Clerodane diterpene derivatives, methods and uses

Abstract

This invention relates to the field of pharmaceutical technology, specifically to a closan-type diterpenoid derivative as shown in general formula (I), a method thereof, and the application of the closan-type diterpenoid derivative in the preparation of antitumor drugs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a closan-type diterpene derivative, its method, and its uses. Background Technology

[0002] *Callicarpa pseudorubella* HTChang is a plant belonging to the genus *Callicarpa* in the family Lamiaceae. Currently, there are few reports on its chemical constituents and pharmacological activities. Cronane-type diterpenes are characteristic components of *Callicarpa* plants, with approximately 300 results reported to date. However, the preparation methods are all quite cumbersome, requiring extraction with methanol or ethanol followed by repeated separation using silica gel column chromatography, reversed-phase chromatography, and gel electrophoresis. Summary of the Invention

[0003] The purpose of this invention is to provide a closan-type diterpene derivative, a method, and its uses.

[0004] The technical problem solved by this invention is achieved by the following technical solution:

[0005] In a first aspect, the present invention provides a closan-type diterpene derivative, said closan-type diterpene derivative being as shown in general formula (I):

[0006]

[0007] in:

[0008] R1 is selected from C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 Alkyne group, 3-6 membered heterocyclic group or 3-6 membered carbocyclic group; the C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 Alkyne, 3-6 membered heterocyclic groups, or 3-6 membered carbocyclic groups are further converted by carbonyl, halogen, cyano, hydroxyl, C 1-6 Alkoxy, C 1-6 Alkyl substituents;

[0009] R2, R3, and R4 are each independently selected from C 1-6 Alkyl, C 2-6 alkenyl, C 2-6 Alkyne, halogen, cyano, hydroxyl or carboxyl.

[0010] In some embodiments of the present invention, wherein:

[0011] R1 is selected from C 1-6 Alkyl, C 2-6alkyl, halo, cyano, hydroxyl, or carboxyl. 1-6 alkyl, halo, cyano, hydroxyl, or carboxyl. 1-6 substituted by substituents of alkyl;

[0012] R2, R3, R4are each independently selected from C 1-6 alkyl, halo, cyano, hydroxyl, or carboxyl.

[0013] In some embodiments of the present application, wherein:

[0014] R1is selected from C 1-4 alkyl, C 2-6 alkyl, C 1-4 alkyl, C 2-6 alkyl, C 1-4 alkyl, C 1-4 substituted by substituents of alkyl;

[0015] R2, R3, R4are each independently selected from C 1-4 alkyl, halo, cyano, hydroxyl, or carboxyl.

[0016] In some embodiments of the present application, wherein:

[0017] R1is selected from C 1-4 alkyl, C 2-6 alkyl, C 1-4 alkyl, C 2-6 alkyl, C 1-4 alkyl, C 1-4 substituted by substituents of alkyl;

[0018] R2, R3, R4are each independently selected from C 1-4 alkyl, or carboxyl.

[0019] In some embodiments of the present application, the structure of the Cembranoid diterpene derivative is:

[0020]

[0021] In a second aspect of the present application, a method for extracting Cembranoid diterpene from R. pseudo-hyalina is provided, comprising the following steps:

[0022] Step one, immerse R. pseudo-hyalina in petroleum ether, and concentrate to get the extract;

[0023] Step two, elute the extract with methanol aqueous solution, and get the Cembranoid diterpene.

[0024] In some embodiments of the present invention, in step one, *Callicarpa rubiginosa* is soaked in petroleum ether 2 to 4 times, each time for 60 to 80 hours.

[0025] In some embodiments of the present invention, step two includes the following steps:

[0026] S21. Divide the extract into 5 portions and elute them sequentially with methanol solvents of 58-62%, 68-72%, 78-82%, 88-92%, and 98-100%, respectively. Eluting with 58-62% methanol solvent yields Fr.1, 68-72% methanol solvent yields Fr.2, 78-82% methanol solvent yields Fr.3, 88-92% methanol solvent yields Fr.4, and 98-100% methanol solvent yields Fr.5.

[0027] S22. Crystallize Fr.2 component in 68-72% methanol solvent to obtain compound 1;

[0028] Fr.3 fraction was eluted by column chromatography with dichloromethane and methanol at a ratio of 1:(0.9-1.1), and then purified by semi-preparative high performance liquid chromatography with 82-88% methanol aqueous solution to obtain compound 2;

[0029] Fr.4 fraction was eluted by column chromatography with dichloromethane and methanol at a ratio of 1:(0.9–1.1), and then purified by semi-preparative high performance liquid chromatography with 62–68% acetonitrile aqueous solution to obtain compounds 3 and 4, respectively.

