A linderane-type sesquiterpene oligomer, its preparation method and use in preparing anti-leukemia drugs

Abstract

The present invention belongs to the field of medical technology and discloses a class of linalool-type sesquiterpene oligomers with a novel skeleton structure, a preparation method thereof, and use thereof in anti-leukemia drugs. The compound provided by the present invention (structure shown in Formula 1 and / or Formula 2) is obtained from the medicinal plant linalool (linalool) of the genus linalool of the family linalool. Chloranthus holostegius var. trichoneurus ) are extracted and isolated from the whole plant. Pharmacological experiments have shown that both can significantly inhibit the proliferation of myeloid leukemia cells MV-4-11 and induce their apoptosis. Therefore, they can be used alone or in combination, or combined with suitable excipients to prepare oral or non-oral dosage forms according to conventional methods for the treatment or adjuvant treatment of leukemia.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, specifically to the novel medical uses of two novel linderane-type sesquiterpene oligomers (1 and 2). More specifically, the use of linderane-type sesquiterpene oligomers with a unique skeleton structure in an extract of the herbaceous plant Chrysanthemi serrata in the treatment of leukemia. The present invention also relates to the extraction and preparation processes of compounds 1 and 2. Technical Background

[0002] Leukemia is a type of hematologic malignancy, a malignant clonal disease caused by mutations in hematopoietic stem or progenitor cells. There are five main clinical types of leukemia, classified by acute or chronic onset and the type of blood cells affected: acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), and acute promyelocytic leukemia (APL). Radiation, viruses, parasites, bacteria, and chemicals can all contribute to the development of leukemia. Currently, leukemia is the sixth most common malignant tumor in my country and the leading cause of cancer death in children and adolescents aged 0-19 years. Although mortality rates have declined in recent years, leukemia remains a global public health problem.

[0003] Natural products have always been an important source of new anti-leukemia drugs, such as paclitaxel, a broad-spectrum anti-cancer drug discovered from Taxus chinensis, harringtonine and homoharringtonine discovered from Cephalotaxus chinensis, and teniposide, a semi-synthetic drug derived from the natural product podophyllotoxin. Chloranthus holostegius var. trichoneur ) belongs to the Chloranthaceae family (Chloranthaceae) Chloranthus This plant is a rare species with a limited distribution. It is primarily found in Yunnan and Guizhou, growing in hillside grasslands or mixed woodlands at altitudes of 1,050-1,600 meters. The second volume of the 2005 edition of the Yunnan Provincial Traditional Chinese Medicine Standards lists this plant as suitable for infusion or external application. It dispels wind and cold, reduces swelling, and relieves pain. It is used to treat colds, headaches and body aches, rheumatic arthritis, injuries from falls and strains, lower abdominal pain, and leucorrhea.

[0004] Chinese Patent No. ZL201610848333.9 relates to a class of linalane-type dimerized sesquiterpenoid compounds, their preparation methods, and their use in the preparation of antimalarial drugs. Twelve of these linalane-type dimerized sesquiterpenoid compounds exhibit significant antimalarial effects. Furthermore, a structure-activity relationship was found between these compounds and their activity, with essential moieties for activity and groups that can be optimized, providing a basis for further activity optimization.

[0005] Publication No. CN103127062A relates to the use of 13'-acetylsilanol C in the preparation of anti-tumor drugs. This disclosure provides the use of 13'-acetylsilanol C in the preparation of anti-tumor drugs. The tumor cells described include liver cancer cells, leukemia cells, gastric cancer cells, and pancreatic cancer cells. 13'-acetylsilanol C is a natural product with minimal toxicity and side effects, high bioavailability, and stable properties, making it clinically valuable. Summary of the Invention

[0006] The invention provides a linderane-type sesquiterpene oligomer, which is a linderane-type sesquiterpene oligomer with a completely new skeleton structure.

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned linderane-type sesquiterpene oligomers.

[0008] Another object of the present invention is to provide the use of the linderane-type sesquiterpene oligomer in drugs for treating leukemia.

[0009] The object of the present invention is achieved by the following scheme: a linderane-type sesquiterpene oligomer, wherein compound 1 is a linderane-type sesquiterpene trimer with a new 3 / 5 / 6 / 6 tetracyclic skeleton, which is formed by a linderane dimer molecule reacting with another linderane sesquiterpene monomer via its tiglic acid side chain. homo -Diels-Alder reaction; and / or, compound 2 is formed by a molecule of linalool dimer and a molecule of long-chain p-benzoquinone compound homo -Diels-Alder addition reaction, the compounds 1 and 2 are heteropolymers with a new carbon skeleton; the structural formulas 1 and 2 of the compounds are as follows:

[0010] .

