Anticancer Potential of Betulonic Acid Derivatives

Clinical trials have evidenced that several natural compounds, belonging to the phytochemical classes of alkaloids, terpenes, phenols and flavonoids, are effective for the management of various types of cancer. Latest research has proven that natural products and their semisynthetic variants may serve as a starting point for new drug candidates with a diversity of biological and pharmacological activities, designed to improve bioavailability, overcome cellular resistance, and enhance therapeutic efficacy. This review was designed to bring an update regarding the anticancer potential of betulonic acid and its semisynthetic derivatives. Chemical derivative structures of betulonic acid including amide, thiol, and piperidine groups, exert an amplification of the in vitro anticancer potential of betulonic acid. With the need for more mechanistic and in vivo data, some derivatives of betulonic acids may represent promising anticancer agents.


Introduction. Natural Compounds in the Management of Different Types of Cancer
The extensive research over the past decades has identified an increased number of natural compounds from various medicinal plants that elicit chemopreventive potential. These phytochemicals represent an important potential source of anticancer molecules as such or after various physico-chemical modulations, which can be successfully used in different therapeutic protocols [1,2]. The analysis of natural compounds has led to the design of novel therapeutics owing to their vast structural diversity, supported by the fact that plants are accessible resources. The main issues are the isolation and purification of bioactive derivatives [3].
Cancer is one of the largest causes of mortality in the world and due to its prevalence, the discovery of novel anticancer drugs has great importance. Plants, microorganisms, and marine organisms are considered valuable sources for the discovery of anticancer drugs [4,5].
Throughout history, natural products and their derivatives have proved to be useful for the development of chemotherapeutics, presenting a great structural diversity as well as molecular and pharmacological properties that favor such development [6]. at this point [27].
Flavonoids are secondary metabolites commonly found in plants [28]. Flavonoids are usually found in citrus fruits, berries, legumes, green tea, as well as in red wine [29]. Genistein along with cholecalciferol has completed phase II clinical trials treating patients with early-stage prostate cancer [30]. Quercetin is classified as a flavonol and is currently involved in an ongoing phase I trial, which is evaluating its impact on green tea polyphenol absorption in the prostate tissue from patients with prostate cancer [31,32]. Moreover, quercetin is also involved in an ongoing phase II clinical trial regarding its chemopreventive activity in the development of squamous cell carcinoma in patients with Fanconi anemia [33].
Although there are many studies on betulin and betulinic acid [34,35], this review focuses on betulonic acid and, more specifically, on its derivatives, which have also shown important therapeutic effects, thus suggesting their further thorough research.

An Overview of the Biologic Activity of Pentacyclic Triterpenes
To date, more than 20,000 triterpenes have been isolated [36]. Triterpenoids are originally synthesized by plants as metabolites and are abundantly present in most medicinal plants in the form of free acids or aglycones. Triterpenes are a diverse group of natural compounds, classified into two main groups: tetracyclic and pentacyclic triterpenes. The class of tetracyclic triterpenes includes compounds such as: euphol, oleandrin, and cucurbitacin. The class of pentacyclic triterpenoids (PT) is richer in structures representative for their biological activity, lupane, oleane, and ursane scaffolds being the main groups of this class. Important phytocompounds due to their biological activity and belonging to the category of pentacyclic triterpenoids are: lupeol, betulin, betulinic acid, betulonic acid (lupane structure), oleanolic acid, maslinic acid (oleanane structure), ursolic acid, and uvaol (ursane structure) (Figure 1), nevertheless other such categories including hopane, serratane, friedelane, and taraxane can also be referred [37][38][39].  Secondary plant metabolites such as PT are generated through the cyclization of squalene [40]. Lupeol, oleanane, and ursane structures were found in a variety of plant parts such as bark, cork, leaf, or fruit cuticular wax [41]. Moreover, pentacyclic triterpenes have been detected in edibles such as mango, apple peel, strawberries, pear peel, green pepper, guava, mulberry, or olives, but also in herbal plants such as rosemary, oregano, basil, and lavender. The individual natural human consumption of triterpenes is reported to be approximately 250 mg per day in the Western world and 400 mg per day in Mediterranean countries [42]. However, small quantities, estimated to be under 0.1% of the dry weight of the plant organ, are found in plants. Even so, few plants are considered to have elevated amounts of pentacyclic triterpenes, above 1% of the dry weight of the plant. The highest pentacyclic terpene content has been noticed in the outer bark of the white birch tree (up to 34% [w/w] in betulin) [40]. Furthermore, the leaves from Rosmarinus officinalis L., Olea europaea L., Coffea arabica L., the sapling from Viscum album L., the bark from Platanus L., and the fruit peels from Malus domestica Mill. produce more than 1% (w/w) pentacyclic triterpenes. Due to this property, these plants are useful for obtaining triterpene dry extracts which contain 50-90% (w/w) triterpenes [43].
