Deuterated crotonane diterpenoid compound as well as preparation method and application thereof

Abstract

The invention provides a deuterated crotonane diterpenoid compound as well as a preparation method and application thereof, and relates to the technical field of medicinal chemistry. The deuterated crotonane diterpenoid compound comprises a compound as shown in the following general formula structure and pharmaceutically acceptable salts thereof: # imgabs0 #, wherein R1 is a deuterated side chain; and R2 is a non-deuterated side chain different from R1. The deuterated crotonane diterpenoid compound provided by the invention can improve pharmacokinetics and reduce administration dosage and possible toxic and side effects through deuterated modification. By activating protein kinase C (PKC), mitochondrial oxidative stress and plasma membrane rupture are caused, tumor cells are induced to generate immunogenic death, and the anti-tumor effect is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal chemistry, and in particular, to a deuterated tigliane diterpenoid compound, a preparation method thereof, and an application thereof. Background Art

[0002] Tigliane diterpenoids are a class of macrocyclic diterpenoid compounds with diverse biological activities and special structures, which are mainly extracted from plants of the Euphorbiaceae and Thymelaeaceae families. According to the differences in functional groups in their molecular structures, tigliane diterpenoids can be divided into multiple subtypes such as phorbol ester type, C-6, C-7 epoxy type, C-12 deoxyphorbol ester type, etc. Most of these compounds have antiviral activities and exhibit significant cytotoxic activities, so they have important potential in drug research and development. Due to the uniqueness of their structures, the research on tigliane diterpenoid compounds in anti-cancer, anti-viral and other aspects has received extensive attention.

[0003] At present, there have been many studies on the development of such natural compounds. For example, since the phorbol ester type compound Prostatin was isolated by the US National Cancer Institute in 1992, it has been used in the research of anti-AIDS. Since 2001, the US AIDS Research Alliance has obtained an exclusive license for the research of this compound. In the clinical research of leukemia, the phorbol ester type compound 12-O-tetradecanoylphorbol-13-acetate (TPA) has also entered phase II clinical trials. In addition, the C-6, C-7 epoxy type compound Tigilanol tiglate, as an activator of protein kinase C (PKC), can enhance the permeability of vascular endothelial cells in tumor tissues, destroy the blood vessels at the tumor site, and thus promote tumor ablation.

[0004] However, there are still significant defects in the application of such compounds. First of all, due to the existence of multiple active sites in the structure of tigliane diterpenoids, this makes them face the problem of instability in the process of drug development. These compounds are extremely easy to degrade in vivo, resulting in their short-lasting efficacy and difficulty in achieving the ideal therapeutic effect. Secondly, although these compounds have powerful biological activities, they themselves have certain toxicity. When using larger doses, the side effects will be more obvious, which may lead to adverse reactions during the treatment process, posing a great challenge to clinical applications.

[0005] In addition to the problems of stability and toxicity, pharmacokinetic properties are also key factors affecting the clinical application of these compounds. Most tigliane diterpenoid compounds are metabolized rapidly in vivo, resulting in their low bioavailability and difficulty in maintaining the efficacy. Due to their easy metabolism, the therapeutic concentration of the drug is difficult to maintain in the body for a long time, which limits their application in long-term treatment. In addition, the problem of large side effects also limits the possibility of these compounds as long-term medications, especially in high-dose treatments, these problems are more prominent.

[0006] In summary, although tigliane diterpenoids have great potential in the fields of anti-cancer, anti-viral, etc., the existing technologies still face many challenges. The poor stability, relatively high toxicity, unsatisfactory pharmacokinetic properties of the compounds, as well as the possible side effects, all limit their wide application in clinical practice. Therefore, how to improve these problems and enhance the efficacy, safety and pharmacokinetic characteristics of these natural compounds remains an important topic in current research and development.

[0007] In view of this, the present invention is specifically proposed. Summary of the Invention

[0008] The first object of the present invention is to provide a deuterated tigliane diterpenoid compound, a preparation method of the deuterated tigliane diterpenoid compound, and an application of the deuterated tigliane diterpenoid compound in the preparation of anti-tumor products. The deuterated tigliane diterpenoid compound improves pharmacokinetic characteristics, enhances bioavailability, reduces the dosing frequency, and has extensive potential for clinical application, especially in the fields of cancer and viral infections, by improving stability, prolonging the drug effect, and reducing side effects.

[0009] In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:

[0010] In a first aspect, the present invention provides a deuterated tigliane diterpenoid compound, which includes a compound shown by the following general formula structure and its pharmaceutically acceptable salt:

[0011]

[0012] Wherein, R1 is a deuterated side chain; R2 is a non-deuterated side chain different from R1.

[0013] In a preferred embodiment, the deuterated side chain includes at least one of deuterated trans-2-methyl-2-butenoic acid substituents and deuterated tetradecanoic acid substituents;

[0014] In a preferred embodiment, the deuterated side chain is trans-2-methyl-2-butene(3,4,4,4-d3) acid.

[0015] In a preferred embodiment, the non-deuterated side chain includes at least one of S-2-methyl-butyric acid substituents and acetic acid substituents;

[0016] In a preferred embodiment, the non-deuterated side chain is S-2-methyl-butyric acid.

[0017] In a preferred embodiment, the compound structure of the deuterated tigliane diterpenoid compound is:

[0018]

[0019] In a second aspect, the present invention provides a method for preparing a deuterated tigliane diterpenoid compound, comprising:

[0020] Obtaining a monoesterified tigliane diterpenoid intermediate by esterifying the hydroxyl group of the tigliane diterpenoid raw material with the carboxyl group of S-2-methyl-butyric acid;

[0021] Esterifying the monoesterified tigliane diterpenoid intermediate with a deuterated side-chain carboxylic acid to obtain a diesterified tigliane diterpenoid intermediate;

[0022] Removing the isopropylidene protecting group of the diesterified tigliane diterpenoid intermediate by using an acid catalyst to obtain the deuterated tigliane diterpenoid compound;

[0023] In a preferred embodiment, the acid catalyst includes at least one of p-toluenesulfonic acid, camphorsulfonic acid, and hydrochloric acid;

[0024] In a preferred embodiment, the acid catalyst is p-toluenesulfonic acid.

[0025] In a preferred embodiment, the step of obtaining a monoesterified tigliane diterpenoid intermediate by esterifying the hydroxyl group of the tigliane diterpenoid raw material with the carboxyl group of S-2-methyl-butyric acid includes:

[0026] Using triethylamine and 4-dimethylaminopyridine as catalysts, carrying out an esterification reaction of the tigliane diterpenoid raw material, S-2-methyl-butyric acid, and a condensing agent in dichloromethane to obtain a crude esterification product;

[0027] Purifying the crude esterification product by silica gel column chromatography to obtain the monoesterified tigliane diterpenoid intermediate;

[0028] In a preferred embodiment, the condensing agent includes (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and / or N,N'-dicyclohexylcarbodiimide;

[0029] In a preferred embodiment, the condensing agent is (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride;

[0030] In a preferred embodiment, the addition amount of 4-dimethylaminopyridine is 0.2 equivalents.

[0031] In a preferred embodiment, the step of esterifying the monoesterified tigliane diterpenoid intermediate with a deuterated side-chain carboxylic acid to obtain a diesterified tigliane diterpenoid intermediate includes:

[0032] Dissolving the monoesterified tigliane diterpenoid intermediate in toluene to obtain a mixture;

[0033] Using triethylamine and 4-dimethylaminopyridine as catalysts, a strong acylating agent and a deuterated side-chain carboxylic acid are added to the mixture, and the deuterated side-chain carboxylic acid reacts with the ester group of the monoesteryl clerodane diterpene intermediate in the mixture to form an esterified substance;

[0034] Quench with an aqueous sodium bicarbonate solution, extract and separate with dichloromethane, and then purify by silica gel column chromatography to obtain the diesteryl clerodane diterpene intermediate;

[0035] In a preferred embodiment, the strong acylating agent is 2,4,6-trichlorobenzoyl chloride;

[0036] In a preferred embodiment, the solvent in the ester group reaction includes at least one of toluene, tetrahydrofuran, and dichloromethane;

[0037] In a preferred embodiment, the solvent for the ester group reaction is toluene.

