An allosteric activator of alkaline phosphatase and methods of making and using the same

Abstract

This invention discloses an allosteric activator of alkaline phosphatase, its preparation method, and its applications. Specifically, it discloses a small chemical molecule and / or its derivatives that bind to alkaline phosphatase, their preparation method, and their applications, belonging to the field of biotechnology. The small chemical molecule and / or its derivatives include one or more compounds with activating activity, such as S2, S3, AP14, AP14-S, AP10, and AP16. The small chemical molecule and / or its derivatives of this invention have a strong binding ability to alkaline phosphatase and can promote the catalytic activity of strong alkaline phosphatase and its mutant proteins. This invention has broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an allosteric activator of alkaline phosphatase, its preparation method and application, specifically to a small chemical molecule that binds to alkaline phosphatase and / or its derivatives, its preparation method and application. Background Technology

[0002] Alkaline phosphatase consists of 524 amino acids. Its N-terminal amino acids 1-17 form the signal peptide sequence, while the C-terminal amino acids 502-524 are cleaved during protein maturation. After proper folding and assembly in the endoplasmic reticulum, the protein forms a functional homodimer. Its structure includes five key domains: a calcium-binding site, a coronal region, a dimer-binding domain, an N-terminal helical domain, and an active catalytic domain. The protein is anchored to the cell membrane via a C-terminal glycosylphosphatidylinositol (GPI), orienting its active site towards the extracellular space.

[0003] Functionally, alkaline phosphatase hydrolyzes inorganic pyrophosphate (PPi) to phosphate, participating in the regulation of hydroxyapatite crystal formation and thus promoting osteoblast mineralization. In patients with hypophosphatase syndrome (HPP), PPi accumulates due to reduced enzyme activity. This excess PPi, along with calcium ions, enters cells via the Ank transmembrane transporter, activating the Ras / MAPK / ERK1 / 2 signaling pathway and inhibiting the Smad signaling pathway. This ultimately leads to upregulation of osteopontin (OPN) expression and downregulation of Runx2 gene expression, hindering hydroxyapatite formation and causing bone mineralization disorders, clinically manifesting as rickets in children or osteomalacia in adults. Furthermore, this enzyme also participates in the hydrolysis of pyridoxal phosphate (PLP) and ethanolamine phosphate (PEA). PLP, as the active form of vitamin B6, is hydrolyzed by alkaline phosphatase to generate pyridoxal, which plays an important role in transamination and neurotransmitter metabolism. Therefore, insufficient enzyme activity in infancy may induce seizures.

[0004] Given the aforementioned mechanisms, enhancing alkaline phosphatase activity has become a key strategy for treating related diseases, including HPP, fractures, osteoporosis, and spinal fusion. Small molecule agonists possess characteristics such as high penetration, low cost, modifiability, and precise targeting, making them particularly suitable for drug therapies requiring "clear targets, long-term use, and local regulation." Their complementarity with macromolecular drugs (antibodies, proteins) not only expands the boundaries of targeted therapy but also provides more flexible and efficient treatment options for complex diseases, making them indispensable tools and therapies in current drug development and basic research. Therefore, developing small molecule agonists capable of specifically activating alkaline phosphatase will provide a new research direction for the treatment of related diseases. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to provide an allosteric activator of alkaline phosphatase, its preparation method, and its application. Specifically, it provides a small chemical molecule that binds to alkaline phosphatase and / or its derivatives, its preparation method, and its application.

[0006] The innovation of this invention lies in the fact that the concept of activating small molecules is proposed for the first time. While there are many small molecules with affinity, those that can both bind to and activate are unknown. Currently, no theoretical mechanism for this activation has been proposed; therefore, this invention is truly innovative.

[0007] This invention provides a small chemical molecule and / or its derivatives, wherein the small chemical molecule and / or its derivatives include, but are not limited to, one or more of the compounds shown below:

[0008] .

[0009] Among them, S2, S3, AP14, AP14-S, AP10, and AP16 are compounds with activating activity.

[0010] This invention also provides a method for preparing S2 as described above, the method comprising: reacting Oridonin and ak2 (4-acetylenic benzoic acid) in a solvent in the presence of EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and DMAP (4-dimethylaminopyridine) to obtain S2. The reaction process of the preparation method is shown in reaction formula (I):

[0011]

[0012] Reaction formula (I)

[0013] In the method, the solvent includes one or more of dichloromethane, chloroform, dichloroethane, toluene, ethyl acetate, and dimethyl sulfoxide; preferably, it is dichloromethane.

[0014] In the method, the molar ratio of Oridonin, ak2, solvent, EDCI, and DMAP is 1 mol: (0.5-5) mol: (1-20) ml: (1-20) mol: (1-5) mol; preferably, it is 1 mol: 1.5 mol: 10 ml: 2 mol: 1.2 mol.

[0015] In the method, the reaction temperature is 5-150 degrees Celsius; preferably, it is room temperature.

[0016] In the method, the reaction time is 0.5-24 hours; preferably, it is 3 hours.

[0017] In the method, the reaction is carried out under one or more of argon, nitrogen, and helium, preferably under argon protection.

[0018] This invention also provides a method for preparing S3 as described above, the method comprising: reacting Oridonin and ak3 (3-ethynylbenzoic acid) in a solvent in the presence of EDCI and DMAP to obtain S3. The reaction process of the preparation method is shown in reaction formula (II):

[0019]

[0020] Reaction (II)

[0021] In the method, the solvent includes one or more of dichloromethane, chloroform, dichloroethane, toluene, ethyl acetate, and dimethyl sulfoxide; preferably, it is dichloromethane.

[0022] In the method, the molar ratio of Oridonin, ak3, solvent, EDCI, and DMAP is 1 mol: (0.5-5) mol: (1-20) ml: (1-20) mol: (1-5) mol; preferably, it is 1 mol: 1.5 mol: 10 ml: 2 mol: 1.2 mol.

[0023] In the method, the reaction temperature is 5-150 degrees Celsius; preferably, it is room temperature.

[0024] In the method, the reaction time is 0.5-24 hours; preferably, it is 3 hours.

[0025] In the method, the reaction is carried out under one or more of argon, nitrogen, and helium, preferably under argon protection.

[0026] This invention also provides a method for preparing Al as described above, the method comprising: reacting Se-16 and ak2 in a solvent in the presence of AgNO3 and NH3·H2O to obtain Al. The reaction process of the preparation method is shown in reaction formula (III):

[0027]

[0028] Reaction (III)

[0029] In the method, the solvent includes one or more of dichloromethane, chloroform, dichloroethane, toluene, ethyl acetate, and dimethyl sulfoxide; preferably, it is dichloromethane.

[0030] In the method, the molar ratio of Se-16, ak2, solvent, AgNO3, and NH3·H2O is 1 mol: (0.5-5) mol: (1-20) ml: (0.001-1) mol: (0.5-5) mol; preferably, it is 1 mol: 1.2 mol: 10 ml: 0.05 mol: 1.5 mol.

[0031] In the method, the reaction temperature is 5-150℃; preferably, it is room temperature.

[0032] In the method, the reaction time is 0.5-24 hours; preferably, it is 2 hours.

[0033] In the method, the reaction is carried out under one or more of argon, nitrogen, and helium, preferably under argon protection.

[0034] This invention also provides a method for preparing A2 as described above, the method comprising: reacting Se-5 and ak3 in a solvent in the presence of AgNO3 and NH3·H2O to obtain A2. The reaction process of the preparation method is shown in reaction formula (IV):

[0035]

[0036] Reaction (IV)

[0037] In the method, the solvent includes one or more of dichloromethane, chloroform, dichloroethane, toluene, ethyl acetate, and dimethyl sulfoxide; preferably, it is dichloromethane.

[0038] In the method, the molar ratio of Se-5, ak3, solvent, AgNO3, and NH3·H2O is 1 mol: (0.5-5) mol: (1-20) ml: (0.001-1) mol: (0.5-5) mol; preferably, it is 1 mol: 1.2 mol: 10 ml: 0.05 mol: 1.5 mol.

[0039] In the method, the reaction temperature is 5-150℃; preferably, it is room temperature.

[0040] In the method, the reaction time is 0.5-24 hours; preferably, it is 2 hours.

[0041] In the method, the reaction is carried out under one or more of argon, nitrogen, and helium, preferably under argon protection.

