4,5-seco-16,17-dinorsclareolide diterpenoids isolated from salvia yunnanensis and uses thereof

Abstract

The present application relates to 4,5-open ring-16,17-doradecane type diterpenoid compounds isolated from Yunnan rat-tail grass and its application, belonging to the field of natural medicinal chemistry. The compound is a compound shown in formula (I) and formula (II). The present application also relates to a pharmaceutical composition comprising the compound. The preparation method of the compound comprises: taking Yunnan rat-tail grass roots, crushing, extracting with an organic solvent, extracting with ethyl acetate and repeatedly column chromatography separation, respectively obtaining formula (I) and formula (II) compounds. Through AlphaLISA method activity test, the inhibition rate of formula (I) compound on LSD1 is 105.8% at 50 muM concentration, IC 50 12.01 muM; the inhibition rate of formula (II) compound is 67.1%. The compound of the present application can be used as an LSD1 inhibitor for preparing a drug for treating diseases related to abnormal LSD1 activity.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry, specifically relating to a 4,5-open-ring-16,17-dinorrosine-type diterpenoid compound from *Salvia yunnanensis*, its preparation method, and its application in LSD1 inhibitors. Background Technology

[0002] Yunnan sage ( Salvia yunnanensis CH Wright), commonly known as Salvia miltiorrhiza, is a species of sage belonging to the genus Salvia. Salvia This plant is a perennial herb, with its roots used medicinally. It is mainly distributed in eastern, central, and western Yunnan, southwestern Sichuan, and western Guizhou, growing on grassy slopes, forest edges, roadsides, or dry ground in sparse forests at altitudes of 1800–2900 meters. This plant has a history of medicinal use in Yunnan for hundreds of years, as recorded in the Ming Dynasty's *Diannan Bencao* (Herbal Materia Medica of Southern Yunnan). The *Yunnan Provincial Standards for Traditional Chinese Medicine (2005 Edition)* records its effects of "clearing heat and detoxifying, reducing swelling and relieving pain," and it is commonly used for lung heat cough, mastitis, boils, and carbuncles.

[0003] Modern chemical research indicates that *Salvia yunnanensis* mainly contains diterpenes, triterpenes, sesquiterpenes, flavonoids, phenolic acids, and diterpenoid alkaloids, with diterpenes and polyphenolic acids being the primary components. However, current research primarily focuses on abietane-type diterpenes and their analogues, such as tanshinone compounds. For the more structurally rare 4,5-open-ring-16,17-dinorabinane-type diterpenes, no reports have yet been found isolated from *Salvia yunnanensis*. These compounds are characterized by ring-opening at the 4,5-position of ring A and the removal of two carbon atoms at C-16 and C-17, forming a unique dinorabinane skeleton. Their chemical diversity and biological activity require further exploration.

[0004] On the other hand, histone demethylase LSD1 (Lysine-Specific Demethylase 1), as the first discovered histone demethylase, is abnormally upregulated in various malignant tumors, promoting tumor cell proliferation, invasion, and metastasis by regulating the transcriptional activity of target genes. LSD1 has become an important target for anti-tumor drug development, but currently reported LSD1 inhibitors are mostly concentrated on synthetic small molecule compounds (such as ORY-1001, GSK-2879552, etc.), while naturally derived LSD1 inhibitors are limited in variety and suffer from drawbacks such as insufficient selectivity and significant toxic side effects. Therefore, searching for novel and highly active LSD1 inhibitors from natural products has significant research value and clinical significance.

[0005] During in-depth research on *Salvia yunnanensis*, the inventors unexpectedly isolated two novel compounds (Formula I and Formula II) from its roots, each possessing a 4,5-open-ring-16,17-dinorpinene diterpenoid skeleton. In vitro activity screening revealed that these compounds exhibited significant inhibitory activity against LSD1, with compound I showing the highest IC50 value. 50 The value reached 12.01 μM. Based on the above findings, this invention provides a novel natural LSD1 inhibitor, offering a new source of lead compounds for the development of antitumor drugs. Summary of the Invention

[0006] The present invention aims to provide 4,5-open-ring-16,17-dinorrosine-type diterpenoids isolated from *Salvia yunnanensis* and their applications.

[0007] The above-mentioned objective of the present invention is achieved through the following technical solution.

[0008] A 4,5-open-ring-16,17-dinorpinene type diterpenoid compound, wherein the compound of the present invention is any one of the compounds shown in formula (I) or formula (II): .

