Artesunate prodrug, preparation method and application thereof

Abstract

This invention discloses an artesunate prodrug, its preparation method, and its application. The artesunate prodrug comprises amphiphilic polymer groups with different functions coupled to a 1,3,5-triazine-2,4,6-triamine or cyanuric chloride backbone molecule as shown in formula (I). The amphiphilic polymer groups are hydrophilic polyethylene glycol derivative groups containing terminal amino or carboxyl groups and artesunate groups. This artesunate prodrug can self-assemble into long-acting liposomes in water, showing potential for treating neutrophilic asthma.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to an artesunate prodrug, its preparation method, and its application. Background Technology

[0002] Asthma is a common chronic inflammatory respiratory disease characterized by variable airflow limitation, bronchial hyperresponsiveness, mucus hypersecretion, and airway inflammation, leading to airway narrowing and symptoms such as wheezing, shortness of breath, and chest tightness. Asthma is generally defined as a chronic inflammatory disease characterized by predominantly eosinophilic infiltration in the airways. However, increasing evidence suggests that neutrophils play a crucial role in severe asthma and glucocorticoid-resistant asthma. Approximately 50% of asthma cases are no longer associated with eosinophils but with neutrophil inflammation; this specific phenotype is known as neutrophilic asthma (NA). Neutrophilic asthma is often associated with severe asthma and poorly treated asthma. Although this group of asthma patients, who are insensitive to hormone therapy, accounts for only 5%–10% of all asthma cases, they constitute a significant medical expenditure burden for asthma prevention and treatment. Therefore, researchers both domestically and internationally have paid special attention to neutrophilic asthma, attempting to find methods to treat it.

[0003] The mainstream treatment for asthma is currently inhaled corticosteroids (ICS). Clinically, budesonide (an ICS) combined with indacaterol (IND) is often used to treat asthma, achieving both anti-inflammatory and long-term bronchodilatory effects. This is crucial for the prevention, control, and treatment of non-ICS resistant asthma. Although ICS is currently the most effective treatment for asthma, it can cause many side effects, such as glaucoma, cataracts, telangiectasia, osteoporosis, hyperglycemia, adrenal suppression, and local candidiasis. Numerous studies have shown that corticosteroids are not very effective for neutrophilic asthma. Although these drugs can effectively promote eosinophil apoptosis, they can lead to an increase in respiratory neutrophils. Furthermore, biotherapy using small molecule antibodies such as anti-IL-17R and anti-TNF-α can treat neutrophilic asthma by interfering with pro-inflammatory signaling pathways, but often results in immune non-response and severe toxic side effects. Therefore, finding new treatment strategies is of great significance for the clinical prevention and treatment of neutrophilic asthma, and it is also an important problem that urgently needs to be solved in the current field of neutrophilic asthma prevention and treatment.

[0004] Artesunate (ART) is an artemisinin derivative commonly used clinically to treat malaria. Studies have found that artesunate can reduce inflammation, fight cancer, alleviate fibrosis, promote repair of ischemia-reperfusion injury, and inhibit rabies virus. However, artesunate has a short half-life (t1 / 2) of approximately 30 minutes after intravenous injection, leading to frequent dosing and poor patient compliance. Furthermore, its poor water solubility results in low bioavailability, severely limiting its further clinical application.

[0005] There are currently no safe and effective drugs for treating neutrophilic asthma caused by neutrophil inflammation. New and effective treatment methods are still needed.

[0006] The challenge in addressing the above problems and shortcomings lies in modifying artesunate and applying it to the treatment of neutrophilic asthma.

[0007] The significance of solving the above problems and defects is that, after the artesunate is improved, its circulation time in vivo can be prolonged, its bioavailability can be improved, and it can be used to improve neutrophil asthma. Summary of the Invention

[0008] To address the current lack of effective treatments for neutrophil-induced asthma caused by neutrophil inflammation, this invention provides an artesunate prodrug, its preparation method, and its applications. This artesunate prodrug is a modified version of artesunate, which prolongs its in vivo circulation time, improves its bioavailability, and can be used to improve and treat neutrophil-induced asthma.

