Application of thienopyrimidine compound as beta2 adrenergic receptor allosteric modulator

Abstract

The invention discloses an application of a thienopyrimidine compound as a beta2 adrenergic receptor (beta2AR) allosteric modulator, and belongs to the field of medicinal chemistry. The thienopyrimidine compound disclosed by the invention is shown as a formula 1, and the structure of the compound comprises a thieno [3, 2-d] pyrimidine ring skeleton. Through deep research on the invention, the inventor finds that the thienopyrimidine compound can be used as a negative allosteric modulator or allosteric antagonist of beta2AR. According to the invention, a GloSensor cAMP content accumulation experiment is adopted to test the functional activity and allosteric regulation mechanism of the compound on beta2AR. Research results show that most of the thienopyrimidine compounds have an allosteric regulation function on beta2AR, namely, the activity of isoproterenol (ISO) serving as an endogenous ligand of beta2AR can be subjected to negative allosteric regulation. Wherein the allosteric regulation function of 6q on beta2AR is 2.7 times that of a positive drug Cmpd-15. Discovery of the thienopyrimidine beta2AR allosteric antagonist provides a new chemical entity for research and development of new drugs targeting beta2AR.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and specifically relates to the application of thienopyrimidine compounds as β2 adrenergic receptor (β2AR) allosteric modulators. Background Art

[0002] β2-adrenergic receptor (β2AR) is a type of class A G-protein-coupled receptor (GPCR). It plays an important regulatory role in the human heart, lungs, immune response and body homeostasis. It is also one of the important drug targets for chronic obstructive pulmonary disease (COPD), cardiovascular and cerebrovascular diseases and digestive system diseases.

[0003] According to the different distribution of β2AR in various organs and tissues of the body, it can be divided into three receptor subtypes: β1, β2 and β3. β2 is expressed in smooth muscle tissues such as vascular smooth muscle, respiratory smooth muscle and digestive tract smooth muscle. Blocking vascular β2 receptors, coupled with reduced cardiac output, reflexively excites the sympathetic nerves, causes vasoconstriction, increased peripheral resistance, reduced organ blood flow, and reduced coronary blood flow; blocking bronchial smooth muscle β2 receptors, contracting bronchi, and increasing tracheal resistance can induce or aggravate asthma in patients with bronchial asthma. In clinical practice, beta-receptor blockers are widely used to treat hypertension, angina pectoris, arrhythmia and hyperthyroidism because they can bind to beta-adrenaline receptors and antagonize the effects of beta receptors. For example, the commonly used non-selective beta-receptor antagonists propranolol and selective beta-1 antagonists metoprolol are all representatives of beta-receptor blockers. In the treatment of bronchial diseases, short-acting β2AR agonists such as salbutamol and long-acting β2AR agonists such as salmeterol are the most commonly used drugs for the treatment of asthma (Allergy 2012, 67, 835-846).

[0004] In 2017, the inventors collaborated with Professor Robert Lefkowitz of Duke University to report the first β2AR allosteric antagonist, Cmpd-15 (Nature, 2017, 548, 480-484.). Figure 1 The figure shows a polypeptide skeleton, which has poor water solubility and drugability. It is urgent to develop new small molecule β2AR allosteric antagonists. Summary of the invention

[0005] The object of the present invention is to provide the use of thienopyrimidine compounds as β2 adrenergic receptor allosteric modulators.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] The present invention provides application of thienopyrimidine compounds as beta 2 adrenergic receptor allosteric modulators.

[0008] Furthermore, the structure of the thienopyrimidine compound is shown in Formula 1:

[0009]

[0010] R1 is one of the following structures: H or CH3-; R2 is one of the following structures:

[0011]

[0012] R3 is one of the following structures:

[0013]

[0014] Furthermore, the thienopyrimidine compound is one of the following structural formulas:

[0015]

[0016] Furthermore, the thienopyrimidine compound specifically acts as a β2 adrenergic receptor allosteric antagonist or negative allosteric modulator.

