Application of gemmarane type sesquiterpene lactone compound in preparation of antitumor drugs

Abstract

The invention provides an application of a gemmarane type sesquiterpene lactone compound in preparation of an antitumor drug. The gemmarane type sesquiterpene lactone compound disclosed by the invention has a relatively strong inhibition effect on proliferation of tumor cells, especially leukemia cells, and is more particularly most sensitive to human erythroleukocyte leukemia cells (HEL).

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of a gemmae-type sesquiterpene lactone compound in the preparation of antitumor (especially leukemia) drugs. Background Technology

[0002] Leukemia, commonly known as blood cancer, is a malignant blood disease caused by abnormal hematopoietic stem cell renewal, differentiation disorders, and inhibited apoptosis. Currently, drugs targeting leukemia stem cells have achieved some success, but their toxicity and severe adverse reactions in patients limit their use. Therefore, discovering innovative drugs that can effectively treat leukemia is imperative, but how to discover such novel drugs is a pressing scientific question that needs to be explored.

[0003] Tobacco tube grass ( Carpesium cernuum L. *Tobacco japonica* is a perennial herb belonging to the genus *Tobacco* in the family Asteraceae. In modern clinical practice, it is used to treat colds with fever, high fever with convulsions, sore throat, boils, carbuncles, mastitis, etc. Its main chemical components include sesquiterpenes, flavonoids, phenols, steroids, lignans, triterpenes, and coumarins. In the Miao ethnic minority region of Guizhou, China, it is used as a traditional medicine, specifically wild tobacco leaves, and is the principal ingredient in the traditional Chinese medicine formula *Shuangyang Houbi Tong Granules*, primarily for its antibacterial and anti-inflammatory properties. However, there are currently no reports of *Tobacco japonica* or its related compounds being used to treat tumors, particularly leukemia. Summary of the Invention

[0004] The inventors have discovered that a gemmaline-type sesquiterpene lactone compound isolated from *Hemiberlesia lataniae* exhibits a strong inhibitory effect on the proliferation of tumor cells, particularly leukemia cells, and is especially sensitive to human erythroleukemia (HEL) cells. Further in-depth research revealed that the gemmaline-type sesquiterpene lactone compound of this invention (e.g., TMJ-21) can selectively inhibit HEL proliferation in vitro and reduce splenomegaly in leukemia model mice in vivo, restoring normal growth in these mice and demonstrating a highly effective anti-leukemic effect.

[0005] Therefore, on the one hand, the present invention provides the use of gemmacone-type sesquiterpene lactone compounds and their analogues or compositions comprising them in the preparation of antitumor drugs, wherein the compounds are selected from: .

[0006] In this invention, the molecular formula of TMJ-21 is C 25 H 36O9, chemically named (3aR,4S,5R,6S,8S,10R,11S,11aR)-5,10-dihydroxy-6,10-dimethyl-11-(((S)-2-methylbutanoyl)oxy)-3-methylene-2-oxododecahydro-5,8-epoxycyclodeca[b]furan-4-yl (Z)-2-methylbut-2-enoate). Its Chinese name is (3aR,4S,5R,6S,8S,10R,11S,11aR)-5,10-dihydroxy-6,10-dimethyl-11-(((S)-2-methylbutanoyl)oxy)-3-methylene-2-oxododecylhydro-5,8-epoxycyclodeca[b]furan-4-yl(Z)-2-methylbut-2-enoate).

[0007] In some implementations, analogues of TMJ-21 may have the following structure: .

[0008] In some embodiments, the pharmaceutical composition may comprise the aforementioned gemmacone-type sesquiterpene lactone compound and pharmaceutically acceptable excipients or carriers.

[0009] In some embodiments, the tumor in this invention may include solid tumors and hematologic malignancies, examples of which include, but are not limited to, colorectal cancer (such as colon cancer, colorectal cancer), lung cancer (such as non-small cell lung cancer), breast cancer, pancreatic cancer, liver cancer, leukemia, etc.

