Racemic total synthesis and evaluation of the biological activities of the isoquinoline–benzylisoquinoline alkaloid muraricine

The first racemic total synthesis of the isoquinoline–benzylisoquinoline alkaloid muraricine is reported herein. Pharmacological characterization identified muraricine as a moderate inhibitor of P‐glycoprotein, a crucial factor of multidrug resistance in cancer. When combined with vincristine, muraricine partly reversed the chemoresistance of vincristine‐resistant leukemia cells at a nontoxic concentration. Furthermore, no cytotoxic effects on noncancerous human cells in therapeutically relevant concentrations were observed.


| INTRODUCTION
Bisbenzylisoquinolines are a large and structurally diverse subclass of the widely distributed benzylisoquinoline alkaloids. [1][2][3] In this chemotype, typically two tetrahydroisoquinoline units are connected by two (or more) diaryl ether (in some cases also biaryl) units to result in rigid, macrocyclic compounds, as exemplified by the structure of tetrandrine (1). [4] Numerous biological activities have been described for bisbenzylisoquinoline and several monomeric benzylisoquinoline alkaloids including anticancer, [5][6][7][8][9] multidrug resistance reversing, [10][11][12][13][14][15] antiviral, [16,17] antimicrobial, [18] and calcium channel-blocking activities. [19,20] Moreover, seco-analogues (so-called seco-bisbenzylisoquinoline alkaloids) have been detected, mainly in plants from the families Berberidaceae and Annonaceae. [21] There is some evidence that these seco-bisbenzylisoquinolines of natural origin are formed by oxidative degradation or by chemical and enzymatic cleavage [22] of parent bisbenzylisoquinoline alkaloids, probably as a first step of alkaloid catabolism in plant cells. [23] A couple of artefacts identical to these alkaloids were obtained by controlled photooxidation of bisbenzylisoquinoline alkaloids. [24,25] In these seco-compounds, one or more bonds of the macrocyclic bisbenzylisoquinoline core are formally broken, with the most typical first scission between C-1 of an isoquinoline ring and the adjacent benzylic methylene group, as shown for seco-isotetrandrine (2). [26] But there are also examples for formal cleavage of a diaryl ether bridge between two isoquinoline units, as shown for berbamunine (3). [27] The tetrahydroisoquinolin-1-one unit of seco-bisbenzylisoquinolines (e.g., 2) can formally be reduced to a tetrahydroisoquinoline, as shown for chenabine (4), but the mechanism of the biosynthesis is still unclear. [28] In certain cases, parts of the molecules are lost, as demonstrated for bargustanine (5), [29] where a complete benzyl unit is missing, and small "dimeric" isoquinolone alkaloids like berbidine (6), [23,30] in which both benzyl residues were cleaved off ( Figure 1).
Based on our recently published racemic synthesis of the bisbenzylisoquinoline alkaloids tetrandrine (1) and isotetrandrine, [32] we herein present the first total synthesis of racemic muraricine (rac-7). Key steps of this approach are N-acyl Pictet-Spengler condensations for the formation of the tetrahydroisoquinoline moieties and a copper-catalyzed Ullmann coupling for the construction of the diaryl ether bridge.
Focus of the pharmacological evaluation of muraricine (rac-7) is the antiproliferative effect against several cancer cell lines, the evaluation of toxicity to noncancerous cells, chemoresistance reversing property by inhibition of the P-glycoprotein (P-gp), calcium channel-blocking activities, and antimicrobial effects.

