Serine/threonine phosphatases, including PP1, PP2A, and PP2B, account for approximately 90% of the dephosphorylation events in the heart. The pronounced toxicity of CTD and its analogs in mammals stems from their elevated affinity and specificity for CTD-binding proteins, identified as PP1 and PP2A, and the efficiency of CTD in inhibiting PP2A is 10 times that of inhibiting PP1 (Neumann et al., 1995). CTD enhances myocardium contractility by regulating the phosphorylation state of proteins, specifically by inhibiting cardiac protein phosphatases, which leads to sustained phosphorylation of PLN (phospholamban). Additionally, it can moderately increase the L-type calcium channel current in guinea-pig cardiomyocytes, thereby exerting positive inotropic effects without increasing cAMP (cyclic adenosine monophosphate) content, distinguishing it from the positive inotropic mode of adrenaline (Neumann et al., 1995). However, this effect is not entirely attributable to calcium ions, as an increase in calcium ions without CTD at the same concentration does not yield equivalent contractile force, suggesting that CTD may increase myocardial contractility through pathways other than calcium current inhibition (Knapp et al., 1998; Neumann et al., 1995). PP2A activity is linked to the activities of a number of key ion channels, including the L-type calcium channel (Cav1.2), ryanodine receptor 2 (RyR2), Na⁺/Ca²⁺ exchanger (NCX), and Na⁺/K⁺ ATPase (El Refaey et al., 2019). CTD inhibits PP2A activity, potentially influencing the above - mentioned receptors. CTD can inhibit the negative inotropic effects induced by various drugs in the human or animal heart, such as the negative inotropic effect caused by carbachol in the isolated human atrium and endothelin-1 in canine ventricular myocardium (Schwarz et al., 2024; Chu et al., 2003).

Moreover, CTD protects heart from ischemia and reduces infarct size through ERK phosphorylation dependency (Yang et al., 2011). However, it fails to alleviate reperfusion-induced damage. CTD can also enhance the contractility of smooth muscle, primarily by inhibiting phosphatase activity in aortic smooth muscle (Knapp et al., 1997) and increasing the phosphorylation state of vascular regulatory proteins. In contrast to its cardiac effects, CTD does not affect calcium homeostasis (Knapp et al., 1998), instead predominantly modulating vascular contraction and regulates the phosphorylation of myosin light chains. In addition, CTD influences coronary artery perfusion while exerting positive inotropic effects, potentially exerting contradictory cardiac impacts (Knapp et al., 1998). In vascular smooth muscle cells, CTD reduces proliferation and migration capabilities by inhibiting inflammatory responses, suggesting its potential application in post-angioplasty neointimal hyperplasia and restenosis (Qiu et al., 2019). Additionally, CTD inhibits vascular endothelial growth factor (VEGF)-induced JAK1/STAT3, ERK, and AKT signaling pathways, suggesting its potential for anti-angiogenic therapy (Wang T. et al., 2015).

Despite its toxicity to many animals, some species such as the Otis tarda consume blister beetles to treat their own bacterial and parasite infections (Bravo et al., 2014). Experimental evidence demonstrates that CTD exerts concentration-dependent inhibitory effects on bacteria such as Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae. Douglas et al. primarily studied by testing the antiparasitic efficacy of both pure CTD and extracts of B. majalis beetles against protozoa (Trichomonas vaginalis), a nematode (Meloidogyne javanica), and a tick (Hyalomma lusitanicum) (Whitman et al., 2019). Previously, CTD has shown good effects against severe Leishmania major infections (Ghaffarifar, 2010), achieving 80% growth inhibition in vitro at a concentration of 50 μg/mL, as well as efficacy in vivo in experimentally infected BALB/c mice. CTD also exerts a potent toxic effect on certain pests. Hong Sun et al. had shown that CTD can control Periplaneta americana (L.) through Serine/Threonine Protein Phosphatase Type 5 (Sun et al., 2020). In agriculture, CTD exhibits potent toxicity against lepidopteran pests (Noctuidae), showing low susceptibility to resistance, thus offering promise for agricultural applications (Li Y. et al., 2021). Additionally, CTD has been reported to possess insecticidal activity against pests such as Plutella xylostella, Helicoverpa armigera, Walker, Fabricius, and Musca domestica (Sun et al., 2022). Collectively, CTD displays activity against a wide range of organisms, including protozoa, nematodes, ticks, and insects (Figure 3). CTD shows great potential in the fields of animal husbandry and agriculture. In the animal field, considering its antibacterial and antiparasitic properties, it may be used in preventing and treating various animal diseases caused by bacteria or parasites. For example, it could be developed into veterinary drugs to protect the health of livestock and poultry, improve their survival rate and productivity. In the agricultural field, its pest-killing characteristic makes it a potential natural pesticide. It can be used to control agricultural pests, reduce the damage of pests to crops, and help increase crop yields. Moreover, compared with some traditional chemical pesticides, CTD is more environmentally friendly, which is in line with the concept of sustainable agricultural development.