[0038] In a preferred embodiment, removing the isopropylidene protecting group of the diesteryl clerodane diterpene intermediate with an acid catalyst to obtain the deuterated clerodane diterpene compound includes:

[0039] Performing deprotection on the diesteryl clerodane diterpene intermediate and an acid catalyst in a mixed solvent of acetonitrile and water to obtain a deprotected product;

[0040] Neutralizing the deprotected product with an aqueous sodium carbonate solution to obtain a neutralized product;

[0041] Extracting the neutralized product with ethyl acetate and purifying by silica gel column chromatography to obtain the deuterated clerodane diterpene compound.

[0042] In a preferred embodiment, the deuterated side-chain carboxylic acid is trans-2-methyl-2-butenoic acid (3,4,4,4-d3);

[0043] In a preferred embodiment, the preparation method of the trans-2-methyl-2-butenoic acid (3,4,4,4-d3) includes:

[0044] Mix triethyl diphosphate and an ether solvent, and then add deuterated acetaldehyde to obtain a crude deuterated intermediate;

[0045] For the crude deuterated intermediate, quench the reaction with water and perform back extraction with ethylene glycol dimethyl ether, and wash to obtain a deuterated intermediate;

[0046] Hydrolyze the deuterated intermediate with an inorganic base, and perform quenching, washing, drying, and filtration and concentration on the reaction solution after hydrolysis with the inorganic base to obtain a crude deuterated side chain;

[0047] The crude deuterated side chain is purified to obtain the trans-2-methyl-2-butenoic acid (3,4,4,4-d3).

[0048] In a preferred embodiment, the ether solvent includes at least one of ethylene glycol dimethyl ether, methyl tert-butyl ether, and diethyl ether;

[0049] In a preferred embodiment, the ether solvent is ethylene glycol dimethyl ether;

[0050] In a preferred embodiment, the inorganic base includes at least one of sodium hydroxide, potassium hydroxide, and potassium carbonate;

[0051] In a preferred embodiment, the inorganic base is sodium hydroxide and / or potassium hydroxide;

[0052] In a preferred embodiment, the purification treatment includes:

[0053] The crude deuterated side chain is successively dissolved by adding an aqueous sodium bicarbonate solution and potassium hydroxide to adjust the pH, and the impurities are extracted with ethyl acetate to obtain the product dissolved in the aqueous layer;

[0054] The pH of the product dissolved in the aqueous layer is adjusted to acidic with hydrochloric acid to free the product, and it is extracted with ethyl acetate to obtain the organic layer material;

[0055] The organic layer material is concentrated and purified by vacuum distillation to obtain the trans-2-methyl-2-butenoic acid (3,4,4,4-d3).

[0056] In a third aspect, the present invention provides an application of a deuterated tigliane diterpenoid compound as described in any one of the foregoing embodiments in the preparation of an anti-tumor product.

[0057] The deuterated tigliane diterpenoid compound provided by the present invention can improve pharmacokinetics, reduce the dosage and possible toxic side effects through deuteration modification. By activating protein kinase C (PKC), it causes mitochondrial oxidative stress and plasma membrane disruption, induces immunogenic death of tumor cells, and significantly improves the anti-tumor effect. Due to these advantages, the deuterated tigliane diterpenoid compound is suitable for diseases that require long-term treatment or high-dose use, and has broad clinical application prospects especially in the fields of cancer and viral infections. In short, these improvements provide greater safety and efficacy potential for the clinical application of deuterated tigliane diterpenoid compounds. Description of the Drawings

[0058] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0059] Figure 1 Synthesis reaction route diagram of trans-2-methyl-2-butenoic acid (3,4,4,4-d3) in Example 1 of the present application;

[0060] Figure 2 Synthesis reaction route diagram of D12-TT in Example 2 of the present application;

[0061] Figure 3 1H NMR spectrum of D12-TT in Example 2 of the present application;

[0062] Figure 4 13C NMR spectrum of D12-TT in Example 2 of the present application;

[0063] Figure 5 MS test result diagram of D12-TT in Example 2 of the present application;

[0064] Figure 6 Synthesis reaction route diagram of TT in Comparative Example 1 of the present application;

[0065] Figure 7 1H NMR spectrum of TT in Comparative Example 1 of the present application;

[0066] Figure 8 MS test result diagram of TT in Comparative Example 1 of the present application;

[0067] Figure 9 Synthesis reaction route diagram of D12-PBE in Comparative Example 2 of the present application;

[0068] Figure 10 Growth curve diagram of the body weight of mice after treatment in Comparative Experiment 1 of the present application;

[0069] Figure 11 Growth curve diagram of the tumor of mice after treatment in Comparative Experiment 1 of the present application;

[0070] Figure 12 Growth curve diagram of the tumor of mice after treatment in Comparative Experiment 2 of the present application. Specific embodiments

[0071] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those not specified in the examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0072] In an embodiment of the present application, a deuterated crotonane diterpene compound is provided. The deuterated crotonane diterpene compound includes a compound represented by the following general formula structure and its pharmaceutically acceptable salts:

[0073] Among them, R1 is a deuterated side chain; R2 is a non-deuterated side chain different from R1.

[0074] Further, the deuterated side chain includes at least one of deuterated trans-2-methyl-2-butenoic acid substituents and deuterated tetradecanoic acid substituents.

[0075] Further, the deuterated side chain is trans-2-methyl-2-butene(3,4,4,4-d3) acid.

[0076] Further, the non-deuterated side chain includes at least one of S-2-methyl-butyric acid substituents and acetic acid substituents.

[0077] Further, the non-deuterated side chain is S-2-methyl-butyric acid.

[0078] As described above, the deuterated side chain is a deuterium-labeled side chain, and at least one of deuterated trans-2-methyl-2-butenoic acid substituents or deuterated tetradecanoic acid substituents can be selected. This indicates that the side chain of the deuterated compound not only has the deuteration function (such as deuterium-labeling hydrogen atoms), but also can select specific chemical group types as needed, such as deuterated trans-2-methyl-2-butenoic acid or deuterated tetradecanoic acid groups.

[0079] The deuterated side chain can preferably be trans-2-methyl-2-butene(3,4,4,4-d3) acid, which is a deuterium-labeled compound. By deuterium-labeling trans-2-methyl-2-butenoic acid, the stability of the fatty chain at the C12 position is improved, and the stability and metabolic properties of the drug can be improved. This deuteration design helps to enhance the pharmacokinetic performance of the drug, such as improving stability and reducing toxicity.

[0080] As described above, the non-deuterated side chain requirements are different from those of the deuterated side chain and include at least one of S-2-methyl-butyric acid substituents and acetic acid substituents. Here, the design of the non-deuterated side chain illustrates the structure of the other side chain in the compound, and these substituents (such as S-2-methyl-butyric acid) are relatively independent of the deuterated part and can play a role in balancing the properties of the compound, such as reducing toxicity or improving the water solubility of the drug.

[0081] In the structure, it is clear that the non-deuterated side chain can be S-2-methyl-butyric acid. This non-deuterated side chain may interact with the deuterated side chain and jointly affect the biological activity, stability, and pharmacokinetic properties of the compound.

[0082] In this example, by introducing a deuterated side chain, the metabolic stability of the tigliane diterpene compound can be improved, and its half-life in the body can be extended. This helps to enhance the efficacy of the drug, especially in long-term treatments such as anti-cancer or anti-viral therapies, improves the bioavailability of the drug, and reduces side effects.

[0083] The design of the non-deuterated side chain (such as S-2-methyl-butyric acid) can regulate the drug properties of the compound, reduce possible toxicity, and form a reasonable structural combination with the deuterated side chain to improve the overall biological activity of the drug.

