Synthesis and cytotoxic evaluation of malachite green derived oleanolic and ursolic acid piperazineamides

The coupling of acetylated piperazinylamide spacered triterpenoic oleanolic acid and ursolic acid with meta or para substituted carboxylated malachite green analogs gave conjugates 10, 11, 15, and 16 that were cytotoxic for several human tumor cell lines. Especially, an oleanolic acid-derived compound 10 was cytotoxic for MCF-7 human breast carcinoma cells (EC50 = 0.7 μM). These derivatives represent first examples of triterpenoic acid derivatives holding a cationic scaffold derived from malachite green.


Introduction
Chemotherapeutic treatment of cancer represents still a scientific challenge. Although nowadays many patients suffering from cancer can be cured or-at least-their life span can be increased. Several types of cancer, however, are difficult to be handled, and the cure rate (Holzel et al. 2017;Laffman-Johnson 2012;Vliek et al. 2018) remained low over all the years despite intensive research.
Recently the potential of scaffolds holding a cationic functional group came into the focus of renewed interest inasmuch as their transport into cells might be facilitated by cation-transporters (Cai et al. 2016;Everett et al. 2013;Qian et al. 2016). Some of these transporters were reported to be overexpressed in malignant cells (Cai et al. 2014;Cai et al. 2016;Everett et al. 2013). Several molecules holding a cationic residue are cytotoxic and seem to target the mitochondria, such as ammonium (Biedermann et al. 2010;Kataev et al. 2014) or phosphonium salts (Spivak et al. 2013(Spivak et al. , 2017) of more complex molecules. Thus, these compounds are mitocans ("mitochondrially targeted anticancer drugs"). Some conjugates of pentacyclic triterpenes are mitocans, too. Thus, hybrids holding an extra cationic functional group have shown promising to excellent cytotoxic results. While "simple" quaternary ammonium salts (Biedermann et al. 2010;Kataev, Strobykina, and Zakharova 2014) were only of moderate cytotoxicity with their EC 50 values being in the same potency range as phosphonium salts (Spivak et al. 2017(Spivak et al. , 2013, hybrids consisting of a suitable pentacyclic triterpene, an amine spacer and a BODIPY-FL group (Brandes et al. 2020;Krajcovicova et al. 2018) held lower EC 50 values against a variety of different human cancer cell lines. Superior cytotoxicity, however, was found for those triterpenoids holding one or two O-acetyl groups on ring A, an amide spacer at C-28 (preferentially a piperazinyl residue), and a rhodamine B moiety attached to this spacer (Sommerwerk et al. 2017). EC 50 values in the low micromolar (Kahnt et al. 2018;Wolfram et al. 2018aWolfram et al. , 2018b and even nano-molar range (Sommerwerk et al. 2017) were reported for these conjugates.