In July 2023, YCANTH™ (CTD 0.7% topical solution) was approved in the United States for the topical treatment of molluscum contagiosum in adult and pediatric patients aged 2 years and older (Keam, 2024). Mechanistically, CTD is absorbed by lipids in keratinocytes, where it activates neutral serine proteases, triggering progressive degeneration of desmosomal dense plaques. This results in selective acantholysis occurring intraepidermally, which heals over time without scarring. Blisters form within 24–48 h and resolve within 4–7 days (Torbeck et al., 2014). Additionally, CTD has been employed as an inflammatory model and in cancer treatment (Naz et al., 2020; Dinh et al., 2011). Sun et al. also reported that carboxylesterases (CarEs), glutathione S-transferases (GSTs), and cytochrome P450 monooxygenases (P450s) act as detoxifying enzymes for CTD, with P450 exhibiting consistent activity recovery comparable to that of phosphatases (Sun et al., 2022). Future research may explore incorporating detoxifying enzymes into treatment protocols to maximize the clinical utility of CTD. By screening for CTD-detoxifying enzymes, genetic testing can be performed on patients prior to clinical administration, aiming to provide guidance for determining clinical drug dosages.

In China, Aidi Injection (Z52020236, approved by the National Medical Products Administration), comprising extracts from Astragalus membranaceus, Acanthopanax senticosus, Panax ginseng C. A. Mey, and CTD from Lytta vesicatoria, has emerged as a widely utilized adjuvant chemotherapy agent for anti-tumor therapy (Zhang et al., 2025). Clinically, it is predominantly employed to treat malignant tumors, including hepatocellular carcinoma, lung cancer, bladder cancer, and colorectal cancer. It is typically administered in combination with other anti-tumor drugs for cancer treatment. For example, when combined with paclitaxel to treat advanced non-small cell lung cancer (NSCLC), its efficacy is comparable to that of the chemotherapy drug cisplatin, yet it exhibits reduced side effects (Xiao et al., 2019). The available evidence (Xiao et al., 2017) indicates that Aidi injection plus gemcitabine and cisplatin (GP) can significantly increase the clinical efficacy and improve the quality of life for patients (QOL) of patients with NSCLC. Meanwhile, CTD is also used to treat certain inflammatory diseases and gout.

