Chemical and pharmacological information on cantharidin-based small molecules was analyzed. The review summarizes new facts about blister beetles' metabolites for the period 2006–2012. General synthetic approaches to cantharidin-based small molecules as well as their chemical transformations and biological activities related to cantharidin, norcantharidin, cantharidimide, and norcantharimide analogs, especially their inhibitory activity of phosphoprotein phosphatases in cancer treatment, were discussed in this mini review, which could help to design new small molecule modulators for other biological models.
With the development in the natural product chemistry, giving many interesting objects to study from the structural, synthetic, and pharmacological standpoints, the organic synthesis had evolved at the same rate with the generation of molecules/molecular assemblies with well-defined biological functions, and within this challenging task in the biology-oriented synthesis field, the design and development of more efficient chemical reactions and methodologies would revolutionize the next generation of chemical and biological research. Secondary metabolites of plants and animals are the main objects to study phytochemistry and organic chemistry, as shown by their centennial history that has successfully promoted the discovery and drug development. According to a recent analysis, in the last 25 years, nearly half of the drugs currently in clinical use belong to drugs of natural origin [1, 2]. Therefore, the success of drug discovery based on nature's secondary metabolites depends on how these natural substances are considered, mainly by medicinal and organic chemists, as source of inspiration rather than target molecules [3, 6].
For centuries, it has well known that many plants (shrubs, trees and grasses) possess a wide spectrum of medicinal properties and that bacterium and fungi are also capable of generating toxic constituents that inhibit the growth of other organisms in their proximity. These kingdoms have provided us more information, regarding the biological interactions of small molecules (SMs), to understand and encourage our investigations in the discovery and development of new SMs with marked physiological activities [7, 8]. In addition, these SMs are also useful for investigating biological systems as effective tools to elucidate the mechanism of important cellular processes that are based purely on (i) the performance of enzymatic reactions and (ii) protein–protein interactions, where the bioactive SMs are called bioprobes, and more importantly, (iii) the fact that they allow the rapid and conditional modulation of biological functions often in a reversible, dose-dependent manner [9, 10].
In contrast to plants and bacteria, the invertebrate animal kingdom, to which belong different insects (about a million species) including beetles, is less studied. However, particular medicinal properties of some beetles stimulated (bio)chemical researchers to study and discover new SM inhibitors with an extremely important protein affinity, which are responsible for cell proliferation. Within this group, cantharidin (CTD), norcantharidin (NCTD), and cantharimide and its analogs represent the simplest natural models that could play an important role in the search for new effective and selective anticancer drugs [11-13].
The vast volume of information concerned with these natural derivatives prevents us from preparing an exhaustive review; rather, in this mini review, we aim to present the key background information related to the chemical and pharmacological information on the SMs that metabolize blister beetles of family Meloidae for the period 2006–2012. We will focus on the biological and synthetic approaches to CTD-based SMs as well as their more recent chemical transformations, hoping that these chemical modifications in the structure of these natural products could be a real and rapid way in developing new drug candidates and could help to design new SM modulators from other biological models.
This revision is divided in the following sections: First, we will survey a minor scienti?c classi?cation of the Meloidae family; this will provide a sense of the origin, history, and the biological role of CTD, NCTD, and cantharimides; then outlines the strategies for the synthesis of CTD and NCTD analogs, while we highlights the current strategies for the synthesis of cantharidinimides and norcantharidinimides. Next, the biological role of CTD, NCTD, and cantharimides is discussed, the most important being their interaction with the protein phosphatases: PP1 and PP2A. Finally, we present some concluding remarks.
Blister Beetles of the Meloidae Family
Blister beetles are insects of the order Coleoptera (beetles) of the family Meloidae that contains about 2500 species, divided among 120 genera and four subfamilies: Eleticinae, Meloinae, Nemognathinae, and Tetraonycinae . As some of these species contain a chemical secretion of a blistering agent, they are known as blister insects, and despite the approximately 7500 species that are widespread throughout the world in warmer and drier areas, they are not considered as pest. But in New Zealand, Antarctic zones, and temperate and arid regions, as well as in the subtropical and tropical savannas, they are not present and cannot survive .
According to their life cycle, the blister beetles undergo hypermetamorphosis, in which first larva stage takes the form of a triungulin, highly mobile to search out a host, while the following stages are more sedentary and remain on or within the occupied host. The adult beetles are phytophagous, which means that their dietary is based on plants of the families Amaranthaceae, Compositae, Leguminosae, and Solanaceae, and they are easily recognized by morphological characteristics such as soft body, bright coloration, rather elongate shape, head deflexed with narrow neck, pronotum not carinate at sides, heteromerous tarsi, and smooth integument .
Many of the species of the Meloidae family are blister beetles whose metabolism provides a poisonous substance, comparable to cyanide and strychnine in toxicity, that is used by the beetles as a defensive chemical weapon to protect them from predators and that displayed severe effects on the gastrointestinal tract, kidney, and ureter in mammals, including humans. Nevertheless, this toxin has rich history and a wide range of biological activities that have affected human health for centuries.
Cantharidin and Norcantharidin: Origin and History
Since the year 1264, China began to use the extracts from the wretched insect Mylabris caragnae to remove wart, time when high incidences of deaths in the treated patients revealed the first evidence of the toxicity in humans and limited the general use of these extracts. Posterior studies revealed that the dried bodies of the Mylabris caragnae possess antitumor properties and an increased number of leukocytes, but once again, the irritant effects on the urinary system reduce their uses . In the Middle Ages, in Europe, the dried bodies of the ‘Spanish fly’ (Lytta vesicatoria), a green, 11- to 21-mm-long beetle that can be found in certain areas of Europe and some eastern regions such as Vietnam, Taiwan, Thailand, Korea, and China, began to be used as an alleged aphrodisiac and abortifacient and in the treatment for malignant tumors as an extended application in the Chinese medicine. But its use resulted in poisoning side effects in humans .
To investigate and explain the mode of action, as well as the acute toxicity, of the extracts of the Spanish fly and based on the observation that when these beetles were pressed, rubbed, or squashed by any external agents, they expel a hemolymph ‘blood’ from the joints in the legs as a chemical defense, giving any would-be predators a foul-tasting appetizer, in 1810, the French pharmacist Pierre Jean Robiquet, by grinding and drying the bodies of the Spanish fly, isolated a crystalline compound, known today as cantharidin 1, whose correct structure was later proposed by Gadamer and his students [18, 19]. Cantharidin, or exo,exo-2,3-dimethyl-7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid anhydride 1, is a monoterpene stored in the insects' blood. It is stable and remains toxic even in beetle carcasses for long periods of time, being classified as a strong vesicant substance that causes blisters on contact with the human skin, giving the fly the name blister beetles. The CTD concentration in the blister beetles differs between species: The highest reported CTD content for a blister beetle is 5.4% in the dry weight bodies of Epicauta vittata, while in the ‘Spanish fly’, the CTD content is about 5%  (Figure 1).