[0042] This invention also provides a method for preparing A3 as described above, the method comprising: reacting Se-16 and ak3 in a solvent in the presence of AgNO3 and NH3·H2O to obtain A3. The reaction process of the preparation method is shown in reaction formula (V):

[0043]

[0044] Reaction (V)

[0045] In the method, the solvent includes one or more of dichloromethane, chloroform, dichloroethane, toluene, ethyl acetate, and dimethyl sulfoxide; preferably, it is dichloromethane.

[0046] In the method, the molar ratio of Se-16, ak3, solvent, AgNO3, and NH3·H2O is 1 mol: (0.5-5) mol: (1-20) ml: (0.001-1) mol: (0.5-5) mol; preferably, it is 1 mol: 1.2 mol: 10 ml: 0.05 mol: 1.5 mol.

[0047] In the method, the reaction temperature is 5-150 degrees Celsius; preferably, it is room temperature.

[0048] In the method, the reaction time is 0.5-24 hours; preferably, it is 2 hours.

[0049] In the method, the reaction is carried out under one or more of argon, nitrogen, and helium, preferably under argon protection.

[0050] This invention also provides a method for preparing AP10 as described above, the method comprising: reacting Se-16 and S2 in a solvent in the presence of AgNO3 and NH3·H2O to prepare AP10. The reaction process of the preparation method is shown in reaction formula (VI):

[0051]

[0052] Reaction (VI)

[0053] In the method, the solvent includes one or more of dichloromethane, chloroform, dichloroethane, toluene, ethyl acetate, and dimethyl sulfoxide; preferably, it is dichloromethane.

[0054] In the method, the molar ratio of Se-16, S2, solvent, AgNO3, and NH3·H2O is 1 mol: (0.5-5) mol: (1-20) ml: (0.001-1) mol: (0.5-5) mol; preferably, it is 1 mol: 1.2 mol: 10 ml: 0.05 mol: 1.5 mol.

[0055] In the method, the reaction temperature is 5-150 degrees Celsius; preferably, it is room temperature.

[0056] In the method, the reaction time is 0.5-24 hours; preferably, it is 2 hours.

[0057] In the method, the reaction is carried out under one or more of argon, nitrogen, and helium, preferably under argon protection.

[0058] This invention also provides a method for preparing AP14 as described above, the method comprising: reacting Se-5 and S3 in a solvent in the presence of AgNO3 and NH3·H2O to obtain AP14. The reaction process of the preparation method is shown in reaction formula (VII):

[0059]

[0060] Reaction formula (VII)

[0061] In the method, the solvent includes one or more of dichloromethane, chloroform, dichloroethane, toluene, ethyl acetate, and dimethyl sulfoxide; preferably, it is dichloromethane.

[0062] In the method, the molar ratio of Se-5, S3, solvent, AgNO3, and NH3·H2O is 1 mol: (0.5-5) mol: (1-20) ml: (0.001-1) mol: (0.5-5) mol; preferably, it is 1 mol: 1.2 mol: 10 ml: 0.05 mol: 1.5 mol.

[0063] In the method, the reaction temperature is 5-150 degrees Celsius; preferably, it is room temperature.

[0064] In the method, the reaction time is 0.5-24 hours; preferably, it is 2 hours.

[0065] In the method, the reaction is carried out under one or more of argon, nitrogen, and helium, preferably under argon protection.

[0066] This invention also provides a method for preparing AP16 as described above, the method comprising: reacting Se-16 and S3 in a solvent in the presence of AgNO3 and NH3·H2O to prepare AP16. The reaction process of the preparation method is shown in reaction formula (VIII):

[0067]

[0068] Reaction formula (VIII)

[0069] In the method, the solvent includes one or more of dichloromethane, chloroform, dichloroethane, toluene, ethyl acetate, and dimethyl sulfoxide; preferably, it is dichloromethane.

[0070] In the method, the molar ratio of Se-16, S3, solvent, AgNO3, and NH3·H2O is 1 mol: (0.5-5) mol: (1-20) ml: (0.001-1) mol: (0.5-5) mol; preferably, it is 1 mol: 1.2 mol: 10 ml: 0.05 mol: 1.5 mol.

[0071] In the method, the reaction temperature is 5-80 degrees Celsius; preferably, it is room temperature.

[0072] In the method, the reaction time is 0.5-24 hours; preferably, it is 2 hours.

[0073] In the method, the reaction is carried out under one or more of argon, nitrogen, and helium, preferably under argon protection.

[0074] This invention also provides a method for preparing AP14-S as described above, the method comprising: reacting S-5 and S3 in a solvent in the presence of AgNO3 and NH3·H2O to obtain AP14-S. The reaction process of the preparation method is shown in reaction formula (IX):

[0075]

[0076] Reaction formula (IX)

[0077] In the method, the solvent includes one or more of dichloromethane, chloroform, dichloroethane, toluene, ethyl acetate, and dimethyl sulfoxide (DMSO); preferably, it is dimethyl sulfoxide.

[0078] In the method, the molar ratio of S3, S-5, solvent, AgNO3, and NH3·H2O is 1 mol: (0.5-5) mol: (1-20) ml: (0.001-1) mol: (0.5-5) mol; preferably, it is 1 mol: 1.2 mol: 10 ml: 0.05 mol: 1.5 mol.

[0079] In the method, the reaction temperature is 20-100 degrees Celsius; preferably, it is 70 degrees Celsius.

[0080] In the method, the reaction time is 0.5-24 hours; preferably, it is 2 hours.

[0081] In the method, the reaction is carried out under one or more of argon, nitrogen, and helium, preferably under argon protection.

[0082] The present invention also proposes a regulator / promoter / activator, which includes small chemical molecules and / or their derivatives as described above.

[0083] The present invention also provides a reagent / kit comprising one or more of the small chemical molecules and / or their derivatives as described above, and the regulators / promoters / activators as described above.

[0084] The present invention also provides a drug / drug composition comprising one or more of the small chemical molecules and / or their derivatives as described above, the modulators / promoters / activators as described above, or the reagents / kits as described above.

[0085] Furthermore, the pharmaceutical composition includes a pharmaceutically acceptable carrier, etc.

[0086] Preferably, the pharmaceutically acceptable carrier refers to a carrier that, when properly administered to animals or humans, does not produce adverse, allergic, or other adverse reactions. Pharmaceutically acceptable carriers include, but are not limited to: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to help stabilize the formulation or to improve its activity or bioavailability or to produce an acceptable taste or smell when taken orally.

[0087] Specifically, the pharmaceutical composition may also contain physiologically compatible excipients, including buffers, diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc.

[0088] Specifically, the drug / drug composition includes liquid dosage forms, gaseous dosage forms, solid dosage forms, and semi-solid dosage forms.

[0089] Specifically, the pharmaceutical composition can be formulated into injections, sterile powders for injection, tablets, pills, capsules, lozenges, liniments, powders, granules, syrups, solutions, tinctures, aerosols, powder inhalers, or suppositories, etc. All of the above dosage forms of the drug / pharmaceutical composition can be prepared according to conventional methods in the pharmaceutical field.

[0090] Specifically, the pharmaceutical composition is administered orally, by injection, nasal administration, transdermal administration, or mucosal administration.

[0091] Specifically, the pharmaceutical composition can be introduced into the body, such as into muscles, intradermal tissues, subcutaneous tissues, veins, or mucous membranes, via injection, spray, nasal drops, eye drops, penetration, absorption, or physical or chemical mediated methods; or it can be introduced into the body after being mixed with or encapsulated by other substances. Preferably, it is administered by injection. The drug / pharmaceutical composition can also be used in combination with other treatment methods, including surgery, radiotherapy, chemotherapy, and targeted therapy.

[0092] The dosage level of the pharmaceutical composition described in this invention can be adjusted according to the amount of composition required to achieve the desired diagnostic or therapeutic outcome. The administration regimen can also be a single injection or multiple injections, or adjustments thereof. The selected dosage level and regimen are subject to reasonable adjustment based on various factors, including the activity and stability (i.e., half-life) of the cellular drug / pharmaceutical composition, the formulation, the route of administration, the combination with other drugs or treatments, the disease or condition to be detected and / or treated, and the health status and prior medical history of the subject to be treated.

