Synthesis, biological evaluation and toxicity of novel tetrandrine analogues

European Journal of Medicinal Chemistry 207 (2020) 1 12810

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Research paper

Synthesis, biological evaluation and toxicity of novel tetrandrine

analogues

Ramona Schütz ¹, Martin Müller ¹, Franz Geisslinger, Angelika Vollmar, Karin Bartel,

Franz Bracher^*

Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-University of Munich, Butenandtstr. 5e13, 81377, Munich, Germany

a r t i c l e i n f o

a b s t r a c t

Article history:

In this work, we present the design and synthesis of novel fully synthetic analogues of the bisbenzyli-

soquinoline 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 un-

derlying mechanisms, however, are controversially discussed. Along this line, novel tetrandrine ana-

logues 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 charac-

terization 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 prop-

erties 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.

Received 3 June 2020

Received in revised form

22 July 2020

Accepted 31 August 2020

Available online 4 September 2020

Keywords:

Tetrandrine

Toxicity

Metabolic toxiﬁcation

CYP metabolism

Multidrug resistance

P-gp inhibition

1. Introduction

and cytotoxic properties against several cancer cell lines [4,9],

arrested cell cycle progression [21], induced pro-apoptotic

The natural product tetrandrine (1) (Fig. 1), isolated from the

plant Stephania tetrandra [1,2], belongs to the class of bisbenzyli-

soquinoline alkaloids. Tetrandrine has a wide range of pharmaco-

logical activities [3,4], most interestingly antiviral [5e8], anticancer

[9,10], multidrug resistance reversing [1 1e15] and calcium channel

blocking [6,16e18] effects.

signaling pathways [22] and reduced migration [23] of tumor

cells. Additionally, tetrandrine was shown to resensitize chemo-

resistant tumors by inhibition of P-glycoprotein (P-gp) [1 1,12,14], a

universal efﬂux pump for xenobiotics, which is a key factor of drug

resistance [24] frequently causing treatment failure of tumor

therapy.

Recently, it was shown that tetrandrine (1) blocks endolysoso-

mal two-pore channels (TPCs, voltage-gated calcium channels) and

thereby reduces cellular entry of Ebolavirus (EBOV) [6], MERS-CoV

[7], SARS-CoV-2 [5] viruses or pseudoviruses into host cells.

Currently, a clinical trial (NCT04308317) is announced to investi-

gate tetrandrine (1) as adjuvant treatment of COVID-19 patients in

China [20]. Moreover, tetrandrine displayed strong antiproliferative

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] hypothe-

* Corresponding author.

sized that an interaction of tetrandrine with p38

activated protein kinase) led to liver injury, whereas several other

a MAPK (mitogen-

E-mail address: franz.bracher@cup.lmu.de (F. Bracher).

1

These authors contributed equally to this work.

https://doi.org/10.1016/j.ejmech.2020.1 12810

2

R. Schütz et al. / European Journal of Medicinal Chemistry 207 (2020) 112810

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 para-

methoxybenzyl 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 investi-

gated. 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.

Fig. 1. Tetrandrine (1) (1S,1⁰S) and isotetrandrine (2) (1R,1⁰S) e Numbering of the

skeleton according to Shamma [19].

studies suggest the involvement of cytochrome P450 (CYP) en-

zymes 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 CYP2E1-

mediated toxicity. In a metabolic study by Jin et al. [26] a CYP3A4-

and 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 evi-

dence for the formation of the para-quinone methide 4.

A considerable number of pharmacologically interesting bis-

benzylisoquinolines and seco analogues such as dauricine (6)

[33e36], berbamine (7) [37e39], fangchinoline (8) [7,15], cepha-

ranthine (9) [40] and muraricine (10) [41,42] contain the discussed

para-methoxybenzyl motif like the one present in tetrandrine (1) or

the equivalent para-hydroxybenzyl moiety (see Fig. 2), which could

potentially result in an unfavorable toxicity proﬁle as well. So it is of

great relevance to clarify if and to which extent this structural motif

causes cytotoxicity after oxidation by CYP3A4/5 enzymes.