[0017] Furthermore, in the case where the thienopyrimidine compound is specifically used as a β2 adrenergic receptor allosteric antagonist, the β2 adrenergic receptor allosteric antagonist includes a ligand or a small molecule compound that inhibits β2AR; wherein the ligand that inhibits β2AR is an endogenous β2AR ligand, isoproterenol (ISO), epinephrine, norepinephrine or a synthetic agonist.

[0018] Furthermore, the final concentration of the thienopyrimidine compound is 50 μM.

[0019] Furthermore, the concentration gradient of the thienopyrimidine compound used to verify the allosteric mechanism is 100 μM, 50 μM, 25 μM, 12.5 μM, and 6.25 μM.

[0020] Furthermore, it includes a pharmaceutical composition; the pharmaceutical composition comprises the thienopyrimidine compound, its pharmaceutically acceptable salt or prodrug, hydrate, solvate, crystal form, tautomer or their metabolite form; optionally, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier or excipient.

[0021] The present invention also provides a method for detecting the biological activity of β2AR allosteric modulators based on the GloSensor cAMP accumulation experiment, which is applied to the above-mentioned application; the method comprises: constructing a β2AR plasmid, using cells to overexpress β2AR and 22FcAMP, stimulating the overexpressing cells with the thienopyrimidine compound to be tested, and then stimulating the cells with the β2AR endogenous ligand isoproterenol, detecting the bioluminescent signal of the stimulated cells, and reflecting the content of cAMP produced by the strength of the bioluminescent signal.

[0022] Furthermore, the overexpression cells are HEK-293T cells.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides for the first time the application of thienopyrimidine compounds that have a good allosteric antagonistic effect on β2AR. The research results of the present invention reveal the connection between thienopyrimidine compounds and β2AR, and further clarify the relationship at the level of allosteric regulation mechanism, providing new potential candidate drugs for the treatment of cardiovascular diseases.

[0025] The present invention has experimentally found that thienopyrimidine compounds can act on β2AR, causing changes in downstream cAMP. In-depth mechanism studies have shown that the target compounds in the present invention are all β2AR allosteric antagonists. At present, β2AR, as the preferred clinical drug target, plays an extremely important therapeutic role in the treatment of cardiovascular system, bronchial and digestive tract related diseases. However, adrenergic receptors can be divided into three receptor subtypes: β1, β2 and β3. Due to the high homology between receptor subtypes, the specificity is not high and it is easy to go off-target. Long-term use of this type of drug will cause obvious side effects. Allosteric modulators have specific targeting to receptors, and allosteric modulators have a saturation limit, so it is expected to develop safer and more effective β2AR allosteric antagonist candidate drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a schematic diagram of the structure of compound Cmpd-15;

[0027] Figure 2 This is the dose-effect curve of compound 6q allosterically regulating ISO;

[0028] Figure 3 This is the concentration dependence curve of compound 6q allosterically regulating ISO;

[0029] Figure 4 This is the concentration dependence curve of compound 10d allosterically regulating ISO. DETAILED DESCRIPTION

[0030] It should be noted that the following detailed description of the contents of the present invention is illustrative only, but the present invention is not limited to these embodiments and specific examples.

[0031] In a specific embodiment of the present invention, the thienopyrimidine or a pharmaceutically acceptable salt thereof is an antagonist allosteric modulator of β2AR.

[0032] In a specific embodiment of the present invention, the thienopyrimidine or a pharmaceutically acceptable salt thereof allosterically regulates β2AR via the Gs-AC-cAMP pathway.

[0033] In a specific embodiment of the present invention, provided is the use of a thienopyrimidine compound or a pharmaceutically acceptable salt thereof in the preparation of a β2AR allosteric antagonist.

[0034] Wherein, the thienopyrimidine compound is an allosteric antagonist or an allosteric inhibitor of β2AR.

[0035] In a specific embodiment of the present invention, the allosteric antagonist of β2AR includes ligands and small molecule compounds that inhibit β2AR.

[0036] In a specific embodiment of the present invention, the ligand is the endogenous ligand ISO of β2AR.

[0037] In another specific embodiment of the present invention, there is provided the use of thienopyrimidine compounds with different concentration gradients as β2AR allosteric antagonists.

