Compound, preparation method and application of compound as orphan receptor GPR99 agonist

Abstract

The invention belongs to the technical field of medicine research and development, and particularly relates to a compound, a preparation method and application of the compound as an orphan receptor GPR99 agonist. The small molecule compound capable of specifically activating the orphan receptor GPR99 is found and identified for the first time, and the action mechanism of the small molecule compound in vascular smooth muscle cell (VSMC) contraction regulation and pathological vasodilation improvement is systematically clarified. The discovery not only fills the blank of the GPR99 agonist in the field of acne rosacea treatment, but also provides a new molecular target and a medicine research and development direction for precise treatment of vascular dysfunction related diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of drug research and development, and particularly relates to a compound, a preparation method and application thereof as an agonist of orphan receptor GPR99. BACKGROUND

[0002] Rosacea is a common chronic inflammatory skin disease, mainly involving the central facial region, and its clinical features include persistent facial erythema, telangiectasia, inflammatory papules and pustules, etc. The disease has the characteristics of repeated attacks and long-term progression, not only affecting the appearance of patients, but also accompanied by symptoms such as burning and tingling, which seriously affects the psychological health and quality of life of patients. According to epidemiological investigations, the prevalence rate of rosacea in the global population is about 5.5%, and the incidence rate is higher in people with lighter skin, which has a high public health burden.

[0003] Current studies have shown that the pathogenesis of rosacea is complex, involving abnormalities in innate immune response, impaired skin barrier function, dysregulation of neurovascular modulation and imbalance of microbiota, etc. Among them, vascular dysfunction is considered to be one of the core factors leading to persistent erythema and vascular dilation of the disease. In particular, the dysfunction of vascular smooth muscle cell (VSMC) contraction can trigger pathological vasodilation, leading to increased blood flow and aggravated erythema in the face. In addition, vascular dysfunction may further exacerbate chronic inflammatory reactions in local tissues through the sustained release of inflammatory factors.

[0004] Current clinical drug treatment methods include external use of alpha-adrenergic receptor agonists (such as brimonidine and oxymetazoline) to constrict blood vessels and improve erythema symptoms. However, such drugs generally have the problems of fast onset but short duration of action, easy rebound of symptoms, tolerance and local irritation after long-term use, etc., making it difficult to achieve long-term stable efficacy. In addition, some patients have poor response to existing drugs or have contraindications, further limiting their application range. Therefore, it is urgent to develop new therapeutic strategies targeting the regulation of vascular function to achieve more durable and safe clinical benefits. SUMMARY

[0005] By clinical metabolomics analysis, we found that alpha-ketoglutarate (alpha-KG) as an endogenous GPR99 ligand was abnormally accumulated in patients with rosacea, and enhanced vascular smooth muscle contraction by activating the GPR99-Gq-MLCK-MYL9 signaling axis, and inhibited pathological vascular dilation. In addition, by activating its specific receptor GPR99, alpha-KG and another endogenous GPR99 agonist itaconic acid (ITA) can prevent abnormal vascular dilation, thereby reducing the erythrotic phenotype of rosacea. In order to further understand the signaling and structural basis of the therapeutic effect of alpha-KG and ITA, we set out to study the structure of GPR99 and the downstream pathway. Based on the structural features of the GPR99 "double acid binding pocket" structure resolved by cryo-EM, we developed the agonist A-1, which significantly activates GPR99 signaling at nanomolar concentrations, promotes MYL9 phosphorylation-dependent vascular contraction. Experimental data show that local application of A-1 can reduce the erythema area score by 60%, reduce inflammatory cell infiltration, and has no cross-activity on the homologous receptor GPR91. Compared with the natural ligand alpha-KG, the half-life of A-1 is significantly prolonged, and at a dose of 80 μM, it can achieve sustained improvement of vascular function.