[0030] The above-mentioned closan-type diterpenoid derivatives are used in the preparation of antitumor drugs.

[0031] This invention offers the following advantages: Four novel closan-type diterpenoid compounds (1-4) were isolated from the petroleum ether fraction of *Callicarpa rubiginosa* using phytochemical methods, including HPLC and recrystallization. Compound 2 contains a succinic anhydride fragment, a relatively rare structure in natural compounds, more commonly found in synthetically produced compounds. Screening of all compounds for antitumor activity revealed that compound 2 exhibited better inhibitory activity against human erythroleukemia leukemia cells (HEL), exceeding the performance of positive control drugs.

[0032] Croane-type diterpenes are characteristic components of plants in the genus *Callicarpa*, with approximately 300 reported to date. However, their preparation methods are generally cumbersome, requiring extraction with methanol or ethanol followed by repeated separation using silica gel column chromatography, reversed-phase chromatography, and gel electrophoresis. In contrast, cloane-type diterpenoid derivatives can be obtained by soaking in petroleum ether, followed by reversed-phase chromatography and recrystallization, eliminating the need for complex preparative purification. Attached Figure Description

[0033] Figure 1The chemical structures of compounds 1-4 are shown.

[0034] Figure 2 The key 1H-1H COSY, HMBC, and NOESY related signals for compounds 1-4 are shown.

[0035] Figure 3 This is a comparison of the measured and calculated ECD spectra of compound 2.

[0036] Figure 4 The diffraction patterns are for single crystals of compounds 1, 3, and 4.

[0037] Figure 5 For compound 1 1 H NMR spectrum.

[0038] Figure 6 For compound 1 13 C10 NMR spectrum.

[0039] Figure 7 This is the DEPT spectrum of compound 1.

[0040] Figure 8 For compound 1 1 H– 1 H COSY spectrum.

[0041] Figure 9 This is the HSQC spectrum of compound 1.

[0042] Figure 10 The image shows the HMBC spectrum of compound 1.

[0043] Figure 11 The image shows the NOESY spectrum of compound 1.

[0044] Figure 12 The image shows the HR-ESI-MS spectrum of compound 1.

[0045] Figure 13 This is the UV spectrum of compound 1.

[0046] Figure 14 This is the IR spectrum of compound 1.

[0047] Figure 15 For compound 2 1 H NMR spectrum.

[0048] Figure 16 For compound 2 13 C10 NMR spectrum.

[0049] Figure 17 This is the DEPT spectrum of compound 2.

[0050] Figure 18 For compound 2 1 H– 1 H COSY spectrum.

[0051] Figure 19 This is the HSQC spectrum of compound 2.

[0052] Figure 20 The image shows the HMBC spectrum of compound 2.

[0053] Figure 21 The image shows the NOESY spectrum of compound 2.

[0054] Figure 22 The image shows the HR-ESI-MS spectrum of compound 2.

[0055] Figure 23 This is the UV spectrum of compound 2.

[0056] Figure 24 This is the IR spectrum of compound 2.

[0057] Figure 25 Compound 3 1 H NMR spectrum.

[0058] Figure 26 For compound 3 13 C10 NMR spectrum.

[0059] Figure 27 This is the DEPT spectrum of compound 3.

[0060] Figure 28 For compound 3 1 H– 1 H COSY spectrum.

[0061] Figure 29 The image shows the HSQC spectrum of compound 3.

[0062] Figure 30 The image shows the HMBC spectrum of compound 3.

[0063] Figure 31 The image shows the NOESY spectrum of compound 3.

[0064] Figure 32 The image shows the HR-ESI-MS spectrum of compound 3.

[0065] Figure 33 This is the UV spectrum of compound 3.

[0066] Figure 34 The image shows the IR spectrum of compound 3.

[0067] Figure 35 For compound 41 H NMR spectrum.

[0068] Figure 36 For compound 4 13 C10 NMR spectrum.

[0069] Figure 37 For compound 4 1 H– 1 H COSY spectrum.

[0070] Figure 38 The image shows the HSQC spectrum of compound 4.

[0071] Figure 39 The image shows the HMBC spectrum of compound 4.

[0072] Figure 40 The image shows the NOESY spectrum of compound 4.

[0073] Figure 41 The image shows the HR-ESI-MS spectrum of compound 4.

[0074] Figure 42 This is the UV spectrum of compound 4.

[0075] Figure 43 This is the IR spectrum of compound 4.