[0038] In another specific embodiment of the present invention, a pharmaceutical composition is provided, which comprises the above-mentioned thienopyrimidine or its solvate or pharmaceutically acceptable salt.

[0039] The technical solution of the present invention is described in detail below.

[0040] Example 1

[0041] The activity of thienopyrimidine compounds was determined using the GloSensor cAMP accumulation assay.

[0042] HEK 293T cells with good growth status and cell confluence of more than 80% were plated at 6.0×10 5 The cells were seeded into a 3.5 cm cell culture dish, shaken crosswise to evenly cover the surface, and then cultured overnight in a constant temperature and humidity cell culture incubator containing 5% CO2 at 37°C.

[0043] After 24 hours, 22F cAMP and human β2AR plasmids were transiently transfected into adherent HEK 293T cells using FuGENE HD transfection reagent (Promega) and cultured in an incubator for another 24 hours.

[0044] HEK 293T cells transfected with the expression plasmid for 24 hours were harvested, diluted with culture medium according to the calculated amount, and then evenly seeded into a transparent 96-well plate coated with poly-lysine (PDL) using a spray gun, and cultured for another 24 hours.

[0045] Wash the cells once with CO2 independent medium, and then add 10 mL of equilibration medium (2% GloSensor TM cAMP Reagent, 10% FBS and 88% CO2 independent medium) in a 96-well plate, incubate in a cell culture incubator for 1 hour, place on a clean bench at room temperature for 1 hour, and immediately collect the bioluminescent signal with a multi-function microplate reader until a stable background signal is obtained.

[0046] After obtaining a stable background baseline signal, the thienopyrimidine compound to be tested (final concentration of 50 μM) was added to the 96-well plate cells containing the substrate at the same time, incubated at 37°C for 30 minutes and then at room temperature for 5 minutes, and different concentrations of agonist ISO were added (final concentration gradient of 1nM, 10nM, 100nM, 1μM, 10μM, 100μM), and the changes in bioluminescence were read with a microplate reader. The allosteric antagonistic activity of thienopyrimidine compounds was compared with that of the lead compound Cmpd-15.

[0047] Table 1 Statistical comparison of the allosteric antagonistic activity of thienopyrimidine compounds and the activity of Compd-15

[0048]

[0049]

[0050]

[0051] The test results in Table 1 show that most thienopyrimidine compounds have different degrees of allosteric antagonism on β2AR, and some of them have significantly better allosteric antagonism on β2AR than the lead compound Cmpd-15, such as 6e, 6g, 6o, 6q, 10b, and 10d, which are 1.22 times, 1.84 times, 1.24 times, 2.72 times, 1.23 times, and 1.31 times of the lead compound, respectively, and are more effective negative allosteric modulators (NAM) of β2AR. 6q has the strongest allosteric antagonism. In addition, the target compound not only significantly reduced the maximum effect value (Emax) of ISO, but also increased the EC of ISO. 50 The value is obviously shifted to the right. For example, 6q shifts ISO's EC 50 Shifted right by 2.8 times, such as Figure 2 The above results show that the thienopyrimidine compounds of the present invention are β2AR allosteric antagonists.

[0052] Example 2

[0053] The GloSensor cAMP accumulation experiment was used to further study the allosteric antagonism mechanism of this class of compounds, and to test whether the target compound can allosterically regulate the functional activity of the endogenous ligand ISO of β2AR to study its mechanism of negative allosteric regulation of β2AR, and further confirm that this class of compounds is a negative allosteric modulator of β2AR. After waiting for the background signal of bioluminescence to stabilize, thienopyrimidine compounds with good antagonistic activity were added to the cells expressing the receptor, and the concentration was multiple. After incubation at 37°C for 30 minutes, the temperature was restored to room temperature, and different concentration gradients of ISO were added to the cells (the final concentration gradient was 1nM, 10nM, 100nM, 1μM, 10μM, 100μM), and the change of the bioluminescence signal at this time was tested to see whether it showed a concentration-dependent limited downward shift trend. From the ISO concentration-dependent curve, it can be found that the dose-effect curve of ISO showed a concentration-dependent limited downward shift after adding different concentrations of the same compound.