In case of large-dose CTD poisoning, myocardial injury manifests earlier than hepatic and kidney functional abnormalities. Excessive exposure can lead to direct cardiotoxic effects, including arrhythmias and arterial occlusion (Rabkin et al., 1979). Severe poisoning elevates cardiac enzyme levels, notably serum creatine kinase (Zhang Y. et al., 2018; Youyou et al., 2020). Previous studies on fatal CTD poisoning have shown ventricular arrhythmias, persistent ST-segment elevation, and T-wave alterations on electrocardiography, accompanied by anatomical evidence of myocardial hemorrhage and patchy myocardial necrosis. These ST-segment and T-wave alterations correlate with focal myocardial degeneration, necrosis, inflammation, and/or fibrosis. However, as with other cardiomyopathies, the precise mechanisms of CTD’s cardiotoxicity remain elusive. Research by multiple author (Rabkin et al., 1979; Shao et al., 2020) has identified dose-dependent mitochondrial swelling, nuclear chromatin clumping, sarcoplasmic reticulum disruption, and myofibrillar degeneration in CTD-poisoned myocardium. These electron microscopic observations suggest severe mitochondrial damage as a key feature of CTD-induced myocardial injury. Fleckenstein et al. (1975) proposed that myocardial necrosis results from excessive calcium influx into myocardial cells, with electron-dense structures observed within mitochondria potentially representing calcium deposits. This offers a plausible explanation for CTD-induced myocardial injury, where CTD alters myocardial cell membranes or mitochondria, leading to calcium overload and ultimately myocardial fiber necrosis. Shi et al. reported that CTD impairs mitochondrial function, evidenced by reduced basal respiration, ATP levels, and spare respiratory capacity, alongside decreased mitochondrial DNA copy numbers and downregulated mRNA levels of cytochrome C oxidase I, II, and III. Additionally, CTD inhibited activities of mitochondrial complexes I and II (Shi et al., 2022). Youyou et al. (2020) previously validated factors related to hypoxia, angiogenesis, and apoptosis, which confirmed that CTD can induce hypoxia, necrosis, and inflammation. Shao et al. (2020) observed early oxidative stress parameters affecting myocardium in CTD poisoning, marked by significant increases in superoxide dismutase and glutathione levels, coupled with a notable decrease in malondialdehyde content. Transcriptomic analysis underscored the roles of Tnc and Myh7 genes in CTD-induced myocardial injury in rats, involving vascular regeneration, myocardial repair, and remodeling (Youyou et al., 2020). Additionally, CTD poisoning may directly impair capillary endothelial cells, leading to widened intercellular spaces and increased vascular permeability. This facilitates the extravasation of plasma components, contributing to myocardial cells and myocardial hemorrhage (Knapp et al., 1999). In conclusion, the mitochondrial damage and cardiomyocyte apoptosis induced by CTD are significant and warrant attention. As a phosphatase inhibitor, CTD disrupts multiple energy pathways in cardiomyocytes, potentially leading to arrhythmia and, in severe cases, sudden death. Shao et al. (2020) provided evidence that L-glutamine could protect cardiac cells against the acute cantharidin-induced cardiotoxicity, and the protective mechanism of glutamine may be related to the myocardial cell membrane or the tricarboxylic acid cycle in the mitochondria.

The liver, as the primary organ responsible for drug metabolism and detoxification, is more susceptible to drug-induced damage compared to other organs. Zhang Y. et al. (2018) reported varying degrees of liver injury in retrospective study of CTD poisoning and associated mortality cases. Serum biochemical markers of liver injury, such as bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP), were significantly elevated in models of different degrees of CTD-induced liver injury and toxicity. Post-mortem examinations of cadavers revealed hepatic infiltration by inflammatory cells, hepatocyte degeneration, and necrosis. In rat models, high doses of CTD triggered multifocal hepatic hemorrhage, focal necrosis, and inflammatory cell infiltration (Yu et al., 2020). These observations were substantiated by Western blot analysis, which revealed increased inflammatory factors and confirmed hepatocyte apoptosis and necrosis following CTD exposure. Jin et al. (2023) outlined that CTD-mediated hepatotoxicity mainly involves endoplasmic reticulum stress (ERS), autophagy, activation of cysteine signaling pathways, mitochondrial dysfunction, and bile acid circulation. CTD induces ERS, mitochondrial swelling and distortion, altering hepatocyte membrane permeability and ultimately cell death, thereby causing liver damage. Metabolomic analysis via UPLC-Q-TOF/MS (Zhu et al., 2019) indicated that CTD treatment disrupts 14 metabolic pathways, indicating its potential to impair various biological processes within the liver. Comparative studies further identified significant perturbations in glutathione metabolism, taurine and hypotaurine metabolism, and the interconversion of pentose and glucuronate. Additionally, metabolic pathway analysis identified CTD-induced disturbances in serum lipid metabolism, pointing to dysregulation in biosynthetic pathways anchored by glycerophospholipids, glycerides, and glycosylphosphatidylinositol (GPI) (Li et al., 2024). Cytochrome P450 enzymes play a crucial role in hepatic drug metabolism, with CTD shown to inhibits CYP2D6 and CYP3A4 while inducing CYP2C9 (Zhou et al., 2015). Consequently, caution is warranted when co-administering CTD with drugs primarily reliant on the cytochrome P450 enzyme system for metabolism. Interestingly, vitamin C (VC) has been found to attenuate liver damage by downregulating CTD-induced mRNA expression of TLR4 and NF-κB in the liver, participating in the inhibition of intrahepatic oxidative stress and the TLR4/NF-κB pathway associated with inflammation, thereby improving hepatocyte metabolic function (Wu et al., 2015). Ginsenoside Rb1 (GRb1) shows potential hepatoprotective effects. Xiong et al. revealed the mechanism of GRb1 against CTD-induced hepatotoxicity by inhibiting apoptosis and endoplasmic reticulum stress (ERS) (Xiong et al., 2024). Similarly, Astragalus polysaccharides (APS) regulated primary bile acid biosynthesis and glycerophospholipid metabolism, thus decreasing the liver damage caused by CTD (Huang et al., 2021). GRb1 and APS are present in many clinically used drugs to reduce the hepatotoxicity caused by CTD (Jin et al., 2023).