[0009] To achieve the objectives of this invention, the following implementation scheme is provided.

[0010] In one embodiment, the present invention provides an artesunate prodrug comprising amphiphilic polymer groups with different functions coupled to a 1,3,5-triazine-2,4,6-triamine or cyanuric chloride backbone molecule as shown in formula (I), wherein the amphiphilic polymer groups are hydrophilic polyethylene glycol derivative groups containing terminal amino or terminal carboxyl groups and artesunate groups.

[0011]

[0012] In the formula, R represents polyethylene glycol derivative groups containing terminal amino or terminal carboxyl groups and artesunate groups.

[0013] In some embodiments, in the artesunate prodrug of the present invention, at least one of R is a polyethylene glycol derivative, and at least one is an artesunate group, wherein the polyethylene glycol derivative group is: or , where n is 4-120.

[0014] The chemical structure of artesunate is as follows: .

[0015] Furthermore, in the artesunate prodrug of the present invention, the skeletal structure of formula (I) can also be other halogenated 1,3,5-triazines with nucleophilic substitution reactivity.

[0016] In some embodiments, the artesunate prodrug of the present invention is a compound represented by Formula II or a pharmaceutical salt thereof:

[0017] In the formula, R1 is an artesunate group, forming an amide bond. R2 is a polyethylene glycol derivative group, selected from... and , where the size of n is between 4 and 120.

[0018] In one specific embodiment, the artesunate prodrug of the present invention, the compound represented by Formula II or a salt thereof, is: , The value of n in the formula is between 4 and 120.

[0019] In another embodiment, the present invention also provides a method for preparing compounds of formulas (I) and (II), comprising: Under nitrogen protection, halogenated 1,3,5-triazine was prepared by stepwise nucleophilic substitution reactions with polyethylene glycol derivatives containing terminal amino or carboxyl groups and artesunate in an organic solvent containing a condensing agent and an acid-binding agent.

[0020] Preferably, in the method of the present invention described above, the halogenated 1,3,5-triazine is selected from 1,3,5-triazine-2,4,6-triamine, cyanuric chloride, and 2,4,6-tribromo-1,3,5-triazine, and the polyethylene glycol derivative containing terminal amino or terminal carboxyl groups has an average molecular weight between 200 Da and 5000 Da.

[0021] Preferably, in the method of the present invention described above, the condensing agent is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole, the acid-binding agent is selected from one or a combination of two of triethylamine, N,N-diisopropylethylamine, sodium carbonate and pyridine, and the organic solvent is selected from one or more of dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide and 1,4-dioxane.

[0022] In one specific embodiment, the preparation method of the artesunate prodrug of the present invention includes the following steps: under nitrogen protection, a halogenated 1,3,5-triazine reacts with a polyethylene glycol derivative containing a terminal amino or carboxyl group and artesunate in an organic solvent containing a condensing agent and an acid-binding agent in a stepwise nucleophilic substitution reaction. Each substitution reaction takes 48 hours, and the reaction temperature is -20 to 25°C. After the reaction is completed, the product is obtained by dialysis and lyophilization. This product is an amphiphilic polymer with a multi-active-site compound such as 1,3,5-triazine-2,4,6-triamine or cyanuric chloride as the backbone, coupled with a polyethylene glycol molecular chain and artesunate.

[0023] In the above method, the halogenated 1,3,5-triazine is selected from 1,3,5-triazine-2,4,6-triamine, cyanuric chloride, or 2,4,6-tribromo-1,3,5-triazine. The polyethylene glycol derivative containing terminal amino or terminal carboxyl groups has an average molecular weight between 200 Da and 5000 Da.

[0024] In the above method, the condensing agent is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole. The acid-binding agent is selected from one or a combination of two of triethylamine, N,N-diisopropylethylamine, sodium carbonate, and pyridine.

