Aphidicolin diterpenoid compound, its preparation method and application in anti-tumor drugs

Abstract

The application relates to the technical field of marine medicines, and particularly discloses an Aphidicolin diterpenoid compound, a preparation method thereof and application of the Aphidicolin diterpenoid compound in anti-tumor medicines. The application is characterized in that a specific culture medium is used to cultivate a deep-sea derived fungus Botryotinia fuckeliana MCCC 3A00494, and a secondary metabolite Aphidicolin diterpenoid compound is obtained from a fermentation product through various chromatography technologies. Twelve new Aphidicolin diterpenoid compounds are separated, and the Aphidicolin diterpenoid compounds are identified to include nine rare nor-diterpenes. Through experimental research, the separated Aphidicolin diterpenoid compounds have obvious inhibiting effects on a human T24 bladder cancer tumor cell line, and can be used for preparing and developing bladder cancer treatment medicines. On the other hand, the application also provides a lead compound for treating bladder cancer, and has good application and development potential.

Description

Technical Field

[0001] This invention belongs to the field of marine pharmaceutical technology, specifically relating to an Aphidicolin diterpenoid compound, its preparation method, and its application in antitumor drugs. Background Technology

[0002] According to the 2018 Global Cancer Statistics Report, there were 18.1 million new cancer cases and 9.6 million cancer deaths worldwide, averaging about 18 people dying from cancer every minute. Bladder cancer is one of the most common malignant tumors of the urinary system, ranking 9th in incidence worldwide. Currently used anti-bladder cancer drugs often suffer from high toxicity, low specificity, and easy inactivation. Therefore, there is an urgent need to develop novel, highly effective, and low-toxicity drugs for the treatment of bladder cancer.

[0003] Natural products are a crucial source of innovative drugs. Currently, over 80% of small-molecule anti-tumor drugs used clinically are derived from natural products or their derivatives. The US FDA has approved nine marine drugs, six of which are anti-tumor drugs. In addition, 27 marine drug candidates are currently in clinical trials, 23 of which are anti-tumor candidates. This greatly highlights the important role of marine natural products in anti-tumor drug development. Deep-sea microorganisms, in particular, possess unique metabolic regulatory pathways different from terrestrial microorganisms and offer advantages such as renewable resource utilization, making them a hot topic in natural product research in recent years. Summary of the Invention

[0004] In response to the problems of existing technologies and the advantages of marine drugs,

[0005] The first aspect of this invention provides an Aphidicolin diterpenoid compound or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, or prodrug thereof, wherein the Aphidicolin diterpenoid compound is shown in formula (A):

[0006]

[0007] The Indicates a single bond or a double bond;

[0008] X1 is selected from C(R1R2) and C(O), wherein R1 and R2 are each independently selected from hydroxyl group and -CH2OH;

[0009] X2 is selected from C(R3R4), C(O), C(R) 11 ), wherein R3 and R4 are each independently selected from hydroxyl, -CH2OH, alkyl, and hydrogen, and R 11 Selected from alkyl groups;

[0010] The X3 is selected from CH, C(R5R6), C(O), wherein R5 and R6 are each independently selected from hydroxyl, -CH2OH, and hydrogen;

[0011] The X4 is selected from CH, C(R7R8), and C(O), wherein R7 and R8 are each independently selected from hydroxyl and hydrogen;

[0012] The X5 is selected from CR 12 C(R9R) 10 ), C(O), wherein R9 and R 10 Each is independently selected from hydroxyl and hydrogen, wherein R 12 Selected from hydroxyl groups;

[0013] The R a Selected from alkyl groups;

[0014] The R b R e Each is independently selected from hydroxyl and hydrogen;

[0015] The R c R d Each is independently selected from hydrogen or does not exist.

[0016] Each of the carbon atoms marked with * has an independent R configuration or S configuration.

[0017] In some specific embodiments of the first aspect, the Aphidicolin diterpenoid compound is characterized as shown in formula (A-1).

[0018]

[0019] The R2 is selected from -CH2OH, or the R2 and R e The atoms connected to them form a ring;

[0020] When the R b When selected from hydroxyl groups, it is related to the R b The bonded carbon atom has an R configuration;

[0021] The X5 is selected from methylene or C(O), wherein the H on the methylene is replaced by a hydroxyl group, and the carbon connected to the hydroxyl group has an S configuration;

[0022] When the above formula (A-1) is When X3 is a single bond, it is selected from methylene or C(O), wherein the H on the methylene is replaced by a hydroxyl group, and the carbon bonded to the hydroxyl group is in the R configuration or S configuration; when X2 is selected from C(R3R4), R3 is selected from -CH2OH, and R4 is selected from hydroxyl groups.

[0023] When the above formula (A-1) is When it is a double bond, X3 is selected from CH; X2 is selected from CR. 11 The R 11 Selected from methyl.

[0024] In some specific embodiments of the first aspect, the Aphidicolin diterpenoid compound is as shown in formula (A-2).