Unlike CTD, (±)-palasonin, the closer demethyl analog, is the only CTD analog found in both plants and beetles. (S)-(-)-palasonin 2 has been isolated from the seeds of an Himalayan plant, Butea frondosa (Leguminosae), while the (R)-(+)-palasonin 3 is present in the dried bodies of the Hycleus oculatus and H. tinctus beetles, a southern African Meloid species [21-25]. Nonetheless, both natural sources provide very small quantity of this molecule  (Figure 1).
Cantharidin is found in the blood of both male and female blister beetles, but oddly enough, it is ‘bio-synthesized’ only by the males, which use it as a male courtship pheromone . It was reported that CTD can act as an excitatory pheromone for male fire-colored beetle Neopyrochroa flabellata, which consumes this molecule and produces a CTD-rich secretion from the cephalic gland during courtship. Thus, CTD has an important function in an aphrodisiac-like capacity in this particular insect, especially during mating . Cantharidin is transmitted to females for the protection of eggs, with an intention to cover and preserve the life of the larvae during their early stages of development [29, 30]. However, not only protection is the primary role of CTD in the life cycle of the beetles, but this chemical weapon with odor and bad taste is released in small doses when the adult specimens are endangered and are swayed by their predators. As has been described earlier, CTD is exclusively biosynthesized by male beetles of the family Meloidae. Although detailed mechanism of biosynthesis of CTD is unknown at present, the above facts can conclude that the production of CTD may be a more complicated process and seem to even cast some doubts on the mevalonoid origin of this compound; however, specialized researches on this have suggested that instead of the two obvious possible routes – the tail-to-tail condensation of two isoprene units and the head-to-tail condensation of two isoprene units followed by a methyl shift – several unknown oxidation processes from farnesol 4 would be involved in CTD biosynthesis [31, 32] (Figure 2).
Once the pharmacological and toxicological properties of CTD become known around the world, the responsibility of development of more selective and bioactive CTD derivatives with lower toxicological profiles lied on organic chemistry. Using the structural modification of CTD as a real and rapid way to develop new chemotherapeutic agents, new CTD derivatives, NCTD 5 and 5,6-dehydronorcantharidin 6, were prepared. To date, any of these derivatives have been isolated or are present in any natural source, but they have been synthesized easily from commercial reagents with the advantage that these analogs have reduced the intrinsic toxicity of CTD, while its biological activities (especially the anticancer activity) are retained [33, 34] (Figure 3).
Cantharimides: Origin and History
At the beginning of the last decade, all the studies performed in other species of the Meloidae family had as the main objective the isolation of CTD and other oxygen analogs (e.g., NCTD). However, when these studies were extended to the Chinese beetles, whose habitats principally are Korea and China, in the dried bodies of Mylabris phalerate PALLAS, besides the presence of CTD and NCTD, a new class of compounds was discovered: the cantharimide 7 and the 5-substituted cantharimide 8; nonetheless, these substances were found in very low concentrations, ≤0.002%  (Figure 4).
The main structural difference between CTD and cantharimides is that the oxygen atom in the anhydride core of CTD is replaced by a nitrogen atom, which may or may not be substituted; this improved the solubility and the cytotoxicity of these derivatives against human hepatocellular carcinoma cell lines . Although the biological role of CTD is well known, the role of cantharimides in the life cycle of the beetle Mylabris phalerata and the process by which CTD is biosynthesized in the insect are still unknown; probably, CTD undergoes a chemical transformation in which oxygen is replaced by nitrogen through some kind of biochemical transformation or by an enzymatic source of nitrogen, although these hypotheses are unconfirmed and will encourage further investigations related to these issues (Figure 5).
Strategies for the Synthesis of Cantharidin and Norcantharidin Analogs
Being anhydrides of the 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid core, the CTD and NCTD molecules can be formally considered as Diels–Alder cycloaddition adducts from furan derivatives and maleic anhydride and presented a well-defined exo-stereochemistry of oxygenated ring and in the 7-oxa-norborn-2-ene system. Once the synthetic protocol for the preparation of these derivatives had been established, the further investigations focused their attention on the structural transformations of the CTD and NCTD cores to increase the bioactivities and reduce the toxicity of these natural products.
Cantharidin and Norcantharidin Synthesis Via Diels–Alder Reaction
The structural analysis of the natural CTD 1 and (±)-palasonin 2 molecules indicated that the most obvious and current protocol that could be adopted for the synthesis of this derivatives is the direct cycloaddition of furan, dimethylmaleic, and/or citraconic anhydrides 9 and 10, followed by the hydrogenation (H2, 10% Pd/C, THF) of the respective adducts 11 and 12, and the unsaturated CTD and palasonin analogs. In general, the reduction of the double bond, besides providing the desired natural products, has to be done because of their high instability; as the predominant aromaticity of furan makes it a poor Diels–Alder diene, the Diels–Alder adducts are not thermally very stable and it is known that they undergo a retro-Diels–Alder reaction, regenerating the original starting materials. As most of the subsequent steps are intended to modify the derivatives 11 and 12, these procedures usually require prolonged reaction times and high reaction temperatures, so the double bond needs to be removed to prevent the retro-Diels–Alder reaction (Figure 6). As an interesting fact, the dimethylmaleic anhydride 12 cannot react itself with another molecule of furan in a subsequent cascade reaction, even at pressures up to 40 kbars, so some authors have suggested that the failure of this is a result of both electronic and steric factors. Nevertheless, this reaction failed in the terms of the ‘classic conditions’ of the Diels–Alder cycloaddition (the use of solvents such as Et2O, CHCl3, benzene, toluene at room temperature or reflux) [37, 38].
The complete synthesis of CTD was performed in the 1950s by Stork [17, 39] and Schenck . Both groups used a linear synthetic tactic, using the Diels–Alder reaction between different dienes and dienophiles. But the breadth and complexity of these synthetic efforts contrast with the simple structure of these molecules. But with the development of high-pressure Diels–Alder reaction (8–15 kbar, 138 h) of furan and anhydride 10, the large-scale synthesis of (±)-palasonin was achieved and the racemic mixture was resolved with the preparation of amides using two equiv of (S)-(-)-α-methylbenzylamine .