[0025] In the above method, the organic solvent is selected from one or more of dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, and 1,4-dioxane.

[0026] In the above method, the molar ratio of the halogenated 1,3,5-triazine to the polyethylene glycol derivative is 1:1, and the molar ratio of the halogenated 1,3,5-triazine to artesunate is between 1:2 and 1:3.

[0027] In yet another embodiment, the present invention provides the use of the above-mentioned artesunate prodrugs of formulas (I) and (II) in the preparation of a drug for treating neutrophil asthma.

[0028] The beneficial technical effects of this invention are as follows: (1) The artesunate prodrug of the present invention is prepared by stepwise nucleophilic substitution reaction. The synthesis method is simple, the structure and function are controllable, and it is easy to synthesize and prepare on a large scale.

[0029] (2) The artesunate prodrug of the present invention is synthesized by chemical bonding between halogenated 1,3,5-triazine and artesunate. It can only be hydrolyzed to release the active unit and exert its efficacy in a high-level reactive oxygen environment, thus avoiding the premature release of the active unit in the body.

[0030] (3) The artesunate prodrug of the present invention increases the water solubility of artesunate by linking it with a halogenated 1,3,5-triazine and a polyethylene glycol derivative.

[0031] (4) The artesunate prodrug of the present invention is an amphiphilic polymer with hydrophilic and hydrophobic ends. It can self-assemble in water to form nanomicelles with a particle size of about 100-200nm. It is easy to target and accumulate at the lesion site, prolong the circulation time in the body, achieve long-term effect, and improve the therapeutic effect.

[0032] (5) The artesunate prodrug of the present invention has good activity and effect on neutrophil asthma and has the potential to be developed into a drug for treating neutrophil asthma. Attached Figure Description

[0033] Figure 1 Infrared spectrum of PMART, the prodrug of artesunate; Figure 2 PMART, a prodrug of artesunate 1 H NMR spectrum; Figure 3 This is a particle size distribution diagram of PMART, the prodrug of artesunate; Figure 4 The zeta potential diagram of PMART, the prodrug of artesunate; Figure 5 Transmission electron microscopy image of PMART, the prodrug of artesunate; Figure 6 A graph showing serum IgE levels in mice with neutrophilic asthma treated with PMART, a prodrug of artesunate. Figure 7 The graph shows the expression levels of pro-inflammatory factors MPO, IL-1β, and TNF-α in the bronchoalveolar lavage fluid of mice treated with the artesunate prodrug PMART for neutrophilic asthma. Figure 8 H&E staining of lung tissue sections from mice with neutrophilic asthma treated with PMART, a prodrug of artesunate. Detailed Implementation

[0034] The following embodiments are provided to describe the present invention in more detail. However, these embodiments are provided only to help further understand the present invention and are not intended to limit the present invention. Those skilled in the art should understand that equivalent substitutions or corresponding improvements made to the content of the present invention still fall within the protection scope of the present invention. In the following embodiments, the English abbreviations and Chinese annotations of the main materials or drugs involved are as follows: Artesunate (ART): Artesunate; 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI): 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; 1-Hydroxybenzotriazole HOBt: 1-Hydroxybenzotriazole; Dimethyl sulfoxide (DMSO): Dimethyl sulfoxide; N,N-Dimethylformamide (DMF): N,N-dimethylformamide; Polyethylene glycol (PEG): Polyethylene glycol; Ovalbumin (OVA): Chicken egg albumin; Lipopolysaccharide (LPS): Lipopolysaccharide.

[0035] Example 1: Synthesis of artesunate prodrug (PMART) 1) Weigh 12.60 mg of 1,3,5-triazine-2,4,6-triamine (0.10 mmol) and dissolve it in 1 mL of DMSO / DMF (w / w=1:1) with stirring to form a 1,3,5-triazine-2,4,6-triamine solution.

