An alkaloid, a process for its preparation and use in the preparation of human carboxylesterase 2 inhibitors

Abstract

This invention discloses an alkaloid, its preparation method, and its application in the preparation of human carboxylesterase 2 inhibitors. The residue of Paeonia lactiflora after ultrasonic extraction was extracted with 95% ethanol and then with ethyl acetate to obtain ethyl acetate extract CS-5. Separation was performed by silica gel column chromatography, using a gradient elution with petroleum ether-acetone mixed solvent and dichloromethane-methanol mixed solvent to obtain six eluent fractions, named A-F. Compound D was separated by silica gel column chromatography, Sephadex LH-20 column chromatography, and semi-preparative HPLC to obtain compound I-2. Compound F was separated by Sephadex LH-20 column chromatography and semi-preparative HPLC to obtain compounds I-1 and I-3. Compound I-1 was separated by a chiral HPLC column to obtain enantiomers I-(+)-1 and I-(–)-1. The four alkaloids of this invention have a pH range of 13.65–14.16. m At concentrations of M, it can significantly inhibit the activity of human carboxylesterase 2. Therefore, it can be applied to the preparation of attenuated and protective agents for anticancer drugs such as irinotecan.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to an alkaloid, its preparation method, and its application in the preparation of human carboxylesterase 2 inhibitors. Background Technology

[0002] Chemotherapy is a crucial treatment for cancer, but the severe toxic side effects commonly associated with chemotherapy drugs often limit their full clinical efficacy. Irinotecan, a topoisomerase I inhibitor widely used in the treatment of solid tumors such as colorectal cancer and lung cancer, is a prodrug that needs to be metabolized in vivo to become the active ingredient SN-38 in order to exert its anti-tumor effect. However, this conversion process exhibits significant organ-specific differences: in the intestine, the reaction is primarily catalyzed by highly expressed human carboxylesterase 2 (hCE2), leading to excessively high local SN-38 concentrations and causing severe delayed diarrhea and enteritis. This toxicity is the most significant dose-limiting toxicity of irinotecan, severely impacting patients' quality of life and treatment adherence, and often forcing clinicians to reduce the dosage or discontinue treatment, thereby weakening its anti-tumor effect.

[0003] To address this issue, one of the main strategies in existing technologies is to develop selective human carboxylesterase 2 (hCE2) inhibitors. By inhibiting hCE2 activity in the intestine, the in-situ activation of irinotecan in the intestine can be significantly reduced, thereby decreasing the direct damage of SN-38 to intestinal epithelial cells and effectively preventing severe diarrhea. Theoretically, this not only improves patient tolerance to irinotecan but also provides the possibility of increasing the therapeutic dose and enhancing efficacy. Currently, some synthetic small-molecule hCE2 inhibitors have entered preclinical or clinical research stages, but overall, existing inhibitors still face challenges in terms of selectivity, pharmacokinetic properties, long-term safety, and development costs. Therefore, finding novel hCE2 inhibitors with novel structures, wide availability, and greater safety potential is of significant clinical importance and market demand for optimizing the clinical application of prodrugs such as irinotecan.

[0004] Natural products, especially the active ingredients of traditional Chinese medicine, have always been an important source of innovative drug discovery due to their structural diversity and rich biological activities. Red peony root (Paeonia lactiflora or Paeonia veitchii) is a plant in the Ranunculaceae family and has the effects of clearing heat and cooling blood, dispersing blood stasis and relieving pain. Modern pharmacological studies have shown that red peony root contains various chemical components such as monoterpenoid glycosides, phenolic acids, and flavonoids, possessing anti-inflammatory, antithrombotic, antitumor, and hepatoprotective pharmacological activities. However, to date, research on the chemical components of red peony root has focused primarily on its glycosides and phenolic acids, with relatively little attention paid to its alkaloid components. Furthermore, its in-depth pharmacological mechanisms, particularly its interaction with specific metabolic enzymes such as carboxylesterase 2, have not been systematically reported. No studies in this field have revealed or suggested that any alkaloid component in red peony root has inhibitory activity against hCE2, nor have they proposed its use to address the toxic side effects of chemotherapy drugs mediated by hCE2.