Today and with the same approach, Diels?Alder reactions at high-pressure, between furan and 2,5-dihydrothiophene-3,4-dicarboxylic anhydride 13, have been used as a straightforward method for the preparation of CTD 1, with the advantage that the dihydrothiophene ring induces the selective formation of the exo-cycloadduct 14, which is then reduced under catalytic conditions with Raney Ni for the formation of CTD 1 in 72% yield, process that has been employed to obtain 1 in large scale  (Figure 7).
It should be noted that the Diels–Alder reaction of furans with dienophiles usually results in the formation of the thermodynamically more stable exo product, while the kinetic endo product, once is formed, undergoes a facile retro-reaction to form the starting materials and is rarely observed in synthetic procedures. However, a dramatic acceleration rate of the Diels–Alder reaction of furans with anhydrides as a dienophiles generates the endo products when a 5 m lithium perchlorate–diethyl ether medium is employed .
Structural Modifications Performed on the Cantharidin and Norcantharidin
The acute toxicity exhibited by CTD and NCTD has motivated the organic chemistry to modify the chemical structure of these natural derivatives and in that way to improve their pharmacological profiles and reduce their toxicological properties. The retro-synthetic analysis of the CTD and NCTD structures revealed in first place that intrinsic modifications on the furan and in the anhydride rings could generate diverse libraries of new CTD analogs that have been the focus of a wide range of bioassays in which have been discovered novel pharmacological agents.
Substitutions on the 7-oxabicyclo[2.2.1]Heptane Core
With the hypothesis that fluorine substitution in several SMs often leads to modification and improvement in the biological activities and/or reduction in the toxicity in comparison with the corresponding non-fluorinated parent compounds , in 2001, Essers et al. reported the synthesis of fluorinated maleic anhydrides 15 and 16 and studied their behavior as a dienophile in the Diels–Alder reaction with furan. In case of anhydride 15, the reaction occurred at 50 °C within 42 h and without any solvent. However, the expected adduct exo-17 was not observed, suggesting a rapid hydrolysis of the cycloadduct when this product enter in contact with moisture, leading to the formation of endothall derivative 18 (Figure 8A). The second fluorinated anhydride 16 reacted with furan at 55 °C within 52 h and once again without using any solvent to give the fluorinated dehydropalasonin analog 19 with an exo,exo stereochemistry  (Figure 8B).
Besides furan, another heterocyclic compound commonly used as a diene for the synthesis of CTD analogs is thiophen, but usually this compound does not undergo the Diels–Alder reaction with maleic anhydride under the same conditions depicted in Figure 8 because thiophen is a very poor diene than furan, due to its aromatic nature; however, the reaction of thiophen with maleic anhydride had been promoted by high pressure in dichloromethane at 40 °C for 71 h to yield the exo-7-thiabicyclo[2.2.1]heptane cycloadduct 20 in 60%  (Figure 9).
2-Substituted furan rings have also been used as a diene in the Diels–Alder reaction with maleic anhydride. Unfortunately, it seems to be that the length of the chain, as well as the presence of withdrawing groups in this chain, has a negative impact during the reaction due to the inductive effect of these groups. This statement can be evidenced when 2-cyanomethyl furan 21 and N-acetyl furfurylamine 22 are used as a dienes, giving the corresponding adducts 23 (19%) and 24 (23%) in very poor yields  (Figure 10).
Based on computational docking experiments, Tatlock et al. modified the C-5 position of the 7-oxabicyclo ring system using 3-substituted furan rings. When 10 different carboxylic acids 26a–j were esterified with 3-hydroxymethyl furan 25, different types of dienes were generated, 27a–j, and those compounds undergo the Diels–Alder reaction with maleic anhydride in diethyl ether at 23 °C. The reactivity of the dienes 27a–j can be compared with the reactivity of dienes 21 and 22, in which products 23 and 24 were obtained in much lower yields than the adducts 28a–j, so it can be concluded that in those cases, the presence of substituents in C-2 position of the furan ring, besides the electronic effects, could generate an steric impediment during the reaction, which affects the final yields, while if the substitution is in the C-3 position of the furan ring, this effect does not represent any negative impact  (Figure 11).
It was reported that the reaction between maleic and furfuryl alcohol at 69 bar and 35 °C proceeded 10 times faster in sc-CO2 compared with reactions carried out in diethyl ether; this obviously illustrates the extraordinary potential of sc-CO2 as synthetic media to access molecules unobtainable using conventional synthetic methodologies .
The avoidance of usage of organic solvents is one of the goals in current efforts toward the design of more environmentally benign chemical processes. Thus, sc-CO2 is non-toxic alternative solvent in organic chemistry. However, its use requires more sophisticated equipment than standard laboratory apparatus. The scope of reaction media for Diels–Alder reactions is rather large, including water , ionic liquids , polyethylene glycols , and mixtures of simple carbohydrates (fructose, sorbitol, glucose, etc.), urea, and inorganic salts .
Reduction and Substitutions of the Carbonyl Function on the Anhydride Core
Tarleton et al. modified the structure of NCTD by reducing one of their carbonyl functions to produce the (3S,3aR,4S,7R,7aS)-3-hydroxyhexahydro-4,7-epoxyisobenzofuran-1(3H)-one 29, and promoted by the structure of this cyclic lactone, different alkylation reactions of the hydroxyl group with short and large chains were performed under microwave irradiation, yielding 18 compounds  (Figure 12).
In the same study and with the hypothesis that the phosphate group of natural fostriecin 31 plays an important role in the broad spectrum of anticancer activities displayed by this molecule , a new series of compounds 32a–d were synthesized with a terminal phosphate moiety employing various chlorophosphates and in the presence of dibutyltin oxide to obtain the desired products in good yields (Figure 13).
Strategies for the Synthesis of Cantharidimide and Norcantharidimide Analogs
Being cyclic imides of the 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid core, cantharidimides and norcantharidimides, as well as CTD and NCTD, can be formally considered as Diels–Alder cycloaddition adducts from furan derivatives and substituted maleimides. However, this methodology has not been useful for the preparation of these derivatives due to the low selectivity to obtain the exo-adduct and to the limited structural diversity of the synthetic maleimides (most of them have to be prepared in a two-step methodology). With the discovery of the condensation of CTD and NCTD with primary amines, a wide range of compounds has been synthesized, setting this methodology as the most powerful strategy for the preparation of cantharidimide and norcantharidimide derivatives, less toxic, and with similar bioactivities exhibited by CTD and NCTD.
