Madecassic Acid—A New Scaffold for Highly Cytotoxic Agents

Due to their manifold biological activities, natural products such as triterpenoids have advanced to represent excellent leading structures for the development of new drugs. For this reason, we focused on the syntheses and cytotoxic evaluation of derivatives obtained from gypsogenin, hederagenin, and madecassic acid, cytotoxicity increased—by and large—from the parent compounds to their acetates. Another increase in cytotoxicity was observed for the acetylated amides (phenyl, benzyl, piperazinyl, and homopiperazinyl), but a superior cytotoxicity was observed for the corresponding rhodamine B conjugates derived from the (homo)-piperazinyl amides. In particular, a madecassic acid homopiperazinyl rhodamine B conjugate 24 held excellent cytotoxicity and selectivity for several human tumor cell lines. Thus, this compound was more than 10,000 times more cytotoxic than parent madecassic acid for A2780 ovarian cancer cells. We assume that the presence of an additional hydroxyl group at position C–6 in derivatives of madecassic, as well as the (2α, 3β) configuration of the acetates in ring A, had a beneficial effect onto the cytotoxicity of the conjugates, as well as onto tumor/non-tumor cell selectivity.


Introduction
Despite all efforts and tremendous achievements in the fight against cancer, in 2020 10 million people worldwide died as a consequence of this disease [1], and 19.3 million new cases were reported by the International Agency for Research on Cancer [2]. Many chemotherapeutic agents hitherto used for the therapy of cancer are natural products or derived from them. Several compounds derived from pentacyclic triterpenes were successfully encountered as highly cytotoxic as well as exhibiting pronounced tumorselectivity [3][4][5][6][7]. A closer look into this class of compounds, however, revealed that research was mainly focused on derivatives of betulinic, ursolic, and oleanolic acid and-to a minor extent-of glycyrrhetinic acid [8][9][10][11][12][13][14][15]. The number of investigations dealing with cytotoxic agents derived from gypsogenin, hederagenin, or madecassic acid, however, remained small over many years [16][17][18][19]. This may be due to the fact that the availability of these compounds is considered to be worse than that of, for example, betulinic acid [20][21][22]. This has changed as gypsogenin can now be obtained in good yields from the corresponding saponin (which is available in large technical quantities). Hederagenin was previously only available from extracts from the pericarp of the soap tree [23,24]. However, it is now even more convenient, obtained by a partial synthesis from also readily available gypsogenin [23]. Furthermore, hederagenin and gypsogenin can be interconverted into each other by a short sequence and in good yields [23].
Recently, we were able to show that acetylated pentacyclic triterpenoid amides are highly cytotoxic for many human tumor cell lines [24][25][26][27], while parent triterpenoic acids possess low cytotoxicity. The cytotoxicity of these compounds, however, intensifies upon acetylation and amidation, and even more by linking them with rhodamine B [28]. Thus, the synthesis of different rhodamine B conjugates has moved into the focus of scientific interest [25][26][27]29]. For example, benzyl-, piperazinyl-, and homopiperazinyl-amides and their resulting rhodamine B conjugates show cytotoxic effects even in nanomolar concentration. The presence of an extra cationic moiety such as a rhodamine B scaffold enhances their cytotoxic activity tremendously. However, many of these conjugates were also less cytotoxic to non-malignant fibroblasts (NIH 3T3) [26][27][28]30]. As a consequence of these promising results, and to have a closer look into this class of compounds, gypsogenin and hederagenin were chosen as starting materials for further studies. For comparison, madecassic acid was also included to investigate the possible influence of the presence of extra hydroxyl groups onto cytotoxic activity and selectivity. During our work, different acetylated triterpenoic derivatives modified at their carboxylic group were accessed, such as substituted benzyl, piperazinyl, and homopiperazinyl amides which were subsequently linked at their distal amino group as an amide to a rhodamine B moiety. Previous studies showed an increase in malignant cells' membrane potential in relation to non-malignant cells. Thus, cationic compounds such as rhodamine B conjugates accumulate in these areas and cause selective cell death [31,32]. This is regarded as the main reason for the selective cell death of tumor cells upon incubation with the rhodamine B conjugates.
Our main objective was to find out whether triterpene amides, as well as spacered triterpene rhodamine conjugates, exhibit good cytotoxicity when the pentacyclic triterpene skeleton holds a vicinal hydroxyl groups in ring A, and optionally another hydroxyl group at another position. These requirements are fulfilled, for example, by the compounds gypsogenin, hederagenin, or madecassic acid. This should also allow a comparison with the conjugates of ursolic, oleanolic, and maslinic acid presented earlier.
Thus, a set of novel terpenoic amides and their rhodamine B conjugates was synthesized, and the cytotoxicity of the compounds was determined by sulforhodamine B (SRB) assays.

