Compounds in Selaginella tamariscina and Their Uses

Abstract

The present invention provides a plurality of new compounds separated and purified from Selaginella truncatula, and also provides the anti-tumor uses of these new compounds, especially in the treatment of triple-negative breast cancer.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry. Background Art

[0002] Selaginella (Selaginella tamariscina (P. Beauv.) Spring) is a terrestrial or rocky fern in the genus Selaginella, family Selaginaceae. Its roots are densely forked and hairy, forming a dense tree-like trunk with the stem and branches. Its branches are sparse and regular, glabrous, and flattened dorsoventrally. Its leaves are alternately arranged, thick, smooth, and green or brown with fine serrations on the margins, with white margins. Its megaspores are light yellow, while its microspores are orange-yellow. It blooms from July to September, and its fruiting period is September to October. Selaginella derives its name from its stems and leaves, which resemble the young branches of a cypress tree and curl inward.

[0003] Selaginella is native to China and is now distributed in provinces such as Hebei, Henan, Hubei, Guangxi, and the southwest of China. It is also found in Siberia, North Korea, Japan, India, and the Philippines. It prefers warm, humid, and semi-shady environments and grows on sunny hillsides or in dry rock crevices. It reproduces by spores.

[0004] The book "Compendium of Materia Medica" records that Selaginella has the effects of promoting blood circulation, relieving menstruation, and removing blood stasis to stop bleeding. It is used to treat amenorrhea, dysmenorrhea, lumps and masses, hematemesis, metrorrhagia, hematochezia, and rectal prolapse. Its lush, verdant branches and leaves make it a delightful indoor miniature bonsai, remaining evergreen year-round. Its shape, resembling a mountain pine, makes it a highly ornamental addition to rockery and large bonsai. With an evolutionary history spanning 400 million years, Selaginella is the oldest group of terrestrial plants to date. Summary of the Invention

[0005] The present invention provides a plurality of new compounds isolated and purified from Selaginella, the specific structures of which are as follows:

[0006]

[0007]

[0008] The present invention also provides use of the compound in preparing anti-tumor drugs.

[0009] Furthermore, the tumor is selected from liver cancer, melanoma, and breast cancer.

[0010] Furthermore, the breast cancer is triple-negative breast cancer.

[0011] Triple-negative breast cancer refers to breast cancer that is negative for estrogen receptor (ER), progesterone receptor (PR), and the proto-oncogene Her-2 in immunohistochemical tests. This type of breast cancer accounts for 10.0% to 20.8% of all breast cancer types and exhibits unique biological behavior and clinicopathological features, resulting in a poorer prognosis than other types. It has been dubbed the "king of breast cancers."

[0012] The present invention provides an antitumor pharmaceutical composition comprising any one or more of the aforementioned compounds 1-5.

[0013] The pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0014] The pharmaceutically acceptable excipients described in the present invention are a general term for all additional materials in a drug other than the main drug. Excipients should have the following properties: (1) be non-toxic to the human body and have almost no side effects; (2) be chemically stable and not easily affected by temperature, pH, storage time, etc.; (3) have no incompatibility with the main drug and do not affect the efficacy and quality inspection of the main drug; (4) do not interact with the packaging material. Excipients in the present invention include but are not limited to fillers (diluents), lubricants (glidants or anti-adhesives), dispersants, wetting agents, adhesives, regulators, solubilizers, antioxidants, antibacterial agents, emulsifiers, disintegrants, etc. Binders include syrup, gum arabic, gelatin, sorbitol, tragacanth, cellulose and its derivatives (such as microcrystalline cellulose, sodium carboxymethyl cellulose, ethyl cellulose or hydroxypropyl methyl cellulose, etc.), gelatin slurry, syrup, starch slurry or polyvinyl pyrrolidone, etc.; fillers include lactose, powdered sugar, dextrin, starch and its derivatives, cellulose and its derivatives, inorganic calcium salts (such as calcium sulfate, calcium phosphate, calcium hydrogen phosphate, precipitated calcium carbonate, etc.), sorbitol or glycine, etc.; lubricants include micropowdered silica gel, magnesium stearate, talc, aluminum hydroxide, boric acid, hydrogenated vegetable oil, polyethylene glycol, etc.; disintegrants include starch and its derivatives (such as sodium carboxymethyl starch, sodium starch glycolate, etc. , pregelatinized starch, modified starch, hydroxypropyl starch, corn starch, etc.), polyvinyl pyrrolidone or microcrystalline cellulose, etc.; wetting agents include sodium lauryl sulfate, water or alcohol, etc.; antioxidants include sodium sulfite, sodium bisulfite, sodium metabisulfite, dibutyl benzoic acid, etc.; antibacterial agents include 0.5% phenol, 0.3% cresol, 0.5% trichlorobutanol, etc.; regulators include hydrochloric acid, citric acid, potassium (sodium) hydroxide, sodium citrate and buffers (including sodium dihydrogen phosphate and disodium hydrogen phosphate), etc.; emulsifiers include polysorbate 80, sorbitan monophosphate, Pluronic F-68, lecithin, soy lecithin, etc.; solubilizers include Tween-80, bile, glycerol, etc.

