P2X7r antagonists

Abstract

A compound having formula I: R1 is hydrogen, hydroxyl, halogen, nitro, amino, alkyl, alkoxy, alkylamino, cycloalkyl, cycloalkylamino, heterocyclic, aryl, heteroaryl, -NR7R8, -CO-R 10 or -NH-CO-R 10 L is a bond, a heterocyclic divalent group, a heteroaromatic divalent group, or an aromatic divalent group; M is a bond, an alkyl group, an aryl group, a heterocyclic divalent group, a heteroaromatic divalent group, or an aromatic divalent group; X is a bond, -O-, -S-, -SO2-, -CO-, -NR9-, -(CH2)m-, or a heterocyclic divalent group, where m is 1, 2, 3, 4, 5, or 6; Y is a bond, -NH-, a heterocyclic divalent group, a heteroaromatic divalent group, a divalent benzyl group, or an aromatic divalent group; and Z is hydrogen, halogen, alkyl, aryl, heterocyclic, heteroaromatic, -NR7R8, -CO-R 10 or -NH-CO-R 10 R7, R8, and R9 are independently hydrogen, hydroxyl, halogen, nitro, amino, alkyl, alkoxy, alkylamino, cycloalkyl, cycloalkylamino, heterocyclic, or heteroaryl; and R 10 It can be -O-tert-butyl, -CH2CH2-phenyl, hydrogen, hydroxyl, halogen, nitro, amino, alkyl, alkoxy, alkylamino, cycloalkyl, cycloalkylamino, heterocyclic or heteroaryl.

Description

[0001] This application claims priority to U.S. Provisional Application No. 62 / 869,040, filed July 1, 2019, which is incorporated herein by reference for all purposes as fully set forth herein. Technical Field

[0002] This invention relates to pharmaceutical chemistry, and more particularly, to purinergic receptor P2X7 (P2X7R) antagonists and their use in pharmaceuticals and diagnostics. Background Technology

[0003] P2X7R is a member of ligand-gated ion channels and is expressed in a variety of cell types. It plays a crucial role in development and normal physiological activity. Compared to other P2X receptors, P2X7R exhibits the most prominent role in various pathological states because it is a key factor in inflammation and immune processes. Its expression in peripheral macrophages and monocytes, and especially in glial cells of the nervous system (microglia, astrocytes, oligodendrocytes, and Schwann cells), makes it a therapeutic target for neurodegenerative diseases and other neuropathological conditions. P2X7R is also expressed in antigen-presenting cells, keratinocytes, salivary gland acinar cells, hepatocytes, erythrocytes, erythroleukemia cells, monocytes, fibroblasts, bone marrow cells, neurons, and glomerular cells. In the brain, high-expression regions of P2X7R have been found in the preolfactory nucleus, cerebral cortex, piriform cortex, lateral septal nucleus, hippocampal pyramidal cell layer (CA1, CA3, and CA4), pontine nuclei, lateral cuneate nucleus, and medial vestibular nucleus. P2X7R messenger RNA hybridization signals have also been observed in motor neurons of the trigeminal motor nucleus, facial nerve nucleus, hypoglossal nucleus, and anterior horn of the spinal cord. Studies have shown that P2X7R acts as a scavenger receptor, promoting phagocytosis by directly binding apoptotic cadavers and foreign debris to its extracellular domain in the absence of extracellular adenosine triphosphate (ATP). P2X7R differs from other P2X receptors because it requires high concentrations of ATP for activation, consistent with its involvement in various pathological states. Upon activation by an agonist, the ion channels formed by P2X7R initially allow only small ions to pass through, but slowly shift to a state permeable to both small and large molecules with sustained agonist presence. The elongated macropore opening of P2X7R channels leads to the release of pro-inflammatory cytokines such as IL-1β, IL-6, and IL-18, the chemokine CCL2, and / or tumor necrosis factor TNFα, which promotes inflammation and may regulate other events leading to cell deterioration or even death. Inhibition of P2X7R could provide a novel perspective on anti-inflammatory therapy.

[0004] Accumulated data have shown that P2X7R is a key factor in inflammation. Inhibiting P2X7R activity with pharmaceuticals or inducing P2X7R deficiency through gene deletion, mutation, or silencing can improve various pathological conditions mediated by P2X7R. The compounds of the present invention for the treatment of humans and lower animals, or pharmaceutically acceptable salts thereof, can be applied to the treatment of identified symptoms and preventative care of P2X7R-mediated conditions, including but not limited to: platelet dysfunction, hyperplasia, bone diseases, cancer, cardiovascular diseases, depression, diabetes, fever, gastrointestinal dysfunction, inflammation and inflammatory symptoms, immune diseases, impotence or erectile dysfunction, renal dysfunction, hepatic dysfunction, neurodegenerative diseases and other neuropathological conditions, and pain and pain-related conditions. The following are examples of conditions where P2X7R involvement has been shown: neuropathological conditions such as Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease, spinal cord injury, cerebral ischemia, head trauma, meningitis, sleep disorders, emotional and anxiety disorders, epilepsy, HIV-induced neuroinflammation and CNS damage, chronic neuropathic and inflammatory pain, as well as peripheral inflammatory conditions and autoimmune diseases, including age-related macular degeneration. Sexual dysfunction, airway hyperresponsiveness, allergic dermatitis, asthma, atherosclerosis, bronchitis, burns, chronic obstructive pulmonary disease, Crohn's disease, diabetes, fatty liver disease, fibrosis, glomerulonephritis, growth and metastasis of malignant cells, irritable bowel syndrome, ischemic heart disease, liver fibrosis, emphysema, muscular dystrophy, myeloid leukemia, osteoporosis, osteoarthritis, psoriasis, rheumatoid arthritis, septic shock, Sjogren's syndrome, skin lesions, and ulcerative colitis. Due to the importance of P2X7R in human health, inventing novel P2X7R antagonists represents an attractive avenue for developing new therapeutic agents. While P2X7R antagonists are described in various patent applications, there is a need for new P2X7R antagonists that are effective and deliverable to different target organs mediated by P2X7R in their pathogenesis. P2X7R antagonists also show promising potential in medical diagnostics or targeted drug delivery. Many 11 C and 18 Radiolabeled P2X7R antagonists have been used as positron emission tomography (PET) imaging tracers to elucidate the location and expression of P2X7R in the nervous system or other lesion regions. The development of P2X7R antagonists for the diagnosis and treatment of various diseases represents an attractive prospective view. Summary of the Invention