Natural PT have a broad range of biological activities. Intense pharmacological researches have been conducted on natural PT in order to exploit their medicinal value and mechanism of action. In general, the bioactivities of these phytocompounds have been shown to include antitumor [40,44,45], antiviral [46], antidiabetic [47], anti-inflammatory [48], antimicrobial [49], antiparasitic [39], cardio-protective [50], hepato-protective [51], wound healing properties [52], and others. For example, oleanolic acid, glycyrrhizin, glycyrrhetinic acid, ursolic acid, betulin, betulinic acid, and lupeol increase the absorption of glucose, enhance insulin secretion, boost glucose uptake in peripheral organs, and contribute to the treatment of diabetes and its vascular complications [53]. Zhang et al. tested the inhibitory activity of oleanane-, ursane-and lupane-type triterpenes against α-amylase and yeast α-glucosidase. Results have shown that, in comparison to the IC 50 value of acarbose (5.3 µM), ursolic acid exerted the highest inhibition of α-amylase, with an IC 50 value of 22.6 µM, followed by corosolic acid and oleanolic acid with IC 50 values of 31.2 µM and 94.1 µM, respectively. Regarding the α-glucosidase inhibition, ursolic acid, betulinic acid, and corosolic acid displayed a stronger inhibitory activity than acarbose (IC 50 = 2479 µM) with IC 50 levels of 12.1 µM, 14.9 µM, and 17.2 µM, respectively. It appears that the variety of structural skeletons of pentacyclic triterpenes had a substantial effect on the inhibition of α-amylase activity. The fact that ursolic acid displayed stronger α-amylase inhibition than oleanolic acid may be attributted to the shift of the C-29 methyl group from C-20 to C-19. The study found that betulinic acid inhibited α-glucosidase with comparable effectiveness to ursolic acid, indicating that the disparity between ursane and lupane had no major effect on the enzyme inhibition [54]. Numerous experiments have shown that different PT have antidiabetic properties in healthy or diabetic animal models. Phanoside (a dammarane-type structure) at a dosage of up to 500 µM exerted significant effects on insulin secretion in rat pancreatic cells. In INS-1 832/13 pancreatic β-cells, oleanolic acid (50 µM) improved insulin secretion. Moreover, in isolated rat islets, oleanolic acid (30 µM) also improved insulin secretion, which was stimulated by high glucose levels [55,56]. The plasma insulin levels of Wistar rats were improved by ginsenoside Rh2 (1.0 mg/kg) and oleanolic acid (5, 10 and 20 mg/kg) by freeing acetylcholine (ACh) from nerve terminals and then activating M3 muscarinic receptors in pancreatic cells [57,58].
Furthermore, PT showed anti-inflammatory, antioxidant, anti-adiposity, and cardioprotective properties in high-carbohydrate high-fat diet-induced metabolic syndrome [59,60]. In addition, the weight-reducing effects of PT are due to the downregulation of proinflammatory cytokines, insulin resistance, oxidative stress, and total fatty acids, all of these contributing to a more efficient glycemic regulation [61]. In the study performed by Tang et al., they have demonstrated that betulin at 3 µg/mL incubated for 6 h suppressed the maturation of sterol regulatory element-binding proteins (SREBP) and reduced cholesterol and fatty acid biosynthesis in rat hepatocytes CRL-1601. At a dosage of 30 mg/kg/day for 6 weeks, betulin has lowered serum and tissue lipid levels, helped improve glucose tolerance, and tended to increase insulin sensitivity in C57BL/6J mice fed with Westernstyle diet. In addition, the application of betulin to the LDLR-knockout mice model of atherosclerosis disease has revealed its ability to decrease the size of the atherosclerotic plaques [62]. As well as betulin, betulinic acid (50 mg/kg for 15 weeks) lowered body weight, blood glucose, abdominal fat, plasma triglycerides, and total cholesterol levels in Swiss mice fed with a high-fat diet. Interestingly, betulinic acid treatment further decreased the circulating level of the orexigenic hormone ghrelin and increased the anorexigenic hormone leptin. The results showed that betulinic acid could represent a candidate to cure obesity through changes in fat and carbohydrate metabolisms [63].