[0015] The tumor described in the present invention includes primary cancer or metastatic cancer.

[0016] The primary cancer mentioned in the present invention refers to normal cells in normal tissues and organs, which gradually transform into malignant tumor cells under the long-term action of various internal and external carcinogenic factors, and then form cancer cell clusters, namely "primary cancer" or "primary malignant tumor".

[0017] Metastatic cancer, as used herein, refers to the process by which tumor cells from the primary site invade lymphatic vessels, blood vessels, or other pathways, are carried elsewhere and continue to grow, forming a tumor of the same type as the primary tumor. This process is called metastasis, and the resulting tumor is called a metastatic tumor or metastatic cancer. Metastasis is a hallmark of malignant tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 HMBC(→) spectrum of compound 1

[0019] Figure 2 HMBC(→) spectrum of compound 2

[0020] Figure 3 HMBC(→) spectrum of compound 3

[0021] Figure 4 HMBC(→) spectrum of compound 4

[0022] Figure 5 HMBC(→) spectrum of compound 5 DETAILED DESCRIPTION

[0023] Example 1

[0024] 13.0 kg of dried whole herb of Selaginella was extracted using a flash extractor with a 75% ethanol-water solution (12:1) for three 2-min extractions. The extract was then concentrated on a rotary evaporator under reduced pressure until the alcohol was free. This yielded 1.2 kg of Selaginella total extract, which was then suspended in water and extracted three times with petroleum ether, dichloromethane, and ethyl acetate. The fractions were collected and the solvent was recovered under reduced pressure to yield 80.5 g of petroleum ether, 160.0 g of dichloromethane, and 180.5 g of ethyl acetate.

[0025] 160.0 g of the dichloromethane layer extract was taken and separated using HP20 macroporous adsorption resin. The mobile phase was methanol-water, and the gradient elution was 50%, 60%, 70%, 80%, and 90%. Each mobile phase had 7 BV. The concentrate was combined to obtain five fractions (D1→D5, which means "D1, D2, D3, D4, and D5 in sequence" and similar expressions are interpreted the same way).

[0026] D2 was eluted by polyamide column chromatography with dichloromethane-methanol (100:0, 50:1, 10:1, 5:1, 2:1, 1:1, v / v) to obtain 6 fractions in sequence. The elution portion of dichloromethane-methanol 10:1 was taken and eluted isocratically with 50% methanol-water by p-HPLC to obtain compound 5 (3.0 mg, t R =21.9min); the elution fraction of 80% methanol-water was subjected to silica gel column chromatography with dichloromethane-methanol 100:0, 100:1, 50:1, 30:1, 20:1, 10:1, 8:1, 5:1, 2:1, 2:3 v / v as the mobile phase to obtain 10 fractions (Fr.1→Fr.10). Among them, the Fr.7 fraction was eluted isocratically with 70% methanol-water by p-HPLC to obtain compound 1 (7.2 mg, t R =23.4min), compound 3 (6.4 mg, t R =33.6min), compound 4 (5.2mg, t R =38.2min).

[0027] 178.0 g of ethyl acetate extract was initially separated by HP20 macroporous adsorption resin using a gradient of 50%, 60%, 70%, 80%, and 90% methanol-water as the elution solvent. Gradient elution was performed with 7 BV for each gradient. After identification by thin layer chromatography, the eluates of the same spot were combined in the order of elution and concentrated to obtain 5 fractions (Fr.1→Fr.5). The Fr.3 fraction was subjected to silica gel column chromatography using a gradient elution system of dichloromethane-methanol (100:0, 50:1, 10:1, 8:1, 5:1, 2:1, and 1:1). After identification by thin layer chromatography, the eluates of the same spot were combined in the order of elution to obtain 7 fractions Fr.3-1→Fr.3-7. Among them, the Fr.3-5 fraction was subjected to p-HPLC using 65% methanol-water as the elution condition to obtain compound 2 (4.6 mg, t R =26.5min).