[0006] These compounds regulate GPR99-mediated vascular homeostasis through precise regulation, providing an innovative treatment strategy for rosacea and related vascular disorder diseases. Its high efficiency not only overcomes the limitations of traditional therapy, but also provides a structural biology basis for local drug design targeting GPCRs, and has great potential for clinical application.

[0007] In a first aspect of the present application, a compound is provided, the chemical structure of which is shown in formula alpha-KG, formula ITA or formula A-1: , , .

[0008] Further, the pharmaceutically acceptable salt, hydrate, solvate or prodrug of the compound.

[0009] In another aspect of the present application, the compound is provided as an agonist of GPR99.

[0010] In another aspect of the present application, the compound is provided as an agonist of GPR99.

[0011] In another aspect of the present application, a synthesis route of the compound A-1 is provided, as follows: .

[0012] Further, the synthesis method of compound A-1: take 100 mL three-necked flask, add compound 5 (1 equiv.), add 20 mL anhydrous DCM, dissolve, add methyl chloroformate (2 equiv.), triethylamine (3 equiv.) in turn under ice bath, nitrogen seal the reaction system, stir from 0℃ to room temperature for 12 h. After the reaction is completed, spin dry, silica gel column chromatography purification, to obtain compound A-1.

[0013] Compared with the prior art, the beneficial effects of the present application are: 1、The present application first discovers and identifies small molecule compounds capable of specifically activating orphan receptor GPR99, and systematically elucidates the mechanism of action thereof in the regulation of vascular smooth muscle cell (VSMC) contraction and improvement of pathological vascular dilation. The discovery not only fills the blank of GPR99 agonists in the field of treatment of rosacea, but also provides a new molecular target and drug research and development direction for precise treatment of diseases related to abnormal vascular function.

[0014] 2、Compared with existing alpha-adrenergic receptor agonists and other vasoconstrictor drugs, the GPR99 small molecule agonist of the present application has the advantages of long-lasting effect, low side effects, not easy to tolerate and rebound. At the same time, the present application provides a suitable synthetic route for industrial production, which is simple, high-yielding, easy to obtain raw materials, and convenient for large-scale preparation and subsequent clinical application transformation. BRIEF DESCRIPTION OF DRAWINGS

[0015] The drawings accompanying the specification of the present application serve to provide further understanding of the present application, and the illustrative embodiments of the present application and the description thereof serve to explain the present application, and do not constitute an improper limitation on the present application.

[0016] Figure 1 Effect of compounds on GPR99 activity.

[0017] Figure 2 Compounds alpha-KG and A-1 promote vascular smooth muscle cell contraction. A. Representative images of gel contraction. Vascular smooth muscle cells were treated with alpha-KG or OXGR1 agonist A-1 (200 nM) for 12 h. B. Quantitative analysis of gel area in A (n = 3).

[0018] Figure 3Compound a-KG and A-1 promote vascular smooth muscle cell expression of smooth muscle contractile marker pMYL-9. A. Immunoblot analysis of p-MLY9 and MYL9 in vascular smooth muscle cell lysates. Vascular smooth muscle cells were treated with a-ketoglutarate (a-KG) or OXGR1 agonist (A-1 200 nM). GAPDH as loading control. p-MLY9 protein levels were analyzed relative to total MLY9. B. Quantitative analysis of p-MLY9 protein levels relative to total MYL9 protein in A (n = 3).

[0019] Figure 4 Improvement of compounds a-KG and A-1 on mouse acne rosacea. A. Schematic diagram of mouse treatment scheme: mice were applied with a-KG or A-1 gel for 10 days (20 μL, once a day), injected with LL37 as shown in the figure, and observed for skin lesions. n = 6 mice per group. B. Representative images of the back skin of mice in the solvent, A-1, a-KG groups (n = 6 mice per group). Images were taken at the indicated time points. C. Assessment of the severity of acne rosacea-like phenotype in each group based on erythema area (n = 6). D. Assessment of the severity of acne rosacea-like phenotype in each group based on erythema score (n = 6).