[0005] In one embodiment, the present invention provides a compound having the following formula I: In Formula I, R1 can be hydrogen, hydroxyl, halogen, nitro, amino, alkyl, alkoxy, alkylamino, cycloalkyl, cycloalkylamino, heterocyclic, aryl, heteroaryl, -NR7R8, or -CO-R. 10 or -NH-CO-R 10 L represents a bond, a heterocyclic divalent group, a heteroaromatic divalent group, or an aromatic divalent group; M represents a bond, an alkyl group, an aryl group, a heterocyclic divalent group, a heteroaromatic divalent group, or an aromatic divalent group; X represents a bond, -O-, -S-, -SO2-, -CO-, -NR9-, or -(CH2). m - or a heterocyclic divalent group, m is 1, 2, 3, 4, 5 or 6; Y is a bond, -NH-, a heterocyclic divalent group, a heteroaromatic divalent group, a divalent benzyl group or an aromatic divalent group; Z is hydrogen, halogen, alkyl, aryl, heterocyclic group, heteroaryl, -NR7R8, -CO-R 10 or -NH-CO-R 10 R7, R8, and R9 are independently hydrogen, hydroxyl, halogen, nitro, amino, alkyl, alkoxy, alkylamino, cycloalkyl, cycloalkylamino, heterocyclic, or heteroaryl; and R 10 It is -O-tert-butyl, -CH2CH2-phenyl, hydrogen, hydroxyl, halogen, nitro, amino, alkyl, alkoxy, alkylamino, cycloalkyl, cycloalkylamino, heterocyclic or heteroaryl; its isomers, its tautomers, its pharmaceutically acceptable solvates, or its pharmaceutically acceptable prodrugs.

[0006] In another implementation, in Equation I, L is And n can be 0, 1, 2, 3, 4 or 5.

[0007] In another embodiment, in Formula I, Y is a divalent phenyl, a divalent naphthyl, a divalent quinolinyl, or a divalent isoquinolinyl.

[0008] In another embodiment, M in Formula I is a bond, and the compound has the following formula II: R1 can be hydrogen, hydroxyl, halogen, nitro, amino, alkyl, alkoxy, alkylamino, cycloalkyl, cycloalkylamino, heterocyclic, aryl, heteroaryl, -NR7R8, or -CO-R. 10 or -NH-CO-R 10 R2, R3, R4, R5, and R6 are independently hydrogen, hydroxyl, halogen, nitro, amino, alkyl, alkoxy, alkylamino, cycloalkyl, cycloalkylamino, heterocyclic, or heteroaryl; L is a bond, a heterocyclic divalent group, a heteroaromatic divalent group, or an aromatic divalent group; X is a bond, -O-, -S-, -SO2-, -CO-, -NR9-, or -(CH2). m-, m is 1, 2, 3, 4, 5 or 6; R7, R8 and R9 are independently hydrogen, hydroxyl, halogen, nitro, amino, alkyl, alkoxy, alkylamino, cycloalkyl, cycloalkylamino, heterocyclic or heteroaryl; and R 10 It can be -O-tert-butyl, -CH2CH2-phenyl, hydrogen, hydroxyl, halogen, nitro, amino, alkyl, alkoxy, alkylamino, cycloalkyl, cycloalkylamino, heterocyclic or heteroaryl.

[0009] In another implementation, in formula II, L is And n can be 0, 1, 2, 3, 4 or 5.

[0010] In another embodiment, in Formula II, R2, R3, R4, R5, and R6 are independently hydrogen, hydroxyl, halogen, nitro, amino, alkyl, alkoxy, alkylamino, cycloalkyl, cycloalkylamino, heteroaryl, etc.

[0011] In another embodiment, in Formula II, the compound is selected from the group consisting of:

[0012]

[0013]

[0014]

[0015] In another implementation, R1-XL- in Formula I is And the compound has the following formula III: M is a bond, alkyl, aryl, heterocyclic divalent group, heteroaromatic divalent group, or aromatic divalent group; and Y is a bond, -NH-, heterocyclic divalent group, heteroaromatic divalent group, divalent benzyl, or aromatic divalent group; Z is hydrogen, halogen, alkyl, aryl, heterocyclic, heteroaromatic, -NR7R8, -CO-R 10 or -NH-CO-R 10 R7 and R8 are independently hydrogen, hydroxyl, halogen, nitro, amino, alkyl, alkoxy, alkylamino, cycloalkyl, cycloalkylamino, heterocyclic, or heteroaryl; and R 10 It can be -O-tert-butyl, -CH2CH2-phenyl, hydrogen, hydroxyl, halogen, nitro, amino, alkyl, alkoxy, alkylamino, cycloalkyl, cycloalkylamino, heterocyclic or heteroaryl.

[0016] In another embodiment, in Formula III, M is a bond; Y is a divalent benzyl group; and Z is hydrogen or a halogen.

[0017] In another embodiment, the compound is selected from the group consisting of:

[0018]

[0019] In another embodiment, in formula III, M is a divalent phenyl group; Y is a bond or -NH-; Z is hydrogen, heterocyclic group, heteroaryl group, -NR7R8, -CO-R 10 or -NH-CO-R 10 R7 and R8 are independently hydrogen or alkyl; and R 10 It can be -O-tert-butyl, -CH2CH2-phenyl, hydrogen, hydroxyl, halogen, nitro, amino, alkyl, alkoxy, alkylamino, cycloalkyl, cycloalkylamino, heterocyclic or heteroaryl.

[0020] In another embodiment, in Formula III, the compound is selected from the group consisting of:

[0021]

[0022] It should be understood that the foregoing overview and the following detailed description are exemplary and illustrative only, and are intended to provide further explanation of the invention as requested. Detailed Implementation

[0023] The embodiments of the present invention will now be described in detail.

[0024] The methods described herein include administering to a subject in need a composition containing a therapeutically effective amount of one or more of the purinergic receptor P2X7 (P2X7R) antagonists described herein, the antagonists comprising their enantiomeric pure forms as well as their pharmaceutically acceptable salts or cocrystals and prodrugs.

[0025] A drug precursor is any compound that, when administered to a mammalian subject, releases an active parent drug according to formulas I-III in vivo. Drug precursors are prepared by modifying functional groups present in compounds of formulas I-III such that the modification is cleaved in vivo to release the parent compound. Drug precursors are prepared by modifying functional groups present in compounds such that the modification can be used by conventional methods or cleaved in vivo to release the parent compound.

[0026] Tautomers are compounds resulting from the transfer of a proton from one atom of a molecule to another. Tautomers also refer to one of two or more structural isomers that are in equilibrium and readily convert from one isomer to another. Those skilled in the art will recognize that other tautomeristic ring arrangements are possible. All such isomers of these compounds are explicitly included in this invention.

[0027] An isomer is a compound that has the same molecular formula but differs in the nature or order of atomic bonding or in the spatial arrangement of atoms. Isomers that differ in the spatial arrangement of atoms are called stereoisomers. Stereoisomers that are not mirror images of each other are called diastereomers, and those that are non-overlapping mirror images of each other are called enantiomers. When a compound has an asymmetric center, for example, it is bonded to four different groups, a pair of enantiomers is possible. Chiral compounds can exist as single enantiomers or mixtures thereof. Unless otherwise indicated, this description is intended to include single stereoisomers as well as mixtures.