| Chemistry
The total synthesis of racemic muraricine (rac-7) requires the construction of an N-methylated 1-benzyltetrahydroisoquinoline subunit, which is connected via a diaryl ether bridge with a 1-unsubstituted N-methylated tetrahydroisoquinoline. Very recently, we reported on a new modular total racemic synthesis [32] of tetrandrine (1) and isotetrandrine, for which we optimized both the diaryl ether synthesis (Ullmann coupling) and the construction of N-methylated 1-benzyltetrahydroisoquinolines using methoxystyrenes as arylacetaldehyde equivalents in N-acyl Pictet-Spengler condensations. Conveniently, one intermediate of this (iso)tetrandrine synthesis (8; compound no. 19 in Ref. [32]) lends itself to be used as a precursor of racemic muraricine (rac-7). Compound 8 is readily available in high-yielding steps starting from two commercially available substituted benzaldehydes (4-(benzyloxy)benzaldehyde, 3-bromo-4,5-dimethoxybenzaldehyde) and one arylethylamine (vanillin-derived 4-hydroxy-3-methoxyphenethylamine). A detailed scheme on the preparation of which in certain cases can be controlled by solvent-directed approaches. [33,34] When conducting N-acyl Pictet-Spengler condensation in polar solvents such as trifluoroethanol or methanol following Kayhan's protocol, [34] the reaction unfortunately proceeded notably slower and was incomplete. From the obtained mixture, the desired isomer 9a was isolated in good yield (68%) readily by flash column chromatography (FCC). This regioisomer 9a could be unambiguously identified by 2D nuclear magnetic resonance (NMR) experiments (heteronuclear multiple bond correlation [HMBC] of 9a and by nuclear Overhauser effect spectroscopy [NOESY] experiments of rac-7, respectively). Finally, bis-carbamate 9a was converted to racemic muraricine (rac-7) in 23% yield by simultaneous reduction of both ethoxycarbonyl groups to N-methyl groups using lithium alanate (Scheme 1). The NMR data of product rac-7 were in full agreement with the data F I G U R E 1 Bisbenzylisoquinoline alkaloids and naturally occurring seco-analogues 2 of 9 | published for the natural alkaloid 7. [31] The proposed structure of isolated muraricine (7) has herewith been confirmed.

| Pharmacology
As already mentioned, the only reported biological activity of muraricine (7) is a moderate butyrylcholinesterase inhibition. [31] As various biological activities have been described for the class of bisbenzylisoquinoline and benzylisoquinoline alkaloids as discussed above, we submitted synthetic rac-7 to our screening systems for antiproliferative/cytotoxic, chemoresistance reversing, and antimicrobial and calcium channel-modulating activities.

| Antiproliferative activity
Antiproliferative effects of rac-7 were investigated against hepatocellular carcinoma (HepG2), colorectal adenocarcinoma (HCT-15), and bladder carcinoma (T24) cell lines and determined by CellTiter-Blue ® assay. The bisbenzylisoquinoline alkaloid tetrandrine (1) was used as a reference compound, whereas tetrandrine (1) showed significant activity against the three tumor cell lines; the truncated analogue rac-7 did only show very low antiproliferative effects and no cytotoxic activity ( Figure 2). These findings add to results of a structure-activity relationship study by Kupchan and Altland. [9] In this investigation, both macrocyclic bisbenzylisoquinolines and seco-analogues which retained a S C H E M E 1 Total synthesis of racemic muraricine (rac-7). DCM, dichloromethane; rt, room temperature; TFA, trifluoroacetic acid; THF, tetrahydrofuran F I G U R E 2 Antiproliferative effects of rac-7 on hepatocellular carcinoma (HepG2), colorectal adenocarcinoma (HCT-15) and bladder carcinoma (T24) cell lines were determined by CellTiter-Blue ® assay. Cells were incubated with the respective concentrations for 72 hr and proliferation is shown as percentage of vehicle control after subtraction of the zero value. Tetrandrine (1) served as control. Line graphs display mean ± SEM of three independent experiments. DMSO, dimethyl sulfoxide; SEM, standard error of the mean diaryl ether bridge connecting both benzyl groups showed antiproliferative/cytotoxic effects, whereas trivial monomeric benzyltetrahydroisoquinolines were virtually inactive.

| Inhibition of P-gp
As pointed out, rac-7 does not have direct antitumor effects. Hence, we aimed to evaluate its potential as an add-on in combination therapy. Natural alkaloids have been shown to improve the response to several antitumor drugs by targeting the P-gp. [10,11,42] P-gp facilitates the efflux of cytostatic drugs and thereby represents a major cause of treatment failure and resistance to common cancer therapeutics. [10] As no clinically approved treatment options targeting P-gp are available so far, there is a need for identifying novel, safe, and efficacious P-gp inhibitors. Inhibition of P-gp is mostly described for macrocyclic bisbenzylisoquinolines like tetrandrine (1) [42,43] and related alkaloids, [10] but also for some seco-analogues, such as dauricine and daurisoline. [44,45] Therefore, we investigated the influence of rac-7 on the accumulation of the P-gp model substrate calcein-acetoxymethyl (AM) in vincristine-resistant CEM (VCR-R CEM) cells, leukemia cells that overexpress P-gp. represents a nontoxic seco-analogue of tetrandrine (1) with the potential to overcome drug resistance in cancer. P-gp-mediated drug efflux is not only implicated in tumor therapy but also represents a hurdle for the treatment of other diseases such as HIV and bacterial infections. [11] Eradication of HIV reservoirs in the central nervous system, for instance, is partly prevented by efflux transporters at the blood-brain barrier, including P-gp. [46] Consequently, this study provides the basis for further investigating the potential of rac-7 to overcome resistance mechanisms by the efflux transporter P-gp.

