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Synthesis, biological evaluation and toxicity of novel tetrandrine analogues

In this work, we present the design and synthesis of novel fully synthetic analogues of the bisbenzylisoquinoline tetrandrine, a molecule with numerous pharmacological properties and the potential to treat life-threatening diseases, such as viral infections and cancer. Its toxicity to liver and lungs and the underlying mechanisms, however, are controversially discussed. Along this line, novel tetrandrine analogues were synthesized and biologically evaluated for their hepatotoxicity, as well as their antiproliferative and chemoresistance reversing activity on cancer cells. Previous studies suggesting CYP-mediated toxification of tetrandrine prompted us to amend/replace the suspected metabolically instable 12-methoxy group. Of note, employing several in vitro models showed that the proposed CYP3A4-driven metabolism of tetrandrine and analogues is not the major cause of hepatotoxicity. Biological characterization revealed that some of the novel tetrandrine analogues sensitized drug-resistant leukemia cells by inhibition of the P-glycoprotein. Interestingly, direct anticancer effects improved in comparison to tetrandrine, as several compounds displayed a markedly enhanced ability to reduce proliferation of drug-resistant leukemia cells and to induce cell death of liver cancer cells. Those enhanced anticancer properties were linked to influences on activation of the kinase Akt and mitochondrial events. In sum, our study clarifies the role of CYP3A4-mediated toxicity of the bisbenzylisoquinoline alkaloid tetrandrine and provides the basis for the exploitation of novel synthetic analogues for their antitumoral potential.

All these multiple pharmacological activities make tetrandrine (1) an interesting lead compound and a potential drug candidate for diverse applications. Unfortunately, its clinical application as a drug is limited by its toxicity. Several toxic side effects have been reported for tetrandrine and other bisbenzylisoquinolines so far [25]. In animal models, mainly a damage of liver and lungs was observed [26e30].
The molecular mechanism of tetrandrine-induced toxicity has not been entirely elucidated yet. Li and coworkers [31] hypothesized that an interaction of tetrandrine with p38a MAPK (mitogenactivated protein kinase) led to liver injury, whereas several other studies suggest the involvement of cytochrome P450 (CYP) enzymes in tetrandrine-induced pulmonary [26,27] and hepatic toxicity [32]. Qi et al. [32] consider CYP2E1 as primary reason for mitochondrial dysfunction of rat hepatocytes after tetrandrine exposure, which they relate to reactive oxygen species (ROS) that were generated in the course of CYP2E1 metabolism. However, it is unknown which molecular mechanisms are connected to CYP2E1mediated toxicity. In a metabolic study by Jin et al. [26] a CYP3A4and CYP3A5-mediated metabolism equally leading to a potentially toxic intermediate is described. Hereby one particular structural element is suggested to be responsible for tetrandrine's toxicity. In the hypothesized metabolic pathway, the methoxy group at C-12 in para-position to a benzylic methylene group is first demethylated to give the corresponding phenol 3. This phenol then undergoes enzymatic oxidation to the para-quinone methide 4, which is an electrophilic intermediate prone to attack bio-nucleophiles (see Scheme 1). After incubation of tetrandrine (1) with human liver mircosomes in the presence of glutathione (GSH), a corresponding GSH conjugate (5) was detected via LC-MS, which provides evidence for the formation of the para-quinone methide 4.
Aim of this study was to investigate if expression of CYP3A4, the most abundant CYP enzyme in human liver [43], substantially contributes to the toxicity of tetrandrine (1) and whether the paramethoxybenzyl moiety is indeed the crucial point. In a combined medicinal chemistry and cell biology approach we evaluated if a reduction of the discussed CYP3A4-mediated toxicity of tetrandrine can be achieved by replacing or eliminating the hypothesized metabolically instable 12-methoxy group. This motivated us to synthesize analogues of tetrandrine which are lacking the critical methoxy group or the entire benzyl unit. Subsequently, the toxicity of the obtained tetrandrine analogues to human hepatocyte like cells (HepaRG™) and human hepatocellular carcinoma cells (HepG2), with or without overexpression of CYP3A4, was investigated. Concurrently, we determined the impact of structural modifications on the biological activity of compounds and focused on inhibition of cancer cell proliferation and interaction with the efflux pump P-gp, as an application in tumor therapy is desired.