[0028] Some compounds of the present invention may exist in both non-solventized and solvated forms, including hydrated forms. A solvate refers to a complex formed by a combination of solvent molecules and compounds of formulas I-III. The solvent may be an organic compound, an inorganic compound, or a mixture thereof.

[0029] The present invention also includes isotopically labeled compounds, which are identical to those of formulas I-III and those listed below, but with one or more atoms replaced by atoms having atomic masses or mass numbers different from those most commonly found in nature. Examples of isotopes that can be incorporated into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, iodine, and chlorine, such as... 2 H, 3 H, 11 C 14 C 18 F, 123 I and 125 I. Compounds of formulas I-III containing the above-described isotopes and / or other isotopes, and pharmaceutically acceptable salts of said compounds, are within the scope of this invention. The isotopically labeled compounds of this invention can be used in medical diagnostic and therapeutic treatments. Utilizing, for example... 2 H, 3 H, 11 C 14 C 18 F, 123 I and 125 Different isotopes of I, the isotope-labeled compounds of the present invention have wide applications in medical diagnosis and treatment. The isotope-labeled compounds of formulas I-III and hereinafter referred to as the present invention can generally be prepared by performing the procedures disclosed in scheme AE and / or the examples below, by replacing non-isotope-labeled reagents with readily available isotope-labeled reagents.

[0030] Pharmaceutically acceptable salts are those that, within medical judgment, are suitable for use in contact with human and lower animal tissues without undue toxicity, irritation, allergic reactions, etc., and are commensurate with a reasonable benefit / risk ratio. They can be obtained during the final separation and purification of the compounds of this invention, or by separately reacting the free base functional with suitable inorganic or organic acids, such as hydrochloric acid, phosphoric acid, or sulfuric acid, and organic acids such as ascorbic acid, citric acid, tartaric acid, lactic acid, maleic acid, malonic acid, fumaric acid, glycolic acid, succinic acid, propionic acid, acetic acid, methanesulfonic acid, etc. The acid functional can react with organic or inorganic bases, such as sodium hydroxide, potassium hydroxide, or lithium hydroxide.

[0031] Therapeutic effective amount means the amount of the compound or composition of the present invention that effectively activates the purinergic receptor P2X7 and produces the desired therapeutic effect.

[0032] As used herein, the term alkyl refers to a monovalent, straight-chain or branched, saturated aliphatic hydrocarbon group having a number of carbon atoms within a specified range. For example, C 1-6 Alkyl refers to hexyl and pentyl isomers, as well as any of the following: n-, iso-, secondary and tert-butyl, n- and isopropyl, ethyl and methyl. Alkyl also includes saturated aliphatic hydrocarbon groups in which one or more hydrogen atoms are replaced by deuterium, such as CD3.

[0033] The term branched alkyl refers to an alkyl group as defined above, except that straight-chain alkyl groups within the specified range are excluded. As defined herein, branched alkyl includes alkyl groups in which the alkyl group is connected to the remainder of the compound via a secondary or tertiary carbon atom. For example, isopropyl is a branched alkyl group.

[0034] The term cycloalkyl refers to any monocyclic alkane having a number of carbon atoms within a specified range. For example, C 3-6 Cycloalkyl refers to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0035] The term halogen refers to fluorine, chlorine, bromine, and iodine (alternatively referred to as fluorinated, chlorinated, bromine, and iodinated).

[0036] The term haloalkyl refers to an alkyl group as defined above in which one or more hydrogen atoms are replaced by a halogen (i.e., F, Cl, Br, and / or I). For example, C 1-6 Halogenated alkyl groups refer to C1- to C6 straight-chain or branched alkyl groups as defined above, having one or more halogen substituents. The term fluoroalkyl has a similar meaning, except that the halogen substituent is limited to fluorine. Suitable fluoroalkyl groups include (CH2). 0-4 CF3 series.

[0037] The terms C(O) or CO refer to a carbonyl group. The terms S(O)2 or SO2 refer to a sulfonyl group. The terms S(O) or SO refer to a thionyl group.

[0038] The term aryl (aromatic group) refers to phenyl, naphthyl, tetrahydronaphthyl, indenyl, dihydroindenyl, and analogs. Of particular interest is the phenyl group.

[0039] The term heteroaryl (heteroaryl) refers to (i) a 5- or 6-membered heteroaryl ring containing 1 to 4 heteroatoms independently selected from N, O, and S, or (ii) a heterobicycle selected from quinolinyl, isoquinolinyl, and quinoxalinyl. Suitable 5- and 6-membered heteroaryl rings include, for example, pyridyl, pyrroloyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, thiophenyl, furanyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, oxtriazolyl, thiazolyl, isothiazolyl, and thiadiazolyl. A class of heteroaryl rings of interest consists of (i) 5- and 6-membered heteroaryl rings containing 1 to 3 heteroatoms independently selected from N, O, and S, and (ii) heterobicycles selected from quinolinyl, isoquinolinyl, and quinoxalinyl. Of particular interest are the heteroaryl groups pyrrole, imidazolyl, pyridyl, pyrazinyl, quinolinyl, isoquinolinyl, and quinoxalyl.

[0040] Examples of 4- to 7-membered saturated heterocycles within the scope of this invention include, for example, acridinel, piperidinyl, morpholinyl, thiomorpholinyl, thiazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyrrolidinyl, imidazolyl, piperazinel, tetrahydrofuranyl, tetrahydrothiophenyl, pyrazolyl, hexahydropyrimidinyl, thiazolyl, thiazolyl, cyclohexylimino, dicyclohexylimino, tetrahydropyranyl, tetrahydrothiaranyl, and dioxyl. Examples of 4- to 7-membered unsaturated heterocycles within the scope of this invention include monounsaturated heterocycles corresponding to the saturated heterocycles listed in the preceding sentence, wherein single bonds are replaced by double bonds (e.g., carbon-carbon single bonds are replaced by carbon-carbon double bonds).

[0041] It should be understood that the specific rings listed above are not a limitation on the rings that can be used in this invention. These rings are merely representative.

[0042] Synthetic methods for preparing the compounds of the present invention are shown in the following schemes, methods, and examples. The raw materials are commercially available or can be prepared according to procedures known in the art or as described herein. The compounds of the present invention are illustrated with the aid of specific examples shown below. However, these specific examples should not be construed as forming only the types considered to be of the present invention. These examples further illustrate details of the preparation of the compounds of the present invention. It will be readily understood by those skilled in the art that known variations of conditions and processes can be used to prepare such compounds.

[0043] The compounds, salts, and solvates of the present invention can be prepared by the methodologies that constitute another aspect of the present invention, as described below.

