Preparation and use of dicarboxylic acid derivatives as fluorescent probes for GPR35 receptors

Abstract

The present invention provides a method for preparing a fluorescent probe molecule 10-methyl-4,6-dioxo-1,4,6,9-tetrahydropyrido[3,2-g]quinoline-2,8-dicarboxylic acid derivative for human G protein-coupled receptor 35 (GPR35) and its application. In the invention, such compounds generally show agonistic activity to GPR35, are specific agonists of human GPR35 receptors, and can be applied to bioluminescence resonance energy transfer (BRET) to test the affinity constants of GPR35 receptors and related ligands. The method has good sensitivity and biocompatibility, solves the problem that general compounds need to be labeled with radioactive ligands in activity tests, can realize the determination of affinity constants of active molecules targeting GPR35, and provides new probe molecules for the study of GPR35 biology, helps to explain the active mechanism of action of related compounds, and provides lead compounds for the study of related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a 10-methyl-4,6-dioxo-1,4,6,9-tetrahydropyrido[3,2-g]quinoline-2,8-dicarboxylic acid derivative which can be used as a GPR35 receptor fluorescent probe molecule, a preparation method and an application thereof.

[0002] The method of the present invention can be used to test the affinity between GPR35 ligand and receptor, so as to explain the active mechanism of related compounds. Technical Background

[0003] G protein coupled receptors (GPCRs) are the most successful drug receptors in the history of drug development. Among the drugs used in modern clinical practice, 30-40% of the drugs use G protein coupled receptors as targets (The Antiallergic Mast Cell Stabilizers Lodoxamide and Bufrolin as the First High and Equipotent Agonists of Human and Rat GPR35. Mol Pharmacol 2014, 85: 91-104). There are several GPCR ligands in the world's 100 best-selling drugs. However, among all G protein coupled receptor families, only 108 receptors have been developed as drug targets. Therefore, such targets still have huge development potential in drug development.

[0004] G protein-coupled receptor 35 (GPR35) is an orphan receptor first discovered in 1998 (O'Dowd, BF, Tonai-Kachi, H., and Ichikawa, K. GPR35 is a functional receptor in rat dorsal root ganglion neurons. Biochem. Biophys. Res. Commun. 2008, 365, 344-348.). Studies to date have shown that this receptor is associated with many diseases. However, the lack of suitable pharmacological tools and the absence of endogenous ligands have restricted physiological studies on this receptor. So far, the only study on functionalized GPR35 ligands was published in 2013, which used chromone as the parent structure of a radioactive GPR35 ligand.

[0005] Ligands that incorporate fluorescent probes into molecules have become a very competitive tool for evaluating ligand-receptor binding, which has advantages over classical radioactive ligands. In addition to practical considerations of convenience, safety, and cost, GPCRs often undergo receptor internalization upon stimulation by agonists, leading to desensitization. Therefore, fluorescent ligands are an attractive tool in GPCR research, providing dynamic information about GPCRs in real time and with spatiotemporal resolution. Summary of the invention

[0006] In view of the above prospects, the first object of the present invention is to provide a 10-methyl-4,6-dioxo-1,4,6,9-tetrahydropyrido[3,2-g]quinoline-2,8-dicarboxylic acid derivative that can be used as a GPR35 receptor fluorescent probe molecule, including a pharmaceutically acceptable salt, hydrate or crystal form thereof, and the general structural formula thereof is as follows:

[0007]

[0008] in,

[0009] linker is selected from Wherein n is 2-10, preferably 3-5 saturated fatty carbon chains; or linker is selected from Where n is 1-5, preferably a PEG chain with a degree of polymerization of one.

[0010] In a specific embodiment, according to the present invention, preferred compounds include:

[0011] 4-(3-(1-(2-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborol-10-yl)phenoxy)ethyl)-1H-1,2,3-triazol-4-ylpropoxy)-6-hydroxy-10-methylpyridyl[3,2-g]quinoline-2,8-dicarboxylic acid (Compound 14)

[0012] 4-(4-(1-(2-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborol-10-yl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)butoxy)-6-hydroxy-10-methylpyridyl[3,2-g]quinoline-2,8-dicarboxylic acid (Compound 15)

[0013] 4-((5-(1-(2-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaboroserine-10-yl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)pentyloxy)-6-hydroxy-10-methylpyridyl[3,2-g]quinoline-2,8-dicarboxylic acid (Compound 16)

[0014] 4-(2-((1-(2-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborolan-10-yl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)-6-hydroxy-10-methylpyridyl[3,2-g]quinoline-2,8-dicarboxylic acid (Compound 17)

[0015] And pharmaceutically acceptable salts, hydrates or crystal forms of the above compounds.

[0016] Furthermore, the pharmaceutically acceptable salts of the above-mentioned compounds are especially lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts and ammonium salts.

[0017] The second object of the present invention is to provide a method for synthesizing 10-methyl-4,6-dioxo-1,4,6,9-tetrahydropyrido[3,2-g]quinoline-2,8-dicarboxylic acid derivatives, which can be prepared by the following reaction route:

[0018]

[0019] Reaction conditions: (a) K2CO3, DMF, 50°C, overnight. (b) Diisopropyl azodicarboxylate, PPh3, 2-(prop-2-en-1-yloxy)ethane-1-ol, 0°C to room temperature, overnight. (c) CuSO4, sodium ascorbate, THF:H2O (1V:1V). (d) LiOH, MeOH:H2O:THF (1V:1V:4V).