Biological Evaluation
To assess their cytotoxicity parent, triterpenoic acids 1-3, their acetates 4-6, the amides 7-18, as well as the respective rhodamine B conjugates 19-24 were subjected to SRB assays employing several human tumor cell lines (A375, HT29, MCF-7, A2780, and HeLa) as well as non-malignant fibroblasts (NIH 3T3). The results from these assays are compiled in Table 1. The EC 50 values in µM from SRB assays were determined after 72 h of treatment, and the values are averaged from three independent experiments performed each in triplicate, confidence interval CI = 95%; mean ± standard mean error).  As a result, and as expected, the parent compounds 1-3 (holding unsubstituted hydroxyl groups) were-by and large-less cytotoxic than their corresponding acetates 4-6.
The former compounds showed some cytotoxicity for all human tumor cell lines and moderate selectivity.
The latter compounds, however, holding one or more acetyl groups at positions 2, 3, and 23, showed an increased cytotoxic activity for all cancer lines, with EC 50 values as low as EC 50 = 3.50 µM (for A375 and compound 6).
Transforming the carboxyl group into an amide led to compounds of increased cytotoxic effects onto the tumor cells. Benzylamides (7, 11, and 15), phenyl amides (8, 12, and 16), piperazinyl amides (9, 13, and 17) and homopiperazinyl amides (10, 14, and 18) held high cytotoxicity and moderate selectivity. In this series of compounds, hederagenin derived phenyl amide 12 was most cytotoxic with an EC 50 value of 0.66 µM for A375 tumor cells. Comparing the cytotoxic effects of phenyl-and benzyl amides conjugates with those measured for the corresponding piperazinyl and homopiperazinyl analogues showed the former of higher cytotoxicity but also of an improved selectivity (malignant vs. non-malignant cells).
Rhodamine B analogues 19-24 showed both an increased cytotoxicity but also improved selectivity. The lowest EC 50 value was determined for 24 with EC 50 = 0.0029 ± 0.0005 for A2780 ovarian tumor cells.
The selectivity between malignant cells and the non-malignant cells was also affected by the kind of spacer linking the triterpene with the rhodamine B moiety. Thereby, a significant difference in cytotoxicity was detected between compounds holding a homopiperazinyl (20,22, and 24) instead of piperazinyl spacer (19, 21, and 23).
As a result, compound 24, the most active as well as selective compound with homopiperazinyl spacer, was nearly 10,000 times more cytotoxic than parent madecassic acid.
The mode of action-as previously shown for several triterpene-rhodamine B conjugates-is to act as mitocans. To demonstrate this also for 24-the most cytotoxic compound of this study-A375 cells were stained with acridine orange (AO), rhodamine 123, and Hoechst 33,342. Thereby, Hoechst 33,342 binds selectively to DNA while rhodamine 123 is usually applied for dying mitochondria. The results of these staining experiments are depicted in Figure 1 and prove that 24 also acts as a mitocan, as this compound is localized within the cells exactly in the same area as rhodamine 123, while a location in the nucleus can be ruled out from the staining experiments using Hoechst 33,342. The influence of an extra hydroxyl group in ring B can best be seen from the cytotoxicity of the analogs of madecassic acid. The results from the SRB assays showed compounds 3, 6, 15, 16, 17, 18, 23, and 24 as cytotoxic with EC 50 values as low as 0.015 ± 0.001 µM; for compound 24 and A2780 cells, a selectivity index SI EC50, NIH 3T3/EC50, A2780 of approximately 20 can be calculated. From these results, we conclude that the presence of an additional hydroxyl group at position C-6 in the triterpenoid skeleton might be responsible for a significantly improved cytotoxicity.
As compounds holding two acetyl groups in ring A with (2α, 3β) configuration (such as 24 or the analogues prepared earlier from maslinic or tormentic acid) are clearly more cytotoxic than those bearing only one acetyl group (such as oleanolic and ursolic, but also glycyrrhetinic, betulinic, or platanic acid), this confirms our original working hypothesis. At present, triterpenes with a larger number of hydroxyl or acetyl groups are being converted to the corresponding spacered rhodamine B conjugates in our laboratories in order to verify whether the trend shown here has general validity.

General
A detailed description of materials and methods can be found in the Supplementary Materials File. For biological screening, the cell lines were obtained from ATCC: A375 (CRL-1619), HT29 (HTB-38), MCF7 (HTP-22), A2780 (HTP-77), HeLa (CCL-2), and NIH 3T3 (CRL-1573) 13 C NMR spectra (Supplementary Materials) were recorded as APT spectra showing CH and CH 3 groups as positive signals and CH 2 groups and quaternary carbons as negative signals.

General Procedure for the Synthesis of of Triterpenoic Amides 7-18 (GPB)
To a solution of 4-6 (1 eq.) in dry DCM (10 mL), oxalyl chloride (4 eq.), DMF (0.24 eq.), and TEA (0.24 eq.) were added. After stirring for 2 h at room temperature, the solvent was removed under reduced pressure, re-evaporated with DCM (3 × 10 mL), and the residue was dissolved in dry DCM (10 mL). A solution of the respective amine or diamine (3 eq.) in dry DCM, TEA (1 eq.), and DMAP (cat.) was added, and stirring at room temperature was continued until completion of the reaction (as indicated by TLC). The solvent was removed under reduced pressure, and the crude product was subjected to column chromatography to yield compounds 7-18.

Madecassic Acid (3)
This compound was obtained from different local suppliers and used as received.

Conclusions
Several amides and rhodamine B conjugates derived from parent triterpenoic acids gypsogenin, hederagenin, and madecassic acid were synthesized and their cytotoxicity was evaluated in SRB assay. While the parent acids exhibited very low cytotoxicity, their corresponding acetates proved better. Acetylated amides, however, gave significantly lower EC 50 values than the acetates. A significant increase in cytotoxicity was observed for the corresponding rhodamine B conjugates obtained from the latter. While rhodamine B is not cytotoxic (cut-off 30 µM), triterpenoic conjugates of rhodamine B holding a piperazinyl or homopiperazinyl spacer were cytotoxic, even in nano-molar concentration, with compound 24 being the most cytotoxic one. In comparison, homopiperazinyl-spacered compounds were more tumor-cell selective than their piperazinyl-spacered analogs. The preparation of benzyl and phenylamides succeeded in generating compounds holding similar bioactivity as previously reported for some analogs derived from betulinic, ursolic, glycyrrhetinic, or oleanolic acid. Our present studies showed spacered conjugates derived from madecassic acid of excellent cytotoxicity in single-digit nanomolar concentration combined with a noteworthy tumor/non-tumor cell selectivity.