[0025]

[0026] The R b Selected from hydroxyl or hydrogen, wherein the carbon atom connected to the hydroxyl group has an R configuration;

[0027] X1 is selected from C(R1R2) and C(O), wherein R1 and R2 are each independently selected from hydroxyl and -CH2OH;

[0028] The X2 is selected from C(R3R4), wherein R3 and R4 are each independently selected from -CH2OH, methyl, and hydrogen;

[0029] The X3 is selected from CH, C(R5R6), and C(O), wherein R5 and R6 are each independently selected from hydroxyl and hydrogen;

[0030] When X3 is selected from C(O), in equation (A-2) It is a double bond.

[0031] In some specific embodiments of the first aspect, the Aphidicolin diterpenoid compound is as shown in formulas (A-3), (A-4), (A-5), and (A-6).

[0032]

[0033] X1 is selected from C(R1R2) and C(O), wherein R1 and R2 are each independently selected from hydroxyl and -CH2OH;

[0034] X2 is selected from C(R3R4), CR 11 Wherein R3 and R4 are each independently selected from hydroxyl, -CH2OH, and R 11 Selected from methyl;

[0035] The X3 is selected from CH, C(R5R6), C(O), wherein R5 and R6 are each independently selected from hydroxyl, -CH2OH, alkyl, and hydrogen;

[0036] X4 is selected from CH, C(R7R8), C(O), wherein R7 and R8 are each independently selected from hydroxyl and hydrogen, or one of R3 and R4 is selected from -CH2OH, and the other is connected to X4 to form a ring;

[0037] The X5 is selected from CR 12 C(R9R) 10 ), C(O), wherein R9 and R 10 Each is independently selected from hydroxyl and hydrogen; the R 12 Selected from hydroxyl groups.

[0038] The second aspect of this invention provides an Aphidicolin diterpenoid compound or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, or prodrug thereof, wherein the Aphidicolin diterpenoid compound specifically has one of the structures shown in compounds 1 to 12:

[0039]

[0040] The third aspect of this invention provides the use of an Aphidicolin diterpenoid compound or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, or prodrug in the preparation of a drug for treating bladder cancer, wherein the Aphidicolin diterpenoid compound has the structure shown in one of compounds 1 to 3.

[0041]

[0042] A fourth aspect of the present invention provides a formulation composition comprising any of the Aphidicolin diterpenoid compounds described in the first or second aspect.

[0043] The fifth aspect of this invention provides for the use of the Aphidicolin diterpenoid compound described in the first or second aspect in the preparation of a medicament for the prevention or treatment of bladder cancer.

[0044] The sixth aspect of this invention provides a method for preparing the Aphidicolin diterpenoid compound described in either the first or second aspect, comprising the following steps:

[0045] S1: Deep-sea fungus Botryotinia fuckeliana MCCC 3A00494 was inoculated into PDB medium to cultivate seed culture;

[0046] S2: The above seed liquid is inoculated into millet fermentation medium for fermentation culture, wherein the millet fermentation medium includes millet, soybean peptone, and seawater;

[0047] S3: The fermentation culture is separated and purified to obtain secondary metabolites, wherein the secondary metabolites are Aphidicolin diterpenoids as described in any one of claims 1 to 5.

[0048] In some specific embodiments of the sixth aspect of the present invention, the seed culture temperature in step S1 is 25–30°C.

[0049] In some specific embodiments of the sixth aspect of the present invention, the culture time of the seed liquid in step S1 is 2 to 4 days.

[0050] In some specific embodiments of the sixth aspect of the present invention, the seed culture in step S1 is cultured at a stirring speed of 100 to 300 rpm.

[0051] In some specific embodiments of the sixth aspect of the present invention, in step S2, 80-120g of millet and 0.40-0.50g of soybean peptone are added to every 120mL of seawater, and the culture temperature in step S2 is 25-30°C.

[0052] The deep-sea fungus *Botryotinia fuckeliana* MCCC 3A00494 of this invention is deposited at the China Marine Microbiological Culture Collection Center (CMC), with accession number MCCC 3A00494. It can be shared collaboratively through this accession number. The website is: https: / / www.mccc.org.cn / BacteriaService / Service1.

[0053] The reagents used in this invention are from the open and legal market, such as Sinopharm Shanghai Pharmaceuticals, Xilong Pharmaceutical, and Aladdin, and have not undergone further purification.

[0054] Advantages of this invention:

[0055] This invention provides a novel aphidicolin diterpenoid compound for preparation from the fermentation product of the deep-sea fungus *Botryotinia fuckeliana* MCCC 3A00494. The extraction and separation process is simple, and the raw material can be obtained through repeated fermentation, offering the advantage of resource renewability. The novel aphidicolin diterpenoid compounds exhibit significant inhibitory activity against human T24 bladder cancer tumor cells. Compounds 1-3 show IC50 values... 50 The values ​​were 9.54, 3.11, and 1.86 μM, respectively. This invention provides candidate compounds for the development of drugs to treat bladder cancer and advances the development and utilization of deep-sea biological resources. Attached Figure Description

[0056] Figure 1 Compound 1 1 H- 1 H COSY, HMBC, and NOESY are related;

[0057] Figure 2 Compound 3 1 H-1 H COSY, HMBC, and NOESY are related;

[0058] Figure 3 Compound 4 1 H- 1 H COSY, HMBC, and NOESY are related.

