1) as condensing agent. Other amides and sulfonamides are prepared from the corresponding acyl chlorides or sulfonyl chlorides in the presence of 4-dimethylaminopyridine (DMAP).

In accordance with an embodiment, the present invention provides pharmaceutically acceptable salts of the compounds described above. Examples of pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulfonic acids. An example of arylsulphonic acid isp- toluenesulphonic acid.

The present invention further provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound as described above. The present invention further provides a composition comprising a pharmaceutically acceptable carrier and an effective (e.g., therapeutically or prophylactically effective) amount of at least one of the compounds set forth above. The pharmaceutically acceptable (e.g., pharmacologically 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, intraperitoneal, intrathecal, rectal, and vaginal adrninistration are merely exemplary and are in no way limiting.

Formulations suitable for oral administration can comprise (a) liquid solutions, such as an effective amount of the compound dissolved in diluents, such as water, saline, or orange juice; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid formulations can include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant, suspending agent, or emulsifying agent. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such carriers as are known in the art.

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 can 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-l,3- dioxolane-4-methanol, ethers, such as polyethyleneglycol) 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 olef n sulfonates, alkyl, olefϊn, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents such as, for example, alkyl-β-aminopropionates, and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof.

The parenteral formulations will typically contain from about 0.5 to about 25% by weight of the active ingredient in solution. Suitable preservatives and buffers can be used in such formulations. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants. The quantity of surfactant in such formulations typically ranges from about 5 to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

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 for example, Pharmaceutics and Pharmacv Practice. J.B. Lippincott Co., Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on Iniectable Drugs. Toissel, 4th ed., pages 622-630 (1986).

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.

One skilled in the art will appreciate that suitable methods of administering the compound of the present invention to an animal are available, and, although more than one route can be used to administer a particular compound, a particular route can provide a more immediate or more effective reaction than another route. Accordingly, the methods described herein are merely exemplary and are in no way limiting. The dose administered to an animal such as a mammal, particularly a human, in the context of the present invention should be sufficient to effect prophylactic or other therapeutic response in the animal over a reasonable period of time. One skilled in the art will recognize that dosage will depend upon a variety of factors including the strength of the particular compound employed, the age, species, condition, and body weight of the animal, as well as the severity/stage of the disease or condition. The size of the dose will also be determined by the route, timing, and frequency of administration of a particular compound and the desired physiological effect. It will be appreciated by one skilled in the art that various conditions or disease states, in particular chronic 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, which 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. For convenience, the total daily dosage may be divided and administered in portions during the day if desired. In proper doses and with suitable administration of certain compounds, the present invention provides for a wide range of responses. Typically the dosages range from about 0.001 to about 1000 mg/kg body weight of the animal being treated/day. Preferred dosages range from about 0.01 to about 10 mg kg body weight day, and further preferred dosages range from about 0.01 to about 1 mg/kg body weight/day.

The present invention further provides a method of preventing or treating a disease, state, or condition in a mammal by the use of the compounds of the present invention. In an embodiment, the method involves preventing a disease, state, or condition. In another embodiment, the method involves treating an existing disease, state, or condition. The present invention further provides a method of blocking, preferably selectively, a P2X₇ receptor in an animal, e.g., a mammal such as a human. The method comprises administering to the mammal a compound of the present invention. The present invention further provides a method of blocking an ATP-induced toxic process, e.g., a process involving release of interleukin-1 (e.g., IL-1/3), in a blood cell in a mammal comprising administering to the mammal a compound as described above. The present invention provides a method for blocking P2X₇ receptor in blood cells, in the brain (on microglial cells), or in the salivary gland. Examples of blood cells include monocytes, macrophages, and lymphocytes.

The antagonists are also useful in treating septic shock or a neurodegenerative diseases, since the P2X₇ receptor activates astrocytes and microglial cells. Modulation of the P2X₇ receptor may also be beneficial in ophthalmic diseases or conditions, e.g., ophthalmic inflammation. The P2X₇ receptor is expressed in high levels in dendritic cells and ATP acting at this site may serve as a signal to down modulate the immune response. The present invention further provides a method of treating or preventing septic shock, inflammation, stroke, or a neurodegenerative disease in a mammal comprising administering to the mammal a compound as described above. The compounds of the present invention are useful in medicine. The compounds of the present invention are useful in the manufacture of a medicament for the treatment or prevention of septic shock, inflammation, stroke, or a neurodegenerative disease. The antagonists of the present invention are useful in preventing apoptosis or programmed cell death. The antagonists of the present invention are useful in preventing the release of TNF-α and other inflammatory cytokines such as interleukin-1. The present invention further provides a method of inhibiting the binding of a ligand to a P2X₇ receptor of a substrate comprising contacting the substrate with a compound of the present invention so that the compound binds to the P2X₇ receptor and inhibits the ligand from binding to the P2X₇ receptor. The contacting can be carried out in vitro or in vivo. The present invention further provides a method of characterizing a P2X₇ receptor site in a substrate comprising contacting the substrate with a compound of the present invention and evaluating the interaction of the compound with the P2X₇ receptor. The receptor site can be characterized pharmacologically to provide valuable information relating to the receptor including its occurrence in the tissue, its density, its activation characteristics, or its ability to couple to proteins. "Characterizing" involves evaluation of the interaction of the antagonist with the receptor. The evaluation can provide qualitative information whether binding has occurred as well as quantitative information as to the extent of binding.

The following examples further illustrate the present invention. The examples, of course, should not be construed as in any way limiting the scope of the present invention.

EXAMPLE 1 This Example illustrates a method of preparation of compounds in accordance with an embodiment of the invention. Synthetic reagents are purchased from Sigma Chemical Co. (St. Louis, MO) and

Aldrich (St. Louis, MO). 1H-NMR spectra are obtained with a Varian Gemini-300 spectrometer using CD₃OD or CDC1₃ as a solvent. Low-resolution CI-NH₃ (chemical ionization) mass spectra are carried out with Finnigan 4600 mass spectrometer and high- resolution El (electron impact) mass spectrometry with a VG7070F mass spectrometry at 6kV. High-resolution FAB (fast atom bombardment) mass spectrometry is performed with JEOL SX102 spectrometer using 6-kV Xe atoms following desorption from a glycerol matrix. Compounds 51 and 55a - c are obtained from Calbiochem- Novabiochem, La Jolla, CA. [N-Fmoc-L-tyrosyl] -Boc-piperazine (52) A mixture of Fmoc-Tyr-OH (51) (0.4 g, 1 mmol) Boc-piperazine (0.186 g, 1 mmol) and BOP-C1 (0.255 g, 1 mmol) in CH₂C1₂ (5 mL) is treated with Et₃N (0.28 mL, 2 mmol) and stirred at rt for 5 h. The solvent is removed and the residue obtained is purified using flash chromatography eluting with 10% MeOH in CHC1₃ to furnish 52 (0.38 g,

67%) as a solid foam.

1H NMR (CDC1₃): δ 7.77 (d, J = 7.3 Hz, 2H), 7.59 (d, J - 7.3 Hz, 2H), 7.40 (t, J = 7.3 Hz, 2H), 7.31 (t, J = 7.3 Hz, 2H), 7.04 (d, J = 7.7 Hz, 2H), 6.74 (d, J = 7.7 Hz, 2H), 5.88

(s, IH), 5.72 (d, J = 8.4Hz, IH), 4.90-4.78 (m, IH), 4.44-4.28 (m, 2H), 4.24-4.16 (m,

2H), 3.6-2.8 (m, 9H), 1.45 (s, 9H).

[L-Tyrosyl] -Boc-piperazine (53)

Compound 52 (0.37 g, 0.65 mmol) is treated with 20% piperidine in DMF (N,N- dimethylformamide) at room temperature for 10 min. for complete reaction. DMF is removed under high vacuum, and the residue obtained is purified using flash chromatography eluting with 15% MeOH in CHC1₃ to furnish 53 (0.19 g , 54%) as a gum.

1H NMR (CDC1₃): δ 6.99 (d, J = 8.2 Hz, 2H), 6.72 (d, J = 8.2 Hz, 2H), 3.9 (t, J - 7.1 Hz, IH), 3.62-3.20 (m, 6H), 3.10-2.74 (m, 4H), 1.45 (s, 9H).

[N,O~Bis-(quinolinesuIfonyl)-L-tyrosyI]-Boc-piperazine and [N,0-Bis-(5- isoquinolinesulfonyl)-L-tyrosyl] -Boc-piperazine (4 and 6)

To a suspension of 8-quinoline sulfonyl chloride or 5-isoquinoline sulfonyl chloride

(0.274 g, 1.2 mmol) in anhydrous CH₂C1₂ (5 mL) is added a solution of 53 (0.175 g, 0.5 mmol) and Et₃N in CH₂C1₂ (3 mL) at 0 °C, and the mixture stirred at room temperature for 4 h. The solvent is removed from the reaction mixture under vacuum, and the residue obtained is purified using flash chromatography using 5% MeOH in CHC1₃ to furnish 0.26 g of 4 and 0.25 g of 6 as solid foam.