2. Results and discussion

2.1. 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 meta-

bolically stabile triﬂuoromethoxy (RMS3-4) or chlorine sub-

stituents (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 A⁰-C⁰) 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.

The second tetrahydroisoquinoline moiety (rings A and B) was

constructed via triﬂuoromethanesulfonic acid-mediated intermo-

lecular N-acyl Pictet-Spengler reaction of arylethylamino carba-

mate intermediate 11 and enol ethers 12a-12d or the aliphatic

aldehyde

12e,

respectively,

to

obtain

the

seco-

Scheme 1. Postulated CYP-enzyme mediated oxidative metabolism of tetrandrine (1) adopted from Jin et al. [26].

R. Schütz et al. / European Journal of Medicinal Chemistry 207 (2020) 112810

3

Fig. 2. Related bisbenzylisoquinolines and seco analogues containing the hypothesized metabolically labile para-methoxy(hydroxy)benzyl motif (highlighted in red). (For inter-

pretation of the references to colour in this ﬁgure legend, the reader is referred to the Web version of this article.)

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 commer-

cially available aromatic aldehydes (Scheme 2), 5-bromopentanal

(12e) was obtained by PCC oxidation of the corresponding pri-

mary alcohol [45]. The intermediates 13a-13e were further pro-

cessed in intramolecular Ullmann-type cross-coupling reactions or

more than 100 bisbenzylisoquinolines [46]. Herein the chemical

shifts of distinctive protons in the NMR spectra of bisbenzyliso-

quinolines can be used for a reliable determination of the relative

conﬁgurations at both asymmetric centers. The relative conﬁgu-

rations 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. Unfor-

tunately, attempts of crystallization for crystallographic analysis

were unsuccessful, and the determination of the stereo-

conﬁguration in this case was therefore not possible.

an S_N2 reaction (for the

u-bromobutyl intermediate 13e) respec-

tively to access the diaryl ether bridges (connecting rings C and C’ in

14a-d) or the aliphatic bridge in 14e, furnishing macrocyclic bis-

benzylisoquinoline 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 dihy-

dride). To circumvent this problem, the carbamate groups were ﬁrst

removed by treatment with methyl lithium and the resulting sec-

ondary amino groups were subsequently N-methylated via reduc-

tive alkylation using formaldehyde/NaBH₃CN. 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 iso-

tetrandrine (2) (Fig. 1) in high diastereomeric ratios (d.r., deter-

mined by HPLC).

2.2. Biological evaluation

2.2.1. Assessment of CYP3A4-mediated toxicity

2.2.1.1. Evaluation of toxicity using non-malignant hepatocyte-like

cells. To assess general toxicity, HepaRG™ cells, non-cancerous

hepatic stem cells which possess various characteristics of

healthy liver cells and which have a high activity of P450 enzymes

[47,48], were treated with 10 and 20

analogues RMS1-10. Unlike expected, none of the new compounds

was less toxic to HepaRG™ cells than tetrandrine (1) at 10

(Fig. 3a). In contrast, RMS4 and RMS8 slightly, but RMS2 strongly

decreased cell viability. Similarly, at 20 M, RMS2, RMS4 and RMS8

were signiﬁcantly more toxic to HepaRG™ cells than tetrandrine

(1) (Fig. 3b). Of note, 20 M of RMS6 and RMS9 were signiﬁcantly

mM of tetrandrine (1) and the

mM

m

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 CYP-

mediated ring hydroxylation), whereas in the triﬂuoromethoxy

compounds RMS3/RMS4 and the chloro compounds RMS7/RMS8

oxidation processes are prevented by metabolically stable

substituents.

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

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 respec-

tive compounds did not signiﬁcantly increase cell death (Fig. 3c). As

observed for the non-transfected HepaRG™ cells, RMS2 still had

the strongest effect on cell viability.