[0011] The above compounds 1 and 2 are novel linderane-type sesquiterpene oligomers with a completely new skeleton structure, and can be isolated from the plant Chrysanthemi tricholoma.

[0012] The present invention also relates to a process for extracting and separating compounds 1 and 2 from Chrysanthemi tricholoma, comprising the following steps:

[0013] The whole herb of Chrysanthemi tricholoma was used as raw material, crushed, extracted and concentrated by immersion in 90% methanol at room temperature; the obtained concentrate was first dispersed with deionized water and extracted with an equal volume of ethyl acetate to obtain an ethyl acetate extract; the obtained ethyl acetate extract was separated by silica gel, microporous adsorption resin MCI resin, ODS and / or Sephadex LH-20 gel column chromatography and reversed-phase semi-preparative HPLC chromatography to obtain linderane-type sesquiterpene oligomers 1 and 2.

[0014] For compounds 1 and 2, in vitro activity screening found that they had significant cytotoxic activity against leukemia cells MV-4-11 (IC 50 3.59 and 5.19 μM, respectively), and significantly induced cell apoptosis.

[0015] The present invention also provides a use of the above-mentioned linderane-type sesquiterpene oligomer in the preparation of an anti-leukemia drug, wherein the drug comprises a compound described in Formula 1 and / or Formula 2, and the leukemia types include but are not limited to acute myeloid leukemia and chronic myeloid leukemia.

[0016] The linderane-type sesquiterpene oligomer compounds 1 and 2 can be used alone or in combination to prepare anti-leukemia drugs.

[0017] The linderane-type sesquiterpene oligomer compounds 1 and 2 are combined with pharmaceutically acceptable carriers or excipients to prepare oral or non-oral dosage forms.

[0018] The drug can be prepared into oral or non-oral dosage forms according to conventional methods and used in the treatment of leukemia.

[0019] The present invention has the following advantages: Compounds 1 and 2 are new skeleton compounds discovered for the first time and have novel chemical structures; Compounds 1 and 2 have significant anti-leukemia activity and have broad application prospects in the treatment of leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 .Compounds 1 and 2 induce apoptosis of leukemia cells MV-4-11, including: A: apoptosis of MV-4-11 cells after treatment with compound 1 for 48 hours; B: apoptosis of MV-4-11 cells after treatment with compound 2 for 48 hours; C: apoptosis of MV-4-11 cells after treatment with DMSO for 48 hours; D: statistical chart of the percentage of apoptotic cells in MV-4-11 cells after treatment with compounds 1 and 2 for 48 hours. DETAILED DESCRIPTION

[0021] The following examples further illustrate the present invention, but these examples are by no means limiting of the present invention. Any changes made by those skilled in the art in the art in light of this specification will fall within the scope of the claims.

[0022] Example 1:

[0023] 28.5 kg of the whole herb of Chrysanthemi tricholoma was sun-dried and ground, then extracted five times with 40 L of 90% methanol at room temperature for 24 hours. The combined extracts were concentrated under reduced pressure to yield 2.25 kg of a brown total extract. The extract was suspended in 5.0 L of distilled water and extracted four times with ethyl acetate and n-butanol (5.0 L each time), yielding 704.2 g of the ethyl acetate fraction. The ethyl acetate fraction was initially fractionated by silica gel column chromatography using a gradient elution system of petroleum ether:ethyl acetate (20:1 to 1:1, v / v), ethyl acetate, ethyl acetate:methanol (10:1), and pure methanol to obtain fractions F1 to F10. UPLC-MS analysis revealed that fraction F6 was rich in sesquiterpenoid polymers. Fraction F6 was fractionated using a microporous resin (MCI) and a gel column (Sephadex LH-20) before purification by semi-preparative HPLC to yield compound 1 (5.0 mg, t R = 20.0 min) and 2(3.5 mg, t R = 28.5 min).