4: 1H NMR (CDC1₃): δ9.43 (s, IH), 9.34 (s, IH), 8.84 (d, J = 5.9 Hz, IH), 8.69 (d, J = 5.4 Hz, IH), 8.52 (d, J = 5.9 Hz, IH), 8.54-8.10 (m, 5H), 7.70-7.52 (m, 2H), 6.85 (d, J =

8.2 Hz, 2H), 6.61 (d, J = 8.2 Hz, 2H), 6.00 (bs, IH), 4.20-4.38 (m, IH), 3.12-2.50 (m,

10H), 1.45 (s, 9H).

6: 1H NMR (CDC1₃): δ 9.25 (d, J = 3.9 Hz, IH), 9.08 (d, J = 3.9 Hz, IH), 8.34-8.20 (m,

4H), 8.13 (d, J - 8.1 Hz, IH), 8.02 (d, J = 8.1 Hz, IH), 7.68-7.52 (m, 4H), 7.17 (d, J = 9.9 Hz, IH), 6.90 (d, J = 8.2 Hz, 2H), 6.79 (d, J = 8.2 Hz, 2H), 4.42-4.58 (m, IH), 3.02-

2.26 (m, 10H), 1.44 (s, 9H).

[N,O-Bis-(quinoIinesulfonyl)-L-tyrosyl]piperazine and [N,O-Bis-(5- isoquinolinesulfonyl)-L-tyrosyl]piperazine (3 and 5)

Compound 4 or 6 (0.17 g, 0.23 mmol) is treated with 10% TFA in CH₂C1₂ at room temperature for 6 h for complete reaction. The solvent is removed under vacuum, and the residue obtained is purified using flash chromatography using 10% MeOH in CHC1₃ to furnish 0.12 g of 3 and 5 as solid foam.

3: 1H NMR (CD₃OD): δ 9.41 (s, IH), 9.30 (s, IH), 8.73 (d, J = 5.9 Hz, IH), 8.55-8.05

(m, 7H), 7.66 (ABq, J = 8.1 Hz, 2H), 6.74 (d, J = 8.4 Hz, 2H), 6.27 (d, J = 8.4 Hz, 2H), 4.38-4.22 (m, IH), 3.80-3.38 (m, 5H), 3.18-2.58 (m, 6H).

5: 1H NMR (CD₃OD): δ 9.14 (d, J = 4.3 Hz, IH), 8.97 (d, J = 4.1 Hz, IH), 8.52 (d, J =

8.4 Hz, IH), 8.44-8.22 (m, 4H), 8.17 (d, J = 8.2 Hz, IH), 7.82-7.58 (m, 4H), 6.94 (d, J =

8.4 Hz, 2H), 6.74 (d, J = 8.4 Hz, 2H), 3.64-2.45 (m, 12H).

[N-Fmoc-O-quinolinesulfonyl-L-tyrosyl]-Boc-piperazine (8) A suspension of 8-quinoline sulfonyl chloride (0.16 g, 0.71 mmol) in CH₂C1₂ is treated with a solution of 52 (0.34 g, 0.60 mmol) and Et₃N in CH₂C1₂ at 0°C, and the mixture is stirred at room temperature for 6 h. The solvent is removed under vacuum, and the crude product is purified using flash chromatography using 5% MeOH in CHC1₃ to furnish 0.35 g of 8. 8: 1H NMR (CDC1₃): δ 9.26 (s, IH), 8.3 (t, J = 6.3 Hz, 2H), 8.12 (d, J = 7.9 Hz, IH),

7.75 (d, J = 7.1 Hz, 2H), 7.64-7.48 (m, 4H), 7.44-7.20 (m, 4H), 7.03 (d, J = 8.3 Hz, 2H),

6.91 (d, J = 8.3 Hz, 2H), 5.61 (d, J = 8.2 Hz, IH), 4.85-4.65 (m, IH), 4.45-4.05 (m, 4),

3.55-3.12 (m, 9H), 1.47 (s, 9H).

[O-Quinolinesulfonyl-L-tyrosyl]-Boc-piperazine (9) Compound 8 (0.3 g, 0.39 mmol) is treated with 20% piperidine in DMF (10 mL) at room temperature for 10 min. The DMF is removed under vacuum, and the residue obtained is purified using flash chromatography using 10% MeOH in CHC1₃ to furnish

0.19 g of 9 as a solid foam.

9: 1H NMR (CDCI₃): δ 9.26 (d, J = 2.8 Hz, IH), 8.42-8.25 (m, 2H), 8.15 (d, J = 7.9 Hz, IH), 7.70-7.56 (m, 2H), 7.03 (d, J = 8.3 Hz, 2H), 6.92 (d, J = 8.3 Hz, 2H), 3.92-2.62 (m,

13H), 1.47 (s, 9H).

General procedure for the synthesis of 7, 10-18

To a solution of respective RiCl (0.11 mmol) in anhydrous CH₂C1₂ (1 mL) at 0°C is added a solution of 9 (0.03g, 0.055mmol), Et₃N (0.015 mL, 0.11 mmol) and DMAP (0.007 g, 0.055 mmol) in CH₂C1₂ (1 mL) and the mixture stirred for 30 min. The solvent is removed and the resulting crude product is purified by preparative TLC using

5% MeOH in CHC1₃ to furnish 7, 10-18.

[N-(5-IsoquinoIinesulfonyl)-O-quinolinesulfonyl-L-tyrosyl]-Boc-piperazine (7) 1H

NMR (CDCI3): δ 9.45-9.20 (m, 2H), 8.40-8.18 (m, 6H), 7.70-7.52 (m, 4H), 6.83 (d, J = 8.5 Hz, 2H), 6.74 (d, J = 8.5 Hz, 2H), 5.99 (d, J = 9.3 Hz, IH), 4.40-4.20 (m, IH), 3.15-

2.49 (m, 10H), 1.45 (s, 9H). [N-Ethy!oxycarbonyl-0-quinolinesulfonyl-L-tyrosyl]-Boc-piperazine (10) 1H NMR (CDC1₃): δ 9.27 (d, J = 2.4 Hz, IH), 8.32 (d, J = 7.4 Hz, 2H), 8.15 (d, J = 7.9 Hz, IH), 7.72-7.52 (m, 2H), 7.04 (d, J = 8.2 Hz, 2H), 6.91 (d, J = 8.2 Hz, 2H), 5.47 (d, J = 7.9 Hz, IH), 4.82-4.64 (m, IH), 4.06 (q, J = 7.1 Hz, 2H), 3.52-2.64 (m, 10H), 1.47 (s, 9H), 1.21 9(t, J = 7.1 Hz, 3H).

[N-Cbz-O-quinolinesuIfonyl-L-tyrosyl]-Boc-piperazine (11) 1H NMR (CDC1₃): δ 9.27 (d, J = 3.9 Hz, IH), 8.31 (d, J = 8.2 Hz, 2H), 8.14 (d, J = 7.9 Hz, IH), 7.68-7.52 (m, 2H), 7.44-7.20 (m, 5H), 7.02 (d, J = 7.9 Hz, 2H), 6.90 (d, J = 7.9 Hz, 2H), 5.63 (d, J =

8.5 Hz, IH), 5.05 (s, 2H), 4.82-4.68 (m, IH), 3.71 (dd, J = 13.7, 6.87 Hz, IH), 3.50-2.60 (m, 9H), 1.48 (s, 9H).

[N-Benzoyl-O-quinolinesulfonyl-Ltyrosyl]-Boc-piperazine (12) ¹H NMR (CDC1₃): δ 9.27 (d, J = 3.8 Hz, IH), 8.31 (d, J = 7.9 Hz, 2H), 8.14 (d, J = 8.2 Hz, IH), 7.74 (d, J =

7.6 Hz, 2H), 7.68-7.54 (m, 2H), 7.54-7.34 (m, 3H), 7.08 (d, J - 8.5 Hz, 2H), 6.92 (d, J -

8.2 Hz, 2H), 5.25 (m, IH), 3.56-2.68 (m, 10H), 1.47 (s, 9H). [N-Methanesulfonyl-0-quinolinesulfonyl-L-tyrosyl]-Boc-piperazine (13) 1H NMR (CDCI₃): δ 9.27 (d, J = 3.9 Hz, IH), 8.33 (d, J = 7.9 Hz, 2H), 8.15 (d, J = 9.0 Hz, IH), 7.70-7.55 (m, 2H), 7.06 (d, J = 8.2 Hz, 2H), 6.97 (d, J = 8.2 Hz, 2H), 5.50 (bs, IH), 4.47 (bs, IH), 3.66-3.44 (m, IH), 3.42-2.80 (m, 9H), 2.62 (s, 3H), 1.47 (s, 9H). [N-Benzenesulfonyl-0-quinolinesulfonyl-L-tyrosyl]-Boc-piperazine (14) 1H NMR (CDCI₃): δ 9.25 (s, IH), 8.36-8.24 (m, 2H), 8.14 (d, J = 7.9 Hz, IH), 7.74 (d, J = 7.4 Hz, 2H), 7.68-7.38 (m, 5H), 6.97 (d, J = 7.9 Hz, 2H), 6.87 (d, J = 7.9 Hz, 2H), 5.80 (d, J =

9.3 Hz, IH), 4.34-4.18 (m, IH), 3.10-2.48 (m, 10H), 1.45 (s, 9H). [N-Toluenesulfonyl-O-quinolinesulfonyl-L-tyrosyl] -Boc-piperazine (15) 1H NMR (CDCI₃): δ 9.26 (d, J = 2.5 Hz, IH), 8.38-8.24 (m, 2H), 8.15 (d, J = 8.2 Hz, IH), 7.70- 7.54 (m, 4H), 7.32-7.18 (m, 2H), 6.96 (d, J = 8.2 Hz, 2H), 6.88 (d, J = 8.2 Hz, 2H), 5.72 (bs, IH), 4.24 (s, IH), 3.18-2.44 (m, 10H), 2.38 (s, 3H), 1.46 (s, 9H). [N-(4-Methoxybenzenesulfonyl)-O-quinolinesulfonyl-L-tyrosyl]-Boc-piperazine (16) 1H NMR (CDCI₃): δ 9.25 (d, J = 3.9 Hz, IH), 8.31 (d, J = 8.2 Hz, 2H), 8.15 (d, J - 8.2 Hz, IH), 7.72-7.56 (m, 4H), 7.06-6.80 (m, 6H), 5.70 (d, J = 12.6 Hz, IH), 4.30-4.14 (m, IH), 3.83 (s, 3H), 3.22-2.42 (m, 10H), 1.46 (s, 9H).