[0044] Certain compounds in this application can be prepared according to the following scheme and with the knowledge of those skilled in the art. All temperatures are in degrees Celsius. Common abbreviations are used.

[0045] Option A

[0046]

[0047] Step A1: The first step is accomplished by reacting compound (1) with sulfonyl chloride (2) in different solvents and at different temperatures using triethylamine or other bases to form sulfonamide (3). Compound (1) can be any of the amines listed as L in formulas I-III.

[0048] Step A2: Remove the protecting group of the amine in (3) by acid treatment with an acid such as trifluoroacetic acid or concentrated hydrochloric acid. In the case of hydrogenolysis with Cbz protection, free amine (4) is provided.

[0049] Step A3: Use a suitable base to react compound (4) with all kinds of acyl chlorides to provide amide (5).

[0050] Step A4: Replace the fluorine atom of compound (5) with a common reaction with an amine, alcohol or thiol to provide compound (6) of scheme A.

[0051] Option B

[0052]

[0053] Step B1: Using reaction conditions similar to those described in step A1 of scheme A, react compound (7) of scheme B with compound (2) to provide sulfonamide (8).

[0054] Step B2: In the presence of a suitable base such as sodium hydride, the alcohol of compound (8) reacts with an alkyl halide or alkyl sulfate to provide compound (9).

[0055] Step B3: In a solvent such as DMSO, at high temperature, replace the fluorine atom of compound (9) in scheme B with an amine, alcohol or thiol to produce compound (10).

[0056] Option C

[0057]

[0058] Step C1: In the presence of a common base, the alcohol of compound (8) in scheme C is converted by methanesulfonyl chloride to provide methanesulfonate (11).

[0059] Step C2: Compound (12) is obtained by reacting methanesulfonate (11) with thiourea and then by alkaline hydrolysis.

[0060] Step C3: Alkylate the sulfur atom of compound (12) with an alkyl halide or methanesulfonate to provide compound (13).

[0061] Step C4: Replace the fluorine atom in compound (13) with a common reaction with an amine, alcohol or thiol to provide compound (14) of scheme C.

[0062] Option D

[0063]

[0064] Step D1: Compound (15) (available at Combi-Blocks, San Diego, CA) was dissolved in an aqueous sodium hydroxide solution and cooled to 0°C, followed by the addition of sulfonyl chloride (2) to dioxane with stirring. The reaction was monitored by HPLC. After the reaction was complete, the mixture was neutralized to pH 7 with 2N HCl. The dioxane was removed and the solid was collected. The solid was washed with water and dried to provide compound (16) of scheme D.

[0065] Step D2: Dissolve compound (16) in dry DMF and add carbonyl diimidazole (CDI) (Combi-Blocks). Stir the mixture for 1 to 2 hours and add amine (Combi-Blocks). Monitor the reaction by HPLC and pour water in once the reaction is complete. Collect the precipitate and wash it with 1N aqueous HCl, aqueous sodium bicarbonate solution, and water, and dry it to provide compound (17) of scheme D.

[0066] Step D3: Dissolve compound (17) in DMSO and add a base, followed by the addition of structural unit R4. Stir the mixture at high temperature. Monitor the reaction by HPLC and cool to room temperature once the reaction is complete. Pour the mixture into water to precipitate. Collect the precipitate and wash with water. Further purify the final compound (18) of scheme D by recrystallization or column chromatography. The addition of other structural units at positions R2, R3, R5, or R6 can be achieved in a similar manner.

[0067] Option E

[0068]

[0069] Step E1: Dissolve compound (19) (Combi-Blocks) in DCM and cool to 0°C. Then add triethylamine to the DCM, followed by dropwise addition of sulfonyl chloride (2). Monitor the reaction by HPLC. Wash the product with 1N HCl aqueous solution and water. Extract the organic layer and dry it with magnesium sulfate. Concentrate the solution to provide compound (20) in scheme E.

[0070] Step E2: Compound (20) was added to TFA at room temperature, and the resulting solution was stirred at 40 degrees to remove the Boc protecting group. Once the reaction was complete, the mixture was cooled to 0 degrees and then made alkaline with alkaline water to provide compound (21).

[0071] Step E3: Compound (21) was dissolved in dioxane and cooled to 0°C. After the addition of triethylamine, acyl chloride was added dropwise. The reaction was monitored by HPLC. The dioxane was removed and the residue was prepared with water to provide the solid compound (22) of scheme E.

[0072] Step E4: Dissolve compound (22) in DMSO and add a base, followed by the addition of structural unit R4. Stir the mixture at high temperature. Monitor the reaction by HPLC and cool to room temperature once the reaction is complete. Collect the solid and purify it by recrystallization or column chromatography to provide the final pure compound (23) for Scheme E. Addition of other structural units at positions R2, R3, R5, or R6 can be achieved in a similar manner.

[0073] The following embodiments illustrate the present invention prepared according to the above scheme and the knowledge of those skilled in the art.

[0074] Example 1:

[0075] Triethylamine (110 mmol) was added to a solution of 100 mmol of 3-Boc-aminopiperidine in 100 mL of dichloromethane at 0°C, followed by dropwise addition of 100 mmol of 4-fluorobenzenesulfonyl chloride to the dichloromethane with stirring. The reaction was monitored by HPLC. Once the reaction was complete, the solution was washed successively with 1N HCl and NaHCO3. The dichloromethane solution was collected and concentrated to provide solids for Example 1.

[0076] Example 2:

[0077] Example 1 (100 mmol) was dissolved in dichloromethane (200 mL), and concentrated HCl (175 mL) was added dropwise while the reaction solution was vigorously stirred. After the addition was complete, stirring was maintained at 40°C until HPLC analysis showed that Example 1 was no longer present. The reaction solution was cooled to room temperature and the aqueous layer was collected. The aqueous layer was neutralized to pH 14 with 4N NaOH solution. The white precipitate was collected, washed with water, and dried to provide Example 2.

[0078] Example 3:

[0079] Example 2 (50 mmol) was dissolved in dichloromethane (100 mL) and cooled to 0°C. Triethylamine (55 mmol) was added, followed by dropwise addition of hydrogenated cinnamoyl chloride (50 mmol) to dichloromethane (30 mL). The reaction was monitored by HPLC. When the reaction was complete, 1N HCl solution (100 mL) was added. The organic layer was collected and washed with sodium bicarbonate solution. The solution was then dried over magnesium sulfate and concentrated to provide a white solid, Example 3.

[0080] Example 4:

[0081] Example 3 (10 mmol) was dissolved in DMSO (50 mL) and potassium carbonate (20 mmol) was added, followed by piperidine (12 mmol). The mixture was stirred at 60 to 100 degrees Celsius for 1 to 2 hours and then cooled to room temperature. Water was added to precipitate the solid. The precipitate was collected and washed with 1N HCl. The solid was then dried and redissolved in ethyl acetate. The ethyl acetate solution was passed through a silica gel pad and concentrated to provide a pure white solid, Example 4, in 80% yield.