2.2.1.2. Evaluation of toxicity using HepG2 liver cancer cells.

Next, transgenic HepG2 cells, a liver cancer cell line frequently used

for recombinant expression of CYP enzymes and hepatotoxicity

studies [49e51], were generated. Stable transfection and

Scheme 2. Synthesis of enol ethers 12a-d and aldehyde 12e. Reagents and conditions:

(a) (Ph)₃P(Cl)CH₂OCH₃, LDA, THF, 0 ^ꢀC to rt (80e87%). (b) PCC, DCM, rt (64%).

4

R. Schütz et al. / European Journal of Medicinal Chemistry 207 (2020) 112810

Scheme 3. Synthesis of tetrandrine analogues. Reagents and conditions: (a) TfOH, DCM, 0 ^ꢀC to rt (23e37% separated diastereomers). (b) for 13a-13d: CuBr^.Me₂S, Cs₂CO₃, pyridine,

110 ^ꢀC (17e77%). (c) for 13e: KI, K₃PO₄, DMF, 100 ^ꢀC (29e60%). (d) for 14a/b/c/e: LiAlH₄, THF, 50 ^ꢀC (62e94%). (e) for 14d: 1) MeLi, THF, 0 ^ꢀC, 2) aq. formaldehyde solution (37%),

NaBH₃CN, MeOH, rt (30% over two steps).

Fig. 3. Toxicity assessment using HepaRG™ and HepG2 cells. (a,b) Differentiated HepaRG™ cells were exposed to (a) 10 mM and (b) 20 mM tetrandrine (1) and RMS1-RMS10 for 24 h

and cell viability was determined by CellTiter-Blue^®cell viability assay. Cell viability was normalized to vehicle control. Bar graphs indicate means SEM of three independent

experiments (One-Way ANOVA followed by Dunnett’s multiple comparison test, relative cell viabilites were compared with that of tetrandrine, *P < 0.05, **P < 0.01, ***P < 0.001).

(c) Differentiated HepaRG™ cells were transfected with pcDNA3-CYP3A4-EGFP or an empty vector control and treated with 10

mM of tetrandrine (1) and RMS1-RMS10 for 24 h. (d)

HepG2 cells stably transfected with pcDNA3-CYP3A4-EGFP or empty vector control were treated with berbamine (7), dauricine (6), tetrandrine (1) and RMS1-RMS10 (15

mM) for

24 h. (c,d) Cell death was determined by propidium iodide staining and ﬂow cytometry. Means SEM of three independent experiments are shown (unpaired t-test with Welch’s

correction, not signiﬁcant). bbm: berbamine, dau: dauricine, Rel.: relative, tet: tetrandrine. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred

to the Web version of this article.)

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.

Interestingly, most tetrandrine analogues (RMS1, RMS2, RMS3,

RMS4, RMS5, RMS7, RMS8) exerted generally increased cytotoxic-

ities against cancerous HepG2 cells, mostly independent of their

R. Schütz et al. / European Journal of Medicinal Chemistry 207 (2020) 112810

5

stereochemistry, in comparison to tetrandrine (1). Surprisingly, the

diastereomers RMS9 and RMS10 slightly, while RMS5 and RMS6

substantially differed in their cytotoxic potencies. Of note, RMS3

and RMS5 inﬂuenced cell viability of HepaRG™ cells similarly to

tetrandrine (1) (Fig. 3aec), but they exerted strongly increased

cytotoxicites to cancerous HepG2 cells (Fig. 3d).

(Fig. 4c). These data indicate that by replacing the para-methox-

ybenzyl moiety of tetrandrine with other substituted or unsub-

stituted 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.

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 alka-

loids berbamine (7) and dauricine (6) were also tested. Both alka-

loids (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-demethyla-

tion. 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 para-

methoxybenzyl 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 mole-

cules 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 sug-

gested 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 toxic-

ities 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 tet-

randrine (1), other proposed mechanisms might play a more sub-

stantial role in toxicity, such as the generation of ROS by CYP2E1

2.2.2.2. Studies on anticancer effects and related pathways. In a next

step, we determined direct antiproliferative effects of the com-

pounds on VCR-R CEM cells, which are cross-resistant to a variety of

chemotherapeutic drugs [55]. Interestingly, most tetrandrine ana-

logues (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 tet-

randrine (1) affect the intrinsic apoptosis pathway and phosphor-

ylation 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 lymphoma-

extra 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 ﬂuo-

rescence 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.

