CN117126176A - Thienopyrimidine compound and dopamine D thereof 2 Use of receptor allosteric antagonists - Google Patents

Abstract

The invention belongs to the field of pharmaceutical chemistry, and particularly discloses a thienopyrimidine compound and a preparation method thereof as dopamine D ₂ Receptor (D) ₂ R) allosteric antagonists. The target compound of the invention comprises thiophene [3,2-d ]]And pyrimidine ring skeleton with the structural formula shown in the formula 1. The invention tests the target compound pair D by a GloSensor cAMP accumulation experiment ₂ Functional Activity of R and elucidation of novel Compounds towards D ₂ The allosteric mechanism of action of R. Pharmacological results show that all target compounds in the invention can negative allostericRegulation D ₂ Functional activity of the R endogenous ligand dopamine, i.e. the target compounds are all D ₂ R-negative allosteric modulators or allosteric antagonists.

Description

A thienopyrimidine compound and its application as a dopamine D2 receptor allosteric antagonist

Technical Field

The invention belongs to the field of medicinal chemistry, and particularly relates to a thienopyrimidine compound and an application thereof as a dopamine _D2 receptor allosteric antagonist.

Background Art

G protein-coupled receptor (GPCR), also known as 7-transmembrane protein, is a class of receptor protein superfamily encoded by the human genome and present on the surface of the cell membrane. GPCR has a 7-transmembrane α-helical structure connected by three extracellular loops and three intracellular loops. Among them, the N-terminus is extracellular and the C-terminus is intracellular.

Dopamine (DA) is an important catecholamine neurotransmitter. It projects to dopamine receptors in the prefrontal cortex, hippocampus, striatum, nucleus accumbens, hypothalamus and other parts through the ventral tegmental nucleus of the midbrain. It is mainly involved in emotional thinking, motor function and cognitive activities. Dopamine receptors are an important member of the A family GPCR. According to their pharmacological and biochemical characteristics, they can be divided into two categories: _D1 -like receptors ( _D1R ) and _D2- like receptors ( _D2 -like). The former includes dopamine _D1 receptors ( _D1R ) and dopamine _D5 receptors ( _D5R ), which are mainly coupled to _Gs proteins and stimulate the production of cAMP after receptor activation; the latter includes dopamine _D2 receptors ( _D2R ), dopamine _D3 receptors ( _D3R ) and dopamine _D4 receptors ( _D4R ), which are coupled to G _i/o proteins and inhibit the production of cAMP after receptor activation. Among them, _D2R is an important target for the treatment of central nervous system disorders (including Parkinson's disease and schizophrenia).

Small molecule compound 1 is a new type of negative allosteric modulator NAM of _D2R recently discovered through virtual screening (J.Med.Chem.2019,62,174-206.). The structure of this compound contains a thienyl[2,3-d]pyrimidine ring skeleton, which has a new structural feature different from the known dopamine ligands. It is also the first time that thienylpyrimidine compounds have been used in the study of _D2R . Fyfe et al. conducted a preliminary structure-activity relationship (SAR) study on compound 1 for the first time and found an open-ring and structurally simplified analogue 2, whose functional activity and synergistic effect are still comparable to those of the lead compound 1 (Eur.J.Med.Chem.2019,168,474-490.). Further structure-activity relationship studies found some essential groups for activity, but the activity of all the derivatives obtained was not significantly improved compared with the lead compound 1, and the allosteric regulatory functional activity was at the micromolar level, which is far from the druggable activity of _D2R ligands. In addition, the research group retained the core skeleton thiophene [2,3-d] pyrimidine ring in the structural modification and transformation of compound 1, and did not conduct structure-activity relationship research on the skeleton, which is also likely to be the reason why the obtained derivatives have low activity.

Scaffold hopping, also known as scaffold migration, is a medicinal chemistry method for molecular scaffold replacement 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 pharmacokinetics (ADME) and other properties, and is one of the important ways to discover and optimize lead compounds. The methods of scaffold hopping mainly include bioisosteres, heterocyclic replacement, ring opening or ring closing, etc. (J. Med. Chem. 2017, 60, 1238-1246.).