Norcantharidimide Synthesis Via Diels–Alder Reaction
The preparation of exo-5,6-dehydrocantharidin 6 and its saturated analog NCTD 5 is more easy achieved using classic reaction conditions (THF or Et2O, 16 h, rt) for Diels–Alder cycloaddition of furan and maleic anhydride. Similar results were obtained when the same reaction conditions were successfully applied to the reaction of furan with N-methylmaleimide 33 to give both diastereomers, N-methyl-7-oxabicyclo[2.2.1]hept-5-ene-2-endo,3-endo-dicarboximide 34 and N-methyl-7-oxabicyclo[2.2.1]hept-5-ene-2-exo,3-exo-dicarboximide 35 in good yields after chromatographic separation  (Figure 14).
Structural Modifications Performed on the Cantharidimide and Norcantharidimide Analogs
Having discovered the existence of cantharimides and norcantharimides, the world found once again in nature, the solution to the acute toxicity problem of CTD and NCTD. These compounds disclose a major solubility and proved to be less toxic, and due to the presence of heterocyclic nitrogen, many structural modifications can be carried out on this atom. As well as in the case of CTD and NCTD, intrinsic modifications on the furan ring could generate diverse libraries of new cantharidimide analogs in addition to those generated by the N-substitution.
Cantharidimides and Norcantharidimides Analogs Synthesized by a Dehydrative Condensation of CTD and NCTD with Primary Amines
One of the first reports related to the synthesis of cantharimides was published by McCluskey et al. in 2001. As the early modification of CTD with primary amines, the reaction of NCTD with different amino acids was performed with triethylamine and anhydrous toluene at 200 °C for 24 h. As a result, 16 new norcantharimides 36a–p were obtained in moderate yields, and the use of both D- and L-amino acids allowed the examination of the effect of these different stereoisomers  (Figure 15).
Using a similar procedure for the synthesis of the related amino acid-substituted N-derivatives 36a–p, 13 analogs of norcantharidimide were prepared using the same reaction conditions described in Figure 15. For the first library, 12 N-alkyl amines were employed having short and long chains, cycloalkanes, and unsaturated groups, for the N-alkylation reaction of NCTD 5 to yield the desired products in moderate-to-excellent yields (31–98%) compared with the ones that exhibit by the series of amino acids. In the second library of nine compounds, different N-alkylated NCTDs were prepared containing in the alkyl chain hydroxyl groups and terminal carboxylic acid groups with moderate-to-good yields (22–80%). The morpholine moiety was included as well in three derivatives when it is bonded to the nitrogen directly and by an alkyl chain of two and three carbons, unfortunately in very low-to-moderate yields (7–43%). A fourth library of seven compounds was prepared from substituted anilines and benzylamines; due to the less reactivity of anilines, these derivatives were obtained in moderate-to-good yields (36–79%) in comparison with the benzyl products (43–91%), in which the substrate is considered more as a primary amine, similar to the ones described for the first library.
Finally, the remarkable compounds synthesized in the study performed by Hill et al.  are the bis-NCTDs 39 and 40 prepared with 1,3-diaminopropane 37 and 1,12-diaminododecane 38 under the standard conditions, described in Scheme 15, afforded the rest of the N-alkylated NCTDs (Figure 16).
With an idea to provide a new direction for novel drug discovery in experimental cancer therapy, it has been established that CTD can be combined with basic hetero-molecules in a single molecule through chemical modifications. Indeed, CTD-containing N-hetero-molecules 41 and 42 were easily prepared via a one-pot condensation reaction from CTD 1 and 2-aminobenzothiazole derivatives 43 and 5-amino-1,3,4-thiadiazole-2-thiol 44 [59-61] (Figure 17).
As was conducted for the preparation of bis-NCTDs 39 and 40, and promoted by the structural diversity and the high yield in which compound 42 was obtained, additional efforts to prepare the dimer of cantharimide 42 has lead the formation of bisnorcantharimide 45 using piperazinium dichromate as a promoter at room temperature, unfortunately the low yield in which 45 was obtained did not encourage the extension of this methodology to prepare other derivatives  (Figure 18).
Previous synthetic efforts have shown that NCTDs can be synthesized using high temperatures and using toxic and corrosive solvents. Recently, alternative approaches to access new NCTD libraries were investigated; from this study, the most notable protocol so far involves the use of microwaves to accelerate the reactions with the use of alternative solvents. With this approach, Thaqi et al.  reported the synthesis of three new libraries of NCTD analogs in which, besides the N-substitution with amino acids and alkyl chains containing or not hydroxyl groups, the 7-oxabicyclo[2.2.1]heptane core was replaced by the hetero-substituted 5,6-ethyl bridge, modification that intended to improve the biological activities of these derivatives, through a reaction carried on water or dimethylacetamide at 170 °C and assisted by microwaves (Figure 19).
One way to combine the NCTD ring with potentially bioactive hetero-molecules, similar to chloroquine, was developed in a two-step methodology that involves the first amidation and formation of open anhydride ring followed by the dehydratation (ring closure) of this intermediate to yield the novel 5,6-dehydronorcantharimide-chloroquine hybrids 56 and 57 in excellent yields, compounds that are being investigated for their antitumoral activity  (Figure 20).
Another study on chemical modifications on the scaffold 5, start with the Wittig reaction between NCTD 5 and the phosphorous ylide 58 in order to modify one of the carbonyl functions of 59 and with the subsequent nucleophilic ring opening of the ester, promoted by the amine, has generated the formation of a new library of 22 compounds 60, which contain the ethereal bridge (7-O) of cantharimide and a wide range of substituents bonded to the nitrogen atom (64), within which are: aliphatic, branched aliphatic cycloaliphatic and aromatic, basically the same substituents used by the same authors in their previous works [54, 62] (Figure 21).
To complement the previous works related to the synthesis of cantharimide analogs from amino acids [57, 62], Cheng et al. highlight in their study that the reaction of NCTD 5 with l-histidine 61 resulted in the formation of L-histidine norcantharimide 62 in 97% yield, avoiding the use of toxic solvents such as toluene and triethylamine as a catalyst, improving the reaction conditions with the use of ethanol and under low ranges of temperatures. However, compound 62, due to the proximity of the carboxylic function with one of the nitrogens of the imidazole ring, forms zwitter-ionic species in equilibrium with derivative 62  (Figure 22).