[0024] The spectra and physicochemical data of compounds 1 and 2 are as follows:

[0025] Compound 1: colorless needle-shaped crystals; [ α ]25 D −100.0 ( c 0.05, MeOH); ECD (MeOH) l max (Δ e ) 327 (−1.3) nm, 254 (+3.2) nm, 212 (−16.8) nm; 1 H NMR data (600 MHz, CDCl3): d 2.13 (ddd, J = 8.0, 5.5, 4.4 Hz, H-1), 1.01 (ddd, J = 8.0, 7.7, 4.3 Hz, H-2 α ),0.27 (ddd, J = 4.4, 4.3, 3.7 Hz, H-2 β ), 1.86 (m, H-3), 3.90 (s, H-6), 3.93 (s, H-9), 1.86 (s, CH3-13), 1.06 (s, CH3-14), 2.59 (ddd, J = 17.0, 5.5, 4.5 Hz, H-15 α ), 2.70 (dd,J = 17.0, 6.9 Hz, H-15 β ), 1.45 (m, H-1′), 0.70 (ddd, J = 8.5, 8.3,5.6 Hz, H-2′ α ), 1.18 (ddd, J = 5.6, 5.4, 5.3 Hz, H-2′ β ), 1.46 (m, H-3′), 1.47(m, H-5′), 2.21 (dd, J = 17.0, 5.7 Hz, H-6′ α ), 2.70 (dd, J = 17.0, 16.9 Hz, H-6′ β ), 1.76 (dd, J = 5.7, 1.3 Hz, H-9′), 4.80 (d, J = 12.7 Hz, H-13′a), 4.77 (dd, J =12.7 Hz, H-13′b), 0.81 (s, CH3-14′), 4.04 (d, J = 11.9 Hz, H-15′a), 3.42 (d, J =11.9 Hz, H-15′b), 2.41 (br qd, J = l7.0, 6.8 Hz, H-3′′), 1.05 (d, J = 7.0 Hz, H-4′′), 1.27 (s, CH3-5′′), 1.94 (ddd, J = 8.0, 6.9, 4.4 Hz, H-1′′′), 0.88 (ddd, J =8.0, 7.7, 4.2 Hz, H-2′′′ α ), 0.11 (ddd, J = 4.4, 4.2, 3.9 Hz, H-2′′′ β ), 1.66(ddd, J = 7.7, 6.9, 3.9 Hz, H-3′′′), 3.34 (dd, J= 3.2, 3.1 Hz, H-6′′′) 3.71 (s,H-9′′′), 2.02 (s, CH3-13′′′) 1.04 (s, CH3-14′′′), 2.59 (dd, J = 17.0, 6.8 Hz, H-15′′′ α ), 1.99 (br d, J = 17.0 Hz, H-15′′′ β ), 3.78 (s, OMe-12), 3.74 (s, OMe-12′), 2.10 (s, 13′-OCOCH3) ppm; 13 13C NMR data (150 MHz, CDCl3): d25.2 (C-1), 15.8(C-2), 24.6 (C-3), 171.2 (C-4), 131.5 (C-5), 40.6 (C-6), 132.3 (C-7), 202.4(C-8),80.8 (C-9), 51.2 (C-10), 147.2 (C-11), 170.5 (C-12), 20.4 (C-13), 15.5(C-14), 25.3 (C-15), 25.0 (C-1′), 12.2 (C-2′), 27.4 (C-3′), 77.9 (C-4′), 60.9(C-5′), 22.2 (C-6′), 142.1 (C-7′), 93.1 (C-8′), 56.1 (C-9′), 44.5 (C-10′),123.7 (C-11′), 172.2 (C-12′), 55.0 (C-13′), 26.3 (C-14′), 70.7 (C-15′), 177.2(C-1′′), 53.2 (C-2′′), 34.6 (C-3′′), 15.1 (C-4′′), 22.6 (C-5′′), 25.5 (C-1′′′), 15.4 (C-2′′′), 24.0 (C-3′′′), 141.8 (C-4′′′), 132.3 (C-5′′′), 35.9 (C-6′′′), 134.1 (C-7′′′), 203.1 (C-8′′′),80.3 (C-9′′′), 51.1 (C-10′′′), 140.5(C-11′′′), 171.8 (C-12′′′), 19.4 (C-13′′′), 15.0 (C-14′′′), 31.7 (C-15′′′),52.7 (C-12-OMe), 52.3(C-12′′′-OMe), 170.5 (13′-O C OMe), 20.5 (13′-OCO Me ) ppm;(+) HRESIMS m / z 973.3988 [M+Na] + (calcd. for C 54 H 62 O 15 Na, 973.3981, Δ = +0.7ppm).