[0093] The therapeutically effective amount of the pharmaceutical composition described in this invention can initially be estimated in cell culture experiments or animal models such as rodents, rabbits, dogs, pigs, and / or primates. Animal models can also be used to determine suitable concentration ranges and routes of administration. These can then be used to determine the effective dose and route of administration in humans. Generally, the determination and adjustment of the effective dose or dosage, and the assessment of when and how to make such adjustments, are known to those skilled in the art.

[0094] For further guidance on formulations, dosages, administration regimens, and measurable treatment outcomes, see Berkow et al. (2000) The Merck Manual of Medical Information and Merck & Co. Inc., Whitehouse Station, New Jersey; Ebadi (1998) CRC Desk Reference of Clinical Pharmacology.

[0095] Furthermore, the pharmaceutical composition may also contain other pharmaceuticals.

[0096] The present invention also provides a method for preventing / inhibiting / alleviating / reducing / treating a disease, the method comprising administering to a subject in need one or more of the following: a small chemical molecule and / or its derivatives as described above, a modulator / promoter / activator as described above, a reagent / kit as described above, a drug / pharmaceutical composition as described above. The method may also be in vitro or non-therapeutic.

[0097] Specifically, the object or individual for preventing / inhibiting / alleviating / reducing / treating the disease is preferably a mammal, including but not limited to humans, primates, livestock (such as sheep, cattle, horses, donkeys, and pigs), pets (such as dogs and cats), laboratory test animals (such as mice, rabbits, rats, guinea pigs, and hamsters), or captured wild animals (such as foxes and deer). Preferably, the object is a primate. Most preferably, the object is a human.

[0098] Specifically, the subject may be a patient with a disease or an individual seeking to prevent disease. One or more of the following can be administered to the subject before, during, or after receiving disease treatment: small chemical molecules and / or their derivatives, modulators / promoters / activators, reagents / kits, protein drug conjugates, and drugs / drug compositions as described above.

[0099] The present invention also provides the use of the chemical small molecules and / or their derivatives as described above, or the regulators / promoters / activators as described above, or the reagents / kits as described above, or the drugs / drug compositions as described above, or the methods as described above, in alkaline phosphatase promotion, alkaline phosphatase and its mutant molecules, osteogenic promotion, cartilage protection, muscle protection, and promotion of the degradation of phosphorus-containing compounds in the body (such as bacterial lipopolysaccharide, pyridoxal phosphate, pyrophosphate, etc.).

[0100] The present invention also provides the application of the chemical small molecules and / or their derivatives as described above, or the regulators / promoters / activators as described above, or the reagents / kits as described above, or the drugs / drug compositions as described above, or the methods as described above, in the preparation of drugs that promote alkaline phosphatase activity, drugs that prepare alkaline phosphatase and its mutant molecules, drugs that promote osteogenic activity, drugs that protect cartilage, drugs that protect muscle, drugs that promote the degradation of phosphorus-containing compounds in the body (such as bacterial lipopolysaccharide, pyridoxal phosphate, pyrophosphate, etc.), and drugs that prevent / inhibit / alleviate / relieve / treat diseases.

[0101] In this invention, the diseases include HPP, fractures, osteoporosis, spinal fusion, neurodegenerative diseases, cardiovascular diseases, etc.

[0102] Compared with the prior art, the beneficial effects of the present invention include: the chemical small molecules and / or their derivatives described in the present invention have a strong binding ability to alkaline phosphatase, and can promote the catalytic activity of strong alkaline phosphatase and its mutant proteins.

[0103] the term

[0104] In this application, unless otherwise stated, the scientific and technical terms used have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used in this application are all conventional procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0105] The three-letter and single-letter codes for amino acids used in this application are as known to those skilled in the art, or as described in J. Biol. Chem, 243, p3558 (1968).

[0106] As used in this application, the terms “comprising” or “including” are intended to mean that a composition and method includes the said elements but excludes other elements, but, depending on the context, also includes the case of “consisting of”.

[0107] In this invention, unless otherwise stated, any concentration range, percentage range, proportion range or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0108] In this invention, the terms "subject," "individual," etc., are used interchangeably herein and refer to any animal or its in vitro and in situ cells suitable for the methods described herein. In some non-limiting embodiments, the patient, subject, or individual is a human being.

[0109] In this invention, the term "disease" refers to a state of health in an animal in which the animal is unable to maintain homeostasis, and if the disease is not improved, the animal's health condition continues to deteriorate. In contrast, an animal's "disorder" is a state of health in which the animal is able to maintain homeostasis, but its health condition is not as good as when it is free from disease. Without treatment, a disorder does not necessarily lead to a further decline in the animal's health condition.

[0110] As used in this invention, the term "and / or" includes any one or more of the related listed items and all combinations thereof.

[0111] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0112] The reagents and raw materials used in this invention are all commercially available.

[0113] The positive and progressive effects of this invention are as follows: the antibody of this invention has a strong binding to alkaline phosphatase, which can improve the activity of alkaline phosphatase, and its use in anti-alkaline phosphatase has better specificity and higher safety. Attached Figure Description

[0114] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0115] Figure 1The effects of different small chemical molecules and their derivatives on the activity of purified alkaline phosphatase protein were investigated. Figure a is a schematic diagram of the chemical structure of the small molecules and their derivatives; Figure b is an SDS-PAGE electrophoresis image of purified ALPL; Figure c shows the promoting effect of different compounds on the enzyme activity of ALPL at a concentration of 50 μM; Figure d shows the promoting effect of different compounds on the enzyme activity of ALPL at a concentration of 25 μM; Figure e is the enzyme reaction rate-substrate concentration curve after the addition of AP14.

[0116] Figure 2 The binding affinity of different small chemical molecules and their derivatives to alkaline phosphatase. Figure a shows the binding affinity of Oridonin to alkaline phosphatase; Figure b shows the binding affinity of AP10 to alkaline phosphatase; Figure c shows the binding affinity of AP14 to alkaline phosphatase; Figure d shows the binding affinity of AP16 to alkaline phosphatase.

[0117] Figure 3 The effects of different small chemical molecules and their derivatives on the activity of human alkaline phosphatase protein mutations in cells were investigated. Figure a shows the effect of AP10 on the activity of human alkaline phosphatase protein mutations in cells; Figure b shows the effect of AP14 on the activity of human alkaline phosphatase protein mutations in cells; Figure c shows the effect of AP16 on the activity of human alkaline phosphatase protein mutations in cells.

[0118] Figure 4 The effects of different concentrations of small chemical molecules and their derivatives on ALP (alkaline phosphatase) staining in cells were investigated. Figure a shows the ALP staining patterns after cell treatment with AP10, AP14, and AP16 at different concentrations; Figure b is a quantitative statistical graph of the alkaline phosphatase intensity in Figure a.

[0119] Figure 5 The effect of AP14 compound on fracture healing in mice. Figure a is a schematic diagram of mouse fracture modeling; Figure b is X-ray image of fractures in mice treated with different concentrations of AP14; Figure c is the callus index curve of mice treated with different concentrations of AP14; Figure d is a micro-CT image after AP14 treatment; Figure e is a statistical graph of bone tissue volume after AP14 treatment; Figure f is a statistical graph of bone volume after AP14 treatment; Figure g is a statistical graph of callus volume ratio after AP14 treatment; Figure h is a statistical graph of trabecular thickness after AP14 treatment; Figure i is a statistical graph of trabecular separation distance after AP14 treatment; Figure j is a statistical graph of the number of trabeculae after AP14 treatment.

[0120] Figure 6AP14 compounds promoted osteoogenesis and improved motor function in mice. Figure a shows HE staining images of callus sites in mice treated with different concentrations of AP14; Figure b shows TRAP staining images of callus sites in mice treated with different concentrations of AP14; Figure c shows Safranin O / Fix Green (SO / FG) staining images of callus areas in control and treatment groups; Figure d shows the ratio of osteoclast area to callus area in mice treated with different concentrations of AP14; Figure e shows the ratio of cartilage area to callus area in mice treated with different concentrations of AP14; Figure f shows gait diagrams of mice treated with different concentrations of AP14; Figure g shows the footprint area of ​​mice treated with different concentrations of AP14; Figure h shows the duty cycle of mice treated with different concentrations of AP14; Figure i shows the ratio of standing time to total time treated with different concentrations of AP14; Figure j shows the maximum contact area of ​​mice treated with different concentrations of AP14; Figure k shows the maximum intensity of mice treated with different concentrations of AP14; Figure l shows the maximum intensity of mice treated with different concentrations of AP14 at maximum contact. Detailed Implementation

[0121] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0122] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0123] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0124] This invention discloses a chemical structure capable of activating alkaline phosphatase. The small chemical molecule of this invention has a strong binding ability to alkaline phosphatase and can promote the catalytic activity of strong alkaline phosphatase and its mutant proteins.