[0076] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0077] As described in this invention, the term "prevention" refers to preventing the occurrence of disease and / or preventing the recurrence of disease. Detailed Implementation

[0078] This specification provides a detailed description of specific embodiments. Those skilled in the art should recognize that the following embodiments are exemplary and should not be construed as limiting the invention. For those skilled in the art, various improvements and modifications can be made to the invention without departing from its principles; such improvements and modifications also fall within the scope of protection of the claims. The beneficial effects of the invention are illustrated below through specific examples.

[0079] Example 1

[0080] The method for extracting closan-type diterpenes from *Callicarpa rubiginosa* involves the following steps:

[0081] 7.8 kg of dried Callicarpa pseudorubella HTChang (a plant belonging to the genus Callicarpa of the family Lamiaceae) was pulverized and soaked in petroleum ether three times, 20 L each time, for three days each time. After recovering the petroleum ether, the extract was concentrated to obtain 92.7 g.

[0082] The petroleum ether extract was separated by reversed-phase silica gel column chromatography, eluted sequentially with 15 L each of 60%, 70%, 80%, 90%, and 100% methanol solvents. After concentrating the eluent, five fractions (Fr.1-Fr.5) were obtained. Fr.2 (7.8 g) precipitated needle-like crystals in a methanol:water ratio of 70:30, which was compound 1. Fr.3 (12.3 g) was eluted by a Sephadex LH-20 gel chromatography column (dichloromethane-methanol 1:1), and then purified by semi-preparative high-performance liquid chromatography (methanol:water = 85:15, Rt = 24.3 min, C18 reversed-phase column) to obtain compound 2. Fr.4 (13.1 g) was purified by elution on a gel chromatography column (dichloromethane:methanol 1:1), and then separated by a semi-preparative high performance liquid chromatography system using a C18 reversed column and an acetonitrile / water ratio of 65:35 as the mobile phase, yielding compound 3 (Rt = 28 min) and compound 4 (Rt = 30 min).

[0083] Compound 1: Colorless crystals (methanol), optical rotation data (c 1.53, methanol); UV (methanol) 218(3.37); IR (cm⁻¹): 2948, 1725, 1698, 1665, 1421, 1283, 1140; High-resolution mass spectrometry ( Figure 12 HR-ESI-MS: m / z 371.2188 [M+Na]+ (calculated value: 371.2192), determine its molecular formula C 21 H 32 O4, with an unsaturation degree of 6. In the 1H-NMR spectrum ( Figure 5 Compound 1 showed four methyl signals δH 0.76 (3H, d, J = 6.6 Hz, H-17), δH 0.77 (3H, s, H-20), δH 1.22 (3H, s, H-19), and δH 2.17 (3H, s, H-16), one methoxy signal δH 3.67 (3H, s), and two olefin proton signals δH 6.80 (1H, t, J = 3.6 Hz, H-3) and δH 5.67 (1H, s, H-14). In the 13C-NMR spectrum (… Figure 6 ) and DEPT135 spectrum ( Figure 7The NMR spectrum of compound 1 shows 21 carbon signals, including 6 quaternary carbons, 4 methyl groups, 6 methylene groups, and 5 methine groups. These signals characterize compound 1 as having a diterpenoid skeleton similar to that of compound (-)-5-epi-hardwickiic acid. Careful comparison of the one-dimensional NMR data of compound 1 and compound (-)-5-epi-hardwickiic acid reveals that the difference is that compound 1 has an additional methoxy group. The HMBC spectrum of compound 1... Figure 10 The significant correlation between δH 3.67 (-OMe) and C-15 (δC 167.4) in the methyl group indicates that the methoxy group is attached at the C-15 position via 1H-1H COSY ( Figure 8 ) and HMBC ( Figure 10 The remaining fragment of compound 1 was identified as being identical to that of compound (-)-5-epi-hardwickiic acid. Thus, the planar structure of compound 1 was determined. Subsequently, we analyzed the NOESY spectrum... Figure 11 Correlation signals of Me-18, Me-19, and H-10 were used to establish the consistency of the configurations of Me-18, Me-19, and H-10. Correlation signals of Me-20 and Me-17 were then used to confirm the consistency of the configurations of Me-20 and Me-17, which are opposite to those of Me-18, Me-19, and H-10. This determined the relative configuration of compound 1. To further determine the stereostructure of compound 1, we cultivated a single crystal of compound 1 for the first time. Figure 4 According to the X-ray single crystal determination (Cu target) results (CCDC number 2298088), the Flack constant is -0.03(4), and its absolute configuration is determined to be 5R,8S,9R,10S. Figure 1 ).

