A thiophene-pyrimidine compound and its application as an allosteric antagonist of dopamine D2 receptor

Abstract

This invention belongs to the field of medicinal chemistry, specifically disclosing a thienopyrimidine compound and its application as an allosteric antagonist of the dopamine D2 receptor (D2R). The target compounds of this invention contain a thieno[3,2-d]pyrimidine ring skeleton, the structural formula of which is shown in Formula 1. This invention uses GloSensor cAMP accumulation experiments to test the functional activity of the target compounds for D2R and to elucidate the allosteric mechanism of action of the new compounds on D2R. Pharmacological results show that all target compounds in this invention can negatively allosterically regulate the functional activity of the D2R endogenous ligand dopamine, i.e., all target compounds are negative allosteric modulators or allosteric antagonists of D2R.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, and specifically relates to a thienopyrimidine compound and its application as an allosteric antagonist of dopamine D2 receptor. Background Technology

[0002] G protein-coupled receptors (GPCRs), also known as seven-transmembrane proteins, are the largest superfamily of receptor proteins encoded by the human genome and located on the cell membrane surface. GPCRs have a seven-transmembrane α-helix structure linked by three extracellular loops and three intracellular loops. The N-terminus is extracellular, and the C-terminus is intracellular.

[0003] Dopamine (DA), an important catecholamine neurotransmitter, projects from the ventral tegmental nucleus of the midbrain to dopamine receptors in the prefrontal cortex, hippocampus, striatum, nucleus accumbens, and hypothalamus. It is primarily involved in emotional thinking, motor function, and cognitive activities. Dopamine receptors are important members of the A family of GPCRs. Based on their pharmacological and biochemical characteristics, they can be divided into two main categories: D1-like receptors and D2-like receptors. The former includes dopamine D1 receptors (D1R) and dopamine D5 receptors (D5R), which mainly interact with GPCRs. s Protein-coupled receptors, upon activation, stimulate the production of cAMP; cAMP includes dopamine D2 receptors (D2R), dopamine D3 receptors (D3R), and dopamine D4 receptors (D4R), which are associated with G... i / o Protein-mediated receptor activation inhibits cAMP production. D2R is an important target for the treatment of central nervous system disorders, including Parkinson's disease and schizophrenia.

[0004] Small molecule compound 1 is a novel D2R negative allosteric modulator, NAM, recently discovered through virtual screening (J. Med. Chem. 2019, 62, 174-206.). This compound contains a thiophene[2,3-d]pyrimidine ring skeleton, exhibiting a completely new structural feature distinct from known dopamine ligands. This is also the first time that thiophenepyrimidine compounds have been used in D2R research. Fyfe et al. conducted the first preliminary structure-activity relationship (SAR) study on compound 1, discovering an open-ring and structurally simplified analog 2, whose functional activity and synergistic effect remain comparable to lead compound 1 (Eur. J. Med. Chem. 2019, 168, 474-490.). Further SAR studies revealed some essential activity groups, but the activity of all the derived compounds was not significantly improved compared to lead compound 1; the allosteric modulatory activity remained at the micromolar level, which is far from the drug-like activity of D2R ligands. In addition, the research group retained the core skeleton thiophene[2,3-d]pyrimidine ring in all structural modifications and alterations of compound 1, and did not conduct a structure-activity relationship study on this skeleton, which may also be the reason for the low activity of the obtained derivative.

[0005]

[0006] Scaffoldhopping, also known as skeletal transition, is a medicinal chemistry method involving molecular skeleton substitution and an important drug design strategy (Drug Discov. Today 2012, 17, 310-324). It can be used to develop new compounds with better activity / selectivity / physicochemical properties and / or improved pharmacokinetic properties (ADME), and is one of the important pathways for lead compound discovery and optimization. Scaffoldhopping methods mainly include bioisosteres, heterocyclic substitution, ring opening, or ring closing (J. Med. Chem. 2017, 60, 1238-1246).

[0007] Summary of the Invention

[0008] This invention focuses on the ring-opening design of small molecule compound 1 mentioned in the background art, and then uses skeletal transition to replace the thiophene [2,3-d]pyrimidine ring with a new thiophene [3,2-d]pyrimidine ring. The influence of substituents on the activity of the new compound is investigated, and a structure-activity relationship study is conducted in order to obtain a new allosteric modulator with improved activity, better selectivity, and D2R targeting.

[0009] The present invention provides a thiophene-pyrimidine compound, the structure of which is shown in Formula 1:

[0010] in:

[0011] R1 can be one of the following structures:

[0012]

[0013] Alkyl or aryl;

[0014] R2 can be one of the following structures:

[0015]

[0016] Fatty amines, aromatic amines, or alkoxy groups;

[0017] R3 can be one of the following structures:

[0018]

[0019] Hydrogen-based, aliphatic amines, aromatic amines, or heterocyclic amines.

[0020] Furthermore, in thiophene-pyrimidine compounds, R1 is -H or -CH3;

[0021] R2 is one of the following groups:

[0022]

[0023]

[0024] R3 is one of the following groups:

[0025]

[0026] Furthermore, the structures of thiophene-pyrimidine compounds are as follows:

[0027]

[0028] This invention also provides a method for synthesizing thiophene[3,2-d]pyrimidine ring compounds of formula 1, specifically,

[0029] The synthetic route for the thiophene[3,2-d]pyrimidine ring compound of Formula 1 is as follows:

[0030]

[0031] The specific synthetic steps of the thiophene[3,2-d]pyrimidine ring compound (Formula 1) are as follows:

[0032] I. Synthetic methods for monosubstituted thiophene[3,2-d]pyrimidine compounds

[0033] The specific synthesis steps are as follows:

[0034] (1) Compound 3 was dissolved in a solvent and heated to 170°C under reflux and stirring. Post-treatment yielded ketone intermediate 4. Compound 3 was ethyl 3-amino-4-methylthiophene-2-carboxylate or ethyl 3-aminothiophene-2-carboxylate, the solvent was formamide, which was both a solvent and a reactant, and was in large excess. The reaction time was 12 h.

