Discovery and synthesis of 3-amino glycyrrhizin derivative containing guanidyl with anti-colorectal cancer activity

Abstract

Colorectal cancer is considered to be a highly immune-related disease, and the severe chronic inflammatory stress in the tumor microenvironment (TME) greatly impedes the effective prognosis and treatment of colorectal cancer. Based on a dual strategy, anti-proliferation and inhibition of inflammatory factor HMGB1, a series of GN-derivatives were synthesized and screened for their anticancer efficacy, and compound 22 showed the strongest anti-proliferative activity against HCT116 cells, with IC₅₀ value 13.1 μmol·L⁻¹. To improve the solubility, compounds 23−40 were synthesized and then evaluated for activities against colon cancer cells. Finally, compounds 23 and 24 with good water solubility were screened as target molecules. In anti-proliferation tests, both displayed the strongest activity on CT26 cells, with IC₅₀ value 1.1 and 1.4 μmol·L⁻¹, respectively; in the cloning tests, they also showed a strong anti-proliferative activity against CT26 cells; moreover, they promoted apoptosis, and blocked cells in G₂/M phase in a concentration-dependent manner; in Western Blot tests, they decreased the levels of cyclin A2, cyclin B1 and CDK1 in a concentration-dependent manner. In anti-inflammation aspect, both them displayed strong anti-inflammation activity; compound 23 reduced the levels of NO in both RAW264.7 cells and CT26 cells in the anti-inflammatory tests, and the IC₅₀ values were 10.3 and 1.1 µmol·L⁻¹, respectively; Western Blot showed 23 decreased the levels of HMGB1, RAGE, TNF-α, IL-6, IL-1 and COX-2 in a dose-dependent manner. In animal tests, compound 23 reduced the volume and weight of tumor more obviously than compound 24 and 5-FU, and its effect was a dose-dependent. In summary, compound 23 has therapeutic effect for the treatment of colorectal cancer in some degree, and it has potential to be a candidate for treatment of colorectal cancer.

Keywords

GN-derivatives / HMGB1 / Anti-cancer / Anti-inflammation

Declaration of competing interest

Supplementary materials can be requested by sending E-mail to the corresponding authors.

Supplementary materials

These authors have no conflict of interest to declare.

References

[1] Walle GT, Kitaw TA, Adane S. Incidence and determinants of mortality among patients with colorectal cancer in oncology centers of Amhara region, Ethiopia, 2024: multicenter retrospective follow up study. BMC Cancer. 2025; 25(1):102. https://doi.org/10.1186/s12885-025-13462-.

[2] Lourenço FC, Sadien ID, Wong K, et al. Decay of driver mutations shapes the landscape of intestinal transformation. Nature. 2026; 649(8097):729-738. https://doi.org/10.1038/s41586-025-09762-.

[3] Sung HYA, Siegel RL, Laversanne M, et al. Colorectal cancer incidence trends in younger versus older adults: an analysis of population-based cancer registry data. Lancet Oncol. 2025; 26(1):51-63. https://doi.org/10.1016/S1470-2045(24)00600-.

[4] Dekker E, Tanis PJ, Vleugels JLA, et al. Colorectal cancer. Lancet. 2019; 394(10207):1467-1480. https://doi.org/10.1016/S0140-6736(19)32319-.

[5] Gray R, Hills R, Stowe R, et al. Adjuvant radiotherapy for rectal cancer: a systematic overview of 8507 patients from 22 randomised trials. Lancet. 2001; 358(9290):1291-1304. https://doi.org/10.1016/S0140-6736(01)06409-.

[6] Chai Y, Liu JL, Zhang S, et al. The effective combination therapies with irinotecan for colorectal cancer. Front Pharmacol. 2024; 15:1356708. https://doi.org/10.3389/fphar.2024.1356708.