The same authors who reported the inclusion of the phosphate group of fostriecin 31 in the chemical structure of NCTD derivatives with the intention to increase the biological properties of these compounds explored the incorporation of the phosphate group in the terminal free -OH moiety of some of the derivatives that they introduced in their work . With similar methods described above, six N-alkyl substituted cantharimides 63a–f were treated with different diethylchlorophosphates in the presence of n-butyltin oxide and triethylamine to give the desired terminally substituted phosphate analogs, for example 64a–f, 65a–f, and 66a–f  (Figure 23).
The dehydrative condensation of CTD and NCTD with primary amines had been the main methodology for the synthesis of cantharidimide derivatives. In addition, two recent reports have been published on the synthesis of 34 derivatives with the same conditions depicted in Figure 15 in poor-to-good yields (5–82%) . The problem of the low reactivity of anilines during this reaction mentioned in Figure 16 was overcome by changing the reaction conditions by Deng et al.; this approach involves the use of triethylamine, acetic anhydride, and manganese (II) acetate as a catalyst. With this methodology, 20 N-phenylnorcantharidimides were prepared in good-to-excellent yields using anilines with both donating and withdrawing groups [67, 68] (Figure 24).
Substitutions on the 7-oxabicyclo[2.2.1]Heptane Core
Substitutions and modifications on the 7-oxabicyclo[2.2.1]heptane core have been achieved by making react the double bond of NCTD derivatives obtained through the Diels–Alder reaction, as well with the use of substituted dienes in this reaction. As an example for the first case, after the synthesis of compound 35 was reported, in 2010 Goksu et al.  converted this adduct into the interesting exo-arylated product through the Heck arylation, generating 5-substituted NCTDs 67a–c in good-to-excellent yields (Figure 25).
With the use of thiophen for the preparation of CTD analogs with the tioetheral bridge (7-S), the methodology developed in that opportunity was extended to the reaction of thiophen with maleimide 68 under the same conditions depicted in Figure 9; high pressures were required and dichloromethane was also used at 40 °C for 71 h to yield the exo-7-thiabicyclo[2.2.1]heptane cycloadduct 69 in 60%  (Figure 26).
One strategy to combine reactive 5,6-dehydronorcantharidin derivatives depicted in Figure 23 with nitrile oximes 70 was developed to generate the 5,6-dehydronorcantharidin-isooxazolidine adducts 71. Through a 1,3-dipolar cycloaddition reaction between those molecules in the presence of tert-butyl hypochloride  or chloramine-T , 20 of these tetracyclic compounds were prepared in good yields with the important pharmacophore group, the 3,4,5-trimetoxyphenyl moiety, responsible for several anticancer activities (Figure 27).
One of the used 2-substituted furan rings that have generated the corresponding adducts 23 and 24 in very poor yields according to Figure 10 was recently used once again as a diene for the Diels–Alder reaction with different N-substituted maleimides 72a–c. This reaction was performed using boric acid as a catalyst with the hypothesis that the yield and the stereoselectivity of the obtain product will be increased through the possible interaction of the boron atom with the carbonyl functions of the maleimides 72a–c. In this order, and also employing a green and mild solvent like polyethylene glycol 400 (PEG-400), the new series of N-(10-oxa-4-azatricyclo[5.2.1.02,6]decan-8-ene-3,5-dione-7-ylmethyl)acetamides 73a–c were obtained in excellent yields  (Figure 28).
Biological Activities Related to Cantharidin, Norcantharidin, Cantharidimide, and Norcantharimide Analogs
Despite many studies on the physiological and biochemical effects of CTD and its analogs, their mechanism of action only could be elucidated until 1992, when Li and Casida demonstrated that CTD and their anhydride analogs can bind, with high affinity, to a specific CTD-binding protein (CBP), whose isolation was reported from the cystol of mouse liver . With the identification of that CBP, a series of studies begun to investigate the nature of this enzyme and the relationship between it and the chemical structure of the CTD and its analogs, natural and synthetic. The acute toxicity exhibited by these SMs delayed their use in the pharmaceutical industry; however, the organic chemistry has provided new and more potent derivatives with high activity against protein phosphatase enzyme and with less toxicity profiles. Although this bioactivity has been deep explored during the last five decades, CTD analogs has been used in the treatment for other important diseases due to the development of new strategies for the oral and intravenous administration of these derivatives.
The Phospho Protein Phosphatases: PP1 and PP2A. The Main Biological Targets of Cantharidin and Cantharidimide Derivatives in Cancer Treatment
Cantharidin and its derivatives act by causing cell death. To explain this fact, many mechanisms of action have been proposed, and the most accepted of them involves a response to DNA damage and apoptosis through the inhibition of PP. With the knowledge that most of these relevant cellular processes involve protein–protein, protein–nucleic acid, protein–SM ligand, and the enzyme–SM interactions, the latter has been recognized as one of the most important relationships for metabolism regulation and plays a key role in initial stages of signal transduction. This pharmacological approach, in which enzyme function (as protein-based catalysts for chemical reactions in living organisms) is modulated by these SMs, represents a milestone in our understanding of life and in our development of cures. Therefore, the study of enzymes is still a central focus of modern biochemical research, and among the enzyme superfamilies, protein kinases (PK) and PP have occupied a preeminent position in this research [72-75].
Nowadays, it is well established that the reversible phosphorylation of proteins is a key regulatory mechanism in animals and higher plants cell division, and the level of phosphorylation of any protein depends on the relative activities of PK and PP . Structurally, PK can be divided into three groups: histidine kinases, cAMP-dependent protein kinase-like kinases, and other PK. While, there are three major classes of PP: tyrosine-specific, serine/threonine-specific, and dual-specificity phosphatases. The serine/threonine family has the ability to influence in the cell cycle control and growth process, in addition to be the enzyme to which CTD and its analogs are bonded. Traditionally, the serine/threonine family has been classified into several groups, with numerous members in each, based on their biological characteristics, sensitivities to specific inhibitors, and substrate specificity: PPP1, PPP2, PPP3, PPP2C, PPP4, PPP5, and PPP6 .