[0059] Figure 4 Compound 5 1 H- 1 H COSY, HMBC, and NOESY are related;

[0060] Figure 5 Experimental ECD and quantum chemical calculation ECD spectra of compound 5;

[0061] Figure 6 Compound 6 1 H- 1 H COSY, HMBC, and NOESY are related;

[0062] Figure 7 Experimental ECD and quantum chemical calculation ECD spectra of compound 9;

[0063] Figure 8 Experimental ECD and quantum chemical calculation ECD spectra of compound 11;

[0064] Figure 9 Compound 12 1 H- 1 H COSY, HMBC, and NOESY are related; Detailed Implementation

[0065] The following embodiments will further illustrate the present invention.

[0066] Example 1: Preparation and structural identification of the Aphidicolin diterpenoid compound involved in this invention.

[0067] 1. Preparation of Aphidicolin diterpenoids

[0068] (1) Preparation of seed fermentation broth: The deep-sea fungus *Botryotinia fuckeliana* MCCC 3A00494 was inoculated into PDA solid medium and cultured at 28°C for 3 days. 100 mL of PDB medium was added to each 250 mL Erlenmeyer flask. After autoclaving, the spores and mycelium were inoculated together into PDB liquid medium and cultured on a shaker (200 rpm, 28°C) for 4 days to obtain the seed fermentation broth.

[0069] (2) Large-scale fermentation and extraction of fermentation products: Millet culture medium was used for large-scale fermentation. 100g of millet, 0.45g of soybean peptone, and 120mL of seawater (taken from the Baicheng sea area of ​​Xiamen) were added to 80 1L Erlenmeyer flasks, respectively. After autoclaving at 121℃ for 30min, 5mL of seed fermentation broth was added to each flask, and then the flasks were placed in a constant temperature chamber at 28℃ for static fermentation for 60 days. After fermentation, ethyl acetate organic solvent was added, the solid fermentation medium was crushed, and 163g of ethyl acetate fermentation extract was obtained after ultrasonic extraction.

[0070] (3) The ethyl acetate extract obtained in step (2) was subjected to normal phase silica gel chromatography with dichloromethane / methanol gradient elution (1:0, 1:1, 0:1, v / v). After analysis and merging, two fractions (Fr.1 and Fr.2) were obtained.

[0071] (4) The fraction Fr.2 obtained in step (3) was subjected to reversed-phase ODS silica gel column chromatography and eluted with methanol / water (10% to 100%, v / v) gradient to obtain 3 sub-fractions (Fr.2-1 to Fr.2-3);

[0072] (5) The fraction Fr.2-2 obtained in step (4) was subjected to normal phase silica gel column chromatography and eluted with a gradient of dichloromethane / methanol (80:1 to 0:1, v / v) to obtain four sub-fractions (Fr.2-2-1 to Fr.2-2-4);

[0073] (6) Subfraction Fr.2-2-1 from step (5) was subjected to reversed-phase ODS silica gel column chromatography with methanol / water gradient elution (80%–100%, v / v) to obtain 6 subfractions. Subfraction Fr.2-2-1-2 was prepared by semi-preparative high-performance liquid chromatography (acetonitrile / water, 35:65, v / v) to obtain compound 5. Subfraction Fr.2-2-1-6 was subjected to reversed-phase ODS silica gel column chromatography with methanol / water gradient elution (40%–100%, v / v) to obtain compounds 6 (6.8 mg) and 11 (1.9 mg).

[0074] (7) The fraction Fr.2-2-2 obtained in step (5) was subjected to reversed-phase ODS silica gel column chromatography and eluted with a methanol / water gradient (80% to 100%, v / v); then compound 7 (1.4 mg) was prepared by semi-preparative high-performance liquid chromatography (acetonitrile / water, 33:67, v / v).

[0075] (8) The sub-fraction Fr.2-2-4 from step (5) was subjected to normal phase silica gel column chromatography and eluted with a gradient of dichloromethane / methanol (20:1 to 0:1, v / v) to obtain 8 sub-fractions Fr.2-2-4-1 to Fr.2-2-4-8;

[0076] (9) The sub-fraction Fr.2-2-4-1 from step (8) was subjected to normal-phase silica gel column chromatography with a gradient elution of dichloromethane / methanol (100:1 to 0:1, v / v) to obtain 11 sub-fractions (Fr.2-2-1-1 to Fr.2-2-1-11). Among them, sub-fraction Fr.2-2-1-4 was further prepared by semi-preparative high-performance liquid chromatography (acetonitrile / water, 20:80, v / v) to obtain compound 8 (2.6 mg);

[0077] (10) The sub-fraction Fr.2-2-1-6 from step (9) was subjected to normal-phase silica gel column chromatography with petroleum ether / ethyl acetate gradient elution (3:1 to 0:1, v / v); the fraction was further separated by normal-phase silica gel column chromatography with dichloromethane / methanol gradient elution (80:1 to 0:1, v / v) to obtain compound 3 (38.1 mg);

[0078] (11) The sub-fraction Fr.2-2-1-8 in step (9) was subjected to normal phase silica gel column chromatography and eluted isocratically with petroleum ether / ethyl acetate (2:1, v / v). Then, compound 9 (1.3 mg) was prepared by semi-preparative high-performance liquid chromatography (acetonitrile / water, 25:75, v / v).