[0084] It should be noted that the deuterated tigliane diterpene compound has high structural flexibility, allowing the performance of the drug to be optimized by selecting different side chains (deuterated and non-deuterated) to meet different clinical needs. Moreover, the compound can also exist in the form of a pharmaceutically acceptable salt, which further enhances its application flexibility in formulations, especially in drug formulations where improved solubility or stability is required.

[0085] In summary, the pharmacokinetic properties of the compound are improved by the design of the deuterated side chain, the drug efficacy is extended, metabolic degradation is reduced, and the non-deuterated side chain is used to regulate the toxicity and biological activity of the compound. This design has great potential in drug development, especially in improving drug stability and reducing side effects.

[0086] Furthermore, the chemical structure of the deuterated tigliane diterpene compound is:

[0087]

[0088] In the embodiment of the present application, a preparation method of a deuterated tigliane diterpene compound is provided, including:

[0089] Step S1, esterifying the hydroxyl group of the tigliane diterpene raw material with the carboxyl group of S-2-methyl-butyric acid to obtain a monoesterified tigliane diterpene intermediate.

[0090] As described above, the structure of the crotonane diterpene raw material used can be a crotonane diterpene compound with exposed hydroxyl groups (such as phorbol type, C6-C7 epoxy type, etc.), which already has one or more hydroxyl groups (-OH). These hydroxyl groups are active groups in the reaction and can participate in the esterification reaction. Among them, S-2-methyl-butyric acid is a compound containing a carboxyl group (-COOH), which reacts with the hydroxyl group at the C13 position of this type of compound to form an ester bond (-COO-), thereby obtaining a crotonane diterpene intermediate with a single ester group.

[0091] The product obtained through this reaction is a crotonane diterpene intermediate with a single ester group, forming a chemically stable ester structure. This esterification reaction protects the active hydroxyl group at the C13 position and helps introduce a deuterated side chain at the C12 position in the subsequent steps.

[0092] In this step, the esterification reaction carried out is simple, rapid and highly selective. It can connect the hydroxyl group at the C13 position of the crotonane diterpene with exposed hydroxyl groups and the carboxyl group of S-2-methyl-butyric acid, and the ester bond is relatively stable, laying a foundation for the subsequent reactions.

[0093] Specifically, it can be carried out through a standard esterification reaction. EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) or DCC (dicyclohexylcarbodiimide) is used as a catalyst to activate the carboxyl group and react with the hydroxyl group in the crotonane diterpene raw material. Usually, additives such as DMAP (4-dimethylaminopyridine) are needed to enhance the selectivity of the reaction.

[0094] In step S2, the crotonane diterpene intermediate with a single ester group is subjected to an esterification reaction with a deuterated side chain carboxylic acid to obtain a crotonane diterpene intermediate with a double ester group.

[0095] As described above, a deuterated side chain carboxylic acid, such as deuterated trans-2-methyl-2-butene (3,4,4,4-d3) acid, is used as an esterification reagent in this step and undergoes an esterification reaction with the crotonane diterpene intermediate with a single ester group.

[0096] In the esterification reaction, the carboxyl group of the deuterated side chain reacts with the hydroxyl group of the monoacyl crotonane diterpene intermediate to generate a new crotonane diterpene intermediate with a double ester group. This reaction introduces deuterium labeling, making the side chain part contain deuterium atoms and improving the pharmacokinetic properties of the drug. Through this step, the crotonane diterpene compound with a deuterated side chain is successfully synthesized. The product not only has the biological activity of crotonane diterpene compounds but also has a deuterated side chain, improving the stability and metabolic properties of the molecule.

[0097] In this step, by introducing a deuterated side chain, the metabolic process of the compound in the body may become slower, thereby reducing the degradation rate and prolonging the drug effect. The acylation reaction can use thionyl chloride or acid anhydride reagents, or amino acid derivatives can be selected as the acylation reagent. In this reaction, triethylamine is usually used as a basic catalyst to help neutralize the acid by-products in the reaction.

[0098] Step S3: Use an acid catalyst to remove the propylidene protecting group of the diester crotonane diterpene intermediate to obtain the deuterated crotonane diterpene compound.

[0099] Under the action of an acid catalyst, the acetonide protecting groups at C5 and C20 will be removed, exposing the hydroxyl groups at C5 and C20, thereby obtaining the deuterated crotonane diterpene compound, which is the target compound of this application. Through a series of reactions, a deuterated crotonane diterpene compound is obtained. This compound has the characteristic structure of crotonane diterpenes, and at the same time, its pharmacokinetic properties (such as delaying metabolism, reducing toxicity, etc.) are improved through deuteration.

[0100] In this step, deprotection can expose specific hydroxyl groups, and the compound has the required biological activity and molecular stability.

[0101] The removal of the acetonide is usually carried out under acidic conditions, using PTSA or other strong acids (such as H2SO4) as catalysts. During the reaction, the reaction is usually carried out in a suitable solvent (such as toluene or tetrahydrofuran) while maintaining a suitable reaction temperature (such as room temperature or slightly higher temperature).

[0102] In some embodiments, the acid catalyst includes at least one of p-toluenesulfonic acid, camphorsulfonic acid, and hydrochloric acid;

[0103] Furthermore, in some preferred embodiments, the acid catalyst is p-toluenesulfonic acid.

[0104] As mentioned above, PTSA is the abbreviation of p-toluenesulfonic acid. The chemical structure of PTSA is p-p-toluenesulfonic acid (i.e., a sulfonic acid group is connected to the para position of the toluene molecule, and the chemical formula is C7H8SO3). It usually exists in the form of crystalline powder or anhydrous form and has strong acidity.

[0105] PTSA (p-toluenesulfonic acid) is used as an acid catalyst to remove the acetonide protecting groups at C5 and C20.

[0106] Furthermore, in step S1, the hydroxyl group of the crotonane diterpene raw material and the carboxyl group of S-2-methyl-butyric acid are subjected to an esterification reaction to obtain a monoester crotonane diterpene intermediate, including:

[0107] Step S11: Using triethylamine and 4-dimethylaminopyridine as catalysts, subject the crotonane diterpene raw material, S-2-methyl-butyric acid, and a condensing agent to an esterification reaction in dichloromethane to obtain a crude esterification product.

[0108] In the above, the raw material contains one or more hydroxyl groups, which are reactive groups of the reaction. Its structure can be phorbol-type crotonane diterpene with exposed hydroxyl groups, C6-C7 epoxy-type crotonane diterpene, or other types of crotonane diterpene compounds.

[0109] S-2-methyl-butyric acid is a compound with a carboxyl group (-COOH), which can react with the hydroxyl groups in the crotonane diterpene raw material to form an ester bond (-COO-), and finally obtain a crotonane diterpene intermediate with an ester group side chain.

[0110] Triethylamine and DMAP (4-Dimethylaminopyridine), as catalysts and basic catalysts, triethylamine neutralizes the acidic by-products generated in the reaction, and DMAP improves the selectivity and rate of the reaction. Especially in the esterification reaction, it can accelerate the activation of the carboxyl group to ensure the reaction efficiency and yield.

[0111] In some embodiments, the addition amount of the 4-dimethylaminopyridine is 0.2 equivalents.

[0112] In some embodiments, the condensing agent includes (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and / or N,N'-dicyclohexylcarbodiimide;

[0113] Further, the condensing agent is (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0114] EDCI, that is, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbamate, is an esterification reagent used to activate the carboxyl group, thereby promoting the reaction with the crotonane diterpene raw material to form an ester bond.

[0115] What is obtained through this step is a crude esterification product, which contains the target compound - monoester crotonane diterpene intermediate. Due to the participation of triethylamine and DMAP, this reaction has a high reaction efficiency and can reduce the formation of by-products.