[0010] In some embodiments, in this invention, the tumor is leukemia.

[0011] In this invention, leukemia can refer to malignant hematological diseases caused by mechanisms such as abnormal hematopoietic stem cell renewal, differentiation disorders, and inhibited apoptosis, such as acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia. Examples include, but are not limited to, human T-lymphoblastic leukemia, human erythroleukocyte leukemia, and human chronic myelogenous leukemia.

[0012] In some embodiments, in this invention, the above-mentioned gemmaeane-type sesquiterpene lactone compounds have one or more of the following effects: (1) Selectively inhibits the proliferation of human erythroleukemia cells (HEL) in vitro; (2) Reduce splenomegaly in leukemia patients in vivo; (3) To restore the normal growth of leukemia patients.

[0013] In some embodiments, the antitumor drug in this invention is an oral or injectable form.

[0014] In some embodiments, in this invention, the oral preparation is selected from any one or more of granules, capsules, tablets, powders, pellets, and sustained-release preparations.

[0015] Compared with the prior art, the beneficial technical effects of the present invention are reflected in: This invention provides compounds derived from natural products that have significant safety and can improve organ damage caused by leukemia; and can achieve significant therapeutic effects on leukemia through novel anti-leukemia mechanisms targeting novel targets. Attached Figure Description

[0016] Figure 1 The results show the effects of TMJ-21 on a mouse model of leukemia. A: Timeline of the experiment; B: Spleen photographs (left) and spleen coefficients (right) of each group of mice; C: Changes in body weight of mice in each group during drug administration; D: Liver coefficients of each group of mice; E: Kidney coefficients of each group of mice. p <0.05, ** p <0.01, compared with the model group; # p <0.05, ## p <0.01, compared with the normal control group.

[0017] Figure 2 The changes in ALT (A) and AST (B) after TMJ-21 was administered at 20 times the effective dose are shown.

[0018] Figure 3 The proteomic identification results of TMJ-21's anti-leukemia effect are shown. A: Fluorescence scans with and without photocrosslinking (+UV) and without photocrosslinking (-UV); B: VENN diagrams of +UV, -UV, and leukemia target sites in the proteomic identification results; C: Proteins labeled by TMJ-21P in HEL cells in a concentration-dependent manner; D: Competition between protein labeling and TMJ-21 in HEL cells; E: Pathway enrichment map of the top 40 proteins shared by +UV and -UV; F: Results of the cell thermal shift assay of TMJ-21 with HSP90AA1, HSPA5, and HSPH1; G: Semi-quantitative diagram of the cell thermal shift assay of TMJ-21 with HSPA5. * p <0.05, ** p <0.01, compared with the DMSO group. Detailed Implementation