[0035] (2) Dissolve the desired compound 4 in a solvent, add the catalyst, slowly add phosphorus oxychloride, heat to 120°C, reflux and stir, and then process to obtain intermediate 5. The solvent is toluene, the catalyst is N,N-dimethylformamide, and the reaction time is 3-5 h. The molar ratio of intermediate 4:catalyst:phosphorus oxychloride is 1:0.3:4.

[0036] (3) Dissolve the desired compound 5 in a solvent, add an acid-binding agent, slowly add the desired amine, heat to 50 degrees Celsius under nitrogen protection, stir the reaction, and then process to obtain the amine target compound 6. The solvent is tetrahydrofuran, the acid-binding agent is triethylamine, the desired amine is any aliphatic or aromatic amine, and the reaction time is 10 h. The molar ratio of intermediate 5: desired amine: acid-binding agent is 1:1.2:3.

[0037] (4) The desired compound 5 was dissolved in a dehydrated solvent, and the base was added under stirring in an ice bath to remove hydrogen. After reacting for half an hour, the ice bath was removed, the desired alcohol was added, and the mixture was stirred at room temperature. The post-treatment yielded the target ether compound 6. The solvent was 1,4-dioxane, the base was NaH, the desired alcohol was methanol or isopropanol, the second reaction time was 5 hours, and the molar ratio of intermediate 5: desired alcohol: NaH was 1:1.5:3.

[0038] II. Synthetic methods for disubstituted thiophene[3,2-d]pyrimidine compounds

[0039] The specific synthesis steps are as follows:

[0040] (1) Compound 3 was dissolved in a solvent, and chloroacetonitrile was slowly added dropwise while hydrogen chloride gas was introduced. The reaction was carried out under heating conditions with stirring. The resulting precipitate was intermediate compound 7. Compound 3 was ethyl 3-amino-4-methylthiophene-2-carboxylate or ethyl 3-aminothiophene-2-carboxylate, the solvent was 1,4-dioxane, the reaction temperature was 50℃, and the reaction time was 6-12 h. The molar ratio of compound 3 to chloroacetonitrile was 1:1.2.

[0041] (2) The desired compound 7 was dissolved in a solvent, an acid-binding agent was added, and the desired amine was slowly added. The mixture was heated to 50 degrees Celsius under nitrogen protection, and the reaction was stirred. After post-treatment, compound 8 was obtained. The solvent was tetrahydrofuran, the acid-binding agent was triethylamine, the desired amine was any aliphatic or aromatic amine, and the reaction time was 10 h. The molar ratio of intermediate 7: desired amine: acid-binding agent was 1:1.2:3.

[0042] (3) Dissolve the desired compound 8 in a solvent, add the catalyst, slowly add phosphorus oxychloride, heat to 120°C, reflux and stir, and then process to obtain intermediate 9. The solvent is toluene, the catalyst is N,N-dimethylformamide, and the reaction time is 3-5 h. The molar ratio of intermediate 4:catalyst:phosphorus oxychloride is 1:0.3:4.

[0043] (4) Dissolve the desired compound 9 in a solvent, add an acid-binding agent, slowly add the desired amine, heat to 50 degrees Celsius under nitrogen protection, stir the reaction, and then process to obtain the target compound 10. The solvent is tetrahydrofuran, the acid-binding agent is triethylamine, the desired amine is any aliphatic or aromatic amine, and the reaction time is 10 h. The molar ratio of intermediate 9: desired amine: acid-binding agent is 1:1.2:3.

[0044] This invention utilizes GloSensor cAMP accumulation assays to test the functional activity of target compounds for D2R and to elucidate the allosteric mechanism of action of novel compounds on D2R. Pharmacological results indicate that all target compounds in this invention can negatively allosterically regulate the functional activity of the D2R endogenous ligand dopamine, meaning that all target compounds are negative allosteric modulators or allosteric antagonists of D2R.

[0045] One or more of the thienopyrimidine compounds provided in this invention can be used as active ingredients and pharmaceutically acceptable carriers to prepare pharmaceutical formulations. More specifically, the application of thienopyrimidine compounds in the preparation of D2R allosteric antagonists.

[0046] The beneficial effects of this invention are as follows:

[0047] Advantages of this invention: The small molecule compound 1 is designed with a ring-opening mechanism, and then a skeletal transition is used to replace the thiophene[2,3-d]pyrimidine ring with a novel thiophene[3,2-d]pyrimidine skeletal structure. The functional activity of the target compound for D2R is tested through cAMP accumulation experiments. Pharmacological results show that the synthesized series of target compounds have good allosteric regulatory functions for D2R; in-depth mechanistic studies have shown that the target compounds in this invention are all D2R allosteric antagonists. Furthermore, D2R is closely related to central nervous system diseases, including Parkinson's disease and schizophrenia. Currently, clinically used D2R drugs for treating Parkinson's disease and schizophrenia bind to the orthomeric site of the receptor. Long-term use of these drugs can cause significant side effects, such as extrapyramidal motor dysfunction, weight gain, or metabolic disorders. Allosteric modulators, however, have a certain saturation limit in regulating receptor function, and will not over-activate or completely block receptor function. Therefore, they can overcome the side effects caused by orthomeric ligand drugs, thus potentially leading to the development of safer and more effective central nervous system drugs. Attached image description:

[0048] Figure 1 The concentration-dependent curve of DA allosterically modulated by compound 6o;

[0049] Figure 2 The concentration-dependent curve of DA by allosteric regulation of compound 6q. Detailed Implementation

[0050] The invention will now be further explained with reference to examples.