The protein phosphorylation–dephosphorylation process consists of two thermodynamically favorable reactions: the transfer of phosphates from a high-energy adenosine triphosphate molecule onto the proteins is catalyzed by PK, while PP, on the other hand, removes the phosphate group by the water-driven hydrolysis of phosphoester bonds. The phosphoryl group transfer is addressed to a nucleophilic acceptor group located on the amino acid side chain(s) of a protein. Typically, the principal acceptor sites are the hydroxyl groups of serine, threonine, and tyrosine; the carboxylate groups of aspartic and glutamic acids; and the nitrogen atoms on the side chains of histidine, lysine, and arginine  (Figure 29).
This reversible phosphorylation–dephosphorylation reaction is a rapid and efficient way to change the properties of proteins in a desired manner in the cellular environment, and any derailment in the action of these enzymatic reactions can lead to numerous diseases. As this switch mechanism plays an important role in signaling pathways that control cell proliferation and carcinogenesis, the design and development of a SM modulator of the PK and PP functions are cornerstones in understanding cancer, Alzheimer's disease, and others. Thus, each new potent SM modulator on these proteins may provide potential drug for cancer (or cystic fibrosis, immunosuppression and, cardiac and neurological disorders) therapy because inhibition of any one of these proteins stops the signaling cascade [77, 79].
Li and Casida  concluded that the enzyme in which CTD is bonded was phosphoprotein phosphatases 1 (PP1) and 2A (PP2A, AC type, EC 126.96.36.199). The special attention is that they might influence secretory processes, phosphatase-linked mechanisms that have been implicated extensively in several endocrine tissues, including islets of langerhans and chromaffin and pituitary cells . PP1 and PP2A exert also their effects by modulating the activity of cyclin-dependent kinases (cdk) and the retinoblastoma protein (pRb), and knowing that the activation of cdk/cyclin complexes requires the phosphorylation of a conserved threonine residue, as well as the removal of inhibitory phosphorylations, these enzymes play a crucial role in other biological process .
PP2A is one of the most studied PP enzymes and its inhibition is associated with the toxicity to most of the organisms, but this property has been useful in cancer chemotherapy, and CTD has demonstrated that that can be used as an inexpensive and readily available probe for analyzing the regulatory phosphorylation–dephosphorylation events mediated by PP2A and other PP. The core enzyme of PP2A consists of a catalytic subunit (PP2Ac) and a regulatory subunit known as A subunit (PP2Aa). Actually, four different families of B subunits have been identified, and they modulate the substrate specificity of PP2A . Interestingly, although PP2A is generally considered to be a cancer repressor, sometimes the inhibition of PP2A has been thought to be cancer-promoting by induction of phosphorylation and activation of several substrate kinases [82, 83]. However, some kinase-dependent growth inhibition pathways that are induced by PP2A inhibitors have recently been reported, suggesting that the activation of kinase pathways may not always be a cancer-promoting agent . Besides their presence in mammals, the enzyme PP2A is also expressed in plants, where CTD possess an herbicidal activity, but its mode of action is not yet clarified and it is presumed that the inhibition of plant PP2A activity is accompanied by a decreased light-induced activation of nitrate reductase .
After the nature of PP2A was established, many synthetic CTD analogs have been prepared and evaluated by McCluskey et al., who published some excellent reviews on this theme [11-13]. The straightforwardness of various (nor)cantharidin analogs prepared via Diels–Alder cycloaddition allowed them to study the relationship between structure and the anticancer activity through the enzymatic inhibitory activity of PP1 and PP2A. The mode of action of CTD and its derivatives is based on causing cell death, and many of the proposed mechanisms involve response to DNA damage and apoptosis through the inhibition of PP [86, 87].
Recently, it was shown that the anhydride rings of CTD and NCTD are hydrolyzed when they bound to the catalytic domain of the human serine/threonine protein phosphatases 5 (PP5c), and the high-resolution crystal structures of PP5c complexed with the corresponding dicarboxylic acid derivatives of the two molecules. However, NCTD shows a unique binding conformation with the catalytically active Mn2PP5c, while CTD is characterized by a double conformation in its binding mode to the enzyme. Different binding modes of NCTD are observed depending on whether the starting ligand is in the anhydride or in the dicarboxylic acid form. All these structures have been provided the basis for the rational design of the CTD-based drugs known so far, and it will be the bases for future design of novel inhibitory agents of PP1 and PP2A [88, 89].
Each synthetic work related to the synthesis of CTD, NCTD, and cantharidimide derivatives is strictly accompanied and complemented with the biological evaluation of these derivatives, more exactly their PP1 and PP2A inhibitory activity. In the following table, for each of the synthetic approaches described in the sections below, the representative CTD-based SMs with the 7-oxabicyclo[2.2.1]heptane core, which have present the highest values of their enzyme inhibitory activity, against the enzyme PP1 and PP2A, are shown (Table 1).
Table 1. The more potent inhibitors of protein phosphatases 1 and 2A among the known CTD, NCTD and cantharidimide derivatives reported so far
From the results derived in Table 1, we can establish a preliminary analysis based on the chemical structure and the inhibitory activity exhibited by active molecules 1–8. The six exchangeable points for further structure?activity relationship studies are marked with arrows in Figure 30, according to what we propose, that would influence future researches related to the synthetic and evaluation of CTD, NCTD, and cantharidimide analogs, it can be observed that (i) molecules that lack substituents at positions C-1 or C-3 are more active in both types of activity; (ii) the saturation of C5-C6 bond appears to affect the inhibitory activity, but not as much as the first factor; (iii) the type of heteroatom in the bridge (X1; position C-7) defines too much the power of studied molecules; (iv) the type of heteroatom (X2)-fused cycle is also important; (v) molecules with methyl groups at positions C-2 and C-3 (just one example, CTD itself) are more potent, but usually more toxic; and (vi) the nature of fragment B considerably defines the type of activity of the CTD derivative.
To validate the statements described above, a similar relationship between the structures of the derivatives depicted in the sections that described the synthetic approaches and their anticancer activity, through the comparison of their growth inhibition (GI50, μm) values in various tumor cell lines, once again the more potent analogs were selected and they are shown in Table 2. In conclusion, the same relation described in Figure 30 can be applied in future designs for the synthesis of CTD derivatives with potent and selective anticancer activities against several human cancer cell lines. In general, due to the possibility to incorporate any kind of substituents in the nitrogen of cantharidimides, these derivatives show more anticancer activities and lead to the construction of versatile derivatives.