CTD poisoning frequently manifests as lumbago and renal dysfunction, evidenced by elevated serum creatinine and urea levels. AS the kidneys metabolized CTD, it may irritate the urinary tract, leading to conditions such as proteinuria, glucosuria, and hematuria (Cotovio et al., 2013). Anatomically, affected kidneys often appear pale, with histological changes including renal capsule hemorrhage, interstitial bleeding, and basal membrane edema (Karras et al., 1996). Common histopathological features include acute tubular necrosis, epithelial cell shedding, and tubular casts. In murine models, CTD exposure induces vacuolar degeneration of renal tubular epithelial cells, glomerular swelling, and interstitial inflammatory cell infiltration. Ultrastructural studies further reveal mitochondrial swelling, disrupted mitochondrial cristae, and increased autophagic vacuoles. The main mechanism likely involves CTD binding to albumin, leading to glomerular injury and acute tubular necrosis upon filtration through the renal glomeruli. CTD triggers endoplasmic reticulum stress (ERS), potentially driving renal toxicity via activation of autophagy and cell apoptosis, mediated through the PERK/CHOP pathway dependent on ERS (He et al., 2022). Transcriptomic studies reveal that CTD alters gene expression related to stress response, cell death induction, transporter proteins, and affects signaling pathways like MAPK, AMPK, and HIF-1 (Liu et al., 2023). These changes contribute to renal oxidative stress, inflammation, autophagy, and apoptosis. Using network pharmacology and UHPLC-QE/MS-based metabolomics, Tianmu He et al. demonstrated that CTD increases Caspase 3 expression levels and the Bax/Bcl-2 ratio in renal cells. Additionally, CTD appears to inhibit glycerophospholipid and sphingolipid pathways, phosphatidylethanolamine, phosphatidylcholine, MAPK3, and PLD2 (He et al., 2020). The recent study (He et al., 2025) showed that CTD induces acute tubular necrosis (ATN), resulting in acute kidney injury (AKI), by activating glycerophospholipid (GP) metabolism and inhibiting sphingolipid (SL)_metabolism in the renal cortex and medulla. In animal models, Ginsenoside Rb1 markedly alleviated cantharidin-induced acute kidney injury by modulating toll-like receptor 4 dimerization and NF-kB/MAPKs signaling pathways (Gao et al., 2020).