[N-(l-Naphthylsulfonyl)-O-quinolinesuIfonyl-L-tyrosyl] -Boc-piperazine (17) ^!H NMR (CDCI₃): δ 9.25 (d, J - 2.5 Hz, IH), 8.52 (d, J = 8.5 Hz, IH), 8.38-8.22(m, 2H), 8.20-8.08 (m, 2H), 8.04 (d, J = 8.2 Hz, IH), 7.90 (d, J = 7.7 Hz, IH), 7.76-7.52 (m, 4H), 7.47 (t, J = 7.7 Hz, IH), 6.83 (d, J - 8.5 Hz, 2H), 6.75 (d, J = 8.5 Hz, 2H), 5.94 (bs, IH), 4.30-4.15 (m, IH), 3.0-2.32 (m, 10H), 1.45 (s, 9H). [N-(2-Naphthylsulfonyl)-O-quinolinesulfonyl-L-tyrosyl]-Boc-piperazine (18) 1H

NMR (CDCI₃): δ 9.25 (d, J = 2.2 Hz, IH), 8.38-8.22 (m, 3H), 8.13 (d, J = 8.2 Hz, IH), 7.96-7.80 (m, 3H), 7.76-7.52 (m, 5H), 6.95 (d, J = 8.2 Hz, 2H), 6.86 (d, J = 8.2 Hz, 2H), 5.83 (bs, IH), 4.28 (m, IH), 3.0-2.70 (m, 5H), 2.70-2.25 (m, 5H), 1.41 (s, 9H). [N-Cbz-L-tyrosyl] -Boc-piperazine (19)

A mixture of Cbz-Tyr-OH (54) (0.4 g, 1.3 mmol, Aldrich, Milwaukee, WI), Boc- piperazine (0.24 g, 1.3 mmol) and BOP-C1 (0.33 g, 1.3 mmol) in anhydrous CH₂C1₂ (5 mL) is treated with Et₃N (0.36 mL, 2.6 mmol), at room temperature and stirred for 5 h. The reaction mixture is concentrated under vacuum, and the crude product obtained is purified using flash chromatography using 10% MeOH in CHC1₃ to furnish 0.4 g of 19. 19: 1H NMR (CDC1₃): δ 7.34 (s, 5H), 7.01 (d, J - 8.2 Hz, 2H), 6.72 (d, J = 8.2 Hz, 2H), 6.16 (bs, IH), 5.70 (d, J = 8.5 Hz, IH), 5.09 (ABq, J = 12.4 Hz, 2H), 4.90-7.77 (m, IH), 3.60-3.40 (m, 2H), 3.40-3.14 (m, 4H), 3.08-2.80 (m, 4H), 1.45 (s, 9H). General procedure for the synthesis of 20-31 To a solution of respective RiCl (0.124 mmol) in anhydrous CH₂C1₂ (1 mL) at 0°C is added a solution of 19 (0.03g, 0.062 mmol), Et₃N (0.017 mL, 0.124 mmol) and DMAP (0.007 g, 0.055 mmol) in CH₂C1₂ (1 mL) and the mixture stirred for 30 min. The solvent is removed, and the crude product is purified by preparative TLC using 5% MeOH in CHC1₃ to provide compounds 20-31. [N-Cbz-0-Methanesulfonyl-L-tyrosyl]-Boc-piperazine (20) 1H NMR (CDC1₃): δ 7.4- 7.3 (m, 5H), 7.23 (d, J = 8.8 Hz, 2H), 7.19 (d, J = 8.8 Hz, 2H), 5.70 (d, J = 8.2 Hz, IH), 5.07 (ABq, J = 12.1 Hz, 2H), 4.92-4.80 (m, IH), 3.60-3.12 (m, 8H), 3.11 (s, 3H), 3.06- 2.72 (m, 2H), 1.45 (s, 9H). [N-Cbz-0-Menzenesulfonyl-L-tyrosyl]-Boc-piperazine (21) 1HNMR (CDC1₃): δ 7.82 (d, J = 7.7 Hz, 2H), 7.62-7.48 (m, 3H), 7.34 (s, 5H), 7.10 (d, J = 7.9 Hz, 2H), 6.88 (d, J = 7.9 Hz, 2H), 5.65 (d, J = 8.5 Hz, IH), 5.07 (ABq, J = 12.4 Hz, 2H), 4.88-4.76 (m, IH), 3.60-2.65 (m, 10H), 1.44 (s, 9H).

[N-Cbz-0-Toluenesulfonyl-L-tyrosyl]-Boc-piperazine (22) 1HNMR (CDC1₃): δ 7.69 (d, J = 7.9 Hz, 2H), 7.42-7.28 (m, 7H), 7.09 (d, J = 7.9 Hz, 2H), 6.88 (d, J = 7.9 Hz, 2H), 5.65 (d, J = 8.5 Hz, IH), 5.07 (ABq, J = 12.6 Hz, 2H), 4.90-4.76 (m, IH), 3.60-3.70 (m, 10H), 2.45 (s, 3H), 1.44 (s, 9H).

[N-Cbz-0-(4-Methoxylbenzenesulfonyl)-L-tyrosyl]-Boc-piperazine (23) 1H NMR (CDCI₃): δ 7.83 (d, J = 8.8 Hz, 2H), 7.50-7.40 (m, 5H), 7.20 (d, J = 8.3 Hz, 2H), 7.09 (d, J - 8.8 Hz, 2H), 6.99 (d, J = 8.8 Hz, 2H), 5.75 (d, J = 8.51 Hz. IH), 5.18 (ABq, J = 12.4 Hz, 2H), 5.0-4.84 (m, IH), 3.99 (s, 3H), 3.66-2.80 (m, 10H), 1.55 (s,9H). [N-Cbz-O-(l-Naphthylsulfonyl)-L-tyrosyl]-Boc-piperazine (24) 1H NMR (CDC1₃): δ 8.81 (d, J = 8.5 Hz, IH), 8.13 (d, J = 8.2 Hz, IH), 8.11-7.94 (m, 2H), 7.92-7.64 (m, 2H), 7.49 (t, J = 7.7 Hz, IH), 7.32 (s, 5H), 6.99 (d, J = 8.2 Hz, 2H), 6.74 (d, J = 8.2 Hz, 2H), 5.62 (d, J - 8.2 Hz, IH), 5.05 (ABq, J= 12.1 Hz, 2H), 4.86-4.70 (m, IH), 3.58-2.64 (m, 10H), 1.47 (s, 9H).

[N-Cbz-O-(2-Naphthylsulfonyl)-L-tyrosyl]-Boc-piperazine (25) 1H NMR (CDC1₃): δ 8.38 (s, IH), 8.05-7.86 (m, 3H), 7.86-7.76 (m, IH), 7.75-7.45 (m, 2H), 7.32 (s, 5H), 5.61 (d, J = 8.5 Hz, IH), 5.05 (ABq, J = 12.6 Hz, 2H), 4.88-4.68 (m, IH), 3.54-2.68 (m, 10H), 1.45 (s, 9H). [N-Cbz-O-(5-Isoquinolinesulfonyl)-L-tyrosyl]-Boc-piperazine (26) 1HNMR

(CDC1₃): δ 9.43 (s, IH), 8.83 (d, J = 6.0 Hz, IH), 8.55 (d, J = 6.0 Hz, 1H), 8.30-8.21 (m, 2H), 7.67 (t, J = 7.7 Hz, IH), 7.37-7.30 (m, 5H), 7.02 (d, J - 8.2 Hz, 2H), 6.74 (d, J = 8.2 Hz, 2H), 5.58 (d, J = 8.8 Hz, IH), 5.05 (ABq, J = 3.3 Hz, 2H), 4.81-4.73 (m, IH), 3.76-2.75 (m, 10H), 1.46 (s, 9H). [N,O-Bis-Cbz-L-tyrosyl]-Boc-piperazine (27) 1H NMR (CDC1₃): δ 7.48-7.28 (m, 10 H), 7.19 (d, J = 8.2 Hz, 2H), 7.19 (d, J = 8.2 Hz, 2H), 5.68 (d, J = 8.5 Hz, IH), 5.25 (s, 2H), 5.08 (ABq, J = 12.36 Hz, 2H), 4.94-3.78 (m, IH), 3.60-2.68 (m, 10H), 1.44 (s, 9H). [N-Cbz-O-Benzoyl-L-tyrosyl]-Boc-piperazine (28) 1H MR (CDC1₃): δ 8.18 (d, J = 7.1 Hz, 2H), 7.68-7.42 (m, 3H), 7.25 (d, J = 8.2 Hz, 2H), 7.13 (d, J = 8.2 Hz, 2H), 5.78 (d, J = 8.2 Hz, IH), 5.10 (ABq, J = 12.4 Hz, 2H), 4.88-4.80 (m, IH), 3.62-2.80 (m, 10H), 1.45 (s, 9H).

