Pentacyclic triterpene derivatives, methods of chemical enzymatic synthesis and uses thereof

Abstract

The application discloses a pentacyclic triterpene derivative shown in a general formula I or a pharmaceutically acceptable salt thereof, and further discloses a method for preparing the pentacyclic triterpene derivative by a chemical enzyme method, and application of the pentacyclic triterpene derivative or the pharmaceutically acceptable salt thereof in preparation of a drug for treating and preventing tumors. The hydroxyl group is used to further derive the B ring and / or the D ring of glycyrrhetinic acid for the first time; the novel pentacyclic triterpene molecule containing multiple Michael reaction acceptor modules in the structure is synthesized by taking the synthetic electrophilic warhead as the basis for the derivation of glycyrrhetinic acid; and through cell toxicity screening of the novel compound in the application, a drug lead compound with good inhibitory effect on human prostate cancer cells PC-3, human breast cancer cells MCF-7, human colon cancer cells SW480 and human cervical cancer cells Hela is found.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to novel pentacyclic triterpenoid derivatives containing multiple Michael response receptor modules in their structure, as well as their chemical enzyme synthesis methods and applications. Background Technology

[0002] In recent years, covalent inhibitors have received increasing attention in drug development. After binding to their biological targets in vivo, covalent inhibitors exert their therapeutic effect by binding to nucleophilic residues of the target protein via electrophilic warheads in their structure. Among commonly used covalent inhibitors targeting cysteine, the most frequently used electrophilic warhead is the Michael receptor. A Michael receptor is a functional group formed by the conjugation of an electron-withdrawing group to an alkene or alkyne. Chemical molecules containing this functional group are called Michael receptor molecules and readily undergo Michael reactions with nucleophiles. These molecules inhibit cysteine-containing proteins during physiological processes and are therefore considered important physiologically active molecules, participating in many biological processes and regulating various signaling pathways in the body.

[0003] Methylbardoxolone (CDDO-Me) and its derivatives are well-known covalent inhibitors of pentacyclic triterpenoids. These molecules possess potent antitumor and anti-inflammatory effects and can act on multiple signaling pathways in vivo. Structure-activity relationship studies have shown that the two Michael receptors in their molecular structure—the α-cyanoenone in ring A and the enone in ring C—are important pharmacological functional groups for their activity. Therefore, structural modification by mimicking the pharmacological functional groups of CDDO-Me series compounds and exploring the potential pharmacological activities of derivatives is currently a research hotspot in pentacyclic triterpenoids.

[0004] Pentacyclic triterpenoids exhibit high structural skeletal similarity, consisting of five fused cyclohexane and / or cyclopentane groups. The C3 hydroxyl group, the olefinic group, and the carboxyl group on the skeletal framework are commonly used chemical handles in structural modifications. While adding more Michael reaction acceptors to the pentacyclic triterpenoid skeletal molecules is crucial for enhancing their pharmacological activity, the lack of effective organic synthetic methods for site- and regioselective activation of the numerous inert CH bonds on their skeletal framework limits the application of pentacyclic triterpenoid molecules containing multiple Michael reaction acceptor modules (multi-Michael reaction acceptor pentacyclic triterpenoid molecules) in natural product medicinal chemistry. Taking glycyrrhetinic acid as an example, the inert CH bonds on its B and D rings have similar chemical environments, lacking organic synthetic means for selective CH activation. Therefore, current structural modifications are mostly limited to the C3 hydroxyl group on the A ring, the 11-oxo-12-ene Michael reaction acceptor on the C ring, and the C30 carboxyl group. Structural modifications and structure-activity relationships on the B and D rings remain to be explored. Summary of the Invention:

[0005] Purpose of the invention: Based on the previously discovered function of cytochrome oxidase CYP161H12 selectively introducing hydroxyl functional groups at the C7β and C15α positions of glycyrrhetinic acid, the technical problem to be solved by the present invention is to provide a novel multi-Michael response receptor pentacyclic triterpenoid molecule for preparation and synthesis.

[0006] Another technical problem to be solved by the present invention is to provide a method for synthesizing pentacyclic triterpenoid derivatives or their pharmaceutically usable salts by modifying the B and D rings of glycyrrhetinic acid using a chemical enzymatic method.

[0007] The final technical problem to be solved by this invention is to provide the application of multi-Michael response receptor pentacyclic triterpenoid molecules in the preparation of drugs for treating / preventing tumors.

[0008] Technical solution: To solve the above-mentioned technical problems, the present invention provides a pentacyclic triterpenoid derivative of general formula I or a pharmaceutically acceptable salt thereof:

[0009]

[0010] Where: A is C or CH; B is C or CH; C is C or CH; D is C or CH; E is C or CH; F is C or CH; R1 is H, I or CN; R2 is O, OH or acetoxy; R3 is O, H or OH; R4 is O, H or OH.

[0011] As a preferred option, its structural formula is shown below:

[0012]

[0013] Where A is C or CH; B is C or CH; R1 is H, I or CN; R2 is OH or acetoxy.

[0014] The derivative or its pharmaceutically acceptable salt includes stereoisomers, hydrates, metabolites, deuterated products, solvates, or pharmaceutically acceptable salts or eutectics.

[0015] Of particular preference of the present invention is a compound having the following structure:

[0016] The structure of a compound containing two Michael response acceptors is as follows:

[0017] 3,7,11,15-Tetraoxo-18β-oleanane-1,12-diene-30-carboxylic acid methyl ester (Example 5)

[0018] 2-Iodo-3,7,11,15-tetraoxo-18β-oleanane-1,12-diene-30-carboxylic acid methyl ester (Example 6)

[0019] 2-Cyano-3,7,11,15-Tetraoxo-18β-oleanane-1,12-diene-30-carboxylic acid methyl ester (Example 7)

[0020] 7,11-Dioxo-3β-acetoxy-18β-oleanolane-5,12-diene-30-carboxylic acid methyl ester (Example 11)

[0021] 7,11-Dioxo-3β-acetoxy-oleanolane-5,12,18-triene-30-carboxylic acid methyl ester (Example 12)

[0022] 7,11-Dioxo-3β-hydroxy-oleanolane-5,12,18-triene-30-carboxylic acid methyl ester (Example 13)

[0023] The structure of a compound containing three Michael response acceptors is as follows:

[0024] 3,7,11-Trioxo-oleanane-1,5,12,18-tetraene-30-carboxylic acid methyl ester (Example 14)

[0025] 2-Iodo-3,7,11-trioxo-oleanane-1,5,12,18-tetraene-30-carboxylic acid methyl ester (Example 15)

[0026] 2-Cyano-3,7,11-trioxo-oleanane-1,5,12,18-tetraene-30-carboxylic acid methyl ester (Example 16)

[0027] The present invention also includes the method for preparing pentacyclic triterpenoid derivatives by the aforementioned chemical-enzymatic method, the preparation method being as follows:

[0028]

[0029] Where: A is C or CH; B is C or CH; C is C or CH; D is C or CH; E is C or CH; F is C or CH; R1 is H, I or CN; R2 is O, OH or acetoxy; R3 is O, H or OH; R4 is O, H or OH.

[0030] This invention provides a method for synthesizing compound I using a chemical-enzymatic approach, with the following three synthetic routes.