While the deleterious effects of CTD on cardiac, hepatic, and renal functions have been well-documented, its reproductive toxicity remains understudied. Historically, CTD was employed in Europe as an aphrodisiac due to its effects on penile congestion and erection in males, and vaginal bleeding in females (Karras et al., 1996). In murine models, CTD exposure drastically reduces testicular index and serum testosterone levels (Xiao et al., 2024). Histological examination reveals a significant decrease in sperm count within seminiferous tubules, accompanied by vacuolization, necrosis of spermatogenic cells, and peripheral infiltration of inflammatory cells. Testicular tissues also exhibit notable oxidative damage and increased mitochondrial autophagy, with suppression of the Nrf2-Keap1 pathway and disruption of the blood-testis barrier. Liu et al. reported that exposure to varying concentrations of CTD induces weight loss and increases testicular coefficient (Liu et al., 2024). Network toxicology and Western blot analyses indicate inhibition of PI3K, AKT, and anti-apoptotic protein Bcl-2 expression, alongside promotion of pro-apoptotic proteins Bax and Caspase-3. Pathway enrichment analysis highlights associations between CTD-induced testicular damage, apoptosis, and the PI3K/AKT and HIF-1 signaling pathways. Regarding CTD’s potential role in abortion, Nae-Fang Twu et al. explored its impact on steroidogenesis in goat corpus luteum cells, revealing a reduction in steroids and progesterone (P4) production, underscoring CTD’s potential reproductive toxicity (Twu et al., 2012). Study had shown that Astragalus polysaccharide can attenuate cantharidin-induced toxic damage, oxidative stress and autophagy in the testes of mice. These protective effects may be closely mediated by regulating the Nrf2-Keap1 signaling pathway, inhibiting autophagy, and restoring the blood-testis barrier (Xiao et al., 2024).

CTD exhibits significant irritant effects on skin and mucous membranes, leading to desquamation (Karras et al., 1996), epidermal vesicles, submucosal edema, hemorrhage, necrosis, and sloughing. Ingestion of beetles containing CTD can cause oral burning sensation, dry retching, vomiting, and diffuse abdominal pain (Cotovio et al., 2013), with potential progression to acute gastrointestinal inflammation or shock. Histopathological findings include esophageal mucosal congestion, swelling, and ulcers, as well as gastric mucosal hyperemia, hemorrhage, and superficial erosions extending to the upper small intestine (Chen and Huang). CTD residues are often detectable in the stomach, which aids in forensic identification of CTD poisoning. Neurological effects resembling Guillain-Barré syndrome have been reported following CTD exposure, suggesting potential neurotoxicity (Harrisberg et al., 1984). Additionally, reversible impairments like cranial nerve paralysis and muscle weakness have been documented, with neuronal recovery typically occurring after treatment (Zouvanis et al., 1994). In studies using hippocampal brain slices, CTD exposure was found to reduce basal synaptic transmission, disrupts synaptic plasticity induction via inconsistent effects on Ca²⁺ signaling across different concentrations, and indicates long-term synaptic transmission reduction through prolonged protein phosphatase inhibition (Koss et al., 2007). CTD also affects the immune system by impairing differentiation and maturation phenotypes of dendritic cells (DCs), as evidenced by downregulation of surface markers CD1a, CD83, and DC-SIGN. Cases of CTD-induced cystitis have been observed (Chen and Teik, 1961), and animal studies have demonstrated severe inflammatory bladder injury with hematuria, possibly mediated by c-Fos and COX-2 overexpression (Huan et al., 2012). In conclusion, while CTD offers considerable therapeutic potential for dermatological and oncological applications, its clinical use demands stringent control over dosage and treatment duration to minimize its toxic side effects.

The toxic effects of CTD in animals are similar to those in humans. Toxicosis occurs when horses consume alfalfa hay or products contaminated with “blister” beetles (Schmitz, 1989). In horses that remain alive for several days, persistence of elevated heart and respiratory rates and increasing serum creatine kinase concentration are associated with a deteriorating condition. Treatment of CTD toxicosis is symptomatic, with gastrointestinal protectants, laxative, intravenous fluids, analgesics, diuretics, calcium gluconate, and magnesium are all included in the treatment regimen. In experiments investigating (Qualls et al., 2013) the use of mineral oil, charcoal, and montmorillonite for treating oral CTD poisoning, it was found that mineral oil exacerbates the toxic effects of CTD. This may be attributed to the fact that CTD is fat-soluble; dissolving it in mineral oil enhances its absorption. This finding also indicates that mineral oil should not be used in the treatment of oral CTD poisoning in animals.