[N-Cbz-O-Ethoxycarbonyl-L-tyrosyl]-Boc-piperazine (29) 1H NMR (CDC1₃): δ 7.34 (s, 5H), 7.19 (d, J = 8.5 Hz, 2H), 7.09 (d, J - 8.2 Hz, 2H), 5.79 (d, J = 8.5 Hz, IH), 5.09 (ABq, J = 12.3 Hz, 2H), 4.94-4.74 (m, IH), 4.29 (q, J = 7.1 Hz, 2H), 3.58-2.70 (m, 10H), 1.44 (s, 9H), 1.38 (t, J = 7.1 Hz, 3H).

[N-Cbz-O-Acetyl-L-tyrosyl]-Boc-piperazine (30) 1H NMR (CDC1₃): δ 7.34 (s, 5H), 7.19 (d, J = 8.2 Hz, 2H), 6.99 (d, J = 8.2 Hz, 2H), 5.70 (d, J = 8.5 Hz, IH), 5.09 (ABq, J - 12.3 Hz, 2H), 4.88-4.84 (m, IH), 3.51-2.76 (m, 10H), 2.27 (s, 3H), 1.44 (s, 9H). [N-Cbz-O-Propionyl-L-tyrosyl]-Boc-piperazine (31) 1H NMR (CDC1₃): δ 7.34 (s, 5H), 7.18 (d, J = 8.2 Hz, 2H), 6.99 (d, J = 8.2 Hz, 2H), 5.71 (d, J = 8.2 Hz, IH), 5.09 (ABq, J = 12.6 Hz, 2H), 4.88-4.84 (m, IH), 3.48-2.78 (m, 10H), 2.57 (q, J = 7.4 Hz, 2H), 1.44 (s, 9H), 1.24 (t, J = 7.4 Hz, 3H). General procedure for the synthesis of 32-40, 42, 43 A solution of 21 (0.03g, 0.057 mmol), Et₃N (0.017 mL, 0.124 mmol) and DMAP (0.007 g, 0.055 mmol) in CH₂C1₂ (1 mL) is added to a solution of the respective RiCl (0.124 mmol) in anhydrous CH₂C1₂ (1 mL) at 0 °C. The mixture is stirred for 30 min. The solvent is removed, and the crude product is purified by preparative TLC using 5% MeOH in CHC1₃ to provide compounds 32-40, 42, 43.

[N-Cbz-O-BenzenesuIfonyI-L-tyrosyl]toluenesulfonylpiperazine (32) 1H NMR (CDC1₃): δ 7.90-7.76 (m, 2H), 7.76-7.64 (m, IH), 7.64-7.48 (m, 4H), 7.42-7.28 (m, 7H), 7.02 (d, J = 8.5 Hz, 2H), 6.81 (d, J = 8.5 Hz. 2H), 5.57 (d, J - 8.5 Hz, IH), 5.03 (s, 2H), 4.80-4.64 (m,

IH), 3.90-3.68 (m, IH), 3.50-2.52 (m, 9H), 2.45 (s, 3H).

[N-Cbz-O-BenzenesulfonyI-L-tyrosyl]methanesulfonylpiperazine (33) 1H NMR

(CDC1₃): δ 7.85 (d, J = 7.4 Hz, 2H), 7.69 (t, J = 7.4 Hz, IH), 7.62-7.52 (m, 2H), 7.40-7.28

(m, 5H), 7.14 (d, J = 8.3 Hz, 2H), 6.95 (d, J = 8.5 Hz, 2H), 5.61 (d, J = 8.8 Hz, IH), 5.08 (t, J = 12.9 Hz, 2H), 4.88-4.72 (m, IH), 3.70-2.80 (m, 9H), 2.73 (s, 3H), 2.60-2.44 (m, IH).

[N-Cbz-O-Benzenesulfonyl-L-tyrosyl]benzenesuIfonylpiperazine (34) ^!H NMR

(CDCI₃): δ 7.83 (d, J = 7.9 Hz, 2H), 7.76-7.50 (m, 8H), 7.40 (m, 5H), 7.01 (d, J = 8.2 Hz,

2H), 6.79 (d, J = 8.2 Hz, 2H), 5.03 (t, J = 12.6 Hz, 2H), 4.78-4.62 (m, IH), 3.84-3.64 (m,

IH), 3.44-2.45 (m, 9H). [N-Cbz-O-benzenesulfonyl-L-tyrosyl]-(4-methoxybenzenesulfonyl)piperazine (35) 1H

NMR (CDCI₃): δ 7.90-7.80 (m, 2H), 7.74-7.52 (m, 5H), 7.38-7.28 (m, 5H), 7.03 (d, J = 9.1

Hz, 4H), 6.82 (d, J -8.5 Hz, 2H), 5.58 (d, J = 8.5 Hz, IH), 5.03 (t, J = 12.4 Hz, 2H), 4.78-

4.64 (m, IH), 3.89 (s, 3H), 3.84-3.70 (m, IH), 3.50-2.52 (m, 9H).

[N-Cbz-O-Benzenesulfonyl-L-tyrosyl]-(l-naphthylsulfonyl)piperazine (36) 1H NMR (CDCI₃): δ 8.78 (d, J - 8.24 Hz, IH), 8.32-8.16 (m, 2H), 8.06 (d, J - 8.2 Hz, IH), 7.92 (d, J

= 8.2 Hz, 2H), 7.84-7.60 (m, 6H), 7.46-7.32 (m, 5H), 7.10 (d, J = 8.2 Hz, 2H), 6.90 (d, J =

8.2 Hz, 2H), 5.64 (d, J = 8.2 Hz, IH), 5.11 (q, J - 12.4 Hz, 2H), 4.88-4.72 (m, IH), 3.94-

2.82 (m, 10H).

[N-Cbz-O-Benzenesulfonyl-L-tyrosyl]-(2-naphthylsulfonyl)piperazine (37) 1H NMR (CDCI₃): δ 8.39 (s, IH), 8.18-8.0 (m, 3H), 7.91 (d, J = 8.5 Hz, 2H), 7.84-7.70 (m, 4H),

7.70-7.62 (m, 2H), 7.46-7.30 (m, 5H), 7.11 (d, J = 8.5 Hz, 2H), 6.90 (d, J = 8.5 Hz, 2H),

5.63 (d, J = 8.5 Hz, IH), 5.08 (ABq, J = 12.4 Hz, 2H), 4.86-4.74 (m, IH), 4.02-3.86 (m,

IH), 3.60-3.15 (m, 4H), 3.10-2.78 (m, 5H).

[N-Cbz-O-Benzenesulfonyl-L-tyrosyl]-Cbz-piperazine (38) 1H NMR (CDC1₃): δ 7.80 (d, J = 7.69 Hz, 2H), 7.69-7.58 (m, IH), 7.58-7.44 (m, 2H), 7.44-7.26 (s, 10H), 7.09 (d, J = 8.2

Hz, 2H), 6.89 (d, J = 8.2 Hz, 2H), 5.62 (d, J = 8.2 Hz, IH), 5.22-5.0 (m, 4H), 4.88-4.74 (m,

IH), 3.62-2.74 (m, 10H).

[N-Cbz-O-Benzenesulfonyl-L-tyrosyl]ethoxycarbonylpiperazine (39) 1H NMR (CDC1₃): δ 7.83 (d, J = 7.4 Hz, 2H), 7.72-7.62 (m, IH), 7.60-7.48 (m, 2H), 7.42-7.28 (m, 5H), 7.10 (d, J 8.5 Hz, 2H), 6.89 (d, J = 8.5 Hz, 2H), 5.63 (d, J = 8.5 Hz, IH), 5.07 (ABq, J = 12.4 Hz, 2H), 4.88- 4.76 (m, IH), 4.13 (q, J = 7.1 Hz, 2H), 3.62-2.78 (m, 10H), 1.25 (t, J = 7.1 Hz, 3H).