[0020] According to the conditions disclosed in the present invention, the 10-methyl-4,6-dioxo-1,4,6,9-tetrahydropyrido[3,2-g]quinoline-2,8-dicarboxylic acid derivatives and / or pharmaceutically acceptable salts thereof disclosed in the present invention can be easily prepared. Based on the synthesis conditions disclosed in the present invention, the scale of the reaction can be enlarged or reduced in equal proportions, or the type or amount of solvent used or the reaction temperature or reaction time can be changed within a reasonable range, or the equivalent of the reaction substrate, catalyst, additive and other substances can be changed within a reasonable range, all of which fall within the protection scope of the present invention.

[0021] The third object of the present invention is to provide a method for evaluating the dissociation constant between the GPR35 receptor and the ligand by using bioluminescence resonance energy transfer (BRET) of 10-methyl-4,6-dioxo-1,4,6,9-tetrahydropyrido[3,2-g]quinoline-2,8-dicarboxylic acid derivatives that can be used as GPR35 receptor fluorescent probe molecules, wherein the dissociation constant includes the equilibrium dissociation constant (K D ) and the dissociation constant (K i ).

[0022] In a specific embodiment, the bioluminescence resonance energy transfer (BRET) system includes a cell target (GPR35-NanoLuc) fused to a bioluminescent reporter, a bioactive agent tethered to a fluorophore (10-methyl-4,6-dioxo-1,4,6,9-tetrahydropyrido[3,2-g]quinoline-2,8-dicarboxylic acid derivative) and a substrate (furimazine) of the bioluminescent reporter. The fusion protein GPR35-NanoLuc is incubated with a concentration gradient of the probe molecule 10-methyl-4,6-dioxo-1,4,6,9-tetrahydropyrido[3,2-g]quinoline-2,8-dicarboxylic acid derivative. The binding degree of probe molecules at different concentrations leads to a change in BRET efficiency to obtain a saturation test curve, and the equilibrium dissociation constant (K) is obtained by fitting the saturation test curve using Graphpad. D ). The dissociation constant (K i ) is to add a fixed concentration of probe molecule 10-methyl-4,6-dioxo-1,4,6,9-tetrahydropyrido[3,2-g]quinoline-2,8-dicarboxylic acid derivatives and a concentration gradient of the ligand of interest to be tested to the fusion protein GPR35-NanoLuc for incubation. The binding of the ligand to be tested changes the degree of binding between GPR35-NanoLuc and 10-methyl-4,6-dioxo-1,4,6,9-tetrahydropyrido[3,2-g]quinoline-2,8-dicarboxylic acid derivatives in the BRET system, thereby resulting in a change in BRET efficiency to obtain a competition binding curve. The dissociation constant (K) is obtained by fitting the competition binding test curve using the competition binding module in the Graphpad software.i ).

[0023] In the invention, this type of compound generally exhibits agonistic activity to GPR35, is a specific agonist of human GPR35 receptor, and can be applied to bioluminescence resonance energy transfer (BRET) to test the affinity constant of GPR35 receptor and related ligands. This method has good sensitivity and biocompatibility, solves the problem that general compounds need to be labeled with radioactive ligands in activity tests, can achieve the determination of affinity constants of active molecules targeting GPR35, provides new probe molecules for GPR35 biology research, helps to explain the active action mechanism of related compounds, and provides lead compounds for the study of related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the general structural formula diagram of the derivative of the present invention;

[0025] Figure 2 The reaction scheme of the method for preparing the derivatives of the present invention;

[0026] Figure 3 Dose dependence curves of compounds 14-17 in dynamic mass distribution experiment (DMR) on HT-29 cells;

[0027] Figure 4 Binding experiments of compounds 14-17 (ad) on CHO-K1 cells expressing GPR35-NanoLuc; DETAILED DESCRIPTION

[0028]

[0029] Reaction conditions: (a) K2CO3, DMF, 50°C, overnight. (b) Diisopropyl azodicarboxylate, PPh3, 2-(prop-2-en-1-yloxy)ethane-1-ol, 0°C to room temperature, overnight. (c) CuSO4, sodium ascorbate, THF:H2O (1V:1V), overnight. (d) LiOH, MeOH:H2O:THF (1V:1V:4V), 1h.

[0030] The starting compounds used in the preparation of the compounds of the present invention are all known, can be prepared according to known methods or are commercially available.

[0031] Various preparations described below are synthetic intermediates useful in preparing compounds of the present invention.

[0032] Synthesis method a:

[0033] 4-Hydroxy-10-methyl-6-(pent-4-yn-1-yloxy)dimethylpyridinium[3,2-g]quinoline-2,8-dicarboxylate (Compound 5)

[0034] 342 mg (1.0 equivalent) of compound 1 (shown in the above reaction formula) was dissolved in 10 mL of DMF, and then 276 mg (2.0 equivalent) of potassium carbonate and 233 mg (1.2 equivalent) of iodopentyne were added thereto, and the mixture was reacted at 50°C for 12 hours, and the LC-MS (liquid chromatography-mass spectrometry) was performed until compound 1 (shown in the above reaction formula) completely disappeared. After the reaction was completed, 30 mL of dilute hydrochloric acid (2M) was added to the system, and then the mixture was extracted twice with 10 mL of ethyl acetate, the ethyl acetate phases were combined, dried over anhydrous sodium sulfate, and silica gel was added to the column for drying, and the elution phase was dichloromethane: methanol (20V: 1V) to obtain the pure target product.