[0009] Furthermore, the compound described in this invention is the compound shown in formula (I): .

[0010] Furthermore, the compound described in this invention is the compound shown in formula (II): .

[0011] The method for preparing the 4,5-open-ring-16,17-dinorpinene type diterpenoid compound of the present invention is characterized by comprising the following steps: taking Yunnan sage Salvia yunnanensis The roots of CH Wright were crushed and then directly cold-soaked or hot-refluxed or ultrasonically or microwave-assisted extracted with organic solvents such as petroleum ether, dichloromethane, ethyl acetate, acetone, methanol, ethanol or water. Alternatively, the roots could be first cold-soaked or refluxed or ultrasonically or microwave-assisted extracted with the above-mentioned organic solvents or water, and then extracted with ethyl acetate to obtain a total extract. The total ethyl acetate extract was repeatedly chromatographically analyzed to obtain the compounds shown in Formula I and Formula II.

[0012] Further, the preparation method of this invention specifically involves: drying the roots of *Salvia yunnanensis* in the shade, pulverizing them to 20-30 mesh, extracting them with 95% ethanol at room temperature, combining the extracts, concentrating the extract under reduced pressure to obtain a total extract, and using solid-phase extraction or liquid-liquid extraction to extract the ethyl acetate fraction with ethyl acetate. The ethyl acetate fraction is dissolved in an appropriate amount of dichloromethane / acetone, mixed with 80-100 mesh silica gel, and then subjected to column chromatography with 200-300 mesh silica gel at a volume ratio of 1:0-0:1 dichloromethane / acetone or... Gradient elution with dichloromethane / methanol at a volume ratio of 1:0-0:1 yielded eight major fractions. The dichloromethane-eluted fraction was subjected to silica gel column chromatography with petroleum ether / ethyl acetate at a volume ratio of 17:1-0:1 to obtain eight sub-fractions Fr.1-Fr.8. Fraction Fr.2 was subjected to silica gel column chromatography with gradient elution with petroleum ether / dichloromethane at a volume ratio of 4:1-0:1 to obtain fractions Fr.2.1-Fr.2.10. Fr.2.7 was further subjected to silica gel column chromatography and RP-18 column chromatography to obtain compounds represented by Formula I and Formula II, respectively.

[0013] As a product, the present invention is a pharmaceutical composition comprising the aforementioned compounds or combinations of compounds.

[0014] Furthermore, the dosage form of the pharmaceutical composition of the present invention is an oral formulation or an injectable formulation.

[0015] As an application, the aforementioned compounds or pharmaceutical compositions of the present invention are used in the preparation of lysine-specific demethylase 1 inhibitors.

[0016] Furthermore, the lysine-specific demethylase 1 inhibitor described in this invention is used to prepare drugs for treating and / or preventing tumor diseases.

[0017] Furthermore, the tumor diseases described in this invention include at least one of leukemia, lung cancer, breast cancer, colorectal cancer, and prostate cancer.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. A novel 4,5-open-ring-16,17-dinorrosine-type diterpenoid compound is provided. This invention isolated compounds of formula (I) and formula (II) from the roots of *Salvia yunnanensis*. Literature search revealed that both are novel compounds, and no reports on their chemical structures, preparation methods, or biological activities have been found in the prior art. These compounds possess a unique 4,5-open ring and 16,17-double-decreasing skeleton, enriching the structural types of diterpenoids.

[0019] 2. The preparation method is simple and has good reproducibility. This invention can obtain the target compound using conventional extraction and separation methods. The process is stable, simple to operate, and requires no special equipment, making it suitable for laboratory-scale preparation and industrial production.

[0020] 3. This is the first time that this type of compound has been found to have significant LSD1 inhibitory activity, but the technical effect is not immediately obvious. This invention is the first to discover that compounds of formula (I) and formula (II) possess LSD1 inhibitory activity. AlphaLISA homogeneous activity testing showed that at a concentration of 50 μM, compound (I) exhibited an inhibition rate of 105.8% ± 0.91% against LSD1, while compound (II) showed an inhibition rate of 67.1% ± 5.81%; the IC50 of compound (I) was... 50 The value was 12.01 μM, which was similar to the positive control drug LSD1 IV (IC50). 50 Although there is a certain gap compared to (0.0037 μM), as a novel lead compound from a natural source, its activity level has reached the starting point for further structural optimization and modification, and it has clear pharmaceutical development value.