Summary of the invention

The present invention conducts a ring-opening design on the small molecule compound 1 mentioned in the background technology, and then uses skeleton transition to replace the thiophene [2,3-d] pyrimidine ring with a new thiophene [3,2-d] pyrimidine ring, and investigates the effect of the substituent on the activity of the new compound, and conducts structure-activity relationship research, in order to obtain a new allosteric modulator with improved activity, good selectivity and targeting _D2R .

The present invention provides a thienopyrimidine compound, the structure of which is shown in Formula 1:

in:

_R1 is one of the following structures:

etc. alkyl or aryl;

_R2 is one of the following structures:

Aliphatic amines, aromatic amines or alkoxy groups;

R ₃ is one of the following structures:

Hydrogen radical, aliphatic amine, aromatic amine or heterocyclic amine.

Further, in the thienopyrimidine compound, R ₁ is -H or -CH ₃ ;

_R2 is one of the following groups:

_R3 is one of the following groups:

Furthermore, the structure of the thienopyrimidine compound is as follows:

The present invention also provides a method for synthesizing a thiophene [3,2-d] pyrimidine ring compound of formula 1. Specifically,

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

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

1. Synthesis of monosubstituted thiophene[3,2-d]pyrimidine compounds

The specific synthesis steps are:

(1) Compound 3 is dissolved in a solvent, heated to 170°C, and refluxed with stirring. Post-treatment is performed to obtain a ketone intermediate 4. Compound 3 is ethyl 3-amino-4-methylthiophene-2-carboxylate or ethyl 3-aminothiophene-2-carboxylate, and the solvent is formamide, which is both a solvent and a reactant and is in large excess. The reaction time is 12 hours.

(2) Dissolve the desired compound 4 in a solvent, add a catalyst, slowly dropwise add phosphorus oxychloride, heat to 120°C, reflux with stirring, and post-treat to obtain an intermediate 5. The solvent is toluene, the catalyst is N,N-dimethylformamide, and the reaction time is 3-5 hours. The molar ratio of intermediate 4: catalyst: phosphorus oxychloride is 1:0.3:4.

(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 to react, and post-treat to obtain the amine target compound 6. The solvent is tetrahydrofuran, the acid-binding agent is triethylamine, the desired amine is various aliphatic amines or aromatic amines, and the reaction time is 10 hours. The molar ratio of intermediate 5: desired amine: acid-binding agent is 1:1.2:3.

(4) The desired compound 5 was dissolved in a solvent except water, and a base was added under ice bath conditions for hydrogen extraction. After half an hour of reaction, the ice bath was removed, and the desired alcohol was added. The mixture was stirred at room temperature, and the ether target compound 6 was obtained after post-treatment. 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.

2. Synthesis of disubstituted thiophene[3,2-d]pyrimidine compounds

The specific synthesis steps are:

(1) Compound 3 is dissolved in a solvent, chloroacetonitrile is slowly added dropwise, and hydrogen chloride gas is introduced, and the reaction is stirred under heating conditions to generate a precipitate, which is intermediate compound 7. Compound 3 is ethyl 3-amino-4-methylthiophene-2-carboxylate or ethyl 3-aminothiophene-2-carboxylate, the solvent is 1,4-dioxane, the reaction temperature is 50°C, and the reaction time is 6-12 hours. The molar ratio of compound 3 to chloroacetonitrile is 1:1.2.

(2) Dissolve the desired compound 7 in a solvent, add an acid-binding agent, slowly add the desired amine, heat to 50 degrees Celsius under nitrogen protection, stir to react, and post-treat to obtain compound 8. The solvent is tetrahydrofuran, the acid-binding agent is triethylamine, the desired amine is various aliphatic amines or aromatic amines, and the reaction time is 10 hours. The molar ratio of intermediate 7: desired amine: acid-binding agent is 1:1.2:3.