[0035] 4-Hydroxy-10-methyl-6-(hex-4-yn-1-yloxy)dimethylpyridinium[3,2-g]quinoline-2,8-dicarboxylate (Compound 6)

[0036] 342 mg (1.0 equivalent) of compound 1 (shown in the above reaction structure) was dissolved in 10 mL of DMF, and then 276 mg (2.0 equivalent) of potassium carbonate and 250 mg (1.2 equivalent) of iodohexyne were added thereto, and the mixture was reacted at 50°C for 12 hours, and the liquid quality control was performed until compound 1 (shown in the above reaction structure) completely disappeared. After the reaction was completed, 30 mL of dilute hydrochloric acid (2M) was added to the system, and then 10 mL of ethyl acetate was used for extraction twice, the ethyl acetate phase was combined, dried over anhydrous sodium sulfate, and silica gel was added to spin dry the column, and the elution phase was dichloromethane: methanol (20V: 1V) to obtain the pure target product.

[0037] 4-Hydroxy-10-methyl-6-(hept-4-yn-1-yloxy)dimethylpyridinium[3,2-g]quinoline-2,8-dicarboxylate (Compound 7)

[0038] 342 mg (1.0 equivalent) of compound 1 (shown in the above reaction structure) was dissolved in 10 mL of DMF, and then 276 mg (2.0 equivalent) of potassium carbonate and 266 mg (1.2 equivalent) of iodoheptyne were added thereto, and the mixture was reacted at 50°C for 12 hours, and the liquid quality control was performed until compound 1 (shown in the above reaction structure) completely disappeared. After the reaction was completed, 30 mL of dilute hydrochloric acid (2M) was added to the system, and then 10 mL of ethyl acetate was used for extraction twice, the ethyl acetate phase was combined, dried over anhydrous sodium sulfate, and silica gel was added to spin dry the column, and the elution phase was dichloromethane: methanol (20V: 1V) to obtain the pure target product.

[0039] Synthesis method b:

[0040] 4-Hydroxy-10-methyl-6-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy]pyridine[3,2-g]quinoline-2,8-dicarboxylic acid dimethyl ester (Compound 8)

[0041] 342 mg (1.0 equivalent) of compound 1 (shown in the above reaction structure) was dissolved in 10 mL of THF, and then 524 mg (2.0 equivalent) of triphenylphosphine and 120 mg (1.2 equivalent) of 2-(prop-2-en-1-yloxy)ethane-1-ol were added thereto, and the air in the system was replaced by nitrogen, and then the temperature was lowered to 0°C. Under nitrogen protection, 410 mg (2.0 equivalent) of diisopropyl azodicarboxylate was added dropwise to the system, and then the temperature was raised to room temperature for reaction for 12 hours. The liquid quality test was performed until compound 1 (shown in the above reaction structure) completely disappeared. After the reaction was completed, 30 mL of water was added to the system, and then 10 mL of ethyl acetate was used for extraction twice. The ethyl acetate phase was combined, dried over anhydrous sodium sulfate, and silica gel was added to spin dry the column. The elution phase was dichloromethane: methanol (20V: 1V) to obtain the pure target product.

[0042] Synthesis method c:

[0043] Dimethyl 4-(3-(1-(2-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborol-10-yl)phenoxy)ethyl)-1H-1,2,3-triazol-4-ylpropoxy)-6-hydroxy-10-methylpyridyl[3,2-g]quinoline-2,8-dicarboxylate (Compound 10)

[0044] 200 mg (1.0 equivalent) of compound 5 (shown in the above reaction formula) was dissolved in 10 mL of a mixed solvent of tetrahydrofuran: water (1V: 1V), and then 210 mg (1.1 equivalent) of compound 9 (shown in the above reaction formula) was added to the system. After stirring evenly, 63 mg (0.5 equivalent) of copper sulfate and 50 mg (0.5 equivalent) of sodium ascorbate were added to the system in sequence, and then reacted at room temperature for 12 hours. The liquid quality monitoring reaction was performed until compound 5 (shown in the above reaction formula) completely disappeared. After the reaction was completed, 50 mL of water was added to the system, and then extracted twice with 20 mL of ethyl acetate, the ethyl acetate phases were combined, dried over anhydrous sodium sulfate, and silica gel was added to spin dry the column. The elution phase was dichloromethane: methanol (50V: 1V) to obtain the pure target product.

