Synthesis and Evaluation of Anticancer Activities of Novel C-28 Guanidine-Functionalized Triterpene Acid Derivatives

Triterpene acids, namely, 20,29-dihydrobetulinic acid (BA), ursolic acid (UA) and oleanolic acid (OA) were converted into C-28-amino-functionalized triterpenoids 4–7, 8a, 15, 18 and 20. These compounds served as precursors for the synthesis of novel guanidine-functionalized triterpene acid derivatives 9b–12b, 15c, 18c and 20c. The influence of the guanidine group on the antitumor properties of triterpenoids was investigated. The cytotoxicity was tested on five human tumor cell lines (Jurkat, K562, U937, HEK, and Hela), and compared with the tests on normal human fibroblasts. The antitumor activities of the most tested guanidine derivatives was lower, than that of corresponding amines, but triterpenoids with the guanidine group were less toxic towards human fibroblasts. The introduction of the tris(hydroxymethyl)aminomethane moiety into the molecules of triterpene acids markedly enhanced the cytotoxic activity of the resulting conjugates 15, 15c, 18b,c and 20b,c irrespective of the triterpene skeleton type. The dihydrobetulinic acid amine 15, its guanidinium derivative 15c and guanidinium derivatives of ursolic and oleanolic acids 18c and 20c were selected for extended biological investigations in Jurkat cells, which demonstrated that the antitumor activity of these compounds is mediated by induction of cell cycle arrest at the S-phase and apoptosis.