(3) Dissolve the desired compound 8 in a solvent, add a catalyst, slowly dropwise add phosphorus oxychloride, heat to 120°C, reflux with stirring, and post-treat to obtain intermediate 9. The solvent is toluene, the catalyst is N,N-dimethylformamide, and the reaction time is 3-5 hours. The molar ratio of intermediate 4: catalyst: phosphorus oxychloride is 1:0.3:4.

(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 to react, and post-treat to obtain the target compound 10. The solvent is tetrahydrofuran, the acid-binding agent is triethylamine, the desired amine is various aliphatic amines or aromatic amines, and the reaction time is 10 hours. The molar ratio of intermediate 9: desired amine: acid-binding agent is 1:1.2:3.

The present invention tests the functional activity of the target compound on _D2R and clarifies the allosteric action mechanism of the new compound on _D2R through the GloSensor cAMP accumulation experiment. The pharmacological results show that all the target compounds in the present invention can negatively allosterically regulate the functional activity of dopamine, the endogenous ligand of _D2R , that is, the target compounds are all negative allosteric modulators or allosteric antagonists of _D2R .

One or more of the thienopyrimidine compounds provided by the present invention can be used as active ingredients and prepared into pharmaceutical preparations with pharmaceutically acceptable carriers. More specifically, the thienopyrimidine compounds are used in the preparation of D ₂ R allosteric antagonists.

The beneficial effects of the present invention are:

The advantages of the present invention are: the small molecule compound 1 is open-loop designed, and then the thiophene [2,3-d] pyrimidine ring is replaced with a new thiophene [3,2-d] pyrimidine skeleton by skeleton transition, and the functional activity of the target compound on D ₂ R is tested by the accumulation experiment of cAMP. Pharmacological results show that the synthesized series of target compounds have good allosteric regulation function on D ₂ R; in-depth mechanism research shows that the target compounds in the present invention are allosteric antagonists of D ₂ R. In addition, D ₂ R is closely related to central nervous system diseases, including Parkinson's disease and schizophrenia. At present, D ₂ R drugs for the treatment of Parkinson's disease and schizophrenia in clinical practice are all bound to the orthosteric site of the receptor. Long-term use of such drugs can cause obvious side effects, such as extrapyramidal motor dysfunction, weight gain or metabolic dysfunction. However, allosteric modulators have a certain saturation limit for the regulation function of receptors, and will not over-activate or completely block the function of receptors. Therefore, they can overcome the side effects caused by orthosteric ligand drugs, thereby hopefully developing safer and more effective central nervous system drugs.

Description of the drawings:

Figure 1 is the concentration dependence curve of compound 6o allosterically regulating DA;

FIG2 is a concentration-dependent curve of compound 6q allosterically regulating DA.

DETAILED DESCRIPTION

The present invention will now be further described with reference to examples.

1. Preparation of monosubstituted thiophene[3,2-d]pyrimidine ring amine compounds:

Example 1

Preparation of N,N-dimethylthieno[3,2-d]pyrimidin-4-amine (Compound 6a)

Step 1: Dissolve 3-aminothiophene-2-carboxylic acid ethyl ester (3 g, 17.54 mmol) in formamide (45 mL), heat to 170 ° C, reflux with stirring for 12 h, and detect with a plate. After the reaction is completed, cool to room temperature, stir in a -20 ° C low temperature box to precipitate the product, wash with ethyl acetate, and dry to obtain the intermediate compound thieno[3,2-d]pyrimidin-4(3H)-one, 2.11 g of off-white solid, and a yield of 79%.

Step 2: The intermediate compound 4-thieno[3,2-d]pyrimidin-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, phosphorus oxychloride (4.82 mL, 52.64 mmol) was slowly added dropwise, heated to 120°C, refluxed for 12 h, and the pH was adjusted to neutral with saturated sodium bicarbonate solution, extracted with ethyl acetate (50 mL×3), and concentrated by column chromatography [eluent: V (petroleum ether): V (ethyl acetate) = 10:1] to obtain the intermediate compound 4-chlorothieno[3,2-d]pyrimidine, 1.63 g of white solid, with a yield of 73%.