[0051] I. Preparation of monosubstituted thiophene[3,2-d]pyrimidine cyclic amine compounds:

[0052] Example 1

[0053] Preparation of N,N-dimethylthiopheno[3,2-d]pyrimidine-4-amine (compound 6a)

[0054]

[0055] Step 1: Ethyl 3-aminothiophene-2-carboxylate (3g, 17.54mmol) was dissolved in formamide (45mL), heated to 170℃, and stirred under reflux for 12h. The mixture was then analyzed by TLC. After the reaction was completed, the mixture was cooled to room temperature and stirred in a -20℃ low-temperature oven to precipitate the product. The product was washed with ethyl acetate and dried to obtain the intermediate compound thieno[3,2-d]pyrimidine-4(3H)-one, 2.11g of grayish-white solid, yield 79%.

[0056] Step 2: The intermediate compound 4-thiopheno[3,2-d]pyrimidine-4(3H)-one (2.0 g, 13.16 mmol) was dissolved in toluene (30 mL), N,N-dimethylformamide (304 μL, 3.95 mmol) was added, and phosphorus oxychloride (4.82 mL, 52.64 mmol) was slowly added dropwise. The mixture was heated to 120 °C and refluxed for 12 h. The pH was adjusted to neutral with saturated sodium bicarbonate solution, and the mixture was extracted with ethyl acetate (50 mL × 3). The solution was concentrated and subjected to column chromatography [eluent: V (petroleum ether): V (ethyl acetate) = 10:1] to give the intermediate compound 4-chlorothiophene[3,2-d]pyrimidine, 1.63 g white solid, yield 73%.

[0057] Step 3: Dissolve the intermediate compound 4-chlorothiophene[3,2-d]pyrimidine (60 mg, 0.35 mmol) in tetrahydrofuran (4 mL), add dimethylamine hydrochloride (34.25 mg, 0.42 mmol), and slowly add triethylamine (194 μL, 1.40 mmol). Heat to 50 °C and stir for 10 h. Detect by TLC. After the reaction is complete, concentrate and precipitate by column chromatography [eluent: V (petroleum ether): V (ethyl acetate) = 5:1] to obtain the target compound N,N-dimethylthiopheno[3,2-d]pyrimidine-4-amine, 42 mg white solid, yield 67%. 1 H NMR (400MHz, CDCl3) δ8.49 (s, 1H), 7.68 (d, J = 5.5Hz, 1H), 7.37 (d, J = 5.5Hz, 1H), 3.39 (s, 6H). 13 C NMR (101MHz, CDCl3) δ160.6,158.8,154.4,131.8,125.0,114.7,39.1.

[0058] Example 2

[0059] Preparation of N,N-diethylthiopheno[3,2-d]pyrimidine-4-amine (compound 6b)

[0060]

[0061] Other conditions were the same as in Example 1, except that dimethylamine hydrochloride was replaced with diethylamine. The product was a white solid with a yield of 71%. 1 HNMR (400MHz, CDCl3) δ8.52(s,1H),7.68(d,J=5.6Hz,1H),7.43(d,J=5.6Hz,1H),3.79(q,J=7.1Hz,4H),1.30(t,J=7.1Hz,6H). 13CNMR (101MHz, CDCl3) δ157.3,154.5,131.4,125.0,113.5,43.7,14.0.

[0062] Example 3

[0063] Preparation of N-methylthiophene[3,2-d]pyrimidine-4-amine (compound 6c)

[0064]

[0065] Other conditions were the same as in Example 1, except that dimethylamine hydrochloride was replaced with methylamine hydrochloride. The product was a white solid with a yield of 67%. 1 H NMR (400MHz, CDCl3) δ8.63 (s, 1H), 7.65 (d, J = 5.3Hz, 1H), 7.38 (d, J = 5.4Hz, 1H), 5.63 (s, 1H), 3.19 (d, J = 4.9Hz, 3H). 13 C NMR (101MHz, CDCl3) δ159.4,158.1,155.1,131.1,125.3,115.4,28.2.

[0066] Example 4

[0067] Preparation of N-ethylthiopheno[3,2-d]pyrimidine-4-amine (compound 6d)

[0068]

[0069] Other conditions were the same as in Example 1, except that dimethylamine hydrochloride was replaced with ethylaminetetrahydrofuran solution. The product was a white solid with a yield of 72%. 1 HNMR (400MHz, CDCl3) δ8.60 (s, 1H), 7.65 (d, J = 5.3Hz, 1H), 7.38 (d, J = 5.4Hz, 1H), 5.37 (s, 1H), 3.71-3.64 (m, 2H), 1.31 (t, J = 7.2Hz, 3H). 13 C NMR (101MHz, CDCl3) δ158.5,156.3,154.0,129.8,124.2,114.1,35.20,14.0.

[0070] Example 5

[0071] Preparation of N-isopropylthiopheno[3,2-d]pyrimidine-4-amine (compound 6e)

[0072]

[0073] Other conditions were the same as in Example 1, except that dimethylamine hydrochloride was replaced with isopropylamine. The product was a white solid with a yield of 73%. 1 HNMR (400MHz, CDCl3) δ8.60 (s, 1H), 7.65 (d, J = 5.3Hz, 1H), 7.38 (d, J = 5.4Hz, 1H), 5.37 (s, 1H), 3.71-3.64 (m, 2H), 1.31 (t, J = 7.2Hz, 3H). 13 C NMR (101MHz, CDCl3) δ159.6,156.7,155.0,130.7,125.4,115.1,43.0,23.0.