Chemistry
As mentioned above, we aimed at investigating the role of the metabolically labile 12-methoxy group of tetrandrine (1). Thus, we designed analogues of tetrandrine, in which this methoxy group is either deleted (target compounds RMS1-2) or replaced by metabolically stabile trifluoromethoxy (RMS3-4) or chlorine substituents (RMS7-8, see Scheme 3). Further we replaced the methoxybenzyl residue by a thienylmethyl (target compound RMS5-6) and by a non-aromatic butylidene unit (RMS9-10). The synthesis of these tetrandrine analogues is based on our recently published racemic total synthesis of tetrandrine and isotetrandrine [44] following the therein described synthetic protocol of "Route 1b". The intermediate 11 (no. 19 in Ref. [44]) of the (iso)tetrandrine synthesis served as a valuable starting material, since it already comprises one tetrahydrobenzylisoquinoline unit (rings A 0 -C 0 ) and ring A of the second half of the bisbenzylisoquinoline scaffold (see Fig. 1). Detailed information for the preparation of intermediate 11 is provided in the Supporting Information.
bisbenzylisoquinolines 13a-13d and bromobutyl analogue 13e (Scheme 3). The required enol ethers 12a-d were conveniently prepared via a Wittig olefination of the corresponding commercially available aromatic aldehydes (Scheme 2), 5-bromopentanal (12e) was obtained by PCC oxidation of the corresponding primary alcohol [45]. The intermediates 13a-13e were further processed in intramolecular Ullmann-type cross-coupling reactions or an S N 2 reaction (for the u-bromobutyl intermediate 13e) respectively to access the diaryl ether bridges (connecting rings C and C' in 14a-d) or the aliphatic bridge in 14e, furnishing macrocyclic bisbenzylisoquinoline analogues. Final step was the simultaneous reduction of both carbamate groups using lithium alanate to give the desired N-methylated compounds RMS1-6 and RMS9-10. In case of the chloroarene variations RMS7-8 this method led to substantial dechlorination, even when using the milder reduction reagent Red-Al® (sodium bis(2-methoxyethoxy)aluminum dihydride). To circumvent this problem, the carbamate groups were first removed by treatment with methyl lithium and the resulting secondary amino groups were subsequently N-methylated via reductive alkylation using formaldehyde/NaBH 3 CN. Since the N-acyl Pictet-Spengler condensations expectedly proceeded with no or only rather poor diastereoselectivity [44] (with a tendency for the formation of the R,R/S,S isomers), we obtained racemic mixtures of diastereomers in every case. Luckily we were able to separate the open-chain diastereomers obtained in the N-acyl Pictet-Spengler reactions by flash column chromatography to provide, in the end, each tetrandrine analogue eventually as racemic compound with the relative stereoconfiguration of either tetrandrine (1) or isotetrandrine (2) (Fig. 1) in high diastereomeric ratios (d.r., determined by HPLC). The relative stereochemistry of the final racemic compounds RMS1-8 was determined by analysis of NMR data utilizing the comprehensive investigation performed by Guinaudeau et al. on more than 100 bisbenzylisoquinolines [46]. Herein the chemical shifts of distinctive protons in the NMR spectra of bisbenzylisoquinolines can be used for a reliable determination of the relative configurations at both asymmetric centers. The relative configurations of all seco-precursors were then assigned retrospectively. For the variations RMS9-10, which contain an alkyl bridge instead of an aromatic ring (ring C), this method is not applicable. Unfortunately, attempts of crystallization for crystallographic analysis were unsuccessful, and the determination of the stereoconfiguration in this case was therefore not possible.  [47,48], were treated with 10 and 20 mM of tetrandrine (1) and the analogues RMS1-10. Unlike expected, none of the new compounds was less toxic to HepaRG™ cells than tetrandrine (1) at 10 mM ( Fig. 3a). In contrast, RMS4 and RMS8 slightly, but RMS2 strongly decreased cell viability. Similarly, at 20 mM, RMS2, RMS4 and RMS8 were significantly more toxic to HepaRG™ cells than tetrandrine (1) (Fig. 3b). Of note, 20 mM of RMS6 and RMS9 were significantly less toxic than tetrandrine, whereas toxic effects of the other compounds did not substantially differ from those of tetrandrine (1). It should be noted that, depending on the substituent at C-12 of ring C, oxidation to a para-quinone methide cannot be excluded for 12-unsubstituted compounds RMS1/RMS2 (via initial CYPmediated ring hydroxylation), whereas in the trifluoromethoxy compounds RMS3/RMS4 and the chloro compounds RMS7/RMS8 oxidation processes are prevented by metabolically stable substituents.