Results and discussion
In extension of these findings and due to the close structural similarity between malachite green (A) and rhodamine B (B, Fig. 1) we became interested in the synthesis and biological evaluation of conjugates holding a cationic triphenylmethane moiety especially of scaffolds of the "malachite green type", i.e., 1 and 2; these scaffolds differ from malachite green by the presence of an additional carboxyl group; the latter is necessary for the attachment to the triterpene-spacer adduct.
Recently conjugates holding this type of a malachite green moiety (Müller et al. 1997;Rassow and Gruber 1915;Yang et al. 2007) have been used for the production of antibodies to be used in environmental analyses (Yang et al. 2007), as part of a fluorophore to label antimicrobial peptides (Zhao et al. 2016) and for the detection of nerve gas simulants by chromogenic chemodosimeters (Costero et al. 2012).
The synthesis of 1 (Scheme 1) started with the reaction of m-formyl-benzoic acid (3) with N, N-dimethylaniline in the presence of zinc chloride to yield 4 (Rassow and Gruber 1915;Sinev et al. 1978). Similarly, from p-formylbenzoic acid (5) compound 6 was obtained in 89% yield as a blue-greenish compound. Reaction of 4 with tetrachloro-p-benzoquinone afforded 41% of 1 while from 6 (Harle et al. 2018;Mueller et al. 1981;Rassow and Gruber 1915) under the same conditions 2 was obtained; both compounds are dark green solids. Compounds of this type are also known as "Mordant Green" or "Mordant Blue". Oleanolic acid (7) was acetylated (Sommerwerk et al. 2017) (Scheme 2) to yield acetate 8; the latter compound was coupled with piperazine as previously reported to yield 9 (Sommerwerk et al. 2017). Compound 1 was activated in situ with oxalyl chloride and coupled with 9 to afford target compound 10. Reaction of 9 with 2 gave 11.
Acetylation of ursolic acid (12) gave acetate 13 followed by its reaction with piperazine to yield amide 14 (Sommerwerk et al. 2017). Coupling of the latter compound with 1 or 2 as described above afforded the hybrids 15 and 16, respectively.
The compounds were screened for their cytotoxic activity using sulforhodamine B assays; the results from these assays are summarized in Table 1. Betulinic acid and doxorubicin were used as standards. While ursolic acid (12) is of similar cytotoxicity as standard betulinic acid, no significant cytotoxicity was found for oleanolic acid (7). Cytotoxicity increased about tenfold for the actylated piperazinylamides 9 and 14. Except for the HT29 human adenocarcinoma cells, the EC 50 values determined for the carboxylated malachite green derivatives 1 and 2 were found in the low one-digit μM range. As far as the coupling products 10, 11, 15, and 16 are concerned, oleanolic acid-derived compounds 10 and 11 were of significantly higher cytotoxicity than ursolic acidderived 15 and 16. The selectivity between tumor cells and the nonmalignant fibroblasts is-by and large-the same as in betulinic acid but significantly better than that of doxorubicin. Furthermore, meta substituted malachite green carboxylates 10 and 15 were about twice as cytotoxic than para substituted analogs 11 and 16, respectively. Thus, this makes oleanolic acid-derived 10 to the most cytotoxic compound of this series holding EC 50 values between 0.7 and 0.9 μM. Furthermore, 10 is~17 times more cytotoxic than standard triterpenoic acid betulinic acid and circa 50 times more cytotoxic than starting material oleanolic acid. Interestingly, while the lowest cytotoxicity was observed for HT29 cells, for an analogous rhodamine B derivative especially for this cell line the highest cytotoxicity has been noted. This seems to prove that both the type of triterpenoic acid, the type of amide linkage, the type of cationic residue, and its substitution pattern are of great importance both with respect to a tumor cell line-specific cytotoxicity and to cytotoxicity in general. Ongoing studies in our laboratory try to gain a deeper insight into these observations.

Conclusion
Coupling of acetylated triterpenoic oleanolic acid and ursolic acid holding a piperazinylamide spacer with meta or para substituted carboxylated malachite green analogs gave conjugates 10, 11, 15, and 16, respectively. These compounds were cytotoxic for several human tumor cell lines. Thereby, oleanolic acid-derived compound 10 was especially cytotoxic for MCF-7 human breast carcinoma cells (EC 50 = 0.7 μM).
Experimental NMR spectra were recorded using the Varian spectrometers Gemini 2000 or Unity 500 (δ given in ppm, J in Hz; typical experiments: H-H-COSY, HMBC, HSQC, NOESY), MS spectra were taken on a Finnigan MAT LCQ 7000 (electrospray, voltage 4.1 kV, sheath gas nitrogen) instrument. The optical rotations were measured on a Perkin-Elmer polarimeter at 20°C; TLC was performed on silica gel (Merck 5554, detection with cerium molybdate reagent); melting points are uncorrected (Leica hot stage microscope), and elemental analyses were performed on a Foss-Heraeus Vario EL (C-HNS) unit. IR spectra were recorded on a Perkin Elmer FT-IR spectrometer Spectrum 1000. The solvents were dried according to usual procedures. The purity of the compounds was determined by HPLC and found to be >96%. Oleanolic acid (7) and ursolic acid (12) were obtained from Betulinines (Stříbrná Skalice, Czech Republic) in bulk quantities.