[0074] Example 6

[0075] Preparation of N-cyclopropylthiopheno[3,2-d]pyrimidine-4-amine (compound 6f)

[0076]

[0077] Other conditions were the same as in Example 1, except that dimethylamine hydrochloride was replaced with cyclopropylamine. The product was a white solid with a yield of 65%. 1 HNMR (400MHz, CDCl3) δ8.59 (s, 1H), 7.38 (d, J = 1.3Hz, 1H), 6.38 (s, 1H), 3.0 3-2.98(m,1H),2.42(d,J=1.2Hz,3H),0.97-0.93(m,2H),0.76-0.72(m,2H). 13 C NMR (101MHz, CDCl3) δ159.7,154.7,133.1,124.9,24.2,9.5.

[0078] Example 7

[0079] Preparation of N-cyclohexylthiopheno[3,2-d]pyrimidine-4-amine (compound 6g)

[0080]

[0081] Other conditions were the same as in Example 1, except that dimethylamine hydrochloride was replaced with cyclohexylamine. The product was a white solid with a yield of 71%. 1 HNMR(400MHz,CD3OD)δ8.34(s,1H),7.90(d,J=5.4Hz,1H),7.25(d,J=5.4Hz,1H),4.13-4.05(m, 1H),1.98(d,J=10.9Hz,2H),1.77(d,J=10.0Hz,2H),1.65(d,J=10.0Hz,1H),1.43-1.15(m,6H).13 C NMR (101MHz, CD3OD) δ159.4,158.0,155.3,133.8,124.7,116.7,51.2,33.8,26.7,26.5.

[0082] Example 8

[0083] Preparation of N-(3-bromophenyl)thiopheno[3,2-d]pyrimidine-4-amine (compound 6h)

[0084]

[0085] Other conditions were the same as in Example 1, except that dimethylamine hydrochloride was replaced with m-bromoaniline. The product was a white solid with a yield of 76%. 1 H NMR (400MHz, CD3OD) δ8.59 (s, 1H), 8.11-8.10 (m, 2H), 7.74-7.69 (m, 1H), 7.42 (d, J = 5.5Hz, 1H), 7.29-7.28 (m, 2H). 13 C NMR (101MHz, CD3OD) δ161.0,157.0,154.9,141.7,135.3,131.2,128.0,126.1,124.9,123.1,121.9,117.6.

[0086] Example 9

[0087] Preparation of N-phenylthiopheno[3,2-d]pyrimidine-4-amine (compound 6i)

[0088]

[0089] Other conditions were the same as in Example 1, except that dimethylamine hydrochloride was replaced with aniline. The product was a white solid with a yield of 72%. 1 HNMR (400MHz, CDCl3) δ8.69 (s, 1H), 7.70 (d, J = 5.4Hz, 1H), 7.56 (d, J = 7.9Hz, 2H), 7.42-7.38 (m, 3H), 7.24-7.22 (m, 1H), 7.13 (s, 1H). 13 C NMR (101MHz, CD3OD) δ160.8,157.5,155.0,139.7,135.1,129.8,126.0,124.7,124.6,117.3,106.2.

[0090] Example 10

[0091] Preparation of N-(3-(trifluoromethyl)phenyl)thiopheno[3,2-d]pyrimidine-4-amine (compound 6j)

[0092]

[0093] Other conditions were the same as in Example 1, except that dimethylamine hydrochloride was replaced with m-aminotrifluorotoluene. The product was a white solid with a yield of 74%. 1 H NMR (400MHz, CD3OD) δ8.72(s,1H),8.29(d,J=5.4Hz,1H),8.18(s,1H),8.01(d,J=7.3Hz,1H),7.59(t,J=8.0Hz,1H),7.51-7.47(m,2H). 13 C NMR (101MHz, CD3OD) δ157.8,155.3,152.6,140.0,138.1,130.8,127.4,122.7,122.3,120.7,117.9.

[0094] Example 11

[0095] Preparation of N,N,7-trimethylthiophene[3,2-d]pyrimidine-4-amine (compound 6k)

[0096]

[0097] Other conditions were the same as in Example 1, except that ethyl 3-aminothiophene-2-carboxylate was replaced with ethyl 3-amino-4-methylthiophene-2-carboxylate. The product was a white solid with a yield of 69%. 1 H NMR (400MHz, CDCl3) δ8.54 (s, 1H), 7.31 (d, J = 1.3Hz, 1H), 3.37 (s, 6H), 2.39 (d, J = 1.2Hz, 3H). 13 C NMR (101MHz, CDCl3) δ159.3,158.8,154.0,133.3,126.8,114.9,39.0,13.2.

[0098] Example 12

[0099] Preparation of N,N-diethyl-7-methylthiophene[3,2-d]pyrimidine-4-amine (compound 6l)

[0100]

[0101] Other conditions were the same as in Example 11, except that dimethylamine hydrochloride was replaced with diethylamine. The product was a white solid with a yield of 72%. 1H NMR (400MHz, CDCl3) δ8.55(s,1H),7.29(s,1H),3.75(q,J=7.1Hz,4H),2.39(s,3H),1.27(t,J=7.1Hz,6H). 13 C NMR (101MHz, CDCl3) δ159.5,157.5,154.2,133.4,126.2,113.7,43.5,14.1,13.4.