[N-Cbz-O-Benzenesulfonyl-L-tyrosyl]benzoylpiperazine (40) ¹H NMR (CDC1₃): δ 7.85 (d, J = 7.7 Hz, 2H), 7.72-7.50 (m, 3H), 7.46-7.28 (m, 10H), 7.12 (d, J = 8.2 Hz, 2H), 6.91 (d, J = 8.2 Hz, 2H), 5.65 (d, J = 8.8 Hz, IH), 5.07 (ABq, J = 12.4 Hz, 2H), 4.82 (bs, IH), 3.65-2.85 (m, 10H).

[N-Cbz-O-BenzenesulfonyI-L-tyrosyl]acetylpiperazine (42) 1H NMR (CDC1₃): δ 7.83 (d, J = 7.7 Hz, 2H), 7.72-7.64 (m, IH), 7.55 (t, J = 7.7 Hz, 2H), 7.34 (s, 5H), 7.13 (t, J = 8.7 Hz, 2H), 6.91 (t, J = 8.7 Hz, 2H), 5.64 (bs, IH), 5.08 (s, 2H), 4.86-4.80 (m, IH), 3.53-2.94 (m, 10H), 2.08 (s, 3H).

[N-Cbz-O-Benzenesulfonyl-L-tyrosyl]propionylpiperazine (43) ¹H NMR (CDC1₃): δ 7.82 (d, J = 7.1 Hz, 2H), 7.66 (t, J = 7.1 Hz, IH), 7.54 (t, J = 7.1 Hz, 2H), 7.33 (s, 5H), 7.12 (t, J = 8.8 Hz, 2H), 6.90 (t, J = 8.8 Hz, 2H), 5.62 (bs, IH), 5.07 (s, 2H), 4.86-4.78 (m, IH), 3.58-2.89 (m, 10H), 2.31 (q, J = 7.4 Hz, 2H), 1.12 (t, J = 7.4 Hz, 3H). [N-Cbz-O-Benzyl-L-tyrosyI]Boc-piperazine (44)

To a mixture of Cbz-Tyr(Bzl)OH (55a) (0.5 g, 1.23 mmol), Boc-piperazine (0.23g, 1.23 mmol), BOP-Cl (0.31 g, 1.23 mmol) in CH₂C1₂ (10 mL) is added Et₃N (0.343 mL, 2.46 mmol) and stirred at room temperature for 5 h. The solvent is removed under vacuum, and the crude material obtained is purified by flash chromatography using 5% MeOH in CHC1₃ to furnish 0.5 g of 44 as a solid foam. 1H NMR (CDC1₃): δ 7.46 (m, 5H), 7.08 (d, J = 8.3 Hz, 2H), 6.87 (d, J - 8.5 Hz, 2H), 5.66 (d, J = 8.5 Hz, IH), 5.08 (ABq, J = 12.1 Hz, 2H), 5.04 (s, 2H), 4.90-4.74 (m, IH), 3.62-2.70 (m, 10H), 1.44 (s, 9H). [N-Boc-O-Benzyl-L-tyrosyl] -Boc-piperazine (45) A mixture of Boc-Tyr(Bzl)OH (55b) (0.03 g, 0.08 mmol), Boc-piperazine (0.015 g, 0.08 mmol), BOP-Cl (0.02 g, 0.08 mmol) in CH₂C1₂ (2 mL) is treated with Et₃N (0.022 mL, 0.16 mmol) and stirred at room temperature for 5 h. The solvent is removed under vacuum, and the crude material obtained is purified using flash chromatography to furnish 0.30 g of 45 as a solid foam. 1H NMR (CDC1₃): δ 7.46-7.28 (m, 5H), 7.09 (d, J = 8.5 Hz, 2H), 6.88 (d, J = 8.5 Hz, 2H), 5.37 (d, J = 8.5 Hz, IH), 5.04 (s, 2H), 4.84-4.66 (m, IH), 3.60-2.70 (m, 10H), 1.44 (s, 9H), 1.42 (s, 9H).

[N-Boc-O~Benzenesulfonyl-L-tyrosyl]-Boc-piperazine (46)

Compound 45 (0.02 g, 0.037 mmol) is hydrogenated using Pd/C (0.005 g), H₂ at 40 psi for 6 h. The reaction mixture is filtered and concentrated to dryness. A solution of this material (0.012 g, 0.026 mmol), Et₃N (0.008 mL, 0.54 mmol) and DMAP (0.003 g, 0.027 mmol) in CH₂C1₂ is treated with benzene sulfonyl chloride (0.007 mL, 0.054 mmol) at 0°C and stirred at room temperature for 30 min. The reaction mixture is concentrated and purified by prep. TLC using 5% MeOH in CHC1₃ to furnish 0.01 g of 46 as a solid foam. 1H NMR (CDC1₃): δ 7.93 (d, J = 8.2 Hz, 2H), 7.84-7.60 (m, 3H), 7.22 (d, J = 8.2 Hz, 2H), 6.99 (d, J = 8.2 Hz, 2H), 5.44 (s, IH), 4.94-4.80 (m, IH), 3.68-3.24 (m, 10H), 1.54 (s, 9H), 1.51 (s, 9H). [N-Boc-O-Benzyl-N-methyl-L-tyrosyl]-Boc-piperazine (47)

A mixture of Boc-MeTyr(Bzl)OH (55c) (0.5 g, 1.29 mmol), Boc-piperazine (0.24 g, 1.29 mmol), BOP-Cl (0.33 g, 1.29 mmol) in CH₂C1₂ (10 mL) is treated with Et₃N (0.36 mL, 2.58 mmol) and stirred at room temperature for 5 h. The solvent is removed under vacuum, and the crude material obtained is purified by flash chromatography using 5% MeOH in CHC1₃ to furnish 0.5 g of 47 as a solid foam. 1H NMR (CDCI₃): δ 7.48-7.22 (m, 5H), 7.14 (d, J = 8.5 Hz, 2H), 6.87 (d, J = 8.5 Hz, 2H), 5.26-5.14 (m, IH), 5.02 (s, 2H), 3.88-2.84 (m, 10H), 2.79 (s, 3H), 1.45 (s, 9H), 1.35 (s, 9H). [N-Cbz-O-Benzyl-N-methyl-L-tyrosyl] -Cbz-piperazine (48) Compound 47 (0.05 g, 0.09 mmol) was treated with 10% TFA in CH₂C1₂ (2 mL) at room temperature for 5 h. The solvent is removed under vacuum and the crude material is purified by preparative TLC to furnish 0.027 g compound. This product (0.027 g, 0.076 mmol) and Et₃N (0.042 ml, 0.18 mmol) and DMAP (0.009 g, 0.073 mmol)) in CH₂C1₂ (2 mL) is cooled to 0°C and is added Cbz-Cl (0.026 mL, 0.18 mmol) and stirred at room temperature for 30 min. The solvent is removed under vacuum, and the crude material is purified by prep. TLC using 5% MeOH in CHC1₃ to furnish 0.025 g of 48 as a solid foam. 1H NMR (CDCI₃): δ 7.48-7.24 (m, 15H), 7.13 (d, J - 8.5 Hz, 2H), 6.86 (d, J = 8.5 Hz, 2H), 5.32-5.19 (m, IH), 5.12 (s, 2H), 5.05 (s, 2H), 5.02 (s, 2H), 3.76-2.98 (m, 10H), 2.92 (s, 3H). [N-Boc-O-Benzyl-D-tyrosy I] -Boc-piperazine (49) This compound is prepared starting from Boc-D-Tyr(Bzl)OH (Bachem Bioscience, Inc., King of Prussia, PA), by the same method as for the corresponding L-enantiomer, 45. [N-Cbz-O-Benzy 1-L-ty rosyl] -Boc-ethy lenediamine (50)

To a mixture of Cbz-Tyr(Bzl)OH (55a) (0.03g, 0.074 mmol), Boc-ethylenediamine (0.012 mL, 0.074 mmol), Bop-Cl (0.019g, 0.074 mmol) in CH₂C1₂ (2 mL) is added Et₃N (0.01 mL, 0.072 mmol) and stirred at room temperature for 5 h. The solvent is removed under vacuum, and the crude material obtained is purified by flash chromatography using 5% MeOH in CHC1₃ to furnish 50 as a solid foam. 1H NMR (CDC1₃): δ 7.46-7.28 (m, 10H), 709 (d, J = 8.2 Hz, 2H), 6.90 (d, J = 8.2 Hz, 2H), 6.23 (bs, IH), 5.40-5.22 (m, IH), 5.18- 4.98 (m, 4H), 4.82-4.64 (m, IH), 4.40-4.22 (m, IH), 3.38-2.85 (m, 6H), 1.41 (s, 9H).

Table la. Synthetic yields and characterization of some of the compounds of the present invention.

36 C₃₇H₃₅N₃O₈S₂.1.5H₂O 70 CHN

37 C₃₇H₃₅N₃O₈S₂.1.8H₂O 70 CHN

38 C₃₅H₃₅N₃O₈S 70 HRMS

39 C₃₀H₃₃N₃O₈S.0.5H₂O 72 CHN

40 C₃₄H₃₃N₃O₇S.H₂O 77 CHN

41 C ₇H₃₄N O₇S 60 HRMS

42 C₂₉H₃₁N₃θ₇S.H₂θ 68 CHN

43 C₃oH₃₃N₃O₇S 69 CHN

44 C₃₃H₃₉N₃O₆.0.2H₂O 71 CHN

45 C₃oH₄₁N₃O₆.0.2H₂O 69 CHN

46 C₂₉H₃₉N₃O₈S.lH₂O 63 CHN

47 C₃₁H₄₃N₃O₆ 70 CHN

48 C₃₇H₃ N₃O₆ 53 HRMS

49 C₃₀H₄₁N₃O₆.0.2H₂O 70 CHN

50 C₃₁H₃₇N₃O₆ 60 CHN

^a Elemental analyses of ±0.4% are considered acceptable. Acceptable tolerance of ±50 ppm is applied to the high resolution mass spectral (HRMS) determination.