Several in vitro and in vivo studies have shown that natural triterpenes such as lupeol, betulin, ursolic acid, and oleanolic acid are successful in reducing hepatotoxicity caused by carbon tetrachloride, acetaminophen, ethanol, and cadmium [64][65][66]. In vitro, betulinic acid has been shown to be a powerful antioxidant agent, which can suppress ethanol-induced activation of hepatic stellate cells mostly by the repression of reactive oxygen species (ROS) and tumor necrosis factor-α (TNF-α) [67]. In the in vivo experiments performed by Yi et al., the authors have highlighted the hepatoprotective effects of betulinic acid, when given orally (0.25, 0.5, and 1.0 mg/kg daily for 14 days) to Kunming mice with induced alcoholic liver disease. Betulinic acid increased hepatic glutathione, superoxide dismutase, glutathione peroxidase, and catalase levels, as well as the levels of malondialdehyde, thereby decreasing the liver's microvesicular steatosis. In addition, the results of the study indicated that betulinic acid-treated mice showed reduced serum levels of triglycerides and total cholesterol, which was attributed to enhanced β-oxidation in the liver and energy consumption. Betulinic acid also reduced the accumulation of visceral adipose tissue in alcohol-treated mice. In particular, this may help prevent secondary complications emerging from elevated levels of hepatic lipids [68].
PT derived from natural products exhibit a wide variety of medicinal properties such as anti-oxidant [69], anti-tumor [70], anti-microbial [71], and anti-inflammatory [72]. It is assumed that the pathways involved in these bioactivities are caused by the regulation of the immune system. The immunomodulatory efficacy of botanical pentacyclic triterpenes extracted from a broad variety of medicinal plant species, based on different in vitro and in vivo experimental models, has been illustrated in several reports [73,74]. In the experiment conducted by Saaby et al., the immunomodulatory effects of various PT obtained from Rosa canina L. have been evaluated in vitro, on lipopolysaccharide (LPS)-activated Mono Mac 6 cells (an in vitro model for human macrophages). The findings showed that oleanolic, betulinic, and ursolic acid mixtures (30:49:21 w/w) inhibit the release of LPS-induced IL-6 in a stronger manner than individual compounds, with an IC 50 value of 21 µmol/L [75]. In the study conducted by Ayatollahi et al., the authors investigated the impact of three phytocompounds (betulinic acid, oleanolic acid and ursolic acid), extracted from Euphorbia microsciadia Boiss, on T-cell proliferation. The T-lymphocytes were isolated from healthy volunteers and analysed using a liquid scintillation counter. Studies have shown that oleanolic acid induces T cell proliferation even at the concentration of 0.5 µg/mL. These findings indicated that oleanolic acid has an important immunostimulating action at very low concentration. Betulinic acid and ursolic acid, on the other hand, were able to suppress the proliferation of T cells with IC 50 values above 50 µg/mL and 3 µg/mL, respectively [76]. Marquez-Martin et al. observed that, at concentrations of 10, 25, 50, and 100 µmol/L, some PT extracted from 'orujo' olive oil such as oleanolic acid and erythrodiol dose-dependently decreased the secretion of IL-1β and IL-6 isolated from peripheral mononuclear blood cells (PBMCs) of healthy volunteers. Erythrodiol exhibited the most potent inhibitory effect (p < 0.05) on the development of IL-1β and IL-6 PBMCs at all doses. At the maximum tested doses (50-100 µM), oleanolic acid substantially (p < 0.05) down-regulated IL-1β and IL-6 release [77].
The molecular mechanisms behind the different biological activities of PT, ranging from inhibition of acute and chronic inflammation, inhibition of tumor cell proliferation, activation of apoptosis, and suppression of angiogenesis and metastasis, have been identified in various research studies [40,78]. PT modulate a wide range of molecular targets (cytokines; chemokines; reactive oxygen intermediates; oncogenes; inflammatory enzymes; anti-apoptotic proteins; and transcription factors such as NF-jB, STAT3, AP-1, and CREB), which mediate tumor cell proliferation, transformation, invasion, angiogenesis, metastasis, chemoresistance, and radioresistance [78]. Previous studies have demonstrated that PT, in particular betulin, betulinic acid, lupeol, and ursolic acid, have caused apoptosis in various forms of cancer cells by stimulation of the mitochondrial pathway (intrinsic pathway) rather than the death receptor pathway (extrinsic way) [79,80].