Currently, there is no specific antidote for CTD poisoning. The treatment after CTD poisoning mainly focuses on protecting the gastrointestinal mucosa, performing blood purification, and safeguarding the functions of the heart, lungs, kidneys, and brain. For those who have ingested CTD orally, gastric lavage should be carried out as early as possible. Patients at risk of acute kidney injury should have their renal function closely monitored, and protective measures such as urine alkalization, the use of diuretics (e.g., furosemide), and avoiding the use of nephrotoxic drugs should be taken (Duan et al., 2025). Given that hemodialysis is ineffective in removing CTD, owing to its binding to circulating albumin and poor aqueous solubility, hemoperfusion and hemofiltration are recommended as alternative approaches. These methods can facilitate the removal of CTD, inflammatory cytokines, and metabolites, while also correcting electrolyte and acid-base disturbances (Xu et al., 2024). In addition, cardiac and liver function indicators should be observed, and targeted treatment should be provided. Although CTD is metabolized relatively quickly in the early stage, the original substance can still be detected in the body after a few days. Therefore, sequelae should also be monitored, and gastroenteritis should be noted in patients who have ingested it orally. In summary, CTD poisoning warrants serious attention. It is advisable to not only administer targeted symptomatic treatment but also closely monitor potential systemic harm to the human body.

Through the study on the structure-activity relationship of modifications at positions 1 and 4 of the CTD structure (Figure 5; Table 2) (McCluskey et al., 2003; Ren and Kinghorn, 2021; McCuskey et al., 2000), it was found that the structural modifications have a significant site-dependence on the phosphatase inhibitory activity. Experimental data indicate that substitution modification at position 1 reduces the inhibitory activity of the derivatives against PP2A, whereas single substitution at position 4 diminishes their broad-spectrum inhibitory capacity against protein phosphatases. Mccluskey et al. (1996) has synthesized a series of CTD analogs, indicating that the 7-hydroxy group is essential for inhibiting PP2A. The derivatives obtained by replacing oxygen atoms with sulfur atoms also lose their inhibitory effect on PPs (Shan et al., 2006). Based on this, these core pharmacophore groups at positions C-1,4 and 7-O should be preferentially retained during the structural optimization of CTD.

Norcantharidin (NCTD) is a synthetic anti-cancer compound derived from CTD. It is produced by removing methyl groups from C-2 and C-3 positions of CTD through hydrolysis, which reduces nephrotoxicity while maintaining anti-cancer activity comparable to that of CTD (Hizartz et al., 2019; Massicot et al., 2005). Compared to CTD, NCTD exhibits lower cytotoxicity and fewer clinical side effects. Since 1984, NCTD has been utilized in China for the treatment of various cancers. It shows stronger inhibition against PP2B compared to CTD, demonstrating its potent activity against tumor cells. NCTD is particularly effective in treating primary liver cancer, where it enhances immune function and reduces tumor size in early to mid-stage cases (Li et al., 2010; Zhao et al., 2019). Research involving BEL-7402 liver cells has shown that NCTD inhibit NF-κB activation, a pathway that protects tumor cells from apoptosis (Zavoral et al., 2011). To further mitigate its toxicity, ongoing studies are exploring the use of thermosensitive hydrogel nanoparticles to encapsulate NCTD, aiming to enhance its efficacy while reducing required dosage (Li XY. et al., 2021).

Essers introduced fluorine groups at the C-2,3 positions, and the derivatives retained the antitumor activity and simultaneously reduced the toxic effects (Essers et al., 2001). In addition, the hydrogen atoms or other groups can also be used for substitution, and the synthesized derivatives show an increased affinity for PPs (Laidley et al., 1999). Furthermore, modifications can tailor the affinity of CTD analogs for different subtypes of serine/threonine phosphatases, enabling targeted enzyme inhibition. Yoshiyasu Baba developed a novel derivative, 1,5-diphenylacetyl oxyethyl substituted NCTD, which selectively inhibits PP2B without affecting PP1 or PP2A (Baba et al., 2003).

Dehydrogenation at the fifth and sixth positions of CTD forms a C=C double bond, yielding a derivative (McCluskey et al., 2000) that exhibits anti-cancer activity comparable to CTD but with reduced toxicity. This derivative enables the introduction of hydrophobic groups such as methyl or ester groups, or epoxy structures. Experimental studies by Thaqi have shown that related analogs lacking the 5,6-ethylene bridge display poorer inhibitory activity, confirming the crucial role of this bridge in biological activity (Thaqi et al., 2010).

