Mitocanic Di- and Triterpenoid Rhodamine B Conjugates

The combination of the “correct” triterpenoid, the “correct” spacer and rhodamine B (RhoB) seems to be decisive for the ability of the conjugate to accumulate in mitochondria. So far, several triterpenoid rhodamine B conjugates have been prepared and screened for their cytotoxic activity. To obtain cytotoxic compounds with EC50 values in a low nano-molar range combined with good tumor/non-tumor selectivity, the Rho B unit has to be attached via an amine spacer to the terpenoid skeleton. To avoid spirolactamization, secondary amines have to be used. First results indicate that a homopiperazinyl spacer is superior to a piperazinyl spacer. Hybrids derived from maslinic acid or tormentic acid are superior to those from oleanolic, ursolic, glycyrrhetinic or euscaphic acid. Thus, a tormentic acid-derived RhoB conjugate 32, holding a homopiperazinyl spacer can be regarded, at present, as the most promising candidate for further biological studies.

Rhodamine B (RhoB) seems to be a privileged scaffold. This fluorescent dye, also known as rhodamine 610, C.I. Pigment violet 1, basic violet 10, and C.I. 45170 [68], was invented in 1888 ("Tetraethyl-rhodamine") by M. Cérésole [69,70], and since then it has been widely used in biology, biotechnology and as a biosensor [71,72]. RhoB exists in an equilibrium [73][74][75][76][77] between an "open" positively charged form A (Figure 1) that is fluorescent and a "closed", non-fluorescent form B. Under acidic conditions, pink-colored A dominates, while colorless B dominates under basic conditions. Further, in less polar organic solvents, the zwitterionic form C undergoes a rapid reversible conversion to B [78][79][80][81]. RhoB is suspected to be carcinogenic [82][83][84][85]. The LD 50 value for orally administered RhoB in rats is >500 mg/kg, and an older report classified RhoB (as well as Rho6G) as possibly carcinogenic in rats [85]. RhoB, however, seems not to be mutagenic in Chinese hamster ovary cells [86], but it presents a genotoxic hazard for mammalian organisms [87]. As far as the RhoB-triterpene conjugates are concerned, two types of compounds have been accessed so far: triterpenes with a RhoB moiety directly attached to the skeleton of the triterpene, and compounds wherein these two units are separated by a suitable spacer.
By means of suitable double-staining experiments, it could be shown that these hybrids are actually effective as mitocans [88], and preliminary molecular modeling studies suggest these compounds might target the mitochondrial NADH dehydrogenase and mitochondrial succinate dehydrogenase [89]. Both enzymes are part of the mitochondrial electron transport chain; this also suggests an increased production of reactive oxygen species (ROS). An increased production of ROS would lead to an oxidative damage of the cell and trigger apoptosis through an intrinsic pathway. Therefore, the integrity of the RhoB basic structure seems to be of crucial importance. It has been shown that derivatives from the triphenylmethane dye malachite green still exhibit increased cytotoxicity as compared to their parent compounds [90]. The cytotoxicity, however, of these hybrids was much lower than those observed for the RhoB derivatives (vide infra). The triterpenoid skeleton is equally important. Here, too, it was shown that "simple" RhoB conjugates 1-9 ( Figure 3) also had lower cytotoxicity than the corresponding triterpenoid analogs, but their tumor cell/non-tumor cell selectivity was also diminished (Table 1) [91].  Of special interest seems the morpholinyl derivative 4 inasmuch as this compound held the highest selectivity of this series with respect to MCF-7 carcinoma cells (S = (EC 50, NIH 3T3 / EC 50, MCF-7 ) > 19.5) and A2780 ovarian cancer cells (S = (EC 50, NIH 3T3 / EC 50, A27807 ) > 18.1) [90].
The highest cytotoxicity was observed for the hexyl ester 2 (EC 50 = 0.15-0.19 µM) for the different tumor cell lines. Interestingly, an eicosyl ester 3 with a lipophilicity similar to that of triterpenoids did not show even moderate cytotoxicity [90], while hydroxycinnamic acid rhodamine B conjugates displayed good cytotoxicity in the low µM range [92].
The importance of the presence of a triterpenoid backbone is also evident from studies concerning RhoB steroid conjugates ( Figure 4) [93]. In these studies, the reaction of the steroids cholesterol, testosterone, prednisone and abiraterone with an activated RhoB chloride furnished ester conjugates holding low EC 50 values (SRB assays with several human tumor cell lines, Table 2). Thus, a testosterone conjugate 10 held EC 50 = 60 nm for MCF-7 cells, but acted by necrosis (20%, A2780 cells). A prednisone conjugate 11 was less cytotoxic (0.2 µM for MCF-7 cells) but acted in A2780 cells mainly by apoptosis (48%) and late apoptosis (14%). In addition, this compound showed a higher selectivity for the A2780 tumor cells (S = 73) than for NIH 3T3 fibroblasts. For comparison, an abiratone conjugate 12 was less cytotoxic and also less selective [93].  A closer look at the cell cycle by FACS (with A2780 cells) showed a decrease of the G1 and G2/M peak with an increase of cells in the S phase. For cells treated with 11, the S phase peak and the subG1/apoptosis peak increased significantly. However, for all compounds the selectivity between tumor cells and non-malignant fibroblasts NIH 3T3 was small and never exceeded 7.3 (11, for MCF-7 cells) [93].
A similar behavior was observed for dehydroabietylamine (DHAA) derivatives 13-16 ( Figure 5, Table 3). These products were easily obtained from dehydroabietylamine by the microwave-assisted multicomponent Ugi reaction using paraformaldehyde, an isocyanide and RhoB with yields between 47 and 50% [94].  Although the cytotoxicity of these compounds was good, their pharmacological potential was restricted by low selectivity values. Interestingly enough, products 16a/16b (Figure 6), having been obtained from a simple Schotten-Baumann reaction with DHAA and RhoB, were not cytotoxic at all [94]. As mentioned above, RhoB conjugates derived from primary amines are able to form intramolecular non-fluorescent spirolactams (here 16a). From a photo-induced ring opening reaction, 16b was obtained from 16a very quickly within 10 s of irradiation either with visible light or with UV light (λ = 254 or 366 nm). This equilibrium is also strongly influenced by changes in temperature, and at room temperature 16a dominates the equilibrium [94].  All of these compounds had EC 50 values between 0.02 and 15.8 µM (Table 4); thereby, the cytotoxicity of benzyl esters 21-24 was lower than the cytotoxicity of the methyl esters 17-20, while the benzyl amides 25-28 were the most cytotoxic compounds of this series. The presence of a benzyl ester group as in 21-24 seems to be disadvantageous, while the opposite is true for the benzyl amides 25-28. Compound 27 was the most cytotoxic compound (EC 50 = 0.02-0.08 µM), but it was not selective for human tumor cells. Extra staining experiments showed this compound to be accumulated in the mitochondria of A2780 cells and to act mainly by apoptosis [95]. Noteworthy in this context is the higher cytotoxicity of the glycyrrhetinic acid derivatives as compared to analogs derived from OA, UA or BA. Extensions in the design of these compounds led to the synthesis of triterpene conjugates with further modifications in the backbone (→ tormentic acid (TA) and euscaphic acid (EA)) as well as to changes in the ring size of the heterocyclic spacer between the backbone of the triterpene and the RhoB moiety ( Figure 8). The significantly higher cytotoxicity (Table 5) of TA-derived 32 seems particularly noteworthy when comparing the different spacers: Thereby, the presence of a homopiperazinyl spacer [96] (as in 32) proved to be clearly superior to the piperazinyl moiety (as in 31). A similar trend was also noted for EA-derived compounds 29 and 30. On the other hand, TA-derived compounds were more cytotoxic than the corresponding EA derivatives. Interestingly, the absolute configuration at C-2 and C-3 in TA corresponds exactly to the configuration found in maslinic acid (MA). Several MA derivatives (for example [97,98], a diacetylated benzylamide EM2, Figure 9) were of higher cytotoxicity and better selectivity than their corresponding OA or UA derivatives.  The same configuration is found in asiatic acid (AA). Again, its acetylated piperazinyl-rhodamine B conjugate 33 was most cytotoxic to many human tumor cell lines, being accumulated in the mitochondria, and it also acted as a mitocan [99]. However, for this compound an unusual non-linear rate of growth was detected for some human tumor cell lines (e.g., colorectal carcinoma HT29 and melanoma 518A2). In a bimodal manner at two different concentrations the tumor cells were killed, a phenomenon that might be due to an accelerated recovery of the mitochondrial membrane potential or due to a modulation of the mitochondrial permeability pores. However, at present a concentration-triggered activation of a metabolizing enzyme cannot completely be ruled out [99].
A graphical comparison of all derivatives (using the target line A2780 as an example) is given in Figure 10 including a comparison of tumor cell/non-tumor cell selectivity (A2780 vs. NIH 3T3) of all compounds.
From Figure 10 the high potential of compound 32 (selectivity for A2780 or FaDu cells, ca. 190) becomes clearly visible, making this compound an interesting drug for advanced testing and biological screening.

