Alkaloid compound as novel topoisomerase I inhibitor and preparation method and application thereof
By isolating and preparing the alkaloid compound PL-9 from camel burdock, the problem of the lack of effective topoisomerase I inhibitors in the prior art has been solved, and drug development with significant anti-cancer effects and reduced side effects has been achieved.
Patent Information
- Application Number
- CN202511047305.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-29
- Publication Date
- 2025-11-14
AI Technical Summary
The lack of effective topoisomerase I inhibitors in existing technologies makes it difficult to develop highly effective anticancer drugs using the active ingredients from plants of the genus *Phyllostachys* in traditional Chinese medicine.
A novel alkaloid compound, PL-9, was isolated and prepared from the traditional Chinese medicine Camel's Fern. The compound with the structure of formula (Ⅰ) was obtained through a multi-step extraction and purification method, and it showed significant topoisomerase I inhibitory activity.
Compound PL-9 exhibits stronger inhibitory activity against topoisomerase I than the positive control drug camptothecin, effectively blocking DNA replication and division in cancer cells, providing a new anti-cancer treatment approach, and reducing drug side effects.
Smart Images
Figure CN120943836A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to an alkaloid compound as a novel topoisomerase I inhibitor, its preparation method, and its application. Background Technology
[0002] Topo I (topoisomerase I) is a key ribozyme that can cleave and rejoin single-stranded DNA, unwinding supercoiled structures and promoting DNA replication and transcription. In tumor cells, the amount of Topo I is much higher than in normal tissues, and they are highly sensitive to Topo I inhibitors. Therefore, targeting Topo I as a drug to regulate its activity in tumor cells, thereby inhibiting DNA replication and subsequent cell division, holds promise as an important approach for treating various cancers.
[0003] The medicinal herb *Peganum harmala* L., belonging to the genus *Peganum* in the family Zygophyllaceae, comprises six species worldwide, mainly distributed along the Mediterranean coast, Central Asia, Mongolia, and North America. my country has three species, primarily distributed in Xinjiang, Inner Mongolia, Tibet, Qinghai, Gansu, and Shaanxi. β-carboline and quinazoline alkaloids are important active components of *Peganum* species, exhibiting diverse structures and broad pharmacological activities, including antitumor, antiviral, and neuroprotective effects. Therefore, identifying Topo I-inhibiting active components from *Peganum harmala* could potentially provide new drugs for cancer treatment. Summary of the Invention
[0004] To overcome the shortcomings of the prior art, this invention provides a novel topoisomerase I inhibitor, an alkaloid compound first discovered and isolated from the traditional Chinese medicine *Camelella asiatica*. This compound can be used to prepare drugs for treating cancer and its complications, as well as for preparing health supplements.
[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0006] The first aspect of this invention provides an alkaloid compound having the structure shown in formula (I):
[0007]
[0008] This invention provides the first-ever preparation of a novel alkaloid compound (abbreviated as PL-9) with the structure shown in formula (Ⅰ) from an extract of *Camelum verum*. Further research by the inventors revealed that this alkaloid compound with the structure shown in formula (Ⅰ) exhibits a significant inhibitory effect on topoisomerase I, and this effect is stronger than that of the positive control drug camptothecin. This technical effect was unexpected by those skilled in the art.
[0009] A second aspect of the present invention provides a camel nut extract, wherein the camel nut extract contains the alkaloid compound described in the first aspect.
[0010] Since the alkaloid compound with the structure shown in formula (I) has excellent topoisomerase I inhibitory activity, those skilled in the art can expect that extracts containing the structure shown in formula (I) will also have topoisomerase I inhibitory activity.
[0011] A third aspect of this invention provides a method for preparing the camel husk extract described in the second aspect, the method comprising the following steps:
[0012] S1. Take dried camel burr medicinal material and crush it, then use ethanol for cold extraction to obtain the total extract;
[0013] S2. The total extract is subjected to "acid extraction and alkali precipitation" treatment to obtain the total alkaloid components: After dispersing the total extract in water, the pH of the solution is adjusted to 2-3 with dilute hydrochloric acid, and then the main acidic substances in the total extract are extracted with dichloromethane; then the pH of the acid solution is adjusted to 9-10 with ammonia water, and the total alkali is extracted with dichloromethane to obtain the total alkaloids.