[0005] Based on this, the inventors, for the first time, focused their research on the underdeveloped alkaloid components of Paeonia lactiflora. Through systematic extraction, separation, structural identification, and activity screening, they discovered that a specific alkaloid can efficiently and selectively inhibit the activity of human carboxylesterase 2. This discovery not only opens up a new direction for the modern pharmacological research and application development of Paeonia lactiflora, but more importantly, it provides a candidate compound derived from traditional Chinese medicine, with a unique structure and great development potential, and a novel technical solution to address the key clinical toxicity issues of prodrugs such as irinotecan. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, the main objective of this invention is to provide an alkaloid, its preparation method, and its application in the preparation of human carboxylesterase 2 inhibitors, thereby solving the problem of toxic side effects caused by chemotherapeutic drugs mediated by hCE2.

[0007] To achieve the aforementioned objectives, the technical solution adopted by this invention includes: An alkaloid compound, namely compounds I-(+)-1, I-(–)-1, I-2, and I-3, has the following structural formula: .

[0008] The method for preparing the alkaloid compound includes the following steps: 1) Red peony root was soaked in distilled water and then extracted by ultrasound. The resulting residue was soaked in 95% ethanol and then extracted by ultrasound. The 95% ethanol extract was concentrated to an alcohol-free concentrate, extracted with ethyl acetate, and concentrated with ethyl acetate to obtain ethyl acetate extract CS-5. 2) The CS-5 was separated by silica gel column chromatography, and the eluent was successively eluted with a gradient of petroleum ether-acetone mixed solvent, and then eluted with a dichloromethane-methanol mixed solvent. The solvent was recovered under reduced pressure to obtain 6 eluent fractions, which were named AF respectively. 3) D was separated by silica gel column chromatography, eluted with a gradient of dichloromethane-methanol mixed solvent, and the solvent was recovered under reduced pressure to obtain five eluent fractions, named D1-D5; D5 was subjected to Sephadex LH-20 column chromatography, eluted with a dichloromethane-methanol mixed solvent, and the solvent was recovered under reduced pressure to obtain one eluent fraction, named D5-1; D5-1 was separated by semi-preparative high performance liquid chromatography to obtain compound I-2; 4) F was subjected to gel Sephadex LH-20 column chromatography, eluted with pure methanol, and the solvent was recovered under reduced pressure to obtain 6 eluent fractions, named F1-F6 respectively; F3 was separated by semi-preparative HPLC to obtain compounds I-1 and I-3; 5) Compound I-1 was further separated by high performance liquid chromatography chiral separation column to obtain enantiomers I-(+)-1 and I-(-)-1.

[0009] In step 1), the volume of distilled water used is 8 times the weight of the medicinal material, the volume of 95% ethanol used is 8 times the weight of the residue, the ultrasonic extraction frequency is 40 kHz, the extraction time is 1 hour, and the extraction is performed 3 times; the volume ratio of ethyl acetate to concentrate is 1:1, and the extraction is performed 5 times.

[0010] In step 2), the silica gel column is 100-200 mesh, the volume ratio of the petroleum ether-acetone mixed solvent is 50:1, 30:1, 20:1, 10:1, and 8:1 respectively, the volume ratio of the dichloromethane-methanol mixed solvent is 20:1, and each eluent elutes five times the column volume.

[0011] In step 3), the silica gel column is 100-200 mesh, the volume ratio of the first dichloromethane-methanol mixed solvent is 50:1, 30:1, 10:1, 1:1, and 1:10, and each eluent elutes five times the column volume; the volume ratio of the second dichloromethane-methanol mixed solvent is 1:1, and it elutes two times the column volume; the semi-preparative high-performance liquid chromatography conditions are: using a C18 semi-preparative column, a detection wavelength of 210 nm, and a mobile phase of acetonitrile-water with a volume ratio of 30:70.

