Synthetic Alkaloids: Cantharidin Derivatives

Abstract

Cantharidin is a naturally occurring cyclic anhydride found in many insect species, particularly Lytta vesicatoria, known as the Spanish fly. Although highly poisonous, dried Spanish fly has been used as an aphrodisiac since ancient Greeks and Romans. Spanish fly has been used in eastern medicine for many years as a natural anticancer agent, especially in the treatment of hepatoma and esophageal carcinoma. Over time, its stotoxicity was determined to be high and its use was limited. Later, alkaloid derivatives with no stotoxic effect were produced synthetically and evaluated as anticancer agents. Since cantharidin obtained from insects is not an alkaloid but its derivatives with lower stotoxicity, cantharimide and norcantharimide are cyclic imides, they can be evaluated in the class of alkaloids. Cantharimide and norcantharimide compounds have gained importance in terms of their stotoxic effect on many cancer cell lines. Many studies have been done on their synthesis and anticancer properties for many years.

1. Introduction

Alkaloids are chemical compounds that contain basic Nitrogen atoms and are produced naturally by various organisms. Alkaloids can contain some groups with neutral [1] or acidic properties [2]. Alkaloids are usually organic bases. They form salts when reacted with acid and form alkaline solutions when dissolved. Primary sources of alkaloids are flowering plants. Plants use alkaloids for defense against herbivores and pathogens. It has been determined that 20% of plants contain alkaloids. Alkaloids are cyclic compounds that contain nitrogen (Figure 1) [3].

Figure 1.

Examples of alkaloids.

Plenty of alkaloids have been used in medicine for ages and even nowadays, they are prominent medical compounds. Since primitive times, alkaloids obtained from plant extracts have been used in medicines and poisons. In ancient times, plant extracts containing alkaloids were used to treat numerous ailments, including snakebite, fever, and insanity. Generally, alkaloids are extremely toxic at low concentrations, even if they have a therapeutic effect. Defense chemicals of plants against microorganisms, insects, and herbivores, as well as other plants using allelopathic active chemicals [4]. Their taste is bitter. They are usually colorless crystals at room temperature and are optically active [5]. Purely isolated plant alkaloids and their synthetic derivatives are used as basic medicinal agents due to analgesic, antispasmodic, and bactericidal effects [6]. Alkaloids generally affect the nervous system in humans (especially acetylcholine, epinephrine, norepinephrine, gamma-aminobutyric acid, dopamine, and serotonin) [7]. Alkaloids such as berberine (in eye medications) and sanguinarine (in toothpaste) are used as antiseptics (Figures 2 and 3) [8].

Figure 2.

Examples of alkaloids acting on the nervous system.

Figure 3.

Chemical formula of berberine and sanguinarine alkaloid.

While the search for new anticancer drugs continue, old drugs are viewed as new options. The dried body of Mylabris, the Chinese bubble beetle, has been the focus of attention for its anticancer properties, as it is known for traditional medicine in China for more than two thousand years, where it has been used as a traditional medicine. The oldest data in China on the use of Mylabris as a medicine dates back to 300–168 BC. In Europe, it was found about 77 AD in a medical article published in Materia Medica. The active ingredient of Mylabris has been identified as cantharidin. Later, it was determined that cantharidin has both anticancer activity and leukocytosis and hemorrhagic cystitis properties [9]. When the natural product cantharidin was purified and used for a long time, its cytotoxic effects began to be observed, and norcantharidin, a demethylated derivative, was synthesized. Subsequently, nitrogen-containing derivatives cantharimide and norcantharimide were synthesized. Later, derivatives of these four analogs were synthesized both in the amide ring and in the cyclohexyl ring, and biological activity studies were carried out (Figure 4).

Figure 4.

The chemical formula of cantharidin, norcantharidin, cantharimide, and norcantharimide.

Norcantharidin slows the proliferation of tumors such as HeLa, CHO, CaEs-17, BEL-7402, SMMC7721 human hematoma, HEP-2, and human epidermoid larynx carcinoma [10, 11, 12]. Norcantharidin has been found to have fewer nephrotoxic and inflammatory effects [13, 14, 15, 16, 17]. Based on the structure–activity relationship, more analogs have been synthesized. For this reason, based on the structure–activity relationship, the researchers synthesized more analogs, and each synthesis was supported by biological activity and anticancer studies [18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28].