[0045] Dimethyl 4-(4-(1-(2-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ4 ,5λ 4 -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborol-10-yl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)butoxy)-6-hydroxy-10-methylpyridyl[3,2-g]quinoline-2,8-dicarboxylate (Compound 11)

[0046] 212 mg (1.0 equivalent) of compound 6 (shown in the above reaction formula) was dissolved in 10 mL of a mixed solvent of tetrahydrofuran: water (1V: 1V), and then 210 mg (1.1 equivalent) of compound 9 (shown in the above reaction formula) was added to the system. After stirring evenly, 63 mg (0.5 equivalent) of copper sulfate and 50 mg (0.5 equivalent) of sodium ascorbate were added to the system in sequence, and then reacted at room temperature for 12 hours. The liquid quality monitoring reaction was performed until compound 5 (shown in the above reaction formula) completely disappeared. After the reaction was completed, 50 mL of water was added to the system, and then extracted twice with 20 mL of ethyl acetate, the ethyl acetate phases were combined, dried over anhydrous sodium sulfate, and silica gel was added to spin dry the column. The elution phase was dichloromethane: methanol (50V: 1V) to obtain the pure target product.

[0047] Dimethyl 4-((5-(1-(2-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaboroserine-10-yl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)pentyloxy)-6-hydroxy-10-methylpyridyl[3,2-g]quinoline-2,8-dicarboxylate (Compound 12)

[0048] 225 mg (1.0 equivalent) of compound 7 (shown in the above reaction formula) was dissolved in 10 mL of a mixed solvent of tetrahydrofuran: water (1V: 1V), and then 210 mg (1.1 equivalent) of compound 9 (shown in the above reaction formula) was added to the system. After stirring evenly, 63 mg (0.5 equivalent) of copper sulfate and 50 mg (0.5 equivalent) of sodium ascorbate were added to the system in sequence, and then reacted at room temperature for 12 hours. The liquid quality monitoring reaction was performed until compound 5 (shown in the above reaction formula) completely disappeared. After the reaction was completed, 50 mL of water was added to the system, and then extracted twice with 20 mL of ethyl acetate, the ethyl acetate phases were combined, dried over anhydrous sodium sulfate, and silica gel was added to spin dry the column. The elution phase was dichloromethane: methanol (50V: 1V) to obtain the pure target product.

[0049] Dimethyl 4-(2-((1-(2-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborolan-10-yl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)-6-hydroxy-10-methylpyridinyl[3,2-g]quinoline-2,8-dicarboxylate (Compound 13)

[0050] 220 mg (1.0 equivalent) of compound 8 (shown in the above reaction structure) was dissolved in 10 mL of a mixed solvent of tetrahydrofuran: water (1:1), and then 210 mg (1.1 equivalent) of compound 9 was added to the system. After stirring evenly, 63 mg (0.5 equivalent) of copper sulfate and 50 mg (0.5 equivalent) of sodium ascorbate were added to the system in sequence, and then reacted at room temperature for 12 hours. The liquid quality monitoring reaction was performed until compound 5 (shown in the above reaction structure) completely disappeared. After the reaction was completed, 50 mL of water was added to the system, and then extracted twice with 20 mL of ethyl acetate, the ethyl acetate phases were combined, dried over anhydrous sodium sulfate, and silica gel was added to spin dry the column. The elution phase was dichloromethane: methanol (50V: 1V) to obtain the pure target product.

[0051] The above compounds are all precursors required for the synthesis of the following examples.

[0052] Example 1

[0053] 4-(3-(1-(2-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborol-10-yl)phenoxy)ethyl)-1H-1,2,3-triazol-4-ylpropoxy)-6-hydroxy-10-methylpyridyl[3,2-g]quinoline-2,8-dicarboxylic acid (Compound 14)

[0054] 100 mg of compound 10 (shown in the above reaction structure) was dissolved in 10 mL of a mixed solvent of methanol: water: tetrahydrofuran (1V: 1V: 4V), stirred evenly, and then 25 mg (4.0 equivalents) of LiOH was added to the system, stirred for 1 hour, and the liquid quality was monitored until compound 10 completely disappeared. After the reaction was completed, 5 mL of dilute hydrochloric acid (2M) was added to the system, and then the tetrahydrofuran and methanol in the system were dried by spin drying, the precipitated solid was filtered out, and then the solid was washed with 1 mL of methanol to obtain 43 mg of pure target product, the product was a yellow solid. The purity of the compound was determined to be >95% by chromatography and mass spectrometry, and the structure was confirmed to be compound 14 by nuclear magnetic resonance and mass spectrometry.

[0055] 1 H NMR (600MHz, DMSO-d6) δ8.87(s,1H),8.06(s,1H),7.45(s,1H),7.16(d,J=3.9Hz,2H),7.07–7.04(m,2H),6.66(s,1H),6.10(s, 2H),4.76(t,J=5.1Hz,2H),4.46–4.42(m,4H),3.03(s,3H),2.94(t,J=7.4Hz,2H),2.42(s,6H),2.32–2.28(m,2H),1.30(s,6H). 13 C NMR(151MHz,DMSO-d6)δ166.90,164.23,163.95,158.93,155.08,151.98,146.83,146.41,143.12,142.36,131.47,129 .60,126.88,124.85,123.34,121.69,118.62,117.58,115.70,99.14,68.72,66.75,49.34,28.53,21.98,14.65,14.59.