[0012] In step 4), the pure methanol elution was performed at five column volumes. The semi-preparative HPLC conditions were as follows: a C18 semi-preparative column was used, the detection wavelength was 210 nm, the mobile phase was acetonitrile-water with a volume ratio of 20:80, and the column temperature was 32℃. In step 5), the high-performance liquid chromatography chiral separation column was Chiral MJ(2), the detection wavelength was 210 nm, the mobile phase was n-hexane-isopropanol with a volume ratio of 88:12, and the column temperature was 32℃.

[0013] A pharmaceutical composition comprising at least one of the alkaloid compounds and a pharmaceutically acceptable salt or a pharmaceutically acceptable carrier or excipient thereof.

[0014] The use of the pharmaceutical composition in the preparation of human carboxylesterase 2 inhibitors.

[0015] The application of the pharmaceutical composition in the preparation of a toxicity-reducing and protective agent for anticancer chemotherapy drugs.

[0016] The anticancer chemotherapy drug mentioned is irinotecan.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention is the first to extract and isolate four novel alkaloid compounds from the traditional Chinese medicine Paeonia lactiflora.

[0018] 2. The alkaloids of this invention have a molecular weight range of 13.65 to 14.16. μ At concentrations M, the activity of human carboxylesterase 2 can be significantly inhibited. Computer-aided molecular docking simulations show that the alkaloids of this invention can also bind to key amino acid residues at the hCE2 active site through multiple hydrogen bonds, thereby inhibiting hCE2 activity. This can further be applied to the preparation of attenuated and protective agents for anticancer drugs such as irinotecan. Attached Figure Description

[0019] Figure 1 For alkaloids I-(+)-1 and I-(–)-1 1 H NMR spectrum; Figure 2 For alkaloids I-(+)-1 and I-(–)-1 13 C NMR spectrum; Figure 3 It is alkaloid I-2 1 H NMR spectrum; Figure 4 It is alkaloid I-2 13 C NMR spectrum; Figure 5 It is alkaloid I-3 1 H NMR spectrum; Figure 6 It is an alkaloid I-3. 13 C NMR spectrum; Figure 7 This is a schematic diagram of a computer simulation of the interaction between alkaloids I-1~I-3 and the active site of the hCE2 protein. Figure 7 a is a schematic diagram illustrating the interaction between the substrate diacetylfluorescein (FD) and the hCE2 protein. Figure 7 b is a schematic diagram of the interaction between alkaloid I-(+)-1 and hCE2 protein; Figure 7c is a schematic diagram of the interaction between alkaloid I-(–)-1 and hCE2 protein; Figure 7 d is a schematic diagram of the interaction between alkaloid I-2 and hCE2 protein; Figure 7 e is a schematic diagram of the interaction between alkaloid I-3 and hCE2 protein. Detailed Implementation

[0020] The embodiments described below are exemplary descriptions of key experimental evidence and are not intended to limit the core content and application scope of this invention due to the amount of evidence. It should be noted that all the accompanying drawings and corresponding descriptions merely illustrate the concept, principles, and representative experimental evidence of the disclosed embodiments of this invention. Where the chain of evidence is complete, it is unnecessary to show all the specific details and extended details of the various embodiments listed in this invention.

[0021] Unless otherwise defined, the technical terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art to which this invention pertains.

