Oxatetracyclic abietane-type terpenoids isolated from salvia yunnanensis and uses thereof

Abstract

This invention discloses oxa-tetracyclohexane-type terpenoid compounds isolated from *Salvia yunnanensis* and their applications, belonging to the field of natural product chemistry. The compounds are novel skeleton compounds represented by formula (I) or formula (II). Their preparation method includes: taking *Salvia yunnanensis* root powder, extracting with organic solvents and ethyl acetate, and then purifying by macroporous adsorption resin column chromatography, silica gel column chromatography, and MCI reversed-phase column chromatography to obtain compounds of formula (I) and formula (II), respectively. Activity experiments show that compound (I) has a half-maximal inhibitory concentration (IC50) of 1 / 3 of LSD1. 50 The concentration was 17.23 μM, and the compound of formula (II) showed an inhibition rate of 69.78% at a concentration of 50 μM. The compounds of this invention can be used as LSD1 inhibitors in the preparation of anti-tumor drugs, providing new lead molecules for epigenetic targeted therapy, and also expanding the medicinal value of Salvia miltiorrhiza.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry and pharmaceutical technology, specifically relating to a class of terpenoid compounds with a novel (6 / 6 / 6 / 5)-oxatetracyclorosine skeleton isolated from Sageia yunnanensis, their preparation method, and the application of such compounds as lysine-specific demethylase 1 (LSD1) inhibitors in the preparation of antitumor drugs. Background Technology

[0002] Yunnan sage ( Salvia yunnanensis *Salvia miltiorrhiza* (CH Wright) is a plant belonging to the genus *Salvia* in the Lamiaceae family. Its roots have been used medicinally in Yunnan Province for centuries as a substitute for *Salvia miltiorrhiza*, possessing properties such as clearing heat and detoxifying, reducing swelling and relieving pain. However, compared to the in-depth and systematic chemical composition and pharmacological research of *Salvia miltiorrhiza*, research on the chemical composition and modern activity of *Salvia miltiorrhiza* from Yunnan remains very weak, with very few reports of novel structural compounds and their targeted activities.

[0003] The development and progression of tumors are closely related to epigenetic regulatory disorders. Histone lysine-specific demethylase 1 (LSD1) is an important epigenetic regulator that regulates gene transcription by catalyzing the demethylation of histones H3K4 and H3K9. LSD1 is highly expressed or abnormally activated in various tumors, promoting tumor cell proliferation, invasion, metastasis, and stemness maintenance not only through its demethylase activity but also through non-catalytic protein interactions. Therefore, LSD1 has become an important target for anti-tumor drug development. Several small molecule inhibitors of LSD1 have entered clinical trials, but they still face challenges such as insufficient selectivity, unsatisfactory pharmacokinetic properties, and limited efficacy against solid tumors. There is an urgent need to develop LSD1 inhibitors with novel structures and unique mechanisms of action.

[0004] Natural products are an important source of lead compounds for innovative drugs. This invention isolated two compounds with a novel (6 / 6 / 6 / 5)-oxatetracyclorosiline skeleton from the root of *Salvia yunnanensis*, and for the first time discovered their significant inhibitory activity against LSD1. This provides novel lead molecules for the development of new LSD1 inhibitors and also expands the medicinal value of *Salvia yunnanensis*. To date, there have been no reports on such compounds and their LSD1 inhibitory activity. Summary of the Invention

[0005] This invention aims to provide oxa-tetracyclorosiline-type terpenoid compounds isolated from *Salvia yunnanensis* and their applications. The above-mentioned objective of this invention is achieved through the following technical solution.

[0006] A (6 / 6 / 6 / 5)-oxatetracyclorosine-type terpene compound, wherein the compound of the present invention is any one of the compounds shown in formula (I) or formula (II): .

[0007] Furthermore, the compound described in this invention is the compound shown in formula (I): .

[0008] Furthermore, the compound described in this invention is the compound shown in formula (II): .

[0009] The preparation method of the (6 / 6 / 6 / 5)-oxatetracyclorosine-type terpenoid compound of the present invention includes the following steps: taking Yunnan sage Salvia yunnanensis The roots of CH Wright were crushed and then extracted directly by cold soaking in organic solvents such as ethanol or water, or by hot reflux, or by ultrasonic or microwave-assisted extraction. Alternatively, the roots could be first extracted by cold soaking in the above-mentioned organic solvents or water, or by reflux, or by ultrasonic or microwave-assisted extraction, and then extracted with ethyl acetate to obtain a total extract. The total ethyl acetate extract was repeatedly chromatographically analyzed to obtain compounds represented by Formula I and Formula II.