[0056] Example 2

[0057] 4-(4-(1-(2-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborol-10-yl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)butoxy)-6-hydroxy-10-methylpyridyl[3,2-g]quinoline-2,8-dicarboxylic acid (Compound 15)

[0058] 100 mg of compound 10 (shown in the above reaction structure) was dissolved in 10 mL of a mixed solvent of methanol: water: tetrahydrofuran (1V: 1V: 4V) by volume, and after stirring evenly, 25 mg (4.0 equivalents) of LiOH was added to the system, and the reaction was stirred for 1 hour. The liquid quality was monitored until the compound 10 (shown in the above reaction structure) completely disappeared. After the reaction was completed, 5 mL of dilute hydrochloric acid (2M) was added to the system, and then the tetrahydrofuran and methanol in the system were dried by spin drying, and the precipitated solid was filtered out, and then the solid was washed with 1 mL of methanol to obtain 58 mg of pure target product, which was a yellow solid. The purity of the compound was determined to be >95% by chromatography and mass spectrometry, and the structure was confirmed to be compound 15 by nuclear magnetic resonance and mass spectrometry.

[0059] 1 H NMR(600MHz,DMSO-d6)δ8.83(d,J=9.7Hz,1H),8.03(s,1H),7.43(s,1H),7.18(d ,J=8.6Hz,2H),7.05(t,J=8.2Hz,2H),6.56(s,1H),6.01(s,2H),4.76(t,J=5.0Hz ,2H),4.45(t,J=5.0Hz,2H),4.38(t,J=6.5Hz,2H),3.01(s,3H),2.79(t,J=7.4Hz ,2H),2.39(s,6H),1.98(dd,J=14.6,6.7Hz,2H),1.92–1.83(m,2H),1.26(s,6H). 13 C NMR(151MHz,DMSO-d6)δ166.94,163.98,163.80,159.00,154.95,151.83,147.05,146.72,143.00,142.21,131.38,129.61,126.88 ,124.79,123.18,121.56,118.41,117.54,115.70,107.19,98.93,69.30,66.94,49.39,28.28,25.96,25.11,14.61,14.55,11.29.

[0060] Example 3

[0061] 4-((5-(1-(2-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaboroserine-10-yl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)pentyloxy)-6-hydroxy-10-methylpyridyl[3,2-g]quinoline-2,8-dicarboxylic acid (Compound 16)

[0062] 100 mg of compound 10 (shown in the above reaction structure) was dissolved in 10 mL of a mixed solvent of methanol: water: tetrahydrofuran (1V: 1V: 4V) by volume, and after stirring evenly, 25 mg (4.0 equivalents) of LiOH was added to the system, and the reaction was stirred for 1 hour. The liquid quality was monitored until the compound 10 (shown in the above reaction structure) completely disappeared. After the reaction was completed, 5 mL of dilute hydrochloric acid (2M) was added to the system, and then the tetrahydrofuran and methanol in the system were dried by spin drying, and the precipitated solid was filtered out, and then the solid was washed with a small amount of methanol to obtain 47 mg of pure target product, which was a yellow solid. The purity of the compound was determined to be >95% by chromatography and mass spectrometry, and the structure was confirmed to be compound 16 by nuclear magnetic resonance and mass spectrometry.

[0063] 1 H NMR(600MHz,DMSO-d6)δ8.88(s,1H),7.99(s,1H),7.45(s,1H),7.19(dd,J=8.2,5.5Hz,2H ),7.06(d,J=8.7Hz,2H),6.63(s,1H),6.11(s,2H),4.74(t,J=5.1Hz,2H),4.44(t,J=5.1Hz ,2H),4.38(t,J=6.4Hz,2H),3.03(s,3H),2.71(t,J=7.5Hz,2H),2.42(s,6H),2.01–1.96( m,2H),1.76(dt,J=15.2,7.6Hz,2H),1.59(dt,J=15.1,7.6Hz,2H),1.32(d,J=10.0Hz,6H). 13 C NMR(151MHz,DMSO-d6)δ165.81,162.94,162.73,157.87,153.99,150.81,146.20,145.69,142.02,141.26,130.38,128.52,125.79,12 3.76,121.88,120.60,117.43,116.50,114.60,106.22,97.92,68.47,65.72,48.20,27.98,27.40,24.48,24.30,13.55,13.52,10.25.

[0064] Example 4

[0065] 4-(2-((1-(2-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ 4 ,5λ 4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborolan-10-yl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)-6-hydroxy-10-methylpyridyl[3,2-g]quinoline-2,8-dicarboxylic acid (Compound 17)

[0066] 100 mg of compound 10 (shown in the above reaction structure) was dissolved in 10 mL of a mixed solvent of methanol: water: tetrahydrofuran (1V: 1V: 4V) by volume, and after stirring evenly, 25 mg (4.0 equivalents) of LiOH was added to the system, and the reaction was stirred for 1 hour. The liquid quality was monitored until the compound 10 (shown in the above reaction structure) completely disappeared. After the reaction was completed, 5 mL of dilute hydrochloric acid (2M) was added to the system, and then the tetrahydrofuran and methanol in the system were dried by spin drying, and the precipitated solid was filtered out, and then the solid was washed with a small amount of methanol to obtain 62 mg of pure target product, which was a yellow solid. The purity of the compound was determined to be >95% by chromatography and mass spectrometry, and the structure was confirmed to be compound 17 by nuclear magnetic resonance and mass spectrometry.