[0102] Example 13

[0103] Preparation of N,7-dimethylthiophene[3,2-d]pyrimidine-4-amine (compound 6m)

[0104]

[0105] Other conditions were the same as in Example 11, except that dimethylamine hydrochloride was replaced with methylamine hydrochloride. The product was a white solid with a yield of 69%. 1 H NMR (400MHz, CDCl3) δ8.61 (s, 1H), 7.22 (s, 1H), 5.39 (s, 1H), 3.12 (d, J = 4.9Hz, 3H), 2.37 (s, 3H). 13 C NMR (101MHz, CDCl3) δ158.4,158.1,154.7,134.0,125.657,115.5,28.1,13.1.

[0106] Example 14

[0107] Preparation of N-ethyl-7-methylthiophene[3,2-d]pyrimidine-4-amine (compound 6n)

[0108]

[0109] Other conditions were the same as in Example 11, except that dimethylamine hydrochloride was replaced with ethylaminetetrahydrofuran solution. The product was a white solid with a yield of 71%. 1 H NMR (400MHz, CDCl3) δ8.65 (s, 1H), 7.29 (s, 1H), 5.11 (s, 1H), 3.71-3.64 (m, 2H), 2.43 (s, 3H), 1.31 (t, J = 7.2Hz, 3H). 13 C NMR (101MHz, CDCl3) δ158.6,157.5,154.8,134.2,125.7,115.4,36.2,15.1,13.2.

[0110] Example 15

[0111] Preparation of N-isopropyl-7-methylthiophene[3,2-d]pyrimidine-4-amine (compound 6o)

[0112]

[0113] Other conditions were the same as in Example 11, except that dimethylamine hydrochloride was replaced with isopropylamine. The product was a white solid with a yield of 70%. 1 H NMR (400MHz, CDCl3) δ8.66 (s, 1H), 7.31 (d, J = 1.3Hz, 1H), 4.65 (s, 1H), 4.56-4.48 (m, 1H), 2.45 (s, 3H), 1.33 (d, J = 6.4Hz, 6H). 13 C NMR (101MHz, CDCl3) δ158.7,156.9,154.9,134.3,125.5,115.4,43.1,23.2,13.2.

[0114] Example 16

[0115] Preparation of N-cyclopropyl-7-methylthiophene[3,2-d]pyrimidine-4-amine (compound 6p)

[0116]

[0117] Other conditions were the same as in Example 11, except that dimethylamine hydrochloride was replaced with cyclopropylamine. The product was a white solid with a yield of 76%. 1 H NMR (400MHz, CDCl3) δ8.59 (s, 1H), 7.38 (d, J = 1.3Hz, 1H), 6.38 (s, 1H), 3.03 -2.98(m,1H),2.42(d,J=1.2Hz,3H),0.97-0.93(m,2H),0.76-0.72(m,2H). 13 C NMR (101MHz, CDCl3) δ159.6,159.4,154.3,133.3,127.9,114.7,24.0,13.0,9.3.

[0118] Example 17

[0119] Preparation of N-cyclohexyl-7-methylthiophene[3,2-d]pyrimidine-4-amine (compound 6q)

[0120]

[0121] Other conditions were the same as in Example 11, except that dimethylamine hydrochloride was replaced with cyclohexylamine. The product was a white solid with a yield of 74%. 1H NMR(400MHz,CD3OD)δ8.39(s,1H),7.52(s,1H),4.15-4.08(m,1H),2.36(s,3H),2.01 (d,J=9.6Hz,2H),1.80(d,J=12.5Hz,2H),1.68(d,J=13.6Hz,1H),1.48-1.16(m,6H). 13 C NMR (101MHz, CD3OD) δ158.4,158.2,155.1,134.0,128.5,117.0,51.1,33.86,26.7,26.,13.0.

[0122] Example 18

[0123] Preparation of N-(3-bromophenyl)-7-methylthiopheno[3,2-d]pyrimidine-4-amine (compound 6r)

[0124]

[0125] Other conditions were the same as in Example 11, except that dimethylamine hydrochloride was replaced with m-bromoaniline. The product was a white solid with a yield of 73%. 1 H NMR(400MHz,DMSO-d6)δ9.76(s,1H),8.68(s,1H),8.21(s,1H),7.88(s,1H),7. 84(d,J=8.0Hz,1H),7.31(t,J=8.0Hz,1H),7.25(d,J=8.9Hz,1H),2.37(s,3H). 13 C NMR (101MHz, DMSO-d6) δ159.4,154.9,153.6,141.2,132.7,130.4,129.1,125.6,123.5,121.3,120.0,116.0,12.6.

[0126] Example 19

[0127] Preparation of N-phenyl-7-methylthiopheno[3,2-d]pyrimidine-4-amine (compound 6S)

[0128]

[0129] Other conditions were the same as in Example 11, except that dimethylamine hydrochloride was replaced with aniline. The product was a white solid with a yield of 69%. 1HNMR (400MHz, DMSO-d6) δ9.65(s,1H),8.61(s,1H),7.83(s,1H),7.80(d,J=8.0Hz,2H),7.36(t,J=7.8Hz,2H),7.10(t,J=7.4Hz,1H),2.36(s,3H). 13 C NMR (101MHz, DMSO-d6) δ159.2,155.3,153.8,139.2,132.7,128.7,128.5,123.5,122.,115.7,12.7.

[0130] Example 20

[0131] Preparation of 7-methyl-N-(3-(trifluoromethyl)phenyl)thieno[3,2-d]pyrimidine-4-amine (compound 6t)

[0132]

[0133] Other conditions were the same as in Example 11, except that dimethylamine hydrochloride was replaced with m-aminotrifluoroaniline. The product was a white solid with a yield of 67%. 1 H NMR (400MHz, CD3OD) δ8.62(s,1H),8.22(s,1H),8.03(d,J=6.0Hz,1H),7.69(s,1H),7.53(t,J=8.0Hz,1H),7.39(d,J=7.8Hz,1H),2.43(s,3H). 13 C NMR (101MHz, CD3OD) δ160.0,157.1,154.7,141.3,134.3,130.5,129.9,126.2,121.2,119.5,118.1,13.0.