[0079] (12) The sub-fraction Fr.2-2-1-11 from step (9) was subjected to reversed-phase ODS silica gel column chromatography with methanol / water (30% to 100%, v / v) gradient elution; then subjected to normal-phase silica gel column chromatography with dichloromethane / methanol (15:1, v / v) isocratic elution; and then compound 12 (7.1 mg) was prepared by semi-preparative high-performance liquid chromatography (methanol / water, 25:75, v / v).

[0080] (13) The sub-fraction Fr.2-2-4-2 from step (8) was subjected to normal-phase silica gel column chromatography with petroleum ether / ethyl acetate gradient elution (5:1 to 0:1, v / v); and then compound 1 (3.4 mg) and 2 (2.0 mg) were prepared by semi-preparative high-performance liquid chromatography (acetonitrile / water, 24:76, v / v).

[0081] (14) The sub-fraction Fr.2-2-4-4 from step (8) was subjected to normal-phase silica gel column chromatography with a gradient elution of dichloromethane / methanol (50:1 to 0:1, v / v); the fraction was further subjected to normal-phase silica gel column chromatography with a gradient elution of petroleum ether / ethyl acetate (3:1 to 0:1, v / v) to obtain compound 4 (2.5 mg);

[0082] (15) The sub-fraction Fr.2-2-4-5 from step (8) was subjected to normal phase silica gel column chromatography and eluted isocratically with dichloromethane / methanol (30:1, v / v); compound 10 (1.1 mg) was then prepared by semi-preparative high-performance liquid chromatography (acetonitrile / water, 27:73, v / v).

[0083] 2. Structural identification of novel Aphidicolin diterpenoid compounds

[0084] Compound 1 is a white powder. The molecular ion peak (m / z) shown in the HR-ESI-MS spectrum is 375.2136 [M+Na]. + And based on NMR data, its molecular formula was determined to be C. 20 H 32 O5, with an unsaturation degree of 5. This compound... 13 The C NMR (150MHz, DMSO-d6) showed two methyl carbon signals, eight methylene carbons, and five methine carbons. 1 ¹H NMR (600MHz, DMSO-d6) showed 2 methyl signals, 6 methylene signals, 2 oxymethylene signals, 2 methine signals, 2 oxymethene signals, 1 olefinic hydrogen signal, and 5 active hydrogen signals. These NMR data are very similar to those of the known compound Aphidicolin A49. Comparison revealed that compound 1 contains an additional oxymethene substituted for one methylene group in Aphidicolin A49. Based on the molecular formula, compound 1 is deduced to be a hydroxylated derivative of Aphidicolin A49. Correlation signals from COSY and HMBC were also observed. Figure 1 The additional hydroxyl group was determined to be located at the C-2 position. Furthermore, the NOESY correlation signal ( Figure 1 The relative configurations of compound 1 are 2S*, 3S*, 4R*, 5R*, 9S*, 10S*, 12R*, 16R*.

[0085] Compound 2 is a white powder, obtained by... 13 C10 NMR data and HR-ESI-MS indicate that compounds 2 and 1 have the same molecular formula C10. 20 H 32 O5. Compound 2 1 H and 13 The C18 NMR data are very similar to those of known Aphidicolin A49, indicating structural similarity. A significant difference lies in the presence of an oxymethyl group in compound 2, which replaces a methylene group in Aphidicolin A49, suggesting that compound 2 is a hydroxyl derivative of Aphidicolin A49. Based on the COSY spectrum of compound 2, the D2O exchangeable proton OH-1 (δ) H3.88) and H-1(δ) H 4.14) correlation indicates that C-1(δ C 65.5) is linked to a hydroxyl group. Furthermore, in the HMBC spectrum, OH-1 is associated with C-1, C-2 (δ... C 35.7) and C-10 (δ C 42.0) related, H3-20(δ) H 0.72) and C-1, C-5 (δc32.1), C-9 (δ C 50.4) and C-10 are related, further confirming that the additional hydroxyl group is located at C-1. Based on the NOESY spectrum of compound 2, H-1 and H-5 (δ) are related. H 2.14) Spatial correlation and the spatial correlation between H3-20 and OH-1 determine that OH-1 is in the β orientation. Therefore, the structure of compound 2 is determined to be a 1β-hydroxylated derivative of Aphidicolin A49.