[0082] Example 5:

[0083] Instead of piperidine in Example 4, pyrrolidine (12 mmol) was used to provide a white solid in 75% yield in Example 5.

[0084] Example 6:

[0085] When morpholine was used instead of piperidine in Example 4, a white solid was obtained in 85% yield in Example 6.

[0086] Example 7:

[0087] When 2-methylpiperidine is used instead of piperidine in Example 4, solid Example 7 is provided in 65% yield.

[0088] Example 8:

[0089] When 3-methylpiperidine was used instead of piperidine in Example 4, Example 8 was obtained in 80% yield.

[0090] Example 9:

[0091] When 4-methylpiperidine was used instead of piperidine in Example 4, a white solid was obtained in 78% yield in Example 9.

[0092] Example 10:

[0093] When 4-phenoxypiperidine (Combi-Blocks) was used instead of piperidine in Example 4, a white solid was obtained in 77% yield in Example 10.

[0094] Example 11:

[0095] When using a reaction similar to that in Example 4, 4-methoxypiperidine was reacted with Example 3, a white solid, Example 11, was provided in 90% yield.

[0096] Example 12:

[0097] When piperidine-4-ol was used instead of piperidine in Example 4, a crude product was given, which was purified by silica gel chromatography to provide pure Example 12 in 45% yield.

[0098] Example 13:

[0099] When 4-trifluoromethylpiperidine (Combi-Blocks) was used in a reaction similar to that in Example 4, Example 13 was produced in 80% yield.

[0100] Example 14:

[0101] Example 14 was produced in 70% yield when the same reaction conditions as in Example 13 were used, but 3-trifluoromethylpiperidine was used instead of 4-trifluoromethylpiperidine.

[0102] Example 15:

[0103] Example 15 was produced in 30% yield when 2-trifluoromethylpiperidine (Combi-Blocks) was used instead of piperidine in Example 4 under sealed tube conditions.

[0104] Example 16:

[0105] When excess butylamine was used instead of piperidine in Example 4, Example 16 was produced in 78% yield.

[0106] Example 17:

[0107] Example 17 was provided in 80% yield when cyclohexylamine was used instead of piperidine in Example 4.

[0108] Example 18:

[0109] When cyclopentylamine was used instead of cyclohexylamine under similar reaction conditions as in Example 17, Example 18 was obtained.

[0110] Example 19:

[0111] Example 19 was provided in 65% yield when furan-2-methylamine (Combi-Blocks) was used instead of piperidine in Example 4.

[0112] Example 20:

[0113] Step 20A: When piperazine-1-carboxylate tert-butyl ester (Combi-Blocks) is used instead of piperidine in Example 4, a Boc-protected compound (compound 20A) is produced in 90% yield.

[0114] Step 20B: Dissolve 100 mg of the Boc-protected material from Step 20A in 5 mL of trifluoroacetic acid and stir at room temperature for 2 hours. Then remove the TFA and make the residue alkaline using a sodium hydroxide solution. Collect the solid, wash with water, and dry to provide Example 20 in 65% yield.

[0115] Example 21:

[0116] When 1-methylpiperazine was used instead of tert-butyl piperidine-1-carboxylate in Example 20, Example 21 was produced directly in 85% yield.

[0117] Example 22:

[0118] Example 22 is produced when 4,4-difluoropiperidine (Combi-Blocks) is used to replace piperidine in Example 4.

[0119] Example 23:

[0120] 2,2,2-trifluoroethanol (30 mmol) was added to a solution of Example 3 (10 mmol) in DMSO (50 mL), followed by sodium hydroxide (30 mmol). The mixture was stirred at 80°C for 3 hours and then cooled to room temperature. Water (100 mL) was added to precipitate the solid. The precipitate was collected and washed with 1N HCl. The solid was then dried and redissolved in ethyl acetate. The ethyl acetate solution was passed through a silica gel pad and concentrated to provide a pure white solid in 80% yield, as described in Example 23.

[0121] Example 24:

[0122] When ethanol was used instead of 2,2,2-trifluoroethanol in Example 23, Example 24 was obtained in 70% yield.

[0123] Example 25:

[0124] Example 25 was provided in 60% yield when isobutanol was used instead of ethanol in Example 24.

[0125] Example 26:

[0126] Benzyl alcohol (12 mmol) was added to the solution of Example 3 (10 mmol) in DMSO (50 mL), followed by sodium hydroxide (20 mmol). The mixture was stirred at 80°C for 3 hours and then cooled to room temperature. Water (100 mL) was added to precipitate the solid. The precipitate was collected and washed with water. The solid was then dried and redissolved in ethyl acetate. The ethyl acetate solution was passed through a silica gel pad and concentrated to provide a pure white solid in 85% yield, as described in Example 26.

[0127] Example 27:

[0128] Example 27 was provided in 90% yield when 2-methoxyethanol was used instead of ethanol in Example 24.

[0129] Example 28:

[0130] When 4-isopropoxypiperidine (Combi-Blocks) was used instead of piperidine in Example 4, Example 28 was produced in 74% yield.

[0131] Example 29:

[0132] When 4-phenoxypiperidine is used instead of piperidine in Example 4, Example 29 is produced.

[0133] Example 30:

[0134] When N-boc-4,4'-dipiperidine (Combi-Blocks) was used instead of piperidine in Example 4 and a similar procedure was followed, a white solid, Example 30, was produced in 78% yield.

[0135] Example 31:

[0136] Solid sodium hydroxide (20 mmol) was added to the solution of Example 3 (10 mmol) in DMF (20 mL). The mixture was stirred at 80°C for 3 hours and then cooled to room temperature. Water (50 mL) was added to precipitate the solid. The precipitate was collected and washed with water. The solid was then dried and redissolved in ethyl acetate. The ethyl acetate solution was passed through a silica gel pad and concentrated to provide a pure white solid, Example 31, in 70% yield.

[0137] Example 32:

[0138] Pyrazole (50 mmol) was mixed with 10 mmol of Example 3 in DMSO (50 mL), and potassium carbonate (20 mmol) was added. The mixture was stirred at 120°C for 3 hours. The reaction mixture was then cooled to room temperature and water was poured in. The solid was collected and washed with water. The solid was purified by recrystallization from methanol, providing the pure material of Example 32 in 65% yield.

[0139] Example 33:

[0140] Using a reaction similar to that in Example 32, but with 4-bromopyrazole (Combi-Blocks) replaced by pyrazole, Example 33 was provided in 70% yield.