[0134] II. Preparation of monosubstituted thiophene[3,2-d]pyrimidine cyclic ether compounds:

[0135] Example 21

[0136] Preparation of 4-methoxythieno[3,2-d]pyrimidine (compound 6u)

[0137]

[0138] The first two steps are the same as in Example 1.

[0139] Step 3: Dissolve the intermediate compound 4-chlorothiophene[3,2-d]pyrimidine (60 mg, 0.35 mmol) in tetrahydrofuran (4 mL), add 40% NaH (42 mg, 1.05 mmol) under ice bath conditions, stir for half an hour, remove the ice bath, add anhydrous methanol (21.2 μL, 0.53 mmol), stir at room temperature for 5 h, and detect by TLC. After the reaction is complete, concentrate and precipitate by column chromatography [eluent: V (petroleum ether): V (ethyl acetate) = 5:1] to obtain the target compound 4-methoxythiophene[3,2-d]pyrimidine, 42 mg white solid, yield 72%. 1 H NMR (400MHz, CDCl3) δ8.74 (s, 1H), 7.83 (d, J = 5.4Hz, 1H), 7.47 (d, J = 5.3Hz, 1H), 4.14 (s, 3H). 13 C NMR (101MHz, CDCl3) δ164.6,161.9,154.5,134.2,124.6,117.8,54.3.

[0140] Example 22

[0141] Preparation of 4-isopropoxythiopheno[3,2-d]pyrimidine (compound 6v)

[0142]

[0143] Other conditions were the same as in Example 21, except that methanol was replaced with isopropanol. The product was a white solid with a yield of 62%. 1 H NMR (400MHz, CDCl3) δ8.65 (s, 1H), 7.75 (d, J = 5.4Hz, 1H), 7.40 (d, J = 5.4Hz, 1H), 5.60-5.50 (d, J = 37.3Hz, 1H), 1.38 (d, J = 6.2Hz, 6H). 13 C NMR (101MHz, CDCl3) δ164.0,162.0,154.5,133.9,124.6,118.2,70.4,22.0.

[0144] Example 23

[0145] Preparation of 4-methoxy-7-methylthiophene[3,2-d]pyrimidine (compound 6w)

[0146]

[0147] Other conditions were the same as in Example 21, except that ethyl 3-aminothiophene-2-carboxylate was replaced with ethyl 3-amino-4-methylthiophene-2-carboxylate. The product was a white solid with a yield of 73%. 1H NMR (400MHz, CDCl3) δ8.77(s,1H),7.45(d,J=1.2Hz,1H),4.15(s,3H),2.47(d,J=1.1Hz,3H). 13 C NMR (101MHz, CDCl3) δ164.7,161.0,154.2,133.5,129.0,118.1,54.19,13.0.

[0148] Example 24

[0149] Preparation of 4-isopropoxy-7-methylthiophene[3,2-d]pyrimidine (compound 6x)

[0150]

[0151] Other conditions were the same as in Example 23, except that methanol was replaced with isopropanol. The product was a white solid with a yield of 64%. 1 H NMR (400MHz, CDCl3) δ8.75 (s, 1H), 7.45 (d, J = 1.2Hz, 1H), 5.64-5.58 (m, 1H), 2.47 (d, J = 1.2Hz, 3H), 1.44 (d, J = 6.2Hz, 6H). 13 C NMR (101MHz, CDCl3) δ164.1,161.0,154.2,133.4,128.8,118.5,70.29,22.0,13.0.

[0152] III. Preparation of disubstituted thiophene[3,2-d]pyrimidine cyclic amine compounds:

[0153] Example 25

[0154] Preparation of 2-(morpholinomethyl)-N-phenylthiopheno[3,2-d]pyrimidine-4-amine (compound 10a)

[0155]

[0156] Step 1: Dissolve 3-aminothiophene-2-carboxylic acid ethyl ester (3 g, 17.54 mmol) in 1,4-dioxane (45 mL), heat to 50 °C, and slowly add chloroacetonitrile (1.33 mL, 21.05 mmol). After the addition is complete, purge with hydrogen chloride gas (20 mL), stir and react for 20 min. After the reaction is complete, cool to room temperature, filter the resulting precipitate, wash with ethyl acetate (50 mL), and dry to obtain the intermediate compound 2-(chloromethyl)thiopheno[3,2-d]pyrimidin-4(3H)-one, 2.74 g of grayish-white solid, yield 78%.

[0157] Step 2: 2-(chloromethyl)thieno[3,2-d]pyrimidin-4(3H)-one (500 mg, 2.50 mmol) was dissolved in tetrahydrofuran (40 mL), morpholine (261 μL, 3.00 mmol) was added, and triethylamine (194 μL, 1.40 mmol) was slowly added dropwise. The mixture was heated to 50 °C and stirred for 10 h. The reaction was detected by TLC. After the reaction was completed, the mixture was concentrated and precipitated by column chromatography [eluent: V(petroleum ether):V(ethyl acetate) = 3:1] to give the intermediate compound 2-(morpholinemethyl)thieno[3,2-d]pyrimidin-4(3H)-one, 526 mg white solid, yield 84%.