[0086] Compound 3 is a colorless oil, and its molecular ion peak (m / z 377.2288 [M+Na]) is shown on HR-ESI-MS. + Based on NMR data, the chemical formula of this compound was determined to be C63. 20 H 34 O5, with an unsaturation degree of 4. This compound... 1 H and 13 The CNMR data are very similar to those of the known compound Aphidicolin, indicating a structural analogue. A significant difference is the presence of an additional hydroxymethyl group replacing the methylene group in Aphidicolin in compound 3, suggesting that compound 3 is a hydroxylated derivative of Aphidicolin. Based on the 2D NMR spectrum of compound 3, H-1(δ) H 4.06) and D2O exchangeable protons OH-1 (δ) H The COSY correlation of 3.74) and the relationship between OH-1 and C-1 (δ) C 66.4), C-2(δ) C 36.2) and C-10(δ) C The HMBC-related signal of 45.2) proves the presence of an additional hydroxyl group at the C-1 position. Figure 2 In the NOESY spectrum, the correlation signals [H-1 and H-5 (δ)] are... H 2.13) Related, OH-1 and H3-20 (δ H [0.84) Correlation] indicates that OH-1 is in the β orientation.

[0087] pass 13 C10 NMR and HR-ESI-MS data: m / z 325.1769 [M+Na]+ (Press C) 19 H 26 O3 Na + Calculations (325.1774) lead to the molecular formula of compound 4 being C. 19 H 26 O3. This compound 1 H and 13 The 13C NMR data are almost identical to those of the known compound Aphidicolin A58, except for the lack of hydroxymethyl and nonprotonated oxygen-containing sp. 3 Besides the carbon atom, compound 4 also contains a ketone carbonyl carbon located at C-16, instead of C-16 and C-17 in Aphidicolin A58, thus forming a novel 17-noraphidicolin-like diterpene. From H2-14 (δ... H 1.70, 1.86) / H2-15(δ H 1.99, 2.66) and H2-11 (δ H 1.57,1.88) / H-12(δ H 2.57) / H2-13(δ H COSY correlation of 1.40, 2.06), ketone carbonyl carbon C-16 (δ C 214.0) is related to the HMBC of H2-11 / H-12 / H2-13 / H2-14 / H2-15 ( Figure 3) ; and compared with the NMR corresponding data of Aphidicolin A58, the deshielded chemical shift C-12 is significantly clear. △ δ C +9.0), C-15 △ δ C +6.5), H-12 △ δ H +0.47), H2-15 ( △ δ H (+0.81,+1.34) proves this point. Therefore, the structure of compound 4 is resolved to 17-nor-16-oxy-aphidicolin A58.

[0088] Based on HR-ESI-MS data, m / z 327.1924 (by C) 19 H 28 O3 Na + Calculations (327.1931) lead to the conclusion that compound 5 has the molecular formula C. 19 H 38 O3 requires six degrees of unsaturation. (Compare) 1 H and 13Analysis of C10 NMR data and 2D NMR data confirmed that part of the structure (ring AC) in compound 5 is identical to part of the structure in Aphidicolin, with the main difference being ring D. Based on H-14 (δ¹⁴) NMR data... H 7.47), H2-11(δ) H 1.78), H-12 (δ) H 2.82) and H2-13 (δ H 1.13, 2.27) to the carbonyl carbon C-16 (δ C The HMBC-related signal of 205.4) combined with δ H 5.99 (dd, J = 10.3, 2.0 Hz) and δ H The vinyl proton at 7.47 (d, J = 10.3 Hz) indicates that at C-14 (δ... C 156.9), C-15 (δ) C There is an α,β-unsaturated ketone group at positions 127.5 and C-16. COSY correlations exist from H2-11 and H-12, and from H-12 to C-9 (δ... C 55.9) and C-15, HMBC correlations from H2-11 to C-14 and H-15 to C-9 and C-12 indicate that ring D is a cyclohexenone containing a ketone group at the C-16 position. Figure 4 Based on experimental and calculated ECD data for compound 5 (). Figure 5 The absolute configurations of C-4 / C-8 / C-10 / C-12 were determined to be R, and C-9 to be S. Therefore, the structure of compound 5 was determined to be 17-nor-14(15)-ene-16-oxy-aphidicolin.

[0089] Based on HR-ESI-MS data, m / z 343.1873 [M+Na] + (Press C) 19 H 28 O4 Na + Calculations (343.1880) suggest that the molecular formula of compound 6 is C6. 19 H 38 O4. Comparison 1 H and 13 Analysis of C10 NMR data and 2D NMR data confirmed that the local structure of the AC ring in compound 6 is partially identical to that of the known compound Aphidicolin A8, while the remaining D ring is identical to that in compound 4. Further analysis based on the 2D NMR correlation signal of compound 6 (…) Figure 6 Its structure was determined to be 17-nor-16-oxy-aphidicolin A8. Compounds 4-6 are the first 17-nor-aphidicolins discovered in nature.