[7] Barriuso J, Nagaraju RT, Belgamwar S, et al. Early adaptation of colorectal cancer cells to the peritoneal cavity is associated with activation of "sternness" programs and local inflammation. Clin Cancer Res. 2021; 27(4):1119-1130. https://doi.org/10.1158/1078-0432.CCR-20-3320.

[8] Xia W, Geng YT, Hu WW. Peritoneal metastasis: a dilemma and challenge in the treatment of metastatic colorectal cancer. Cancers. 2023; 15(23):15235641. https://doi.org/10.3390/cancers15235641.

[9] Liu WX, Kuang TR, Liu L, et al. The role of innate immune cells in the colorectal cancer tumor microenvironment and advances in anti-tumor therapy research. Front Immunol. 2024; 15:1407449. https://doi.org/10.3389/fimmu.2024.1407449.

[10] Ganesh K, Stadler ZK, Cercek A, et al. Immunotherapy in colorectal cancer: rationale, challenges and potential. Nat Rev Gastroenterol Hepatol. 2019; 16(6):361-375. https://doi.org/10.1038/s41575-019-0126-.

[11] Schmitt M, Greten FR. The inflammatory pathogenesis of colorectal cancer. Nat Rev Immunol. 2021; 21(10):653-667. https://doi.org/10.1038/s41577-021-00534-.

[12] Wang H, Tian T, Zhang JH. Tumor-associated macrophages (TAMs) in colorectal cancer (CRC): from mechanism to therapy and prognosis. Int J Mol Sci. 2021; 22(16):8470. https://doi.org/10.3390/ijms22168470.

[13] Shah SC, Itzkowitz SH. Colorectal cancer in inflammatory bowel disease: mechanisms and management. Gastroenterology. 2022; 162(3):715-730.e713. https://doi.org/10.1053/j.gastro.2021.10.035.

[14] Wang SM, Zhang Y. HMGB1 in inflammation and cancer. J Hematol Oncol. 2020; 13(1):116. https://doi.org/10.1186/s13045-020-00950-.

[15] Schiraldi M, Raucci A, Muñoz LM, et al. HMGB1 promotes recruitment of inflammatory cells to damaged tissues by forming a complex with CXCL12 and signaling via CXCR4. J Exp Med. 2012; 209(3):551-563. https://doi.org/10.1084/jem.20111739.

[16] Chen RC, Kang R, Tang DL. The mechanism of HMGB1 secretion and release. Exp Mol Med. 2022; 54(2):91-102. https://doi.org/10.1038/s12276-022-00736-.

[17] Yang H, Antoine DJ, Andersson U, et al. The many faces of HMGB1: molecular structure-functional activity in inflammation, apoptosis, and chemotaxis. J Leukoc Biol. 2013; 93(6):865-873. https://doi.org/10.1189/jlb.1212662.

[18] Wang YY, Yu ZM, Yuan H, et al. Structure-based design of glycyrrhetinic acid derivatives as potent anti-sepsis agents targeting high-mobility group box-1. Bioorg Chem. 2021; 106:104461. https://doi.org/10.1016/j.bioorg.2020.104461.

[19] Qiang X, Peng YJ, Wang ZY, et al. Synthesis of glycyrrhizin analogues as HMGB1 inhibitors and their activity against sepsis in acute kidney injury. Eur J Med Chem. 2023; 259:115696. https://doi.org/10.1016/j.ejmech.2023.115696.

[20] Zhang QP, Wang YN, Wang ZY, et al. Synthesis and anti-inflammatory activities of glycyrrhetinic acid derivatives containing disulfide bond. Bioorg Chem. 2022; 119:105542. https://doi.org/10.1016/j.bioorg.2021.105542.

[21] Martin Sahn AG, Lucie Heller, Wajid Hussain, et al. Synthesis and cytotoxicity of 3-amino-glycyrrhetinic acid derivatives. Mediterr J Chem. 2018; 7(1):39-55. https://doi.org/10.13171/mjc71/01804111430-cesu.