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), slowly add triethylamine (194 μL, 1.40 mmol), heat to 50°C, stir to react for 10 h, and detect with a spot plate. After the reaction is completed, concentrate and column chromatograph [eluent: V (petroleum ether): V (ethyl acetate) = 5:1] to obtain the target compound N, N-dimethylthieno[3,2-d]pyrimidine-4-amine, 42 mg of white solid, with a yield of 67%. ¹ H NMR (400MHz, CDCl ₃ ) δ 8.49 (s, 1H), 7.68 (d, J = 5.5 Hz, 1H), 7.37 ( d, J = 5.5 Hz, 1H), 3.39 ( s, 6H). ¹³ C NMR ( 101 MHz, CDCl ₃ ) δ 160.6, 158.8, 154.4, 131.8, 125.0, 114.7,39.1.

Example 2

Preparation of N,N-diethylthieno[3,2-d]pyrimidin-4-amine (Compound 6b)

The 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 (400 MHz, CDCl ₃ ) δ8.52 (s, 1H), 7.68 (d, J=5.6 Hz, 1H), 7.43 (d, J=5.6 Hz, 1H), 3.79 (q, J=7.1 Hz, 4H), 1.30 (t, J=7.1 Hz, 6H). ¹³ CNMR (101 MHz, CDCl ₃ ) δ157.3, 154.5, 131.4, 125.0, 113.5, 43.7, 14.0.

Example 3

Preparation of N-methylthiophene[3,2-d]pyrimidin-4-amine (Compound 6c)

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

Example 4

Preparation of N-ethylthieno[3,2-d]pyrimidin-4-amine (Compound 6d)

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

Example 5

Preparation of N-isopropylthieno[3,2-d]pyrimidin-4-amine (Compound 6e)

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

Example 6

Preparation of N-cyclopropylthieno[3,2-d]pyrimidin-4-amine (Compound 6f)

Other conditions were the same as in Example 1, except that dimethylamine hydrochloride was replaced by cyclopropylamine. The product was a white solid with a yield of 65%. ¹ H NMR (400 MHz, CDCl ₃ ) δ8.59 (s, 1H), 7.38 (d, J=1.3 Hz, 1H), 6.38 (s, 1H), 3.03-2.98 (m, 1H), 2.42 (d, J=1.2 Hz, 3H), 0.97-0.93 (m, 2H), 0.76-0.72 (m, 2H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ159.7, 154.7, 133.1, 124.9, 24.2, 9.5.

Example 7

Preparation of N-cyclohexylthieno[3,2-d]pyrimidin-4-amine (Compound 6g)

Other conditions were the same as those 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, CD ₃ OD) 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, CD ₃ OD) δ 159.4, 158.0, 155.3, 133.8, 124.7, 116.7, 51.2, 33.8, 26.7, 26.5.

Example 8

Preparation of N-(3-bromophenyl)thieno[3,2-d]pyrimidin-4-amine (Compound 6h)

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 (400 MHz, CD ₃ OD) δ8.59 (s, 1H), 8.11-8.10 (m, 2H), 7.74-7.69 (m, 1H), 7.42 (d, J=5.5 Hz, 1H), 7.29-7.28 (m, 2H). ¹³ C NMR (101 MHz, CD ₃ OD) δ161.0, 157.0, 154.9, 141.7, 135.3, 131.2, 128.0, 126.1, 124.9, 123.1, 121.9, 117.6.