[0116] This reaction method uses EDCI and DMAP as catalysts, has high reaction selectivity, few by-products, and can be carried out at room temperature with mild reaction conditions. By using triethylamine as a basic reagent, acidic by-products can be effectively neutralized, avoiding side reactions and ensuring the high efficiency of the reaction.

[0117] Specifically, the reaction can be carried out in dichloromethane. Since dichloromethane is a solvent with relatively weak polarity, it can effectively dissolve S-2-methyl-butyric acid and the cembrane diterpene raw material. EDCI is used to activate the carboxyl group in S-2-methyl-butyric acid, while DMAP improves the reaction rate and selectivity.

[0118] Step S12: Esterify the mono-ester cembrane diterpene intermediate with the deuterated side-chain carboxylic acid to obtain a bis-ester cembrane diterpene intermediate.

[0119] Silica gel column chromatography is a commonly used method for separation and purification, which is used to separate the target product from the reaction mixture. In this step, through silica gel column chromatography, unreacted raw materials, by-products and other impurities can be effectively removed, and a mono-ester cembrane diterpene intermediate with an ester group side chain and relatively high purity can be obtained.

[0120] In summary, the above steps include two key treatment parts: First, connect the cembrane diterpene raw material with S-2-methyl-butyric acid through an esterification reaction to form a mono-ester cembrane diterpene intermediate with an ester group side chain; then use silica gel column chromatography to purify and obtain the pure target compound. These steps effectively improve the selectivity, purity and reaction efficiency of the reaction. Especially after introducing the deuterated side chain, the finally synthesized deuterated cembrane diterpene compound has better pharmacokinetic properties and biological activities. By precise catalyst selection and control of reaction conditions, the high efficiency of each step of the reaction can be ensured, providing a reliable process route for the subsequent synthesis of deuterated cembrane diterpene compounds.

[0121] Further, step S2, in which the mono-ester cembrane diterpene intermediate is esterified with the deuterated side-chain carboxylic acid to obtain a bis-ester cembrane diterpene intermediate, includes:

[0122] Step S21: Dissolve the mono-ester cembrane diterpene intermediate in toluene to obtain a mixture.

[0123] First, dissolve the mono-ester cembrane diterpene intermediate obtained in the previous reaction step in toluene. Among them, toluene is an organic solvent that can effectively dissolve many non-polar and weakly polar organic compounds, including diterpene compounds. The purpose of this dissolution step is to create a homogeneous solution, which is convenient for the addition and reaction of various reagents in the subsequent reaction. The dissolved mixture provides a homogeneous reaction environment for the next acylation reaction.

[0124] Toluene has good solubility as a solvent, can maintain good dispersion of reactants and reagents, and ensure the smooth progress of the reaction.

[0125] Step S22: Using triethylamine and 4-dimethylaminopyridine as catalysts, add a strong acylating agent and a deuterated side-chain carboxylic acid to the mixture, and react the deuterated side-chain carboxylic acid with the ester group of the monoester crotonane diterpene intermediate in the mixture to form an esterified substance.

[0126] Among them, the strong acylating agent can be 2,4,6-trichlorobenzoyl chloride;

[0127] In some embodiments, the solvent in the ester group reaction includes at least one of toluene, tetrahydrofuran, and dichloromethane;

[0128] Furthermore, the solvent for the ester group reaction is toluene.

[0129] In the dissolved mixture, add triethylamine and 4-dimethylaminopyridine (DMAP). Among them, triethylamine serves as a basic catalyst, and its function is to neutralize the acidic by-products generated during the reaction, maintain the alkalinity of the reaction environment, and contribute to improving the rate and selectivity of the acylation reaction. DMAP serves as a catalyst and can promote the reaction by enhancing the nucleophilicity of the carboxyl group. Especially in the acylation reaction, it can significantly improve the reaction efficiency.

[0130] 2,4,6-Trichlorobenzoyl chloride (strong acylating agent) generates a mixed anhydride with the deuterated carboxylic acid in the presence of a base. With the help of DMAP, the mixed anhydride undergoes an esterification reaction with the alcohol. During the process, 2,4,6-trichlorobenzoyl chloride leaves in the form of 2,4,6-trichlorobenzoic acid. This esterification reaction connects the deuterated side-chain carboxylic acid to the C12 hydroxyl group of the monoester crotonane diterpene intermediate. The deuterated side-chain carboxylic acid refers to a side-chain with deuterium labeling (such as deuterated trans-2-methyl-2-butenoic acid or deuterated myristic acid side-chain), which will react with the ester group of the monoester crotonane diterpene intermediate to form a deuterated esterified product.

[0131] After this step, a double-ester-substituted substance, that is, a precursor of the deuterated crotonane diterpene compound, is obtained. The introduction of the deuterated side-chain can improve the stability of the compound and enhance the pharmacokinetic properties, showing obvious advantages.

[0132] Step S23: Quench with an aqueous sodium bicarbonate solution, extract and separate using dichloromethane, and then purify by silica gel column chromatography to obtain the double-ester crotonane diterpene intermediate.

[0133] In the above steps, the purpose of adding an aqueous sodium bicarbonate (NaHCO3) solution (which can be a saturated solution) is to neutralize the acidic substances (such as hydrogen chloride) that may be generated during the reaction, so that the reaction products are not affected by the acidic environment. Through liquid-liquid extraction with dichloromethane, the target product in the organic phase can be extracted from the aqueous solution and separated from the impurities.

[0134] The diesteryl crotane diterpene intermediate is further purified by silica gel column chromatography. Silica gel chromatography is a separation and purification technique that can effectively separate the target compound from impurities by adjusting the solvent system, resulting in a product with higher purity. Through column chromatography, a high-purity diesteryl crotane diterpene intermediate is obtained, providing high-quality raw materials for subsequent reactions.

[0135] Further, in step S3, the propylene acetal protecting group of the diesteryl crotane diterpene intermediate is removed using an acid catalyst to obtain the deuterated crotane diterpene compound, including:

[0136] Step S31: The diesteryl crotane diterpene intermediate and the acid catalyst are subjected to deprotection in a mixed solvent of acetonitrile and water to obtain a deprotected product.

[0137] The diesteryl crotane diterpene intermediate is obtained through a previous esterification reaction and contains a deuterated side chain, a non-deuterated side chain, and the parent ring part of crotane diterpene. PTSA is a strong acid catalyst used to promote certain chemical reactions such as esterification and deprotection. In this reaction, PTSA acts as an acid catalyst to remove the acetone acetal protecting groups at C5 and C20, exposing the hydroxyl groups at C5 and C20, thereby obtaining the deuterated crotane diterpene compound.

[0138] Acetonitrile is a polar organic solvent that can dissolve many organic compounds; the addition of water is usually to adjust the acidity of the reaction or help dissolve PTSA. Using this solvent system helps to improve the solubility and reaction efficiency of the reaction.

[0139] Step S32: The deprotected product is neutralized with an aqueous sodium carbonate solution to obtain a neutralized product.

[0140] As a weak basic reagent, the aqueous sodium carbonate solution can neutralize acidic by-products generated during the reaction, such as hydrogen chloride. The purpose of this step is to remove acidic substances to avoid their further influence on the product. After neutralization, the product becomes more stable, and the acidic by-products are effectively removed. Through neutralization with sodium carbonate, the obtained product is neutral or nearly neutral, suitable for subsequent extraction and purification steps.

[0141] This step can efficiently remove acidic by-products in the reaction, ensure the purity of the product, and provide a good basis for subsequent separation and purification.

[0142] Step S33: The neutralized product is subjected to extraction with ethyl acetate and purification by silica gel column chromatography to obtain the deuterated crotane diterpene compound.

[0143] Ethyl acetate is used to extract organic compounds from aqueous solutions. In this step, ethyl acetate is used to extract the target product from the aqueous phase into the organic phase to separate impurities. Column chromatography is a common method for separating and purifying compounds. By using silica gel as the packing material and adjusting the solvent system, the target product can be separated according to the polarity differences of the compounds. After extraction with ethyl acetate and purification by silica gel column chromatography, the purified deuterated crotonane diterpenoid compound, namely the target compound, is obtained.