[0031] Among them, the first synthetic route:

[0032]

[0033] The specific steps are as follows:

[0034] Step a: Mix substrate 1, oxidase, cofactor, and coenzyme regeneration system to carry out an enzymatic reaction;

[0035] Step b: Mix substrate 2, potassium carbonate, iodomethane and solvent to react and obtain the methyl ester product;

[0036] Step c: The substrate 3,2-iodobenzoic acid and solvent are mixed and reacted to obtain the oxidation product;

[0037] Step d: Mix substrate 4, elemental iodine, 4-dimethylaminopyridine and solvent to react and obtain the iodine-substituted product;

[0038] Step e: Mix substrate 5, potassium iodide, cuprous cyanide and solvent, and react under a protective atmosphere to obtain the cyano-substituted product.

[0039] Synthesis Route 2:

[0040]

[0041] Specifically, the steps include the following: the steps include steps a and b, and also include the following steps:

[0042] Step f: Mix substrate 9, Jones reagent and solvent to react and obtain the oxidation product;

[0043] Step g: The substrate 10, elemental bromine, hydrogen bromide and solvent are mixed and reacted to obtain a mixed intermediate;

[0044] Step h: The intermediate mixture from step g is mixed with chlortetramine to undergo an elimination reaction to obtain the product.

[0045] Synthesis Route 3:

[0046]

[0047] The steps include steps a, b, and f, steps c, d, and e, and also include the following steps:

[0048] Step i: Mix substrate 11, pyridinium tribromide and solvent to react and obtain the oxidation product.

[0049] Step j: Mix substrate 12, potassium hydroxide and solvent to react and obtain a mixture of deacetylated and partially demethylated products.

[0050] Preferably, the substrate 1 in step a includes compound 1 or compound 7; the oxidase is CYP161H12-RHFRed; the cofactors are NADP+ and Na2HPO3; and the coenzyme regeneration system is phosphite dehydrogenase.

[0051] Preferably, the oxidase-CYP161H12-RHFRed in step a participates in the reaction in the form of cell lysate, using 1-4 L of TB culture medium to ferment the obtained cells. The specific preparation method is described in Example 1. The coenzyme regeneration system-phosphite dehydrogenase is used in the form of cell lysate, using 0.5-2 L of TB culture medium to ferment the obtained cells. The specific preparation method is described in Example 2. The molar ratio of substrate 1, sodium NADP, and Na2HPO3 is 1:0.5:50. The enzyme-catalyzed reaction temperature is 20℃. The enzyme-catalyzed reaction time is 20 h.

[0052] Wherein, the substrate 2 mentioned in step b includes chemical 2 or compound 8 or a mixture of deacetylated and partially demethylated products from step j, the molar ratio of substrate 2, potassium carbonate and iodomethane is 1:3-4:1.3-2, the solvent is 4-10 mL N,N-dimethylformamide, the reaction temperature is 18-35°C, and the reaction time is 2-3 h;

[0053] Preferably, the substrate 3 in step c includes compound 3, compound 10, or compound 13, the molar ratio of substrate 3 to 2-iodoacyl group is 1:4 to 8, the solvent is 2 to 15 mL of dimethyl sulfoxide, the reaction temperature is 100 to 110 °C, and the reaction time is 10 to 15 h.

[0054] Preferably, the substrate 4 in step d includes compound 4 or compound 14, the molar ratio of the substrate, elemental iodine and 4-dimethylaminopyridine is 1:3:0.1-0.2, the solvent is a mixed solvent of pyridine and carbon tetrachloride in a volume ratio of 1:1, the reaction temperature is 90-100°C, and the reaction time is 12-15 h.

[0055] Preferably, the substrate 5 in step e includes compound 5 and compound 15, the molar ratio of the substrate, potassium iodide and cuprous cyanide is 1:0.2:1.5-2, the protective atmosphere is nitrogen or argon, the reaction temperature is 120°C, and the reaction time is 3.5 h.

[0056] Preferably, the molar volume ratio of substrate 9 and Jones reagent in step f is 0.33 mmol: 0.4-0.5 mL, the solvent is 4-5 mL, the reaction temperature is 0℃-35℃, and the reaction time is 1-2 h.

[0057] Preferably, the substrate 10 in step g is compound 10, the molar ratio of substrate 10, elemental bromine, and hydrogen bromide is 1:10:1.8–2, the solvent is 3–4 mL of N,N-dimethylformamide, and the reaction temperature is 18–35 °C.

[0058] Preferably, the substrate 11 in step i is compound 10, the molar ratio of the substrate 11 to pyridinium tribromide is 1:2-3, the solvent is 2-3 mL acetonitrile, the reaction temperature is 37-40 °C, and the reaction time is 36-48 h.

[0059] Preferably, the substrate 12 in step j is compound 12, the molar ratio of substrate 12 to potassium hydroxide is 1:85-100, the solvent is 8-10 mL of methanol, the reaction temperature is 18-35°C, and the reaction time is 2-3 h.

[0060] The present invention also includes the use of the aforementioned pentacyclic triterpenoid derivatives or their pharmaceutically usable salts in the preparation of drugs for treating / preventing tumors.

[0061] For the particularly preferred pentacyclic triterpenoid molecules represented by Formula I, in vitro activity screening revealed that these molecules exhibited significant antitumor activity. Specifically, a pentacyclic triterpenoid molecule containing three Michael receptors (Example 14) with ring A containing an iodoenone structure and rings B and C having unsaturated enones, and a pentacyclic triterpenoid molecule containing two Michael receptors (Example 5) with ring A containing a cyanoenone structure and ring C having unsaturated enones, showed good antitumor activity. Furthermore, a pentacyclic triterpenoid molecule containing three Michael receptors (Example 15) with ring A containing a cyanoenone structure and rings B and C having unsaturated enones exhibited the most significant antitumor activity and has the potential to serve as a drug lead compound.

[0062] Beneficial Effects: Compared with the prior art, the present invention has the following advantages: The present invention utilizes the selective CH bond hydroxylation of glycyrrhetinic acid by cytochrome CYP161H12 to synthesize glycyrrhetinic acid derivatives with B-ring C7β and / or D-ring C15α hydroxylation that cannot be obtained by traditional chemical synthesis. Furthermore, the present invention utilizes hydroxyl groups to further derivatize the B-ring and / or D-ring of glycyrrhetinic acid. The present invention uses the synthesis of electrophilic warheads as the basis for glycyrrhetinic acid derivatization, synthesizing novel pentacyclic triterpenoid molecules with multiple Michael response receptors. Through cytotoxicity screening of the novel compounds in the present invention, drug lead compounds with good inhibitory effects on human prostate cancer cells PC-3, human breast cancer cells MCF-7, human colon cancer cells SW480, and human cervical cancer cells HeLa were discovered. Attached Figure Description

[0063] Figure 1 The proton NMR spectrum of compound 3 ( 1 H NMR spectrum,pyridine-d5);

[0064] Figure 2 Carbon NMR spectrum of compound 3 ( 13 C NMR spectrum,pyridine-d5);

[0065] Figure 3 The proton NMR spectrum of compound 4 ( 1 H NMR spectrum (CDCl3);

[0066] Figure 4 Carbon NMR spectrum of compound 4 13 C NMR spectrum (CDCl3);

[0067] Figure 5 The proton NMR spectrum of compound 5 ( 1 H NMR spectrum (CDCl3);

[0068] Figure 6 Carbon NMR spectrum of compound 5 13 C NMR spectrum (CDCl3);

[0069] Figure 7 The proton NMR spectrum of compound 6 1 H NMR spectrum (CDCl3);