[0036] 2) Weigh out 150mg mPEG 2000 -COOH (0.08 mmol) was dissolved in 1 mL of DMSO / DMF mixed solvent, and 47.90 mg EDCI (0.25 mmol), 33.75 mg HOBT (0.25 mmol), and 0.15 mmol triethylamine were added to prepare a PEG solution, which was then activated in an ice bath for 1 h.

[0037] 3) Under nitrogen protection and ice-salt bath conditions, the activated solution from step 2) was added dropwise to the solution from step 1), and the reaction was carried out for 48 hours. The reaction product was dialyzed at 1000 Da and then freeze-dried to obtain the freeze-dried product.

[0038] 4) Weigh 76.80 mg of artesunate ART (0.20 mmol) and dissolve it in 1 ml of DMSO / DMF. Stir, add 23.96 mg of EDCI (0.125 mmol), 16.38 mg of HOBT (0.125 mmol), and 0.15 mmol of triethylamine, mix and dissolve to form an artesunate solution, and activate in an ice bath for 1 h. 5) Dissolve the lyophilized product obtained in step 3) in 1 ml of DMSO / DMF and mix well. Add artesunate solution from step 4) dropwise and react for 48 h. After dialysis at 2000 Da, freeze dry the reaction solution and collect the lyophilized product, which is the target product PMART.

[0039] Through FT-IR infrared and 1 Its structure was determined by H NMR nuclear magnetic resonance. The results are as follows: Figure 1 and Figure 2 As shown. Infrared ( Figure 1 ) and nuclear magnetic resonance (NMR) Figure 2 Analysis confirmed that the synthesized PMART is a product of coupling one 1,3,5-triazine-2,4,6-triamine backbone with one methoxy polyethylene glycol and two ART molecules. The chemical structure of PMART is as follows:

[0040] PMART Example 2: Preparation and characterization of PMART micelles PMART was dissolved in water and sonicated. 1 mg of PMART was added to 1 mL of deionized water and sonicated for 20 min (40 Hz). The particle size and potential of the PMART micelles were measured using a Malvern particle size analyzer.

[0041] The morphology of PMART micelles was observed using transmission electron microscopy (TEM). Specific procedure: 10 μL of micelle solution was pipetted onto a copper grid, allowed to stand overnight to air dry, and then imaged using a TEM.

[0042] The results showed that the PMART micelle size distribution was relatively narrow, with an average particle size of approximately 119 ± 2.6 nm (e.g., Figure 3 The average potential is -28.4 ± 1.2 mV (e.g. Figure 4 Electron microscopy reveals that the micelles are quasi-spherical (e.g.) Figure 5 ).

[0043] Example 3: Application of artesunate prodrug in the treatment of neutrophilic asthma A mouse model of neutrophilic asthma was established and the therapeutic effect of artesunate prodrug was tested.

[0044] The specific steps are as follows: Mice were randomly divided into a normal control group, a model group, a 10 mg / kg ART group, a 1 mg / kg PMART group, and a 10 mg / kg PMART group (calculated based on ART dose). Specifically, mice were sensitized with OVA and LPS on days 0 and 7. From days 14 to 18, all mice were nebulized with 1% OVA for 1 hour, while the control group was treated with PBS. One hour before OVA stimulation on days 14 to 18, mice were injected with either PBS solution or drug solution via the tail vein.

[0045] On day 19, peripheral blood was collected from mice and left at room temperature for 2 hours. The mice were then centrifuged at 3000 rpm and 4°C for 10 minutes. The supernatant was carefully aspirated and the IgE level was measured according to the ELISA kit instructions.

[0046] After blood collection, the mice were euthanized, and 3×1mL of PBS solution was injected into their lungs to collect bronchoalveolar lavage fluid (BALF).