[0022] The present invention relates to instruments and materials as follows: Instrument: Ultraviolet spectrometer: JASCO J-810; CD measuring instrument: JASCO J-810; Optical polarimeter: Perkin-Elmer 343 polarimeter; Infrared spectrometer: Nicolet Impact 400 Fourier Transform Infrared Spectrometer; Nuclear magnetic resonance (NMR) spectrometers: Mercury-300, Mercury-400, Inova-500, and SYS-600 NMR spectrometers; measurement temperature: 297 K; solvent peak signal used as reference; Varian, USA Mass spectrometer: Agilent UPLC Q-TOF type; Rotary evaporator: Búchi Rotavapor R-205; High Performance Liquid Chromatograph (HPLC): Waters 600 HPLC system (Waters 2487 dual-wavelength detector, binary high-pressure gradient pump, Empower workstation, Waters Corporation, USA). Ultraviolet Analyzer: WFH-203B Three-Purpose Ultraviolet Analyzer; Materials: C18 semi-preparative column, 5 μm, 250 × 10 mm; The chromatographic column was a Chiral MJ (2), 5 μm, 250 × 4.6 mm. Silica gel column chromatography: Column chromatography silica gel (100-200 mesh), produced by Qingdao Marine Chemical Plant; Gel column chromatography: Sephadex LH-20, manufactured by Amersham Pharmacia, Sweden; Thin-layer chromatography: silica gel GF254, produced by Qingdao Marine Chemical Plant, specifications: 50×100mm; thickness 0.2-0.25mm.

[0023] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0024] Example 1 Experimental steps: 50 kg of dried red peony root slices were thoroughly soaked in 8 times their volume of distilled water and then ultrasonically extracted (40 kHz) for 1 hour, repeating the extraction three times. The extracts were combined, and the residue was separated. The remaining residue (30 kg) was soaked in 8 times its volume of 95% ethanol and ultrasonically extracted (40 kHz) three times, 1 hour each time. The concentrated extracts were combined. The 95% ethanol extract was concentrated until alcohol-free and then extracted five times with an equal volume of ethyl acetate to obtain an ethyl acetate phase solution. The ethyl acetate phase was concentrated under reduced pressure to obtain 580 g of ethyl acetate extract CS-5. CS-5 was mixed with 100-200 mesh silica gel at a mass ratio of 1:1 and separated by chromatographic chromatography on a 100-200 mesh silica gel column. The column was eluted sequentially with a petroleum ether-acetone mixed solvent gradient (volume ratios of 50:1, 30:1, 20:1, 10:1, and 8:1, respectively), followed by elution with a dichloromethane-methanol mixed solvent (volume ratio of 20:1). Each eluent was used to elute five times the column volume. Thin-layer chromatography was performed at UV wavelengths of 254 nm and 365 nm, with dichloromethane:methanol (15:1) as the developing solvent. Identical components were combined, and the solvent was recovered under reduced pressure to obtain six eluent fractions, which were named AF.

[0025] D (39 g) was mixed with 100-200 mesh silica gel at a mass ratio of 1:1 and separated by chromatographic chromatography on a 100-200 mesh silica gel column. The sample was eluted with a gradient of dichloromethane-methanol mixed solvent (volume ratios of 50:1, 30:1, 10:1, 1:1, and 1:10, respectively), with each eluent eluting five times the column volume. Thin-layer chromatography was performed at UV wavelengths of 254 nm and 365 nm, with dichloromethane:methanol (15:1) as the developing solvent. The same components were combined, and the solvent was recovered under reduced pressure to obtain five eluent fractions, which were named D1-D5.

[0026] D5 (1.4 g) was eluted on a Sephadex LH-20 column with a dichloromethane-methanol mixture (volume ratio 1:1) for 2 column volumes. Thin-layer chromatography was performed at UV wavelengths of 254 nm and 365 nm. The developing solvent was dichloromethane:methanol (15:1). The solvent was recovered under reduced pressure to obtain one eluent fraction, named D5-1 (36 mg).

[0027] D5-1 (36 mg) was subjected to semi-preparative high-performance liquid chromatography [column C18, 5] μ m, 250 × 10 mm, detection wavelength 210 nm, mobile phase acetonitrile-water (30:70), column temperature: 32℃;] compound I-2 (0.9 mg, t) was isolated and obtained. R = 13.5 min).