Example 9

Preparation of N-phenylthieno[3,2-d]pyrimidin-4-amine (Compound 6i)

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

Example 10

Preparation of N-(3-(trifluoromethyl)phenyl)thieno[3,2-d]pyrimidin-4-amine (Compound 6j)

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 (400 MHz, CD ₃ OD) δ8.72 (s, 1H), 8.29 (d, J = 5.4 Hz, 1H), 8.18 (s, 1H), 8.01 (d, J = 7.3 Hz, 1H), 7.59 (t, J = 8.0 Hz, 1H), 7.51-7.47 (m, 2H). ¹³ C NMR (101 MHz, CD ₃ OD) δ157.8, 155.3, 152.6, 140.0, 138.1, 130.8, 127.4, 122.7, 122.3, 120.7, 117.9.

Embodiment 11

Preparation of N,N,7-trimethylthiophene[3,2-d]pyrimidin-4-amine (Compound 6k)

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

Example 12

Preparation of N,N-diethyl-7-methylthiophene[3,2-d]pyrimidin-4-amine (Compound 61)

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

Example 13

Preparation of N,7-dimethylthiophene[3,2-d]pyrimidin-4-amine (Compound 6m)

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 (400 MHz, CDCl ₃ ) δ8.61 (s, 1H), 7.22 (s, 1H), 5.39 (s, 1H), 3.12 (d, J=4.9 Hz, 3H), 2.37 (s, 3H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ158.4, 158.1, 154.7, 134.0, 125.657, 115.5, 28.1, 13.1.

Embodiment 14

Preparation of N-ethyl-7-methylthiophene[3,2-d]pyrimidin-4-amine (Compound 6n)

Other conditions were the same as in Example 11, except that dimethylamine hydrochloride was replaced with ethylamine tetrahydrofuran solution. The product was a white solid with a yield of 71%. ¹ H NMR (400 MHz, CDCl ₃ ) 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＝7.2 Hz, 3H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ158.6, 157.5, 154.8, 134.2, 125.7, 115.4, 36.2, 15.1, 13.2.

Embodiment 15

Preparation of N-isopropyl-7-methylthiophene[3,2-d]pyrimidin-4-amine (Compound 6o)

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 (400 MHz, CDCl ₃ ) δ8.66 (s, 1H), 7.31 (d, J=1.3 Hz, 1H), 4.65 (s, 1H), 4.56-4.48 (m, 1H), 2.45 (s, 3H), 1.33 (d, J=6.4 Hz, 6H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ158.7, 156.9, 154.9, 134.3, 125.5, 115.4, 43.1, 23.2, 13.2.

Example 16

Preparation of N-cyclopropyl-7-methylthiophene[3,2-d]pyrimidin-4-amine (Compound 6p)

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

Embodiment 17

Preparation of N-cyclohexyl-7-methylthiophene[3,2-d]pyrimidin-4-amine (Compound 6q)

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%. ¹ H NMR (400 MHz, CD ₃ OD) δ8.39 (s, 1H), 7.52 (s, 1H), 4.15-4.08 (m, 1H), 2.36 (s, 3H), 2.01 (d, J = 9.6 Hz, 2H), 1.80 (d, J = 12.5 Hz, 2H), 1.68 (d, J = 13.6 Hz, 1H), 1.48-1.16 (m, 6H). ¹³ C NMR (101 MHz, CD ₃ OD) δ158.4, 158.2, 155.1, 134.0, 128.5, 117.0, 51.1, 33.86, 26.7, 26., 13.0.

Embodiment 18

Preparation of N-(3-bromophenyl)-7-methylthieno[3,2-d]pyrimidin-4-amine (Compound 6r)

The other conditions were the same as those 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-d ₆ ) 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-d ₆ ) 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.

Embodiment 19

Preparation of N-phenyl-7-methylthieno[3,2-d]pyrimidin-4-amine (Compound 6s)

Other conditions were the same as in Example 11, except that dimethylamine hydrochloride was replaced by aniline. The product was a white solid with a yield of 69%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ9.65 (s, 1H), 8.61 (s, 1H), 7.83 (s, 1H), 7.80 (d, J=8.0 Hz, 2H), 7.36 (t, J=7.8 Hz, 2H), 7.10 (t, J=7.4 Hz, 1H), 2.36 (s, 3H). ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ159.2, 155.3, 153.8, 139.2, 132.7, 128.7, 128.5, 123.5, 122., 115.7, 12.7.