[0021] Currently, there are very few reports on naturally derived LSD1 inhibitors, and most known LSD1 inhibitors are synthetic irreversible inhibitors, whose structural types are fundamentally different from the 4,5-open-ring-16,17-dinorrosine diterpenoid skeleton of this invention. When faced with the technical problem of "finding novel LSD1 inhibitors from natural products," those skilled in the art have no motivation to select *Salvia yunnanensis* and specifically isolate compounds with this rare skeleton for activity screening; therefore, this invention is a non-obvious discovery.

[0022] 4. It has the potential for further development into an anti-tumor drug. Based on the above-mentioned LSD1 inhibitory activity, the compound of the present invention can be used as an effective active ingredient of LSD1 inhibitor to prepare drugs for treating tumor diseases related to abnormal LSD1 activity, providing a novel lead compound for the development of anti-tumor drugs.

[0023] The present invention will be further explained below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

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

[0025] Figure 2 For compound 1 13 C-NMR spectrum.

[0026] Figure 3 The image shows the HR-ESI-MS spectrum of compound 1.

[0027] Figure 4 For compound 2 1 H-NMR spectrum.

[0028] Figure 5 For compound 2 13 C-NMR spectrum.

[0029] Figure 6 The image shows the HR-ESI-MS spectrum of compound 2. Detailed Implementation

[0030] 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.

[0031] 4,5-Octopylene-16,17-Dinorpinene-type diterpenoids isolated from *Salvia yunnanensis* and their applications, wherein the compounds include either compound 1 (Formula I) or compound 2 (Formula II).

[0032] .

[0033] The preparation method of compounds (I) and (II) involves taking the root of *Salvia yunnanensis*, crushing it to 20-30 mesh, and directly cold-soaking or hot-refluxing or ultrasonically or microwave-assisted extraction with organic solvents such as petroleum ether, dichloromethane, ethyl acetate, acetone, methanol, ethanol, or water. Alternatively, it can be first cold-soaked or refluxed or ultrasonically or microwave-assisted extracted with the above organic solvents or water, 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).

[0034] 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 or hot reflux or ultrasonic or microwave-assisted extraction with petroleum ether, dichloromethane, ethyl acetate, acetone, methanol, ethanol or water. The ethyl acetate extract is then extracted with ethyl acetate extract. The ethyl acetate extract is repeatedly subjected to column chromatography to obtain compounds of formula (I) and formula (II).

[0035] B: The roots of Sage in Yunnan are extracted by direct cold soaking, hot reflux, ultrasonic or microwave-assisted extraction with organic solvents (such as petroleum ether, methanol, ethanol, acetone, dichloromethane, etc.) to obtain total extract. The total extract can be subjected to repeated column chromatography to obtain compounds of formula (I) and formula (II).

[0036] 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 the extracts were concentrated under reduced pressure to obtain a paste. The paste was then extracted using solid-phase extraction or liquid-liquid extraction with ethyl acetate as the extraction solvent to obtain the ethyl acetate extract. The ethyl acetate extract fraction was dissolved in an appropriate amount of dichloromethane / acetone, mixed with 80-100 mesh silica gel, and then subjected to column chromatography with 200-300 mesh silica gel for fractional partitioning. Gradient elution with 1:0-0:1 dichloromethane / acetone or 1:0-0:1 dichloromethane / methanol yielded 8 major fractions. The dichloromethane eluted fraction was subjected to silica gel column chromatography with petroleum ether / ethyl acetate at a ratio of 17:1-0:1 to obtain 8 subfractions (Fr.1-Fr.8). Fraction Fr.2 was subjected to silica gel column chromatography with gradient elution of petroleum ether / dichloromethane (4:1-0:1) to obtain fraction Fr.2.1- Fr.2.10 and Fr.2.7 were then subjected to silica gel column chromatography and RP-18 column chromatography to obtain compounds 1 and 2 of formula (I) and formula (II), respectively.

[0037] This invention protects the use of the described compound in the preparation of LSD1 inhibitors, which are used to treat and / or prevent diseases including, but not limited to, cancer.

[0038] This invention protects an LSD1 inhibitor drug, wherein the LSD1 inhibitor comprises the chemical structure of the 4,5-open-16,17-dinorrosine diterpenoid compound.