[0020] Figure 5 Compounds a-KG and A-1 improved the vascular dilation of acne rosacea-like dermatitis mice. A. Representative images of CD31 immunohistochemical staining of skin lesions in each group of mice. Scale bar = 100 μm. B. Calculation of the perimeter of blood vessels in A (78-103 blood vessels were selected from 6 independent mice in each group).

[0021] Figure 6 Compound A-1 1 H NMR.

[0022] Figure 7 Compound A-1 13 C NMR. DETAILED DESCRIPTION

[0023] The following detailed description of the embodiments of the present application is made on the premise of the technical solutions of the present application, and detailed embodiments and specific operation processes are given, but the protection scope of the present application is not limited to the following embodiments.

[0024] Example 1 Effect of compound on GPR99 activity: Experimental method: the applicant used IP-One experiment to detect the activation of Gq protein downstream of GPR99 under the stimulation of different agonists. HEK293 cells were transiently transfected with GPR99 receptor plasmid. 24 hours after transfection, the cells were seeded at 1 × 10 4Cells were seeded at a density of one cell per well in 384-well microplates and incubated at 37 °C for 24 hours. The inositol 1 -phosphate (IP1) level was determined by IP-One assay kit (Cisbio Bioassays, #62IPAPEB, Bedford, MA) following the manufacturer’s guideline. Subsequently, cells were stimulated with HEM-PSS buffer containing different concentrations of a-ketoglutarate (a-KG), itaconic acid (ITA) or A-1 (final concentration of 10 -10 to 10 -3 M) at 37 °C for 20 minutes. After stimulation, cells were washed twice with HBSS buffer containing Ca²⁺ and Mg²⁺. Then, 7 μΐ of washed cells were transferred to 384-well plates, and 3 μΐ of D2-labeled IP1 and 3 μΐ of cryptate-labeled anti-IP1 monoclonal antibody were added to each well. After incubation at room temperature for 1 hour, the fluorescence resonance energy transfer (FRET) signal was detected using a Berthold Tristar 5 detector with an excitation wavelength of 330 nm and emission wavelengths of 620 nm and 665 nm. The FRET signal was calculated by the emission ratio (665 nm / 620 nm).

[0025] Experimental results: As shown in Figure 1 , by further viability testing of a-KG, ITA and small molecule A-1 obtained by screening, it is confirmed that the three small molecules including a-KG, ITA and A-1 show strong Gq agonism on GPR99, and the above results suggest that we can develop a biased ligand for the constitutively activated orphan GPCR to selectively regulate its function.

[0026] Example 2 Compounds a-KG and A-1 promote vascular smooth muscle cell contraction: Experimental method: Isolate mouse primary aortic vascular smooth muscle cells, count after digestion and resuspension. Take an equal amount of cells mixed with collagenase type I, sodium hydroxide solution, seed in a 24-well plate, incubate in a constant temperature incubator for 20 min, carefully separate the gel and the well wall, then add complete medium containing 200 nmol A-1 or its carrier solvent (DMSO), incubate in a constant temperature incubator for 12 hours, then observe the area change of the gel in each well, take photos and use ImageJ to analyze the gel area size of each group.

[0027] Experimental results: As shown in Figure 2 , the application of compounds a-KG and A-1 significantly reduces the gel area and promotes the contraction of vascular smooth muscle cells.

[0028] Example 3 Compounds a-KG and A-1 promote vascular smooth muscle cells to express smooth muscle contraction marker pMYL-9: Experimental method: Isolate mouse primary aortic vascular smooth muscle cells, add 200 nmol A-1 or its carrier solvent (DMSO) for 15 min, collect the protein of each group of cells, and perform Western Blot to detect the level of smooth muscle contraction marker pMYL-9.

[0029] Experimental results: As shown in Figure 3 , compounds α-KG and A-1 promote the expression of smooth muscle contraction marker pMYL-9 in vascular smooth muscle cells.