Embodiment 20

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

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

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

Embodiment 21

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

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

Step 3: The intermediate compound 4-chlorothiophene[3,2-d]pyrimidine (60 mg, 0.35 mmol) was dissolved in tetrahydrofuran (4 mL), and 40% NaH (42 mg, 1.05 mmol) was added under ice bath conditions. After stirring for half an hour, the ice bath was removed, and anhydrous methanol (21.2 μL, 0.53 mmol) was added. Stir at room temperature for 5 hours, and the plate was tested. After the reaction was completed, the concentration column was chromatographed [eluent: V (petroleum ether): V (ethyl acetate) = 5:1] to obtain the target compound 4-methoxythiophene[3,2-d]pyrimidine, 42 mg of white solid, and a yield of 72%. ¹ H NMR (400MHz, CDCl ₃ ) δ 8.74 (s, 1H), 7.83 (d, J = 5.4Hz, 1H), 7.47 (d, J = 5.3Hz, 1H), 4.14 (s, 3H). ¹³ C NMR ( 101MHz, CDCl ₃ ) δ 164.6, 161.9, 154.5, 134.2, 124.6, 117.8,54.3.

Example 22

Preparation of 4-isopropoxythieno[3,2-d]pyrimidine (Compound 6v)

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 (400 MHz, CDCl ₃ ) δ8.65 (s, 1H), 7.75 (d, J=5.4 Hz, 1H), 7.40 (d, J=5.4 Hz, 1H), 5.60-5.50 (d, J=37.3 Hz, 1H), 1.38 (d, J=6.2 Hz, 6H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ164.0, 162.0, 154.5, 133.9, 124.6, 118.2, 70.4, 22.0.

Embodiment 23

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

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

Embodiment 24

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

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 (400 MHz, CDCl ₃ ) δ8.75 (s, 1H), 7.45 (d, J=1.2 Hz, 1H), 5.64-5.58 (m, 1H), 2.47 (d, J=1.2 Hz, 3H), 1.44 (d, J=6.2 Hz, 6H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ164.1, 161.0, 154.2, 133.4, 128.8, 118.5, 70.29, 22.0, 13.0.

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

Embodiment 25

Preparation of 2-(Morpholinomethyl)-N-phenylthieno[3,2-d]pyrimidin-4-amine (Compound 10a)

Step 1: Dissolve ethyl 3-aminothiophene-2-carboxylate (3 g, 17.54 mmol) in 1,4-dioxane (45 mL), heat to 50 ° C, slowly add chloroacetonitrile (1.33 mL, 21.05 mmol), after the addition is completed, introduce hydrogen chloride gas (20 mL), stir and react for 20 minutes, after the reaction is completed, cool to room temperature, filter the precipitate, wash with ethyl acetate (50 mL), and dry to obtain the intermediate compound 2-(chloromethyl)thieno[3,2-d]pyrimidin-4(3H)-one, 2.74 g of off-white solid, yield 78%.

Step 2: Dissolve 2-(chloromethyl)thieno[3,2-d]pyrimidin-4(3H)-one (500 mg, 2.50 mmol) in tetrahydrofuran (40 mL), add morpholine (261 μL, 3.00 mmol), slowly add triethylamine (194 μL, 1.40 mmol), heat to 50°C, stir to react for 10 h, and detect with a spot plate. After the reaction is completed, concentrate and column chromatograph [eluent: V (petroleum ether): V (ethyl acetate) = 3:1] to obtain the intermediate compound 2-(morpholinomethyl)thieno[3,2-d]pyrimidin-4(3H)-one, 526 mg of white solid, yield 84%.