[0070] Figure 8 Carbon NMR spectrum of compound 6 13 C NMR spectrum (CDCl3);

[0071] Figure 9 The proton NMR spectrum of compound 9 ( 1 H NMR spectrum (CDCl3);

[0072] Figure 10 Carbon NMR spectrum of compound 9 ( 13 C NMR spectrum (CDCl3);

[0073] Figure 11 The proton NMR spectrum of compound 10 ( 1 H NMR spectrum (CDCl3);

[0074] Figure 12 Carbon NMR spectrum of compound 10 ( 13 C NMR spectrum (CDCl3);

[0075] Figure 13 The proton NMR spectrum of compound 11 ( 1H NMR spectrum (CDCl3);

[0076] Figure 14 Carbon NMR spectrum of compound 11 ( 13 C NMR spectrum (CDCl3);

[0077] Figure 15 The proton NMR spectrum of compound 12 ( 1 H NMR spectrum (CDCl3);

[0078] Figure 16 Carbon NMR spectrum of compound 12 ( 13 C NMR spectrum (CDCl3);

[0079] Figure 17 The proton NMR spectrum of compound 13 ( 1 H NMR spectrum (CDCl3);

[0080] Figure 18 Carbon NMR spectrum of compound 13 ( 13 C NMR spectrum (CDCl3);

[0081] Figure 19 The proton NMR spectrum of compound 14 ( 1 H NMR spectrum (CDCl3);

[0082] Figure 20 Carbon NMR spectrum of compound 14 13 C NMR spectrum (CDCl3);

[0083] Figure 21 The proton NMR spectrum of compound 15 ( 1 H NMR spectrum (CDCl3);

[0084] Figure 22 Carbon NMR spectrum of compound 15 ( 13 C NMR spectrum (CDCl3);

[0085] Figure 23 The proton NMR spectrum of compound 16 ( 1 H NMR spectrum (CDCl3);

[0086] Figure 24 Carbon NMR spectrum of compound 16 13 C NMR spectrum (CDCl3). Detailed Implementation

[0087] Example 1: Preparation of cell lysate for CYP161H12-RHFRed

[0088] To prepare CYP161H12-RHFRed cell lysate, the recombinant expression vector pET28a-CYP161H12-RHFRed synthesized by Anhui General Biotechnology Co., Ltd. (pET28a is linked to the nucleotide sequence of CYP161H12-RHFRed, where the amino acid and nucleotide sequences of CYP161H12-RHFRed are shown in SEC ID NO 1 and SEQ ID NO 2) was transformed into Escherichia coli BL21. The heterologously expressed BL21 was inoculated into 5 mL of LB medium and cultured at 37℃ and 220 rpm for 10 h. This was the seed culture. Seed culture was added to sterile TB medium at an inoculum volume of 0.3% (v / v), along with 0.1% (v / v) 50 mg / mL kanamycin and 0.1% (v / v) trace metal salt solution (components including 50 mM FeCl3, 20 mM CaCl2, 10 mM MnSO4, 10 mM ZnSO4, 2 mM CoSO4, 2 mM CuCl2, 2 mM NiCl2, 2 mM Na2MoO4, and 2 mM H3BO3). The medium was incubated at 37°C and 220 rpm until the OD value reached 0.8. After cooling in an ice-water bath for 20 min, 0.01% (v / v) 1M isopropyl-β-D-thiogalactoside (IPTG) and 0.1% (v / v) 0.5M 5-aminolevulinic acid (5-ALA) were added to induce bacterial expression of the CYP161H12-RHFRed fusion protein. The cells were induced and cultured at 18℃ and 200 rpm for 20 h. The cells were collected by centrifugation at 3750 rpm for 15 min, resuspended in phosphate buffer to OD600 = 30, and sonicated at 60% power for 5 min (1 s on / 5 s off).

[0089] The formulation of TB medium is as follows: 11.8g tryptone, 23.6g yeast extract, dissolved in 0.95L distilled water in 4mL glycerol, autoclaved at 121℃ for 20 minutes, cooled to 60℃, and 0.05L sterile phosphorus source is added to obtain 1LTB medium; wherein, the sterile phosphorus source includes: 2.2g KH2PO4 and 12.3g K2HPO4·3H2O dissolved in 0.05L distilled water, autoclaved at 121℃ for 20 minutes;

[0090] The phosphate buffer formulation is as follows: 14.25g K2HPO4·3H2O and 5.1g KH2PO4 are dissolved in 1L of distilled water to obtain a phosphate buffer with pH=7 and a concentration of 0.1M.

[0091] Example 2: Preparation of cell lysate for phosphorylated dehydrogenase

[0092] To prepare the phosphorylated dehydrogenase cell lysate, the recombinant expression vector pETDuet-opt13 (obtained by linking the pETDuet and opt13 genes, with the amino acid sequence of opt13 shown in SEQ ID NO 3 and the nucleotide sequence shown in SEQ ID NO 4) synthesized by Anhui General Biotechnology Co., Ltd. was transformed into *E. coli* BL21. The heterologously expressed BL21 was inoculated into 5 mL of LB medium and cultured at 37°C and 220 rpm for 10 h; this was the seed culture. The seed culture was added to sterile TB medium at an inoculation rate of 0.3% (v / v), along with 0.1% (v / v) 100 mg / mL ampicillin. The medium was cultured at 37°C and 220 rpm until the OD600 value reached 0.8. After cooling in an ice-water bath for 20 min, 0.01% (v / v) 1M isopropyl-β-D-thiogalactoside (IPTG) was added to induce bacterial expression of opt13 protein. The cells were then cultured at 18°C ​​and 200 rpm for 20 h. The cells were collected by centrifugation at 3750 rpm for 15 min, resuspended in phosphate buffer to OD600 = 30, and sonicated at 60% power for 5 min (1 s on / 5 s off).

[0093] Example 3: Enzymatic preparation of a single oxidation product: 11-oxo-3β,7β,15α-trihydroxy-18β-oleanol-12-ene-30-carboxylic acid

[0094]

[0095] This embodiment requires two consecutive enzymatic catalysis steps to obtain a single oxidation product, 11-oxo-3β,7β,15α-trihydroxy-18β-oleanol-12-ene-30-carboxylic acid.

[0096] For the first enzymatic reaction, 1.00 g (2.13 mmol) of glycyrrhetinic acid was dissolved in 5% (v / v) DMSO, and 0.80 g (0.5 equiv.) of NADP sodium salt and 22.87 g (50 equiv.) of Na2HPO3·3H2O were added to 0.1 M phosphate buffer at pH 7. In this cell-free catalytic system, the catalyst was the bacterial cells obtained from CYP161H12-RHFRed culture medium prepared in Example 1, resuspended in phosphate buffer to OD600 = 30, and the bacterial cells obtained from phosphite dehydrogenase opt13 obtained in Example 2 culture medium were resuspended in phosphate buffer to OD600 = 30. The amount of catalyst used was 0.18 mol%. The reaction was carried out at 23°C and 220 rpm for 20 h. The enzyme reaction solution was extracted with ethyl acetate to remove the organic solvent. The solution was then eluted by silica gel column chromatography (dichloromethane:methanol = 50:1) to obtain 0.48 g of intermediate and 0.50 g of compound 2.