[0010] Further, the present invention includes the following steps: The roots of *Salvia yunnanensis* are dried in the shade, pulverized to 20-30 mesh, and extracted with 95% ethanol at room temperature. The extracts are combined, and the extracts are concentrated under reduced pressure to obtain a total extract. The total extract is extracted with ethyl acetate using solid-phase extraction or liquid-liquid extraction to obtain the ethyl acetate extract fraction. The ethyl acetate extract fraction is loaded onto a D101 macroporous adsorption resin, and then subjected to gradient elution with methanol aqueous solutions of concentrations of 50%, 70%, 80%, 90%, and 100% to obtain five main fractions. The 80% fraction is then... The methanol elution fraction was subjected to silica gel column chromatography with petroleum ether / acetone at a ratio of 18:1-0:1 to obtain six sub-fractions Fr.1-Fr.6. Fraction Fr.3 was subjected to MCI reversed-phase column chromatography with gradient elution of methanol / water at a volume ratio of 1:1-0:1 to obtain fractions Fr.3.1-Fr.3.10. Fr.3.10 was subjected to repeated silica gel column chromatography with petroleum ether / ethyl acetate at a volume ratio of 20:1-0:1 and dichloromethane / methanol at a volume ratio of 300:1-0:1 to obtain compounds 1 and 2 of formula (I) and formula (II).

[0011] As a product, the present invention is a pharmaceutical composition comprising the aforementioned compounds or combinations of compounds.

[0012] Furthermore, the dosage form of the pharmaceutical composition of the present invention is an oral formulation or an injectable formulation.

[0013] As an application, the aforementioned compounds or pharmaceutical compositions of the present invention are used in the preparation of lysine-specific demethylase 1 inhibitors.

[0014] Furthermore, the lysine-specific demethylase 1 inhibitor described in this invention is used to prepare drugs for treating and / or preventing tumor diseases.

[0015] Furthermore, the tumor diseases described in this invention include at least one of leukemia, lung cancer, breast cancer, colorectal cancer, and prostate cancer.

[0016] Compared with the prior art, the present invention has the following significant advantages: (1) This invention provides a class of rosinane-type terpenoid compounds (Formula I and Formula II) with a novel (6 / 6 / 6 / 5)-oxatetracyclic skeleton. According to literature search, this type of skeleton structure has not been reported to date. It belongs to a completely new compound entity, which enriches the structural diversity of natural products and provides a novel lead molecule template for medicinal chemistry research.

[0017] (2) This invention is the first to discover that this type of compound has significant LSD1 inhibitory activity. Activity assay results show that the half-maximal inhibitory concentration (IC50) of compound (I) against LSD1 is... 50 The concentration of the inhibitory concentration (ICM) was 17.23 μM, and the inhibitory rate of compound (II) reached 69.78% at a concentration of 50 μM, both of which showed concentration dependence. This suggests that these compounds can serve as lead molecules for LSD1 inhibitors, providing novel chemical entities for subsequent structural optimization and anti-tumor drug development.

[0018] (3) The compounds of this invention are derived from Yunnan sage, which has abundant raw material resources. The extraction and separation process is simple, mild, and reproducible, making it suitable for large-scale preparation. At the same time, these compounds have good physicochemical stability and clear spectroscopic identification characteristics, which facilitates quality control and the establishment of quality standards.

[0019] (4) This invention provides new scientific basis for the development of the medicinal value of Yunnan sage, expands the application prospects of this traditional medicinal material in the field of targeted epigenetic regulation, and is of great significance for the in-depth development of Yunnan's characteristic plant resources and the research and development of innovative drugs. The invention will be further explained below with reference to the accompanying drawings and specific embodiments.

[0020] The present invention will be further explained below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0021] Figure 1 For compound 1 1 H-NMR spectrum.

[0022] Figure 2 For compound 1 13 C-NMR spectrum.

[0023] Figure 3 The image shows the HR-ESI-MS spectrum of compound 1.

[0024] Figure 4 For compound 2 1 H-NMR spectrum.

[0025] Figure 5 For compound 2 13 C-NMR spectrum.

[0026] Figure 6 The image shows the HR-ESI-MS spectrum of compound 2. Detailed Implementation

[0027] The following embodiments are only a part of the technical solutions of the present invention and are not intended to limit all the technical solutions of the present invention. The embodiments of the present invention are provided to further explain and illustrate the details of the technical solutions of the present invention.