| Calcium imaging
To determine whether rac-7 is an inhibitor of the calcium channel, two-pore channel 2 (TPC2), Ca 2+ -imaging experiments were performed using the recently published TPC2 activator TPC2-A1-N. [47] The response of TPC2-A1-N in HEK293 cells stably expressing TPC2 L11A/L12A was not blocked by rac-7 compared with a dimethyl sulfoxide (DMSO) control, whereas tetrandrine was able to block the activation (Figure 5a,b). Hence, rac-7 does not show inhibitory activity on TPC2.

| Antimicrobial activity
We tested rac-7 for antimicrobial activity in a standard agar diffusion assay against Gram-positive (Streptococcus entericus, Staphylococcus equorum) and Gram-negative bacteria (Escherichia coli, Pseudomonas marginalis), and against fungi (Yarrowia lipolytica, Saccharomyces cerevisiae). There was no zone of inhibition observed in any experiment; consequently, rac-7 does not exhibit antimicrobial activity against the above-listed germs.

| CONCLUSION
In summary, we have developed the first total synthesis of racemic muraricine (rac-7) in a total of 10 steps. The overall yield amounts to F I G U R E 3 Acute toxicity to noncancerous human umbilical vein endothelial cells is shown. Cells were treated for 6 hr with rac-7 and tetrandrine (1) (10, 20, 50 µM) for 6 hr. Cell viability was determined by quantifying the cellular adenosine triphosphate content applying a CellTiter-Glo ® cell assay and is displayed as percentage of vehicle control as mean ± standard error of the mean of three independent experiments (one-way analysis of variance followed by Tukey's multiple comparison test; **p < .01, ***p < .001) 3.8% along the longest sequence. We further investigated biological activities of this natural product and hereby focused on targets that are already known for the class of (bis)benzylisoquinoline alkaloids. Neither antiproliferative, antimicrobial, nor TPC2-blocking activity in Ca 2+ -imaging experiments could be detected, whereas racmuraricine could be identified as an inhibitor of P-gp, an important factor in the mechanism of multidrug resistance of tumors. Although the inhibitory effect of muraricine on this efflux pump is only moderate, significantly increased apoptosis rates were observed in combination with VCR. Hence, rac-muraricine was able to overcome drug resistance to a commonly used chemotherapeutic drug. Moreover, no acute cytotoxicity to healthy human cells (HUVEC) in concentrations higher than that required for reversing chemoresistance was detected for rac-muraricine. These findings suggest that murar-

| Acute toxicity assay
HUVECs were seeded at a density of 10 × 10 3 cells/well of a 96-well plate and allowed to adhere overnight. On the following day, cells were treated with the indicated concentrations of the respective compounds for 6 hr. Cellular ATP content was quantified by CellTiter-Glo ® cell viability assay (Promega) as indicated by the manufacturer. Luminescence at 560 nm was recorded with an Orion II Microplate Luminometer (Berthold Detection Systems).

| Calcein-AM retention assay
The protocol was adapted from Robey et al. [42] Briefly

| Apoptosis assay
VCR-R CEM cells were seeded at a density of 0.125 × 10 6 cells/ well of a 24-well plate and incubated for 4 hr. Treatment was performed with the indicated concentrations for 48 hr. Apoptosis was determined by propidium iodide staining as described before [49] on FACSCalibur.

| 7 of 9
Thermo Fisher Scientific) in HEPES-buffered solution (HBS) comprising 138 mM NaCl, 6 mM KCl, 2 mM MgCl 2 , 2 mM CaCl 2 , 10 mM HEPES, and 5.5 mM D-glucose (adjusted to pH 7.4 with NaOH). After loading, cells were washed with HBS and mounted in an imaging chamber. All recordings were performed in HBS. Ca 2+ imaging was performed using a Leica DMi8 live cell microscope. Fura-2 was excited at 340/380 nm.
Emitted fluorescence was captured using a 515-nm long-pass filter.
Compounds were prediluted in DMSO and stored as 10 mM stock solutions at −20°C, not exceeding 3 months. Working solutions were prepared directly before using by dilution with HBS.

| Agar diffusion assay
The bacteria and fungi, purchased from DSMZ, were cultivated on an AC agar (Sigma-Aldrich). Six-millimeter paper discs, impregnated with

| Statistical analysis
The GraphPad Prism software was used for statistical analysis. For some experiments, results were normalized to controls as indicated.