Introduction
Among natural products of plant origin that are considered as abundant sources of lead structures for the discovery of new drugs, the pentacyclic lupane, ursane, and oleanane triterpenoids occupy a prominent place [1,2]. Triterpene acids (betulinic, ursolic, and oleanolic acids, Figure 1) are of interest for pharmacological research, as they exhibit a variety of biological activities including antimicrobial, antiparasitic, antitumor, and antiviral, in particular, anti-HIV, types of activity [3][4][5][6]. Among these properties of triterpenoids, of special interest is their anticancer activity and the ability to trigger the mitochondrial apoptosis pathway in various types of human cancer cells [5][6][7][8][9]. Thus, betulinic acid is capable of inducing apoptosis in tumor cells, such as melanoma, adenocarcinoma, neuroblastoma, medulloblastoma, glioblastoma and neuroectodermal tumors [7][8][9]. The in vivo anticancer activity of betulinic acid was identified using xenograft models [10,11]. The ursolic acid can also induce apoptosis, autophagy, and cell cycle arrest through various pathways, such as inhibition of DNA replication, stimulation of reactive oxygen species (ROS) production, and affecting the balance between proapoptotic and antiapoptotic proteins [6,12,13]. However, the cytotoxic activity of the phosphonium salts was comparable with their cytotoxic activity against normal peripheral blood cells. In continuation of the search for efficient and selective antitumor agents, we have investigated novel cationic derivatives of pentacyclic triterpenoids containing guanidine groups, which are readily protonated at a physiological pH level. The introduction of hydrophilic guanidine groups into hydrophobic triterpene acid molecules may enhance their transmembrane transport and physicochemical characteristics. Meanwhile, the new hybrid molecules may preserve the selectivity of cytotoxic action against normal cells inherent in the natural triterpene acids. The guanidine group is a common key unit in various natural and synthetic compounds demonstrating antimicrobial, antiviral, and antitumor activities [26]. High symmetry of the Y-shaped guanidinium group promotes the formation of two parallel hydrogen bonds with the biologically relevant counterparts. Unlike ammonium groups, in which the charge is localized on one nitrogen atom (hard cations), guanidinium groups with a delocalized charge actively interact through hydrogen bonds with soft ions such as phosphates and sulfates. This feature of the guanidinium cation induce the efficient transport of biologically active substances through liposomal and cell membranes [27][28][29]. Furthermore, because of high basicity (pKa 13.5), the guanidinium group is important for selective delivery of cytotoxic molecules to tumor cells. Guanidine derivatives can be accumulated in the mitochondria of tumor cells, thus destroying the mitochondrial potential and inhibiting the mitochondrial respiratory chain [29,30].
Polyamines, which are precursors of aminoalkylguanidines, are also used to develop chemotherapeutic agents, including antibacterial and antitumor compounds [31,32]. Structurally, polyamine molecules contain positively charged nitrogen atoms at physiological pH value and can serve as electrostatic bridges between negatively charged phosphates. They are able to bind to negatively charged DNA macromolecules. However, some of physiological diamines, polyamines, and their synthetic analogues have exhibited high toxicity toward normal cells. A large body of data has now been accumulated on the biological activity of polyaminosterols, among which squalamine, trodusquemine, and their synthetic analogues are best known [33][34][35][36]. The synthesis and biological properties of polyamino triterpene acids are described in several publications [6,[37][38][39][40]; the effect of introduction of the guanidine group into triterpenoid molecules has not been studied so far. Here we However, the cytotoxic activity of the phosphonium salts was comparable with their cytotoxic activity against normal peripheral blood cells. In continuation of the search for efficient and selective antitumor agents, we have investigated novel cationic derivatives of pentacyclic triterpenoids containing guanidine groups, which are readily protonated at a physiological pH level. The introduction of hydrophilic guanidine groups into hydrophobic triterpene acid molecules may enhance their transmembrane transport and physicochemical characteristics. Meanwhile, the new hybrid molecules may preserve the selectivity of cytotoxic action against normal cells inherent in the natural triterpene acids. The guanidine group is a common key unit in various natural and synthetic compounds demonstrating antimicrobial, antiviral, and antitumor activities [26]. High symmetry of the Y-shaped guanidinium group promotes the formation of two parallel hydrogen bonds with the biologically relevant counterparts. Unlike ammonium groups, in which the charge is localized on one nitrogen atom (hard cations), guanidinium groups with a delocalized charge actively interact through hydrogen bonds with soft ions such as phosphates and sulfates. This feature of the guanidinium cation induce the efficient transport of biologically active substances through liposomal and cell membranes [27][28][29]. Furthermore, because of high basicity (pKa 13.5), the guanidinium group is important for selective delivery of cytotoxic molecules to tumor cells. Guanidine derivatives can be accumulated in the mitochondria of tumor cells, thus destroying the mitochondrial potential and inhibiting the mitochondrial respiratory chain [29,30].
Polyamines, which are precursors of aminoalkylguanidines, are also used to develop chemotherapeutic agents, including antibacterial and antitumor compounds [31,32]. Structurally, polyamine molecules contain positively charged nitrogen atoms at physiological pH value and can serve as electrostatic bridges between negatively charged phosphates. They are able to bind to negatively charged DNA macromolecules. However, some of physiological diamines, polyamines, and their synthetic analogues have exhibited high toxicity toward normal cells. A large body of data has now been accumulated on the biological activity of polyaminosterols, among which squalamine, trodusquemine, and their synthetic analogues are best known [33][34][35][36]. The synthesis and biological properties of polyamino triterpene acids are described in several publications [6,[37][38][39][40]; the effect of introduction of the guanidine group into triterpenoid molecules has not been studied so far. Here we describe the synthesis and comparative evaluation of the cytotoxic and apoptosis-inducing activities of new guanidine derivatives of pentacyclic lupane, ursane, and oleanane triterpenoids describe the synthesis and comparative evaluation of the cytotoxic and apoptosis-inducing activities of new guanidine derivatives of pentacyclic lupane, ursane, and oleanane triterpenoids and their precursors-С-28 conjugates of triterpene acids with some linear and branched mono-, di-, and triaminoalkanes.

