RAS gene family (HRAS, NRAS and KRAS) have emerged as prominent oncogenes in human cancers. These mutations can activate signaling pathways that promote tumor growth, survival, and invasion. These three oncogene subtypes display varying mutation frequencies, with KRAS mutations being the most prevalent, accounting for 85 % of cases [1]. Notably, KRAS mutations were predominantly identified in pancreatic cancer (88 %), colorectal cancer (50 %), and non-small cell lung cancer, particularly in lung adenocarcinoma (32 %) [2]. Among KRAS mutations, 83 % were localized to codon 12, primarily involving G12C, G12D and G12V mutations [3].

KRAS oscillates between binding to the inactive guanosine diphosphate (GDP) state (‘off’) and the active guanosine triphosphate (GTP) state (‘on’), and the state of KRAS in healthy individuals switches normatively. In mutated cells, KRAS becomes ‘locked’ in the ‘on’ state, resulting in the abnormal activation of KRAS proteins [4], [5]. The abnormal KRAS ultimately leads to the dysregulation of multiple effectors and a loss of control over downstream signaling pathways. This, in turn, causes abnormal cell proliferation, ultimately resulting in the occurrence and development of tumors [6], [7], [8].

In the past, KRAS mutations have been classified as “non-druggable” targets because they lack binding pockets on their smooth surface. This poses a significant obstacle and one of the most challenging issues faced by the scientific community. However, in recent years, there have been significant advancements. Several compounds, such as AMG-510 and MRTX849, specifically targeting KRAS G12C mutations, have already become available in the market [9], [10]. Otherwise, there are currently no approved drugs for the treatment of KRAS G12D mutations. Promising inhibitors such as MRTX1133 (Fig. 1) showed potent and specific activity against KRAS G12D cells in vitro (IC50 = 6 nM) and in vivo [11], and TH-Z835 demonstrated significant antitumor efficacy in vitro with an IC50 value below 2.5 μM and in vivo, which reduced pERK levels in PANC-1(KRASG12D) [12]. It's worth noting that most KRAS G12D inhibitors have a similar structure to KRAS G12C inhibitors, which may lead to similar side effects such as drug resistance. Thus, the development of new inhibitors specific to the KRAS G12D mutation holds substantial scientific significance and potential commercial implications.

Natural products are the abundant resources for drug discovery. Genipin, a natural product also derived from the Gardenia jasminoides J. Ellis, has many pharmacological effects, such as anti-anxiety, neuroprotective activity, anti-Alzheimer’s disease, antithrombosis effects, anti-tumor effects and hypoglycemic activity. In our previous work genipin derivatives demonstrate a low level of toxicity [13], indicating a favorable safety profile. Molecular docking analysis of genipin binding to the KRAS G12D protein (PDB ID: 7RPZ) indicates that the moiety of genipin locates in the catalytic active pocket of KRAS G12D and forms hydrogen bonds with residues ARG68 and ARG69 (shown in Fig. 2).

Based on our previous safety evaluations and docking outcomes, there is potential for developing new scaffolds that could serve as highly effective and selective KRAS G12D inhibitors by incorporating pharmacophores modules into genipin core structure. Herein, we report the design, synthesis, and evaluation of genipin derivatives as a novel class of potent and selective KRAS G12D inhibitors. By understanding of structure–activity relationships (SAR) for KRAS G12D inhibitors, chemical modifications to genipin derivatives have been implemented. Aromatic moieties were incorporated at positions R1 and R2, establishing hydrophobic interactions with specific amino acid residues within KRAS G12D (including TYR96 and HIS95). Further modifications introduced a piperazine ring by transforming the ester group in genipin and a covalent carbonyl functional group was attached to this piperazine ring to facilitate binding through hydrogen bonding with the protein's amino acid residues (LYS88 and ARG68). Nineteen compounds were specifically designed and synthesized as potential inhibitors against KRAS G12D. The majority of these compounds showed potent antiproliferative effects on KRAS G12D mutation cells (CT26 and A427), while exhibiting low cytotoxicity towards normal cells. Through a series of assays, SK12 demonstrated significant antitumor effects against KRAS G12D mutation cells (CT26) both in vitro and in vivo. These results indicate that genipin derivatives, exemplified by SK12, have the potential as next-generation KRAS G12D inhibitors with a novel scaffold that is distinct from other G12D inhibitors.