[0084] Compound 2: Colorless powder, optical rotation data (c 0.33, methanol); CD(MeOH)λmax(Δε): 217(7.81), 239(-13.04)nm; UV(methanol) 221(3.39), IR(cm-1): 2960, 2360, 1714; High-resolution mass spectrometry ( Figure 22 The molecular ion peak is shown at m / z 369.1669 [M+Na]+ (calculated value 369.1673). Combined with the 1D-NMR spectrum, the molecular formula of compound 2 is determined to be C20H26O5, with an unsaturation degree of 8. In the 1H-NMR spectrum (…), Figure 15Compound 2 showed proton signals of δH 6.80 (1H, t, J = 3.6 Hz, H-3) and δH 6.29 (1H, s, H-14) for the two olefins, and δH 1.25 (3H, s, H-10), δH 0.82 (3H, s, H-20), and δH 0.79 (3H, d, J = 6.6 Hz, H-19) for the three methyl groups. In 13C-NMR (… Figure 16 ) spectrum and DEPT135 spectrum ( Figure 17 The NMR spectrum shows 20 carbon signals, including 7 quaternary carbons, 3 methyl groups, 6 methylene groups, and 4 methine groups. These signals characterize compound 2 as having a similar carbon skeleton to compound 1. A careful comparison of the one-dimensional NMR data of compound 2 and compound 1 reveals that the difference is that one methoxy group in compound 1 is replaced by a carboxyl group. In the HMBC spectrum (…),… Figure 20 In the NOESY spectrum, two sets of correlation signals were observed: H-12 (δH 2.32) with C-13 (δc 152.2), C-1 (δc 127.0), C-16 (δc 171.6), and H-14 (δH 6.29) with C-16 (δc 171.0), C-15 (δc 170.6). These correlations further verified the presence of a rare natural furan-2,5-dione unit in the planar structure of compound 2. Therefore, the planar structure of compound 2 was determined. Subsequently, we further analyzed the NOESY spectrum... Figure 21 The relative configuration of compound 2 was established by analyzing two sets of correlation signals: Me-20 and Me-17, and Me-19 and H-10 and H-8. Subsequently, we compared the measured and calculated ECD spectra of compound 2. Figure 3 The absolute configuration of compound 2 was established as 5R,8S,9R,10S. Figure 1 ).

[0085] Compound 3: Optical rotation data (c 0.15, methanol); CD(MeOH)λmax(Δε): 200(7.82), 243(-20.45)nm; UV(methanol) 210(2.67)IR (cm⁻¹): 3286, 2959, 1741, 1681, 1645, 1448, 1385, 1268; High-resolution mass spectrometry ( Figure 32 The molecular ion peak is shown at m / z 371.1828 [M+H]+ (calculated value is 371.1829). Compound 4: Optical rotation data (c 0.11, methanol); UV (methanol) 264 (4.52); IR (cm⁻¹): 2961, 1740, 1680, 1267; High-resolution mass spectrometry ( Figure 41 The molecular ion peak is shown at m / z 371.1821 [M+H]+ (calculated value is 371.1829). Combined with the 1D-NMR spectrum, the molecular formulas of compounds 3 and 4 are determined to be C4. 20 H 28 O5, with an unsaturation degree of 7.

[0086] Careful comparison of the one-dimensional NMR data of compounds 3 and 4 with those of compound 2 reveals that compounds 3 and 4 are also very similar to compound 2. The only difference is that a carbonyl group in compound 2 is replaced by a hydroxyl group in compounds 3 and 4. Furthermore, the HMBC spectra from H-16 to C-12, C-13, C-14, and C-15 are also shown. Figure 30 , Figure 39 Correlation was used to assign the hemiacetal group to C-16. Furthermore, the planar structures of compounds 3 and 4 were determined by analysis of their two-dimensional NMR information. To determine their absolute configurations, we cultivated single crystals of compounds 3 and 4 for the first time. Figure 3 According to the X-ray single crystal determination (Cu target) results (CCDC number 2298086), the Flack constant is 0.11 (13), and the absolute configurations of compounds 3 and 4 are determined to be 5R, 8S, 9R, 10S, 16S and 5R, 8S, 9R, 10S, 16R, respectively. Figure 1 ).