[0028] Oxalide-type terpenoids isolated from *Salvia yunnanensis* and their applications, wherein the compounds include either compound 1 (Formula I) or compound 2 (Formula II): .

[0029] The preparation method of compounds (I) and (II) involves taking the root of *Salvia yunnanensis*, crushing it to 20-30 mesh, and directly extracting it with organic solvents such as ethanol or water by cold soaking, hot reflux, ultrasound, or microwave. Alternatively, it can be extracted with the above-mentioned organic solvents or water by cold soaking, reflux, ultrasound, or microwave, and then extracted with ethyl acetate to obtain ethyl acetate extract. The ethyl acetate extract is repeatedly subjected to column chromatography to obtain compounds (I) and (II).

[0030] The method for preparing the compounds of formula (I) and formula (II) of the present invention is more specifically described using: A: The total extract of the root of Sage in Yunnan is obtained by direct cold soaking with methanol, ethanol or water, or by hot reflux, or by ultrasonic or microwave-assisted extraction. The extract is then extracted with ethyl acetate to obtain ethyl acetate extract. Repeated column chromatography of the ethyl acetate extract yields compounds of formula (I) and formula (II).

[0031] B: The total extract of Yunnan sage is obtained by direct cold soaking, hot reflux, ultrasonic or microwave-assisted extraction of the root by organic solvents (such as methanol, ethanol, acetone, etc.). The total extract can be obtained by repeated column chromatography to obtain compounds of formula (I) and formula (II).

[0032] More specifically, the preparation method of the compounds of formula (I) and formula (II) involves drying the roots of *Salvia yunnanensis* in the shade, pulverizing them to 20-30 mesh, and extracting them four times with 95% ethanol at room temperature, each time for 24 minutes. h, the extracts were combined and concentrated under reduced pressure to obtain the total extract. The total extract was extracted with ethyl acetate using solid-phase extraction or liquid-liquid extraction to obtain the ethyl acetate extract fraction. The ethyl acetate extract fraction was loaded onto D101 macroporous adsorption resin and then eluted with a gradient of methanol-water solution (50%, 70%, 80%, 90%, 100%) to obtain 5 major fractions. The 80% methanol elution fraction was subjected to silica gel column chromatography with petroleum ether / acetone 18:1-0:1 to obtain 6 sub-fractions (Fr.1-Fr.6). Fraction Fr.3 was subjected to MCI reversed-phase column chromatography with a gradient elution of methanol / water (1:1-0:1) to obtain components Fr.3.1-Fr.3.10. Fr.3.10 was subjected to repeated silica gel column chromatography (petroleum ether / ethyl acetate 20:1-0:1 and dichloromethane / methanol 300:1-0:1) to obtain compounds (I) and (II) 1 and 2.

[0033] Example 1: Extraction, separation, and purification of compounds 1 and 2 of the present invention: The roots of *Salvia yunnanensis* were dried in the shade, pulverized to 20-30 mesh, and extracted four times with 95% ethanol at room temperature for 24 hours each time. The extracts were combined and concentrated under reduced pressure to obtain a total extract. The total extract was extracted with ethyl acetate using solid-phase extraction or liquid-liquid extraction to obtain the ethyl acetate extract fraction. The ethyl acetate extract fraction was loaded onto D101 macroporous adsorption resin and then eluted with a gradient of methanol-water solution (50%, 70%, 80%, 90%, 100%) to obtain five major fractions. The 80% methanol eluent fraction was subjected to silica gel column chromatography with petroleum ether / acetone at a ratio of 18:1 to 0:1 to obtain six sub-fractions (Fr.1-Fr.6). Fraction Fr.3 was subjected to MCI reversed-phase column chromatography with a gradient elution of methanol / water (1:1-0:1) to obtain fractions Fr.3.1-Fr.3.10. Fr.3.10 was subjected to repeated silica gel column chromatography (petroleum ether / ethyl acetate 20:1-0:1 and dichloromethane / methanol 300:1-0:1) to obtain compounds (I) and (II) 1 and 2.