Step 3: The intermediate compound 2-(morpholinemethyl)thieno[3,2-d]pyrimidin-4(3H)-one (500 mg, 1.99 mmol) was dissolved in toluene (20 mL), N,N-dimethylformamide (46 μL, 0.60 mmol) was added, phosphorus oxychloride (729 μL, 7.96 mmol) was slowly added dropwise, heated to 120°C, refluxed for 12 h, and the pH was adjusted to neutral with saturated sodium bicarbonate solution, extracted with ethyl acetate (10 mL×3), and concentrated by column chromatography [eluent: V (petroleum ether): V (ethyl acetate) = 1:1] to obtain the intermediate compound 4-(4-chlorothienyl[3,2-d]pyrimidin-2-yl)methyl)morpholine, 474 mg brown solid, yield 88%.

Step 4: The intermediate compound 4-(4-chlorothiophene[3,2-d]pyrimidin-2-yl)methyl)morpholine (80 mg, 0.30 mmol) was dissolved in tetrahydrofuran (4 mL), aniline (33 μL, 0.36 mmol) was added, triethylamine (125 μL, 0.9 mmol) was slowly added dropwise, heated to 50°C, stirred for 10 h, and the plate was tested. After the reaction was completed, the concentration column was chromatographed [eluent: V (dichloromethane): V (methanol) = 20:1] to obtain the target compound 2-(morpholinemethyl)-N-phenylthieno[3,2-d]pyrimidin-4-amine, 71 mg of white solid, and the yield was 73%. ¹ H NMR (400MHz, CD ₃ OD) 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, CD ₃ OD) 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.

Embodiment 26

Preparation of N-(3-bromophenyl)-2-(morpholinomethyl)thieno[3,2-d]pyrimidin-4-amine (Compound 10b)

The other conditions were the same as those in Example 25, except that aniline was replaced by m-bromoaniline. The product was a white solid with a yield of 74%. ¹ HNMR (400MHz, CD ₃ OD) 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,CD ₃ OD)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.

Embodiment 27

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

The other conditions were the same as those in Example 25, except that aniline was replaced by m-aminotrifluorotoluene. The product was a white solid with a yield of 71%. ¹ H NMR (400MHz, CD ₃ OD) 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, CD ₃ OD) 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.

Embodiment 28

Preparation of N-(3-bromophenyl)-2-((diethylamino)methyl)thieno[3,2-d]pyrimidin-4-amine (Compound 10d)

Other conditions were the same as those in Example 26, except that morpholine was replaced with diethylamine. The product was a white solid with a yield of 73%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 10.33 (s, 1H), 8.35 (d, J = 5.4 Hz, 1H), 8.09 (t, J = 1.9 Hz, 1H), 7.87 (dt, J = 7.6, 1.9 Hz, 1H), 7.52 (d, J = 5.3 Hz, 1H), 7.36-7.29 (m, 2H), 4.42 (s, 2H), 3.27 (d, J = 7.3 Hz, 4H), 1.31 (t, J = 7.2 Hz, 6H). ¹³ C NMR (101 MHz, DMSO-d ₆ )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.

Embodiment 29

Preparation of N-phenyl-2-(pyrrolidin-1-ylmethyl)thieno[3,2-d]pyrimidin-4-amine (Compound 10e)

The 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 (400 MHz, CD ₃ OD) δ8.10 (d, J＝5.4 Hz, 1H), 7.69-7.67 (m, 2H),

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,CD ₃ OD)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.

Embodiment 30

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

Other conditions were the same as in Example 25, except that morpholine was replaced by piperidine. The product was a white solid with a yield of 69%. ¹ H NMR (400 MHz, DMSO-d ₆ ) 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 (101 MHz, DMSO-d ₆ ) 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.

Embodiment 31

Preparation of 7-methyl-2-(morpholinomethyl)-N-phenylthieno[3,2-d]pyrimidin-4-amine (Compound 10g)

The other conditions were the same as those in Example 25, except that 3-aminothiophene-2-carboxylic acid ethyl ester was replaced with 3-amino-4-methylthiophene-2-carboxylic acid ethyl ester. The product was a white solid with a yield of 71%. 2 .43(s,3H). ¹³ C ^NMR (101MHz, CDCl ₃ ) _{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} .