[0028] F (6 g) was subjected to gel Sephadex LH-20 column chromatography, eluted with pure methanol for five column volumes, and detected by thin-layer chromatography at UV wavelengths of 254 nm and 365 nm. The developing solvent was dichloromethane:methanol (15:1). Identical components were combined, and the solvent was recovered under reduced pressure to obtain six eluents, which were named F1-F6.

[0029] F3 (260 mg) was subjected to semi-preparative HPLC [column C18, 5]. μ m, 250 × 10 mm, detection wavelength 210 nm, mobile phase acetonitrile-water (20:80), column temperature: 32℃] to separate I-1 (2 mg, t R =16 min) and I-3 (2 mg, t) R =20 min).

[0030] Alkaloid I-1 was further separated by high-performance liquid chromatography (HPLC) using a chiral separation column [Chiral MJ (2), 5 μm, 250 × 4.6 mm, detection wavelength 210 nm, mobile phase n-hexane-isopropanol (88:12); column temperature: 32℃]. The t values ​​were collected. R =16 min fraction, yielding enantiomer I-(+)-1 (0.4 mg); t R =17 min, yielding enantiomer I-(–)-1 (0.4 mg).

[0031] Compounds I-1 to I-3 1 1H NMR spectrum (solvent: deuterated methanol, 600 MHz) and 13The C NMR spectrum (solvent: deuterated methanol, 150 MHz) is shown in the figure below. Figure 1-6 As shown.

[0032] Alkaloid I-1: Yellow amorphous powder; UV (MeOH)λmax (log ε) 209 (1.13), 271.8 (0.37), 310.8 (0.17) nm; IR (cm 1 ) 3347.70, 2951.65, 2922.95, 1693.64, 1627.66, 1466.73, 1436.55, 1375.11, 1273.71, 1203.71, 1168.55, 1140.11, 1107.25, 1005.85, 877.66, 770.52, 715.03; 1 H NMR (CD3OD, 600 MHz) and 13 C NMR (CD3OD, 150 MHz) data are shown in Table 1; HRESIMS m / z 292.1191 [MH] - (calcd for C 15 H 19 O5N - , m / z 292.1190).I-(+)-1:[α] 20 D 20.5 (c 0.04, MeOH); ECD (MeOH) 211 (Δε + 19.04), 237 (Δε – 14.74), 269 (Δε + 3.26); I-(-)-1: [α] 20 D –20.5 (c 0.04, MeOH); ECD(MeOH) 211 (Δε – 20.40), 237 (Δε + 15.60), 269 (Δε – 3.51); Alkaloid I-2: White amorphous powder; [α] 20 D –7.25 (c0.08, MeOH); UV(MeOH)λ max (logε)215.8 (1.18), 256.6 (0.6), 277.8 (0.5), 307 (0.5) nm;IR (cm 1) 3213.80,2959.30, 2928.43, 2855.85, 1712.14, 1684.67, 1622.11, 1534.33, 1465.14,1443.01, 1363.65, 1338.18, 1307.31, 1279.91, 1209.04, 1184.05, 1144.54,1059.42, 1005.89, 978.88, 880.95, 857.79, 802.07, 759.35, 725.50, 699.49,620.13. 1 H NMR (CD3OD, 600 MHz) and 13 C NMR (CD3OD, 150 MHz) data, see Table1;HRESIMS m / z 262.1084 [M+H] + (calcd forC 14 H 15 O4N + , m / z The data for 262.1074 is shown in Table 1. Alkaloid I-3: White amorphous powder; [α] 20 D –12.26 (c1.63, MeOH); UV(MeOH)λ max (logε)215.8 (0.69), 256.2 (0.42), 277 (0.32), 307 (0.32) nm;IR (cm 1 ) 3247.22,2928.30, 1736.42, 1623.42, 1532.48, 1473.60, 1463.92, 1443.99, 1366.46,1337.43, 1305.19, 1286.46, 1210.93, 1187.14, 1146.68, 1168.48, 1007.23,975.11, 876.23, 846.51, 802.18, 764.04, 725.94,711.56, 639.75, 620.62. 1 H NMR (CD3OD, 600 MHz) and 13 C NMR (CD3OD, 150 MHz) data, see Table1; HRESIMS m / z276.1243. [M+H] - (calcd forC 15 H 17 O4N + , m / z The data for 276.1230 is shown in Table 1.