[0019] The present invention will be described in detail below by way of examples. However, the examples provided herein are for illustrative purposes only and are not intended to limit the invention.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0021] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0022] The main instruments used in this study include a Multiskan FC microplate reader (Thermo Scientific, USA); a P50002F high-speed refrigerated centrifuge (Eppendorf, Germany); a BS223S electronic balance (Sartorius Instruments Co., Ltd., Beijing, China); a KQ5200E ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd., China); a ZYCGF-II-10T ultrapure water system (Sichuan Zhuoyue Water Treatment Equipment Co., Ltd.); a TGL16M benchtop high-speed centrifuge; a JXFSTPRP CL cryogenic grinder (Shanghai Jingxin Industrial Development Co., Ltd.); and a DYY-6C electrophoresis apparatus (Beijing Liuyi Instrument Factory). Retrovirus expressing F-MuLV clone 57 vector in NIH3T3 cells (Liu X, Wang B, Mou Y, Sun Y, Rao Q, Song J, Huang Y, Wen M, Huang L, Li Y. Mechanism of ShuangyangHoubitong granules against acute erythroleukemia through PI3K / AKT and JAK / STAT signaling pathways. J Ethnopharmacol. 2025 Jun 26;350:119979. doi:10.1016 / j.jep.2025.119979. Epub 2025 May 15. PMID: 40381820.) was a gift from the collaborating professor; the ALT and AST kits were purchased from Nanjing Jiancheng Biotechnology Institute, batch numbers 20250311 and 20250307 respectively; the 90kDa heat shock protein AA1 (HSP90AA1), 105kDa / 110kDa heat shock protein 1 (HSPH1), and 70kDa heat shock protein 5 (HSPA5) antibodies were purchased from Huaan Biotechnology Co., Ltd., batch numbers NBP1-05054, ET1612-88, and ER1706-50 respectively; the GAPDH antibody was purchased from Affinity Pharmaceuticals, USA, batch number T0004; high-efficiency RIPA lysis buffer, BCA protein concentration assay kit, SDS-PAGE gel preparation kit, Rainbow 180 broad-spectrum protein marker, and HRP Substrate Peroxide were also included. Solution's hypersensitive luminescent liquid was purchased from Beijing Solarbio Technology Co., Ltd., with batch numbers R0010, PC0020, P1200, PR1910, and PE0010 respectively; the polyvinylidene fluoride (PVDF) membrane was purchased from Merck GmbH, Germany, with batch number P2938; all reagents were of analytical grade or standard laboratory specifications, and the water was ultrapure water.

[0023] For experimental needs, neonatal mice were purchased for experiments. BALB / c mice (Changsha Tianqin Biotechnology Co., Ltd., production license number SCXK(Xiang) 2024-0021) were paired and caged. Their body weight was 18-22 g, and they were raised in a well-ventilated environment with a temperature of 18-25 °C and a relative humidity of 40%-70%, with a 12 h / 12 h light-dark cycle. The relevant animal experiments passed the review of the Experimental Animal Ethics Committee of Guizhou Medical University (approval number 2000069).

[0024] Example 1: Preparation of TMJ-21 and its analogs TMJ-21 and its analogs were isolated from our laboratory according to the following references.