To specifically determine the influence of CYP3A4 activity on toxicity in vitro, CYP3A4 with a C-terminal EGFP tag was cloned (CYP3A4-EGFP) and its physiological function was successfully validated by conversion of a proluminogenic CYP3A4 substrate in transiently transfected HepG2 cells, which was strongly reduced by the CYP3A4 inhibitor ketoconazole [49] (Suppl. Fig. 1a). Of note, treatment of CYP3A4 overexpressing HepaRG™ cells with respective compounds did not significantly increase cell death (Fig. 3c)  overexpression of CYP3A4 were confirmed by PCR methods (Suppl. Fig. 1b and c). Again, no correlation between the level of cellular CYP3A4 expression and cytotoxicity was observed.
To exclude that the observed similar cytotoxic effects on vector and CYP3A4-EGFP transfected cells were caused by insufficient initial demethylation that is required for tetrandrine (1) for being oxidized to a putatively toxic para-quinone methide, related alkaloids berbamine (7) and dauricine (6) were also tested. Both alkaloids (Fig. 2) bear a para-hydroxybenzyl moiety at the region of interest, which theoretically can be directly oxidized by CYP3A4 to a para-quinone methide with no need for previous O-demethylation. Similarly, for both berbamine (7) and dauricine (6), cell death was not significantly increased by cellular CYP3A4 overexpression (Fig. 3d). Taken together, the toxicity of tetrandrine (1) was not considerably decreased or even increased by variation of the paramethoxybenzyl moiety in several cellular models. However, no influence of the level of cellular CYP3A4 expression was found. Consequently, we conclude that the proposed CYP3A4-mediated generation of a para-quinone methide [26] does not substantially contribute to the hepatotoxicity of tetrandrine (1).
To decipher the correlation between the structure of quinone methides and their liver toxicity, Thompson and coworkers [52] investigated the toxicity of a series of 4-alkyl-2-methoxyphenols using an in vitro hepatotoxicity model. Although all tested molecules were converted into quinone methides, only little correlation between the rate of quinone methide formation in microsomes and relative toxicities of the alkylphenols was found [52]. It was suggested that primarily the reactivity of the quinone methides being formed and their stability towards solvolysis are the determining factors for their toxicity. These findings support the results of a former in vivo study [53] which observed differences in the toxicities of 2-methoxy-quinone methides that could be explained by their relative reactivities [54]. Thus, formation of quinone methides does not necessarily lead to toxicity in vitro and in vivo. For tetrandrine (1), other proposed mechanisms might play a more substantial role in toxicity, such as the generation of ROS by CYP2E1 [32] or the proposed interaction with p38a MAPK, a promotor of inflammatory processes in the liver [31], or additional effects that remain to be elucidated.

2.2.2.
Evaluation of anticancer effects 2.2.2.1. Chemosensitization of drug-resistant leukemia cells by Pglycoprotein inhibition. As modification of the para-methoxybenzyl moiety of tetrandrine (1) led, depending on the kind of chemical modification and relative stereochemistry, to decreased, similar or elevated cytotoxicities to liver cells, we aimed to decipher whether and to which extent the modifications influenced known biological effects of tetrandrine on cancer cells. Firstly, their interactions with the efflux transporter P-gp [11] were investigated. Except for RMS9 and RMS10 (analogues lacking aromatic ring C), all tetrandrine analogues were able to prevent efflux of the model substrate calcein-AM from P-gp overexpressing, vincristine-resistant (VCR-R) CEM cells [55] equally to the parental molecule (Fig. 4a). P-gp surface expression, evaluated by flow cytometry, was not diminished by any of the compounds at the same conditions (Fig. 4b), suggesting that the observed increase in calcein fluorescence (Fig. 4a) was caused by direct interaction with P-gp. Subsequently, the compounds were used at 1 mM in combination with varying doses of vincristine (VCR). As expected, all but RMS9 and RMS10 were able to sensitize VCR-R CEM cells to VCR, indicated by strongly increased apoptosis rates (Fig. 4c). RMS8 was the most potent analogue as it significantly increased apoptosis at 0.01 mM VCR (Fig. 4c). These data indicate that by replacing the para-methoxybenzyl moiety of tetrandrine with other substituted or unsubstituted aromatic residues, the potency to inhibit P-gp is maintained, while replacement with an alkyl chain (RMS9 and RMS10) strongly reduces it. Thereby, we add new information to structure-activity relationships of bisbenzylisoquinoline alkaloids with regards to their interference with the major efflux transporter P-gp.