[0090] pass 13 C10 NMR and HR-ESI-MS data: m / z 325.1624 [M+Na] + Compound 7 has the molecular formula C 19 H 26 O3. Its 1 H and 13 The C13 NMR data are very similar to those of the rare 19-nor-aphidicolin, Aphidicolin A66, except for C-13 (δ) in compound 7. C 84.3) and C-17 (δ) C The chemical shift of 79.1) is similar to that of C-13 (δ) in Aphidicolin A66. C 72.3) and C-17 (δ) C 67.9). Considering that the molecular weight of compound 7 is 18 smaller than that of Aphidicolin A66, and from H-13 (δ H 4.86) to C-17 (δ) C The HMBC correlation of 79.1) confirms that an ether bridge connects C-13 and C-17 to form a tetrahydrofuran ring. Therefore, the structure of compound 7 is resolved to be 13α,17α-epoxyaphidicolin A66.

[0091] based on 13 359.1823 m / z C NMR and HR-ESI-MS data (by C NMR) 19 H 28 O5Na + Calculations (359.1829) lead to the conclusion that compound 8 has the molecular formula C. 19 H 28 O5. Its 1 H and 13 The 1 / 2C NMR data are very similar to those of the known compound Aphidicolin A54. The obvious difference is the presence of an additional D2O exchangeable proton (δ¹⁰). H 3.80) and an oxygen-containing nonprotonated sp 3 Carbon (δ) C 73.3) replaces one methyl group and aproticized sp in Aphidicolin A54. 3 The carbon atom indicates that the hydroxyl group at C-4 in compound 8 replaces the singlet methyl group at C-19 in Aphidicolin A54, forming a rare 19-nor-aphidicolin diterpene. Based on the carbon atom from H2-18 (δ... H 3.25, 3.42) to C-3(δC 71.0), C-4(δ) C 73.3) and C-5 (δ) C 32.4) and OH-4 (δ H 3.80) with C-3, C-4, C-5 and C-18 (δ) C This is proven by the correlation with 66.8). Furthermore, based on the relationship between OH-4 and H-3 (δ... H 3.63) and H3-20 (δ H The NOESY correlation (0.94) confirms that OH-4 is in the β orientation. Therefore, the structure of compound 8 is resolved to be 4β-hydroxy-19-nor-aphidicolin A54.

[0092] based on 13 C10 NMR and HR-ESI-MS data: m / z 341.1717 [M+Na] + (Press C) 19 H 26 O4Na + Calculations (359.1829) lead to the conclusion that the molecular formula of compound 9 is C. 19 H 26 O4. 2D NMR data analysis confirmed that a portion of the structure (ring BD) of compound 9 is identical to that of the known compound Aphidicolin A43, the difference being ring A. Based on the methyl proton H3-18 (δ) of the alkene... H 2.01) to C-3(δ C 125.1) / C-4(δ C 164.5) / C-5(δ C 39.4), from the olefin methine proton H-3 (δ H 5.89) to C-1(δ C 46.5), from H2-1(δ H 2.14, 2.68) to C-2(δ C 201.3) and H3-20 (δ H 0.89) to C-1 / C-5 / C-9 (δ C 50.1) / C-10(δ C The HMBC correlation of 40.2) confirmed the presence of an α,β-unsaturated ketone group at C-2, C-3, and C-4 of the cyclohexenone ring A. The experimental ECD data for compound 9 are very close to the calculated ECD data for (5S,9R,10S,12S,13R,16R)-9. Figure 7 Thus, its absolute configuration is determined to be S at C-5 / C-10 / C-12 and R at C-9 / C-13 / C-16.

[0093] Based on HR-ESI-MS data, m / z 325.1768 (by C) 19 H 26 O3Na + Calculations (325.1774) lead to the molecular formula of compound 10 being C. 19 H 26 O3, which has one less oxygen atom than compound 9. The 1D and 2D NMR data of compound 10 are very similar to those of compound 9, with the main difference being the presence of a C-8 / C-13 double bond and the absence of a 13-oxygen-containing methine in compound 10. According to H2-11 (δ... H 1.83) / H-12(δ H 2.61) / H-13(δ H 5.53), H2-6(δ) H 1.41,1.96) / H2-7(δ H COSY correlation (2.03, 2.61) and C-8 (δ) C The correlation between 143.9) and HMBC of H2-11 / H-12 confirms this.

[0094] based on 13 C10 NMR and HR-ESI-MS data (m / z 327.1929, [M+Na]) + The molecular formula of compound 11 is C10. 19 H 28 O3. The NMR data of compound 11 are very similar to those of the known compound Aphidicolin A33. Careful comparison reveals that compound 45 exhibits a doublet methyl group instead of a singlet and lacks an oxygen-containing methylene signal. Based on H3-19 (δ... H 1.08) and H-4 (δ) H 2.31) COSY correlation and H3-19 to C-3 (δ C 213.1), C-4(δ) C 45.8) and C-5 (δ C The HMBC correlation of 47.0 indicates that the C-18 hydroxymethyl group in the known compound Aphidicolin A33 is eliminated in compound 11. Furthermore, the NOESY spatial correlation signal from H-4 to H3-20 confirms that CH3-19 is in the α-orientation. Based on a comparison of experimental and calculated ECD data, the absolute configuration of compound 11 is deduced to be 4S, 5S, 9S, 10S, 12S, 16R. Figure 8 Therefore, the structure of compound 11 is determined to be 19-noraphidicolin A33.

