A compound, a method of preparation and use as an agonist of the orphan receptor GPR99

Abstract

The application belongs to the technical field of drug research and development, and particularly relates to a compound, a preparation method and application of the compound as an agonist of orphan receptor GPR99. The application first discovers and identifies a small-molecule compound capable of specifically activating the orphan receptor GPR99, and systematically clarifies the mechanism of the small-molecule compound in the regulation of vascular smooth muscle cell (VSMC) contraction and the 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 a drug research and development direction for precise treatment of diseases related to abnormal vascular function.

Description

Technical Field

[0001] This invention belongs to the field of drug development technology, specifically relating to a compound, its preparation method, and its application as an orphan receptor GPR99 agonist. Background Technology

[0002] Rosacea is a common chronic inflammatory skin disease that primarily affects the central facial area. Its clinical features include persistent facial erythema, telangiectasia, inflammatory papules, and pustules. The disease is characterized by recurrent flare-ups and long-term progression, not only affecting the patient's appearance but also causing burning and stinging sensations, significantly impacting their mental health and quality of life. Epidemiological surveys indicate that the prevalence of rosacea in the global population is approximately 5.5%, with a higher incidence in lighter-skinned individuals, posing a significant public health burden.

[0003] Current research indicates that the pathogenesis of rosacea is complex, involving multiple aspects such as abnormal innate immune responses, impaired skin barrier function, neurovascular regulatory disorders, and microbiome imbalance. Among these, vascular dysfunction is considered one of the core factors leading to persistent erythema and vasodilation. In particular, dysregulation of the contractile function of vascular smooth muscle cells (VSMCs) can trigger pathological vasodilation, resulting in increased facial blood flow and exacerbation of erythema. Furthermore, abnormal vasomotor function may further aggravate the chronic inflammatory response in local tissues through the continuous release of inflammatory factors.

[0004] Currently, commonly used clinical drug treatments include topical alpha-adrenergic receptor agonists (such as brimonidine and oxymetazoline) to constrict blood vessels and improve erythema symptoms. However, these drugs generally suffer from problems such as rapid onset but short duration of action, easy symptom rebound, tolerance and local irritation with long-term use, making it difficult to achieve long-term stable efficacy. In addition, some patients do not respond well to existing drugs or have contraindications, further limiting their application. Therefore, there is an urgent need to develop novel treatment strategies that target vascular function regulation to achieve more lasting and safer clinical benefits. Summary of the Invention

[0005] Through clinical metabolomics analysis, this invention revealed that α-ketoglutarate (α-KG), as an endogenous GPR99 ligand, abnormally accumulates in rosacea patients. It enhances vascular smooth muscle contraction and inhibits pathological vasodilation by activating the GPR99-Gq-MLCK-MYL9 signaling axis. Furthermore, by activating its specific receptor GPR99, α-KG and another endogenous GPR99 agonist, itaconic acid (ITA), abnormal vasodilation can be prevented, thereby alleviating the erythematous phenotype of rosacea. To further understand the signal transduction and structural basis of the therapeutic effects of α-KG and ITA, we investigated the structure and downstream pathways of GPR99. Based on the "dual-acid-binding pocket" structural feature of GPR99 resolved by cryo-electron microscopy, we developed the agonist A-1, which significantly activates GPR99 signaling at nanomolar concentrations, promoting MYL9 phosphorylation-dependent vasoconstriction. Experimental data showed that topical application of A-1 reduced erythema area score by up to 60%, decreased inflammatory cell infiltration, and exhibited no cross-activity with its homologous receptor GPR91. Compared to the natural ligand α-KG, A-1 has a significantly longer half-life, and can achieve sustained improvement in vascular function at a dose of 80 μM.

[0006] These compounds offer an innovative treatment strategy for rosacea and related vascular disorders by precisely regulating GPR99-mediated vascular homeostasis. Their high efficacy not only overcomes the limitations of traditional therapies but also provides a structural biology basis for the design of GPCR-targeted local drugs, holding significant potential for clinical application.

[0007] In a first aspect, the present invention provides a compound having a chemical structure as shown in formula α-KG, formula ITA, or formula A-1: , , .

[0008] Furthermore, the compound may be a pharmaceutically acceptable salt, hydrate, solvate, or prodrug.

[0009] In another aspect, the present invention provides the use of said compound as an agonist of GPR99.

[0010] In another aspect, the present invention provides the use of the compound in the preparation of medicaments for the prevention and / or treatment of rosacea.