[0144] Further, the deuterated side-chain carboxylic acid is trans-2-methyl-2-butene(3,4,4,4-d3) acid;

[0145] Further, the preparation method of the trans-2-methyl-2-butene(3,4,4,4-d3) acid comprises:

[0146] (1) After mixing triethyl diphosphate and an ether solvent, deuterated acetaldehyde is added to obtain a crude deuterated intermediate;

[0147] In some embodiments, the ether solvent includes at least one of ethylene glycol dimethyl ether, methyl tert-butyl ether, and diethyl ether;

[0148] Further, the ether solvent can be ethylene glycol dimethyl ether.

[0149] Triethyl diphosphate, as a phosphonium ylide reagent, can undergo a Witting-Horner reaction with deuterated acetaldehyde to form an alkene. In this reaction, the carbanion stabilized by triethyl diphosphate adds to deuterated acetaldehyde to form β-hydroxyphosphine oxide, which then reacts with sodium hydride to undergo an elimination reaction to obtain the crude deuterated intermediate.

[0150] As the ether solvent, ethylene glycol dimethyl ether has a high solubility and can help triethyl diphosphate and deuterated acetaldehyde dissolve fully and participate in the reaction.

[0151] Deuterated acetaldehyde (d2-Aldehyde) is a deuterium-labeled aldehyde compound, and the deuterium (D) therein will be introduced into the target molecule. The purpose of this step is to react deuterated acetaldehyde with triethyl diphosphate to form a deuterated intermediate, that is, to introduce deuterium into the target molecule.

[0152] The product of this step is the crude deuterated intermediate, which provides a deuterium-labeled molecular structure for subsequent synthesis steps. By this reaction, deuterium labeling can be stably introduced, which helps to improve the pharmacokinetic properties of the compound. Using ethylene glycol dimethyl ether as the solvent provides a good dissolution environment and promotes the smooth progress of the reaction.

[0153] (2) For the crude deuterated intermediate, the reaction is quenched with water, and back-extraction is carried out with ethylene glycol dimethyl ether, and after washing, the deuterated intermediate is obtained.

[0154] To terminate the reaction, adding water can rapidly stop the chemical reaction in the reaction mixture and prevent side reactions or over-reactions. The counter-extraction step using ethylene glycol dimethyl ether is to separate the target product in the organic phase from the aqueous phase. Ethylene glycol dimethyl ether is used for extraction because it can effectively dissolve organic substances and separate the target product. Through this step, the deuterated intermediate is separated and obtained.

[0155] (3) Hydrolyze the deuterated intermediate with an inorganic base, and perform quenching washing, drying treatment, and filtration concentration treatment on the reaction solution after hydrolysis with the inorganic base to obtain a crude deuterated side chain. In some embodiments, the inorganic base includes at least one of sodium hydroxide, potassium hydroxide, and potassium carbonate;

[0156] Further, the inorganic base is sodium hydroxide and / or potassium hydroxide;

[0157] In the above steps, a water / ethanol mixed solution is used to dissolve the deuterium-labeled intermediate, and the strong alkalinity of an inorganic base (such as sodium hydroxide, etc.) is used to hydrolyze the ester group to obtain a reaction solution of trans-2-methyl-2-butene (3,4,4,4-d3) acid.

[0158] Adjust the pH of the hydrolysis reaction product to acidic with hydrochloric acid, and extract with ethyl acetate to obtain an organic layer substance; concentrate the organic layer substance to obtain the crude trans-2-methyl-2-butene (3,4,4,4-d3) acid.

[0159] (4) Purify the crude deuterated side chain to obtain the trans-2-methyl-2-butene (3,4,4,4-d3) acid.

[0160] The crude deuterated side chain is further purified by appropriate methods, usually using methods such as acid-base treatment and vacuum distillation for purification. As an acidic substance, trans-2-methyl-2-butene (3,4,4,4-d3) acid can be separated from compounds with different pH values by acid-base treatment, and compounds with different boiling points can be effectively separated by vacuum distillation to remove impurities in the product and ensure high purity of the target product.

[0161] The purification step can be acid-base treatment and vacuum distillation to separate the target product.

[0162] Further, the purification treatment includes:

[0163] (1) The crude deuterated side chain is successively dissolved by adding an aqueous sodium bicarbonate solution and adjusting the pH with potassium hydroxide, and the product dissolved water layer is obtained after extracting impurities with ethyl acetate;

[0164] (2) Dissolve the product in the aqueous layer, adjust the pH to acidic with hydrochloric acid to free the product, and extract with ethyl acetate to obtain the organic layer substance;

[0165] (3) Concentrate the organic layer substance, purify it by vacuum distillation to obtain the trans-2-methyl-2-butenoic acid (3,4,4,4-d3).

[0166] In the above steps, the organic layer substance is dried with anhydrous sodium sulfate and then concentrated to obtain the acid-base reversed treatment product; the acid-base reversed treatment product is subjected to vacuum distillation to obtain the target trans-2-methyl-2-butenoic acid (3,4,4,4-d3).

[0167] In the embodiments of the present application, there is provided an application of a deuterated crotonane diterpene compound as described in any one of the foregoing embodiments in the preparation of an anti-tumor product.

[0168] The present invention will be further illustrated by specific examples below, but it should be understood that these examples are only for more detailed illustration and should not be construed as limiting the present invention in any form.

[0169] Example 1: Preparation of trans-2-methyl-2-butenoic acid (3,4,4,4-d3)

[0170] In this example, the synthesis of the target compound trans-2-methyl-2-butenoic acid (d2-R12) was carried out.

[0171] Target compound: Named d2-R12 in this example.

[0172] Experimental method:

[0173] The synthetic reaction route refers to Figure 1 .

[0174] (1) Add 18.6 g of ethylene glycol dimethyl ether and triethyl diphosphate (0.078 mol) to a four-necked flask. After dropping, stir for 0.5 h. After 0.5 h, dropwise add 0.078 mol of deuterated acetaldehyde; after dropping, react at 0 °C for 2 h. After 2 h, add water to quench, back-extract the aqueous layer with ethylene glycol dimethyl ether, combine the organic layers, wash with brine, and dry and concentrate the organic layer to obtain the crude product of d2-R12-1;

[0175] (2) Continue to add 5.2 g of potassium hydroxide / 50 mL of water and 100 mL of ethanol to the crude product, and react at room temperature overnight;

[0176] (3) After monitoring the reaction by TLC and finishing, adjust the pH to acidic with hydrochloric acid, extract twice with ethyl acetate, and combine the ethyl acetate layers and concentrate to dryness in mL;

[0177] (4) adding 100 mL of saturated sodium bicarbonate aqueous solution to dissolve, adjusting the pH to 8-9 with potassium hydroxide aqueous solution, extracting impurities with ethyl acetate, and after the extraction, adjusting the pH of the aqueous layer to 3-4, and extracting twice with 100 mL of ethyl acetate;

[0178] (5) The organic layer was dried and concentrated over anhydrous sodium sulfate, and purified by vacuum distillation to obtain 2.1 g of trans-2-methyl-2-butenoic acid (3,4,4,4-d3), i.e., the deuterated side chain carboxylic acid d2-R12.

[0179] Example 2: Synthesis of D12-TT

[0180] In this embodiment, the target deuterated crotonane diterpene compound was synthesized:

[0181] The chemical structure of the target compound is:

[0182] In this embodiment, it is named D12-TT.

[0183] Experimental methods:

[0184] Synthesis reaction route reference Figure 2 .

[0185] (1) Synthesis of P11:

[0186] The structure of the target compound is Named P11.

[0187] Reference: Practical synthesis of the therapeutic leads tigilanoltiglate and its analogues to obtain the target compound P11, 15g.

[0188] The quality test result of P11 is [M+Na] + :459.2013.