Biological Evaluation
The in vitro cytotoxic activity of triterpene acids (dihydrobetulinic, ursolic and oleanolic acids), twelve guanidinium salts, and some of their precursors, primary amines 4-6, 8a, and 15, was evaluated on five human tumor cell lines: Jurkat (T-lymphoblastic leukemia), K562 (chronic myeloid leukemia), U937 (histiocytic lymphoma), HEK 293 (embryonic kidney), and HeLa (cervical cancer). The possible cell toxicity was assessed against normal human fibroblasts. Most of the tested compounds showed moderate or significant activity against Jurkat, K562, and U937 cells as compared to triterpenoic acids (Table 1). Amino derivatives 4-6 and 8a showed cytotoxic activity against all tumor cell cultures with IC 50 values of 1.3-8 µM. However, these compounds were also cytotoxic against fibroblasts. Contrary to our expectations, guanylation of the terminal amino groups of lupane triterpenoids with C-28 linear aminoalkane chains (compounds 9b-11b, 13a, and 14) or the branched tris-aminoethyl moiety (compounds 12a,b) did not considerably enhance the cytotoxic action. Of the listed guanidinium salts, only compounds 9b, 11b, and 14 showed an approximately 3-fold increase in the cytotoxic activity against Jurkat cells in comparison with dihydrobetulinic acid 2 and also showed selectivity towards Jurkat cells with an SI of 8.5, 7.3, and 5.6 respectively (SI = IC 50 fibroblasts/IC 50 Jurkat cells). The introduction of the tris(hydroxymethyl)aminomethane moiety into the molecules of triterpene acids 3, 16 and 17 markedly enhanced the cytotoxic activities for the resulting conjugates 15, 15c, 18b,c, and 20b,c, irrespective of the triterpene skeleton type. The C-28 esters of dihydrobetulinic, ursolic, and oleanolic acids with amino and guanidine groups in the ester side chain had from moderate to good activity against Jurkat and K562 cell lines. For example, the IC 50 values of compounds 15c and 18c were 3.1 and 3.8 µM for T-lymphoblastic leukemia cells and 2.3 and 11.0 µM for chronic myeloid leukemia cells. The most pronounced differences in the antitumor activity were found for oleanolic acid and its conjugates 20b,c. Indeed, the IC 50 values of oleanolic acid, 20b, and 20c for Jurkat cells were 271, 6.7, and 7.6 µM, respectively. It is worth noting that amine 15, its guanidine derivative 15c, and guanidinium salts of ursolic and oleanolic acids 18b,c and 20b,c showed acceptable selectivity towards Jurkat tumor cell with an SI from 6.9 to 13.4.
The identified lead compounds with the highest cytotoxicity characteristics, 15, 15c, 18c, and 20c, were further evaluated for the possible apoptosis induction in tumor cell cultures. The measurements were done by flow cytometry (Figure 2).
Molecules 2018, 23, x FOR PEER REVIEW 6 of 22 moderate to good activity against Jurkat and K562 cell lines. For example, the IC50 values of compounds 15с and 18с were 3.1 and 3.8 µM for T-lymphoblastic leukemia cells and 2.3 and 11.0 µM for chronic myeloid leukemia cells. The most pronounced differences in the antitumor activity were found for oleanolic acid and its conjugates 20b,с. Indeed, the IC50 values of oleanolic acid, 20b, and 20с for Jurkat cells were 271, 6.7, and 7.6 µM, respectively. It is worth noting that amine 15, its guanidine derivative 15с, and guanidinium salts of ursolic and oleanolic acids 18b,c and 20b,c showed acceptable selectivity towards Jurkat tumor cell with an SI from 6.9 to 13.4. The identified lead compounds with the highest cytotoxicity characteristics, 15, 15с, 18с, and 20с, were further evaluated for the possible apoptosis induction in tumor cell cultures. The measurements were done by flow cytometry (Figure 2). The highest percentage of late apoptosis (91.7%) was detected upon the treatment of Jurkat cells with the test compound 15 at IC50 concentration exposure for 48 h as depicted in Figure 2E. Compounds 15c, 18c and 20c also showed apoptotic mode of cell death on Jurkat cells line, but, in this case, the apoptotic effect of these guanidine derivatives was notably weaker. Thus, after treatment of Jurkat cells with compound 15c at IC50 concentration (4 µM) the number of vital cells is decreased from 96.2% (control) to 54.5%. Total number of apoptotic cells population constituted 23.7% (7.2% and 16.5% of early and late apoptotic cells, respectively) and number of necrotic cells The highest percentage of late apoptosis (91.7%) was detected upon the treatment of Jurkat cells with the test compound 15 at IC 50 concentration exposure for 48 h as depicted in Figure 2E. Compounds 15c, 18c and 20c also showed apoptotic mode of cell death on Jurkat cells line, but, in this case, the apoptotic effect of these guanidine derivatives was notably weaker. Thus, after treatment of Jurkat cells with compound 15c at IC 50 concentration (4 µM) the number of vital cells is decreased from 96.2% (control) to 54.5%. Total number of apoptotic cells population constituted 23.7% (7.2% and 16.5% of early and late apoptotic cells, respectively) and number of necrotic cells was 21.7%. Comparable results were obtained with the guanidine derivative of ursolic acid 18c (6.8, 14.3, and 20.7% of early, late apoptotic cells and necrotic cells, respectively). Treatment of the Jurkat cells with 20c resulted in about 15.5% apoptotic cells and 6.1% necrotic cells, with 78.4% of the cells still being considered vital ( Figure 2K). Next, we analyzed the ability of dihydrobetulinic acid to stimulate apoptosis. Our results showed that dihydrobetulinic acid triggers apoptosis in Jurkat cells at higher doses as compared to derivatives 15, 15c, and 18c. The number of apoptotic cells on treatment with dihydrobetulinic acid (59 µM) for 48 h constituted around 24% (4.0% of early-stage and 20.5% of secondary necrotic/late-stage apoptotic), while the number of vital cells was 73.1% ( Figure 2C).
In summary, our results indicate that the apoptosis is induced in Jurkat cells by all test compounds. However, we observed a higher rate of necrosis after 15c, 18c and 20c incubation compared to derivative 15.
DNA flow cytometry was also used to analyze the cell cycle kinetics in Jurkat cells pre-incubated with dihydrobetulinic acid and derivatives 15, 15c, 18c, or 20c at their IC 50 concentration for 24 and 48 h (Figure 3).
Molecules 2018, 23, x FOR PEER REVIEW 7 of 22 was 21.7%. Comparable results were obtained with the guanidine derivative of ursolic acid 18c (6.8, 14.3, and 20.7% of early, late apoptotic cells and necrotic cells, respectively). Treatment of the Jurkat cells with 20c resulted in about 15.5% apoptotic cells and 6.1% necrotic cells, with 78.4% of the cells still being considered vital ( Figure 2K). Next, we analyzed the ability of dihydrobetulinic acid to stimulate apoptosis. Our results showed that dihydrobetulinic acid triggers apoptosis in Jurkat cells at higher doses as compared to derivatives 15, 15c, and 18c. The number of apoptotic cells on treatment with dihydrobetulinic acid (59 µM) for 48 h constituted around 24% (4.0% of early-stage and 20.5% of secondary necrotic/late-stage apoptotic), while the number of vital cells was 73.1% ( Figure 2C). In summary, our results indicate that the apoptosis is induced in Jurkat cells by all test compounds. However, we observed a higher rate of necrosis after 15c, 18c and 20c incubation compared to derivative 15.
DNA flow cytometry was also used to analyze the cell cycle kinetics in Jurkat cells pre-incubated with dihydrobetulinic acid and derivatives 15, 15с, 18с, or 20с at their IC50 concentration for 24 and 48 h (Figure 3).   Figure 3D,F). Considering these results, we assume that dihydrobetulinic acid and compounds 15, 15c, 18c, or 20c are able to trigger programmed cell death, including apoptotic mechanisms and cell cycle arrest in the S-phase.