Betulonic Acid and Its Derivatives. An Overview of In Vitro and In Vivo Active Compounds
Betulin (lup-20(29)-ene-3β,28-diol) and betulonic acid (lup-20(29)-en-3-oxo-28-oic) ( Figure 2) are the most important pentacyclic lupane-structure triterpenoids of natural origin. These compounds are found in a broad range of medicinal plants, but they are often extracted from birch bark through sublimation or extraction with organic solvents including ethanol, acetone, and chloroform [37,89]. Betulonic acid has a keto group at C-3 and a carboxyl group at C-28, with a terminal double bond at C-29; the only structural difference compared to betulinic acid lies at the C-3 position where betulonic acid bears a ketone instead of a β-configured hydroxyl [83].
The content of betulonic acid in plants is generally low, so it is frequently obtained by a semisynthetic process, which consists in the oxidation of betulin (Figure 2), the main constituent (22-30%) found in the outer part of birch bark [80].
The content of betulonic acid in plants is generally low, so it is frequently obtained by a semisynthetic process, which consists in the oxidation of betulin (Figure 2), the main constituent (22-30%) found in the outer part of birch bark [80].
Betulonic acid and its derivatives have been found to possess several medicinal properties, such as anti-viral [96][97][98], antimicrobial [99,100], anti-Human cytomegalovirus (HCMV) [3], anti-inflammatory [101][102][103]antioxidant [104], hepatoprotective [103,105], immunostimulant [106], and anticancer effects [107,108]. Modern research has proved that natural products and their semisynthetic variants may serve as the starting point of new drug candidates with a diversity of biological and pharmacological activities. From 1981-2014, natural products accounted for 25% of all newly licensed medicines [109,110]. Betulonic acid is an example of a natural compound that was found to be a promising candidate as an antitumor agent since it can inhibit the growth of different types of tumor cell lines [83]. Betulonic acid is soluble in organic solvents, but its solubility in water is weak. Chemical modifications are used to obtain derivatives with higher bioavailability and special selectivity to cellular targets [111]. Several derivatives were synthesized and examined against different cancer cell lines for antitumor activity (Table 1).  Betulonic acid and its derivatives have been found to possess several medicinal properties, such as anti-viral [96][97][98], antimicrobial [99,100], anti-Human cytomegalovirus (HCMV) [3], anti-inflammatory [101][102][103] antioxidant [104], hepatoprotective [103,105], immunostimulant [106], and anticancer effects [107,108]. Modern research has proved that natural products and their semisynthetic variants may serve as the starting point of new drug candidates with a diversity of biological and pharmacological activities. From 1981-2014, natural products accounted for 25% of all newly licensed medicines [109,110]. Betulonic acid is an example of a natural compound that was found to be a promising candidate as an antitumor agent since it can inhibit the growth of different types of tumor cell lines [83]. Betulonic acid is soluble in organic solvents, but its solubility in water is weak. Chemical modifications are used to obtain derivatives with higher bioavailability and special selectivity to cellular targets [111]. Several derivatives were synthesized and examined against different cancer cell lines for antitumor activity (Table 1).
III [113] n = 2 (A) n = 3 (B) [113]     The group conducted by Shintyapina et al. examined the inhibitory action of selecte amides of betulonic acid on the growth and potential apoptosis induction of MT-4, MOL 4, CEM (lymphoblastic leukemia), and Hep G2 (liver cancer) tumor cell lines. Betulon acid amides (compounds I (A-D)) inhibited cell growth at low concentrations (50% inh [107] The group conducted by Shintyapina Table 2. The length of the methylene moiety (CH 2 ) n in the amide residue did not affect the inhibitory activity, however, the introduction of the second amide residue increased the inhibition of the tumor cell growth (I (D)). The apoptosis-inducing activity of betulonic acid amides was higher than that of betulonic acid for MT-4 and MOLT-4 cell lines. Thus, the introduction of amide function at the position C28 of PT led to the formation of effective inducers of apoptosis [112].     ), MCF-7, and Bcap-37 (breast adenocarcinoma) cell lines. The compounds substituted with a bromoalkyl moiety (II (A), II (B)) demonstrated weak antitumor activity compared with the parent compound betulonic acid. Results have shown that (4-(piperidin-1-yl) butyl) 3-oxo-20(29)-lupen-28-oate (III) was one of the most active compounds, presenting IC 50 values of 3.6-7.8 µM on the five screened cancer cell lines, whereas compounds substituted with fluorophenyl group (IV) exhibited high and moderate antiproliferative activities, as shown in Table 2. (4-(piperidin-1-yl)butyl) 3-oxo-20(29)-lupen-28-oate has also been shown to induce apoptosis of MGC-803 cells via the mitochondrial intrinsic pathway, which include suppression of the expressions p53, Bax, caspase 9, and caspase 3 [113].