[0028] Compound 1

[0029]

[0030] Compound 1 is a red amorphous powder (MeOH). HR-ESI-MS spectrum shows a quasi-molecular ion peak of m / z 527.1877 [M+H] + , indicating that the molecular weight is 526 and the molecular formula is C 35 H 26 O5, unsaturation is 23. UV spectrum has maximum absorption at 267, 299 and 431nm. 1 H-NMR spectrum data (Table 1) show that δH 7.73 (1H, d, J = 8.1 Hz) and 7.37 (1H, d, J = 8.1 Hz) are AB spin systems on the aromatic ring. H 7.08 and 6.78 (each 2H, d, J = 8.8 Hz), δ H 6.83 and 6.53 (each 2H, d, J = 8.5 Hz), δ H 7.16 and 6.48 (each 4H, d, J = 9.1 Hz) are the proton signals of the four para-substituted spin coupling systems, which are the proton signals of the B ring, E ring and the symmetrically substituted C ring and D ring. In addition, 1 H-NMR spectrum suggests the presence of a hydroxymethyl δ H 4.94 (2H, s), one methoxyl group δ H 3.75 (3H, s) hydrogen signal. 13 The C-NMR spectrum showed 35 carbon signals, including a carboxyl carbon (δ C 177.5), two alkynyl carbons (δ C 98.6,83.7), an oxygen-containing methylene carbon (δ C 62.0), a methoxy carbon (δ C 54.3) and 30 aromatic carbon signals.

[0031] The structure of compound 1 was further confirmed by HMBC spectrum. H 7.08) and C-27 (δ C 98.6) suggest that there is a correlation between alkynyl (δ C 98.6,83.7) connected to the B ring. H-20 / 24 (δ H 6.83) and C-18 (δ C The correlation between H-3, H-5 / C-7 and H-8, H-12 / C-7 indicates that the connection between the C and D rings is located at C-7 (δ C 166.7).

[0032] Thus, C-19 in the A ring is connected to C-7. H 3.75 and C-30 (δ C 160.0) is related to the methoxy proton signal at the C-30 position, indicating that the methoxy group is connected to C-30. H 3.75 (3H, s) and δ C 54.3], compound 1 1 H-NMR and 13The C-NMR data were similar to those reported for Selaginellin (8).

[0033] In summary, compound 1 is a new compound not reported in the literature and is named Selariscinin H.

[0034] Compound 2

[0035]

[0036] Compound 2 is a red amorphous powder. High-resolution mass spectrometry gives a quasi-molecular ion peak of m / z 555.1810 [M+H] + , its molecular formula is deduced to be C 36 H 26 O6, the degree of unsaturation is 24. The UV spectrum shows maximum absorption at 265, 300 and 425 nm, which is the characteristic absorption of phenol in Selaginella.

[0037] Compound 2 1 H-NMR and 13 The C-NMR spectrum showed structural features similar to those of Selaginellin (8), except for an additional acetyl group at the 34-hydroxymethyl position [δ H 2.14(3H,s),δ C 19.4,δ C 171.2], and the rest of the data are basically consistent. In the HMBC spectrum, δ H 2.14 (H-36) and δ C 171.2(C-35), δ H 5.43 (H-34) and δ C The presence of a correlation between 171.2 (C-35) further suggests that -CH2OH at C-15 is replaced by -CH2OOCCH3.

[0038] In summary, compound 2 is a new compound not reported in the literature and is named Selariscinin I.

[0039] Compound 3

[0040]

[0041] Compound 3 is a red amorphous powder (MeOH). In HR-ESI-MS, at m / z 541.1676 [M+H] + The quasi-molecular ion peak is shown at 35 H 24 O6, and showed that the degree of unsaturation was 24. The UV spectrum showed maximum absorption at 265, 292 and 431 nm.1 H-NMR and 13 The C-NMR spectrum also showed similar characteristics to compound 1, with the methoxy carbon signal at δ H 3.75 disappears and at δ H 5.92 (2H, s) and δ C A methylenedioxy group appears at 101.4. The positive Labat reaction further indicates that the structure contains a methylenedioxy group.