Viability and Apoptosis
Apoptosis was determined by flow cytometric analysis of Annexin V and 7-aminoactinomycin D staining. Briefly, 200 µL of Guava Nexin reagent (Millipore, Bedford, MA, USA) was added to 5 × 105 cells in 200 µL, and the cells were incubated with the reagent for 20 min at room temperature in the dark. The plates were treated with compounds 15, 15c, 18c, 20c and dihydrobetulinic acid at IC 50 concentration (4, 8 and

Cell Cycle Analysis
Cell cycle was analyzed using the method of propidium iodide staining. Briefly, cells were plated in 24-well round bottom plates at density 10 × 105 cells per well, centrifuged at 450× g for 5 min, and fixed with ice-cold 70% ethanol for 24 h at 0 • C. Cells were then washed with PBS and incubated with 250 µL of Guava Cell Cycle Reagent (Millipore, Burlington, MA, USA) for 30 min at room temperature in the dark. Samples were analyzed on NovoCyte TM 2000 FlowCytometry System (ACEA, San Diego, CA, USA).

Conclusions
Novel betulinic, ursolic, and oleanolic acid derivatives, containing a guanidine moiety have been designed and synthesized in an attempt to develop potent antitumor agents. These compounds and their precursors, monoamine, diamine and triamine derivatives, were tested for cytotoxic activity on various human tumor cell lines. Guanidine-functionalized triterpenoids demonstrated higher cytotoxicity in Jurkat cells, compared with original triterpenoic acids. Most of the tested guanidine derivatives showed higher IC 50 values than amines, but were less toxic to human fibroblasts. The lead molecules-dihydrobetulinic acid amine 15, its guanidine derivative 15c, and guanidinium salts of ursolic and oleanolic acids 18c and 20c were selected for extended biological testing by using flow cytometry analysis. Our results showed that the antitumor activity of compounds 15, 15c, and 18c is caused by apoptotic processes and induction of cell cycle arrest in the S-phase. Nevertheless, addition information concerning the molecular mechanisms and targets of these triterpene acid derivatives is needed.

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