[0025] The references are as follows: 1. TMJ-8, TMJ-9, TMJ-10: Yan C, Long Q, Zhang YD, et al. Germacranolidesesquiterpenes from Carpesium cernuum and their anti-leukemia activity[J]. ChinJ Nat Medicines, 2021, 19(7): 528-535. 2. TMJ-12: Zhang T, Si JG, Zhang QB, et al. New highly oxygenatedgermacranolides from Carpesium divaricatum and their cytotoxic activity[J]. SciRep, 2016, 6(1): 1-8. 3. TMJ-21: Baruah RN, Sharma RP, Thyagarajan G, et al. Unusualgermacranolides from Inula eupatorioides [J]. J Org Chem, 1980, 45(24): 4838-4843. 4. TMJ-28: Huang YS, Mao JX, Zhang L, et al. Antiprostate canceractivity of ineupatolide isolated from Carpesium cernuum L.[J]. Biomed Res Int,2021: 1-9. 5.TMJ-38:Yan C, Zhang WQ, Sun M, et al. Carpescernolides A and B,rare oxygen bridge-containing sesquiterpene lactones from Carpesium cernuum [J]. Tetrahedron Lett, 2018, 59(46): 4063-4066. 6.TMJ-18:Cheng XR, Zhang SD, Wang CH, et al. Bioactive eudesmane andgermacrane derivatives from Inula wissmanniana Hand. Mazz[J]. Phytochemistry,2013, 96: 214-222. 7.TMJ-55:Kim DK, Lee KR, Zee OP. Sesquiterpene lactones from Carp spread apart [J]. Phytochemistry, 1997, 47(7): 1245-1247. 8.TMJ-60:Chung IM, Moon HI. Antiplasmodial activities ofsesquiterpene lactone from Carpesium cernuum [J]. J Enzyme Inhib Med Chem,2009, 24(1): 131-135. 9.TMJ-102:Kim DK, Baek NI, Choi SU, et al. Four new cytotoxicgermacranolides from Carpesium divaricatum [J]. J Nat Prod, 1997, 60(11): 1199-1202. 10.TMJ-104:Liu QX, Yang YX, Zhang JP, et al. Isolation, structureelucidation, and absolute configuration of highly oxygenated germacranolidesfrom Carpesium cernuum[J]. J Nat Prod, 2016, 79(10): 2479-2486. 11.TMJ-69:Moon HI, Zee O. Antiproliferative effect from sesquiterpenelactones of Carpaceae roseata MlQ consumed in south korea on the five humancancer cell lines[J]. Rec Nat Prod, 2010, 4(3): 149-155. Example 2: Effects of TMJ-21 on a mouse model of leukemia Establishment of a mouse model of leukemia and experimental procedures: Mice were used to induce a leukemia mouse model by intraperitoneal injection (ip) of F-MuLV retrovirus within 48 hours of birth, or by intraperitoneal injection of physiological saline as a normal control group. After injection, mice were continued to be fed for 4 weeks. In week 4, mice were weaned, and the mice injected with physiological saline served as the normal control group. The mice injected with the virus were randomly divided into a model group, a low-dose TMJ-21 group (1.0 mg / kg), and a high-dose TMJ-21 group (2.0 mg / kg), with 6 mice in each group. After grouping, mice were continued to be fed for 1 week. Mice in the treatment groups were injected intraperitoneally with TMJ-21 (1.0 mg / kg and 2.0 mg / kg) at 9:00 AM daily for 3 consecutive weeks. Mice in the other groups received an equal volume of physiological saline. Before the last administration, mice were fasted for 24 hours but allowed free access to water. One hour after the last administration, mice were anesthetized by intraperitoneal injection of sodium pentobarbital. Blood was collected from the orbital sinus, allowed to stand at room temperature for 2 hours, centrifuged at 3500 r / min for 15 min, and the serum was collected and stored at -20 ℃ for later use. After blood collection, mice were euthanized by cervical dislocation. The liver, kidneys, and spleen were dissected and weighed. The organ index was calculated (organ weight (g) / body weight (g) × 100%). Then, part of the spleen tissue was fixed in 4% paraformaldehyde for later use, and another part of the spleen tissue was stored in a -80℃ refrigerator for later use.

[0026] Experimental results are as follows Figure 1 As shown, compared with the model group, the spleen coefficient in the TMJ-21 treatment group (1.0 mg / kg) was significantly reduced (p < 0.01), and this showed a dose-dependent effect. Figure 1 (B) The liver coefficient in the model group was slightly increased, but the difference was not statistically significant. Figure 1 In the middle D group, there was no significant change in the kidney coefficient among the mice. Figure 1(E). Mice in the model group exhibited depression (reduced activity, judged as depression), piloerection (judged by appearance), arched back posture (judged by appearance), and significant weight loss, while mice treated with TMJ-21 maintained stable weight and good activity levels. Figure 1 (C)

[0027] Example 3: TMJ-21 has no hepatotoxicity Serum ALT and AST were measured 24 hours after intraperitoneal injection of 20 times the dose of TMJ-21 (20 mg / kg) into BALB / c mice. The results are as follows: Figure 2 As shown, the serum ALT and AST levels in TMJ-21-treated mice were similar to those in the solvent control group, with no statistically significant difference, suggesting that TMJ-21 is not toxic to the liver.

[0028] Figure 2 The changes in serum ALT and AST were shown in BALB / c mice 24 hours after administration of TMJ-21 (20 mg / kg).