[0011] In another aspect, the present invention provides a synthetic route for said compound A-1, as follows: .

[0012] Further, the synthesis method of compound A-1 was as follows: Compound 5 (1 equiv.) was added to a 100 mL three-necked flask and dissolved in 20 mL of anhydrous DCM. Methyl chloroformate (2 equiv.) and triethylamine (3 equiv.) were added sequentially under ice bath conditions. The reaction system was sealed with nitrogen and stirred for 12 h from 0 °C to room temperature. After the reaction was complete, the mixture was evaporated to dryness and purified by silica gel column chromatography to obtain compound A-1.

[0013] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention is the first to discover and identify small molecule compounds that can specifically activate the orphan receptor GPR99, and systematically elucidates their mechanism of action in regulating vascular smooth muscle cell (VSMC) contraction and improving pathological vasodilation. This discovery not only fills the gap in the treatment of rosacea with GPR99 agonists, but also provides new molecular targets and drug development directions for the precision treatment of diseases related to vascular dysfunction.

[0014] 2. Compared with existing vasoconstrictor drugs such as α-adrenergic receptor agonists, the GPR99 small molecule agonist of this invention has the advantages of long-lasting effect, low side effects, and low tolerance and rebound. Furthermore, this invention provides a synthetic route suitable for industrial production, with a simple process, high yield, and readily available raw materials, facilitating large-scale preparation and subsequent clinical application. Attached Figure Description

[0015] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0016] Figure 1 The effect of compounds on the activity of GPR99.

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

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

[0019] Figure 4 The effects of compounds α-KG and A-1 on rosacea in mice. A. Schematic diagram of mouse treatment protocol: Mice were treated with α-KG or A-1 gel for 10 days (20 μL, once daily), and LL37 was injected as shown in the diagram. Skin lesions were continuously observed. Each group had n=6 mice. B. Representative images of the dorsal skin of mice in the solvent, A-1, and α-KG groups (n=6 mice per group). Images were taken at specified time points. C. Severity of rosacea-like phenotype in each group was assessed based on erythema area (n=6). D. Severity of rosacea-like phenotype in each group was assessed based on erythema score (n=6).

[0020] Figure 5 Compounds α-KG and A-1 improved vasodilation in mice with rosacea-like dermatitis. A. Representative images of CD31 immunohistochemical staining of skin lesions from each group of mice. Scale bar = 100 μm. B. Calculation of vascular circumference in A (78-103 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 Implementation

[0023] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0024] Effect of Compound 1 on GPR99 Activity: Experimental Methods: The applicant used IP-One assays to detect the activation of GPR99 downstream Gq protein under different agonist stimuli. HEK293 cells were transiently transfected with the GPR99 receptor plasmid. 24 hours after transfection, cells were seeded at 1 × 10⁶ cells per well. 4Cells were seeded at a density of 1,000 cells per well in 384-well microplates and incubated at 37 °C for 24 hours. Inositol 1-phosphate (IP1) levels were determined according to the manufacturer's instructions using the IP-One assay kit (Cisbio Bioassays, #62IPAPEB, Bedford, MA). Subsequently, at 37 °C, cells were analyzed using assays containing different concentrations of α-ketoglutarate (α-KG), itaconic acid (ITA), or A-1 (final concentration 10). -10 Up to 10 -3 Cells were stimulated for 20 minutes with HEM-PSS buffer (M). After stimulation, cells were washed twice with HBSS buffer containing Ca²⁺ and Mg²⁺. Then, 7 μl of washed cells were transferred to 384-well plates, and 3 μl of D2-labeled IP1 and 3 μl of latently fluorescently labeled anti-IP1 monoclonal antibody were added to each well. After incubation at room temperature for 1 hour, fluorescence resonance energy transfer (FRET) signals were detected using a Berthold Tristar 5 detector at an excitation wavelength of 330 nm and emission wavelengths of 620 nm and 665 nm. The FRET signal was calculated as the emission ratio (665 nm / 620 nm).

[0025] Experimental results: such as Figure 1 As shown, further activity tests on α-KG, ITA, and the screened small molecule A-1 confirmed that the three small molecules, including α-KG, ITA, and A-1, exhibited strong Gq agonistic effects on GPR99. These results suggest that we can develop preferred ligands for constitutively activated orphan GPCRs to selectively regulate their function.