[0033] Table 1 NMR Data ( δ ) of Compounds 1-3 in MeOH d 4

[0034] The structures of alkaloids I-(+)-1 to I-3 were identified using physicochemical constants and modern spectroscopic techniques (MS, NMR, CD), as shown in the following formulas: .

[0035] Example 2 Pharmacological experiments (1) Assay of the inhibitory activity of alkaloids I-(+)-1~I-3 on human carboxylesterase 2 Instrument: Microplate reader Materials: Human liver microsomes (50 doses, HLM): Celsis. Diacetylfluorescein (FD): TCI (Tokyo, Japan). Loperamide hydrochloride (LPA): TCI (Tokyo, Japan). 0.1 M phosphate-buffered saline PBS (pH 7.4).

[0036] Experimental steps: 1. Establishment of hCE2 inhibitory activity screening system The total volume of the system is 200. μ L. Add 196 to the 96-well plate. µ L PBS buffer (pH 7.4), 2 µ L HLM (1 μ g / mL, final concentration) and 1µL alkaloids (set concentration gradient 1) µ M, 5 µ M, 12.5 µ M, 25 µ M, 50 µ M), vortex to mix and pre-incubate at 37°C for 10 minutes, add 1 µ L substrate FD (1.5) µM (final concentration), incubated at 37°C for 30 minutes, then inactivated with an equal volume of ice-cold acetonitrile. Three parallel lines were set up. Three control groups were also set up: one group without alkaloids (with an equal volume of dimethyl sulfoxide) as a 100% control for enzyme activity; the second group without enzyme (with an equal volume of PBS) used for measuring background fluorescence; and the third group was a positive control group (loperamide hydrochloride LPA). The 96-well plates were placed in a microplate reader (Varioskan Flash type, Thermo Fisher Scientific, USA) for fluorescence analysis under the following conditions: Ex 480 nm / Em 525 nm. Alkaloids I-(+)-1~I-3 and substrate FD were dissolved in dimethyl sulfoxide, and the final proportion of dimethyl sulfoxide in the system should ideally be less than 1% (V / V). The remaining enzyme activity (%) was calculated as follows: fluorescein intensity in the presence of alkaloids / negative control (without alkaloids). 100.

[0037] 2. Kinetic analysis and determination of optimal substrate concentration Assuming the total volume of the reaction system remains unchanged, the final HLM concentration is 1. μ The substrate concentration was set at g / mL, and the incubation time was 30 min. A series of different concentration gradients were established, namely 1.25 g / mL. μ M, 2.5 μ M, 5.0 μ M, 10.0 μ M, 20.0 μ M was used to obtain the corresponding reaction rates for different substrate concentrations. Three parallel experiments were conducted. The Michaelis-Menten equation curves were obtained using GraphPadPrism 8.0 software through nonlinear fitting to determine the values ​​of Km and Vmax.

[0038] Following the above steps, the activity of the obtained alkaloids I-(+)-1~I-3 in inhibiting human carboxylesterase 2 was tested, and the results are shown in Table 2.