2.2.2.2. Studies on anticancer effects and related pathways. In a next step, we determined direct antiproliferative effects of the compounds on VCR-R CEM cells, which are cross-resistant to a variety of chemotherapeutic drugs [55]. Interestingly, most tetrandrine analogues (RMS1-RMS5, RMS7-8) displayed a markedly enhanced ability to inhibit proliferation of VCR-R leukemia cells ( Fig. 5a and  b). RMS6, RMS9 and RMS10 were nearly inactive. Thus, in contrast to the lead structure tetrandrine (1), RMS1-RMS5, RMS7 and RMS8 were also effective when applied as monotherapy and therefore represent potential candidates to treat multidrug resistant cancers. Notably, RMS6 had no direct effect on proliferation ( Fig. 5a and b), but successfully sensitized VCR-R CEM cells to VCR (Fig. 4c) through inhibition of P-gp (Fig. 4a).
To gain more detailed insights into the underlying mechanisms, the influence of tetrandrine (1) and RMS1-RMS10 on pathways related to cell survival and apoptosis was investigated. Wan and coworkers [56] illustrated that tetrandrine and the multikinase inhibitor sorafenib have synergistic antitumor effects by reducing expression of anti-apoptotic proteins and activation of the kinase Akt. This prompted us to investigate how RMS1-RMS10 and tetrandrine (1) affect the intrinsic apoptosis pathway and phosphorylation of Akt, which is a key factor for proliferation and cell survial [56]. Upon treatment with 5 mM, phosphorylation of Akt and expression levels of the anti-apoptotic proteins B-cell lymphomaextra large (Bcl-xL) and myeloid cell leukemia sequence-1 (Mcl-1) were barely affected by any of the compounds (Fig. 5c). However, PARP cleavage, a marker for cells undergoing apoptosis [56], was detected for several RMS compounds (RMS1, RMS3, RMS4, RMS8). Mitochondrial depolarization is an indicator for early stage apoptosis [57] through the intrinsic, mitochondria-initiated pathway and mitochondrial health can be visualized with the fluorescent dye JC-1. Along this line, a shift towards green fluorescence was observed for RMS2, RMS3, RMS4, RMS5 and RMS8, indicating disruption of the mitochondrial membrane potential DJm, whereas tetrandrine (1), RMS1, RMS6, RMS7, RMS9 and RMS10 had no such an effect ( Fig. 5d and e). Thus, these findings indicate that impaired mitochondrial functions partially account for the enhanced antiproliferative and cytotoxic effects of numerous tetrandrine analogues in comparison to tetrandrine (1) on VCR-R CEM cells.
In HepG2 cells, p-Akt levels were reduced by RMS4 and RMS5, whereas no PARP cleavage was detected (Suppl. Fig. 2a) by any of the other tested compounds. Additionally, RMS5 slightly diminished the expression of the anti-apoptotic Bcl-2 family proteins Bcl-XL and Mcl-1. In accordance with the absence of PARP cleavage, none of the compounds significantly disrupted DJm at 5 mM (Suppl. Fig. 2b). Taken together, amendment or replacement of the 12-methoxy group of tetrandrine (1) by metabolically stable substituents can lead to enhanced induction of the intrinsic apoptosis pathway or activation of Akt, depending on the cancer cell line.