[0141] Example 34:

[0142] Step 34A: 1-N-Boc-piperidin-3-amine (Combi-Blocks) (100 mmol) was dissolved in DCM and cooled to 0°C. Triethylamine (110 mmol) was added, followed by dropwise addition of hydrogenated cinnamoyl chloride (100 mmol) with stirring. The reaction was monitored by HPLC. The mixture was washed with 1N aqueous HCl solution, sodium bicarbonate solution, and water. The dichloromethane solution was removed and dried over magnesium sulfate. The DCM was removed, yielding a white solid N-[3-(1-Boc-piperidinyl)]hydrogenated cinnamoyl amide (compound 34A) in 95% yield.

[0143] Step 34B: Compound 34A (80 mmol) was dissolved in TFA and stirred at 40°C. The reaction was monitored by HPLC. The mixture was brought to alkalinity (pH 14) by adding an aqueous sodium hydroxide solution. The solid was collected, washed with water, and dried under vacuum to provide a gel of N-(3-piperidinyl)hydrocinnamic acid amide (compound 34B).

[0144] Step 34C: Compound 34B (50 mmol) was dissolved in dioxane (100 mL), and triethylamine (60 mmol) was added. The mixture was cooled to 0°C, and 3-chloro-4-fluorobenzenesulfonyl chloride (Combi-Blocks) (50 mmol) was added fractionally. The reaction was monitored by HPLC. The reaction mixture was concentrated by rotary evaporation, and the residue was prepared with water to provide a solid. The solid was further purified by recrystallization in methanol, giving a white solid in 90% yield, Example 34.

[0145] Example 35:

[0146] Example 34 (1 mmol) and piperidine (3 mmol) were added to DMSO (15 mL). The mixture was stirred at 100°C for 2 hours and cooled to room temperature. Water was added and the precipitate was collected. The solid was purified by recrystallization in methanol, providing Example 35 in 78% yield.

[0147] Example 36:

[0148] Example 2 (50 mmol) was dissolved in DCM (150 mL), and triethylamine (55 mmol) was added. While the mixture was cooled to 0°C, phenylacetyl chloride (50 mmol) was added dropwise. The reaction was monitored by HPLC. The reaction mixture was washed with 1N HCl aqueous solution, followed by sodium bicarbonate aqueous solution, and dried over magnesium sulfate. The solution was concentrated, and the residue was purified by recrystallization in methanol to provide Example 36.

[0149] Example 37:

[0150] Example 36 (1 mmol) was dissolved in DMSO (15 mL), and piperidine (3 mmol) was added. The mixture was then stirred at 100°C for 2 hours. The mixture was cooled and poured into water. The precipitate was collected and purified by recrystallization in methanol, providing Example 37 in 85% yield.

[0151] Example 38:

[0152] Step 38A: 3-Benzenesulfonyl acridine (Combi-Blocks) (1 mmol) was dissolved in DMF (10 mL) and cooled to 0°C. Triethylamine (1.2 mmol) was then added, followed by 4-fluorobenzenesulfonyl chloride (1 mmol). The reaction was monitored by HPLC. 1N HCl aqueous solution was added and the organic layer was extracted. The organic solution was dried over magnesium sulfate and concentrated to provide N-(4-fluorobenzenesulfonyl)-3-benzenesulfonyl acridine (compound 38A) of Example 38.

[0153] Step 38B: Compound 38A (0.1 mmol) from Step 38A was dissolved in DMSO (5 mL), and piperidine (0.5 mmol) was added. The mixture was then stirred at 80°C for 2 hours and cooled to room temperature. The mixture was poured into water, and the solid was collected and washed with water. The solid was dried and purified by column chromatography to provide a white solid in 65% yield, Example 38.

[0154] Example 39:

[0155] Compound 38A (0.1 mmol) from Example 38 was dissolved in DMSO (5 mL), and morpholine (0.5 mmol) was added. The mixture was stirred at 100°C for 3 hours. The reaction mixture was then cooled to room temperature and water was added. The solid was collected and purified by column chromatography to provide Example 39.

[0156] Example 40:

[0157] Compound 34B (3-piperidinyl) hydrogenated cinnamamide (0.1 mmol) from Example 34 was dissolved in DCM, and triethylamine (0.11 mmol) was added. The mixture was cooled to 0°C, and benzyl sulfonyl chloride (Combi-Blocks) (0.1 mmol) was added. The mixture was then purified by conventional processing and recrystallization in methanol, providing Example 40 in 95% yield.

[0158] Example 41:

[0159] Instead of benzyl sulfonyl chloride in Example 40, Example 41 is provided using 4-bromobenzyl sulfonyl chloride (Combi-Blocks) in the same proportion.

[0160] Example 42:

[0161] Step 42A: Sodium 4-fluorobenzenesulfinate (Combi-Blocks) (1 mmol) was mixed in DMF (15 mL), and 2-iodopropane (5 mmol) was added. The mixture was then stirred at 60°C for 5 hours under argon. The mixture was cooled and water was added, followed by extraction with DCM. The DCM extract was washed with water and dried over magnesium sulfate. The DCM was removed to provide crude 1-fluoro-4-isopropylsulfonylbenzene (compound 42A), which was sufficiently pure for the next step.

[0162] Step 42B: Crude 1-fluoro-4-isopropylsulfonylbenzene (0.1 mmol) was dissolved in DMSO (15 mL), and compound 34B (3-piperidinyl) hydrogenated cinnamamide from Example 34 (0.1 mmol) was added, followed by potassium carbonate (0.5 mmol). The reaction mixture was stirred at 100°C for 3 hours. The mixture was cooled to room temperature and water was added. The solid was collected and further purified by recrystallization in methanol, providing Example 42 in 78% yield.

[0163] Example 43:

[0164] Example 43 is provided when a procedure similar to that in Example 42 is used, but 1-fluoro-4-methylsulfonylbenzene (Combi-Blocks) is used instead of 1-fluoro-4-isopropylsulfonylbenzene in Example 42.

[0165] Example 44:

[0166] At room temperature, a solution of Example 4 (0.1 mmol) in THF (20 mL) was added to a suspension of lithium aluminum hydride (0.1 mmol) in THF (20 mL). The resulting mixture was refluxed for 1 hour and cooled to room temperature. The reaction mixture was quenched by adding an aqueous sodium hydroxide solution. The resulting mixture was filtered and the THF solution was concentrated. The residue was purified by column chromatography to provide Example 44.

[0167] Example 45:

[0168] Step 45A: Add 1 mmol of 4-fluorobenzenesulfonyl chloride to a solution of diisopropylamine (2 mmol) in DCM at 0°C. Then wash the reaction mixture with 1N HCl aqueous solution. The DCM is then concentrated to provide a white solid N,N-diisopropyl-4-fluorobenzenesulfonamide (compound 45A).

[0169] Step 45B: A mixture of N,N-diisopropyl 4-fluorobenzenesulfonamide (0.1 mmol) and compound 34B (3-piperidinyl) hydrogenated cinnamonamide from Example 34 was stirred in DMSO at 100°C in the presence of potassium carbonate (0.2 mmol). The reaction was monitored by HPLC. The mixture was cooled and poured into water. The solid was collected, washed with water, and further purified by recrystallization in methanol to provide a white solid, Example 45.