Anhydride-Modified derivatives of CTD through anhydride modifications yield various analogues (Wang Y. et al., 2015). These derivatives, modified in the anhydride moiety, can be categorized into two categories: those that retain the five-membered anhydride ring and those that involve five-membered ring opening.

McCluskey demonstrated through research that introducing a nitrogen atom into the five-membered ring can lead to the loss of the effect on PP2A. However, introducing a sulfur atom results in an inhibitory effect on PP2A similar to that of CTD and NCTD (McCuskey et al., 2000). Research has shown that maintaining the five-membered ring while substituting with specific groups can produce compounds like cantharimides, a novel class of CTD analogues exhibiting superior inhibition of PP1 and PP2A compared to NCTD (McCluskey et al., 2001). These cantharimides also possess improved solubility and feature electron-withdrawing groups on the aromatic ring, which enhance cytotoxicity against cancer cell lines.

Regarding the analogs with an opened five - membered ring, metal salt analogs are currently a hot research topic. NCT sodium salt is a newly developed drug, and the sodium atom can be substituted with various metal atoms. These NCT metal salt analogs have a strong inhibitory effect on tumors and hold broad application prospects (Zhao et al., 2019). Additionally, a series of ring-opened CTD analogues with only one free carboxylate has been developed, retaining inhibitory activity against PP1 and PP2A while slightly increasing selectivity towards PP2A (McCluskey et al., 2001). The above two derivatives with an opened five - membered ring are mainly modified to address the weaknesses of cantharidin, such as its poor bioavailability and water solubility (Zhao et al., 2019). The water solubility of the derivatives is increased, their inhibitory effect on tumors is also enhanced, and their toxic effects are reduced. Notably, studies by Ji-Yeon Lee explored N-Benzylcantharidinamide, demonstrating its ability to inhibit MMP-9 expression by reducing MMP-9 mRNA stability without affecting MMP-9 transcriptional activity (Lee et al., 2014). This finding suggests potential for preventing tumor invasion and metastasis. Modifications to the anhydride five-membered ring do not universally enhance PP2A inhibition, in some cases, they may even negate the original activity (McCuskey et al., 2000). Conversely, research by Adam McCluskey has shown that derivatives capable of facile ring opening of the anhydride moiety retain significant PP2A inhibition and anti-cancer activity (McCuskey et al., 2000). However, the degree of inhibition on PP2A has decreased.

In addition, researches had also been conducted on the carriers of CTD and its derivatives. Drug-loaded liposomes have attracted much attention due to their advantages such as enhancing activity, improving bioavailability, having low toxicity, and enhancing targeting (Wang et al., 2021). Multiple studies have been carried out to explore the efficacy of carriers for CTD, NCTD and their analogs, such as liposomes, nanoparticles, and micelles (Zhu et al., 2018; Zhou et al., 2019; Zhang H. et al., 2018; Yao et al., 2020). These carriers enhance targeting and reduce toxic side effects. Moreover, as mentioned earlier, when some other substances are used in combination with CTD, such as APS and L-glutamine (Shao et al., 2020; Huang et al., 2021), they can reduce the liver and heart toxicity caused by CTD.

In summary, current modifications of CTD primarily focus on its six-membered and five-membered rings, with the first, fourth, and seventh hydroxy groups on the six-membered ring being crucial factors, modifications at these positions should be avoided in the future. Removing the methyl groups at the C-2 and C-3 positions of cantharidin or substituting them with other groups can significantly reduce the toxic effects and result in better selectivity. Similarly, the C-5, 6 ethyl bridge position is crucial. Forming a C=C double bond or introducing hydrophobic groups at this position can reduce toxic side effects and enhance the inhibitory effect on tumor cells. For the five-membered ring, introducing a carboxylic acid group or a metal salt can increase the water solubility of CTD and its derivatives, enhance absorption, and reduce toxicity. Moreover, after opening the five-membered ring, the inhibitory effect on PP2A is lower compared to the closed-ring form, which is a point worthy of attention. Moreover, “packaging” strategies for CTD, such as liposome encapsulation, have shown promising anti-proliferative effects against cancer cells.