[0125] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores.

[0126] Sequence information of alkaline phosphatase (ALPL) antigen (refer to the protein sequence of Uniprot number P05186).

[0127] >ALPL-DNA (nucleotide sequence of the gene encoding ALPL, SEQ ID NO: 1)

[0128]

[0129] >ALPL-protein (Amino acid sequence of ALPL protein, SEQ ID NO: 2)

[0130] (SEQ ID NO: 2)

[0131] In this invention, the recombinant expression and purification of alkaline phosphatase is as follows: overexpression was achieved in mammalian cells (Yu Y et al. Nat Commun, 2023;14(1):4048.), and the human wild-type ALPL gene (amino acid residues 18-500) was cloned into a modified pcDNA3.4 vector (Invitrogen, USA) in a similar manner. This vector contains a Pre-Scission protease recognition sequence, a Flag tag, and a 10× histidine tag at the C-terminus, and an HA signal peptide at the N-terminus. Expi293F cells (Thermo Fisher Scientific, A14527) were cultured in Union-293 medium (Union-Biotech, UM293-01) with a defined chemical composition at 37°C, 120 rpm, and 5% CO2. When the cell density reached 2.5×10⁻⁶ cells / year, the expression was completed. 6At a cell / mL concentration, the expression plasmid based on the pcDNA3.4 vector was transiently transfected using a PEI MAX (Polysciences, 24765). Cells were collected 96 hours after transfection for protein purification. The cell culture supernatant was collected by centrifugation (Eppendorf 5424R) and subjected to affinity chromatography using nickel chelating resin (Smart-Lifesciences, China). The supernatant was added to a gasketed affinity column. 1) The beads were washed with 10-20 column volumes of deionized water. 2) 10-20 column volumes of equilibration buffer (lysis buffer, composed of 150 mM NaCl, 20 mM HEPES (pH 7.5), and 10% glycerol) were slowly passed through the column. The protein supernatant from the previous step was added to the column and allowed to flow slowly. 3) Subsequently, 10-20 column volumes of washing buffer (lysis buffer with 50 mM imidazole) were slowly passed through the column to wash away some non-specifically bound proteins. 4) Add 3-5 column volumes of elution buffer (lysate with 300 mM imidazole) to elute the protein. Collect the protein eluent in 15 mL centrifuge tubes. Collect the eluent containing the target protein, remove the imidazole by dialysis, concentrate by ultrafiltration, and aliquot. Take 20 μL of the protein eluent, add 5 μL of 5 × SDS-Loading solution, boil at 95℃ for 10 min, and then perform SDS-PAGE electrophoresis at 280 V for 30 min. After electrophoresis, stain with instant blue solution, destain with distilled water, and observe and analyze the protein gel results. The recombinant alkaline phosphatase showed a single band in the SDS-PAGE assay, indicating purity meeting the detection requirements.

[0132] Example 1: Preparation of the small chemical molecules and their derivatives of the present invention

[0133] Unless otherwise specified, the following reactions are carried out in a round-bottom flask under argon protection.

[0134] 1. Preparation of S2

[0135]

[0136] Reaction formula (a)

[0137] Oridonin (Aladdin, catalog number O111382, 364 mg, 1 mmol) and ak2 (4-ethynylbenzoic acid, Aladdin, catalog number E137978, 147 mg, 1.5 mmol) were dissolved in 10 mL of dichloromethane. EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, Maclean, catalog number N742558, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) (282 mg, 2 mmol) and DMAP (4-dimethylaminopyridine, Aladdin, catalog number O111382, D109207, 4-Dimethylaminopyridine) (147 mg, 1.2 mmol) were added and stirred at room temperature for 3 minutes. After the reaction was completed, the mixture was concentrated by rotary evaporation and extracted three times with DCM (dichloromethane, Aladdin, catalog number D116143). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, evaporated under reduced pressure, and purified by normal-phase column chromatography (PE:EA = 2:3) to obtain a white solid S2 (354 mg, 72%). 1 H NMR (500 MHz, DMSO-d6) δ7.88 – 7.78 (m, 2H), 7.58 (d, J = 8.1 Hz, 2H), 6.07 (d, J = 3.7 Hz, 3H), 5.94(d, J = 10.2 Hz, 1H), 5.64 (s, 1H), 4.45 (d, J = 10.0 Hz, 2H), 4.15 (d, J =10.2 Hz, 1H), 3.94 – 3.82 (m, 1H), 3.53 (dd, J = 10.3, 6.8 Hz, 1H), 3.38 –3.32 (m, 1H), 3.14 (d, J = 9.7 Hz, 1H), 2.60 – 2.53 (m, 1H), 2.17 (qd, J =13.4, 8.0 Hz, 1H), 1.91 (dd, J = 13.0, 5.8 Hz, 1H), 1.76 (dt, J = 13.6, 6.3Hz, 1H), 1.50 (dddt, J = 30.2, HRMS calculation [C29 H 33 O7] + [M+H] + The mass-to-charge ratio is 493.2221, and the mass spectrometry shows it to be 493.2227.

[0138] 2. Preparation of S3

[0139]

[0140] Reaction formula (b)

[0141] Oridonin (364 mg, 1 mmol) and ak3 (3-ethynylbenzoic acid, Aladdin, catalog number E165692, 219 mg, 1.5 mmol) were dissolved in 10 mL of dichloromethane. EDCI (282 mg, 2 mmol) and DMAP (147 mg, 1.2 mmol) were added, and the mixture was stirred at room temperature for 3 h. After the reaction was completed, the mixture was concentrated by rotary evaporation and extracted three times with DCM. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, evaporated under reduced pressure, and purified by normal-phase column chromatography (PE:EA = 2:3) to obtain a pale yellow solid S3 (345 mg, 70%). 1H NMR (500 MHz, DMSO) 6.12 (s, 1H), 6.09– 6.05 (m, 2H), 5.94 (d, J = 10.3 Hz, 1H), 5.65 (s, 1H), 4.45 (d, J = 5.1 Hz,1H), 4.30 (s, 1H), 4.15 (d, J = 10.2 Hz, 1H), 3.90 – 3.84 (m, 1H), 3.54 (dd,J = 10.3, 6.8 Hz, 1H), 3.41 – 3.37 (m, 1H), 3.16 (d, J = 9.7 Hz, 1H), 2.56 (dt, J = 13.7, 8.9 Hz, 1H), 2.17 (qd, J = 13.4, 8.0 Hz, 1H), 1.92 (dd, J =13.0, 5.8 Hz, 1H), 1.76 (dt, J = 13.6, 6.4 Hz, 1H), 1.58 – 1.42 (m, 3H), 1.32(dt, J = 13.4, 3.4 Hz, 1H), 1.24 (dd, J = 13.7, 3.4 Hz, 1H), 1.18 (t, J = 7.2Hz, 1H), 1.01 (d, J = 2.0 Hz, 6H). 13 C NMR (126 MHz, DMSO) δ 207.71, 164.35, 151.18, 136.37, 132.80, 131.43, 130.25, 129.54, 122.42, 120.04, 96.33, 82.97, 82.30, 75.10, 74.46, 72.06, 63.08, 62.49, 59.69, 54.33, 41.84, 40.96, 38.82, 33.82, 33.18, 30.78, 29.82, 22.09, 20.22. HRMS calculation [C 29 H 33 O7] + [M+H] + The mass-to-charge ratio is 493.2221, and the mass spectrometry shows it to be 493.2225.