Additional toxicity studies
Considering both the antitumoral potential and the toxicity profile, RMS3 and RMS5 have markedly enhanced antiproliferative and cytotoxic effects on cancer cells (Figs. 3d and 5), while their toxicity to non-malignant hepatocytes is equal in comparison with tetrandrine ( Fig. 3a and b). Consequently, additional toxicity studies with primary cells were conducted to further elucidate their therapeutic potential. In line with the observations made with Hep-aRG™ cells ( Fig. 3a and b), toxicities of RMS3 and RMS5 to human umbilical vein endothelial cells (HUVECs) and peripheral blood mononuclear cells (PBMCs) were similar to those caused by tetrandrine and only minor differences were found ( Fig. 6a and b). While RMS3 had a slightly stronger effect on the viability of HUVECs than tetrandrine at 10 mM (Fig. 6a), the highest percentage of dead PBMCs was detected after tetrandrine exposure at 5 and 10 mM (Fig. 6b). Taken together, the toxicity profile of the tetrandrine analogues RMS3 and RMS5 is largely equal to that of tetrandrine, but the improved anticancer properties theoretically enable dose reduction, providing incentive for further in vivo investigations.

Conclusion
The plant bisbenzylisoquinoline alkaloid tetrandrine (1) has diverse pharmacological properties [3,4], including antiviral [5e8], anticancer [9,10], multidrug resistance reversing [11e15] and calcium channel blocking [6,16e18] activities, making this alkaloid an attractive drug candidate. Unfortunately, its clinical application is limited by its toxicity, mainly affecting liver and lungs [26e30]. Five diastereomeric pairs of novel analogues of tetrandrine and its natural diastereomer isotetrandrine, respectively, were synthesized and biologically evaluated with respect to their toxicity, their antiproliferative and multidrug resistance reversing activity. The design of these new analogues was driven by the published hypothesis that one particular structural element, the methoxy group at C-12 in para-position to a benzylic methylene group, is responsible for toxicity due to its susceptibility to undergo enzymatic oxidation to a reactive (toxic) para-quinone methide, which in turn can react with bio-nucleophiles. The shape of the new compounds was intended to prevent this oxidative toxification process. Although all of the analogues lack the putative problematic structure motif, no significant decrease of toxicity could be observed without a concurrent reduction of anticancer activity. Compounds RMS2, RMS4 and RMS8 were found to be even more toxic to healthy cells than tetrandrine (1). Moreover, CYP3A4 overexpression in two cellular models that are established for recombinant hepatotoxicity studies suggested that the propagated mechanism of CYP3A4-mediated toxification involving the paraquinone methide 4 (see Scheme 1) is no primary cause for the cytotoxicity of tetrandrine and related (bis)benzylisoquinoline alkaloids. Consequently, the structure variations made in our approach are not a suitable approach in order to reduce the alkaloid's toxicity, and different strategies remain to be investigated in this respect. On the contrary, further biological characterization revealed distinct differences between tetrandrine and the synthesized analogues. While P-gp inhibitory potency was maintained by most of the performed structure variations, seven compounds exerted strongly enhanced antiproliferative potential against drugresistant leukemia cells and increased cytotoxicities to liver cancer cells as compared with tetrandrine (1). Mechanistically, those compounds acted on the intrinsic, mitochondria-initiated apoptosis pathway and/or on activation of the kinase Akt. Considering both their toxicity and the pharmacological profile, compounds RMS3 and RMS5 were found to be most promising, since they possess stronger anticancer properties, while displaying no increased toxicity to healthy liver cells. Similar toxicities to primary cells were confirmed using non-malignant blood and endothelial cells. Side effects might therefore be reduced by lowering the required therapeutic dose and, therefore, our study strongly suggests their investigation using in vivo models. Moreover, based on these encouraging data and facilitated by our recent work on effective total synthesis of the bisbenzylisoquinoline alkaloids tetrandrine and isotetrandrine [44], further investigations  the compounds was assessed by CellTiter-Blue ® cell viability assay. Fluorescence intensities were normalized to vehicle control. Bar graph indicates means ± SEM of three independent experiments (Two-Way ANOVA followed by Dunnett's multiple comparison test, *P < 0.05). (b) Cell death of peripheral blood mononuclear cells (PBMCs) from a healthy donor upon treatment with tetrandrine (1), RMS3 and RMS5 was determined by propidium iodide staining and flow cytometry. Experiment was performed in triplicate (Two-Way ANOVA followed by Dunnett's multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) regarding structure-activity relationships in this chemical class should be part of future medicinal chemistry projects. HUVECs were cultured for a maximum of six passages. All cells were cultured at 37 C with 5% CO 2 with constant humidity are proven to be mycoplasma-free on a quarterly basis.