[0026] Example 2: Compounds α-KG and A-1 promote vascular smooth muscle cell contraction. Experimental Methods: Primary mouse aortic vascular smooth muscle cells were isolated, digested, resuspended, and counted. Equal volumes of cells were mixed with type I collagenase and sodium hydroxide solution, seeded in 24-well plates, and incubated for 20 min in a constant temperature incubator. The gel was carefully separated from the plate walls, and complete culture medium containing 200 nmol A-1 or its carrier solvent (DMSO) was added. After culturing in a constant temperature incubator for 12 hours, the area change of the gel in each well was observed, photographs were taken, and ImageJ was used to analyze the gel area of ​​each group.

[0027] Experimental results: such as Figure 2 As shown, the application of compounds α-KG and A-1 significantly reduced the gel area and promoted the contraction of vascular smooth muscle cells.

[0028] Example 3: Compounds α-KG and A-1 promote the expression of the smooth muscle contraction marker pMYL-9 in vascular smooth muscle cells. Experimental methods: Primary mouse aortic vascular smooth muscle cells were isolated and treated with 200 nmol A-1 or its carrier solvent (DMSO) for 15 min. Cells from each group were collected to extract proteins, and the level of smooth muscle contraction marker pMYL-9 was detected by Western blotting.

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

[0030] Example 4: The ameliorative effect of compounds α-KG and A-1 on rosacea in mice: Experimental Methods: Seven-week-old female Balb / c mice were treated with α-KG and A-1 (80 μmol) or a solvent (50% glycerol, 25% ethanol, 20% octyldodecanol, and 5% water) on their backs once daily for 10 days. Three days after administration, a mouse rosacea-like model was established by intradermal injection of 50 μL (640 μM) of PBS-prepared LL37 peptide or an equivalent amount of solvent into the treated area on the back. Injections were given 12 hours apart, for a total of four injections. Twelve hours after treatment, the skin erythema at the injection sites was observed and photographed. Daily administration and photographs were taken every other day thereafter until the erythema subsided. The severity of the erythema was scored based on its color intensity, with 1 point for the lightest color and 5 points for the reddest. The area of ​​the erythema was statistically analyzed using ImageJ based on photographs taken under a stereomicroscope.

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

[0032] Example 5: Effects of compounds α-KG and A-1 on vasoconstriction in mice: Experimental Methods: Skin lesions on the back of mice were fixed, dehydrated, and embedded in 10% neutral formalin, followed by paraffin embedding. Paraffin sections of 3-5 μm were then prepared. The paraffin sections were dewaxed with xylene, rehydrated with graded alcohols, and retrieval using microwave antigen retrieval. After washing with PBS, the sections were blocked with 10% goat serum (NGS, prepared with PBS) for 1 hour. They were then incubated overnight with CD31 (a marker of vascular endothelial cells) antibody, followed by secondary antibody incubation and DAB staining according to the manufacturer's instructions. After hematoxylin counterstaining, the sections were dehydrated and mounted. Bright-field high-power images were taken using a fluorescence microscope, and the circumference of blood vessels in the mouse skin lesion area was calculated using ImageJ software.

[0033] Experimental results: such as Figure 5 As shown, the application of compounds α-KG and A-1 improved vasodilation in mice with rosacea-like dermatitis.

[0034] Example 61: Preparation of methyl 1,1-dioxane-2,3-dihydro-3-thienylcarbamate A-1: In a 100 mL three-necked flask, compound 5 (1 equiv.) was added and dissolved in 20 mL of anhydrous DCM. Methyl chloroformate (2 equiv.) and triethylamine (3 equiv.) were added sequentially under ice bath conditions. The reaction system was sealed with nitrogen and stirred for 12 h from 0 °C to room temperature. After the reaction was complete, the mixture was evaporated to dryness and purified by silica gel column chromatography to obtain compound A-1. Figure 6 For compound A-1 1 H NMR, Figure 7 For compound A-1 13 C NMR.

Claims

1. A compound characterized by its chemical properties. The structure is shown in equation A-1: 。 2. The use of the compound of claim 1 in the preparation of a drug as an agonist of GPR99.

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

4. The synthetic route for the compound of formula A-1 as described in claim 1 is as follows: 。 5. The synthetic route as described in claim 4, characterized in that, In a 100 mL three-necked flask, add 1 equivalent of compound 5 and 20 mL of anhydrous DCM to dissolve it. Under ice bath conditions, add 2 equivalents of methyl chloroformate and 3 equivalents of triethylamine in sequence. Seal the reaction system with nitrogen and stir from 0 °C to room temperature for 12 h. After the reaction is complete, evaporate to dryness and purify by silica gel column chromatography to obtain compound A-1.

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