[0097] The second enzymatic reaction used 0.48 g (1 mmol) of the intermediate as a starting material, dissolved in 5% v / v DMSO. The catalyst CYP161H12-RHFRed prepared in Example 1 was used as the culture medium for 1 LTB of cells, and the phosphorous acid dehydrogenase opt13 prepared in Example 2 was used as the culture medium for 0.5 LTB of cells. All other reaction conditions were consistent with the first enzymatic reaction. The enzyme reaction solution was extracted with ethyl acetate to remove the organic solvent, and then eluted by 200-300 mesh silica gel column chromatography (dichloromethane:methanol = 50:1) to obtain 0.42 g of compound 1b. After two enzymatic reactions, 0.92 g of compound 2 (white powder, yield: 85%) was finally obtained. Mass spectrometry, 1H NMR, and 1C NMR analyses showed that the obtained structural data were consistent with previously reported structural data.

[0098] 1 ¹H NMR (300MHz, Pyridine-d5)δ H 6.18(s,1H,H-12),4.68(m,1H,H-15),4.51(m,1H,H-7),3.51(m,1H,H-3),1.74,1.46,1.42,1.35,1.28,1.09,0.89(s,3H×7); 13 C NMR (75MHz, Pyridine-d5)δ C198.8,179.0,170.2,129.1,77.5,71.4,66.4,62.4,51.9,51.7,50.3,49.7,43.9,41.3,39.4,39.4,3 7.9,37.7,35.9,32.2,31.3,29.2,28.6,28.5,28.0,27.8,18.7,16.5,16.4,13.2; HRESIMS(m / z)[M+H] + Calculated C 30 H 47 O6 503.3373,found503.3408.

[0099] Example 4: Synthesis of methyl 11-oxo-3β,7β,15α-trihydroxy-18β-oleanolane-12-ene-30-carboxylate

[0100]

[0101] 0.37 g of compound 2 (0.74 mmol) was dissolved in 10 mL of DMF, and 0.41 g (4 equiv.) of potassium carbonate was added and stirred at room temperature for 30 min. 93 μL (2 equiv.) of iodomethane was added to the reaction solution, and the mixture was reacted at room temperature for 2.5 h. After rotary evaporation, water was added and a large amount of white solid was precipitated. The solid was filtered, the filter cake was collected and dried to obtain 0.36 g of compound 3 (white powder, yield 94%).

[0102] 1 ¹H NMR (400MHz, pyridine-d5)δ H 6.06(s,1H,H-12),4.64(dd,J=11.5,5.0Hz,1H,H-15),4.49(dd,J=10.8,4.5Hz,1H,H-7),3.66(s, 3H,OCH3),3.51(dd,J=11.6,4.5Hz,1H,H-3),1.67,1.44,1.41,1.27,1.15,1.08,0.86(s,3H,CH3); 13 C NMR (101 MHz, pyridine-d5) δ C198.7,176.6,169.6,129.1,77.5,71.4,66.3,62.4,51.8,51.6,51.5,50.3,49.7,43.9,40.9,39.3,39. 3,37.6,37.6,35.8,32.1,31.2,29.1,28.5,28.0,27.9,27.8,18.6,16.5,16.4,13.2.HRESIMS(m / z)[MH] - Calculated C 31 H 47 O6 515.3373, found 515.3387.

[0103] Example 5: Synthesis of methyl 3,7,11,15-tetraoxo-18β-oleanane-1,12-diene-30-carboxylate

[0104]

[0105] 0.36 g of compound 3 (0.58 mmol) was dissolved in 15 mL of DMSO, and 1.56 g (8 equiv.) of diiodobenzoic acid was added. The mixture was refluxed at 100 °C for 10 h. After cooling, the reaction solution was quenched with 5% NaHCO3 to obtain a white suspension. The suspension was extracted three times with ethyl acetate, and the organic layers were combined. The mixture was washed successively with water, saturated sodium bicarbonate, and saturated sodium chloride, dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation. The solution was then eluted by silica gel column chromatography (petroleum ether:ethyl acetate = 4:1) to give 0.27 g of compound 4 (white solid, yield 76%).

[0106] 1 H NMR (400MHz, CDCl3)δ H 7.58(d,J=10.2Hz,1H,H-1),5.94(s,1H,H-12),5.83(d,J=10.2Hz,1H,H-2) ,3.71(s,3H,OCH3),1.76,1.61,1.46,1.21,1.12,1.11,0.74(s,3H,CH3×7); 13 C NMR (101MHz, CDCl3)δ C205.9,205.3,202.7,195.2,176.3,167.4,158.9,128.0,125.3,58.2,57.5,57.0,52.4,52.1,49.5,45.0,4 3.9,43.6,40.9,39.3,36.8,36.7,35.6,31.3,28.7,28.1,27.2,22.5,21.4,20.2,17.8; HRESIMS(m / z)[M+H] + calculatedC 31 H 41 O6 509.2903, found 509.2907.

[0107] Example 6: Synthesis of methyl 2-iodo-3,7,11,15-tetraoxo-18β-oleanane-1,12-diene-30-carboxylate

[0108]

[0109] 0.19 g of compound 4 (0.39 mmol), 0.30 g (3 equiv) of elemental iodine, and 0.095 g (0.2 equiv.) of 4-dimethylaminopyridine were dissolved in 4 mL of a mixture of pyridine and CCl4 (1:1). The mixture was refluxed at 90 °C for 12 h. After cooling, the organic solvent was removed by rotary evaporation, and the reaction was quenched by adding 20% ​​sodium thiosulfate solution to give a golden yellow liquid. The liquid was extracted three times with ethyl acetate, and the organic layers were combined. The mixture was washed successively with water, saturated sodium bicarbonate, and saturated sodium chloride, dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation. The mixture was then eluted by silica gel column chromatography (petroleum ether: ethyl acetate = 4:1) to give 0.15 g of compound 5 (pale yellow solid, yield 61%).

[0110] 1 H NMR (400MHz, CDCl3)δ H 8.41(s,1H,H-1),6.00(s,1H,H-12),3.75(s,3H,OCH3),1.81,1.67,1.50,1.26,1.21,1.21,0.78(s,3H,CH3×7); 13 C NMR (101MHz, CDCl3)δ C205.3,205.2,196.0,194.9,176.4,167.8,166.8,127.9,101.6,58.2,57.4,56.5,52.1,52.1,49.6,45.4,4 3.9,43.6,43.6,40.9,36.8,36.7,35.6,31.4,28.7,28.3,28.2,22.5,22.0,20.0,17.9; HRESIMS(m / z)[M+H] + Calculated C 31 H 39 IO6 635.1870, found 635.1872.

[0111] Example 7 Synthesis of methyl 2-cyano-3,7,11,15-tetraoxo-18β-oleanane-1,12-diene-30-carboxylate

[0112]

[0113] 63.0 mg of compound 5 (0.1 mmol) was dissolved in 2 mL of DMF, and 3.3 mg (0.2 equiv.) of potassium iodide and 17.8 mg (2 equiv.) of cuprous cyanide were added. The gas in the reflux reaction system was replaced with nitrogen. Under nitrogen protection, the mixture was refluxed at 120 °C for 3.5 h. After cooling, the reaction solution was quenched with water and extracted three times with ethyl acetate. The organic layers were combined and washed successively with water, saturated sodium bicarbonate, and saturated sodium chloride. The mixture was dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation. The mixture was then eluted by silica gel column chromatography (petroleum ether: ethyl acetate = 3:1) to give 34.7 mg of compound 6 (pale yellow solid, yield 65%).