[0026] Compound 2: yellow oil; α 25 D −29.6 ( c0.25, MeOH); ECD (MeOH) l max (Δ e ) 401(+2.7) nm, 330 (−3.5) nm, 265 (+3.8) nm, 225 (−8.0) nm; 1 1H NMR data (600 MHz, CDCl3): d 1.98 (m, H-1), 0.74 (ddd, J J = 8.4, 8.3, 6.3 Hz, H-2 α ), 0.43 (ddd, J J = 8.4, 4.8, 4.4 Hz, H-2 β ), 1.61 (m, H-3), 6.59 (s, H-6), 4.32 (d, J J = 3.4 Hz, H-9), 2.15 (s, CH3-13), 0.86 (s, CH3-14), 1.95 (m, H-15 α ), 2.34 (d, J J = 14.0 Hz, H-15 β ), 1.95 (m, H-1′), 0.83 (ddd, J J = 7.9, 7.8, 4.0 Hz, H-2′ α ), 0.24 (ddd, J J = 4.0, 3.9, 3.7 Hz, H-2′ β ), 1.60 (m, H-3′), 3.13 (br s, H-6′), 4.35 (d, J J = 1.2 Hz, H-9′), 1.26 (s, CH3-13′), 1.21 (s, CH3-14′), 2.25 (m, H-15′a), 1.95 (d, J J = 14.0 Hz, H-15′b), 6.41 (q, J J = 1.2 Hz, H-3′′), 3.62 (d, J J = 4.6 Hz, H-5′′), 1.99 (d, J J = 1.2 Hz, H-7′′), 2.79 (dd, J J = 14.9, 3.2 Hz, H-8′′a), 2.23 (dd, J= 14.9, 9.9 Hz, H-8′′b), 4.96 (br dd, J = 9.9, 3.2 Hz, H-9′′), 1.95 (m, H2-11′′), 2.02(m, H2-12′′), 5.04 (br t, J = 6.7 Hz, H-14′′), 1.63 (br s, H-15′′), 1.66 (s, H-16′′), 1.59 (s, H-17′′), 3.85 (s, OMe-12), 3.68(s, OMe-12′), 3.79 (br s, OH-9), 2.98 (br s, OH-9′) ppm; 13 13C NMR data (150 MHz, CDCl3): d26.3 (C-1), 10.6 (C-2), 28.1 (C-3), 84.7 (C-4), 162.4 (C-5), 118.2 (C-6), 126.9 (C-7), 198.6 (C-8),79.8 (C-9), 47.4 (C-10), 138.6 (C-11), 171.2 (C-12), 17.6 (C-13), 18.9 (C-14), 44.1 (C-15), 25.7 (C-1′), 14.8 (C-2′), 24.8 (C-3′), 135.9 (C-4′), 136.7(C-5′), 32.2 (C-6′), 105.4 (C-7′), 148.6 (C-8′), 73.5 (C-9′), 52.6 (C-10′), 43.7 (C-11′), 175.8 (C-12′), 25.5 (C-13′), 15.4 (C-14′), 33.9 (C-15′), 202.0(C-1′′), 145.7 (C-2′′), 137.0 (C-3′′), 200.0 (C-4′′), 51.9 (C-5′′), 54.4 (C-6′′), 16.3 (C-7′′), 30.8 (C-8′′), 119.1 (C-9′′), 139.0 (C-10′′), 40.0 (C-11′′), 26.7 (C-12′′), 124.0 (C-13′′), 131.6 (C-14′′), 16.2 (C-15′′), 25.7 (C-16′′), 17.6 (C-17′′), 52.5 (C-12-OMe), 51.9(C-12′-OMe) ppm; (+) HRESIMS m / z 807.4104 [M+H] + (calcd. for C 49 H 59 O 10 , 807.4103, Δ = +0.1 ppm).

[0027] Example 2:

[0028] Anti-leukemia cytotoxic activity test:

[0029] Leukemia MV-4-11 cells were purchased from the Cell Bank of the Chinese Academy of Sciences. The culture medium was RMPI-1640 medium supplemented with 10% (v / v) fetal bovine serum (FBS), 1% penicillin and 1% sodium pyruvate. The cells were incubated at 37°C in a 5% CO2 atmosphere. The cells were seeded in 96-well plates (seeding density: 2.5×10 4 The next day, the 10 mM compound stock solution in DMSO was diluted 5 times to 20 m L complete medium, 20 m L of drug-containing culture medium was added with 80 m After incubation in the cell culture box for 24h and 48h, 10 m LCCK-8 solution was added and incubated for 3 h. The absorbance of each well was finally measured at 450 nm. All experimental results were statistically analyzed using GraphPad Prism 7.00 software (IBM). Mean ± SD represents quantitative data. The comparison of rates was performed using the chi-square test, and the comparison of means was performed using the t Inspection, IC 50 The calculation used the "[Inhibitor] vs normalized response-Variableslope" model, with p < 0.05 indicating statistical significance. Using parthenolide and DMAPT as positive controls, the test results are shown in Table 1:

[0030] .