[0014] S3. Separate the total alkaloids onto a D101 macroporous resin column to obtain the macroporous resin eluent: After loading the total alkaloids onto a D101 macroporous resin column, elute with a 10:90 volume ratio of ethanol / water mixed solvent and discard the eluent; then elute again with a 30:70 volume ratio of ethanol / water mixed solvent, collect the eluent, and then concentrate and dry it to obtain the final product.
[0015] S4. Separate the macroporous resin elution fraction onto an ODS column to obtain the ODS elution fraction: After loading the macroporous resin elution fraction onto an ODS column, first elute with a methanol / water solvent with a volume ratio of 80:20, and discard the eluent; then elute again with a methanol / water solvent with a volume ratio of 90:10, collect the eluent, concentrate and dry it to obtain the final product.
[0016] S5 and ODS elution fractions were then processed by HPLC to obtain camel thorn extract containing the alkaloid compound described in claim 1: HPLC was performed using a C18 reversed-phase preparative column with methanol-water-ammonia as the mobile phase. Methanol and water were mixed at a volume ratio of methanol:water = 90:10, and 0.2% ammonia was added to prepare the eluent for isocratic elution.
[0017] Preferably, the ethanol in S1 is 95% ethanol.
[0018] The fourth aspect of this invention provides a method for preparing the alkaloid compound described in the first aspect, namely, after obtaining the camel husk extract using the preparation method described in the third aspect, collecting the extract for a retention time t.R The fraction corresponding to the chromatographic peak at 10.1 min yields the alkaloid compound described in the first aspect.
[0019] This invention is the first to isolate an alkaloid compound with the structure shown in formula (Ⅰ) from camel burdock; and provides a novel method for the preparation of an alkaloid compound with the structure shown in formula (Ⅰ).
[0020] The fifth aspect of this invention provides the use of the alkaloid compound described in the first aspect or the camel nut extract described in the second aspect in the preparation of a product having topoisomerase I inhibitory activity.
[0021] The sixth aspect of this invention provides the use of the alkaloid compound described in the first aspect or the camel nut extract described in the second aspect in the preparation of products that inhibit the growth of tumor cells.
[0022] Preferably, the tumor cells include non-small cell lung cancer cells, colorectal cancer cells, and pancreatic ductal carcinoma cells.
[0023] Preferably, the product comprises an effective dose of the alkaloid compound described in the first aspect or the camel nut extract described in the second aspect, as well as pharmaceutically acceptable excipients.
[0024] More preferably, the excipients include at least one of the following: excipients, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, antioxidants, adsorbents, filter aids, and release inhibitors.
[0025] Preferably, the product includes a drug or health supplement.
[0026] Preferably, the dosage form of the product includes powder, pill, tablet, capsule, oral liquid, aerosol or injection.
[0027] Compared with the prior art, the beneficial effects of the present invention are:
[0028] This invention isolates a novel alkaloid compound from the traditional Chinese medicine *Camel's Footprint*. Studies show that this alkaloid compound exhibits significant inhibitory activity against topoisomerase I, exceeding that of the positive control drug camptothecin. Since topoisomerase I plays a crucial role in DNA replication and division in tumor cells, inhibiting this enzyme can block cancer cell proliferation. Therefore, using this compound as an active ingredient in the preparation of anticancer drugs can provide a new material basis for drug development, improve efficacy, and reduce side effects. Furthermore, it can serve as a lead compound, allowing for the derivation of highly active anticancer molecules through structural optimization, thus possessing significant application value in the field of cancer treatment. Attached Figure Description
[0029] Figure 1 This is a high-resolution mass spectrum of compound PL-9.
[0030] Figure 2 The image shows the UV spectrum of compound PL-9 (soluble in methanol).
[0031] Figure 3 The outer infrared spectrum of compound PL-9 (KBr pellet).