Table 2. Lowest GI50 values in μm of CTD, NCTD and cantharidimide derivatives reported so far against a panel of human cancer cell lines
Tumor cell line
A2780: Human ovarian carcinoma; G401: Human kidney carcinoma; H460: Human lung carcinoma; L1210: Murine leukemia; HT29: Human colorectal carcinoma; SW480: Human colon carcinoma; MCF-7: Human breast carcinoma; A431: Human skin carcinoma; DU145: Human prostate carcinoma; BE2-C: Human neuroblastoma; SJ-G2: Human glioblastoma; N.D.: Not determined.
Because CTD represents the simplest synthetic target among all known naturally occurring toxins (okadaic acid 74, tautomycin 75, microcystin LR 76, fostriecin 31 among others; Figure 31), the synthesis of numerous CTD-like molecules has been performed to improve PP1 and PP2A binding properties (PP1/PP2A inhibition selectivities) as a strategy for the discovery of novel potential anticancer agent [11-13]. However, CTD and NCTD are the archetypal SM protein phosphatase inhibitors [11, 36]; the simple and rigid CTD (7-oxabicyclo [2.2.1] heptane) structure gives a little possibility to modify structurally this tricyclic molecule, although all synthetized CTD analogs have, at best, maintained similar levels of PP2A selectivity and potency.
Further developments toward a new class of CTD analogs allowed to discover more potent enzymatic inhibitors, for example cantharimides with a basic amino acid residue that facilitates inhibition of PP1 and PP2A or ring-opened CTD analogs with only one free carboxylate group, that not only retains inhibitory activity against PP1 and PP2A, but also increases slightly the selectivity toward PP2A, which mediated most cellular processes and was recognized as a potential target for medical research, especially for cancer treatment (Tables 1 and 2).
Cantharidin and Cantharidimide Poisoning and Toxicity
The cases of CTD poisoning have been documented in humans due to the ingestion of the blister beetles (especially the Spanish fly, Lytta vesicatoria). Poisoning usually results after CTD has been ingested as aphrodisiac, diuretic, to induce abortions or for topical uses for dermatologic treatments, and this goes back to the ancient medicine . In addition to this occurrence of self-poisoning, several cases of poisoning have been also described in horses, sheep, and chickens due to the ingestion of wild blister beetles, interestingly in wild birds that eat this beetles; the CTD poisoning was only described recently in a male great bustard (Otis tarda) .
Some studies have concluded unequivocally that these derivatives constitute a potential risk for the population that lives around this beetle and/or consume drugs where the active ingredient is a CTD derivative, whose effect goes from mild poisoning to death. The main symptoms of the population that is poisoned by these beetles are cramping, abdominal pain, vomit, and dysphagia, and recent studies intend to identify the main metabolites, in rat's liver, of this compound using gas chromatography/mass spectrometry . Unfortunately, to date, any kind of antidote to treat the poisoning of this toxin has not been discovered .
One of the most relevant cases of CTD poisoning was reported in a 4-year-old girl from Zimbabwe when she ingested the blister beetle Mylabris dicincta. The girl presented many of the classic signs and symptoms of CTD poisoning including hematuria, diarrhea, and abdominal pains, and after 9 days in the hospital, she was managed conservatively and got recovered during this period of time .
Cantharidin is extremely toxic for mammals, the fact that counters its potent anticancer activity; the fatal dose is estimated in a range from 10 to 65 mg, while its median lethal dose (LC50) is <0.5 mg/kg. The death of the poisoned individual results from renal failure and several injuries in the gastrointestinal tract; surprisingly, NCTD and some cantharidimides, which not have been involved in any cases of human poisoning, do not have this renal toxicity, and this fact has driven the design and synthesis of more NCTD and cantharidimide derivatives than CTD-based molecules [92, 97].
Additional Bioactivities and Pharmacological Uses of Cantharidin and Cantharidimides
Besides the inhibition of PP1 and PP2A, as well as their anticancer activity, CTD, NCTD, and cantharidimide derivatives might possess additional activities in which these SMs can have a positive biological response through other metabolic ways and/or might interact with other proteins or enzymes and in that order and lead the discovery of new biological targets or roles of these natural molecules. Some studies (two so far) that intend to redirect the pharmacological uses of CTD, NCTD, and cantharidimide derivatives and contribute to the little known bioactivities of these natural molecules, besides their anticancer effects, have been published, and the immunomodulatory activity of CTD against dendritic cells and the antiplasmodial activities against different Plasmodium falciparum Malaria strains would be the activities in which future studies will focus their attention [98, 99]. However, other preliminary studies have been performed on some other diseases where the effect of different doses of CTD on Leishmania major (MRHO/IR/75/ER) was investigated both in vitro (promastigote and amastigote viability) and in vivo in experimentally infected BALB/c mice (skin lesions) using ointment or soluble CTD . This report showed that CTD at concentrations of 0.5, 1, 2, 5, 10, 20, and 50 μg/mL inhibited the growth of L. major promastigotes after 24 h and the resultant inhibition levels were 39.22%, 41.95%, 49.88%, 54.78%, 58.01%, 68.30%, and 80.04%, respectively, and that 2 weeks of topical treatment with 0.1% CTD ointment was an effective method for treating cutaneous leishmaniasis in infected BALB/c mice.
Exhibiting exquisite PP-inhibitor activity, some CTD analogs could be designed and tailored to specifically inhibit selected STPs of nematodes . Preliminary investigation has shown that some CTD analogs 76a–c (Figure 32), which have no toxic effect on human cell lines, kill larvae of the trichostrongylid nematodes Haemonchus contortus, displaying 99–100% lethality to this parasite in the larval development assay, with LD50 values in the range of 25–40 μm .
Current Strategies Developed to Increase the Bioavailability of Cantharidin Derivatives
Toxicity is not the only problem to reject the biological benefits of CTD and its derivatives. As many of the natural and synthetic molecules tested in different bioassays and clinical trials, CTD presents problems of administration, solubility, bioavailability, and metabolism that have also boosted the study and synthesis of other derivatives.
The pH in mammal's blood and tissues is usually maintained within a narrow pH range around 7.4, and this slightly alkaline pH is mainly maintained through the regulation of respiration and renal acid extrusion. Nevertheless, as a consequence of lactate accumulation, in the central regions of solid tumors, the extracellular pH decreases at values below 6.5, and this is one of the major problems in the cancer treatment because the activity of many of the chemotherapeutic agents fails under this conditions; nonetheless, this might be one of the reasons why CTD is an excellent anticancer agent because recently it has been found that CTD has higher efficacy under acidic conditions .