In a similar approach, Ledeţi et al. described the cytotoxic effects (MTT assay) of four amino derivatives of betulonic acid against certain tumor cell lines, including HeLa (cervix adenocarcinoma), A431 (skin carcinoma), A2780 (ovarian carcinoma) and MCF-7 (breast adenocarcinoma), as shown in Table 2. Guanylhydrazone (V) had lower cytotoxic activity on all cell lines relative to betulin at both tested concentrations (10, 30 mM). The oxime (VI) was found to have strong cytotoxic effects on the A2780 cell line when used at elevated doses, whereas the cytotoxic effect on the A431 cells was almost negligible when the lower concentration was used. The strongest activity manifested at the lowest concentration of butyl imine (VII) occurred on HeLa cell lines, as opposed to the higher concentration that was relevant for A2780 cell line. Moreover, the thiosemicarbazone (VIII) demonstrated significant cytotoxic effects against HeLa, A2780, and MCF-7 cell lines included in this study, having a comparable inhibitory activity to betulin. Regarding the inhibitory activity against A431 cell line, there was no evidence of it [114].
The inhibitory effects of C(2)-C(3)-fused triazine derivatives of betulonic acid on the proliferation of murine leukemia cells (L1210), human cervix carcinoma cells (HeLa), and human T-lymphoblast cells (CEM) were evaluated by Dinh Ngoc et al. The 1,2,4-triazine derivative of betulonic acid (IX) did not elicit a significant cytostatic activity, while Salkylated triazine derivatives (X, XI) strongly inhibited tumor cell proliferation, especially in case of human T-lymphoblast cells [3].
Kommera et al. demonstrated the in vitro cytotoxic activity of the betulonic acid derivative, 2-amino-3-hydroxy-2-(hydroxymethyl) propyl betulonate (XII), on eight different tumor cell lines: 518A2 (melanoma), A253 (head and neck tumor), A431 (cervical), A2780 (ovarian), A549 (lung), HT-29 (colon), MCF-7 (breast), and SW1736 (anaplastic thyroid tumor) using the sulforhodamine B colorimetric assay. As shown in Table 2, IC 50 values were found to be in the range of 9.44-10.83 µM. A comparison regarding the cytotoxicity of betulonic acid and compound XII showed a loss of specificity towards some cancer cell lines presented by the parent compound. It has been observed that the derivate also induced apoptosis on HT-29 cell line, which has been confirmed by annexin V staining experiments and DNA fragmentation assay [115].
Following the study conducted by Saxena et al., it has been reported that N-Boclysinated-betulonic acid (XIII) elicits positive in vitro effects against prostate cancer cell lines (LNCaP, DU-145, PC-3). The growth of the LNCaP cells was inhibited by lysinatedbetulonic acid at 10 µM following 72 h of incubation to 40%, whereas at a concentration of 100 µM, the inhibitory activity increased to 80% (for the compound dissolved in DMSO) and by 96% (for compound dissolved in phosphate-buffered saline [PBS]). Using the MTT assay and various incubation times, the research group showed a significant inhibitory effect on DU-145 and PC-3 cancer cells treated with 100 µM of lysinated-betulonic, as shown in Table 2  and ID 50 = 37 µM derivative (XV (F)), respectively. Regarding the inhibitory activities of compounds (XV (A-F)), ID 50 values were much higher than those of betulonic acid, suggesting that these derivatives had low cytotoxicity (ID 50 ranged from 28 to 120 µM) as shown in Table 2.
The cytotoxicity of these compounds was significantly influenced by the nature of the substitutes at oxadiazole C-5 [117].