[0170] Example 46:

[0171] When aniline is used instead of diisopropylamine in Example 45, Example 46 is provided using a procedure similar to that in Example 45.

[0172] Example 47:

[0173] To produce Example 47, cyclohexylamine was used instead of diisopropylamine and a procedure similar to that in Example 45 was employed.

[0174] Example 48:

[0175] To produce Example 48, furan-2-methylamine was used instead of diisopropylamine and a procedure similar to that in Example 45 was employed.

[0176] Example 49:

[0177] When ammonia was used instead of diisopropylamine, a similar procedure to that in Example 45 was used to produce Example 49 in 40% yield.

[0178] Example 50:

[0179] In order to provide Example 50 in 80% yield, piperidine was used instead of diisopropylamine in Example 45 and a similar procedure as in Example 45 was also adopted.

[0180] Example 51:

[0181] In order to provide Example 51 in 80% yield, pyrrolidine was used instead of piperidine in Example 50 and a similar procedure was adopted.

[0182] Example 52:

[0183] Step 52A: Piperidine-4-carboxylic acid (100 mmol) was dissolved in 2N aqueous sodium hydroxide solution (200 mmol), and dioxane (100 mL) was added. The mixture was cooled to 0°C, and 4-fluorobenzenesulfonyl chloride (100 mmol) was added fractionally. The mixture was stirred at 0°C until all the sulfonyl chloride disappeared. The mixture was then acidified with 4N aqueous HCl solution. The solid was collected, washed with water, and dried to provide N-(4-fluorobenzenesulfonyl)piperidine-4-carboxylic acid (compound 52A) of Example 52.

[0184] Step 52B: Compound 52A (50 mmol) was dissolved in DMF at 0°C, and CDI (55 mmol) was added. The mixture was stirred for 1 hour, and phenylethylamine (50 mmol) was added. The reaction was monitored by HPLC. After the reaction, water was added, the precipitate was collected, and washed with water. The solid was dried to provide Example 52 in 92% yield.

[0185] Example 53:

[0186] Example 52 (0.1 mmol) was dissolved in DMSO (10 mL), and morpholine (0.3 mmol) was added. The mixture was stirred at 80°C for 2 hours. The mixture was cooled to room temperature and water was added. The solid was collected and purified by recrystallization in methanol to provide Example 53 in 85% yield.

[0187] Example 54:

[0188] In order to provide Example 54 in 82% yield, 1-methylpiperazine was used instead of morpholine in Example 53 and a similar procedure to that in Example 53 was adopted.

[0189] Example 55:

[0190] In order to provide Example 55 in 55% yield, butylamine was used instead of morpholine and a similar procedure to that in Example 53 was adopted.

[0191] Example 56:

[0192] Piperidine-3-carboxylic acid (100 mmol) was used instead of piperidine-4-carboxylic acid in Example 52, and a similar procedure as in Example 52 was also used to provide a white solid in 83% yield in Example 56.

[0193] Example 57:

[0194] Using Example 56 (0.1 mmol) and following a similar procedure to Example 53, a white solid of Example 57 was provided at 78%.

[0195] Example 58:

[0196] Example 58 was produced in 81% yield by replacing morpholine with piperidine in Example 57.

[0197] Example 59:

[0198] Step 59A: Compound 19 (100 mmol) was dissolved in DCM (200 mL), and triethylamine (110 mmol) was added. The mixture was cooled to 0°C, and hydrogenated cinnamoyl chloride (100 mmol) was added dropwise. The reaction mixture was stirred at 0°C for 1 hour and washed with 1N aqueous HCl solution and sodium bicarbonate solution. The DCM solution was dried over magnesium sulfate and concentrated to provide compound 59A in 95% yield.

[0199] Step 59B: Compound 59A (90 mmol) was dissolved in trifluoroacetic acid (50 mL) and stirred at 40°C for 2 hours to remove the trifluoroacetic acid. The residue was brought to alkalinity to pH 14 with 2N sodium hydroxide at 0°C and extracted with DCM. The DCM extract was dried and concentrated to provide compound 59B in 75% yield.

[0200] Step 59C: Compound 59B (50 mmol) was dissolved in DCM (100 mL), and triethylamine (55 mmol) was added. The mixture was cooled to 0°C, and 4-fluorobenzenesulfonyl chloride (50 mmol) was added fractionally. The mixture was then stirred at 0°C for 2 hours, and Example 59 was provided in 90% yield using conventional processing procedures.

[0201] Example 60:

[0202] Example 59 (0.1 mmol) was dissolved in DMSO (10 mL), and morpholine (0.3 mmol) was added. The reaction was stirred at 100°C for 2 hours. Example 60 was provided in 80% yield using a similar procedure to that in Example 53.

[0203] Example 61:

[0204] Using a procedure similar to that of Example 60, Example 61 was obtained in 78% yield when piperidine was used instead of morpholine.

[0205] Example 62:

[0206] Using a similar procedure to that of Example 60, Example 62 was obtained in 75% yield when pyrrolidine was used instead of morpholine.

[0207] Example 63:

[0208] Using a procedure similar to that of Example 60, a white solid of Example 63 was obtained in 86% yield when 1-methylpiperazine was used instead of morpholine.

[0209] Example 64:

[0210] Using a procedure similar to that in Example 53, Example 64 was obtained in 85% yield when pyrrolidine was used instead of morpholine.

[0211] Example 65:

[0212] When pyrrolidine is used instead of morpholine and a procedure similar to that in Example 57 is employed, Example 64 is provided in 80% yield.

[0213] Pharmacological Examples

[0214] Examples of the inventions that have undergone biological studies are listed in Table 1. The biological assays used to characterize these compounds are described below.