[0031] Example 3:

[0032] Annexin V / PI double staining to detect cell apoptosis:

[0033] Leukemia cells (MV-4-11) were cultured at a density of 2.5 × 10 5 Cells were seeded in 6-well plates overnight. Compounds 1 and 2 were then incubated with MV-4-11 cells at a final concentration of 4 μM for 48 hours. MV-4-11 cells were then treated with the Annexin V / PI Apoptosis Detection Kit, and their apoptosis rate was determined. The results were analyzed using CytExpert software.

[0034] The results of flow cytometry apoptosis showed that ( Figure 1), after 48 h of treatment with compound 2, the proportion of MV-4-11 cells decreased significantly (n = 3, P<0.05), and the proportion of apoptotic cells (including early and late apoptotic cells) increased significantly (n = 3, P<0.05). After 48 h of treatment with compound 1, the proportion of MV-4-11 cells decreased significantly (n = 3, P<0.0001), and the proportion of apoptotic cells increased significantly (n = 3, P<0.0001). The apoptotic rate of cells after treatment with compound 1 was significantly higher than that after treatment with compound 2.

[0035] The above experimental results show that compounds 1 and 2 can promote the apoptosis of MV-4-11 cells, among which compound 1 promotes the apoptosis of MV-4-11 cells more significantly.

Claims

1. A linderane-type sesquiterpene oligomer, characterized in that: Selected from Compound 1 and / or Compound 2, wherein the corresponding structural formulas of Compound 1 and Compound 2 are as follows: 。 2. The method for preparing the linderane-type sesquiterpene oligomers according to claim 1, characterized in that: The method comprises the following preparation steps: The whole herb of Chrysanthemi tricholoma is used as raw material, crushed, extracted and concentrated by immersion in 90% methanol at room temperature; the obtained concentrate is first dispersed with deionized water solution and then extracted with an equal volume of ethyl acetate to obtain an ethyl acetate extract; the obtained ethyl acetate extract is separated by silica gel, microporous resin MCI, ODS and / or Sephadex LH-20 gel column chromatography and reversed-phase semi-preparative HPLC chromatography to prepare the linderane-type sesquiterpene oligomers.

3. The method for preparing the linderane-type sesquiterpene oligomer according to claim 2, wherein: Prepare as follows: 28.5 kg of the whole herb of Chrysanthemi pubescens was sun-dried and ground, and extracted with 40 L of 90% methanol at room temperature for 24 hours, for a total of 5 times. The combined extracts were concentrated under reduced pressure to obtain a total brown extract of 2.25 kg. The extract was suspended in 5.0 L of distilled water and extracted four times with 5.0 L of ethyl acetate and n-butanol, yielding 704.2 g of ethyl acetate extract. The ethyl acetate extract was initially fractionated by silica gel column chromatography using a gradient elution system using petroleum ether:ethyl acetate (v / v) ratios of 20:1 to 1:1, ethyl acetate, ethyl acetate:methanol (v / v) ratio of 10:1, and pure methanol to obtain fractions F1-F10. UPLC-MS analysis showed that fraction F6 was rich in sesquiterpene polymers. Fraction F6 was separated multiple times using microporous resin MCI and gel column Sephadex LH-20, and then purified using semi-preparative HPLC to obtain 5.0 mg, tR = 20.0 min of compound 1 and 3.5 mg, tR = 28.5 min of compound 2.

4. Use of the linderane-type sesquiterpene oligomer according to claim 1 in the preparation of an anti-leukemia drug, characterized in that: The drug comprises a compound described in Formula 1 and / or Formula 2.

5. Use of the linderane-type sesquiterpene oligomer according to claim 4 in the preparation of an anti-leukemia drug, characterized in that: The leukemia type is selected from acute myeloid leukemia and chronic myeloid leukemia.

6. The use according to claim 4 or 5, characterized in that The linderane-type sesquiterpene oligomers are used alone or in combination to prepare anti-leukemia drugs.

7. The use according to claim 4 or 5, characterized in that The linderane-type sesquiterpene oligomer is combined with a pharmaceutically acceptable carrier or excipient to prepare an oral or non-oral dosage form.

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