Conclusions
OA-derived RhoB conjugates appear to be superior to analog UA-derived compounds in the majority of cases with respect to their cytotoxicity. Although AKBA-derived derivatives have good cytotoxicity properties, they were found to be less cytotoxic compared to other triterpene carboxylic acid derivatives, but they often showed better tumor cell/non-tumor cell selectivity. So far, the best cytotoxicity properties have been found for MA-, EA-and TA-derived derivatives. These allowed the transition to compounds of nano-molar activity, while many other triterpene carboxylic acid derivatives were cytotoxic only on a micro-molar concentration range. MA-derived derivatives seem to be approximately equivalent to EA-derived compounds. They are currently only surpassed in many tumor cell lines only by the analogous derivatives from TA. From results available so far, it can be concluded that compounds holding a homopiperazinyl spacer are superior to those with a piperazinyl spacer. This underlines the importance of the spacer for obtaining good cytotoxicity properties. Replacement of the secondary amide derived spacer by a primary amine like ethylenediamine has invariably led to RhoB conjugates of insignificant cytotoxicity (EC 50 > 30 µM) due to the formation of a spirolactam holding no positive charge in the RhoB part.
However, the presence of a distal cation is not sufficient to obtain compounds with excellent cytotoxicity, as has been shown for several quaternary ammonium compounds or compounds where the RhoB part has been replaced by, for example, malachite green, a BODIPY residue or a safirinium group. In addition, the latter compounds do not act as mitocans, since their primary target is the endoplasmic reticulum.
A statement on the extent to which the replacement of the RhoB group with another rhodamine has a positive effect on biological activity cannot be made at present. The cytotoxic properties of these compounds, other spacers and other triterpene carboxylic acids are currently the subject of further investigation. The combination of the "correct" parent structure, the "correct" spacer and the "correct" RhoB seems to be decisive for the ability of the conjugate to accumulate in mitochondria. So far, a tormentic acid acid-derived RhoB conjugate 32 holding a homopiperazinyl spacer can be regarded as the most promising candidate for further biological studies. At present, no extended investigations have been carried out on the precise mode of action of these molecules.