[0067] 1 H NMR (600MHz, DMSO-d6) δ8.88(s,1H),8.28(s,1H),7.47(s,1H),7.19–7.15(m,2H),7.04(d,J=8.0Hz,2H),6.59(s,1H),6.07(s,2H),4.84–4 .77(m,2H),4.72(d,J=9.9Hz,2H),4.52(d,J=18.6Hz,2H),4.47(t,J=4.7Hz,2H),4.04–4.00(m,2H),3.02(s,3H),2.41(s,6H),1.28(s,6H). 13 C NMR(151MHz,DMSO-d6)δ166.92,163.95,163.59,158.88,155.07,151.83,146.71,144.41,143.11,142.26,137.95,131.40,129.58 ,126.87,125.10,124.83,121.66,118.32,117.77,115.67,106.99,99.05,68.98,68.15,66.74,64.14,49.54,14.62,14.55,11.25.

[0068] Example 5

[0069] Pharmacological Characterization – DMR Screening Model

[0070] Materials HT-29 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, Minchuanning was purchased from Sigma, and ML145 was purchased from Tocris. The detection platform was Corning's third-generation The imager detects the wavelength shift caused by the dynamic mass resetting (DMR) of the cell.

[0071] HT-29 cells in the logarithmic growth phase were inoculated in 384-well Epic dedicated cell culture plates. The inoculation volume of each well was 40 μL (the culture medium was McCoy's 5A containing 10% fetal bovine serum by volume), and the number of cells inoculated in each well was 3.2×10 4 The inoculated cell plate was placed in 5% CO2 (air containing 5% CO2 by volume concentration). After 20 hours of culture, the cells grew into a stable and tight monolayer cell film at the bottom of the biological microplate.

[0072] Agonist activity test, taking compound 14 as an example. Before the assay, the cell culture medium in the well was aspirated, 30 μL HBSS buffer solution was added, and the culture plate was placed on the epic instrument and incubated at room temperature for 1 hour. After the cell state reached stability (baseline stability), the concentration gradient of compound 14 (final concentrations of 10000nM, 5000nM, 2500nM, 1250nM, 625nM, 312nM, 156nM, 78nM, 39nM, 20nM, 10nM, 5nM, 2.5nM, 1.25nM, each concentration corresponding to three replicate wells) was applied to HT-29 cells, and the DMR signal caused by the agonist was monitored in real time. The detection time was 1 hour (compound 14 was added to different wells where the cells were located).

[0073] Continue to use the desensitization analysis method to verify the specificity of these compounds on the GPR35 receptor. For the desensitization experiment, two steps are required. Before the measurement, the cell culture medium in the well is aspirated, 30 μL HBSS buffered saline solution is added, and the culture plate is placed on the epic instrument and incubated at room temperature for 1 hour. After the cell state reaches stability (baseline stability), the first step is to pretreat the cells with compound 14 (final concentrations of 10000nM, 5000nM, 2500nM, 1250nM, 625nM, 312nM, 156nM, 78nM, 39nM, 20nM, 10nM, 5nM, 2.5nM, 1.25nM, each concentration corresponds to three replicates) for 1 hour. In the second step, a fixed concentration of 1 μM of Minchuanning is added to each well, and the DMR signal caused by the agonist is monitored in real time. The detection time is 1 hour.

[0074] The specificity of compound 14 on GPR35 was verified by antagonistic analysis. Before the assay, the cell culture medium in the well was aspirated, 30 μL HBSS buffer solution was added, and the culture plate was placed on the epic instrument for incubation at room temperature for 1 h. After the cell state reached stability (baseline stability), HT-29 cells were pretreated with ML-145 with a concentration gradient (final concentrations of 8 μM, 4 μM, 2 μM, 1 μM, 0.5 μM, 0.25 μM, 0.125 μM, 0.063 μM, 0.031 μM, 0.015 μM, 0.008 μM, 0.004 μM, and each concentration corresponded to three replicate wells) for 5 min, and then compound 14 was added to each well at a final concentration of 200 nM, and the DMR signal caused by the agonist was monitored in real time for 1 h.

[0075] Compounds 15, 16, and 17 were also tested in sequence according to the above method (the difference is that compounds 15, 16, and 17 were used instead of compound 14), and the activity results obtained are shown in Table 1.

[0076] Table 1. Activity data of compounds of the general formula in the DMR screening model (HT-29).

[0077]

[0078]

[0079] The synthesized compounds all have good GPR35 agonist activity

[0080] Pharmacological characterization - determination of receptor-ligand equilibrium constants

[0081] Materials CHO-K1 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; transfection reagent lipo3000 was purchased from ThermoFisher Scientific; furimazine was purchased from Glpbio; zaprinast was purchased from Sigma; ML145 was purchased from Tocris, and ML194 was purchased from Sigma-Aldrich. The detection platform was the BioTekCytation 5 cell imaging multifunctional detection system, and the detected signal was the fluorescence intensity under 485nm and 528nm filters.

[0082] CHO-K1 cells were seeded in 96-well plates at a density of 40,000 cells per well (the culture medium was F12K containing 10% fetal bovine serum by volume). After 24 hours of growth, CHO-K1 cells were transiently transfected with 100 ng of plasmid DNA (GPR35-NanoLuc fusion) per well according to the instructions of the transfection kit. 48 hours after transfection, the original culture medium in the wells was replaced with HBSS buffer.