[0189] (2) Synthesis of P12:

[0190] The structure of the target compound is: Named P12.

[0191] Add dichloromethane (19 mL), S-2-methyl-butyric acid (545 mg, 5.193 mmol, 3 eq), EDCI (995 mg. 5.193 mmol, 3 eq), triethylamine (524 mg, 5.193 mmol, 3 eq), and DMAP (42 mg, 0.3462 mmol, 0.2 eq) into a 100 mL single-necked bottle and stir evenly;

[0192] Dissolve P11 (755 mg, 1.731 mmol, 1 eq) in 19 mL of tetrahydrofuran and add it to the above system; react at room temperature. After 2 h, monitor the reaction by TLC until it is complete, then add 2 mL of methanol and 50 mL of water to quench the reaction.

[0193] Extract the product with dichloromethane multiple times, combine the organic layers, dry over anhydrous sodium sulfate, filter and concentrate, and perform silica gel column chromatography (PE / EA = 10 / 1 - 1 / 2) to obtain 700 mg of P12, with a yield of 93%.

[0194] (3) Synthesis of D12 - P13:

[0195] The structure of the target compound is: It is named D12 - P13.

[0196] Add P12 (300 mg, 0.574 mmol, 1 eq) to a 50 mL single - necked flask, add 3 mL of toluene and stir to dissolve; add trans - 2 - methyl - 2 - butene(3,4,4,4 - d3) acid (the deuterated side - chain carboxylic acid d2 - R12 prepared in Example 1, 89.6 mg, 0.861 mmol, 1.5 eq), triethylamine (232 mg, 2.296 mmol, 4 eq), and 2,4,6 - trichlorobenzoyl chloride (280 mg, 1.148 mmol, 2 eq) and stir with 3 mL of toluene for 1 - 2 h, then add them to the above system, and finally add DMAP (182 mg, 1.492 mmol, 2.6 eq); react at room temperature. After 2 h, monitor the reaction by TLC until it ends, then add 20 mL of saturated sodium bicarbonate aqueous solution to quench the reaction; extract the product with dichloromethane multiple times, combine the organic layers, dry over anhydrous sodium sulfate, filter and concentrate, and perform silica gel column chromatography (PE / EA = 10 / 1 - 1 / 2) to obtain 270 mg of D12 - P13, with a yield of 82%.

[0197] The 1H NMR data of D12 - P13 are as follows: 11H-NMR (400 MHz, CDCl3) δ 7.60 (dd, J = 2.7, 1.5 Hz, 1H), 5.95 (s, 1H), 5.44 (d, J = 9.9 Hz, 1H), 4.14 (s, 1H), 4.07 - 4.01 (m, 1H), 3.94 (d, J = 12.9 Hz, 1H), 3.61 (d, J = 12.9 Hz, 1H), 3.37 (d, J = 1.1 Hz, 1H), 3.17 (d, J = 6.7 Hz, 1H), 3.01 (d, J = 1.0 Hz, 1H), 2.38 (dt, J = 13.7, 6.8 Hz, 1H), 1.99 - 1.89 (m, 1H), 1.82 (s, 3H), 1.75 (dd, J = 2.8, 1.3 Hz, 4H), 1.50 (s, 3H), 1.48 (s, 4H), 1.30 (s, 3H), 1.27 (d, J = 6.7 Hz, 1H), 1.25 (d, J = 2.7 Hz, 3H), 1.14 (d, J = 7.1 Hz, 3H), 0.94 (t, J = 7.5 Hz, 3H), 0.84 (d, J = 6.5 Hz, 3H).

[0198] The mass spectrometry detection result of D12 - P13 is: MS [M + Na] + : 629.3310.

[0199] (4) Synthesis of D12 - TT:

[0200] Add D1213 - P13 (270 mg, 0.463 mmol, 1 eq) to a 25 mL single - necked flask. After adding 2.7 mL of acetonitrile and 0.8 mL of water and stirring to dissolve, add PTSA (220 mg, 1.158 mmol, 2.5 eq);

[0201] React at room temperature. After 12 h, monitor the reaction by TLC until it is complete. Add 20 mL of saturated sodium bicarbonate aqueous solution to quench the reaction; Extract the product with ethyl acetate multiple times, combine the organic layers, dry over anhydrous sodium sulfate, filter and concentrate, and perform silica gel column chromatography (PE / EA = 10 / 1 - 1 / 2) to obtain 221 mg of D1213 - TT with a yield of 85.3%.

[0202] The 1H - NMR data of D12 - TT is (refer to Figure 3 ): 1H-NMR(400 MHz, CDCl3) δ 7.74 (s, 1H), 5.45 (d, J = 9.9 Hz, 1H), 4.23 (s, 1H), 4.08 (t, J = 2.8 Hz, 1H), 3.91 - 3.77 (m, 2H), 3.29 (s, 1H), 3.19 (d, J = 6.6 Hz, 1H), 2.40 (q, J = 7.0 Hz, 1H), 1.98 (dt, J = 9.7, 6.3 Hz, 1H), 1.82 (s, 3H), 1.78 - 1.68 (m, 4H), 1.47 (dq, J = 14.3, 7.4 Hz, 1H), 1.29 (d, J = 4.5 Hz, 1H), 1.27 (d, J = 2.9 Hz, 3H), 1.25 (d, J = 4.1 Hz, 3H), 1.14 (d, J = 7.0 Hz, 3H), 0.94 (t, J = 7.5 Hz, 3H), 0.87 (d, J = 6.6 Hz, 3H).

[0203] The carbon spectrum data of D12-TT is (refer to Figure 4 ): 13 C-NMR(101 MHz, CDCl3, 30 peaks total) δ 209.9, 178.9, 167.5, 164.7, 137.6, 133.5, 128.5, 77.3, 76.7, 72.3, 71.4, 65.5, 65.3, 64.5, 61.7, 48.8, 45.9, 41.2, 36.3, 36.0, 26.6, 26.2, 23.7, 17.2, 16.1, 15.1, 14.2, 12.2, 11.6, 9.9.

[0204] The mass spectrometry detection result of D12-TT is (refer to Figure 5 ): MS[M+Na] + : 589.3025.

[0205] Comparative Example 1: Synthesis of non-deuterated Tigilanol tiglat (TT)

[0206] In this Comparative Example 1, the synthesis of non-deuterated Tigilanol tiglat was carried out.

[0207] The structure of the target compound is: It is named TT in this example.

[0208] Experimental method:

[0209] The synthetic reaction route refers to Figure 6 . The synthetic method is basically the same as that in Example 2, except that:

[0210] In the synthesis of step (3) D12-P13, the added deuterated side chain carboxylic acid was replaced with S-2-methyl-butyric acid in this example.

[0211] The 1H NMR data of TT is (refer to Figure 7 ): 1 1H-NMR (400 MHz, CDCl3) δ 7.74 (dd, J = 2.7, 1.5 Hz, 1H), 6.82 (dddd, J = 8.5, 7.1, 5.8, 1.6 Hz, 1H), 5.45 (d, J = 9.9 Hz, 1H), 4.23 (d, J = 1.1 Hz, 1H), 4.08 (q, J = 2.8 Hz, 1H), 3.92 - 3.76 (m, 2H), 3.29 (d, J = 1.2 Hz, 1H), 3.19 (d, J = 6.6 Hz, 1H), 2.40 (h, J = 7.0 Hz, 1H), 1.97 (dq, J = 10.0, 6.5 Hz, 1H), 1.88 - 1.81 (m, 3H), 1.80 (dd, J = 7.0, 1.3 Hz, 3H), 1.78 - 1.70 (m, 4H), 1.47 (dd, J = 14.0, 7.0 Hz, 1H), 1.29 (d, J = 4.3 Hz, 1H), 1.27 (d, J = 4.7 Hz, 3H), 1.24 (s, 3H), 1.14 (d, J = 7.1 Hz, 3H), 0.94 (t, J = 7.5 Hz, 3H), 0.87 (d, J = 6.5 Hz, 3H).