[0095] based on 13322.1509 mNMR and HR-ESI-MS data (m / z 322.1509 [M+Na]) + Press C 19 H 24 O5Na + Calculations (355.1516) lead to the conclusion that compound 12 has the molecular formula C. 19 H 24 O5. Its 1 H and 13 The 12C NMR data are very similar to those of the known compound Aphidicolin A67. The significant difference lies in the presence of two additional olefinic carbons (δ¹⁸O₂) in compound 12. C 123.8,δ C 145.7) replaces two methylene groups in the compound Aphidicolin A67. According to H3-20 (δ... H 1.25) to C-1(δ C 123.8), C-5(δ) C 157.6), C-9(δ) C 49.6) and C-10 (δ C 47.6) and from the double bond proton H-1 (δ H 6.22) to C-1 and C-3 (δ) C The HMBC correlation of 182.4) confirms additional double bonds located at C-1 and C-2 (δ). C 145.7) on ( Figure 9 Furthermore, protons (δ) can be exchanged via an additional D2O. H The presence of 6.38), the deshielding effect of C-2, and its molecular weight suggest the attachment of an additional hydroxyl group to C-2. Furthermore, the NOESY-related signal of compound 12 is very similar to that of Aphidicolin A67, indicating that they have the same relative configuration.

[0096] The hydrogen and carbon NMR data of the above 12 new Aphidicolin diterpenoids are shown in Tables 1 to 3.

[0097] Table 1. Compounds 1-6 1 1H NMR (600MHz) NMR data (δin ppm, J in Hz in Parentheses)

[0098]

[0099]

[0100]

[0101] Compounds 7-12 in Table 2 1 1H NMR (600MHz) NMR data (δin ppm, J in Hz in Parentheses)

[0102]

[0103]

[0104] Table 3 Compounds 1-12 13 C NMR (150MHz) data

[0105]

[0106]

[0107] Example 2: Evaluation of the inhibitory activity of Aphidicolin diterpenoids on human T24 bladder cancer cells

[0108] The MTT assay was used to evaluate the in vitro activity of human T24 bladder cancer cell lines. Aphidicolin diterpenoids were initially screened at a single concentration of 10 μM, and diterpenoids with inhibition rates greater than 50% were further tested for their IC50. 50 Value. The specific steps are as follows:

[0109] (1) The human T24 tumor cell line was cultured in RPMI 1640 medium in a cell culture incubator (5% CO2) at 37°C.

[0110] (2) Cells in the logarithmic growth phase were digested with trypsin, counted by trypan blue staining, and their viability was determined to be greater than 95% by trypan blue rejection assay. Then, 10,000 cells per well were seeded into 96-well plates with 180 μL of cell suspension per well. The cells were incubated at 37°C for 24 hours in a 5% CO2 incubator.

[0111] (3) After the cells adhered for 24 hours, different concentrations of Aphidicolin diterpenoids were added, and the cells were cultured in a 37℃ 5% CO2 incubator for 48 hours.

[0112] (4) Then add 10 μL of MTT solution to each well.

[0113] 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5mg / mL), continued at 5%

[0114] Incubate in a CO2 incubator (37℃) for 1 hour.

[0115] (5) Discard the supernatant, add 150 μL of DMSO to each well, and shake for 10 minutes to fully dissolve the crystals. Then, measure the absorbance at 490 nm using a microplate reader and calculate the IC50 using GraphPad Prism 8.0 software. 50 value.

[0116] Experimental results: Compounds 1, 2, and 3 showed an inhibition rate of over 50% against T24 tumor cells at an initial concentration of 10 μM. Further IC50 analysis of the three compounds... 50 Value determination revealed its IC50 value. 50 The concentrations were 9.54, 3.11, and 1.86 μM, respectively, demonstrating significant inhibitory activity against T24 human bladder cancer tumor cells. In particular, the novel compound 3 exhibited the strongest inhibitory activity and can be used in the development of drugs for bladder cancer treatment. Therefore, this invention provides lead compounds for the development of drugs for bladder cancer treatment and provides a scientific basis for the development and utilization of deep-sea biological resources.

[0117] Based on the disclosure and teachings of the foregoing specification, those skilled in the art can make appropriate changes and modifications to the above embodiments. Therefore, the present invention is not limited to the specific embodiments disclosed and described above, and some modifications and changes to the present invention should also fall within the protection scope of the claims of the present invention. Furthermore, although some specific terms are used in this specification, these terms are only for convenience of explanation and do not constitute any limitation on the present invention.