[0054] Taking compound 6q as an example, Figure 3As shown in the figure, the red ISO curve shows a certain degree of downward shift as the concentration gradient of compound 6q increases from 6.25μM (green curve), 12.5μM (dark blue curve), and 25μM (orange curve) by 2 times. When the concentration of 6q increases to 50.0μM (yellow curve), the ISO curve shows a significant downward shift, and when the concentration of 6q increases to 100.0μM (purple curve), the ISO concentration curve shows a very weak downward shift. This phenomenon explains that with the increase of the concentration of compound 6q, the concentration-dependent curve of ISO shows a limited downward shift, indicating that compound 6q can negatively allosterically regulate the functional activity of β2AR endogenous ligand ISO, that is, compound 6q is a β2AR allosteric antagonist or negative allosteric modulator.

[0055] Taking compound 10d as an example, Figure 4 As shown in the figure, the red ISO dose-effect curve also shows a limited downward shift as the concentration of compound 10d increases, indicating that compound 10d is also a β2AR allosteric antagonist. This allosteric regulation phenomenon is consistent with the reported allosteric antagonistic regulation mechanism, indicating that the target compound can negatively allosterically regulate the functional activity of β2AR endogenous ligand ISO, that is, thienopyrimidine compounds are allosteric β2AR antagonists or negative allosteric modulators.

[0056] Those skilled in the art can understand that the above-mentioned embodiments are specific examples for implementing the present application, and in practical applications, various changes can be made to them in form and detail without departing from the spirit and scope of the present application. Any person skilled in the art can make their own changes and modifications without departing from the spirit and scope of the present application, so the scope of protection of the present application shall be based on the scope defined in the claims.

Claims

1. Application of thienopyrimidine compounds as allosteric modulators of β2 adrenergic receptors.

2. The use according to claim 1, characterized in that The structure of the thienopyrimidine compound is shown in Formula 1: R1 is one of the following structures: H or CH3-; R2 is one of the following structures: R3 is one of the following structures:

3. The use according to claim 2, characterized in that The thienopyrimidine compound is one of the following structural formulas:

4. The use according to claim 2 or 3, characterized in that The thienopyrimidine compounds are specifically used as β2 adrenergic receptor allosteric antagonists or negative allosteric modulators.

5. The use according to claim 4, characterized in that When the thienopyrimidine compound is specifically used as a β2 adrenergic receptor allosteric antagonist, the β2 adrenergic receptor allosteric antagonist includes a ligand or a small molecule compound that inhibits β2AR; wherein the ligand that inhibits β2AR is an endogenous β2AR ligand, isoproterenol, epinephrine, norepinephrine or a synthetic agonist.

6. The use according to claim 4, characterized in that The final concentration of the thienopyrimidine compound is 50 μM.

7. The use according to claim 4, characterized in that The concentration gradient of the thienopyrimidine compound used to verify the allosteric mechanism is 100 μM, 50 μM, 25 μM, 12.5 μM, and 6.25 μM.

8. The use according to claim 2, 3 or 4, characterized in that It includes a pharmaceutical composition; the pharmaceutical composition contains the thienopyrimidine compound, its pharmaceutically acceptable salt or prodrug, hydrate, solvate, crystal form, tautomer or their metabolite form; optionally, the pharmaceutical composition also contains a pharmaceutically acceptable carrier or excipient.

9. A method for detecting the biological activity of β2AR allosteric modulators based on GloSensor cAMP accumulation assay, characterized in that: The method is applied in the application as claimed in claim 1; the method comprises: constructing a β2AR plasmid, using cells to overexpress β2AR and 22F cAMP, stimulating the overexpressing cells with the thienopyrimidine compound to be tested, and then stimulating the cells with the β2AR endogenous ligand isoproterenol, detecting the bioluminescent signal of the stimulated cells, and reflecting the content of cAMP produced by the strength of the bioluminescent signal.

10. The method according to claim 9, characterized in that The overexpression cells are HEK-293T cells.

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