[0114] 1 H NMR (400MHz, CDCl3)δ H 8.41(s,1H,H-1),6.01(s,1H,H-12),3.74(s,3H,OCH3),1.79,1.69,1.51,1.25,1.23,1.19,0.77(s,3H,CH3×7); 13 C NMR (101MHz, CDCl3)δ C205.2,204.7,196.1,194.6,176.3,169.8,168.4,127.7,114.4,114.1,58.2,57.5,55.7,52.1,51.2,49.6,45. 3,43.9,43.6,40.9,40.0,36.8,36.3,35.6,31.3,28.7,28.1,27.2,22.4,21.4,19.8,18.0; HRESIMS(m / z)[M+H] + Calculated C 32 H 40 NO6534.2586, found 534.2975.

[0115] Example 8: Enzyme-catalyzed preparation of 11-oxo-3β-acetoxy-7β-hydroxy-18β-oleanol-12-ene-30-carboxylic acid

[0116]

[0117] 1.25 g (2.44 mmol) of 3β-acetoxyglycyrrhetinic acid was dissolved in 5% (v / v) DMSO. Cells obtained from the catalyst CYP161H12-RHFRed prepared according to the method of Example 1 were cultured in 4 L TB medium. Cells obtained from the phosphorous dehydrogenase opt13 prepared according to the method of Example 2 were cultured in 2 L TB medium, with the catalyst dosage being 0.34 mol%. All other reaction conditions were consistent with the first enzyme catalysis conditions in Example 3. The enzyme reaction solution was extracted with ethyl acetate to remove the organic solvent, and then eluted by 200-300 mesh silica gel column chromatography (petroleum ether:acetone = 2:1) to obtain 1.12 g of compound 8 (white solid, yield 87%). Mass spectrometry, 1H NMR, and 1C NMR spectroscopy revealed structural data consistent with previously reported structural data.

[0118] 1 H NMR (400MHz, CDCl3)δ H 5.78(s,1H,H-12),4.52(dd,J=11.4,4.9Hz,1H,H-3),4.10(dd,J=10.8,4.9Hz,1H, H-7),2.07(s,3H,CH3C=O),1.46,1.25,1.18,1.16,0.91,0.89,0.86(s,3H,CH3×7); 13 C NMR (101MHz, CDCl3)δ C199.3,181.7,171.1,169.3,128.6,80.3,72.4,61.9,51.7,50.5,48.8,44.5,43.9,41.0,38.4,37.8,37.6,3 7.0,31.8,30.9,30.1,28.7,28.5,28.5,28.0,26.6,23.7,23.5,21.3,16.7,16.1,12.3; HRESIMS(m / z)[M+H] + Calculated C 32 H 49 O6 529.3529,found 529.3533.

[0119] Example 9: Synthesis of methyl 11-oxo-3β-acetoxy-7β-hydroxy-18β-oleanol-12-ene-30-carboxylate

[0120]

[0121] 0.20 g of compound 8 (0.38 mmol) was dissolved in 4 mL of DMF. 0.21 g (4 equiv.) of potassium carbonate was added, and the mixture was stirred at room temperature for 30 min. 47 μL (2 equiv.) of iodomethane was added to the reaction solution, and the mixture was reacted at room temperature for 2.5 h. The mixture was then rotary evaporated, and water was added until a large amount of white solid precipitated. The solid was filtered, the filter cake was collected and dried, and the filter cake was eluted by 200-300 mesh silica gel column chromatography (petroleum ether:acetone = 5:1) to give 0.18 g of compound 9 (white powder, 88% yield).

[0122] 1 H NMR (400MHz, CDCl3)δ H 5.70(s,1H,H-12),4.50(dd,J=11.5,4.9Hz,1H,H-3),4.06(dd,J=10.8,4.9Hz,1H,H-7),3.6 8(s,3H,OCH3),2.05(s,3H,CH3C=O),1.43,1.16,1.15,1.14,0.89,0.88,0.81(s,3H,CH3×7); 13 C NMR (101MHz, CDCl3)δ C199.1,176.9,171.0,169.2,128.6,80.3,72.3,61.9,51.8,51.7,50.4,49.0,44.5,44.1,41.2,38.4,37.8,37. 6,36.9,31.8,31.1,30.0,28.7,28.5,28.3,28.0,26.6,23.6,23.5,21.3,16.7,16.1,12.2; HRESIMS(m / z)[M+H] + Calculated C 33 H 51 O6 543.3686, found 543.3688.

[0123] Example 10 Synthesis of methyl 7,11-dioxo-3β-acetoxy-18β-oleanol-12-ene-30-carboxylate

[0124]

[0125] 0.18 g of compound 9 (0.33 mmol) was dissolved in 4 mL of tetrahydrofuran. 0.42 mL of Jones reagent was added under ice bath conditions. The ice bath was removed, and the mixture was reacted at room temperature for 1 h. The mixture was quenched with water to give a dark green suspension. The suspension was filtered, and the filter cake was collected, dried, and eluted with 200-300 mesh silica gel column chromatography (petroleum ether: acetone = 10:1) to give 0.14 g of compound 10 (white powder, yield 77%).

[0126] 1 H NMR (400MHz, CDCl3)δ H 5.74(s,1H,H-12),4.55(dd,J=11.3Hz,5.0Hz,1H,H-3),3.70(s,3H,OCH3),2 .07(s,3H,CH3C=O),1.39,1.35,1.32,1.17,0.91,0.86,0.80(s,3H,CH3×7); 13 C NMR (101MHz, CDCl3)δ C 213.1,197.8,176.9,170.8,167.2,128.1,79.9,61.7,59.0,51.8,51.1,50.3,44.6,44.1,40.1,37.8,37.7,37. 4,37.2,36.4,32.2,31.0,28.4,28.4,27.7,27.4,26.3,23.5,23.3,21.2,17.6,15.9,15.7; HRESIMS(m / z)[M+H] +Calculated C 33 H 49 O6 541.3529, found 541.3532.

[0127] Example 11 Synthesis of methyl 7,11-dioxo-3β-acetoxy-18β-oleanolane-5,12-diene-30-carboxylate

[0128]

[0129] 0.14 g of compound 10 (0.24 mmol) was dissolved in 3 mL of acetic acid. 18.2 μL (0.44 equiv.) of HBr and 29.1 μL (2.4 equiv.) of elemental bromine were added dropwise. After reacting at 37 °C for 24 hours, the reaction was quenched with 20% sodium thiosulfate solution. Acetic acid was removed by rotary evaporation, and the mixture was extracted three times with ethyl acetate. The combined organic layers were washed successively with water, saturated sodium bicarbonate, and saturated sodium chloride, dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation. The mixture was then purified by silica gel column chromatography (petroleum ether:acetone = 10:1) to obtain the molecular weight [M+H] in high-resolution mass spectrometry. + The mixture of compounds undergoing bromination at the C6 position (619.2634) was eluted isocratically with 90% acetonitrile and water by preparative high-performance liquid chromatography to give 96.8 mg of C6-brominated product-1 and 44.3 mg of C6-brominated product-2 (both white solids, yield 96%).