[0032] Figure 4 Nuclear magnetic resonance of compound PL-9 1 H spectrum (600MHz, dissolved in DMSO).
[0033] Figure 5 Nuclear magnetic resonance of compound PL-9 13 C spectrum (150MHz, dissolved in DMSO).
[0034] Figure 6 The NMR spectrum of compound PL-9 is DEPT-135 (150 MHz, dissolved in DMSO).
[0035] Figure 7 The NMR 1H-1H COSY spectrum of compound PL-9 (150 MHz, dissolved in DMSO).
[0036] Figure 8 The HSQC NMR spectrum of compound PL-9 (150 MHz, dissolved in DMSO).
[0037] Figure 9 The NMR HMBC spectrum of compound PL-9 (150 MHz, dissolved in DMSO).
[0038] Figure 10 A represents the inhibitory activity of PL-9 against Topo I. Figure 10 B represents the prediction of the interaction between PL-9 and Topo I through molecular docking. Detailed Implementation
[0039] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.
[0040] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.
[0041] Example 1: Isolation and extraction of alkaloid compounds
[0042] (1) Take 30.0 kg of dried camel thorn seeds and crush them (about 80 mesh). Use 95% ethanol for cold extraction (extract three times at room temperature, each extraction lasts 2 days, and each extraction uses 50L of 95% ethanol). A total of 2.3 kg of total extract was prepared.
[0043] (2) To further concentrate the alkaloid components, the total extract was subjected to an "acid extraction and alkali precipitation" process, ultimately yielding 812.3 grams of total alkaloids. The specific procedure for the "acid extraction and alkali precipitation" was as follows: the total extract was dispersed in 2L of water, and the pH of the solution was adjusted to 2 with dilute hydrochloric acid. Then, twice the volume of dichloromethane was added, and the mixture was extracted three times at room temperature, one day each time. The extract was discarded, and the acid solution was retained. Subsequently, the pH of the acid solution was adjusted to 9 with 25% ammonia, and twice the volume of dichloromethane was added again. The mixture was extracted three times at room temperature, yielding 812.3 grams of extract, which constituted the total alkaloids.
[0044] (3) The total alkaloids were loaded onto a D101 macroporous resin column and eluted with a mixed solvent of ethanol / water at a volume ratio of 10:90. The eluent was discarded. Then, the column was eluted with a mixed solvent of ethanol / water at a volume ratio of 30:70. The eluent was collected and then concentrated and dried to obtain the macroporous resin elution fraction.
[0045] (4) The macroporous resin eluent fraction is loaded onto an ODS column. First, it is eluted with a methanol / water solvent with a volume ratio of 80:20 and the eluent is discarded. Then, it is eluted with a methanol / water solvent with a volume ratio of 90:10. The eluent is collected, concentrated and dried to obtain the ODS eluent fraction.
[0046] (5) The ODS elution fraction was subjected to HPLC again (using a C18 reversed-phase preparative column, with methanol-water-ammonia as the mobile phase, and isocratic elution at a volume ratio of methanol:water (0.2% ammonia) = 90:10 (0.2%), and the retention time t was collected). R The fraction corresponding to the chromatographic peak at 10.1 min was used to prepare compound PL-9, which is an alkaloid compound with the structure shown in Formula I.
[0047]
[0048] The structure of compound PL-9 was identified as follows:
[0049] The structure of compound PL-9 was obtained by analyzing high-resolution mass spectrometry (HRESIMS), infrared spectroscopy (IR), and one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy (NMR).
[0050] The molecular weight of compound PL-9 was determined to be 384.1709 [M+H] using high-resolution mass spectrometry (HRESIMS: m / z). + (Theoretical value: 384.1707), indicating the molecular formula is C. 24 H 21 N3O2, with an unsaturation degree of 16 ( Figure 1 ).
[0051] Figure 2 The UV spectrum of CH3OH shows maximum absorption peaks at 205, 223, 280, 310, and 432. Infrared spectroscopy data (…) Figure 3 The results showed that compound PL-9 contained amino groups (3216 cm⁻¹). -1 ) and benzene ring (1629, 1563 cm) -1 ).