Table lb. Chemical analysis of some of the compounds of the present invention based on High Resolution Mass Spectrometry

EXAMPLE 2

This Example illustrates some of the properties of the compounds in accordance with an embodiment of the present invention. This Example also illustrates a method of measuring the properties of the compounds.

P2X₇ Receptor Channel Activation: All experiments are performed using adherent HEK293 cells stably transfected with cDNA encoding the human P2X₇ receptor. Adherent cells on 12-well polylysine-coated plates are incubated at 37°C in 1 mL physiological salt solution (125 mM NaCl, 5 mM KC1, 1 mM MgCl₂, 1.5 mM CaCl₂, 25 mM NaHEPES (pH 7.5), 10 mM D-glucose, 1 mg/mL BSA). Antagonists are added from lOOOx stock solutions dissolved in DMSO. Cells are pre-incubated with antagonists for 15 min prior to stimulation for 10 min with 3 mM ATP (final concentration). Reactions are terminated by rapid aspiration of the extracellular medium in each well. The adherent cells in each well are then extracted overnight with 1 ml 10% HNO₃. K⁺ content in these nitric acid extracts is assayed by atomic absorbance spectrophotometry. Duplicate or triplicate wells are run for all test conditions in each separate experiment. Antagonist function is measured by the percent inhibition of the K⁺ release triggered by 3 mM ATP in paired wells in the absence of antagonist. Data points represent the mean ± SD of values; the number of separate experiments is indicated in parentheses.

The effects of substitution at R_l5 R₂, and R₃ on inhibition of P2X₇ receptor- mediated ion flux (Table 2) are compared. At the Ri position there is a preference for large hydrophobic groups, linked to the α-amino position through a carbamate, amide or sulfonamide group. Within the group of 6 - 18, containing quinoline sulfonyl at R₂ and Boc at R₃, the derivatives containing quinoline sulfonyl, 6, and Cbz, 11, at the N^α- position are preferred over all other acyl groups and sulfonamides examined and inhibited by >50%. There appears to be a sensitivity of the percent inhibition to the structure at the Ri position. For example, a toluene sulfonamide, 15, produces a greater inhibition than the corresponding benzene sulfonamide.

At the R₂ position, both aryl sulfonyl and benzoyl groups lead to antagonism, while in the unsubstituted case a sulfonyl group is preferred over an acyl group (cf. 21 and 28). A benzyl ether, 44, having the same substituents at Ri and R₃ inhibits to a comparable degree. A variety of substitution of aryl sulfonates (21 - 26), including bicyclics, are generally tolerated for antagonism. The approximate rank order of percent inhibition for aryl sulfonates is p-tolyl, 22; p-methoxyphenyl, 23; phenyl, 21 > α- naphthyl, 24; β-naphthyl, 25. A benzoyl ester at the R₂ position, 28, inhibits to an intermediate degree.

At the R₃ position, a Boc group, present in many of the derivatives including 6 - 31, is well tolerated at the receptor site. Subsequently, for the compounds in which the R₃ position is systematically varied, the order of potency is: benzoyl, 40 ≥Boc, 21, Cbz, 38 > ethyoxycarbonyl, 39. Full concentration-response curves also provide a way of comparison among some selected, potent compounds, including the reference compound 1 (Figure 1). A di- isoquinolinyl, piperazinyl-Boc derivative, 4, displays an IC50 of ~40 nM as an antagonist of P2X₇ receptor-mediated ion flux (Figure IB), and appears to be more potent than the reference compound, 1. N^α-Cbz-Boc derivatives, 11 and 22, are nearly as potent as 1 as a P2X₇ receptor antagonist, with IC50 values of approximately 200 and 300 nM, respectively. Compound 41 also is nearly as potent as 1. The IC50 values of 1 and 41 are approximately 100 and 200 nM, respectively.

Several compounds are tested in preliminary experiments as antagonists at the murine P2X₇ receptor, expressed natively in Bacl.2F5 mouse macrophages. The degree of inhibition by is generally greater at murine than at human P2X₇ receptors. The percent inhibition measured at 3 μM in the mouse macrophages is: 35 (32), 34 (33), 74

(38), 80 (39), 94 (40), 27 (42), and 28 (43).

Several novel ligands displaying high percent inhibition (>40% at 3 μM) are identified, e.g., in order of decreasing potency: 41, 40, 4, 22, 23, 6, 21, 11. The three positions selected for modification, i.e., Ri (at N^α-amine), R₂ (on Tyr side chain), and R₃ (an extension of the exposition), are amenable to substitution with various acyl-type groups. The most potent P2X₇ receptor antagonists contain Cbz at the

Ri position, an aryl sulfonate at the R₂ position, and various acyl groups at the R₃ position. At Ri and R₂, aryl substituents are preferred over alkyl. At R₃, the structural requirements are the most restrictive of the three positions. Carbonyl attachment to the piperazinyl ring is allowed, and t-butyloxycarbonyl- and benzoyl groups are preferred.

Table 2. Antagonistic Effects of Tyrosine Derivatives On Function of Human P2X₇ Receptors Expressed in HEK293 Cells (R is R R₂, or R₃)

R =

H H

Tl COC₆H₄-p-CH₃

Bn CH₂CeH5

32 ZBsTs 0 (1)

33 ZBsMs 0 (1)

34 ZBsBs 0 (1)

35 ZBsMo 0 (1)

36 ZBsN 0 (1)

37 ZBsNb 0 (1)

38 ZBsZ 48 ± 29 (3)

39 ZBsE 24 ± 11 (3)

40 ZBsBz 78 ± 22 (3)

41 ZlqBz 85 ± 10 (3)

42 ZBsA 0 (1)

43 ZBsP 0 (1)

44 ZBnB 45 (1)

45 BBnB^c 14 ± 18 (3)

46 BBsB 6 (1)

47 BBnB^b'^c O ± O (3)

48 ZBnZ^b,c 8 ± 7 (3)

49 BBnB^b,c 4 ± 3 (3)

50 ZBnB^b 0 (1)

47 = N^α-methyl derivative of 45. 48 = N^α-methyl derivative. 49 = D-isomer of 45.

50 = Boc-ethylene diamine (instead of Boc-piperazine) derivative of 44. ^c no inhibition detected at 30 μM.

EXAMPLE 3 This Example illustrates the antagonistic properties, i.e., effects on the function of human P2X₇ receptors expressed in HEK293 cells, of some of the compounds in accordance with an embodiment of the present invention. The antagonistic properties are measured as in Example 2.

Compound Ri = X-CO, R₂=Y-benzene R₃ = Z-CO, % wherein X = sulfonyl, wherein wherein Z = Inhibition Y =

103 Ph-CH=€H H Ph —50

(trans)

104 2-OCH₃-Ph- CH₃ Ph 30 CH=CH (trans)

105 2-OCH₃-Ph- CH₃ Ph 20 CH=CH (cis)

106 3-Cl-Ph- CH₃ Ph 70 CH-CH (trans)

107 2,4,6-(OCH₃)₃-Ph- CH₃ Ph 0 CH-CH (trans)

108 4-NO₂-Ph- H Ph —50 CH=CH (trans)

109 Ph-CH-CH NO₂ Ph >80

(trans)

110 Ph-CH=CH H Ph-4-NO₂ —50

(trans)

111 4-NO₂-Ph- NO₂ Ph 59 CH-CH (trans)

112 4-NO₂-Ph- H Ph-4-NO₂ —50 CH=CH (trans) 113 Ph-CH=CH NO, Ph-4-NO₂ >80

(trans)

114 4-NO₂-Ph- NO₂ Ph-4-NO₂ -0

CH=CH (trans)

115 Ph-CH₂O CH₃ Ph 85

116 3-Cl-Ph-CH₂O CH₃ Ph 50

117 4-NO₂-Ph-CH₂O CH₃ Ph 60

118 3-NO₂-Ph-CH₂O CH, O-C(CH₃)₃

119 3-NO?-Ph-CH,O CH₃ Ph

120a 3-NH₂-Ph-CH₂O CH₃ Ph

120b 3-CH₃CONH-Ph- CH₃ Ph CH₂O

121 3-NCS-Ph-CH₂O CH₃ Ph

122 H₂N(CH₂)₂NH- CH₃ Ph

CSNH-Ph-CH₂O

123 Ph-CH,O NO, O-C(CH₃)₃ —50

124 Ph-CH₂O NH, O-C(CH₃)₃ 85

125 Ph-CH₂O NHCOCH₃ O-C(CH₃)₃

127 Ph-CH₂O NH, Ph

128 Ph-CH₂O NHCOCH3 Ph

129 Ph-CH₇O NCS Ph 30-40

130 Ph-CH₂O H₂N(CH₂)₂NH- Ph CSNH-

131 Ph-CH₂O CH₃ Ph-4-NO₂

132 Ph-CH₂O CH₃ Ph-4-NH₂ >80

133 Ph-CH₂O CH₃ Ph-4-NH₂COCH₃ —50

134 Ph-CH₂O CH₃ Ph-4-NCS 30-40

135 Ph-CH₂O CH₃ Ph-4-NHCS- NH(CH₂)₂NH₂

DIMERS

223 R₂R₃ 20

Table 3. Antagonistic Effects of Tyrosine Derivatives On

Function of Human P2X₇

Receptors Expressed in HEK293 Cells.³

Compound Ri R R₃ % Inhibition n =

Ph-CH=CH H Ph 62+4

MRS 2439 (trans)