[0130] 35.5 mg of the C6-brominated product-2 (0.06 mmol) was dissolved in 1 mL of collidine and refluxed at 80 °C for 2 h. The reaction was quenched with 2N hydrochloric acid, and the mixture was extracted three times with ethyl acetate. The organic layers were combined and washed with 2N hydrochloric acid, saturated sodium bicarbonate, and saturated sodium chloride solutions, respectively. The mixture was dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation. The mixture was then purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1) to give 30.6 mg of compound 11 (white solid, 99% yield).

[0131] 1 H NMR (400MHz, CDCl3)δ H 6.09(s,1H,H-6),5.80(s,1H,H-12),4.67(dd,J=11.8,4.4Hz,1H,H-3),3.71(s,3H, OCH3),2.10(s,3H,CH3C=O),1.54,1.35,1.30,1.23,1.17,1.15,0.85(s,3H,CH3×7); 13 CNMR (101MHz, CDCl3)δ C207.5,197.7,177.0,171.0,166.3,128.5,79.4,59.5,58.7,58.7,51.8,49.8,49.6,45.6,44.0,40.2,39.3,38. 6,37.6,35.4,32.5,31.0,30.3,28.4,28.2,27.5,26.2,25.7,22.9,21.2,20.8,17.14,16.9; HRESIMS(m / z)[M+H] + Calculated C 33 H 47 O6 539.3373, found 539.3375.

[0132] Example 12 Synthesis of methyl 7,11-dioxo-3β-acetoxy-oleanolane-5,12,18-triene-30-carboxylic acid

[0133]

[0134] 0.2 g of compound 10 (0.37 mmol) was dissolved in 3 mL of acetonitrile, and 0.24 mg (2 equiv.) of PyHBr3 was added. The reaction was carried out at 37 °C for 36 h, and then quenched with 20% sodium thiosulfate solution. After removing acetic acid by rotary evaporation, the mixture was extracted three times with ethyl acetate. The organic layers were combined and washed successively with water, saturated sodium bicarbonate, and saturated sodium chloride. The mixture was dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation. The mixture was purified by silica gel column chromatography (petroleum ether: acetone = 10:1) with 200-300 mesh. The purified mixture was then eluted isocratically with 85% acetonitrile and water by preparative high performance liquid chromatography to give 0.12 g of compound 12 (a white solid, yield 60%).

[0135] 1 H NMR (400MHz, CDCl3)δ H 6.06(s,1H,H-6),5.81(s,1H,C-12),5.74(s,1H,C-19),4.65(dd,J=11.8,4.4Hz,1H,H-3),3.70(s,3H, OCH3),3.14(s,1H,C-9),2.10(s,3H,CH3C=O),1.58,1.38,1.31,1.23,1.14,1.08,0.89(s,3H,CH3×7); 13 C NMR (101MHz, CDCl3)δ C202.6,198.1,176.8,174.7,170.6,162.0,143.1,130.4,125.4,123.9,77.4,58.0,52.3,52.3,46.1,44.3,41.5,3 7.8,35.7,35.7,35.3,34.6,27.9,27.1,26.0,25.2,24.5,24.2,23.7,22.8,21.2,18.4,16.7; HRESIMS(m / z)[M+H] + Calculated C 33 H 47 O6 537.3216,found537.3222.

[0136] Example 13 Synthesis of methyl 7,11-dioxo-3β-hydroxy-oleanolane-5,12,18-triene-30-carboxylic acid

[0137]

[0138] 101 mg of compound 12 (0.19 mmol) was dissolved in 10 mL of methanol, and 650 mg of potassium hydroxide was added. After stirring at room temperature for 2 h, the organic solvent was removed by rotary evaporation. The pH was adjusted to 4-5 with 10% hydrochloric acid, and the mixture was extracted with dichloromethane / ethyl acetate at a ratio of 1:4. The organic layers were combined, washed with saturated sodium bicarbonate and saturated sodium chloride solutions, dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation to obtain a white crude product. The crude product and 100 mg (4 equiv.) of potassium carbonate were dissolved in 2 mL of LDM, and stirred at room temperature for 30 min. 24 μL of iodomethane was added, and the mixture was reacted at room temperature for 2.5 h. After rotary evaporation, water was added to precipitate a large amount of white solid. The solid was filtered, the filter cake was collected and dried, and the filter cake was eluted by 200-300 mesh silica gel column chromatography (petroleum ether: ethyl acetate = 4:1) to give 89 mg of compound 13 (white powder, yield 88%).

[0139] 1 H NMR (400MHz, CDCl3)δ H 6.10(s,1H,H-6),5.82(s,1H,H-12),5.73(s,1H,H-19),3.71(s,3H,OCH3),3.43(dd,J=11.7 ,4.4Hz,1H,H-3),3.12(s,1H,C-9),1.57,1.39,1.31,1.27,1.16,1.00,0.99(s,3H,CH3×7); 13 C NMR (101MHz, CDCl3)δ C202.8,198.3,176.8,176.0,162.0,143.3,130.3,125.3,123.9,76.1,58.1,52.3,52.2,46.1,44.3,42.9,3 7.9,36.1,35.7,35.3,34.7,27.9,27.2,26.4,26.1,25.2,24.5,23.6,22.9,18.4,16.7; HRESIMS(m / z)[M+H] + Calculated C 31 H 43 O5 495.3110, found 495.3092.

[0140] Example 14 Synthesis of methyl 3,7,11-trioxo-oleanane-1,5,12,18-tetraene-30-carboxylate

[0141]

[0142] 62 mg of compound 13 (0.12 mmol) was dissolved in 2 mL of DMSO, and 275 mg (8 equiv.) of diiodobenzoic acid was added. The mixture was refluxed at 100 °C for 10 h. After cooling, the reaction solution was quenched with 5% NaHCO3 to obtain a white suspension. The suspension was extracted three times with ethyl acetate, and the organic layers were combined. The mixture was washed successively with water, saturated sodium bicarbonate, and saturated sodium chloride, dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation. The solution was then eluted by silica gel column chromatography (petroleum ether:ethyl acetate = 4:1) to give 50 mg of compound 14 (white solid, yield 81%).

[0143] 1 H NMR (400MHz, CDCl3)δ H 7.53(d,J=10.4Hz,1H,H-1),6.11(s,1H,H-6),5.99(d,J=10.4Hz,1H,H-2),5.89(s,1H,H-12),5.76(s, 1H,H-19),3.71(s,3H,OCH3),3.31(s,1H,H-9),1.83,1.48,1.46,1.40,1.30,1.08,1.01(s,3H,CH3×7); 13 C NMR (101MHz, CDCl3)δ C205.9,205.3,202.7,195.2,176.3,167.4,158.9,128.0,125.3,58.2,57.5,57.0,52.4,52.1,49.5,45.0, 43.9,43.6,40.9,39.3,36.8,36.7,35.6,31.3,28.7,28.1,27.2,22.5,21.4,20.2,17.8; HRMS(m / z)[M+H] + Calculated C 31 H 41 O6 491.2797,found491.2798.