[0052] The proton NMR spectrum of compound PL-9 1 The HNMR data and attribution are shown in Table 1 and Figure 4 As shown. Data shows that compound PL-9 has 7 aromatic proton signals [δ]. H 7.91 (1H,d,J=8.6Hz), 7.53 (1H,d,J=8.6Hz), 7.13 (1H,s), 7.10 (1H,d,J=2.2Hz), 6.92 (1H,d,J=2.2Hz), 6.77 (1H,dd,J=8.6,2.2Hz), 6.74 (1H,dd,J=8.6,2.2Hz)], 2 methylene proton signals [δ H 4.92 (2H,t,J=7.2Hz), 3.26 (2H,t,J=7.2Hz)], 2 oxygen-bound methyl proton signals [δ H 3.84(3H,s), 3.83(3H,s)], a methyl proton signal [δ H 2.82(3H,s)].
[0053] Carbon NMR spectrum of compound PL-9 13 The C NMR data and their attribution are shown in Table 1 and Figure 5As shown. Data shows that compound PL-9 has two methoxy groups, one methyl group, two methylene groups, seven methine groups, and twelve quaternary carbons. It contains two sets of AMX system benzene ring carbon signals (δ). C 158.9, 157.6, 154.0, 139.4, 122.5, 120.2, 117.7, 112.8, 110.6, 107.5, 100.3, 94.4), two methylene groups (δ C 44.2, 19.1), two oxygen-bound methyl groups (δ C 55.3, 55.1), a methyl signal (δ) C 19.6), a total of 24 carbon signals.
[0054] The data and binding relationships of the two-dimensional nuclear magnetic resonance spectrum of compound PL-9 are shown in Table 1 and Figure 6-9 As shown. The structure of compound PL-9 can be obtained from this. Specifically, the molecular numbering of compound PL-9 is shown below:
[0055]
[0056] Table 1. One-dimensional and two-dimensional NMR spectra of compound PL-9 (DMSO-d6, δin ppm, Jin Hz)
[0057]
[0058]
[0059] Experimental Example 2: Cytotoxicity Experiment of Alkaloid Compound PL-9
[0060] A549, H1299, HCT116, and PANC-1 cells were seeded in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours to allow cell adhesion and stable growth. Subsequently, using 0.1% DMSO as a control, different concentration gradients of the test compound (50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.5625 μM) were added to each well, with five replicates per concentration. After addition, incubation was continued for 24 hours under the same conditions. Next, 10 μL of MTT solution (5 mg / mL) was added to each well, and the plate was incubated at 37°C for 4 hours until a purple formazan precipitate formed. After incubation, the culture medium in the wells was discarded, and 100 μL of DMSO was added to each well to dissolve the precipitate, gently shaking for 10 minutes to ensure complete dissolution. Finally, the absorbance of each well was measured at a wavelength of 490 nm using an ELISA reader.
[0061] As shown in Table 2, the cytotoxic activity of compound PL-9, which has the structure shown in Formula I, against four human cancer cell lines (A549, H1299, HCT116, and PANC-1) was tested. It was found that compound PL-9 has significant growth-inhibiting activity against all cell lines, and its cytotoxic activity is significantly better than that of the positive control drugs camptothecin (CPT) and cisplatin. The effect of this technology is beyond the expectations of those skilled in the art.
[0062] Table 2 Cytotoxic activity of compound PL-9
[0063]
[0064] Experimental Example 3: Topo I-mediated DNA loosening assay
[0065] Add 2 μL of DNA Topo I buffer (35 mM Tris-HCl pH 8.0, 72 mM KCl, 25 mM MgCl2, 5 μM dithiotetrol, 5 mM spermidine), 2 μL of 0.1% BSA, 0.2 μL of the test compound (dissolved in DMSO), and 0.5 μL of LPBR322 plasmid DNA (0.125 μg, purchased from Takara) to 10 μL of deionized water, then add Topo I (0.5 U). Finally, add deionized water to the reaction solution to a final volume of 20 μL. Incubate the reaction solution at 37°C for 30 minutes, then add the loading buffer (0.9% sodium dodecyl sulfate (SDS), 0.05% bromophenol blue, 50% glycerol). The resulting sample was electrophoresed on a 0.8% agarose gel at 110 V in 1×TAE (Tris-acetate EDTA) for 40 minutes. The samples were then stained with ethylenediamine bromide (0.5 μg / mL) for 30 min, and the DNA bands under ultraviolet light were observed. Finally, the electrophoresis results were analyzed using a gel imaging system.