[0039] Table 2. Inhibition of human carboxylesterase 2 activity by alkaloids I-1~I-3

[0040] (2) Computer simulation study on the interaction between alkaloids I-1~I-3 and the active site of hCE2 protein Instruments: Computer, Autodock molecular docking software Experimental steps: The interaction between alkaloids and the active site of hCE2 protein was studied using a computer-simulated molecular docking method. First, alkaloids I-(+)-1 to I-3 were optimized and their conformations searched to obtain different conformations. hCE2 crystal data (PDB: 6QP9) from the PDB database were selected and subjected to dehydration and hydrogenation treatment. The active site region was constructed using an inhibitor co-crystallized with the alkaloids as a template. The docking tool in Autodock molecular docking software was used to virtually dock the different conformations of each alkaloid and the substrate diacetylfluorescein (FD) with the active site region, ultimately obtaining the optimal docking mode. The molecular docking of each alkaloid and substrate FD with the target protein is shown in the figure below. Figure 7 As shown: Figure 7 a is a schematic diagram illustrating the interaction between the substrate diacetylfluorescein (FD) and the hCE2 protein. Figure 7 b is a schematic diagram of the interaction between alkaloid I-(+)-1 and hCE2 protein; Figure 7 c is a schematic diagram of the interaction between alkaloid I-(–)-1 and hCE2 protein; Figure 7 d is a schematic diagram of the interaction between alkaloid I-2 and hCE2 protein; Figure 7 e is a schematic diagram of the interaction between alkaloid I-3 and hCE2 protein.

[0041] Alkaloids I-(+)-1~I-3 can interact with key amino acid residues in the active site of hCE2 protein through multiple hydrogen bonds, indicating that alkaloids I-(+)-1~I-3 act as effective competitive inhibitors of hCE2-mediated FD hydrolysis by competing with the substrate FD for binding to the active site of hCE2.

[0042] The alkaloids or alkaloid salts (e.g., hydrochloride, acetate) of the present invention, prepared by conventional techniques with pharmaceutically acceptable carriers and excipients, into formulations suitable for oral or injectable applications, such as tablets, capsules, powders, syrups, injections, etc. These formulations exhibit human carboxylesterase 2 inhibitory activity.

[0043] Currently, the mechanisms of action of discovered alkaloids (such as Rocaglamide) are similar to those of irinotecan in reducing toxicity. Based on cutting-edge research on Rocaglamide and other alkaloids, the application of alkaloids in anticancer drugs such as irinotecan is gradually shifting towards a new direction of "selective protectants." This approach utilizes specific alkaloids (such as Rocaglamide) to precisely intervene in key cellular pathways, reducing dose-limiting toxicities such as myelosuppression after chemotherapy-induced DNA damage. This upstream intervention, unlike traditional antidiarrheal drugs that only relieve diarrhea symptoms, aims to broaden the therapeutic window of chemotherapy drugs from the root cause. Although this strategy is currently mostly in the preclinical research stage, it has indeed verified the scientific feasibility of alkaloids as "chemotherapy protectors" to reduce side effects and improve treatment efficacy, representing an important potential pathway for the future development of highly effective and low-toxicity combination therapies.

[0044] The alkaloids or alkaloid salts (e.g., hydrochloride, acetate) of the present invention, prepared using conventional techniques with pharmaceutically acceptable carriers and excipients, into formulations suitable for oral or injectable applications, such as tablets, capsules, powders, syrups, injections, etc. They can be used to prepare attenuated and protective agents for anticancer chemotherapy drugs such as irinotecan.

[0045] The above embodiments only describe a portion of the specific implementation methods of the present invention in detail, and are not limited to the embodiments disclosed herein. Furthermore, the substantive content protected by the present invention is not limited thereto. Any other modifications, equivalent substitutions, improvements, etc., made based on the principles and techniques of the present invention without departing from its design scope are all within the protection scope of the present invention.