For the CTD to reach the tumor tissue from its application site (oral or injected), it has to be administered in high doses because of its poor solubility, which leads to an intense irritation at the injection site and is responsible for the renal toxicity. Moreover, CTD has displayed poor intestinal absorption, with a bioavailability of 26.7%, being one of the main factors that must be solved to use CTD as a chemotherapeutic agent without affecting the life of the treated patient.
One of the first attempts to increase the biological activities and the bioavailability of CTD and its derivatives, were made through the structural modification, mainly on cantharidimides, by preparing a series of organoantimony (V) derivatives 77, based on the structure of the 5,6-dehydrocantharidimide and with the hypothesis that of some organoantimony (III) derivatives have shown significant antitumor activities and good bioavailability (104). Additionally, the preparation of several norcantharimide alkylammonium salts 78, that besides having several levels of dynamin GTPase inhibition, they are been using as a room temperature ionic liquids (RTILs)  (Figure 33).
With the hypothesis that rare earth ions can cleave pBR322 plasmid DNA and with the idea that complexes of these ions can increase the solubility of CTD derivatives, three novel complexes of rare earth ions with NCTD were synthesized. Complexes with lanthanum (Ln), erbium (Er), and ytterbium (Yb) demonstrated, by UV-spectra, fluorescence spectra, viscosity, and gel electrophoresis, that they could bind to DNA through non-classical intercalation and that they could cleave pBR322 plasmid DNA effectively .
Despite the less toxicity, NTCD has a short half-life and has to be administrated in high doses in order to maintain the high level of NCTD in the circulatory system. With this idea, Zeng and Sun, to avoid the side effects of NCTD, proposed an alternative dosage form for this compound using biodegradable polymeric nanoparticles, incorporating NCTD into poly (lactide-co-glycolide) (PLGA) to form nanospheres with 150 nm of diameter. The authors determinate that NCTD entrapment efficiency reaches up to 95% in PLGA nanoparticles and that this delivery system shows a biphasic profile with an initial rapid and a following slower release from this phase for more than 10 days. In addition, this system not only exhibits low toxicity, but also presents high efficacy in antitumor growth assays .
Perhaps, one of the most relevant works in this field was reported by Yun-jie and Chun-yan in 2012. In their research, they were faced with one of the common pharmaceutics challenges related with the water solubility of CTD. The author's work was based on the concept of solid dispersion, in which CTD will disperse in an inert hydrophilic carrier, to enhance the dissolution rate, solubility, and oral absorption of poorly water-soluble CTD. The solid dispersion with CTD was prepared in polyethylene glycol-4000 (CA-PEG4000) to study the permeability behavior of this dispersion with the one related with CTD itself. After the evaluation of the oral bioavailability of both systems in rats, the in vivo results showed that the solid dispersion CA-PEG4000 had a higher bioavailability (295.4%) than when free CTD was administrated via oral (26.7%). This improvement obviously established a new starting point for future tests in mammals due that the new CTD, NCTD, and cantharidimide derivatives could be well absorbed by the gastrointestinal tract and more realistic ADME-Tox results can be obtained .
The many varied and new chemical and pharmacological information on CTD-based SMs discussed herein provide substantial evidence related to the synthetic approaches of CTD-based SMs, and their chemical transformations and potential biological applications were also conferred. Furthermore, the advances in the field have provide strong evidence that CTD analogs, as metabolites of blister beetles (fam. Meloidae), are still promising, simple biomodels and that their potent PP-inhibitor activities will be the main objective for future pharmacological research. Therefore, the design and development of CTD analogs could generate other SMs that will stimulate the progress of specific and selective inhibitors of these targets enzymes. Highlighting the synthetic power of the Diels–Alder reactions, as well as simplicity of synthetic green procedures, to obtain these novel and diverse CTD-based SMs should be taken into consideration for the development of potential therapeutic agents. Finally, we hope that this mini review will aid the organic chemistry community to start new designs of novel molecules that could contribute to medicinal and biological chemistry and in this way contribute to the knowledge in this interestingly field.
Financial support from Patrimonio Autónomo Fondo Nacional de Financiamiento para la Ciencia, la Tecnología y la Innovación, Francisco José de Caldas, contract RC-0572-2012, is gratefully acknowledged. C.E.P.G. and L.Y.V.M thank COLCIENCIAS for the respective fellowships.
Carlos E. Puerto Galvis was born in 1986 in the small town of Duitama, BOY. He received a B.S. in Chemistry from the Universidad Industrial de Santander in 2010, where he performed undergraduate research in the laboratories of Professor Vladimir V. Kouznetsov, where he began his Master studies in 2011. His research in the kouznetsov group has focused on the synthesis and chemistry of N-benzylcantharimides, N-benzyl and N-phenylethyl cinnamamides, spirooxoindole 1'-nitropyrrolizidines, as well as the development of the zebrafish embryo model to screening small molecules.
Leonor Y. Vargas Méndez was born in Bucaramanga, Colombia. She received her B.Sc (1998), M.Sc (2001), and Laureate Ph.D. (2009) from Industrial University of Santander (Bucaramanga-Colombia) working on the synthetic approach to analogues of dendrobatid alkaloid 241D, antifungal quinoline derivatives, and dihydrospiro-[piperidine-4,2'-quinolines] chemistry, respectively. She is Titular Professor at Santo Tomás University and the head of the Environmental and Sustainable Development Research Group. Currently, Dr. Vargas works on the development of agrochemicals inspired on molecules obtained from natural sources and semi-synthethic processes.
Vladimir V. Kouznetsov was born and raised in Murmansk, Russia, in 1957. He studied Chemistry at the Russian Peoples Friendship University, Moscow where received his Laureate M.Sc. (1981), Ph.D. (1986) and D.Sc. (1994) degrees in Chemistry, working with Professor Nikolai S. Prostakov and Associate Professor Ludmila A. Gayvoronskaya. He spent postdoctoral fellowship (1990?1992) with Professor José M. Barluenga at the University of Oviedo (Spain), working on synthetic approach to hetaryl substituted polyamines and tetraponerine alkaloids. He became Associate Professor in 1993 at the Russian Peoples Friendship University. In 1994 he moved to Colombia through a Colciencias' program of scientific mobilization. Very soon he became Professor Titular at the Industrial University of Santander (Bucaramanga-Colombia), where he founded the Laboratorio de Síntesis Fina. Now he is director of Laboratorio de Química Orgánica y Biomolecular. Kouznetsov' research interests focus on heterocyclic diversity-oriented and target-oriented synthesises; including natural product synthesis, interface between chemistry and biology.