[0144] Example 15 Synthesis of methyl 2-iodo-3,7,11-trioxo-oleanane-1,5,12,18-tetraene-30-carboxylate

[0145]

[0146] 50.0 mg of compound 14 (0.1 mmol), 76.1 mg (3 equiv.) of elemental iodine and 2.4 mg (0.2 equiv.) of 4-dimethylaminopyridine were dissolved in 1.6 mL of pyridine / CCl solution. 4＝ In a 1:1 mixture, the mixture was refluxed at 90°C for 12 hours. After cooling, the organic solvent was removed by rotary evaporation, and the reaction was quenched by adding 20% ​​sodium thiosulfate solution to obtain a golden yellow liquid. The mixture was extracted three times with ethyl acetate, and the organic layers were combined. The layers were washed successively with water, saturated sodium bicarbonate, and saturated sodium chloride, dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation. The mixture was then eluted by silica gel column chromatography (petroleum ether:ethyl acetate = 4:1) to give 47.5 mg of compound 15 (pale yellow solid, yield 74%). 1 H NMR (400MHz, CDCl3)δ H 8.30(s,1H,H-1),6.10(s,1H,H-6),5.92(s,1H,H-12),5.79(s,1H,H-19),3.72(s,3 H,OCH3),3.35(s,1H,H-9),1.84,1.50,1.47,1.46,1.31,1.08,1.00(s,3H,CH3×7); 13 C NMR (101MHz, CDCl3)δ C200.8,196.9,193.5,176.6,167.7,163.2,159.9,142.9,131.2,125.7,123.5,99.2,53.6,53.4,52.3,49.4 ,46.7,44.4,44.1,35.6,35.4,34.6,30.4,27.9,27.1,25.2,25.1,24.6,24.2,18.4,17.1; HRMS(m / z)[M+H] + Calculated C 31 H 38 IO5 617.1764, found 617.1783.

[0147] Example 16 Synthesis of methyl 2-cyano-3,7,11-trioxo-oleanane-1,5,12,18-tetraene-30-carboxylate

[0148]

[0149] 45.5 mg of compound 15 (0.07 mmol) was dissolved in 1.5 mL of DMF. 2.4 mg (0.2 equiv.) of potassium iodide and 13.2 mg (2 equiv.) of cuprous cyanide were added. The gas in the reflux reaction system was replaced with nitrogen. Under nitrogen protection, the mixture was refluxed at 120 °C for 3.5 h. After cooling, the reaction solution was quenched with water. The mixture was extracted three times with ethyl acetate. The organic layers were combined and washed successively with water, saturated sodium bicarbonate, and saturated sodium chloride. The mixture was dried over anhydrous sodium sulfate. After removing the organic solvent by rotary evaporation, the mixture was eluted by silica gel column chromatography (petroleum ether: ethyl acetate = 3:1) to give 25.1 mg of compound 16 (pale yellow solid, yield 66%).

[0150] 1 H NMR (400MHz, CDCl3)δ H 8.32(s,1H,H-1),6.13(s,1H,H-12),5.93(s,1H,H-12),5.79(s,1H,H-19),3.72(s, 3H,OCH3),3.33(s,1H,H-9),1.88,1.49,1.49,1.45,1.30,1.07,1.01(s,3H,CH3×7); 13 CNMR (101MHz, CDCl3)δ C200.2,196.4,193.0,176.5,165.7,163.7,163.2,142.7,131.5,126.4,123.2,113.7,113.2,53.7,53.0,52.3, 49.2,46.8,44.4,40.5,35.5,35.4,34.6,29.9,27.9,27.1,25.1,24.8,24.6,22.9,18.4,17.2; HRMS(m / z)[M+H] + Calculated C 32 H 38 NO5 516.2750, found 516.2770.

[0151] Example 17: Pharmacological Study of Pentyl Triterpenoid Molecules from MultiMichael Reaction Receptors

[0152] The antiproliferative activity of compounds against tumor cells was determined using the CCK-8 assay. The CCK-8 assay for cell proliferation inhibition is based on the property that WST-8 in the CCK-8 kit can be reduced to an orange-yellow, water-soluble formazan in the presence of an electron coupling reagent. The more and faster the cell proliferation, the darker the color; conversely, the less proliferating the cells, the lighter the color. Within a certain cell number range, the absorbance of CCK-8 at 450 nm is directly proportional to the number of viable cells, thus reflecting the drug's inhibitory effect on cell proliferation.

[0153] Reagents: DMEM medium (Glibco), fetal bovine serum (BI), trypsin (biosharp), antibiotics (containing 100 U / mL penicillin and 100 U / mL streptomycin, biosharp), PBS buffer (0.01M, pH=7.4, biosharp), DMSO (Sangon Biotech), CCK-8 kit (biosharp).

[0154] Cell lines: human prostate cancer cells PC-3, human breast cancer cells MCF-7, human colon cancer cells SW480, and human cervical cancer cells HeLa (all purchased from the Cell Bank of the Chinese Academy of Sciences).

[0155] Preparation of compound solutions of different concentrations: A certain amount of the compound was weighed and dissolved in DMSO to prepare a 20 mM stock solution. The stock solution was then diluted with complete culture medium to obtain compound solutions of different concentrations. The compound solution concentrations in this invention include 200 μM, 80 μM, 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, and 0.313 μM.

[0156] Cell culture: Tumor cells were cultured in a complete medium containing 10% (v / v) heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin in high-glucose DMEM. All cells used in the experiments were cultured in a 37°C incubator with 5% CO2 saturated humidity. Cells were in the exponential growth phase when the cell density in the culture dish was approximately 80%.

[0157] Cell proliferation inhibition activity: Cells were seeded at a density of 3000 cells / well in 96-well plates, with 100 μL of complete culture medium containing cells added to each well (three wells were set up as a blank control group with 100 μL of cell-free complete culture medium, and three wells were set up as a negative control group with 100 μL of complete culture medium containing cells). After incubation at 37°C and 5% CO2 for 24 hours, the cells adhered, and 100 μL of the prepared compound solution was added (since the final volume per well was 200 μL, the measured compound concentration was 1 / 2 of the concentration of the prepared compound solution). Three replicates were set up for each concentration of each compound. At this time, 100 μL of complete culture medium was added to the blank control group and the negative control group. After culturing the 96-well plates at 37°C in a 5% CO2 saturated humidity incubator for 48 hours, 20 μL of CCK-8 assay reagent was added to each well under dark conditions, gently shaken, and incubated at 37°C for 1.5 h. The absorbance of each well at 450 nm was measured using a microplate reader, and the inhibition rate of the compounds on cells was calculated according to the following formula. The average of three initial screening results was the final inhibition rate, as shown in Table 1. For compounds with inhibition rates higher than 50%, further concentration gradient screening was performed. The half-maximal inhibitory concentration (IC50) was calculated using GraphPad Prism9, and the results of three replicate experiments were the final IC50 values ​​of the tested compounds, as shown in Table 2.

[0158] Inhibition rate (IR; %) = [(OD value of negative control group - OD value of drug treatment group)] / [(OD value of negative control group - OD value of blank group)] × 100%

[0159] First, the CCK-8 assay was used to evaluate the inhibitory activity of the positive control drug methyldopasolone (provided by Professor Cheng Li's research group at China Pharmaceutical University), parent compound 1, and the specially selected pentacyclic triterpenoid compounds 4, 5, 6, 11, 12, 13, 14, 15, and 16 (particularly preferred in this invention) against the proliferation of human cervical cancer cells HeLa at a concentration of 40 μM. Based on the results in Table 1, compounds 6, 15, and 16, which showed a tumor cell inhibition rate >50% at 40 μM, were selected for further IC50 determination. The results are shown in Table 2. The in vitro antitumor activity of the selected compounds was significantly better than that of parent compound 1, and the antitumor activity of compound 16 was comparable to that of the positive control drug CDDO-Me.

[0160] Table 1. Inhibitory effect of the compound on HeLa cells at a concentration of 40 μM.