[0212] The mass spectrometry detection result of TT is (refer to Figure 8 ): MS[M+Na] + : 585.2770.

[0213] Comparative Example 2: Synthesis of D12-PBE

[0214] In this Comparative Example 2, the synthesis of D12-PBE was carried out.

[0215] The structure of the target compound is: It is named D12-PBE in this example.

[0216] Experimental method: The synthetic reaction route refers to Figure 9 .

[0217] (1) Extraction of phorbol

[0218] Refer to Patent CN118908818A to extract phorbol, and 20 g of the target compound was obtained.

[0219] (2) Synthesis of PBE-1:

[0220] Add phorbol (19 g, 0.052 mol, 1 eq) to a 500 mL four-necked glass bottle, add 380 mL of DMF and stir to dissolve. Add imidazole (5.32 g, 0.078 mol, 1.5 eq), protect with nitrogen, and cool to 0 °C in an ice bath; add TBDMSCl (11.8 g, 0.078 mol, 1.5 eq) in portions, and maintain the reaction at 0 °C; after 2 h, monitor by TLC until the raw materials disappear, and the reaction ends. Slowly pour the reaction solution into 2 L of water, stir to crystallize, filter after 0.5 h, and rinse the solid with 500 mL of water; dissolve the solid in 200 mL of ethyl acetate, dry over anhydrous sodium sulfate, filter and concentrate; add 200 mL of petroleum ether to the concentrated solid and slurry for 1 h, filter after 1 h, and dry in vacuo to obtain 18 g of PBE-1, with a yield of 72%. MS [M+Na] + : 501.28.

[0221] (3) Synthesis of PBE-2:

[0222] Add dichloromethane (100 mL), S-2-methyl-butyric acid (3.4 g, 32.37 mmol, 3 eq), EDCI (6.2 g, 32.37 mmol, 3 eq), triethylamine (3.3 g, 32.37 mmol, 3 eq), and DMAP (4.0 g, 2.16 mmol, 0.2 eq) to a 250 mL single-necked flask and stir evenly; dissolve PEB-1 (5 g, 10.79 mmol, 1 eq) in 19 mL of tetrahydrofuran and add it to the above system; react at room temperature, monitor the reaction by TLC after 2 h until it is complete, add 5 mL of methanol and 250 mL of water to quench; extract the product with dichloromethane multiple times, combine the organic layers, dry over anhydrous sodium sulfate, filter and concentrate, and perform silica gel column chromatography (PE / EA = 10 / 1 - 1 / 2) to obtain 5.9 g of PEB-2, with a yield of 93%. MS [M+Na] + : 585.35.

[0223] (4) Synthesis of D12-PBE-3:

[0224] Add PBE-2 (5 g, 8.84 mmol, 1 eq) to a 250 mL single-necked flask, add 100 mL of toluene and stir to dissolve; add trans-2-methyl-2-butene (3,4,4,4-d3) acid (1.4 g, 13.27 mmol, 1.5 eq), triethylamine (3.6 g,

[0225] 35.38 mmol, 4 eq), 2,4,6-trichlorobenzoyl chloride (4.3 g, 17.69 mmol, 2 eq) were stirred in 14 mL of toluene for 1 - 2 h and then added to the above system. Finally, DMAP (2.8 g, 22.99 mmol, 2.6 eq) was added; the reaction was carried out at room temperature. After 2 h, the reaction was monitored by TLC and completed. Then, 200 mL of saturated sodium bicarbonate aqueous solution was added to quench the reaction; the product was extracted multiple times with dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated. Column chromatography on silica gel (PE / EA = 10 / 1 - 1 / 2) gave 4.9 g of D12 - PBE - 3 with a yield of 82%. MS [M + Na] + : 671.41.

[0226] (5) Synthesis of D12 - PBE:

[0227] Pyridine (12 mL, 3 v) was added to a tetrafluoride reaction kettle and cooled to 0 °C. Hydrogen fluoride pyridine complex (8 mL, 2 v) was added slowly in batches; D12 - PBE - 3 (4 g) was dissolved in 20 mL of tetrahydrofuran and 20 mL of methanol and added to the above system; the reaction was carried out at room temperature. The reaction progress was monitored by TLC. After 2 h, the reaction was completed. The reaction solution was slowly poured into a mixed solution of 300 mL of saturated sodium bicarbonate / 300 mL of dichloromethane to quench the reaction. After stirring for 0.5 h, the insoluble substances were filtered off and separated; the organic layer was washed with 1 M hydrochloric acid aqueous solution (200 mL); after separation, the organic layer was dried over anhydrous sodium sulfate and concentrated. After concentration to dryness, crystallization with n - hexane gave 2.9 g of PBE with a yield of 89.4%. MS [M + Na] + : 557.33.

[0228] Comparative experiment 1: Pharmacological investigation

[0229] In this example, for the compound D12 - TT prepared in Example 2 and the compound TT prepared in Comparative Example 1, pharmacological experiments were carried out respectively to investigate the anti - tumor effects of different compounds.

[0230] Experimental method:

[0231] 1. Preparation of reagents:

[0232] Target samples to be measured: The compound D12 - TT prepared in Example 2 and the compound TT prepared in Comparative Example 1.

[0233] Solution preparation: Accurately weigh 2 mg of the target sample to be tested (taking TT as an example), add 133 μL of propylene glycol, and prepare a mother solution of TT at 15 mg / mL, then vortex and mix well. Before the experiment on the same day, pipette 60 μL of the 15 mg / mL TT mother solution into 540 μL of propylene glycol to prepare a 1.5 mg / mL TT solution. Pipette 900 μL of 30 mM sodium acetate (pH = 4.2) into 600 μL of the 1.5 mg / mL TT solution to obtain a 0.6 mg / mL TT solution.

[0234] 2. Establishment of animal model:

[0235] (1) Before inoculating the mice, shave the hair on their ventral and dorsal parts to form an exposed area of about 2 cm × 2 cm. Centrifuge the mouse melanoma B16F10 cell line at 1000 r / min for 5 minutes.

[0236] (2) Detect the cell viability by 0.4% trypan blue exclusion method (viability > 90%). Wash the cells 3 times with PBS and centrifuge at 1000 r / min for 5 minutes. Inject 100 μL of B16F10 per mouse subcutaneously into the left lumbar and dorsal part of the mouse routinely, so that the inoculation quantity per animal is 2×10 6 B16F10. 6 cells.

[0237] (3) Observe the tumor formation in mice on the 4th and 7th days after inoculation. When the average tumor volume is about 100 - 150 mm 3 , group the mice, and select mice with appropriate tumor volumes and randomly assign them to each treatment group. See Table 1 for details of the administration route, dose, and regimen. Measure the tumor volume of the mice every 2 days after administration.

[0238] 3. Determination of animal body weight and tumor volume:

[0239] (1) Measure the animal body weight 3 times a week and detect the weight change, and draw the body weight curve of the tumor. After inoculating the tumor cells, visually observe the inoculation site every day, and record the time of tumor appearance when the mass can be palpated. At the same time, record the tumor color, texture, shape, and location.

[0240] (2) Measure the tumor volume once every 2 days. Use a precision vernier caliper to measure the longest diameter (a) and the shortest diameter (b) perpendicular to it of the tumor, and calculate the approximate volume of the tumor according to the formula V = 0.5×ab 2 ;

[0241] (3) After administering TT (the same treatment for other target samples to be tested), continue to monitor the change in tumor size until the end of the experiment, and draw the growth curve of the tumor.

[0242] Experimental results:

[0243] Table 1. Administration design table for each group

[0244] Group Number of animals Injected drug Dose administered Route of administration G1 6 Vehicle(40%PG) 30ug Intratumorally G2 6 TT 30ug Intratumorally G3 6 D12-TT 30ug Intratumorally

[0245] (1) As Figure 10 shown, the Vehicle group (control group, 40% PG, 40% propylene glycol), TT group, and D12-TT group had no significant effect on the body weight of mice, indicating that neither TT nor D12-TT had significant toxicity.