Disodium cantharidate and norcantharidin derivatives, among the compounds synthesized analogously, are among the derivatives synthesized in the earliest period by Wang et al. [10]. Because of their found stronger antihepatoma activities, these derivatives were more popular than cantharidin itself. However, they also cause minimal urinary irritation (Figure 5) [10].

Cantharidin has many derivatives besides disodium cantharidat and norcantharidin, some of which are hydrocantharidimite, methyl cantharidimite, and dehydronorkantharidin (Figure 5). Chinese scientists are collecting their medical literature by researching cantharidin and its derivatives, but there are still large gaps in researchers’ knowledge about these drugs and their effects [29, 30, 31, 32]. According to current information, norcantharidin, a demethylated analog of cantharidin, is stated to slow down the proliferation of tumors such as HeLa, CHO, esophageal carcinoma (CaEs-17), hepatoma (BEL-7402 and SMMC-7721), epidermoid larynx carcinoma (HEP2), and human epidermoid larynx carcinoma [10, 11, 12].

Figure 5.

Examples of early synthesized derivatives of cantharidin.

Two main methods were used in the synthesis of cantharimide and norcantharimides. One of them is the addition of furan and (2,3-dimethyl)maleic anhydride and then its conversion to the imide derivative. The other (2,3-dimethylmaleimide) is made by initially synthesizing maleimides and adding Diels-Alder with furan (Figure 6).

Figure 6.

General synthesis methods of cantharimide and norcantharimides (R′: Primary amine containing the desired derivative to be synthesized).

2. Cantharimides

Cantharidine is found in Cantharis vesicatory, Lytta caraganae, Mylabris phalerata, Meloidae, Oedemeridae, and various other insect species. Derivatives of cantharidin are synthesized to reduce their blistering effects on the skin, reduce their toxic properties, and benefit from their antitumor activity. Since the 1950s, it has been determined that various anhydride and imide derivatives exhibit a wide range of biological activities (antidepressant, anticonvulsant) as well as antitumor properties [33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43]. For this reason, cantharimide, which has an imide structure, attracted the attention of Guang-Sheng WANG reported that he synthesized cantharimide and N-methyl cantharimide in an article he prepared in 1989 and conducted an activity study in KB cell culture. As a result of his study, he stated that both compounds showed antitumor activity [10].

Pen-Yuan Lin and his group synthesized 10 different cantharimide derivatives using various tryptamine, indolyl, naphthyl, and pyridyl amines [34]. Lin used Zhang’s method in his synthesis. In this method, in the presence of triethyl amine at high pressure, the related amine compound gives cantharimides as a result of an addition reaction under pressure at 200°C (Figures 7 and 8) [44].

Figure 7.

Synthesis of tryptamine, indolyl, naphthyl, and pyridyl canthrimidines [44].

Figure 8.

Synthesis of cantharimides at high pressure [44].

Another of the cantharimide derivatives is the derivatives obtained by the addition of heterocyclic groups. These derivatives were synthesized for cytotoxicity tests against human hepatocellular carcinoma cells. In this synthesis, N-thiazolyl and N-thiadiazolyl cantharimides were synthesized by the method used above. According to the results of the study, they observed that the side groups attached to N-Thiazolyl and N-Thiadiazolyl amine compounds showed higher cytotoxicity than cantharidin in the area of electron-withdrawing (such as -NO₂), whereas a decrease in cytotoxic activity was observed in methyl-substituted compounds (Figure 9) [45].

Figure 9.

The synthesis of the N-thiazolyl- and N-thiadiazolyl cantharimides [45].

In 2007, a new cantharimide derivative was synthesized by Chan et al. Its structure was elucidated and its cytotoxicity on SK-Hep-1 hepatoma cells was determined. In this study, two cantharimides were synthesized with 2-amino benzothiazole derivatives. Cantharidin and 2-amino benzothiazole derivatives were added to a tube containing dry toluene and triethylamine (TEA), and related derivatives were synthesized at 200°C [45]. As a result of cytotoxic studies of the study group, it was determined that the compounds showed inhibition on SK-Hep-1 hepatoma. Using the results from the study, the group is also designing new (Figure 10) molecules [46].

Figure 10.

The synthesis of the (Me/-OMe)-2-amino benzothiazole cantharimides [46].