[0083] For the equilibrium dissociation constant (K D ), taking compound 14 as an example. Minchuanning (final concentration of 10 μM, used to determine nonspecific binding) or DMSO of corresponding concentration (used to determine total binding) was added to the above-mentioned transient cell wells, and incubated at room temperature for 30 minutes; the concentration gradient of fluorescent probe compound 14 (final concentrations of 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM, 1.5625nM, 0.78125nM, each concentration corresponds to three replicate wells) was added to different wells where the cells were located, and incubated at room temperature for 30 minutes. The fluorescent reporter NanoLuc substrate furazolidinone was added to the wells where the cells were located (final concentration 10 μM), and the fluorescence intensity was immediately measured under 485 nm and 528 nm filters on a BioTek Cytation 5 microplate reader. The ratio of the fluorescence intensity at 528 nm to the fluorescence intensity at 485 nm (vertical axis) was plotted against the concentration (horizontal axis). The curve was fitted using the binding-saturation curve module (provided in the software) in the Prism software to obtain the equilibrium dissociation constant (K) of compound 14 on GPR35. D ) is the value of .

[0084] Compounds 15, 16, and 17 were tested in the same manner as above (the difference was that compounds 15, 16, and 17 were used to replace compound 14), and K D The results are as follows Figure 4 shown.

[0085] For the dissociation constant (K i ) was measured, and the test compound was taken as an example of Minchuanning. A fixed concentration of fluorescent probe compound 15 (final concentration 25nM) and the corresponding concentration of DMSO (blank) were added to the cell wells and incubated at room temperature for 30 minutes. Minchuanning with a concentration gradient (final concentrations of 200μM, 50μM, 12.5μM, 3.125μM, 0.78125μM, 0.1953μM, 0.0488μM, 0.0122μM, each concentration corresponding to three replicate wells) was added to different wells where the cells were located and incubated at room temperature for 30 minutes. After the fluorescent reporter NanoLuc substrate furazolidinone was added to the wells where the cells were located (final concentration 10 μM), the fluorescence intensity was immediately measured under 485 nm and 528 nm filters on the BioTek Cytation 5 microplate reader. The ratio of the fluorescence intensity at 528 nm to the fluorescence intensity at 485 nm (vertical axis) was plotted against the logarithmic concentration (horizontal axis). After deducting the blank group data, the Prism software binding-competition curve module (provided with the software) was used to fit the curve to obtain the dissociation constant (K) of chloranil. i ) is the value of .

[0086] Compounds Pamer's acid, 4,6-dihydroxy-10-methylpyridine [3,2-g] quinoline-2,8-dicarboxylic acid, ML-145, ML-194, and sodium cromoglycate were tested in the same manner as above (the difference was that Pamer's acid, 4,6-dihydroxy-10-methylpyridine [3,2-g] quinoline-2,8-dicarboxylic acid, ML-145, ML-194, and sodium cromoglycate were used to replace fenvalerate, respectively). The K i The results are shown in Table 2.

[0087] Table 2 K values ​​of known GPR35 ligands tested by BRET method i

[0088]

[0089]

[0090] Measured pK i The values ​​are in the same order of magnitude as those reported in the literature using the radioligand method. Spectral properties characterization - absorption and emission spectrometry

[0091] 10-Methyl-4,6-dioxo-1,4,6,9-tetrahydropyrido[3,2-g]quinoline-2,8-dicarboxylic acid derivatives 14, 15, 16, and 17 were dissolved in n-octanol solution at a concentration of 2 μM. The maximum absorption wavelength of the compound was determined using a Cary 60UV-Vis spectrophotometer; the maximum emission wavelength of the compound was determined using a Cary Eclipse fluorescence spectrophotometer.

[0092] Table 3 Spectral properties of compounds

[0093]

[0094] The spectral properties of the above compounds were tested in n-octanol.

Claims

1. A dicarboxylic acid derivative comprising one or more of 10-methyl-4,6-dioxo-1,4,6,9-tetrahydropyrido[3,2-g]quinoline-2,8-dicarboxylic acid derivatives and / or pharmaceutically acceptable salts thereof, characterized in that: The general structural formula of 10-methyl-4,6-dioxo-1,4,6,9-tetrahydropyrido[3,2-g]quinoline-2,8-dicarboxylic acid derivatives is as follows: , where linker is selected from , where n is 2-10; or linker is selected from , where n is 1-5.

2. The dicarboxylic acid derivative according to claim 1, characterized in that: The linker is selected from , where n is 3-5; or linker is selected from , where n is 1.

3. A dicarboxylic acid derivative, characterized in that: The derivatives include one or more of the following compounds 14-17 and / or their pharmaceutically acceptable salts, the specific names of which are: (1) 4-(3-(1-(2-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborol-10-yl)phenoxy)ethyl)-1H-1,2,3-triazol-4-ylpropoxy)-6-hydroxy-10-methylpyridyl[3,2-g]quinoline-2,8-dicarboxylic acid, i.e., compound 14; (2) 4-(4-(1-(2-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborol-10-yl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)butoxy)-6-hydroxy-10-methylpyridyl[3,2-g]quinoline-2,8-dicarboxylic acid, i.e., compound 15; (3) 4-((5-(1-(2-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaboroserine-10-yl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)pentyloxy)-6-hydroxy-10-methylpyridyl[3,2-g]quinoline-2,8-dicarboxylic acid, i.e., compound 16; (4) 4-(2-((1-(2-(4-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ 4 ,5λ 4 -dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborolan-10-yl)phenoxy)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)-6-hydroxy-10-methylpyridinyl[3,2-g]quinoline-2,8-dicarboxylic acid, i.e., compound 17.