Biological activity test

GloSensor cAMP accumulation assay was used to test the functional activity of target compounds on _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 with 500,000 cells per well. The next day, 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 incubated with equilibration medium containing 2% v/v GloSensor cAMP reagent stock solution (dissolved in 10% FBS in CO2-independent medium). After incubation at 37°C for 1 h, bioluminescence signal was 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 constant temperature cell incubator for 30 min. Then, DA with a concentration gradient (final concentration 1 nM-10 μM) was added and incubated at room temperature for 5 min. Then, 10 μM Forskolin was added to the cells, and the changes in bioluminescence were read using an enzyme-labeled instrument.

Table 1 Statistical table of the comparison of the allosteric antagonistic activity of the target compound and the activity of positive drug 2

Allosteric antagonistic activity screening

The GloSensor cAMP accumulation test was applied, with DA of different concentration gradients as blank control (final concentration gradient of 1nM-10μM), and compound 2 (final concentration of 100μM) as positive control, to compare the difference between the allosteric antagonistic activity of the target compound (final concentration of 100μM) and positive drug 2. The test results (Table 1) show that the compounds of the present invention all have different degrees of allosteric antagonism to D ₂ R. In general, the allosteric antagonistic activity is relatively better when there is a methyl substitution on the thiophene ring, and some of the compounds have significantly better allosteric antagonistic activity to D ₂ R than positive drug 2, such as 6d, 6e, 6f, 6j, 6n, 6o, 6q, 6s, 10d; among them, the activities of compounds 6e, 6o, 6q, 6s, 10d are 1.28 times, 1.39 times, 1.15 times, 1.14 times, and 1.38 times that of positive drug 2, respectively.

Allosteric mechanism studies

The GloSensor cAMP accumulation experiment was used to further study whether this type of compound can allosterically regulate the functional activity of dopamine (DA), the endogenous ligand of D ₂ R, and further confirmed that this type of compound is a negative allosteric modulator (NAM) of D ₂ R. Taking compound 6o as an example (as shown in Figure 1), when the concentration of compound 6o reached 60μM, the dose-effect curve of DA showed a significant downward shift and almost reached the maximum downward shift limit of the dose-effect regulation of DA functional activity, which indicates that the IC ₅₀ value of compound 6o may be less than 60μM, which is specifically manifested in the sudden drop of the dose-effect curve. When the concentration of 6o increased from 60μM to 120μM, the downward shift of the dose-effect curve of DA was very weak. This phenomenon explains that with the increase of the concentration of compound 6o, the concentration-dependent curve of DA showed a limited downward shift, indicating that compound 6o can effectively negatively allosterically regulate the functional activity of DA, the endogenous ligand of D ₂ R. This allosteric regulation phenomenon is consistent with the reported D ₂ R allosteric antagonistic regulation mechanism. The concentration dependence curve of compound 6q allosterically regulating DA (as shown in FIG2 ) is consistent with the result of 6o, that is, the target compounds of the present invention are all negative allosteric modulators (NAM) or allosteric antagonists of D ₂ R.

Claims (5)

1. A thienopyrimidine compound characterized in that: the structure of the thienopyrimidine compound is shown as formula 1:

wherein R is ₁ Is one of the following groups:

R ₂ is one of the following groups:

R ₃ is one of the following groups:

2. the thienopyrimidines of claim 1, wherein: r is R ₁ is-H or-CH ₃ ；R ₂ Is one of the following groups:

R ₃ is one of the following groups:

3. the thienopyrimidines of claim 1, wherein: the thienopyrimidine compound has the following structure:

4. use of a thienopyrimidine compound according to any one of claims 1 to 3, characterized in that: one or more of the thienopyrimidine compounds are used as active ingredients and are prepared into a pharmaceutical preparation with a pharmaceutically acceptable carrier.

5. The use of the thienopyrimidines according to claim 4, wherein: the thienopyrimidine compound is used for preparing dopamine D ₂ Use of receptor allosteric antagonists.