2-OCH₃-Ph- 4-CH₃ Ph 27+2

MRS 2405 CH=CH (trans)

2-OCH₃-Ph- 4-CH₃ Ph 17+1

MRS 2406 CH=CH (cis)

3-Cl-Ph- 4-CH₃ Ph 57+5

MRS 2383 CH=CH (trans)

2,4,5-(OCH₃)₃-Ph- 4-CH₃ Ph -2+6

MRS 2407 CH=CH (trans)

4-NO₂-Ph- H Ph 43+20

MRS 2437 CH=CH (trans)

Ph-CH=CH 4-NO, Ph 72+4

MRS 2435 (trans)

Ph-CH=CH H PI1-4-NO2 41+1

MRS 2440 (trans) 4-NO₂-Ph- 4-NO₂ Ph 58+0.6 MRS 2433 CH=CH (trans)

4-NO₂-Ph- H Ph-4-NO₂ 30+2 MRS 2438 CH=CH (trans)

Ph-CH=CH 4-NO₂ Ph-4-NO₂ 43±14 MRS 2436 ^(trans)

4-NO₂-Ph- 4-NO₂ Ph-4-NO, 4+9 MRS 2434 CH=CH (trans)

Ph-CH₂O 4-CH₃ Ph 71+21 MRS 2427

2-Cl-Ph-CH₂O 4-CH3 Ph 43+17

MRS 2385

4-NO₂-Ph-CH₂O 4-CH₃ Ph 69+25

MRS 2386

3-NO₂-Ph-CH₂O 4-CH₃ O-C(CH₃)₃ 94±28

MRS 2464

3-NO₂-Ph-CH₂O 4-CH₃ Ph 72+6

MRS 2465

3-NH₂-Ph-CH₂O 4-CH₃ Ph 50+6

MRS 2466

3-CH₃CONH-Ph- 4-CH₃ Ph 9+13

MRS 2479 CH₂O

3-NCS-Ph-CH₂O 4-CH₃ Ph -4+5

MRS 2467 3-[H₂N(CH₂)₄NH- 4-CH₃ Ph 17+4 MRS 2482 CSNH]-Ph-CH₂O

Ph-CH₂O 3-NO, P-C(CH₃)₃ 74+2

MRS 2472

Ph-CH₂O 4-NO₂ O-C(CH₃)₃ 30±28

MRS 2442

Ph-CH₇O 4-NH₂ O-C(CH₃)₃ 64±17

MRS 2444

Ph-CH₂O 4-NHCOCH₃ O-C(CH₃)₃ 64±27

MRS 2445

Ph-CH₇O 3-NO, Ph 75+10

MRS 2473

MRS 2447

Ph-CH₂O 4-NH, Ph 56+9 2

MRS 2450

Ph-CH₂O 4-NHCOCH₃ Ph 31+41 2

MRS 2451

Ph-CH₇O 4-NCS Ph 72+1

MRS 2449

Ph-CH₂O 4- Ph 80+11 3

MRS 2483 [H₂N(CH₂)₄NH-

CSNH]- Ph-CH₂O 4-CH₃ Ph-3-NO₂ 60±16

MRS 2468

Ph-CH₂O 4-CH₃ PI1-4-NO2 61+6 MRS 2446

Ph-CH₂O 4-CH₃ Ph-4-NH₂ 93+3

MRS 2452

Ph-CHoO 4-CH₃ Ph-4-NHCOCH₃ 45+21

MRS 2456

Ph-CH₇O 4-CH₃ Ph-4-NCS 30+16

MRS 2448

Ph-CH₂O 4-CH, Ph-4-NHCS- 14+19

MRS 2475 NH(CH₂)₂NH₂

Ph-CH₂O 4-CH, Ph-4-NHCS- 63+16

MRS 2484 NH(CH₂)₄NH₂ DIMERS

R1R1 -5+4

MRS 2474

R3R3 26 1

MRS 2453

R2R3 61+29 2

MRS 2454

R2R2 54+42 3

MRS 2455 a All experiments were performed using adherent HEK293 cells stably transfected with cDNA encoding the human P2X₇ receptor. Adherent cells on 12-well polylysine-coated plates were incubated at 37°C in 1 ml physiological salt solution (125 mM NaCl, 5 mM KCl, 1 mM MgCl , 1.5 mM CaCl₂, 25 mM NaHEPES (pH 7.5), 10 mM D-glucose, 1 mg/ml BSA). Antagonists (3 μM final concentration) were added from lOOOx stock solutions dissolved in DMSO. Cells were pre-incubated with antagonists for 15 min prior to stimulation for 10 min with 3 mM ATP (final concentration). Reactions were terminated by rapid aspiration of the extracellular medium in each well. The adherent cells in each well were then extracted overnight with 1 ml 10% HNO₃, and the K⁺ content in the extracts was assayed by atomic absorbance spectrophotometry. Duplicate or triplicate wells were run for all test conditions in each separate experiment and the measured K⁺ contents were averaged. Antagonist function was measured by the percent inhibition of the K⁺ release triggered by 3 mM ATP in paired wells in the absence of antagonist. Data points represent the mean ± SD of values obtained.

Table 4. Antagonistic Effects of Tyrosine Derivatives On Function of Human P2X₇ Receptors Expressed in HEK293 Cells.^a

* -.-configuration, unless noted. Compound Ri R₇ R₃ X,Y= % n =

Inhibitio n

MONOMERI C

MRS2427 H 4-CH₃-Ph Ph 71+21

MRS 2509 H 4-CH₃-Ph Ph Z-Me

MRS 2535 H 4-CH₃-Ph Ph Y-l-R-Me

MRS 2536 H 4-CH₃-Ph Ph Y=l-S-Me, Z = Me

MRS 2534 H 4-CH₃-Ph Ph Y = 2-R-Me

MRS 2531 H 4-CH₃-Ph Ph Y = 2-S-Me

MRS 2540 H 4-CH₃-Ph Ph X = 2,6- (Me)2

MRS 2476 3-SCN- 4-CH₃-Ph Ph -4±5

MRS 2482 3- 4-CH₃-Ph Ph 17+4 2

[H₂N(CH₂)₄

NH-CSNH]- MRS 2546 3- 4-CH₃-Ph Ph [H₂O₃P(CH₂)₂

NH-CSNH]-

.2NH₃

MRS ??? H 4-CH₃-Ph O-C(CH₃)₃

MRS 2505 H 4-CH₃-Ph O-C(CH₃)₃ D-Tyrϋ

MRS 2547 H 4-F-Ph Ph

MRS 2548 H 4-Cl-Ph Ph

MRS 2549 H 4-Br-Ph Ph

MRS 2550 H 4-I-Ph Ph

MRS 2551 H 4-CH₃-Ph 4-F

MRS 2552 H 4-CH₃-Ph 4-C1

MRS 2553 H 4-CH₃-Ph 4-Br

MRS 2554 H 4-CH₃-Ph 4-1 MRS 2451 H 4-NHCOCH₃-Ph Ph 31+41

MRS 2449 H 4-NCS-Ph Ph 72+1

MRS 2498 H Ph

[CH₃CONH(CH₂)

₂NH-CSNH]-Ph

MRS 2483 H 4-[H₂N(CH₂)₄NH- Ph 80+11 CSNH]-Ph

MRS 2499 H 4- Ph [CH₃CONH(CH₂)