[0161]

[0162] Table 2. In vitro antiproliferative effects of compounds 5, 15, 16 and CDDO-Me on four human cancer cell lines.

[0163]

[0164] The following conclusions can be drawn from Tables 1 and 2:

[0165] (1) Overall, the novel multi-Michael response receptor pentacyclic triterpenoid compounds 4, 5, 6, 13, 14, 15, and 16 involved in this invention exhibit superior antitumor activity compared to the parent compound 1.

[0166] (2) Compound 6 has significant antitumor activity against SW480 and Hela, while compounds 15 and 16 have excellent antiproliferative effects against PC-3, MCF-7, SW480 and Hela tumor cell lines.

[0167] (3) Compound 16, which has three Michael response receptor molecules in its structure, has antitumor activity comparable to that of the positive drug CDDO-Me.

[0168] (4) Compared with compound 6, which also contains a cyano fragment, compound 16, which contains three Michael response receptor modules, has significantly better antitumor activity than compound 6, which contains two Michael response receptor modules.

Claims

1. Pentacyclic triterpenoid derivatives or their pharmaceutically usable salts: characterized in that, Its structural formula is shown below: , or Where A is C or CH; B is C or CH; R1 is H, I or CN; and R2 is OH or acetoxy.

2. The pentacyclic triterpenoid derivative or its pharmaceutically usable salt according to claim 1, characterized in that, The pentacyclic triterpenoid derivatives include methyl 3,7,11,15-tetraoxo-18β-oleanolane-1,12-diene-30-carboxylate, methyl 2-iodo-3,7,11,15-tetraoxo-18β-oleanolane-1,12-diene-30-carboxylate, methyl 2-cyano-3,7,11,15-tetraoxo-18β-oleanolane-1,12-diene-30-carboxylate, methyl 7,11-dioxo-3β-acetoxy-18β-oleanolane-5,12-diene-30-carboxylate, and methyl 7,11-dioxo-3 methyl β-acetoxy-oleanolane-5,12,18-triene-30-carboxylate, methyl 7,11-dioxo-3β-hydroxy-oleanolane-5,12,18-triene-30-carboxylate, methyl 3,7,11-trioxo-oleanolane-1,5,12,18-tetraene-30-carboxylate, methyl 2-iodo-3,7,11-trioxo-oleanolane-1,5,12,18-tetraene-30-carboxylate, or methyl 2-cyano-3,7,11-trioxo-oleanolane-1,5,12,18-tetraene-30-carboxylate.

3. The chemical enzymatic method for preparing the pentacyclic triterpenoid derivative according to any one of claims 1 to 2, characterized in that, The following synthetic routes are included: ， The specific steps are as follows: Step a: Mix compound 1, oxidase, cofactor, and coenzyme regeneration system for enzymatic reaction; the oxidase is CYP161H12-RHFRed; the cofactor is sodium NADP and Na2HPO3; the coenzyme regeneration system is phosphite dehydrogenase; Step b: Mix compound 2, potassium carbonate, iodomethane and solvent to react and obtain the methyl ester product; Step c: The compound 3,2-iodobenzoic acid and solvent are mixed and reacted to obtain the oxidation product; Step d: Compound 4, elemental iodine, 4-dimethylaminopyridine and solvent are mixed and reacted to obtain the iodine-substituted product; Step e: Compound 5, potassium iodide, cuprous cyanide and solvent are mixed and reacted under a protective atmosphere to obtain the cyano-substituted product.

4. The chemical enzymatic method for preparing the pentacyclic triterpenoid derivative according to any one of claims 1 to 2, characterized in that, The following synthetic routes are included: ； Specifically, the steps include the following: The steps include steps a and b as described in claim 3, and also include the following steps: Step f: Compound 9, Jones reagent, and solvent are mixed and reacted to obtain the oxidation product; Step g: The mixture of compound 10, elemental bromine, hydrogen bromide and solvent is reacted to obtain a mixed intermediate; Step h: The intermediate mixture from step g is mixed with chlortetramine to undergo an elimination reaction to obtain the product.

5. The chemical enzymatic method for preparing the pentacyclic triterpenoid derivative according to any one of claims 1 to 2, characterized in that, The following synthetic routes are included: ； The steps include steps a, b, and f as described in claim 4, and steps c, d, and e as described in claim 3, and further include the following steps: Step i: Compound 10, pyridinium tribromide, and solvent are mixed and reacted to obtain the oxidation product; Step j: Compound 12, potassium hydroxide and solvent are mixed and reacted to obtain a mixture of deacetylated and partially demethylated products.

6. The chemoenzymatic preparation method of the pentacyclic triterpenoid derivative according to claim 3, characterized in that, The molar ratio of compound 2, potassium carbonate, and iodomethane in step b is 1:3~4:1.3~2, the solvent is 4~10 mL of N,N-dimethylformamide, the reaction temperature is 18~35℃, and the reaction time is 2~3 h.

7. The chemical enzymatic preparation method for pentacyclic triterpenoid derivatives according to claim 3, characterized in that, The molar ratio of compound 3 and 2-iodoacyl group in step c is 1:4~8, the solvent is 2~15 mL of dimethyl sulfoxide, the reaction temperature is 100~110℃, and the reaction time is 10 h~15 h.

8. The chemical enzymatic preparation method of the pentacyclic triterpenoid derivative according to claim 3, characterized in that, The molar ratio of compound 4, iodine and 4-dimethylaminopyridine in step d is 1:3:0.1~0.2, the solvent is a mixed solvent of pyridine and carbon tetrachloride in a volume ratio of 1:1, the reaction temperature is 90~100℃, and the reaction time is 12~15 h.

9. The chemical enzymatic preparation method for pentacyclic triterpenoid derivatives according to claim 3, characterized in that, The molar ratio of compound 5, potassium iodide, and cuprous cyanide in step e is 1:0.2:1.5~2, the protective atmosphere is nitrogen or argon, the reaction temperature is 120℃, and the reaction time is 3.5 h.

10. The chemoenzymatic preparation method of the pentacyclic triterpenoid derivative according to claim 4, characterized in that, The molar volume ratio of compound 9 and Jones reagent in step f is 0.33 mmol: 0.4~0.5 mL, the solvent is 4~5 mL, the reaction temperature is 0℃~35℃, and the reaction time is 1~2 h.

11. The chemical enzymatic preparation method of the pentacyclic triterpenoid derivative according to claim 4, characterized in that, In step g, the molar ratio of compound 10, elemental bromine, and hydrogen bromide is 1:10:1.8~2, the solvent is 3~4 mL of N,N-dimethylformamide, and the reaction temperature is 18~35℃.

12. The chemical enzymatic preparation method for pentacyclic triterpenoid derivatives according to claim 5, characterized in that, The molar ratio of compound 10 and pyridinium tribromide in step i is 1:2~3, the solvent is 2~3 mL acetonitrile, the reaction temperature is 37~40℃, and the reaction time is 36~48 h.

13. The chemical enzymatic preparation method for pentacyclic triterpenoid derivatives according to claim 5, characterized in that, The molar ratio of compound 12 to potassium hydroxide in step j is 1:85~100, the solvent is 8~10 mL methanol, the reaction temperature is 18~35℃, and the reaction time is 2~3 h.

14. The use of the pentacyclic triterpenoid derivative or its pharmaceutically acceptable salt as described in any one of claims 1 to 2 in the preparation of a drug for treating / preventing tumors.

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