[0029] Example 4: TMJ-21 has a selective inhibitory effect on the growth of leukemia cells. Human colon cancer cells (SW480), human non-small cell lung cancer cells (A549), human breast cancer cells (MDA-MB-231), human colorectal adenocarcinoma cells (HCT116), human pancreatic cancer cells (Panc-1), human liver cancer cells (HepG2), human orthotopic pancreatic adenocarcinoma cells (Bxpc-3), human breast cancer cells (MCF-7), human liver cancer cells (HuH7), human T-lymphoblastic leukemia cells (Jurkat), human erythroleukemia cells (HEL), and human chronic myeloid leukemia cells (K562) were purchased from ATCC (Manassas, VA, USA). Primary hepatocytes and primary leukemia cells were extracted in our laboratory. Cells were cultured at 37°C in a 5% CO2 incubator in RPMI 1640 medium (Gibco, USA) or DMEM medium (Gibco, USA) containing 5% fetal bovine serum. Once the cells reached the logarithmic growth phase, they were cultured at 6-8 × 10⁶ cells / year. 3 Cells were seeded at a density of 100 cells / well in 96-well plates and cultured for 24 h. Cells were then treated with different concentrations of TMJ-21 (1-100 μM), with 0.1% DMSO as a control. 24 h after treatment, 10 μL of CCK8 was added to the cells and incubation continued for 4 h. Absorbance (OD) values ​​were measured at 450 nm using a microplate reader. The inhibition rate was calculated using the following equation: Inhibition rate = (Control OD - Treated OD) / Control OD × 100%.

[0030] Table 1. Toxicity of TMJ-21 against solid tumors and leukemia cells ( (n=6)

[0031] The results are shown in Table 1. TMJ-21 showed IC50 activity against various solid tumor cells. 50 The effective concentration range was 23.8-96.5 μM. Among them, TMJ-21 showed the strongest inhibitory effect on HEL cell proliferation, with an IC50 concentration of 23.8-96.5 μM. 50 The concentration was 5.28 μM, indicating its selectivity for HEL cells. Furthermore, TMJ-21 showed IC50 activity against primary hepatocytes. 50 The concentration was 96.2 μM, and the IC50 value for primary leukemia cells was [missing value]. 50 The concentration ranged from 8.62 to 11.7 μM, indicating that TMJ-21 has a relatively safe therapeutic window and inhibits the growth of primary leukemia cells.

[0032] Example 5: TMJ-21 and its analogues have a selective inhibitory effect on HEL cell growth. Thirteen tests were conducted on the effects of TMJ-21 and similar compounds on the proliferation of HEL, K562, and Jurkat cells. Cell culture and manipulation were the same as in Example 4.

[0033]

[0034] Table 2. Toxicity of TMJ-type compounds to leukemia cells ( (n=6)

[0035] The results are shown in Table 2. TMJ-21 and its similar compounds have an effect on HEL cell IC50. 50 The effective concentration range is 5.28-111.8 μM, and the IC50 value for K562 cells is [missing information]. 50 The effective range is 5.01-95.4 μM, and the IC50 concentration for Jurkat cells is [missing information]. 50 The effective concentration range was 10.13-134.6 μM. Among them, TMJ-21 and TMJ-69 showed strong inhibitory effects on HEL cell proliferation, with an IC50 concentration of 10.13-134.6 μM. 50 The concentrations were 5.28 and 5.10 μM, respectively. Because TMJ-21 has a high content in *Hedysarum heterotropoides*, subsequent experiments selected TMJ-21 to explore its mechanism of action against leukemia.

[0036] Example 6: Protein Spectroscopy Identification Results Design and synthesize TMJ-21-P for target protein fishing. The design of the TMJ-21 probe must meet several prerequisites: First, the ideal synthetic strategy is to introduce modifications at a modifiable site. Second, derivatization at this site should not affect the activity of TMJ-21. Finally, the corresponding probe should be separable from any bulky markers that would affect its interaction with the target protein during protein labeling. Based on this, we selected a bifunctional probe, TMJ-21-P (TMJ-21-Probe), which binds to both a photocrosslinker and a terminal alkyne. In this structure, the compact alkyldiazeline acts as the photoreactive functional group, generating a highly reactive carbene intermediate under UV irradiation and rapidly forming a covalent bond with the target protein. The terminal alkyne is modified with a labeled biotinylate via a click chemistry reaction, causing the target protein to be pulled down and identified using proteomics methods.