[0142] 3. Preparation of A1

[0143]

[0144] Reaction formula (c)

[0145] Se-16 (0.2 mmol, 1 equiv) and AgNO3 (0.01 mmol, 0.05 equiv) were dissolved in 2 mL of DCE, followed by the addition of ak2 (0.24 mmol, 1.2 equiv) and NH3·H2O (0.3 mmol, 1.5 equiv). The reaction mixture was stirred at room temperature for 2 h. The solution was then removed by evaporation under reduced pressure, and purified by normal-phase column chromatography to obtain a white flocculent solid A1 (96 mg, 92%). 1 H NMR (400 MHz, CDCl3) δ 8.21 (d, J = 7.6 Hz, 1H), 8.01 (d, J = 8.4Hz, 2H), 7.57 (d, J = 8.4 Hz, 2H), 7.53 – 7.47 (m, 2H), 7.33 – 7.24 (m, 1H), 6.96 (d, J = 8.4 Hz, 2H), 6.79 (d, J = 7.6 Hz, 1H), 6.74 (d, J = 8.4 Hz, 2H), 5.71 (s, 1H), 5.04 (dt, J = 7.2, 5.4 Hz, 1H), 3.93 (s, 3H), 3.79 (s, 3H),3.27 – 3.12 (m, 2H). 13 C NMR (151 MHz, CDCl3) δ 172.00, 166.77, 166.62, 155.09, 134.34, 132.53, 131.30, 130.46, 130.31, 129.75, 129.53, 129.38, 128.01, 127.19, 126.89, 126.32, 115.61, 103.71, 78.35, 53.80, 52.62, 52.26, 36.95. HRMS calculation of C 27 H 24 NO6Se + ([M+H)) + The mass-to-charge ratio of 451.2127 was shown in the mass spectrometry as 451.2156.

[0146] 4. Preparation of A2

[0147]

[0148] Reaction formula (d)

[0149] Se-5 (0.2 mmol, 1 equiv) and AgNO3 (0.01 mmol, 0.05 equiv) were dissolved in 2 mL of DCE, followed by the addition of ak3 (0.24 mmol, 1.2 equiv) and NH3·H2O (0.3 mmol, 1.5 equiv). The reaction mixture was stirred at room temperature for 2 h. The solution was then removed by evaporation under reduced pressure, and purified by normal-phase column chromatography to obtain a white flocculent solid A2 (93 mg, 94%). 1 H NMR (400 MHz, CDCl3) δ 8.23 ​​– 8.17 (m, 2H), 7.99 (dt, J = 8.0, 1.6Hz, 1H), 7.69 (dt, J = 7.6, 1.6 Hz, 1H), 7.52 – 7.42 (m, 2H), 7.46 – 7.38 (m,1H), 7.27 (td, J = 7.6, 0.8 Hz, 1H), 5.92 (s, 1H), 3.93 (s, 3H), 2.13 (s,9H), 1.78 – 1.67 (m, 6H). 13 C NMR (151 MHz, CDCl3) δ 166.69, 166.39, 135.73, 133.72, 132.69, 131.99, 131.75, 130.40, 130.34, 129.18, 128.45, 126.31, 125.98, 123.96, 102.72, 76.26, 52.82, 52.26, 41.60, 36.26, 29.46. HRMS calculated [C 27 H 28 NO3Se] + [M+H] + The mass-to-charge ratio is 494.1229, and the mass spectrometry shows it to be 494.1235.

[0150] 5. Preparation of A3

[0151]

[0152] Reaction formula (e)

[0153] Se-16 (0.2 mmol, 1 equiv) and AgNO3 (0.01 mmol, 0.05 equiv) were dissolved in 2 mL of DCE, followed by the addition of ak3 (0.24 mmol, 1.2 equiv) and NH3·H2O (0.3 mmol, 1.5 equiv). The reaction mixture was stirred at room temperature for 2 h. The solution was then removed by evaporation under reduced pressure, and purified by normal-phase column chromatography to obtain a white flocculent solid A3 (100 mg, 92%). 1 H NMR (400 MHz, CDCl3) δ 8.22 (dd, J = 8.4, 1.2 Hz, 1H), 8.20 (t, J =1.6 Hz, 1H), 8.00 (dt, J = 8.0, 1.2 Hz, 1H), 7.70 (dt, J = 7.6, 1.2 Hz, 1H),7.54 – 7.46 (m, 2H), 7.43 (t, J = 7.6 Hz, 1H), 7.33 – 7.25 (m, 1H), 6.96 (d,J = 8.4 Hz, 2H), 6.77 (d, J = 7.6 Hz, 1H), 6.74 (d, J = 8.4 Hz, 2H), 5.51 (s, 1H), 5.04 (dt, J = 7.2, 5.4 Hz, 1H), 3.93 (s, 3H), 3.79 (s, 3H), 3.29 – 3.12(m, 2H). 13 C NMR (150 MHz, CDCl3) δ 171.96, 166.78, 166.47, 155.01, 135.78, 134.50, 132.74, 132.53, 130.50, 130.42, 130.38, 129.79, 129.33, 128.52, 127.32, 126.85, 126.25, 123.83, 115.60, 103.22, 75.58, 53.80, 52.61, 52.32, 36.95. HRMS calculated [C 27 H 24 NO6Se] + [M+H] + The mass-to-charge ratio is 538.0763, and the mass spectrometry shows it to be 538.0767.

[0154] 6. Preparation of AP10

[0155]

[0156] Reaction (f)

[0157] Se-16 (0.2 mmol, 1 equiv) and AgNO3 (0.01 mmol, 0.05 equiv) were dissolved in 2 mL of DCE, followed by the addition of S2 (0.24 mmol, 1.2 equiv) and NH3·H2O (0.3 mmol, 1.5 equiv). The reaction mixture was stirred at room temperature for 2 h. The solution was then removed by evaporation under reduced pressure, and purified by normal-phase column chromatography to obtain a white flocculent solid, AP10 (154 mg, 89%). 1H NMR (500 MHz, DMSO-d6) δ 9.27 – 9.16 (m, 2H), 8.11 (d, J = 8.1 Hz,1H), 8.05 (dd, J = 7.8, 1.4 Hz, 1H), 7.86 (d, J = 8.2 Hz, 2H), 7.64 (dd, J =11.7, 7.6 Hz, 3H), 7.47 (t, J = 7.5 Hz, 1H), 7.13 – 7.04 (m, 2H), 6.69 – 6.63(m, 2H), 6.10 – 6.03 (m, 3H), 5.93 (d, J = 10.2 Hz, 1H), 5.64 (s, 1H), 4.61(ddd, J = 10.1, 7.7, 5.2 Hz, 1H), 4.43 (d, J = 4.9 Hz, 1H), 4.15 (d, J = 10.2Hz, 1H), 3.87 (d, J = 10.2 Hz, 1H), 3.66 (s, 3H), 3.54 (dd, J = 10.3, 6.8 Hz,1H), 3.39 (s, 0H), 3.15 (d, J = 9.7 Hz, 1H), 3.09 (dd, J = 13.9, 5.2 Hz, 1H),3.01 (dd, J = 13.9, 10.1 Hz, 1H), 2.54 (s, 1H), 2.17 (tq, J = 13.2, 7.9, 7.4Hz, 1H), 1.92 (dd, J = 12.9, 5.8 Hz, 1H), 1.76 (dt, J = 13.8, 6.2 Hz, 1H),1.51 (dtt, J = 23.4, 15.3, 8.5 Hz, 3H), 1.35 – 1.21 (m, 2H), 1.17 (d, J = 6.8Hz, 1H), 1.00 (d, J = 2.9 Hz, 6H). 13C NMR (126 MHz, DMSO) δ 207.69, 172.32,167.43, 164.52, 156.44, 151.23, 133.36, 133.29, 131.63, 130.45, 130.10,129.76, 129.62, 129.01, 127.87, 127.31, 127.01, 119.99, 115.56, 103.91,96.34, 79.58, 74.95, 74.51, 72.05, 63.08, 62.51, 59.69, 55.30, 54.33, 52.55, 41.89, 40.96, 38.82, 35.84, 33.82, 33.18, 30.77, 29.82, 22.10, 20.23. HRMS calculates [C 46 H 48 NO 11 Se] + [M+H] + The mass-to-charge ratio is 870.2387, and the mass spectrometry shows it to be 870.2395.