Cantharimide derivatives were also synthesized using aliphatic primary amines, phenethyl amines, aniline, and pyridine components by high-pressure addition reaction method. In the synthesis, the product was obtained with a yield of 29—96%. It is thought that the primary factor affecting the efficiency is the inductive effect of electron-negative groups. Another factor affecting the yield is the nucleophilic and basicity strength of the amines. Again, cytotoxicity studies of the compounds obtained in this synthesis were performed in human hepatocellular carcinoma (Hep G2) and myeloid leukemia cells (HL-60). In the evaluation, it was determined that compounds (10 and 16) with electron-withdrawing NO₂ groups in the pyridyl and benzene rings showed strong inhibitory effects in both cell lines. Methyl-containing compounds (6, 7, and 9) have less effect, while halogen-containing compound (8) has a moderate effect. Compound 10 with the 2-(3-nitro pyridyl) group showed stronger cytotoxicity. Para-Pyridyl imide 4 showed greater potency than ortho- or meta-pyridyl imide. 3-Pyridyl (5) and N-phenyl imides (14 and 16) also showed strong cytotoxicity. Compounds with planar side chains (12, 14, and 20) and N-azaethyl or N-aryl compounds (17, 18, 19, and 21) showed moderate cytotoxicity on both Hep G2 and HL-60. However, aliphatic chain imides (1, 2, and 3) were found to have very low effects in the studied cell lines (Hep G2, HL-60) (Figure 11) [33].

Figure 11.

Cantharimides are synthesized by the high-pressure addition reaction method [33].

Until 2017, about thirty cantharidin derivativeshave been isolated from insects of the genus Mylabris and Hycleus. As cantharimide compounds are known to exist in different insects from Mylabris and Hycleus species, thirteen new cantharidin derivatives have been isolated from the whole body of Mythicomyia phalerata as part of studies to discover new potential antitumor agents [34]. During this purification, dimeric cantharimides were also obtained. The cytotoxic effects of the isolated derivatives against HepG2, MDA-MB-231, and A-549 cell lines were investigated, and it was determined that all isolated compounds showed high activity, except for the compound called Canthaminomide F (Figure 12) [34].

Figure 12.

Dimeric-canthraimides isolated from M. Phaletata [34].

Due to the presence of dimeric products in cantharimides obtained from natural sources, some researchers have included these compounds in their synthesis. In a study conducted in this way, cantharimide dimers were synthesized and the structure was determined. The dimers synthesized in the same study were also isolated from natural sources. Along with these dimers, two different cantharimides were purified (Figure 13) [35].

Figure 13.

Cantharimide dimers from the Chinese Blister Beetle [35].

3. Norcantharimides

Studies on norcantharimides are examined by dividing them into two groups distribution methods and medicinal chemicals. Compounds such as norcantharimide [36] and norcantharidin dimer [37] belong to the class of medicinal chemicals. Norcantharimides are made from either the imide ring [38] or the cyclohexyl ring [39] in their medicinal chemical derivative synthesis. Then, new derivatives [40] were obtained by opening the imide ring in these derivatives (Figures 14 and 15).

Figure 14.

Examples of norcantharimide as a medicinal chemical [36, 37, 38, 39, 40].

Figure 15.

(A) Reagents and conditions: (a) Et₂O, rt., 48 h; (b) acetone, 10% Pd–C, H₂(g) 50 psi, 18 h; (c) RNH₂, PhCH3, reflux, 24–36 h (d) RNH₂, THF, rt [41]. (B) Reagents and conditions: (a) CH₃OH, rt. 30 min; (b) PhCH₃, sealed tube 200°C, 36 h.

In addition, when dimer products were detected in cantharimide derivatives purified in natural sources, dimer structures in norcantharimide derivatives were included in the synthesis [36]. Norcantharidin-dimer analogs as analogs of norcantharidin, norcantharidin with lactose acid, norcantharidin with amantadine, water-soluble norcantharidin with chitosan analogs, esterification of norcantharidin, amino acid norcantharimides, N-substituted dehydronortharimid analogs, immune liposomes, and modifications of norcantharidin show potential in the anticancer field [36].

Some norcantharidin analogs are known to have very good PP1 and PP2A inhibitory activity [22, 41, 42, 43]. In addition, studies by McCluskey et al. reported that some norcantharidin analogs, which have no toxic effect on human cell lines, kill trichostrongylid nematode Trichostrongylus vitrinus and Haemonchus contortus larvae [47].