[0039] 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, concentrated under reduced pressure to obtain a paste, and then extracted with ethyl acetate using solid-phase extraction or liquid-liquid extraction to obtain an ethyl acetate extract. The ethyl acetate extract was dissolved in an appropriate amount of dichloromethane / acetone, mixed with 80-100 mesh silica gel, and then subjected to column chromatography with 200-300 mesh silica gel for fractional separation. Gradient elution with 1:0-0:1 dichloromethane / acetone or 1:0-0:1 dichloromethane / methanol yielded eight major fractions. The dichloromethane-eluted fraction was further eluted with petroleum ether / ethyl acetate at a ratio of 17:1-0:1 to obtain eight subfractions (Fr.1-Fr.8). Fraction Fr.2 was further eluted with petroleum ether / dichloromethane at a ratio of 4:1-0:1 to obtain fraction Fr.2.1-. Fr.2.10 and Fr.2.7 were then subjected to silica gel column chromatography and RP-18 column chromatography to obtain compounds 1 and 2 of formula (I) and formula (II), respectively.

[0040] Example 2: Physical and spectroscopic data of compound 1 of the present invention: Yellow powder, IR: 3348.1, 3040.6, 2961.3, 2921.1, 2884.0, 1634.1, 1525.7, 1446.0, 1379.3, 1353.0, 1248.5, 1218.3, 1137.6, 999.3, 878.7, 846.1, 796.1 cm⁻¹ -1 HR-ESI-MS: m / z 255.1748 [M+H] + (calcd for C 18 H 23 O + 255.1743); 1 H-NMR (600MHz, CDCl3) δ : 2.97 (2H, m, H-1), 2.27 (2H, m, H-2), 5.32 (1H, m, H-3), 7.12(1H, d, J = 8.2 Hz, H-6), 7.48 (1H, d, J = 8.2 Hz, H-7), 7.29 (1H, s, H-11), 7.54 (1H, s, H-14), 2.41 (3H, s, H-15), 1.59 (3H, s, H-18), 1.73 (3H, s, H-19), 2.46 (3H, s, H-20); 13 C-NMR (150 MHz, CDCl3) δ : 29.2 (t, C-1), 28.4 (t, C-2), 124.3 (d, C-3), 132.2 (s, C-4), 132.6 (s, C-5), 127.1 (d, C-6), 125.2 (d,C-7), 128.4 (s, C-8), 132.3 (s, C-9), 133.7 (s, C-10), 105.7 (d, C-11), 152.9 (s, C-12), 124.6 (s, C-13), 130.2 (d, C-14), 16.3 (q, C-15), 17.8 (q, C-18), 25.8 (q, C-19), 20.3 (q, C-20).

[0041] Physical and spectral data of compound 2 of this invention: Yellow oil, [α]20 D +87.4 ( c = 0.5, MeOH); IR: 3729.3, 3701.6, 3625.7, 2854.4, 1741.2, 1713.8, 1512.7, 1452.7, 1424.2, 1219.4, 499.3, 449.1, 420.6cm -1 ; HR-ESI-MS: m / z 293.1509 [M+Na] + (calcd for C 18 H 22 O2Na + 293.1512); 1 H-NMR (600 MHz, CDCl3) δ : 3.12 (1H, m, H-1a), 2.97 (1H, m, H-1b), 1.81 (2H, m, H-2), 4.25 (1H, m, H-3), 7.11 (1H, d, J = 8.2 Hz, H-6), 7.47 (1H, d, J = 8.2Hz, H-7), 7.34 (1H, s, H-11), 7.53 (1H, s, H-14), 2.40 (3H, s, H-15), 5.04 (1H, s, H-18a), 4.90 (1H, s, H-18b), 1.77 (3H, s, H-19), 2.45 (3H, s, H-20); 13 C-NMR (150 MHz, CDCl3) δ : 24.9 (t, C-1), 34.6 (t, C-2), 76.2 (d, C-3), 147.6 (s, C-4), 132.4 (s, C-5), 126.9 (d, C-6), 125.3 (d, C-7), 128.2 (s, C-8), 132.2 (s, C-9), 133.2 (s, C-10), 105.6 (d, C-11), 153.3 (s, C-12), 125.1 (s, C-13), 130.1 (d, C-14), 16.4 (q, C-15), 111.1 (t, C-18), 18.2 (q, C-19), 20.2 (q, C-20).

[0042] a Measured using a Bruker Avance III-600 MHz nuclear magnetic resonance spectrometer; chemical shift values ​​are expressed in ppm.