[0047] The specific procedure is as follows: Carefully open the mouse's chest cavity to fully expose the lungs. Cut open the neck skin and bluntly dissect the mouse's trachea. After making a small incision in the trachea, insert the syringe needle about 0.3 cm into the trachea and secure it with a knotted thread to prevent it from falling off. Attach the syringe to the needle and slowly inject 1 mL of pre-cooled PBS to irrigate the lungs three times, holding each in place for 10 seconds before slowly aspirating. Place the extracted bronchoalveolar lavage fluid on ice for later use.

[0048] Take 1 mL of BALF, centrifuge at 16000 g for 10 min at 4℃, and collect the supernatant. Measure the levels of MPO, IL-1β, and TNF-α according to the ELISA kit instructions.

[0049] One lung tissue sample was taken from each group, washed thoroughly with PBS, fixed in 4% paraformaldehyde, embedded, and sectioned. Hematoxylin-eosin (H&E) staining was then performed.

[0050] The experimental results show that serum IgE (such as...) Figure 6 The level of [a specific substance] significantly increased in the model group and significantly decreased after drug intervention. According to [the relevant data]... Figure 7 It was observed that the expression levels of MPO, IL-1β, and TNF-α in BALF were significantly increased in the model group, while drug intervention could significantly reduce the expression levels of these classic pro-inflammatory factors. Additionally, H&E staining of lung tissue (such as...) Figure 8 As can be seen, drug intervention can significantly improve inflammatory cell infiltration and tracheal wall thickening in the model group. Furthermore, in the above results, PMART was more effective than free ART.

Claims

1. An artesunate prodrug comprising amphiphilic polymer groups with different functions coupled to a 1,3,5-triazine-2,4,6-triamine or cyanuric chloride backbone molecule as shown in formula (I), wherein the amphiphilic polymer groups are hydrophilic polyethylene glycol derivative groups containing terminal amino or terminal carboxyl groups and artesunate groups. In the formula, R represents a polyethylene glycol derivative group containing a terminal amino or a terminal carboxyl group and an artesunate group.

2. The artesunate prodrug as described in claim 1, wherein at least one of R is a polyethylene glycol derivative, and at least one is an artesunate group, wherein the polyethylene glycol derivative group is... or ,in, n is an integer between 4 and 120.

3. The artesunate prodrug as described in claim 1, wherein the artesunate is: 。 4. The artesunate prodrug as described in claim 1, wherein the skeletal structure of Formula I may also be another nucleophilically substituted reactive halogenated 1,3,5-triazine.

5. The artesunate prodrug as described in claim 1 is a compound of formula II or a pharmaceutically acceptable salt thereof. In the formula, R1 is an artesunate group, forming an amide bond. R2 is a polyethylene glycol derivative group, selected from... and ,in, The size of n is between 4 and 120.

6. The artesunate prodrug as described in claim 5, wherein the compound represented by formula (II) or a salt thereof is: ， In the formula, the value of n is between 4 and 120.

7. A method for preparing the artesunate prodrug according to claims 1-6, comprising: Under nitrogen protection, 1,3,5-halogenated triazine was prepared by reacting it with polyethylene glycol derivatives containing terminal amino or carboxyl groups, and by artesunate by stepwise nucleophilic substitution in an organic solvent containing a condensing agent and an acid-binding agent.

8. The method of claim 7, wherein the halogenated 1,3,5-triazine is selected from 1,3,5-triazine-2,4,6-triamine, cyanuric chloride, and 2,4,6-tribromo-1,3,5-triazine, and the polyethylene glycol derivative containing terminal amino or terminal carboxyl groups has an average molecular weight between 200 Da and 5000 Da.

9. The method of claim 7, wherein the condensing agent is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole, the acid-binding agent is selected from one or a combination of two of triethylamine, N,N-diisopropylethylamine, sodium carbonate and pyridine, and the organic solvent is selected from one or more of dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide and 1,4-dioxane.

10. The use of the artesunate prodrug according to any one of claims 1-6 in the preparation of a drug for treating neutrophilic asthma.

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