Kazakova et al. have reported the biological activity of C-28-imidazolides of betulonic acid, containing 3-oxo-, 3-hydroxyimino-, and 2-cyano-2,3-seco-4(23)-ene fragments in cycle A. Firstly, they assessed the cytotoxicity of the imidazolides (cycle A) using different human tumor cell lines (lungs, colon, central nervous system, ovary, renal, prostate, mammary gland, melanoma, leukemia), dyed with sulforhodamine B, and treated with 10 µM of the derivatives for 48 h. The results have shown that the imidazolide of 3-hydroxyimino-lup-20(29)-en-28-carboxylic acid (cycle A) (XXII) was the most efficient in inhibiting the cell growth of all six lung cancer cell lines, all seven large intestine cancer cell lines, all six leukemia cell lines, all six melanoma cell lines, all four breast cancer cell lines, two nervous system cancer cell lines, one prostate cancer cell line, three ovarian cancer cell lines, and five renal cancer cell lines. The cell survival rates are depicted in Table 2. Moreover, cell death was found in three cell lines: lung cancer cell line (NCI-H460), colon cancer cell line (COLO 205), and leukemia cell line (HL-60[TB]) [107]. An overview of the anticancer activity of betulonic acid derivatives is presented in Figure 2.
Owing to the hydrophobic properties of betulonic acid, its in vivo anti-cancer effectiveness has not been intensively studied (Figure 3). In a research performed by Saxena et al., they investigated the in vivo activity of hydrophilic lysinated betulonic acid on LNcaP prostate cancer cells xenografts in athymic mice (Table 3). In contrast to the control group, the lysinated betulonic acid injected mice displayed a 92% inhibition of tumor development. Moreover, histological analyses of the tumors revealed the lack of dividing cells, demonstrating the anticancer activity of the lysinated-betulonic acid [116].

pound Experimental Animal Model Injected Tumor Cells Concentration Conclusion Reference
Boc-lysinated-betu-  Based on the remarkable results obtained for the imidazolide of 3-hydroxyiminolup-20(29)-en-28-carboxylic acid (XXII), Kazakova et al. have studied its antineoplastic activity in vivo on two ascites tumors (Ehrlich tumor and P388 lympholeukemia) and three solid tumors (breast adenocarcinoma [Ca755], colon adenocarcinoma [AKATOL], and Lewis lung cancer [LLC]) inoculated in BDF1 hybrids (DBA2 × C57B1/6J), BALB/C, and DBA mice. This chemical derivative injected intraperitoneally, five times daily at a dosage of 70 mg/kg, had no antineoplastic effect neither on the Ehrlich ascites tumor nor P388 lympholeukemia (at a dose of 30 and 50 mg/kg), respectively. Interestingly, the administration of 50 mg/kg through the same procedure suppressed the development of breast adenocarcinoma (Ca755) by 56-77%. The result was maintained without an improvement in life duration until day eight of the study. In mice with large intestinal adenocarcinoma treated by the same procedure, a 53% decrease in tumor growth was seen only after 5 days of therapy without any change in the mice's lifespan [107].
The research study conducted by Zhukova et al. examined the effects of betulonic acid and its methyl esters derivatives on the pathological modifications in the kidneys of the C57BL/6 mice, presenting Lewis pulmonary adenocarcinoma. Compounds were administered intraperitoneally in a dosage of 50 mg/kg for 8 days. After 8 days, mice were decapitated under ethereal anesthesia and sections of the treated kidneys were analyzed using periodic acid-Schiff reaction-hematoxylin and eosin-orange G dyeing. These chemical derivatives had a beneficial impact on the course of paraneoplastic nephropathy; they decreased the volume density of epithelial cell necrosis at 56%. On average, the number of cells with edematic cytoplasm, vesicular lipid infiltrate, and disrupted brush lymbus fell by 8-13% in comparison to untreated tumoral cells [120].

Conclusions
Data presented in this work show that following structural modifications, which involved the introduction of amide, thiol, and piperidine groups, amplification of the in vitro anticancer potential of betulonic acid can be achieved. Studies published in the state of the art literature revealed that betulonic acid derivatives possess an important in vitro cytotoxic and pro-apoptotic activity. However, the number of in vivo studies is limited. The screening of scientific literature on this topic indicates a demand for in-depth in vivo data regarding the effect and mechanism of action of the betulonic acid derivatives in order to benefit from these structures in different therapeutic protocols designated for the management of various types of cancer.

Conflicts of Interest:
The authors declare no conflict of interest.