[0158] 7. Preparation of AP14

[0159]

[0160] Reaction formula (g)

[0161] Se-5 (0.2 mmol, 1 equiv) and AgNO3 (0.01 mmol, 0.05 equiv) were dissolved in 2 mL of DCE, followed by the addition of S3 (0.24 mmol, 1.2 equiv) and NH3·H2O (0.3 mmol, 1.5 equiv). The reaction mixture was stirred at room temperature for 2 h. The solution was then removed by evaporation under reduced pressure, and purified by normal-phase column chromatography to obtain a white flocculent solid, AP14 (155 mg, 92%). 1H NMR (500 MHz, DMSO-d6) δ 8.07 (dd, J = 8.1, 1.1 Hz, 1H), 8.03 –7.96 (m, 2H), 7.93 (d, J = 1.8 Hz, 1H), 7.86 (dt, J = 7.8, 1.5 Hz, 1H), 7.78(dt, J = 7.6, 1.5 Hz, 1H), 7.60 (td, J = 7.7, 1.4 Hz, 1H), 7.54 (t, J = 7.8Hz, 1H), 7.40 (td, J = 7.5, 1.1 Hz, 1H), 6.12 (s, 1H), 6.09 – 6.04 (m, 2H),5.94 (d, J = 10.3 Hz, 1H), 5.65 (s, 1H), 4.43 (d, J = 5.1 Hz, 1H), 4.18 –4.11 (m, 1H), 3.87 (dd, J = 10.2, 1.6 Hz, 1H), 3.54 (dd, J = 10.3, 6.8 Hz,1H), 3.38 (dd, J = 11.0, 5.4 Hz, 1H), 3.16 (d, J = 9.7 Hz, 1H), 2.60 – 2.52(m, 1H), 2.23 – 2.13 (m, 1H), 2.09 (d, J = 2.6 Hz, 9H), 1.93 (dd, J = 12.9,5.8 Hz, 1H), 1.81 – 1.72 (m, 1H), 1.67 (s, 6H), 1.57 – 1.44 (m, 3H), 1.32(dt, J = 13.3, 3.4 Hz, 1H), 1.24 (dt, J = 13.7, 3.9 Hz, 1H), 1.17 (d, J = 6.8Hz, 1H), 1.00 (d, J = 2.2 Hz, 6H). 13C NMR (126 MHz, DMSO) δ 207.71, 167.05,164.43, 151.21, 135.94, 132.58, 132.36, 132.05, 131.49, 129.92, 129.60,129.38, 129.20, 126.67, 123.31, 120.03, 102.89, 96.34, 77.67, 75.11, 74.47,72.04, 63.09, 62.51, 59.68, 54.32, 52.66, 41.85, 41.27, 41.15, 40.96, 40.58, 38.81, 36.47, 33.82, 33.19, 30.79, 29.82, 29.35, 22.10, 20.22. HRMS calculates [C 46 H 52 NO8Se] + [M+H] + The mass-to-charge ratio is 826.2835, and the mass spectrometry shows it to be 826.2859.

[0162] 8. Preparation of AP16

[0163]

[0164] Reaction formula (h)

[0165] Se-16 (0.2 mmol, 1 equiv) and AgNO3 (0.01 mmol, 0.05 equiv) were dissolved in 2 mL of DCE, followed by the addition of S3 (0.24 mmol, 1.2 equiv) and NH3·H2O (0.3 mmol, 1.5 equiv). The reaction mixture was stirred at room temperature for 2 h. The solution was then removed by evaporation under reduced pressure, and purified by normal-phase column chromatography to obtain a white flocculent solid AP16 (154 mg, 91%). 1H NMR (500 MHz, DMSO-d6) δ 9.20 (d, J = 7.8 Hz, 2H), 8.09 (d, J = 8.1Hz, 1H), 8.05 (d, J = 7.8 Hz, 1H), 7.94 (d, J = 1.8 Hz, 1H), 7.87 (d, J = 7.9Hz, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.66 (t, J = 7.6 Hz, 1H), 7.54 (t, J = 7.8Hz, 1H), 7.47 (t, J = 7.5 Hz, 1H), 7.09 (d, J = 8.2 Hz, 2H), 6.67 (d, J = 8.1Hz, 2H), 6.13 (s, 1H), 6.07 (t, J = 1.8 Hz, 2H), 5.95 (d, J = 10.2 Hz, 1H),5.65 (s, 1H), 4.61 (ddd, J = 10.3, 7.7, 5.2 Hz, 1H), 4.44 (s, 1H), 4.15 (d, J= 10.2 Hz, 1H), 3.87 (d, J = 10.2 Hz, 1H), 3.67 (s, 3H), 3.55 (dd, J = 10.3,6.8 Hz, 1H), 3.16 (d, J = 9.7 Hz, 1H), 3.10 (dd, J = 13.9, 5.2 Hz, 1H), 3.01(dd, J = 13.9, 10.1 Hz, 1H), 2.56 (dt, J = 13.7, 8.9 Hz, 1H), 2.17 (ddd, J =21.1, 13.5, 6.9 Hz, 1H), 1.93 (dd, J = 12.9, 5.8 Hz, 1H), 1.76 (dt, J = 15.5,6.3 Hz, 1H), 1.61 – 1.11 (m, 7H), 1.00 (s, 6H). 13C NMR (126 MHz, DMSO) δ207.73, 172.35, 167.44, 164.43, 156.46, 151.20, 135.97, 133.45, 133.30,132.38, 131.50, 130.46, 129.99, 129.81, 129.60, 129.01, 127.88, 126.95,123.21, 120.05, 115.57, 103.41, 96.34, 77.08, 75.13, 74.47, 72.05, 63.10,62.51, 59.69, 55.30, 54.32, 52.55, 41.85, 40.96, 38.81, 35.85, 33.82, 33.19, 30.78, 29.83, 22.10, 20.22. HRMS calculates [C 46 H 48 NO 11 Se] + [M+H] + The mass-to-charge ratio is 870.2387, and the mass spectrometry shows it to be 870.2390.

[0166] 9. Preparation of AP14-S:

[0167]

[0168] S-5 (0.24 mmol, 1.2 equiv) and AgNO3 (0.01 mmol, 0.05 equiv) were dissolved in 2 mL of DMSO, followed by the addition of S3 (0.2 mmol, 1.0 equiv) and NH3·H2O (0.3 mmol, 1.5 equiv). The reaction mixture was stirred at 70 °C for 2 h. The solution was then removed by vacuum evaporation, and the solution was purified by normal-phase column chromatography to obtain a white, foamy solid, AP14-S (126 mg, 81%). 1H NMR (500 MHz, DMSO-d6) δ 7.95 – 7.93 (m, 2H), 7.86 (d, J= 8.5 Hz, 2H), 7.79 (td, J = 8.0, 1.5 Hz, 1H), 7.64 (dd, J = 7.5, 1.5 Hz,1H), 7.61 – 7.56 (m, 1H), 7.54 (t, J = 7.5 Hz, 1H), 7.34 (td, J = 7.5, 1.0Hz, 1H), 6.12 (s, 1H), 6.05 (s, 2H), 5.93 (d, J = 10.0 Hz, 1H), 5.64 (s, 1H),4.44 (d, J = 5.0 Hz, 1H), 4.14 (d, J = 10.0 Hz, 1H), 3.86 (d, J = 10.0 Hz,1H), 3.53 (dd, J = 10.0, 7.0 Hz, 1H), 3.40 – 3.38 (m, 1H), 3.15 (d, J = 10.0Hz, 1H), 2.60 – 2.52 (m, 1H), 2.23 – 2.11 (m, 1H), 2.09 – 2.03 (m, 9H), 1.92(dd, J = 12.5, 5.5 Hz, 1H), 1.79 – 1.72 (m, 1H), 1.66 (s, 6H), 1.56 – 1.45(m, 3H), 1.34 – 1.29 (m, 1H), 1.25 – 1.21 (m, 1H), 1.16 (d, J = 6.5 Hz, 1H),1.00 (s, 3H), 0.99 (s, 3H). 13C NMR (200 MHz, DMSO-d6) δ 207.26, 166.15,163.94, 150.73, 135.57, 133.97, 132.37, 132.02, 131.19, 131.08, 129.70,129.20, 128.46, 126.82, 126.18, 122.28, 119.58, 95.88, 79.22, 74.72, 74.03,71.59, 62.64, 62.06, 59.24, 53.87, 51.90, 41.39, 40.75, 40.51, 38.35, 36.05, 33.36, 32.72, 30.32, 29.35, 28.89, 21.63, 19.75. HRMS calculates [C 46 H 52 NO8S] + [M+H] + The mass-to-charge ratio is 778.3408, and the mass spectrometry result is 778.3406.