[0034] Example 2: Physical and spectroscopic data of compound 1 of the present invention: Green needle-like crystals, [α]25 D -94.35 ( c = 0.12, MeOH); UV (MeOH) l max (log e):216 (0.69), 232 (0.97), 246 (0.90), 261 (0.99), 341 (0.12) nm; ECD (MeOH) λ max (Δ e ): 217 (-1.25), 264 (-0.19) nm; IR (KBr) n max 3398, 2928, 1706 cm −1 ; HR-ESI-MS: m / z 377.1365 [M+Na] + (calcd for C 21 H 22 O 54 Na + 377.1359); 1 H-NMR (400 MHz,CDCl3) d : 3.45 (2H, m, H-1), 1.87 (2H, m, H-2a), 1.72 (2H, t, J = 7.0 Hz, H-2b), 1.87 (2H, m, H-3a), 1.72 (2H, t, J = 7.0 Hz, H-3b), 7.77 (1H, d, J = 8.8Hz, H-6), 8.23 (1H, d, J = 8.8 Hz, H-7), 1.72 (3H, s, H-17), 1.36 (3H, s, H-18), 1.39 (3H, s, H-19), 2.74 (3H, s, H-22); 13 C-NMR (100 MHz, CDCl3) d: 30.5(t, C-1), 19.6 (t, C-2), 38.1 (t, C-3), 35.1 (s, C-4), 150.1 (s, C-5), 131.3(d, C-6), 123.1 (d, C-7), 130.7 (s, C-8), 124.6 (s, C-9), 136.2 (s, C-10), 159.7 (s, C-11), 106.6 (s, C-12), 103.0 (s, C-13), 169.2 (s, C-14), 198.6 (s, C-16), 22.8 (q, C-17), 31.9 (q, C-18), 31.9 (q, C-19), 204.1 (s, C-20), 108.9(s, C-21), 31.4(q, C-22).

[0035] Physical and spectral data of compound 2 of this invention: Yellow oily, [α]25 D -19.72 ( c = 0.10, MeOH); UV (MeOH) l max (log e ): 2220(2.00), 235 (2.13), 295 (0.41) nm; ECD (MeOH) λ max (Δ e ): 211 (-2.37), 232(0.21), 239 (-0.41) nm; IR (KBr) n max 3445, 2941, 1777, 1708 cm −1 HR-ESI-MS: m / z 405.1317 [M+Na] + (calcd for C 22 H 22 O6Na + 405.1309); 1 H-NMR (600 MHz, CDCl3) d :3.28 (1H, m, H-1a), 2.84 (1H, t, J = 5.6 Hz, H-1b), 1.96 (1H, m, H-2a), 1.77(1H, m, H-2b), 2.50 (2H, m, H-3), 7.90 (1H, d,J = 8.3 Hz, H-6), 7.54 (1H, d, J = 8.3 Hz, H-7), 2.07 (3H, s, H-17), 5.52 (1H, s, H-18a), 5.08 (1H, s, H-18b), 3.42 (1H, s, H-21a), 3.00 (1H, s, H-21b), 2.21 (3H, s, H-23), 3.22 (3H,s, 14-OCH3); 13 C-NMR (150 MHz, CDCl3) d : 27.8 (t, C-1), 23.4 (t, C-2), 32.4 (t,C-3), 143.0 (s, C-4), 138.5 (s, C-5), 129.4 (d, C-6), 122.6 (d, C-7), 135.1(s, C-8), 130.4 (s, C-9), 139.4 (s, C-10), 197.9 (s, C-11), 74.6 (s, C-12), 151.9 (s, C-13), 102.6 (d, C-14), 127.4 (s, C-15), 171.4 (s, C-16), 9.9 (q,C-17), 110.9 (t, C-18), 50.2 (t, C-21), 205.2 (s, C-22), 31.3 (q, C-23), 52.1 (q, 14-OCH3).

[0036] a Chemical shift values ​​were measured using Bruker Avance III-600 MHz and Bruker Avance III-400 MHz nuclear magnetic resonance spectrometers and are expressed in ppm.

[0037] Example 3: Detection of LSD1 inhibitory activity of the compounds of the present invention: LSD1 activity assay was performed using the AlphaLISA method, with LSD1IV as the positive control (IC50). 50= 3.7 nM), using LSD1 enzyme (Cat. No. 31426) and H3K4me2 peptide from ACTIVE MOTIF as substrates. The experimental buffer was a 1× assay buffer containing 50 mM Tris-HCl (pH 7.5), 0.005% Tween-20, 1 mM DTT, and 0.005% BSA. The compound was dissolved in 100% DMSO and diluted to a final concentration of 100×. It was then serially diluted 3-fold to 10 concentration points, and 100 nL was transferred to a 384-well plate using an Echo 550 Liquid Handler, with a final DMSO concentration of 1%. Then, 5 μL of enzyme solution (or 1× assay buffer as a low control) was added, centrifuged at 1000 rpm for 1 min, and incubated at room temperature for 15 min. Then, 5 μL of substrate solution was added to start the reaction, centrifuged at 1000 rpm for 1 min, and reacted at room temperature for 40 min. After the reaction, add 15 μL of 25 / 15× detection solution (containing AlphaLISA Anti-unmodified Histone H3 Lysine 4 Acceptor beads and Strep-Tactin® Alpha Donor beads, with final concentrations of 25 μg / mL and 15 μg / mL, respectively), centrifuge at 1000 rpm for 1 min, incubate at room temperature in the dark for 60 min, and finally read the plate using EnSpire (Alpha mode). The inhibition rate is calculated as Inh% = (Max – Signal) / (Max – Min) × 100, IC50 is calculated as follows: 50 The equation Y = Bottom + (Top – Bottom) / (1 + (IC) is adopted. 50 / X)^HillSlope) fitting.