CYP3A4 P450-Glo™ assay
Metabolic activity of CYP3A4-EGFP was confirmed using a CYP3A4 P450-Glo™ assay (Promega, Madison, USA). HepG2 cells were seeded at a density of 0.75 Â 10 6 cells per well into a 24 well plate and allowed to adhere overnight. On the following day, cells were transfected with either pcDNA3-CYP3A4-EGFP or pcDNA3-EGFP using the Lipofectamine™ 3000 (Invitrogen, Waltham, USA) transfection reagent according to the manufacturer's instructions. On the subsequent day, cells were incubated with luciferin-IPA (3 mM) in the presence or absence of ketoconazole (Santa Cruz biotechnology, Dallas, USA) (10 mM) for 60 min. Luminescence was detected using the nonlytic method as described by the manufacturer.

Toxicity assays
For assessing the toxicity of tetrandrine and the analogues, several cellular models and approaches were used: differentiated HepaRG™ cells without transfection, differentiated HepaRG™ cells with transient transfection, HepG2 cells with stable transfection, human umbilical vein endothelial cells (HUVECs) and peripheral blood mononuclear cells (PBMCs).
Differentiated HepaRG TM cells without transfection: Hep-aRG™ cells were differentiated in Williams' medium E supplemented with GlutaMAX and HepaRG Maintenance/Metabolism Medium Supplement (Maintenance/Metabolism Working Medium) (all purchased from Life Technologies) as indicated by the manufacturer. After 24 h treatment with the respective compound concentrations, cell viability was determined by CellTiter-Blue ® cell viability assay as described by the manufacturer.
Differentiated HepaRG TM cells with transient transfection with pcDNA3-CYP3A4-EGFP: HepaRG™ cells were differentiated as described above. 16 h prior to stimulation, cells were transfected with either pcDNA3-CYP3A4-EGFP or pcDNA3-EGFP using Lip-ofectamine™ 3000 (Invitrogen) transfection reagent according to the manufacturer's instructions. Cells were treated as indicated for 24 h. Cell death was assessed by propidium iodide (5 mg/mL in PBS; Carl Roth, Karlsruhe, Germany) staining and flow cytometry using a BD FACS Canto II (BD Biosciences, Becton Dickinson, Franklin Lakes, USA). Data were analyzed using FlowJo 7.6 (BD Biosciences). No FSC/SSC gating was performed. Determination of the percentage of PI-A positive cells was conducted as stated below. Specific cell death was calculated as follows: specific cell death ð%Þ ¼ cell death ðxÞ % À cell death ðcontrolÞ%.
HepG2 cells with stable transfection with CYP3A4-EGFP: HepG2 cells were transfected with either pcDNA3-CYP3A4-EGFP or pcDNA3-EGFP using Lipofectamine™ 3000 (Invitrogen) transfection reagent according to the manufacturer's instructions. Transfected cells were constantly cultivated in the presence of 0.5 mg/mL G418 (Sigma Aldrich, St. Louis, USA) for four weeks. Presence of plasmids was confirmed by PCR (pcDNA3-forward: 5 0 -TACATCAATGGGCGTGGATAG-3 0 , pcDNA3-reverse: 5 0 -AGGAAGG-GAAGAAAGCGAAAG-3 0 ). Primers were purchased from metabion. HepG2 cells stably expressing either CYP3A4-EGFP or pcDNA3-EGFP were seeded at a density of 0.1 Â 10 6 cells per well of a 24well plate and allowed to adhere overnight. Treatment, flow cytometry and data analysis were performed as described for HepaRG™ cells. The natural product tetrandrine was kindly donated by Prof. P. Pachaly and used as free base in all assays. Berbamine (dihydrochloride) was purchased from Sigma-Aldrich (now Merck, Darmstadt, Germany) and Dauricine (free base) from Carbosynth (Compton, Berkshire, United Kingdom).