₄NH-CSNH]-Ph

MRS 2493 H 4-[(4-SCN- Ph C₆H₄)NHCSNH

(CH₂)₄NH-CSNH] -Ph

MRS 2490 H 4-[(4-SCN- Ph

(CH₂)₄NH- CSNH]-

MRS 2511 H 4-[H₂N(CH₂)₁₂ Ph NH-CSNH]-Ph

MRS 2545 H 4-[H₂O₃P(CH₂)₂ Ph

NH-CSNH]-Ph

.2NH₃ MRS 2537 H 4-[(C₂H₅)₂O₃P Ph

(CH₂)₂ NH-CSNH]-Ph

MRS 2542 H 4-CH₃-Ph Ph-4-N(CH₃)₂

MRS 2541 H 4-CH₃-Ph Ph-4-CF₃

MRS 2544 H 4-CH,-Ph Ph-4-F

MRS 2543 H 4-CH₃-Ph Ph-2,4,5-F

MRS 2456 H 4-CH₃-Ph Ph-4-NHCOCH₃ 45±21

MRS 2448 H 4-CH,-Ph Ph-4-NCS 30+16

MRS 2475 H 4-CH₃-Ph Ph-4-NHCS- 14+19

NH(CH₂)₂NH₂

MRS 2555 H 4-CH₃-Ph Ph-4-NHCS-

NH(CH₂)₂PO₃H₂

.2NH₂

DIMERIC Linkage

MRS 2510 MRS 2512

Others

MRS 2489 Binary/ A2A 2 methylene agonist spacer

MRS 2492 Binary/ A2A 4 methylene agonist spacer

a All experiments were performed using adherent HEK293 cells stably transfected with cDNA encoding the human P2X₇ receptor. Cells were pre- incubated with antagonists (3 μM final concentration) for 15 min prior to stimulation for 10 min with 3 mM ATP (final concentration). Antagonist function was measured by the percent inhibition of the K⁺ release triggered by 3 mM ATP in paired wells in the absence of antagonist. Data points represent the mean ± SD of values obtained. 15, MRS 2427; 18, MRS 2464; 26b, MRS 2447; 30, MRS 2483; 32, MRS 2452; 35b, MRS 2484; 66, MRS 2454; 67, MRS 2455.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations of those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above- described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

WHAT IS CLAIMED IS: 1. A compound of the formula

which may be monomeric or dimeric; wherein Ri-Rs, each independently is selected from the group consisting of H, alkyl sulfonyl, alkoxy sulfonyl, aryl sulfonyl, heteroaryl sulfonyl, arylalkenyl sulfonyl, arylalkoxy sulfonyl, alkyl carbonyl, alkoxy carbonyl, alkoxyalkoxy carbonyl, arylalkoxy carbonyl, aryloxy carbonyl, aryl carbonyl, arylalkyl carbonyl, arylalkyl, and arylalkenyl carbonyl; wherein the aryl or heteroaryl portions of Ri-R₃ may have 1-3 aromatic rings, and each of the aromatic ring may be optionally substituted with one or more substituents selected from the group consisting of alkyl, alkoxy, halo, haloalkyl, nitre, amino, isothiocyanato, acetylamino, thioureido, and aminoalkylthioureido; the alkyl and alkoxy portions of Ri-R₃ may have 1-20 carbon atoms; and the heteroaryl includes one or more of N, O, and S atoms; and R₄ is H or alkyl having 1-6 carbon atoms.

2. The compound of claim 1, wherein R₄ is H.

3. The compound of claim 1 or 2, wherein Ri is selected from the group consisting of heteroaryl sulfonyl, aryl sulfonyl, aryl carbonyl, arylalkoxy carbonyl, alkoxy carbonyl; and arylalkenyl carbonyl; R₂ is selected from the group consisting of aryl sulfonyl and arylalkyl; and R₃ is selected from the group consisting of H, alkoxy carbonyl, and arylalkoxy carbonyl.

4. The compound of claim 3, wherein the heteroaryl sulfonyl is quinoline sulfonyl or isoquinoline sulfonyl; the aryl sulfonyl is benzene sulfonyl, /?-toluene sulfonyl, or naphthalene sulfonyl; the aryl carbonyl is benzoyl or toluoyl; arylalkoxy carbonyl is benzyloxy carbonyl, or fluorenylmethoxy carbonyl; the alkoxy carbonyl is t-butoxy carbonyl or ethoxy carbonyl; the arylalkyl is benzyl; and the arylalkenyl carbonyl is phenylethenyl carbonyl.

5. The compound of claim 4, wherein Ri is isoquinoline 5-sulfonyl; R₂is isoquinoline 5-sulfonyl or quinoline 8-sulfonyl; and R₃ is H or t-butoxy carbonyl.

6. The compound of claim 4, wherein _t and R₂ are quinoline 8-sulfonyl and R₃ is H or t-butoxy carbonyl.

7. The compound of claim 4, wherein Ri is ethoxy carbonyl; R₂ is quinoline 8- sulfonyl; and R₃ is t-butoxy carbonyl.

8. The compound of claim 4, wherein Ri is benzyloxy carbonyl or benzoyl; R₂ is quinoline 8-sulfonyl; and R₃ is t-butoxy carbonyl.

9. The compound of claim 4, wherein R^ is benzene sulfonyl, -toluene sulfonyl, p- methoxy benzene sulfonyl, or 1 -naphthalene sulfonyl; R₂ is quinoline 8-sulfonyl; and R₃ is t-butoxy carbonyl.

10. The compound of claim 4, wherein Ri is benzyloxy carbonyl; R₂ is benzyl, benzene sulfonyl, p-to iene sulfonyl, >-methoxy benzene sulfonyl, naphthalene 1- sulfonyl, or naphthalene 2-sulfonyl; and R₃ is t-butoxy carbonyl.

11. The compound of claim 4, wherein Ri is benzyloxy carbonyl; R₂ is benzene sulfonyl; and R₃ is t-butoxy carbonyl, ethoxy carbonyl, or benzoyl.

12. The compound of claim 4, wherein Ri is benzyloxy carbonyl; R₂ is isoquinoline 5-sulfonyl; and R₃ is benzoyl.

13. The compound of claim 4, wherein R_t is benzyloxy carbonyl; R₂ is benzyl; and R₃ is t-butoxy carbonyl.

14. The compound of claim 4, wherein Ri is traws-phenylethenyl carbonyl; R₂ is benzene sulfonyl or -nitrobenzene sulfonyl; and R₃ is benzoyl or 4-nitrobenzoyl.

15. The compound of claim 4, wherein Ri is tra«s-4-nifrophenylethenyl carbonyl; R₂ is benzene sulfonyl or 4-nitrobenzene sulfonyl; and R₃ is benzoyl or 4-nitrobenzoyl.

16. The compound of claim 4, wherein Ri is tr «s-2-methoxyphenylethenyl carbonyl or trans-3-chlorophenylethenyl carbonyl; R₂ isp-toluene sulfonyl; and R₃ is benzoyl.

17. The compound of claim 4, wherein Ri is benzyloxy carbonyl; R₂ is/?-toluene sulfonyl, 4-nitrobenzene sulfonyl, 4-aminobenzene sulfonyl, or 4- isothiocyanatobenzene sulfonyl; and R₃ is benzoyl, t-butoxy carbonyl, 4- aminobenzoyl, 4-acetylamino benzoyl, or 4-isothiocyanato benzoyl.

18. The compound of claim 4, wherein R_x is 3-chlorobenzyloxy carbonyl or 4-nitro- benzyloxy carbonyl; R₂ is j?-toluene sulfonyl; and R₃ is benzoyl.

19. The compound of any of claims 1-18, which is monomeric.

20. The compound of any of claims 1-18, which is dimeric.

21. The compound of claim 20, wherein the dimeric compound includes two monomeric compounds such that R_l3 R₂, or R₃ of one of the two monomeric compounds is linked to R_ls R₂, or R₃ of the other of the two monomeric compounds through a linker.

22. The compound of claim 21 , wherein the linker comprises a thiourea group.

23. The compound of claim 21 or 22, wherein R₂ of one of the monomeric compounds is linked to R₂ of the other of the two monomeric compounds.

24. The compound of claim 21 or 22, wherein R₂ or R₃ of one of the two monomeric compounds is linked to the R₃ of the other of the two monomeric compounds.

25. The compound of claim 23, which has the formula:

26. The compound of claim 25, wherein Ri is benzyloxy carbonyl, R₃ is benzoyl, and R₄ is H.

27. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of any of claims 1-26.

28. A method of blocking a P2X₇ receptor in a mammal comprising administering to the mammal a compound of any of claims 1-26.

29. A method of blocking an ATP-induced toxic process in a blood cell in a mammal comprising administering to the mammal a compound of any of claims 1-26.

30. The method of claim 29, wherein the toxic process involves release of interleukin-1.

31. A method of treating or preventing septic shock in a mammal comprising administering to the mammal a compound of any of claims 1-26.

32. A method of treating or preventing inflammation in a mammal comprising administering to the mammal a compound of any of claims 1-26.

33. The method of claim 32, wherein the inflammation is ophthalmic inflammation.

34. A method of treating or preventing stroke in a mammal comprising administering to the mammal a compound of any of claims 1-26.

35. A method of treating or preventing a neurodegenerative disease in a mammal comprising administering to the mammal a compound of any of claims 1-26.

36. A method of inhibiting the binding of a ligand to a P2X₇ receptor of a substrate comprising contacting the substrate with a compound of any of claims 1-26 so that the compound binds to the P2X₇ receptor and inhibits the ligand from binding to the P2X₇ receptor.

37. The method of claim 36, wherein the contacting is carried out in vitro.

38. The method of claim 36, wherein the contacting is carried out in vivo.

39. A method of characterizing a P2X₇ receptor site in a substrate comprising contacting the substrate with a compound of any of claims 1-26 and evaluating the interaction of the compound with the P2X₇ receptor.

40. The use of a compound of any of claims 1-26 in medicine.

41. The use of a compound of any of claims 1-26 in the manufacture of a medicament for the treatment or prevention of septic shock, inflammation, stroke, or a neurodegenerative disease.