4. The dicarboxylic acid derivative according to claim 1 or 3, characterized in that: The compound exists in its crystalline form; The pharmaceutically acceptable salt includes one or more of lithium salt, sodium salt, potassium salt, calcium salt, magnesium salt, or ammonium salt of dicarboxylic acid derivatives.

5. A method for synthesizing a dicarboxylic acid derivative according to any one of claims 1 to 4, characterized in that: The synthetic route of the compound described in claim 2 is as follows: 。 6. Use of the dicarboxylic acid derivative according to any one of claims 1 to 4 as a GPR35 agonist or a GPR35 receptor fluorescent probe molecule, wherein the use is not suitable for diagnosis and treatment of diseases.

7. A GPR35 agonist comprising one or more of the dicarboxylic acid derivatives according to any one of claims 1 to 4.

8. Use of the dicarboxylic acid derivative according to any one of claims 1 to 4 for characterizing whether a compound to be determined can bind to a target receptor GPR35; Or, a dicarboxylic acid derivative according to any one of claims 1 to 4 is used to determine the dissociation constant K between the ligand to be tested and the target receptor GPR35 i Purpose; Or, a method for determining the equilibrium dissociation constant K between the dicarboxylic acid derivative according to any one of claims 1 to 4 and a target receptor D method, The uses and methods described are not suitable for diagnosis and treatment of diseases.

9. The use or method according to claim 8, using a method for determining the affinity constant of a ligand of interest targeting a receptor based on bioluminescence resonance energy transfer (BRET), wherein the BRET system comprises a cell target fused to a bioluminescent reporter, a bioactive agent tethered to a fluorophore, and a substrate for the bioluminescent reporter, wherein (1) the cell target portion fused to the bioluminescent reporter comprises a luciferase fused to a target receptor (TR), which is expressed intracellularly as a fusion; (2) The bioactive agent tethered to the fluorophore is a dicarboxylic acid derivative according to any one of claims 1 to 3, comprising a ligand L capable of targeting and binding to TR, and a fluorophore capable of accepting energy from the Luc by resonance energy transfer in the presence of a suitable Luc substrate, wherein the absorption spectrum of the fluorophore overlaps with the emission spectrum of the bioluminescent reporter; (3) Among them, The binding of the ligand to be tested to TR changes the degree of binding between TR and L in the BRET system, thus leading to changes in BRET efficiency; Where Luc is nanoluciferase; The luciferase substrate is furazolidinone; wherein the TR is G protein-coupled receptor 35; The ligand to be tested is any ligand molecule that may target GPR35 that is to be assayed.

10. The use or method of claim 9, wherein the bioactive agent tethered to the fluorophore, the ligand to be detected, and the luciferase substrate are added to the cell culture system; A. Using bioluminescence resonance energy transfer to detect the equilibrium dissociation constant K of the 10-methyl-4,6-dioxo-1,4,6,9-tetrahydropyrido[3,2-g]quinoline-2,8-dicarboxylic acid derivative described in any one of claims 1 to 3 D , the method comprises the steps of: (1) transiently transfecting cells to express a fusion of the cell target GPR35 and the bioluminescent reporter NanoLuc; (2) incubating the cells with two or more dicarboxylic acid derivatives at different concentration gradients; (3) incubating the cells with a substrate of the bioluminescent reporter; (4) Collecting fluorescence at a first wavelength of 485 nm, the emission wavelength of the bioluminescent reporter NanoLuc, and a second wavelength of 528 nm, the emission wavelength of the bioactive agent tethered to the fluorophore, wherein the ratio of the fluorescence values ​​at the second wavelength to the first wavelength indicates the dynamic binding process of the dicarboxylic acid derivative on the cell target, and obtaining a saturated binding curve with the fluorescence intensity ratio as the ordinate and the concentration as the abscissa. According to the saturated binding curve, the equilibrium dissociation constant K is obtained by fitting using Graphpad D ; or B. Use bioluminescence resonance energy transfer to detect the dissociation constant K of the ligand compound of interest i , the method comprises the steps of: (1) transiently transfecting cells to express a fusion of the cell target GPR35 and the bioluminescent reporter NanoLuc; (2) incubating the cells with a fixed concentration of a dicarboxylic acid derivative of a bioactive agent tethered to a fluorophore; (3) incubating the cells with a concentration gradient of the ligand of interest; (4) incubating the cells with a substrate of the bioluminescent reporter; (5) Collecting fluorescence at a first wavelength of 485 nm, the emission wavelength of the bioluminescent reporter NanoLuc, and a second wavelength of 528 nm, the emission wavelength of the bioactive agent tethered to the fluorophore, wherein the ratio of the fluorescence value at the second wavelength to the first wavelength indicates the competitive binding effect between the ligand of interest and the dicarboxylic acid derivative on the cell target, and obtaining a competition binding curve with the fluorescence intensity ratio as the ordinate and the logarithmic concentration as the abscissa. According to the competition binding test curve, the dissociation constant K is obtained by fitting using the competition binding module in the Graphpad software. i .

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