PBMCs: PBMCs were isolated from anticoagulated whole blood from healthy donors by density gradient centrifugation using Ficoll-Paque PLUS density gradient medium (GE Healthcare, Chicago, USA) as described by the manufacturer. Isolated PBMCs were cultivated in RPMI 1640 supplemented with 20% FCS and 1% penicillin/streptomycin (all purchased from PAN Biotech). 4 h after seeding, cells were treated as indicated for 48 h. Cell death was analyzed by propidium iodide (Carl Roth, Karlsruhe, Germany; 5 mg/ mL in PBS) staining and flow cytometry using a BD FACS Canto II (BD Biosciences). For evaluation, cell debris was excluded and propidium iodide positive cells were determined using FlowJo 7.6 (BD Biosciences).
HUVECs: HUVECs were seeded at a density of 0.125 Â 10 3 cells per well of a 96-well plate and allowed to adhere overnight. After 24 h treatment with the respective compound concentrations, cell viability was determined by CellTiter-Blue® cell viability assay as described by the manufacturer.

Calcein-AM retention assay
The calcein-AM retention assay was performed as described previously [42].

Apoptosis assay
VCR-R CEM cells were seeded at a density of 0.125 Â 10 6 cells per well of a 24-well plate and incubated for 4 h. Treatment was performed with the indicated concentrations for 48 h. Apoptosis was determined by propidium iodide staining as described before [60] on a BD FACS Canto II (BD Biosciences).

Western blot analysis and antibodies
HepG2 were seeded at a density of 0.5 Â 10 6 cells per well of a 6well plate and allowed to adhere overnight. VCR-R CEM cells were seeded at a density of 2 Â 10 6 cells per well of a 6-well plate 4 h prior to treatment. Afterwards, cells were treated with DMSO, tetrandrine or the respective RMS compounds (5 mM) for 24 or 48 h as indicated. Cell lysis was performed with RIPA (radioimmunoprecipiation) buffer as described before [58]. Separation of proteins by SDS-PAGE, transfer to Hybond PVDF (polyvinylidene difluoride) membranes (Amersham Bioscience, Little Chalfont, United Kingdom), incubation with primary and secondary antibodies and detection of chemiluminescence on a Chemidoc™ Touch Imaging System (Bio-Rad) were performed as described previously. The following antibodies were used: phospho-Akt  1:1000, CST, #4572). The stain free technology (Bio-Rad, Hercules, USA) [61] was used to confirm equal protein loading using Image Lab 6.0.1 software (Bio-Rad).

Mitochondrial membrane potential
The cationic dye JC-1 (Thermo Fisher) was used to measure mitochondrial membrane potential (DJm). When DJm is intact, JC-1 accumulates in mitochondria, yielding a red fluorescence. In contrast, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity. HepG2 cells were seeded at a density of 0.

General
All solvents and reagents were purchased from commercial suppliers and were used without further purification, unless mentioned otherwise. TLC was carried out on 0.2 mm silica gel polyester plates with a fluorescence indicator (POLYGRAM SIL G/ UV254, Macherey-Nagel). NMR spectra were recorded with a 400 MHz (400 MHz for 1 H and 101 MHz for 13 C), 500 MHz (500 MHz for 1 H and 126 MHz for 13 C) or 800 MHz Bruker Biospin Avance spectrometer (800 MHz for 1 H and 201 MHz for 13 C). Peak assignments were based on 2D NMR experiments using standard pulse programs (COSY, HSQC/HMQC, DEPT, HMBC and NOESY). Chemical shifts were referenced to the residual solvent signal (CDCl 3 : d H ¼ 7.26 ppm, d C ¼ 77.16 ppm). For the characterization of rotamers a temperature program was employed for recording both 1D and 2D spectra. Hereby chemical shifts were referenced to the signal of tetramethylsilane in deuterated tetrachloroethane (Tcl 2 [100 C]: d H ¼ 5.92 ppm, d C ¼ 74.0 ppm). IR spectra were recorded using a Jasco FT/IR-4100 (type A) instrument equipped with a diamond ATR unit (Jasco PRO450-S). High resolution mass spectra (HR-MS) were recorded using a Jeol Mstation 700 or JMS GCmate II Jeol instrument for electron impact ionisation (EI). Thermo Finnigan LTQ was used for electrospray ionisation (ESI). Reaction monitoring by mass spectrometry was performed by atmospheric pressure solids analysis probe (ASAP) via atmospheric-pressure chemical ionisation (APCI) on an Advion expression L CMS device. Purification by flash column chromatography (FCC) was performed using Merck silica gel 60 (0.040e0.063 mm, 230e400 mesh ASTM). For the determination of purity HPLC was performed on a HP Agilent 1100 system equipped with an Agilent 1100/1200 Diode Array Detector and an Agilent Zorbax Eclipse Plus C18-column (5.0 mm, 150 Â 4.6 mm) using following method: flow 0.8 mL/min; temperature 50 C; eluent system for compounds RMS1-10, 13c-d and 14c-e: 80% MeOH, 20% water, NaOH buffer pH 9; for compounds 13a and 14a: 50% ACN, 49.9% water, 0.1% THF; for compounds 13b and 14b: 70% ACN, 30% water). The purity of all final compounds was >95%.