[0158] Step 3: Dissolve the intermediate compound 2-(morpholinomethyl)thiopheno[3,2-d]pyrimidin-4(3H)-one (500 mg, 1.99 mmol) in toluene (20 mL), add N,N-dimethylformamide (46 μL, 0.60 mmol), slowly add phosphorus oxychloride (729 μL, 7.96 mmol), heat to 120 °C, reflux for 12 h, adjust the pH to neutral with saturated sodium bicarbonate solution, extract with ethyl acetate (10 mL × 3), concentrate and column chromatography [eluent: V(petroleum ether):V(ethyl acetate) = 1:1], to obtain the intermediate compound 4-(4-chlorothiophene[3,2-d]pyrimidin-2-yl)methyl)morpholine, 474 mg brownish-yellow solid, yield 88%.

[0159] Step 4: Dissolve the intermediate compound 4-(4-chlorothiophene[3,2-d]pyrimidin-2-yl)methyl)morpholine (80 mg, 0.30 mmol) in tetrahydrofuran (4 mL), add aniline (33 μL, 0.36 mmol), and slowly add triethylamine (125 μL, 0.9 mmol). Heat to 50 °C and stir for 10 h. Detect by TLC. After the reaction is complete, concentrate and precipitate by column chromatography [eluent: V(dichloromethane):V(methanol) = 20:1] to obtain the target compound 2-(morpholinemethyl)-N-phenylthiopheno[3,2-d]pyrimidin-4-amine, 71 mg white solid, yield 73%. 1 H NMR (400MHz, CD3OD) δ8.14(d,J=5.4Hz,1H),7.65(d,J=7.5Hz,2H),7.45-7.41( m,3H),7.25(t,J=7.4Hz,1H),4.50(s,2H),3.95(t,J=4.9Hz,4H),3.52(s,4H). 13 C NMR (101MHz, CD3OD) δ161.3,157.8,157.0,139.4,136.0,129.9,126.5,125.1,124.9,116.4,65.1,61.4,54.2.

[0160] Example 26

[0161] Preparation of N-(3-bromophenyl)-2-(morpholinomethyl)thiopheno[3,2-d]pyrimidine-4-amine (compound 10b)

[0162]

[0163] Other conditions were the same as in Example 25, except that aniline was replaced with m-bromoaniline. The product was a white solid with a yield of 74%. 1 HNMR(400MHz,CD3OD)δ8.18(d,J=5.4Hz,1H),8.01(s,1H),7.67(d,J=7.6Hz,1H),7.48(d ,J=5.4Hz,1H),7.34(d,J=26.0Hz,2H),4.55(s,2H),3.98(t,J=4.9Hz,4H),3.55(s,4H). 13 CNMR(101MHz,CD3OD)δ161.7,157.4,157.0,141.4,136.2,131.4,128.7,126.9,125.2,123.1,122.8,65.1,61.6,54.1.

[0164] Example 27

[0165] Preparation of 2-(morpholinomethyl)-N-(3-(trifluoromethyl)phenyl)thieno[3,2-d]pyrimidine-4-amine (compound 10c)

[0166]

[0167] Other conditions were the same as in Example 25, except that aniline was replaced with m-aminotrifluorotoluene. The product was a white solid with a yield of 71%. 1 H NMR (400MHz, CD3OD) δ8.19(d,J=5.5Hz,1H),8.08(d,J=8.5Hz,2H),7.61(t,J=7 .9Hz,1H),7.51-7.46(m,2H),4.58(s,2H),3.99(t,J=4.8Hz,4H),3.57(s,4H). 13 C NMR (101MHz, CD3OD) δ162.6,161.8,157.3,156.9,140.8,136.2,130.8,127.2,125.3,121.9,117.0,65.0,61.6,54.1.

[0168] Example 28

[0169] Preparation of N-(3-bromophenyl)-2-((diethylamino)methyl)thiopheno[3,2-d]pyrimidine-4-amine (compound 10d)

[0170]

[0171] Other conditions were the same as in Example 26, except that morpholine was replaced with diethylamine. The product was a white solid with a yield of 73%. 1 H NMR (400MHz, DMSO-d6) δ10.33(s,1H),8.35(d,J=5.4Hz,1H),8.09(t,J=1.9Hz,1H),7.87(dt,J=7.6,1.9Hz ,1H),7.52(d,J=5.3Hz,1H),7.36-7.29(m,2H),4.42(s,2H),3.27(d,J=7.3Hz,4H),1.31(t,J=7.2Hz,6H). 13 C NMR (101MHz, DMSO-d6) δ160.5,156.3,155.1,140.5,136.0,130.5,126.3,124.4,124.1,121.2,121.0,115.4,54.8,48.0,9.2.

[0172] Example 29

[0173] Preparation of N-phenyl-2-(pyrrolidone-1-ylmethyl)thiopheno[3,2-d]pyrimidine-4-amine (compound 10e)

[0174]

[0175] Other conditions were the same as in Example 25, except that morpholine was replaced with tetrahydropyrrole. The product was a white solid with a yield of 64%. 1 HNMR(400MHz,CD3OD)δ8.10(d,J=5.4Hz,1H),7.69-7.67(m,2H),

[0176] 7.44-7.38(m,3H),7.22(t,J=7.4Hz,1H),4.56(s,2H),3.55(s,4H),2.13(s,4H). 13 C NMR (101MHz, CD3OD) δ161.7,158.2,157.7,139.6,135.7,129.9,126.2,125.0,124.9,116.3,60.0,56.2,24.2.

[0177] Example 30

[0178] Preparation of N-phenyl-2-(piperidin-1-ylmethyl)thieno[3,2-d]pyrimidine-4-amine (compound 10f)

[0179]

[0180] Other conditions were the same as in Example 25, except that morpholine was replaced with piperidine. The product was a white solid with a yield of 69%. 1 H NMR (400MHz, DMSO-d6) δ7.31(s,1H),6.85(s,2H),6.61(s,3H),6.41(s,1H),4.10(s,4H),3.58(s,2H),1.08(s,4H),0.90(s,2H). 13 C NMR (101MHz, DMSO-d6) δ161.7,157.6,157.3,139.5,135.8,129.9,126.3,125.0,125.0,116.4,61.5,55.4,24.3,22.6.