[0169] Example 2: Effects of different small chemical molecules and their derivatives on alkaline phosphatase activity

[0170] This example (experiment) evaluates the effects of various small chemical molecules and their derivatives on alkaline phosphatase activity. The activity was assessed using p-nitrophenyl phosphate (pNPP; Sangon Biotech (Shanghai) Co., Ltd.) as the phosphatase substrate, which is hydrolyzed by the enzyme to produce a yellow product (with a maximum absorption peak at 405 nm). Reaction initiation method: 50 μL of substrate solution (containing 10 mM pNPP lysis buffer: 150 mM NaCl, 20 mM HEPES, pH 7.5) was added to wells containing 50 μL of a 1:100 diluted ALPL sample (diluted using the same buffer system). The absorbance was then measured at 405 nm at 2-minute intervals using an Enspire microplate reader (PerkinElmer) at 37°C.

[0171] Conclusion: Figure 1 As shown, different small chemical molecules and their derivatives have different effects on alkaline phosphatase activity. At concentrations of 50 μM and 25 μM, compounds AP14, AP16, and AP10 exhibit the best activity. Replacing the Se atom with the S atom weakens the activity of AP14, and it only significantly activates the protein at a concentration of 50 μM. The fragment compounds show relatively weak activity, with S2 and S3 exhibiting partial activity, indicating that S2 and S3 are the smallest structural units that activate the ALPL protein, and modification of these units can enhance their activity. Figure 1 As shown in Figure d, after AP14 compound activates ALPL protein, the protein's km decreases and Vmax increases, indicating that the activation mechanism of AP14 compound belongs to allosteric activators. In summary, this explains the mechanism and the difference between AP14-S converted to S and Se.

[0172] Example 3: Affinity of different small chemical molecules and their derivatives to alkaline phosphatase

[0173] This embodiment (this experiment) utilizes surface plasmon resonance to detect the affinity of different small chemical molecules and their derivatives for alkaline phosphatase. First, the optimal pH of the coupling buffer solution was determined: the pH of the coupling buffer solution should be at least higher than the isoelectric point (3.5) of the protein ligand to allow the protein to carry a positive charge. The dextran on the surface of the CM5 chip (Bioco, Series S Sensor Chip) carries a negative charge, and the two are coupled through electrostatic adsorption. Using "pH Scouting," different pH values ​​(4.0, 4.5, 5.0) were selected for diluting ALPL with 10 mM sodium acetate. Preliminary experimental analysis determined the pH of the coupling buffer solution to be 4.5. Then, the protein ligand was coupled: the protein ligand concentration was 30... g / mL, added to 100 g using a standard amine coupling kit (GE Healthcare, BR100050) L 0.1M EDCI and 100 L 0.1 M sNHS and 1 M ethanolamine blocking solution at pH 8.0 were manually coupled and cured onto the surface of a CM5 sensor chip (GE Healthcare, 29-1049-88). Since DMSO was used as the solvent, solvent correction was required, i.e., preparing 0.5% DMSO (in PBS-T80) and 2.0% DMSO (in PBS-T80) correction solutions to monitor fluctuations in the DMSO concentration of the SPR mobile phase. Finally, in the Biacore T200 software, the LWM multi-cycle kinetics / affinity module was selected. A serially diluted small molecule was added to a U-bottom 96-well plate. Using the low molecular weight multi-cycle kinetics / affinity analysis method provided by GE Healthcare, the compound solution, diluted twice, was injected at a flow rate of 30 µL / min for 90 seconds to initiate the binding reaction, followed by dissociation for 90 seconds. Solvent correction was performed before and after each analysis cycle using eight different concentrations of DMSO solution. The binding affinity (Kb) of oridonin was calculated using Biacore 8K assessment software (GE Healthcare) with a 1:1 binding model fitting of the data. DThe binding kinetic parameters of compounds AP10, AP14, and AP16 were calculated using Biacore evaluation software (Cytiva), including ka (binding rate constant) and kd (dissociation rate constant). D The value is calculated based on the ratio of kd / ka.

[0174] Conclusion: Figure 2 As shown, different small chemical molecules and their derivatives have different binding forces to alkaline phosphatase. Among them, the fragment Oridonin has partial activity, but the affinity of compound AP14 is the highest at 10.5 μM, compound AP10 has an affinity of 13.4 μM, and compound AP16 has an affinity of 39.3 μM.

[0175] Example 4: Effects of different small chemical molecules and their derivatives on the activity of different alkaline phosphatase mutations in cells.

[0176] HEK293T cells were used at a rate of 1×10 5 Cells were seeded at a standard density of 1 cell / well in 24-well tissue culture plates (CCP-Nunc, 144530) to achieve 70-80% cell confluence at transfection. After 24 hours of culture in complete DMEM medium containing 10% FBS at 37°C and 5% CO2 to allow adherence, transfection was performed using PEI MAX according to the manufacturer's instructions. A constant ratio was maintained for all experimental conditions: 1 μg of ALPL mutant plasmid DNA per well to 2 μL of transfection reagent per well. Transfection efficiency was assessed 24 hours after transfection using a fluorescence microscope (Nikon Eclipse Ti) for the green fluorescent protein-labeled constructs.

[0177] Before protein extraction, the culture medium was aspirated and the cells were washed twice with ice-cold PBS (pH 7.4). Cell lysis was performed using RIPA lysis buffer (Absin, abs9231, 100 ml) supplemented with 1× protease inhibitor mixture (Sigma-Aldrich, P8215-1 ml). After incubation on ice for 30 min with intermittent vortexing, the lysis buffer was clarified by centrifugation at 13,000 × g for 10 min at 4°C. The supernatant was aliquoted and stored at -80°C or used immediately for enzyme activity assay.

[0178] Cellular enzyme activity assay: Cell lysis buffer was added to 96-well flat-bottom plates (Sigma-Aldrich, M0661-1CS) in triplicate (10 μL / well) and AP10, AP14 or AP16 compounds (prepared using lysis buffer containing 5% DMSO, final concentration 10 µM) were added respectively, and the plates were treated at 25°C for 30 minutes.

[0179] Conclusion: Figure 3As shown, different small chemical molecules and their derivatives have different effects on promoting the activity of human alkaline phosphatase protein mutations in cells. The compounds AP10, AP14 and AP16 significantly enhanced the activity of A133G, R136P, R152H, T167M and R450C at a concentration of 10 μM.

[0180] Example 5: Cellular alkaline phosphatase staining analysis

[0181] Bone marrow-derived mesenchymal stromal cells (BMSCs) were used at a rate of 1×10 4 The cells were seeded at a density of cells / well in 48-well plates (CCP-Nunc, 150787). The control group was cultured in complete growth medium (α-MEM supplemented with 10% FBS and 1% penicillin-streptomycin (MedChemexpress, HY-K0014-10mL); the experimental group was cultured in osteogenic induction medium (complete medium supplemented with 50 μg / mL ascorbic acid, 10 mM β-glycerophosphate sodium and 100 nM dexamethasone) (Sigma, SCM121), with different concentrations (0, 0.8, 1.6 or 3.2 μM) of the test compounds AP10, AP14 or AP16. The medium was replaced with fresh medium every three days.

[0182] After 7 days of culture, cells were washed twice with PBS and fixed for 15 minutes at room temperature with 4% paraformaldehyde (Alfa Aesar, J61899.AK). ALP activity was detected using the BCIP / NBT chromogenic assay kit (Beyotime, C3206): cells were incubated at 37°C for 30 minutes in the dark, washed three times with PBS, and air-dried at room temperature. Images were acquired using a Cytation 5 cell imaging multifunction detection system (BioTek Instruments, USA). Density quantification was performed using ImageJ software (NIH, version 1.53).

[0183] Conclusion: Figure 4 As shown, compounds AP10, AP14, and AP16 exhibited gradient-dependent enhancement of ALP alkaline phosphatase staining in cells at concentrations of 0.8 μM, 1.6 μM, and 3.2 μM, indicating osteogenic promotion.

[0184] Example 6: Promoting effect of agonists in a mouse fracture model

[0185] Establishment of a mouse femoral fracture model: Eight-week-old male C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All mice were housed in a specific pathogen-free (SPF) environment with a constant temperature of (22±2)℃ and humidity of (55±10%), using a 12-hour light-dark cycle. Animals were given free access to standard rodent feed and drinking water. Under isoflurane inhalation anesthesia, the skin of the femoral region was incised with a scalpel blade, and the muscle tissue was bluntly dissected to expose the femur. The femoral condyle was exposed through lateral patellar dislocation. After medullary reaming with a 25G needle (BD), an intramedullary nail of the same diameter was implanted. A midshaft femoral osteotomy was performed using a dental drill, retaining the intramedullary nail in situ for fixation to stabilize the fracture ends and ensure postoperative mobility. The muscle tissue was layered and the skin incision was sutured with 4 / 0 nylon sutures. The fracture model mouse model was thus established.