[0066] The inhibitory activity of compound PL-9 against Topo I was detected by DNA loosening assay. The results showed that ( Figure 10 A) Compound PL-9 exhibits superior activity compared to the positive control drug camptothecin (CPT). Meanwhile, Figure 10 B discovered through molecular docking virtual screening that compound PL-9 binds well to Topo I, which acts on DNA replication, an effect that was unexpected by those skilled in the art.
[0067] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.
Claims
1. An alkaloid compound, characterized in that, The alkaloid compound has the structure shown in formula (Ⅰ):
2. A camel nut extract, characterized in that, The camel husk extract contains the alkaloid compound as described in claim 1.
3. The method for preparing the camel husk extract according to claim 2, characterized in that, Includes the following steps: S1. Take dried camel burr medicinal material and crush it, then use ethanol for cold extraction to obtain the total extract; S2. The total extract is subjected to "acid extraction and alkali precipitation" to obtain the total alkaloids: After dispersing the total extract in water, the pH of the solution is adjusted to 2-3 with dilute hydrochloric acid, and then the main acidic substances in the total extract are extracted with dichloromethane; then the pH of the acid solution is adjusted to 9-10 with ammonia, and the total alkali is extracted with dichloromethane to obtain the total alkaloids. S3. Separate the total alkaloids onto a D101 macroporous resin column to obtain the macroporous resin eluent: After loading the total alkaloids onto a D101 macroporous resin column, elute with a 10:90 volume ratio of ethanol / water mixed solvent and discard the eluent; then elute again with a 30:70 volume ratio of ethanol / water mixed solvent, collect the eluent, and then concentrate and dry it to obtain the final product. S4. Separate the macroporous resin elution fraction onto an ODS column to obtain the ODS elution fraction: After loading the macroporous resin elution fraction onto an ODS column, first elute with a methanol / water solvent with a volume ratio of 80:20, and discard the eluent; then elute again with a methanol / water solvent with a volume ratio of 90:10, collect the eluent, concentrate and dry it to obtain the final product. S5 and ODS elution fractions were then processed by HPLC to obtain camel thorn extract containing the alkaloid compound described in claim 1: HPLC was performed using a C18 reversed-phase preparative column with methanol-water-ammonia as the mobile phase. Methanol and water were mixed at a volume ratio of methanol:water = 90:10, and 0.2% ammonia was added to prepare the eluent for isocratic elution.
4. The method for preparing the alkaloid compound according to claim 1, characterized in that, After obtaining the camel husk extract using the preparation method described in claim 3, the retention time t is collected. R The fraction corresponding to the chromatographic peak at 10.1 min yields the alkaloid compound described in claim 1.
5. The use of the alkaloid compound of claim 1 or the camel husk extract of claim 2 in the preparation of a product having topoisomerase I inhibitory activity.
6. The use of the alkaloid compound of claim 1 or the camel husk extract of claim 2 in the preparation of products that inhibit tumor cell growth.
7. The application according to claim 6, characterized in that, The tumor cells include non-small cell lung cancer cells, colorectal cancer cells, and pancreatic ductal carcinoma cells.
8. The application according to claim 5 or 6, characterized in that, The product comprises an effective dose of the alkaloid compound of claim 1 or the camel nut extract of claim 2, and pharmaceutically acceptable excipients.
9. The application according to claim 5 or 6, characterized in that, The products mentioned include medicines or health supplements.
10. The application according to claim 5 or 6, characterized in that, The dosage forms of the products include powders, pills, tablets, capsules, oral liquids, aerosols, or injections.