[0043] Example 3: Detection of the inhibitory activity of the compounds of the present invention against LSD1: LSD1 activity assay was performed using the homogeneous AlphaLISA method, with LSD1IV as the positive control. The LSD1 enzyme (Cat. No. 31426) derived from ACTIVEMOTIF and H3K4me2 peptide were used as substrates. The reaction was carried out in 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 first prepared as a 100× maximum concentration stock solution using 100% DMSO. Ten concentration points were obtained by serially diluting the compound 3-fold in a 384-well Echo plate. Two wells each were set up as a compound-free control (Max) and an enzyme-free control (Min). 100 nL of the solution was transferred to each well of the Perkin Elmer 384-well assay plate using an Echo 550 Liquid Handler to achieve a final DMSO concentration of 0.5%. Subsequently, 5 μL of LSD1 enzyme solution was added to the assay plate (1× assay buffer was used instead of the low control wells). After centrifugation at 1000 rpm for 1 min, pre-incubate at room temperature for 15 min, then add 5 μL of substrate solution to start the reaction, centrifuge at 1000 rpm for 1 min, and react at room temperature for 40 min. After the reaction, add 15 μL of 25 / 15× detection solution prepared by AlphaLISA Anti-unmodified H3K4 Acceptor beads and Strep-Tactin® Alpha Donor beads in 1× AlphaLISA detection buffer, centrifuge at 1000 rpm for 1 min, and incubate at room temperature in the dark for 60 min. Finally, read the endpoint signal using EnSpire (Alpha module). The inhibition rate was calculated using the formula Inh% = (Max − Signal) / (Max − Min) × 100, and the data were analyzed using XL-Fit software according to the four-parameter logistic regression equation Y = Bottom + (Top − Bottom) / (1 + (IC 50 IC was obtained by fitting / X)^HillSlope) 50 value.

[0044] Activity data are shown in Tables 1 and 2.

[0045] Table 1. LSD1 inhibitory activity data of compounds 1 and 2 at 50 μM (x ± 0.5%) s , n = 3)

[0046] Table 2 shows the LSD1 inhibition IC of compound 1. 50 (μM)

[0047] As shown in the table above, compounds 1 and 2 obtained in Example 1 of this invention exhibit good inhibitory activity against LSD1, with compound 1 showing the best inhibitory activity against LSD1, and its IC50 value being [value missing]. 50 The value is 12.01 μM.

[0048] 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 4,5-open-ring-16,17-dinorrosine-type diterpenoid 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 4,5-open-ring-16,17-dinorrosine-type diterpenoid compound according to any one of claims 1 to 3, characterized in that, The steps include: Collecting Yunnan sage Salvia yunnanensis The roots of CH Wright were crushed and then directly cold-soaked or hot-refluxed or ultrasonically or microwave-assisted extracted with organic solvents such as petroleum ether, dichloromethane, ethyl acetate, acetone, methanol, ethanol or water. Alternatively, the roots could be first cold-soaked or refluxed or ultrasonically or microwave-assisted extracted with the above-mentioned organic solvents or water, and then extracted with ethyl acetate to obtain a total extract. The total ethyl acetate extract was repeatedly chromatographically analyzed to obtain the compounds shown in Formula I and Formula II.

5. The preparation method according to claim 4, characterized in that, Includes the following steps: Yunnan sage Salvia yunnanensis The roots of *Chlorophytum comosum* were dried in the shade, pulverized to 20-30 mesh, and extracted with 95% ethanol at room temperature. 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 dissolved in an appropriate amount of dichloromethane / acetone, mixed with 80-100 mesh silica gel, and then subjected to column chromatography with 200-300 mesh silica gel for fractional separation using a dichloromethane / acetone volume ratio of 1:0-0:1 or 1:0-0:

1. Gradient elution with dichloromethane / methanol yielded eight major fractions. The dichloromethane-eluted fraction was subjected to silica gel column chromatography with a petroleum ether / ethyl acetate volume ratio of 17:1-0:1 to obtain eight sub-fractions Fr.1-Fr.

8. Fraction Fr.2 was subjected to silica gel column chromatography with a gradient elution of petroleum ether / dichloromethane at a volume ratio of 4:1-0:1 to obtain fractions Fr.2.1-Fr.2.

10. Fr.2.7 was further subjected to silica gel column chromatography and RP-18 column chromatography to obtain compounds represented by Formula I and Formula II, respectively.

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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