[0030] Example 4: Improvement effect of compounds α-KG and A-1 on mouse acne rosacea: Experimental method: α-KG and A-1 (80 μmol) or solvent (50% glycerol, 25% ethanol, 20% octyldodecanol and 5% water) were applied to the back of 7-week-old female Balb / c mice once a day for 10 days. After 3 days of administration, the mouse acne rosacea model was established by intradermal injection of PBS-prepared LL37 polypeptide (50 μL, 640 μM) or the same amount of solvent to the mouse back. Each injection was 12 hours apart, and a total of 4 times. 12 hours after the end of treatment, the skin erythema formed at the injection site of the mouse was observed and photographed. And after that, continue to give medicine and take pictures every other day until the erythema subsides. According to the color of the erythema, the severity score is 1 for the lightest color and 5 for the reddest color; according to the photos taken by the body microscope, the size of the erythema area is counted using Image J.

[0031] Experimental results: As shown in Figure 4 , the application of α-KG and A-1 significantly improved the acne rosacea-like dermatitis of female Balb / c mice and reduced the severity and area of erythema. During the treatment period, the application of α-KG and A-1 significantly accelerated the recovery progress of acne rosacea-like dermatitis in female Balb / c mice.

[0032] Example 5: Effect of compounds α-KG and A-1 on mouse vasoconstriction: Experimental method: Take the mouse back lesions, immerse in 10% neutral formalin for fixation, dehydration, paraffin embedding, and then prepare 3-5 μm paraffin sections. After dewaxing with xylene, rehydrating with gradient alcohol, and microwave antigen repair, the paraffin sections were washed with PBS and blocked with 10% goat serum (NGS, prepared with PBS) for 1 h. Then incubate with CD31 (marker of vascular endothelial cells) antibody overnight, incubate secondary antibody according to the instructions and develop with DAB, and then dehydrate and mount after hematoxylin restain the nucleus. Use a fluorescence microscope to take photos of the high-power field of view, and use ImageJ software to count the vascular circumference of the mouse lesion area.

[0033] Experimental results: As shown inFigure 5 As shown, the application of compound α-KG and A-1 improved the vascular dilation of the mice with rosacea-like dermatitis.

[0034] Example 61, 1-dioxo-2,3-dihydro-3-thienylcarbamic acid methyl ester A-1 was prepared: Into a 100 mL three-necked flask, compound 5 (1 equiv.) was dissolved in 20 mL anhydrous DCM, and then methyl chloroformate (2 equiv.), triethylamine (3 equiv.) were added successively under ice bath. The reaction system was sealed with nitrogen and stirred from 0 °C to room temperature for 12 h. After the reaction was completed, it was rotary evaporated and purified by silica gel column chromatography to obtain compound A-1, Figure 6 H NMR, 1 H NMR, Figure 7 H NMR, 13 C NMR.

Claims

1. A compound characterized by its chemical properties. Structures as shown in Formula α-KG, Formula ITA or Formula A-1: ， ， 。 2. The compound of claim 1, wherein a pharmaceutically acceptable salt, hydrate, solvate or prodrug of the compound.

3. The compound of claim 1 for use as an agonist of GPR99.

4. The use of the compound of claim 1 in the preparation of a medicament for the prevention and / or treatment of rosacea disease.

5. The synthetic route of the compound of Formula A-1 as shown in claim 1, as follows: 。 6. The synthetic route of claim 5, wherein, Into a 100 mL three-necked flask, compound 5 (1 equiv.) was dissolved in 20 mL of anhydrous DCM, and then methyl chloroformate (2 equiv.) and triethylamine (3 equiv.) were added successively under ice bath. The reaction system was sealed with nitrogen and stirred from 0 °C to room temperature for 12 h. After the reaction was completed, the solvent was removed by rotary evaporation, and the product was purified by silica gel column chromatography to obtain compound A-1.

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