McCluskey synthesizes a large number of norcantharimide in his studies and conducts various activity studies. In one of these studies, he synthesized 54 norcantharimide compounds and conducted a toxicity study against H. Contortus, which showed serine–threonine phosphatases (PP1 and PP2A) [48]. As a result of the study, it was determined that three of the 54 analogs synthesized were almost completely lethal against H. contortus and showed at least five times more inhibition than the control compounds. McCluskey’ reported that he synthesized 54 compounds by the methods given below Figure 14 [47].

In another study published by McCluskey in 2011, eighteen phosphate esters of the side group attached to the imide nitrogen were synthesized. It has bioscreened them in nine human cancer cell lines (HT29 ve SW480, MCF-7, A2780, H460, A431, DU145, BE2-C, SJ-G2). As a result of the study, he stated that he obtained a new series of norcantharidin analogs with broad-spectrum antiproliferative activity [41]. Another important finding from the study is the relative ease of Phosphate ester hydrolysis. Of the phosphates studied, diphenyl and bis-trichloroethyl analogs showed the highest level of cell death (Figure 16) [49].

Figure 16.

Reagents and conditions: (a) H₂N–X–OH, D, 18 h; (b) ClP(O)(OR)₂, n-Bu₂O, Et₃N, rt [49].

McCluskey also conducted biological activity studies by synthesizing norcantharidin-dimer analogs. McCluskey explained the relationship between heterocyclic substituted (nor)cantharidin analogs and PP1 and PP2A as a result of his studies. McCluskey et al. synthesized many norcantharidin analogs, including two bis-norcantharimides. Among the compounds it synthesized were compounds containing 10, 12, and 14 alkyl chains attached to the imide nitrogen, 1,2-diol units, and two norcantharimide attached to dodecyl. Among these groups, two norcantharimide (Bis-Norcantharimide) bound to dodecyl showed the highest activity in the cell line [42].

Evaluating the results obtained from this study, Cuifang Cai showed that the compound containing N-C₁₄H₂₉ (Figure 17) side group has a longer half-life and higher volume of distribution (Vss) compared to norcantharidin, as a result of the pharmacokinetic study performed by injecting it into rats. The results obtained from this study suggested that the formulation to be prepared with N-C₁₄H₂₉ is a promising alternative with its high encapsulation efficiency, significant physical and chemical stability, and long half-life (Figure 17) [50].

Figure 17.

N-C₁₄H₂₉ and norcantharimide, whose physical and chemical stability was determined by Cuifang Cai [50].

Dimeric compounds norcantharidin compounds have been synthesized and studied by many researchers for the treatment of cancer, HIV, Alzheimer’s, malaria, and various parasitic diseases. Some of the scientists who carried out these studies are Gervais [51], McCluskey [42], T. Nakatani [35], Tang [52], and Tan [38].

When Xue-Jie Tan and his group realized that good results were obtained both in extract analysis and in many synthesis and activity studies, they used four dimeric norcantharimide directly synthesized in their studies. Two of these compounds were synthesized for the first time by Xue-Jie Tan and his group, while the remaining two were synthesized by Dominic V. McGrath’s group. However, Xue-Jie Tan and his group presented the crystal structure, spectroscopic properties, and anticancer activities of these four unsaturated dimeric norcantharimides to the knowledge of researchers (Figure 18) [38].

Figure 18.

Dimer-norcantharimides synthesized by Tan et al. [38].

4. Conclusion

Alkaloids are defined as amine compounds naturally produced by plants. In addition, alkaloids are defined as secondary metabolites with important biological properties. It is also known that some alkaloids are beneficial for some diseases. In light of this information, we can evaluate cantharimide and norcantharimide derivatives in the alkaloid class, even if they are not of plant origin. Considering the information, we have briefly compiled above, the starting compound is a product of natural origin. Later, all of the synthesized derivatives contained nitrogen atoms, and positive test results were obtained on many disease-causing agents. After obtaining a large number of alkaloid cantharimide derivatives by extraction, it has also begun to be synthesized as a medicinal chemical. Researchers at the time sought to improve previous methods. In this review, it was concluded that medicinal chemical-synthesized derivatives of an insect-derived alkaloid can be used for pharmaceutical purposes.

Acknowledgments

We would like to thank Atatürk University (Project number: 2012/470 and FBG-2018-6476) for its financial support of this work.