[0087] Example 2

[0088] Experimental methods and procedures for antitumor activity;

[0089] The methods for assessing antitumor activity were performed according to the literature [M. Bai, YY Zhang, SHDong, H. Ren, JJ Chen, GD Yao, QB Liu, B. Lin, XX Huang, SJSong, Targeted isolation of cytotoxic germacranolide sesquiterpenes from Elephantopus scaber L. using small molecule accurate recognition technology, Bioorg. Chem. 104(2020)104314]. The cytotoxicity of compounds 1-4 against human erythroleukemia cells was evaluated using a CCK-8 assay kit (cell proliferation and toxicity assay kit).

[0090] HEL cells were cultured in RPMI 1640 medium [containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin]. Cells were passaged at 37°C in a 5% CO2 cell culture incubator, and cells in the logarithmic growth phase were used for experiments. The experiment included an experimental group (containing cell culture medium + CCK-8 + compound (1-4) or 5-fluorouracil), a control group (containing cell culture medium + CCK-8), and a blank group (without cell culture medium + CCK-8). 5-fluorouracil (5-FU) served as a positive control, with three replicates per group. HEL cells in the logarithmic growth phase were cultured at a rate of 3 × 10⁻⁶ cells / year. 3 90 μL of reagent was seeded per well in a 96-well plate and incubated for 24 h. Subsequently, 10 μL of compound 1 or 5-fluorouracil was added to each well in experimental groups at concentrations of (50, 25, 12.5, 6.25, 3.13, 1.56 μM) and the plates were incubated for another 48 h. Then, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated for 1 h. The absorbance was measured at 450 nm. The inhibition rate was calculated as follows: Inhibition rate (%) = (D... 对照组 -D 实验组 ) / (D 对照组 -D 空白组 ()×100%. (Compounds 2-4 were tested using the same method). The results of the activity data of compounds 1-4 in inhibiting human erythroleukemia cells (HEL) are shown in Table 1.

[0091] Table 1 shows the inhibitory activity of compounds 1-4 on human erythroleukemia cells (HEL).

[0092] Compd. IC 50 (μM) 1 43.21±1.04 2 14.02±0.77 3 23.3±0.26 4 32.4±0.71

[0093] As shown in Table 1, compounds 1-4 have good inhibitory activity against the proliferation of erythroleukemia leukemia (HEL).

[0094] Four new closan-type diterpenoid compounds (1-4) were isolated from the petroleum ether fraction of *Callicarpa rubiginosa*. Compound 2, in particular, contains a succinic anhydride fragment, a relatively rare structure in natural compounds. Screening for antitumor activity of all compounds revealed that compound 2 exhibited good inhibitory activity against human erythroleukemia leukemia cells (HEL).

[0095] This invention specification provides a detailed description of specific embodiments. Those skilled in the art should recognize that the above embodiments are exemplary and should not be construed as limiting the invention. For those skilled in the art, various improvements and modifications can be made to the invention without departing from its principles, and the resulting technical solutions also fall within the scope of protection of the claims of this invention.

Claims

1. A closan-type diterpene derivative, characterized in that, The structure of the closan-type diterpene derivative is as follows: or .

2. A method for extracting closan-type diterpenes from *Callicarpa rubiginosa*, characterized in that, Includes the following steps: Step 1: Soak the *Callicarpa rubiginosa* in petroleum ether 2-4 times, 60-80 hours each time, and concentrate to obtain an extract; Step 2: Purification and preparation of closan-type diterpenes, including the following steps: S21. Divide the extract into 5 portions and elute them sequentially with methanol solvents of 58-62%, 68-72%, 78-82%, 88-92%, and 98-100%, respectively. Elute with 58-62% methanol solvent to obtain Fr.1, elute with 68-72% methanol solvent to obtain Fr.2, elute with 78-82% methanol solvent to obtain Fr.3, elute with 88-92% methanol solvent to obtain Fr.4, and elute with 98-100% methanol solvent to obtain Fr5. S22. Crystallize Fr.2 component in 68-72% methanol solvent to obtain compound 1; Fr.3 fraction was eluted by column chromatography with dichloromethane and methanol at a ratio of 1:(0.9~1.1), and then purified by semi-preparative high performance liquid chromatography with 82~88% methanol aqueous solution to obtain compound 2; The Fr.4 fraction was eluted by column chromatography with dichloromethane and methanol at a ratio of 1:(0.9~1.1), and then purified by semi-preparative high performance liquid chromatography with 62~68% acetonitrile aqueous solution to obtain compounds 3 and 4, respectively. The structure of compound 1 is as follows: The structure of compound 2 is as follows: The structure of compound 3 is as follows: The structure of compound 4 is as follows: .

3. The use of the closan-type diterpene derivative according to claim 1 or 2 in the preparation of a medicament for treating leukemia.

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