[0186] Drug intervention protocol: The above-mentioned fracture model mice were randomly divided into three groups (n=3 in each group). Intramuscular injections (200 μL per dose) were administered on postoperative days 3, 7, 10, and 14: Group 1 (Control group) received phosphate-buffered saline (PBS), Group 2 (Treatment group) received 10 μM AP14 compound, and Group 3 (Treatment group) received 20 μM AP14 compound. Fracture healing was evaluated using the aforementioned X-ray, micro-CT imaging, tissue staining, and gait analysis methods.

[0187] X-ray imaging assessment was performed using a digital radiographic imaging system (MultiFocus 10x15, Faxitron Bioptics, LLC) to monitor the healing process of femoral fractures. Mice with the fracture model were anesthetized with isoflurane inhalation and fixed in a prone position on the scanning platform with the affected limb abducted and the knee joint flexed at 90°. High-resolution digital images were acquired. For micro-CT, fresh bone samples were fixed in 4% paraformaldehyde at 4°C for 24-48 hours to preserve microstructure. After fixation, the samples were transferred to PBS solution containing 0.01% sodium azide to inhibit bacterial growth. The samples were fixed in a cylindrical sample holder and cushioned with foam for shock absorption, and scanned using a high-resolution micro-CT scanner (μCT-100, SCANCOMedical AG, Switzerland). The original projection data were reconstructed using the modified Feldkamp algorithm in SCANCO micro-CT software (v6.5-3), and 3D reconstruction and subchondral bone parameter analysis were performed using μCT-100 software (v6.5-3, SCANCO Medical AG) according to standard procedures. For histological analysis, fractured femoral samples from 8-week-old male C57BL6 / J mice (the aforementioned fracture model mice) were fixed for 1 hour in 4% paraformaldehyde (prepared with 0.1 M phosphate buffer, pH 7.4). Sequential dehydration with a gradient of ethanol (70%, 80%, 95%, and 100% v / v, 1 hour per concentration) was performed to reduce tissue shrinkage artifacts. For bone components, samples were decalcified using 0.5 M EDTA (pH 7.4) (solution changed daily). After decalcification, the tissue was cleared with xylene and embedded in paraffin under standardized anatomical orientation (paraffin melting point 56-58°C). 5 µm thick serial sections were prepared using a rotary microtome (Leica, RM2235) and attached to poly-L-lysine-coated slides. The slides were baked at 45°C for 24 hours to ensure tissue adhesion. Histological analysis was performed using a multiple staining protocol: 1) Hematoxylin-eosin (H&E) staining – standard protocol for paraffin tissue sections; 2) Safranin O / Fix Green (SO / FG) staining – to distinguish sulfated glycosaminoglycans (red) from collagen (green) in the cartilage matrix; 3) Tartrate-resistant acid phosphatase (TRAP) staining – using naphthol AS-BI phosphate substrate to visualize enzyme activity and identify osteoclast resorption areas. Gait analysis was performed using a CatWalk XT system (Noldus Information Technology, Wageningen, Netherlands) according to the manufacturer's instructions. A simplified procedure was as follows: mice were placed individually on an illuminated glass walkway and allowed to walk freely from one end to the other. A high-speed color camera (100 Hz) located below the platform records the dynamic light contact area between the claw and the glass surface.Signal strength (0-255 units) is related to the contact area and increases proportionally with increasing claw pressure. Only continuous motion sequences meeting preset speed standards (5-25 cm / s) are collected for analysis. The claw recognition algorithm equipped in CatWalk Software v10.6 automatically quantifies spatiotemporal parameters (including claw area, pressure distribution, and gait kinematic data).

[0188] Conclusion: Figure 5 As shown, compound AP14 significantly improved fracture healing in mice. (a) Schematic diagram of the AP14 compound injection protocol in the fracture healing study. Male C57BL / 6J mice were locally injected with solvent control, 10 µM, or 20 µM AP14 compound at different time points after femoral fracture surgery. (b) X-ray images of fractured femurs in mice treated with PBS (control group) and AP14 compound (10 µM or 20 µM) at different post-fracture fractional fracture (DPF) days. Scale bar = 1 mm (n = 5). (c) Callus formation quantified by the callus index at different DPF days. The callus index is defined as the ratio of the maximum diameter of the callus to the diameter of the bone shaft. (d) Representative μCT images of fractured femurs in mice at 20 DPF days. Scale bar = 2 mm. (ej) Quantitative analysis of callus volume, bone volume, bone volume fraction (BV / TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N) based on μCT images (n=5).

[0189] Figure 6 The results show that the AP14 compound promotes osteogenesis and improves motor function in mice. (a, b) H&E and TRAP staining results of callus tissue from mice in the control and treatment groups, with black dashed lines indicating callus areas. (c) Quantitative analysis results of callus sections, scale bar = 5 mm, n = 5 per group. (d) Safranin O / Fix Green (SO / FG) staining images of callus areas from mice in the control and treatment groups, with black dashed lines indicating callus extent. (e) Quantitative analysis results of callus sections, scale bar = 5 mm, n = 5 per group. (f) Representative paw print images (left) and corresponding pressure distribution heatmaps (right) of the hind limb obtained 14 days after fracture using the CatWalk XT system (Noldus), with white arrows indicating the surgically fractured right hind limb. Each dataset includes three biological replicates. (g) Quantitative analysis of gait parameters, including claw mark area (ratio of affected side to contralateral limb, %), standing phase duration (standing phase duration / gait cycle, %), and maximum contact area (contact area of ​​affected limb / total contact area of ​​contralateral limb, %).

[0190] The results show that the AP14 compound can significantly promote fracture healing and improve the motor function of mice.

[0191] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of this invention are included in this invention and are protected by the appended claims.

Claims

1. A small chemical molecule and / or a derivative thereof, characterized in that, The chemical small molecule and / or its derivatives are selected from one or more of the following compounds: 。 2. A modulator, characterized in that, The regulator is selected from the small chemical molecules and / or their derivatives as described in claim 1.

3. A reagent / kit characterized in that, The reagents / kits are selected from one or more of the small chemical molecules and / or their derivatives as described in claim 1, and the regulators as described in claim 2.

4. A drug / drug composition, characterized in that, The drug / drug composition is selected from one or more of the small chemical molecules and / or their derivatives as described in claim 1, the modifiers as described in claim 2, or the reagents / kits as described in claim 3.

5. A method for preparing AP10, characterized in that, The preparation method is selected from: reacting Se-16 and S2 in a solvent in the presence of AgNO3 and NH3·H2O to prepare AP10; the reaction process of the preparation method is shown in reaction formula (VI): ； Reaction formula (VI).

6. A method for preparing AP14, wherein the preparation method is selected from: reacting Se-5 and S3 in a solvent in the presence of AgNO3 and NH3·H2O to obtain AP14; the reaction process of the preparation method is shown in reaction formula (VII): ； Reaction formula (VII).

7. A method for preparing AP16, characterized in that, The preparation method is selected from: reacting Se-16 and S3 in a solvent in the presence of AgNO3 and NH3·H2O to prepare AP16; the reaction process of the preparation method is shown in reaction formula (VIII): ； Reaction formula (VIII).

8. A method for preparing AP14-S, characterized in that, The preparation method is selected from: reacting S-5 and S3 in a solvent in the presence of AgNO3 and NH3·H2O to prepare AP14-S; the reaction process of the preparation method is shown in reaction formula (IX): ； Reaction formula (IX).

9. The use of the small chemical molecule and / or its derivatives as described in claim 1, or the regulator as described in claim 2, or the reagent / kit as described in claim 3, or the drug / drug composition as described in claim 4, or the method as described in claims 5-8 in the preparation of a drug that promotes alkaline phosphatase activity, or in the preparation of a drug for the prevention / treatment of diseases, wherein, The diseases mentioned are selected from HPP, fractures, osteoporosis, spinal fusion, neurodegenerative diseases, and cardiovascular diseases.

10. The application as described in claim 9, characterized in that, The drugs used to prepare alkaline phosphatase-promoting drugs are selected from drugs that promote osteogenic formation, drugs that protect cartilage, and drugs that protect muscle.

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