[0037]

[0038] The results are as follows Figure 3 As shown, comparing ultraviolet (UV) irradiation (+UV) and no UV irradiation (-UV), 25 proteins were common to both groups. Intersecting these 25 proteins with leukemia targets revealed 7 common targets: HSP90AA1, HSPA5, HSPH1, HRNR, FLNA, CLTC, and JUP. Figure 3 (A / B). Next, the ability of TMJ-21P to label proteins was investigated, and the results showed that multiple proteins were labeled by TMJ-21P in a concentration-dependent manner. Figure 3 (C), and subsequently TMJ-21P (25 μM) was selected for a competitive experiment. The results are as follows. Figure 3 As shown in Figure D, co-incubation with an excess of TMJ-21 can specifically compete for its target protein, indicating that TMJ-21P and TMJ-21 exert their pharmacological effects on the same target protein. Pathway enrichment analysis showed that the Proteinprocessing in endoplasmic reticulum pathway was most significantly enriched, involving 11 targets in total. Figure 3 In the VENN diagram, among the 11 targets, HSP90AA1, HSPA5, and HSPH1 are consistent with 7 targets. Cellular thermal shift analysis of these 3 candidate targets showed that... Figure 3 Among the F / G ratios, TMJ-21 exhibits the strongest thermostability against the HSPA5 protein, suggesting that HSPA5 is a potential target for TMJ-21 to exert its therapeutic effect.

[0039] It should be noted that all data were statistically analyzed using SPSS 19.0 software, and the data are presented in [data format]. This indicates that one-way ANOVA was used for comparisons among multiple groups, and LSD-t test was used for pairwise comparisons between groups. The significance level was set at α=0.05.

[0040] The above activity test results show that TMJ-21 can not only selectively inhibit the proliferation of human erythroleukemia cells (HEL) in vitro, but also reduce splenomegaly in leukemia model mice in vivo and restore normal growth, demonstrating a very effective anti-leukemic effect. More importantly, the direct target of TMJ-21's anti-leukemic effect is heat shock protein 5 (HSPA5). In conclusion, TMJ-21 and its analogues have good potential for application in tumor treatment, especially leukemia. The direct target of TMJ-21's anti-leukemic effect is HSPA5, providing a monomeric drug, TMJ-21, isolated from the natural plant *Hypericum perforatum*, for the treatment of leukemia.

[0041] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. The use of a gemmacone-type sesquiterpene lactone compound and its analogues, or a composition comprising the same, in the preparation of an antitumor drug, wherein the compound is selected from: 。 2. The application according to claim 1, wherein, The pharmaceutical composition further comprises pharmaceutically acceptable excipients or carriers.

3. The application according to claim 1, wherein, The tumors include solid tumors and hematologic malignancies.

4. The application according to claim 3, wherein, The tumors include colorectal cancer (such as colon cancer, colorectal cancer), lung cancer (such as non-small cell lung cancer), breast cancer, pancreatic cancer, liver cancer, and leukemia.

5. The application according to claim 4, wherein, The tumor is leukemia.

6. The application according to claim 5, wherein, The leukemias mentioned include acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia.

7. The application according to claim 5, wherein, The leukemias mentioned include human T-lymphoblastic leukemia, human erythroleukocyte leukemia, and human chronic myelogenous leukemia.

8. The application according to any one of claims 5 to 7, wherein, The gemmae-type sesquiterpene lactone compound has one or more of the following effects: (1) Selectively inhibits the proliferation of human erythroleukemia cells in vitro; (2) Reduce splenomegaly in leukemia patients in vivo; (3) To restore the normal growth of leukemia patients.

9. The application according to any one of claims 1-8, wherein, The antitumor drug is an oral or injectable form.

10. The application according to claim 9, wherein, The oral preparation is selected from any one or more of granules, capsules, tablets, powders, pellets, and sustained-release preparations.

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