[0215] Using a Yo-Pro-1 uptake assay (Yo-Pro-1 assay), it was found that the embodiments of the invention are P2X7R antagonists. resistance

[0216] Yo-Pro-1 iodide (Fisher Scientific, Cat. Y3603) is a fluorescent DNA-binding dye with a molecular weight (MW) of 629 Da. This method is based on the fact that Yo-Pro-1 enters cells via an expanded or "macrowell" P2X7R and binds to intracellular DNA, thereby increasing its fluorescence intensity (J. Pharmacol. Exp. Ther. 308, 1053-1061). The dye has an absorption spectrum compatible with argon laser excitation at 488 nm, and its emission wavelength is in the 515-575 nm range. Yo-Pro-1 uptake was measured in HEK-293 cells transiently transfected with P2X7R (J. Biol. Chem. 290, 7930-7942). Briefly, on day 1, HEK-293 cells were seeded in growth medium at approximately 20,000 cells / well in 96-well plates. On day 2, cells were transfected with P2X7R DNA plasmid at a concentration of 1.7 μg / ml using Lipofectamine 2000. The transfection solution was replaced with growth medium 4 hours later. Fluorescence assays were performed on day 4. On day 4, the culture medium was removed, and cells were washed with phosphate-buffered saline (PBS) (Fisher Scientific, Cat. MT21040CV). Then, 25 μl of PBS was first added to each well, followed by 25 μl of each of the various test compounds. A third 25 μl solution contained a mixture of Yo-Pro-1 and the stimulant bzATP (2'(3')-O-(4-benzoylbenzoyl)adenosine-5'-tris(triethylammonium)triphosphate). The final concentration of Yo-Pro-1 was 2.5 μM, and the final concentration of bzATP was 30 μM. Fluorescence enhancement was measured every 30 minutes in a microplate apparatus using excitation at 475 nm and emission wavelengths in the 500–550 nm range. PPADS (pyridoxine-6-azophenyl-2',4'-disulfonate tetrasodium) was used as a positive control. Wells containing only PBS and Yo-Pro-1 without the test compound and bzATP served as negative controls (NContl). The positive control (P Contl) wells were the same as N Contl, but in this case contained the stimulant bzATP.

[0217] Data used for analysis were derived from microplate readings 1 hour after bzATP stimulation. Results for each well were first normalized to their corresponding baseline microplate reading. The baseline microplate reading was the first reading after the addition of all components. The negative control signal was then subtracted. In the negative control, the test compound and stimulus were replaced with PBS. The average of the six highest values ​​of the plate (AVG) was considered 100%, and the value for each well was divided by the AVG and presented as a percentage relative value.

[0218] Using 30 μM bzATP as the stimulus, the half-maximal inhibitory concentration (IC50) of the antagonist compounds in Equations I-III was estimated based on dose-response curves spanning several log units and generated by Prism software. 50 The results are listed in Table 1.

[0219] To compare the antagonistic efficacy of the compounds of the present invention in the P2X receptor, measurements were performed at 5 μM. Of the 55 compounds tested, 29 reduced P2X7R activity to 15% or less of normal activity (Table 2).

[0220] Table 1. Compound names, IDs, and estimated IC50 values. 50

[0221]

[0222]

[0223]

[0224] Table 2 shows the inhibitory efficacy of the 5 μM compound when applied to P2X1R, P2X2R, P2X4R, and P2X7R.

[0225]

[0226]

[0227]

[0228] Using a receptor-mediated calcium entry assay (calcium assay), it was found that the embodiments of the invention were ineffective against P2X1R.

[0229] The human P2X1 receptor (P2X1R) is a member of the P2X family. Preliminary studies showed that the Yo-Pro-1 uptake assay could not be used to measure P2X1R activity. Therefore, a calcium assay was used. In the calcium assay, P2X receptor activation leads to the opening of ion channels, which increases intracellular calcium concentration. The increased intracellular calcium concentration can be detected by calcium indicator dyes such as Fluo-4 or Fluo-8. Molecular probes are used in this assay. TMFluo-4, AM (Fisher Scientific, Cat. F14201). The procedure was similar to that described in the Yo-Pro-1 assay until day 4. The P2X1R construct used for transient transfection of HEK-293 cells was sponsored by Dr. Richard J. Evans' laboratory (J. Biol. Chem. 279:9043-9055). Following transfection and expression, on day 4, cells were washed twice with Ca-HBSS (composed of NaCl 135, HEPES 10, D-glucose 10, KCl 5.4, CaCl2 2, and MgCl2 1, pH adjusted to 7.4 with NaOH) and incubated for 70 minutes at 37°C in dye-loaded solution (containing 2.5 μM Fluo-4, 1 mM probenecid, and 1× PowerLoad in Ca-HBSS (Fisher Scientific, Cat. P10020)). The extracellular dye was then washed off, and the cells were incubated for 30 minutes in Ca-HBSS containing 1 mM probenecid. Probenecid was washed off with Ca-HBSS before the final step. Assays were performed by adding 25 μl of Ca-HBSS followed by 25 μl of each test compound. At this point, the baseline fluorescence level—the baseline microplate reading for this assay—was obtained via microplate readings. Finally, a stimulant (for P2X1R, a final concentration of 20 μM ATP) was added to activate P2X1R. Activation of P2X1R opened the channel to allow calcium influx, which was represented by fluorescence enhancement measured by a microplate apparatus with excitation at 475 nm and emission wavelengths in the 500–550 nm range. Data were analyzed in the same manner as described above: the results for each well were first normalized to their corresponding baseline microplate reading. Then, the negative control signal was subtracted. In the negative control, the test compound and stimulant were replaced with Ca-HBSS. The positive control was the same as most wells but contained no test compound. The average of the six highest values ​​on the plate (AVG) was considered as 100%, and the value for each well was divided by the AVG and presented as a percentage relative value. The results are listed in Table 2. At 5 μM, none of the compounds tested completely inhibited P2X1R activity or reduced the activity to 15% of the normal activity.

[0230] Using a Yo-Pro-1 uptake-based assay, it was found that the embodiments of the invention were ineffective for P2X2R.

[0231] Human P2X2 receptor (P2X2R) constructs (J. Biol. Chem. 290, 7930-7942) were transiently transfected into HEK-293 cells, and Yo-Pro-1 assays were performed as described above. An increase in receptor-mediated activity was observed within 10 minutes when 5 μM ATP was used as a stimulus. The inhibitory results of the tested compounds of formulas I-III against P2X2R are listed in Table 2. At 5 μM, none of the compounds completely inhibited P2X2R activity or reduced it to 15% of normal activity.

[0232] Using a receptor-mediated calcium entry assay, the embodiments of the invention were found to be ineffective for P2X2R.

[0233] When P2X2R was measured using the calcium assay method described above, the results were essentially the same as those of the Yo-Pro-1 assay. Their results are listed in Table 2.

[0234] Using a receptor-mediated calcium entry assay, the embodiments of the invention were found to be ineffective against P2X4R.

[0235] Human P2X4 receptor (P2X4R) constructs (J. Biol. Chem. 290, 7930-7942) were transiently transfected into HEK-293 cells, and calcium assays were performed as described above. With 1 mM ATP as the stimulus, an increase in receptor-mediated activity leading to elevated intracellular calcium was observed within less than 10 minutes. The inhibitory results of compounds of formulas I-III on P2X4R are listed in Table 2. At 5 μM, none of the compounds completely inhibited P2X4R activity or reduced it to 15% of normal activity.

[0236] Those skilled in the art will understand that various modifications and variations can be made to this invention without departing from the spirit or scope thereof. Therefore, this invention is intended to cover modifications and variations thereof, provided they fall within the scope of the appended claims and their equivalents.

Claims

1. A compound with the structure shown below, or selected from the group consisting of the following compounds: ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， 。

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