[22] Wang R, Yang W, Fan YQ, et al. Design and synthesis of the novel oleanolic acid-cinnamic acid ester derivatives and glycyrrhetinic acid-cinnamic acid ester derivatives with cytotoxic properties. Bioorg Chem. 2019; 88:102951. https://doi.org/10.1016/j.bioorg.2019.102951.

[23] Xu Q, Deng H, Li XT, et al. Application of amino acids in the structural modification of natural products: a review. Front Chem. 2021; 9:650569. https://doi.org/10.3389/fchem.2021.650569.

[24] Cui HW, He Y, Wang J, et al. Synthesis of heterocycle-modified betulinic acid derivatives as antitumor agents. Eur J Med Chem. 2015; 95:240-248. https://doi.org/10.1016/j.ejmech.2015.03.048.

[25] Zhang CZ, Yan WQ, Zhao R, et al. Design, synthesis and evaluation of new ligustrazine derivatives as potential plasma-stable neuroprotective agents. Medchemcomm. 2017; 8(3):652-656. https://doi.org/10.1039/c7md00003.

[26] Falbo F, Gemma S, Koch A, et al. Synthetic derivatives of natural cinnamic acids as potential anti-colorectal cancer agents. Chem Biol Drug Des. 2024; 103(1):e14415. https://doi.org/10.1111/cbdd.14415.

[27] Cabello CM, Bair III WB, Lamore SD, et al. The cinnamon-derived Michael acceptor cinnamic aldehyde impairs melanoma cell proliferation, invasiveness, and tumor growth. Free Radic Biol Med. 2009; 46(2):220-231. https://doi.org/10.1016/j.freeradbiomed.2008.10.025.

[28] He R, Dong T, Chu J, et al. Phaser-trim: a phase separation based genetically encoded reporter for H3K9 trimethylation in living cells. Anal Chem. 2024; 96(32):13007-13014. https://doi.org/10.1021/acs.analchem.4c00992.

[29] Jiang M, Qi F, Zhang K, et al. MARCKSL1-2 reverses docetaxel-resistance of lung adenocarcinoma cells by recruiting SUZ12 to suppress HDAC1 and elevate miR-200b. Mol Cancer. 2022; 21(1):150. https://doi.org/10.1186/s12943-022-01605-.

[30] Barcia JJ. The Giemsa stain: its history and applications. Int J Surg Pathol. 2007; 15(3):292-296. https://doi.org/10.1177/1066896907302239.

[31] Crowley LC, Marfell BJ, Waterhouse NJ. Analyzing cell death by nuclear staining with Hoechst 33342. Cold Spring Harb Protoc. 2016; 2016(9): 087205. https://doi.org/10.1101/pdb.prot087205.

[32] Ragonese F, Monarca L, De Luca A, et al. Resveratrol depolarizes the membrane potential in human granulosa cells and promotes mitochondrial biogenesis. Fertil Steril. 2021; 115(4):1063-1073. https://doi.org/10.1016/j.fertnstert.2020.08.016.

[33] Trachootham D, Alexandre J, Huang P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat Rev Drug Discov. 2009; 8(7):579-591. https://doi.org/10.1038/nrd2803.

[34] Carneiro BA, El-Deiry WS.Targeting apoptosis in cancer therapy. Nat Rev Clin Oncol. 2020; 17(7):395-417. https://doi.org/10.1038/s41571-020-0341-.

[35] Xu X, Piao HN, Aosai F, et al. Arctigenin protects against depression by inhibiting microglial activation and neuroinflammation via HMGB1/TLR4/NF-κB and TNF-α/TNFR1/NF-κB pathways. Br J Pharmacol. 2020; 177(22):5224-5245. https://doi.org/10.1111/bph.15261.

[36] van Beijnum JR, Buurman WA, Griffioen AW. Convergence and amplification of toll-like receptor (TLR) and receptor for advanced glycation end products (RAGE) signaling pathways via high mobility group B1 (HMGB1). Angiogenesis. 2008; 11(1):91-99. https://doi.org/10.1007/s10456-008-9093-5.