Claims

1. Aphidicolin diterpenoids or pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, cis-trans isomers, isotope-labeled compounds, or prodrugs thereof, wherein the Aphidicolin diterpenoids are shown in formula (A): The Indicates a single bond or a double bond; X1 is selected from C(R1R2) and C(O), wherein R1 and R2 are each independently selected from hydroxyl group and -CH2OH; X2 is selected from C(R3R4), C(O), C(R) 11 ), wherein R3 and R4 are each independently selected from hydroxyl, -CH2OH, alkyl, and hydrogen, and R 11 Selected from alkyl groups; The X3 is selected from CH, C(R5R6), C(O), wherein R5 and R6 are each independently selected from hydroxyl, -CH2OH, and hydrogen; The X4 is selected from CH, C(R7R8), and C(O), wherein R7 and R8 are each independently selected from hydroxyl and hydrogen; The X5 is selected from CR 12 C(R9R) 10 ), C(O), wherein R9 and R 10 Each is independently selected from hydroxyl and hydrogen, wherein R 12 Selected from hydroxyl groups; The R a Selected from alkyl groups; The R b R e Each is independently selected from hydroxyl and hydrogen; The R c R d Each is independently selected from hydrogen or does not exist; Each of the carbon atoms marked with * has an independent R configuration or S configuration.

2. The Aphidicolin diterpenoid compound according to claim 1, characterized in that, As shown in equation (A-1), The R2 is selected from -CH2OH, or the R2 and R e The atoms connected to them form a ring; When the R b When selected from hydroxyl groups, it is related to the R b The bonded carbon atom has an R configuration; The X5 is selected from methylene or C(O), wherein the H on the methylene is replaced by a hydroxyl group, and the carbon connected to the hydroxyl group has an S configuration; When the above formula (A-1) is When X3 is a single bond, it is selected from methylene or C(O), wherein the H on the methylene is replaced by a hydroxyl group, and the carbon bonded to the hydroxyl group is in the R configuration or S configuration; when X2 is selected from C(R3R4), R3 is selected from -CH2OH, and R4 is selected from hydroxyl groups. When the above formula (A-1) is When it is a double bond, X3 is selected from CH; X2 is selected from CR. 11 The R 11 Selected from methyl.

3. The Aphidicolin diterpenoid compound according to claim 1, characterized in that, As shown in equation (A-2), The R b Selected from hydroxyl or hydrogen, wherein the carbon atom connected to the hydroxyl group has an R configuration; X1 is selected from C(R1R2) and C(O), wherein R1 and R2 are each independently selected from hydroxyl and -CH2OH; The X2 is selected from C(R3R4), wherein R3 and R4 are each independently selected from -CH2OH, methyl, and hydrogen; The X3 is selected from CH, C(R5R6), and C(O), wherein R5 and R6 are each independently selected from hydroxyl and hydrogen; When X3 is selected from C(O), in equation (A-2) It is a double bond.

4. The Aphidicolin diterpenoid compound according to claim 1, characterized in that, As shown in equations (A-3), (A-4), (A-5), and (A-6), X1 is selected from C(R1R2) and C(O), wherein R1 and R2 are each independently selected from hydroxyl and -CH2OH; X2 is selected from C(R3R4), CR 11 Wherein R3 and R4 are each independently selected from hydroxyl, -CH2OH, and R 11 Selected from methyl; The X3 is selected from CH, C(R5R6), C(O), wherein R5 and R6 are each independently selected from hydroxyl, -CH2OH, alkyl, and hydrogen; X4 is selected from CH, C(R7R8), C(O), wherein R7 and R8 are each independently selected from hydroxyl and hydrogen, or one of R3 and R4 is selected from -CH2OH, and the other is connected to X4 to form a ring; The X5 is selected from CR 12 C(R9R) 10 ), C(O), wherein R9 and R 10 Each is independently selected from hydroxyl and hydrogen; the R 12 Selected from hydroxyl groups.

5. An Aphidicolin diterpenoid compound or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, cis-trans isomer, isotope label, or prodrug thereof, wherein the Aphidicolin diterpenoid compound specifically has one of the structures shown in compounds 1 to 12:

6. The use of Aphidicolin diterpenoids or pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, cis-trans isomers, isotope-labeled compounds, or prodrugs thereof in the preparation of drugs for treating bladder cancer, wherein the Aphidicolin diterpenoids have the structure shown in one of compounds 1 to 3; 7. A formulation composition comprising any one of the Aphidicolin diterpenoid compounds according to claims 1 to 5.

8. The use of the Aphidicolin diterpenoid compound according to any one of claims 1 to 5 in the preparation of a medicament for the prevention or treatment of bladder cancer.

9. A method for preparing the Aphidicolin diterpenoid compound according to any one of claims 1 to 5, characterized in that, Includes the following steps: S1: Deep-sea fungus Botryotinia fuckeliana MCCC 3A00494 was inoculated into PDB medium to cultivate seed culture; S2: The above seed liquid is inoculated into millet fermentation medium for fermentation culture, wherein the millet fermentation medium includes millet, soybean peptone, and seawater; S3: The fermentation culture is separated and purified to obtain secondary metabolites, wherein the secondary metabolites are Aphidicolin diterpenoids as described in any one of claims 1 to 5.

10. The method according to claim 9, characterized in that, The seed culture temperature in step S1 is 25–30°C, and / or the culture time for the seed culture in step S1 is 2–4 days, and / or the seed culture in step S1 is cultured at a stirring speed of 100–300 rpm, and / or 80–120 g of millet and 0.40–0.50 g of soybean peptone are added to every 120 mL of seawater in step S2, and the culture temperature for step S2 is 25–30°C.

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