General procedures
General procedure 1: Wittig olefination: A suspension of (methoxymethyl)triphenylphosphonium chloride (1.2 equiv.) in anhydrous THF (2 mL per mmol aromatic aldehyde) was cooled to 0 C under nitrogen atmosphere. A solution of lithium diisopropylamide (1.4 equiv., 2.0 M solution in THF) was added dropwise and the resulting mixture stirred for 45 min. A solution of the aromatic aldehyde (1.0 equiv.) in anhydrous THF (2 mL per mmol) was added with stirring. The mixture was allowed to warm up to ambient temperature and stirred for 4 h. The reaction was then quenched with deionized water and extracted 3x with ethyl acetate. The combined organic phases were washed with brine, dried over anhydrous Na 2 SO 4 and concentrated in vacuo to afford the crude product which was purified by column chromatography.
General procedure 2: N-Acyl Pictet-Spengler condensation: A solution of carbamate (1.0 equiv.) and enol ether (1.2e2.0 equiv.) in dichloromethane (10 mL per mmol carbamate) was cooled to 0 C under nitrogen atmosphere. Trifluoromethanesulfonic acid (TfOH, 0.1 equiv., 0.113 mol/L in acetonitrile) was added dropwise, the reaction mixture was then allowed to warm up to ambient temperature and stirred for 6e12 h. A saturated NaHCO 3 solution was then added for neutralization and the mixture extracted 3x with dichloromethane. The combined organic phases were dried over anhydrous Na 2 SO 4 and concentrated in vacuo to afford the crude product which was purified by column chromatography.
General procedure 3: Ullmann-type CeO coupling reaction: According to a modified procedure of Wang et al. [62], bromoarene (1.0e1.2 equiv.), phenol (1.0e2.0 equiv.), CuBr$Me 2 S (1.0 equiv.) and Cs 2 CO 3 (3.0 equiv.) were placed in a pressure tube or a flask closed with a screwcap with septum inlet and sealed with PTFE tape. Anhydrous pyridine (the reaction was carried out in a concentration of 0.02 mM) was added and after 5 min of pre-stirring the reaction mixture was heated to 110 C for 2e7 days under nitrogen atmosphere. The reaction mixture was concentrated in vacuo, diluted with ethyl acetate and filtered over a small plug of silica gel in order to remove the catalyst and the excess base, followed by washing with ethyl acetate. The filtrate was concentrated in vacuo to afford a brown oil as crude product which was purified by column chromatography.
General procedure 4: Carbamate reduction: Lithium alanate (12 equiv.) was suspended in 1 mL anhydrous THF under nitrogen atmosphere. A solution of carbamate (1.0 equiv.) in anhydrous THF (1 mL per 0.02 mmol carbamate) was added dropwise and the resulting mixture was heated at 50 C for 4e12 h. The reaction mixture was cooled to 0 C and slowly quenched with water. After alkalizing to pH 12e14 with a 2.0 M sodium hydroxide solution, the mixture was extracted 3x with ethyl acetate. The combined organic phases were dried over anhydrous Na 2 SO 4 and concentrated in vacuo to afford the crude product which was purified by column chromatography.