[0181] Example 31

[0182] Preparation of 7-methyl-2-(morpholinomethyl)-N-phenylthiopheno[3,2-d]pyrimidine-4-amine (compound 10 g)

[0183]

[0184] Other conditions were the same as in Example 25, except that ethyl 3-aminothiophene-2-carboxylate was replaced with ethyl 3-amino-4-methylthiophene-2-carboxylate. The product was a white solid with a yield of 71%. 1 H NMR (400MHz, CDCl3) δ7.59 (d, J = 7.3Hz, 2H), 7.38 (t, J = 7.9Hz, 2H), 7.30 (s, 1H), 7. 23(t,J=7.4Hz,1H),3.83(s,2H),3.78(t,J=4.7Hz,4H),2.70(s,4H),2.43(s,3H). 13 C NMR (101MHz, CDCl3) δ162.6,160.9,156.2,137.8,133.8,129.1,127.6,125.6,124.0,113.7,67.1,65.1,54.0,13.2.

[0185] Bioactivity test

[0186] The GloSensor cAMP accumulation assay was used to test the functional activity of target compounds for D2R and to elucidate the allosteric mechanism of action of novel compounds. Briefly, HEK 293T cells stably expressing D2R were seeded into 6-well plates at a density of 500,000 cells per well. The next day, the GloSensor-22F cAMP plasmid was transfected into HEK 293T D2R cells using FuGene transfection reagent (Promega). Transfected cells were washed with CO2-independent medium and then incubated with equilibration medium containing 2% v / v GloSensor cAMP stock solution (dissolved in 10% FBS in CO2-independent medium). After incubation at 37°C for 1 h, bioluminescence signals were detected until a steady-state baseline signal was obtained. Next, 100 μM of the test compound was added to the cells, and the cells were incubated in a 37°C cell incubator for 30 min. Then, a concentration gradient of DA (final concentration 1 nM-10 μM) was added and the cells were incubated at room temperature for 5 min. Finally, 10 μM of Forskolin was added to the cells, and the changes in bioluminescence were read using a microplate reader.

[0187]

[0188] Table 1. Statistical comparison of the allosteric antagonistic activity of the target compound and the activity of positive control drug 2.

[0189]

[0190]

[0191]

[0192] Allosteric antagonistic activity screening

[0193] Using GloSensor cAMP accumulation assays, different concentration gradients of DA were used as blank controls (final concentration gradient 1 nM-10 μM), and compound 2 (final concentration 100 μM) was used as a positive control. The allosteric antagonistic activity of the target compound (final concentration 100 μM) was compared with that of positive control 2. The test results (Table 1) show that all compounds in this invention have different degrees of allosteric antagonistic activity against D2R. In general, the allosteric antagonistic activity is relatively better when there is a methyl substitution on the thiophene ring. Some of these compounds have significantly better allosteric antagonistic activity against D2R than positive control 2, such as 6d, 6e, 6f, 6j, 6n, 6o, 6q, 6s, and 10d; among them, the activities of compounds 6e, 6o, 6q, 6s, and 10d reached 1.28 times, 1.39 times, 1.15 times, 1.14 times, and 1.38 times that of positive control 2, respectively.

[0194] Allosteric mechanism of action study

[0195] Further investigation using GloSensor cAMP accumulation assays revealed whether these compounds could allosterically modulate the functional activity of the D2R endogenous ligand dopamine (DA), further confirming that these compounds are negative allosteric modulators (NAMs) of D2R. Taking compound 6o as an example (e.g.) Figure 1 As shown in the figure, when the concentration of compound 6o reaches 60 μM, the dose-response curve of DA shows a significant downward shift, almost reaching the lower limit of the maximum downward shift in dose-response regulation of DA activity. This indicates that the IC50 of compound 6o is relatively high. 50 The value may be less than 60 μM, specifically manifested in the sharp drop in the dose-response curve. Furthermore, when the concentration of 6o increases from 60 μM to 120 μM, the downward shift in the DA dose-response curve is very slight. This phenomenon explains that with increasing concentration of compound 6o, the concentration-dependent curve of DA shows a limited downward shift, indicating that compound 6o can effectively negatively allosterically regulate the functional activity of the D2R endogenous ligand DA. This allosteric regulation phenomenon is consistent with previously reported D2R allosteric antagonistic regulatory mechanisms. The concentration-dependent curve of DA allosterically regulated by compound 6q (e.g.) Figure 2 The results shown are consistent with those of 6o, indicating that the target compounds in this invention are all negative allosteric modulators (NAM) or allosteric antagonists of D2R.

Claims

1. Use of a thienopyrimidine compound for the manufacture of a dopamine D2 receptor allosteric antagonist, characterized in that: The structure of the thienopyrimidine compound is shown in formula 1: ， R2 is one of the following groups: 、 、 、 、 、 、 、 、 、 ； When R1 is -H, R3 is one of the following groups: 、 、 、 、 ； When R1 is -CH3, R3 is H.

2. Use of a thienopyrimidine compound according to claim 1 for the manufacture of a dopamine D2 receptor allosteric antagonist, characterized in that: The structure of the thienopyrimidine compound is as follows: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 3. Use of a thienopyrimidine compound according to claim 1 for the manufacture of a dopamine D2 receptor allosteric antagonist, characterized in that: The thienopyrimidine compound is prepared into a pharmaceutical preparation as an active ingredient together with a pharmaceutically acceptable carrier.

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