[0042] In the HMBC spectrum, the typical methylenedioxy 1 H-NMR signal (δ H 5.92) respectively with [δ C 148.4 (C-30) and δ C 147.5 (C-31)] 13 The C-NMR signal correlation showed that the methylenedioxy groups were located at C-30 and C-31. In summary, the compound was a new compound not reported in the literature, and compound 3 was named Selariscinin J.

[0043] Compound 4

[0044]

[0045] Compound 5

[0046]

[0047] Compound 4 and compound 5 are both red amorphous powders. 1 H-NMR and 13 The C-NMR data were similar to the structures of (S)-selaginellin U and (S)-selaginellin V, with only some differences in the positions of the symmetrical substituents on the C- and D-rings. This suggests that compounds 4 and 5 are derivatives of (S)-selaginellin U and (S)-selaginellin V synthesized through tautomerism of the (R) and (S) forms.

[0048] The absolute configurations of compounds 4 and 5 were determined by optical rotation and Cotton Effect (CE) analysis. In the CD spectrum, +CE was observed at 206 and 214 nm, and -CE was observed at 210 and 220 nm. The absolute configurations of compounds 4 and 5 were (R), which is opposite to the (S) configuration reported in the literature (-CE at 206 and 214 nm, +CE at 210 and 220 nm).

[0049] Table 1 Compounds 1-3 1 H NMR (600 MHz) and13 C NMR (150 MHz) nuclear magnetic resonance spectrum

[0050]

[0051]

[0052] Table 2 Compounds 4-5 1 H NMR (600 MHz) and 13 C NMR (150 MHz) nuclear magnetic resonance spectrum

[0053]

[0054] Example 2: Screening study on the anti-tumor activity of the Chinese herbal medicine Selaginella in vitro

[0055] 1.1 Experimental Materials

[0056] The tested monomer compounds were all homemade in the laboratory with a purity of over 98%.

[0057] Table 3 Sources of tumor cell lines

[0058]

[0059] 1.2 Experimental methods

[0060] CCK-8 assay was used to detect the inhibitory activity of Selaginella monohydrate compounds on human triple-negative breast cancer MDA-MB-231 and MDA-MB-468 cells

[0061] Human triple-negative breast cancer MDA-MB-231 and MDA-MB-468 cells were seeded in culture flasks and cultured in DMEM medium (L-15 medium containing 10% fetal bovine serum, 100 units / mL penicillin and 100 μg / mL streptomycin) in a 37°C, 0.1% CO2 incubator.

[0062] Take the human triple-negative breast cancer MDA-MB-231 and MDA-MB-468 cells in the logarithmic growth phase, discard the culture medium, add 2mL of PBS buffer to wash twice, discard the PBS, add 1mL of trypsin, gently blow the cells, add 2mL of culture medium to stop digestion, centrifuge at 1500rmp for 5min, discard the supernatant, add an appropriate amount of fresh culture medium and count the cells, mix the cells, add 100μL of cell suspension to each well, inoculate in a 96-well plate (1.5×103 / well), add different concentrations of the test monomer compound or 5-fluorouracil to each well, set 4 replicates for each drug, place in a CO2 incubator and continue to culture for 48h, and set up a blank control group. After 48 hours, add CCK-8 reagent, place in the incubator, and continue to culture for 1.5h. Then take out the cells, turn on the microplate reader, detect at 450nm, record the absorbance (A) value, and calculate the cell proliferation inhibition rate. Observe the inhibition rate at different concentrations and determine its IC 50 value.

[0063] 1.3 Experimental Results

[0064] Inhibitory activity of Selaginella monohydrate compounds against human triple-negative breast cancer MDA-MB-231 and MDA-MB-468 cells

[0065] Table 4 Inhibitory activity of compounds 1-5 on triple-negative breast cancer cells

[0066]

[0067] According to the above IC 50 The values indicate that the compounds of the present invention have certain anti-triple-negative breast cancer activity, among which compound 2 has the best activity, which is similar to that of the positive control 5-Fu.

Claims

1. One of the following compounds:

2. Use of the compound according to claim 1 in the preparation of anti-breast cancer drugs.

3. The use according to claim 2, characterized in that The breast cancer is triple-negative breast cancer.

4. The use according to claim 2, characterized in that The compound is compound 2.

5. An anti-breast cancer pharmaceutical composition, characterized in that: It comprises any one or more of the compounds described in claim 1.

6. The pharmaceutical composition according to claim 5, characterized in that The compound is selected from Compound 2.

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