[0038] Activity data are shown in Tables 1 and 2.

[0039] Table 1. LSD1 inhibitory activity data of compounds 1 and 2 at 50 μM (x ± 0.5%) s , n = 3)

[0040] Table 2 shows the inhibitory effect of compound 1 on LSD1 IC50. 50 (μM) value.

[0041] As can be seen from the table above, compounds 1 and 2 prepared in Example 1 of this invention have good inhibitory activity against LSD1.

[0042] The above descriptions are merely some specific embodiments of the present invention. Commonly known details or common knowledge in the solutions are not described in detail here (including but not limited to abbreviations, acronyms, units commonly used in the art, experimental methods, parameter conditions, etc.). It should be noted that the above embodiments do not limit the present invention in any way. For those skilled in the art, any technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A (6 / 6 / 6 / 5)-oxatetracyclorosine-type terpene compound, characterized in that, The compound is any one of the compounds shown in formula (I) or formula (II): 。 2. The compound according to claim 1, characterized in that, The compound is the compound shown in formula (I): 。 3. The compound according to claim 1, characterized in that, The compound is the compound shown in formula (II): 。 4. The method for preparing the (6 / 6 / 6 / 5)-oxatetracyclorosine-type terpene compound according to any one of claims 1 to 3, characterized in that, Includes the following steps: Take Yunnan sage Salvia yunnanensis The roots of CH Wright were crushed and then extracted directly by cold soaking in organic solvents such as ethanol or water, or by hot reflux, or by ultrasonic or microwave-assisted extraction. Alternatively, the roots could be first extracted by cold soaking in the above-mentioned organic solvents or water, or by reflux, or by ultrasonic or microwave-assisted extraction, and then extracted with ethyl acetate to obtain a total extract. The total ethyl acetate extract was repeatedly chromatographically analyzed to obtain compounds represented by Formula I and Formula II.

5. The preparation method according to claim 4, characterized in that, The process includes the following steps: The roots of *Salvia yunnanensis* are dried in the shade, pulverized to 20-30 mesh, and extracted with 95% ethanol at room temperature. The extracts are combined and concentrated under reduced pressure to obtain a total extract. The total extract is then extracted with ethyl acetate using solid-phase extraction or liquid-liquid extraction to obtain the ethyl acetate extract fraction. This ethyl acetate extract fraction is loaded onto a D101 macroporous adsorption resin and then eluted with a gradient of 50%, 70%, 80%, 90%, and 100% methanol aqueous solutions to obtain five major fractions. The 80% methanol eluent fraction is then... Six sub-fractions, Fr.1-Fr.6, were obtained by silica gel column chromatography with petroleum ether / acetone at a ratio of 18:1-0:

1. Fraction Fr.3 was subjected to MCI reversed-phase column chromatography with gradient elution of methanol / water at a volume ratio of 1:1-0:1 to obtain fractions Fr.3.1-Fr.3.

10. Fr.3.10 was subjected to repeated silica gel column chromatography with petroleum ether / ethyl acetate at a volume ratio of 20:1-0:1 and dichloromethane / methanol at a volume ratio of 300:1-0:1 to obtain compounds 1 and 2 of formulas (I) and (II).

6. A pharmaceutical composition, characterized in that, It includes the compound according to any one of claims 1 to 3.

7. The pharmaceutical composition according to claim 6, characterized in that, The dosage form of the pharmaceutical composition is an oral formulation or an injectable formulation.

8. Use of the compound according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 6 in the preparation of a lysine-specific demethylase 1 inhibitor.

9. The application according to claim 8, characterized in that, The lysine-specific demethylase 1 inhibitor is used to prepare drugs for the treatment and / or prevention of tumor diseases.

10. The application according to claim 9, characterized in that, The tumor diseases include at least one of leukemia, lung cancer, breast cancer, colorectal cancer, and prostate cancer.

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