[0246] (2) Figure 11 The results showed that after 1 intratumoral injection of the drug, 3 out of 6 mice in the TT group had complete tumor remission, and the complete remission rate CR was 50%. All 6 mice in the D12-TT group had complete tumor remission, and the complete remission rate CR was 100%.

[0247] Comparative experiment 2: Detection of the anti-tumor effect of the comparative example D12-PBE

[0248] Experimental method:

[0249] 1. Preparation of reagents: Accurately weigh 2 mg of the target sample to be measured D12-PBE (the product in comparative example 2), add 133 μL of propylene glycol, and prepare a 15 mg / mL D12-PBE stock solution, vortex and mix well. Before the experiment on the same day, pipette 60 μL of the 15 mg / mL D12-PBE stock solution into 540 μL of propylene glycol to prepare a 1.5 mg / mL D12-PBE solution. Pipette 900 μL of 30 mM sodium acetate (pH = 4.2) into 600 μL of the 1.5 mg / mL D12-PBE solution to obtain a 0.6 mg / mL D12-PBE solution.

[0250] 2. Establishment of animal models and determination of animal tumor volume: The same as in comparative experiment 1.

[0251] Experimental results:

[0252] Reference Figure 12 , after 1 intratumoral injection of the drug, it had a certain inhibitory effect on the tumor in the early stage, but the tumors would all relapse. This indicates that D12-PBE has no anti-tumor effect, and this result proves that if the parent ring structure is changed, deuteration at the 12th position has no anti-tumor effect.

[0253] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A deuterated crotonane diterpenoid compound, characterized in that, The deuterated tigliane diterpenoid compound includes a compound represented by the following general formula structure and a pharmaceutically acceptable salt thereof: Wherein, R1 is a deuterated side chain; R2 is a non-deuterated side chain different from R1.

2. The deuterated tigliane diterpenoid compound according to claim 1, wherein, The deuterated side chain includes at least one of deuterated trans-2-methyl-2-butenoic acid substituents and deuterated myristic acid substituents; Preferably, the deuterated side chain is trans-2-methyl-2-butene(3,4,4,4-d3) acid.

3. The deuterated tigliane diterpenoid compound according to claim 1, wherein The non-deuterated side chain includes at least one of S-2-methyl-butyric acid substituents and acetic acid substituents; Preferably, the non-deuterated side chain is S-2-methyl-butyric acid.

4. The deuterated tigliane diterpenoid compound according to claim 1, wherein, The compound structure of the deuterated tigliane diterpenoid compound is:

5. A preparation method of a deuterated tigliane diterpenoid compound, characterized in that, Including: Esterifying the hydroxyl group of the tigliane diterpenoid raw material with the carboxyl group of S-2-methyl-butyric acid to obtain a monoester tigliane diterpenoid intermediate; Esterifying the monoester tigliane diterpenoid intermediate with a deuterated side chain carboxylic acid to obtain a diester tigliane diterpenoid intermediate; Removing the isopropylidene protecting group of the diester tigliane diterpenoid intermediate with an acid catalyst to obtain the deuterated tigliane diterpenoid compound; Preferably, the acid catalyst includes at least one of p-toluenesulfonic acid, camphorsulfonic acid, and hydrochloric acid; Preferably, the acid catalyst is p-toluenesulfonic acid.

6. The preparation method of the deuterated tigliane diterpenoid compound according to claim 5, wherein, The step of esterifying the hydroxyl group of the tigliane diterpenoid raw material with the carboxyl group of S-2-methyl-butyric acid to obtain a monoester tigliane diterpenoid intermediate includes: Using triethylamine and 4-dimethylaminopyridine as catalysts, carrying out an esterification reaction on the tigliane diterpenoid raw material, S-2-methyl-butyric acid, and a condensing agent in dichloromethane to obtain a crude esterification product; Purifying the crude esterification product by silica gel column chromatography to obtain the monoester tigliane diterpenoid intermediate; Preferably, the condensing agent includes (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and / or N,N'-dicyclohexylcarbodiimide; Preferably, the condensing agent is (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; Preferably, the addition amount of 4-dimethylaminopyridine is 0.2 equivalents.

7. The preparation method of the deuterated tigliane diterpenoid compound according to claim 5, wherein The step of esterifying the monoester tigliane diterpenoid intermediate with a deuterated side chain carboxylic acid to obtain a diester tigliane diterpenoid intermediate includes: Dissolving the monoester tigliane diterpenoid intermediate in toluene to obtain a mixture; Using triethylamine and 4-dimethylaminopyridine as catalysts, adding a strong acylating agent and a deuterated side chain carboxylic acid to the mixture, and reacting the deuterated side chain carboxylic acid with the ester group of the monoester tigliane diterpenoid intermediate in the mixture to form an esterification substance; Quenching with an aqueous sodium bicarbonate solution, extracting and separating with dichloromethane, and then purifying by silica gel column chromatography to obtain the diester tigliane diterpenoid intermediate; Preferably, the strong acylating agent is 2,4,6-trichlorobenzoyl chloride; Preferably, the solvent in the ester group reaction includes at least one of toluene, tetrahydrofuran, and dichloromethane; Preferably, the solvent for the ester group reaction is toluene.

8. The preparation method of the deuterated tigliane diterpenoid compound according to claim 5, characterized in that, The step of removing the isopropylidene protecting group of the diester tigliane diterpenoid intermediate with an acid catalyst to obtain the deuterated tigliane diterpenoid compound includes: The double-ester crotonane diterpene intermediate and an acid catalyst are deprotected in a mixed solvent of acetonitrile and water to obtain a deprotected product; The deprotected product is neutralized with an aqueous sodium carbonate solution to obtain a neutralized product; The neutralized product is purified by ethyl acetate extraction and silica gel column chromatography respectively to obtain the deuterated crotonane diterpene compound.

9. The preparation method of the deuterated tigliane diterpenoid compound according to claim 5, wherein, The deuterated side-chain carboxylic acid is trans-2-methyl-2-butene(3,4,4,4-d3) acid; Preferably, the preparation method of the trans-2-methyl-2-butene(3,4,4,4-d3) acid includes: After mixing triethyl diphosphate and an ether solvent, deuterated acetaldehyde is added to obtain a crude deuterated intermediate; Regarding the crude deuterated intermediate, the reaction is quenched with water, and back-extracted with ethylene glycol dimethyl ether. After washing, a deuterated intermediate is obtained; The deuterated intermediate is hydrolyzed with an inorganic base, and the reaction solution after hydrolysis with the inorganic base is quenched, washed, dried and filtered and concentrated to obtain a crude deuterated side chain; The crude deuterated side chain is purified to obtain the trans-2-methyl-2-butene(3,4,4,4-d3) acid; Preferably, the ether solvent includes at least one of ethylene glycol dimethyl ether, methyl tert-butyl ether and diethyl ether; Preferably, the ether solvent is ethylene glycol dimethyl ether; Preferably, the inorganic base includes at least one of sodium hydroxide, potassium hydroxide and potassium carbonate; Preferably, the inorganic base is sodium hydroxide and / or potassium hydroxide; Preferably, the purification treatment includes: The crude deuterated side chain is successively dissolved by adding an aqueous sodium bicarbonate solution and potassium hydroxide to adjust the pH. After impurities are extracted with ethyl acetate, a product-dissolved aqueous layer is obtained; The pH of the product-dissolved aqueous layer is adjusted to acidic with hydrochloric acid to free the product, and the product is extracted with ethyl acetate to obtain an organic layer substance; The organic layer substance is concentrated and purified by vacuum distillation to obtain the trans-2-methyl-2-butene(3,4,4,4-d3) acid.

10. Use of a deuterated crotonane diterpene compound according to any one of claims 1-4 in the preparation of an anti-tumor product.

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