Claims

1. An alkaloid compound, characterized in that, The compounds are I-(+)-1, I-(–)-1, I-2, and I-3, with the specific structural formulas shown below: 。 2. The method for preparing the alkaloid compound according to claim 1, characterized in that, The method includes the following steps: 1) Red peony root was soaked in distilled water and then extracted by ultrasound. The resulting residue was soaked in 95% ethanol and then extracted by ultrasound. The 95% ethanol extract was concentrated to an alcohol-free concentrate, extracted with ethyl acetate, and concentrated with ethyl acetate to obtain ethyl acetate extract CS-5. 2) The CS-5 was separated by silica gel column chromatography, and the eluent was successively eluted with a gradient of petroleum ether-acetone mixed solvent, and then eluted with a dichloromethane-methanol mixed solvent. The solvent was recovered under reduced pressure to obtain 6 eluent fractions, which were named AF respectively. 3) D was separated by silica gel column chromatography, eluted with a gradient of dichloromethane-methanol mixed solvent, and the solvent was recovered under reduced pressure to obtain five eluent fractions, named D1-D5; D5 was subjected to Sephadex LH-20 column chromatography, eluted with a dichloromethane-methanol mixed solvent, and the solvent was recovered under reduced pressure to obtain one eluent fraction, named D5-1; D5-1 was separated by semi-preparative high performance liquid chromatography to obtain compound I-2; 4) F was subjected to gel Sephadex LH-20 column chromatography, eluted with pure methanol, and the solvent was recovered under reduced pressure to obtain 6 eluent fractions, named F1-F6 respectively; F3 was separated by semi-preparative HPLC to obtain compounds I-1 and I-3; 5) Compound I-1 was further separated by high performance liquid chromatography chiral separation column to obtain enantiomers I-(+)-1 and I-(–)-1.

3. The method for preparing the alkaloid compound according to claim 2, characterized in that, In step 1), the volume of distilled water used is 8 times the weight of the medicinal material, the volume of 95% ethanol used is 8 times the weight of the residue, the ultrasonic extraction frequency is 40kHz, the extraction time is 1 hour, and the extraction is performed 3 times; the volume ratio of ethyl acetate to concentrate is 1:1, and the extraction is performed 5 times.

4. The method for preparing the alkaloid compound according to claim 2, characterized in that, In step 2), the silica gel column is 100-200 mesh, the volume ratio of the petroleum ether-acetone mixed solvent is 50:1, 30:1, 20:1, 10:1, and 8:1 respectively, the volume ratio of the dichloromethane-methanol mixed solvent is 20:1, and each eluent elutes five times the column volume.

5. The method for preparing the alkaloid compound according to claim 2, characterized in that, In step 3), the silica gel column is 100-200 mesh, the volume ratio of the first dichloromethane-methanol mixed solvent is 50:1, 30:1, 10:1, 1:1, and 1:10, and each eluent elutes five times the column volume; the volume ratio of the second dichloromethane-methanol mixed solvent is 1:1, and elution is performed on two times the column volume; the semi-preparative high-performance liquid chromatography conditions are: using a C18 semi-preparative column, a detection wavelength of 210 nm, and a mobile phase of acetonitrile-water with a volume ratio of 30:

70.

6. The method for preparing the alkaloid compound according to claim 2, characterized in that, In step 4), the pure methanol elution is performed at five column volumes. The semi-preparative HPLC conditions are as follows: a C18 semi-preparative column is used, the detection wavelength is 210 nm, the mobile phase is acetonitrile-water (volume ratio 20:80), and the column temperature is 32 °C. In step 5), the high-performance liquid chromatography chiral separation column is Chiral MJ2, the detection wavelength is 210 nm, the mobile phase is n-hexane-isopropanol (volume ratio 88:12), and the column temperature is 32 °C.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises at least one of the alkaloid compounds of claim 1 and its pharmaceutically acceptable salt or pharmaceutically acceptable carrier or excipient.

8. Use of the pharmaceutical composition of claim 7 in the preparation of a human carboxylesterase 2 inhibitor.

9. The use of the pharmaceutical composition of claim 7 in the preparation of a toxicity-reducing and protective agent for anticancer chemotherapy drugs.

10. The use of the pharmaceutical composition according to claim 9 in the preparation of a toxicity-reducing and protective agent for anticancer chemotherapy drugs, characterized in that, The anticancer chemotherapy drug mentioned is irinotecan.

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