[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 P-

glycoprotein 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 [1 1] 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 dimin-

ished 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,

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 dimin-

ished 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 sub-

stituents can lead to enhanced induction of the intrinsic apoptosis

pathway or activation of Akt, depending on the cancer cell line.

the compounds were used at 1

mM in combination with varying

2.2.3. Additional toxicity studies

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

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

analogue as it signiﬁcantly increased apoptosis at 0.01

mM VCR

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R. Schütz et al. / European Journal of Medicinal Chemistry 207 (2020) 112810

Fig. 4. P-gp inhibition and chemosensitization. (a) The inﬂuence of tetrandrine (1) and its analogues on the retention of the model substrate calcein-AM by the efﬂux transporter P-

gp was assessed by ﬂow cytometry. Cells were incubated with calcein-AM (200 nM) together with the potential P-gp inhibitors (0.1 and 1

M) for 30 min at 37 ^ꢀC. After a washing

step, cells were incubated without calcein-AM for another 60 min at 37 ^ꢀC before ﬂow cytometric analysis. Geometric means of ﬂuorescence intensities (gMFI) were normalized to

the calcein-AM control. (b) No reduction of P-gp cell surface expression was observed upon exposure of VCR-R CEM cells to tetrandrine (1) or RMS1-RMS10 (1 M) for the conditions

used in (a), as determined by ﬂow cytometry. gMFI values were normalized to DMSO control. (c) VCR-R CEM cells were incubated in the absence or presence of tetrandrine and

RMS1-RMS10 (1 M) with increasing concentrations of VCR for 48 h and apoptosis was determined by propidium iodide staining and ﬂow cytometry. (a,c) Bar graphs indicate

m

means SEM of three independent experiments (Two-Way ANOVA followed by Dunnett’s multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). (b) Bar

graphs indicate means SEM of three independent experiments (One-Way ANOVA followed by Dunnett’s multiple comparison test, *P < 0.05). -: solvent control, Rel.: relative, tet:

tetrandrine, VCR: vincristine.

tetrandrine (Fig. 3a and b). Consequently, additional toxicity studies

with primary cells were conducted to further elucidate their ther-

apeutic 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 tet-

randrine and only minor differences were found (Fig. 6a and b).

While RMS3 had a slightly stronger effect on the viability of

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 enzy-

matic 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 re-

combinant hepatotoxicity studies suggested that the propagated

mechanism of CYP3A4-mediated toxiﬁcation involving the para-

quinone methide 4 (see Scheme 1) is no primary cause for the

cytotoxicity of tetrandrine and related (bis)benzylisoquinoline al-

kaloids. Consequently, the structure variations made in our

approach are not a suitable approach in order to reduce the alka-

loid’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 synthe-

sized analogues. While P-gp inhibitory potency was maintained by

most of the performed structure variations, seven compounds

exerted strongly enhanced antiproliferative potential against drug-

resistant 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. Consid-

ering both their toxicity and the pharmacological proﬁle,

HUVECs than tetrandrine at 10

of dead PBMCs was detected after tetrandrine exposure at 5 and

10 M (Fig. 6b). Taken together, the toxicity proﬁle of the tetran-

mM (Fig. 6a), the highest percentage

m

drine analogues RMS3 and RMS5 is largely equal to that of tet-

randrine, but the improved anticancer properties theoretically

enable dose reduction, providing incentive for further in vivo

investigations.

3. Conclusion

The plant bisbenzylisoquinoline alkaloid tetrandrine (1) has

diverse pharmacological properties [3,4], including antiviral [5e8],

anticancer [9,10], multidrug resistance reversing [1 1e15] and cal-

cium 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

R. Schütz et al. / European Journal of Medicinal Chemistry 207 (2020) 112810

7

Fig. 5. RMS compounds inhibit proliferation of VCR-R CEM cells and inﬂuence the intrinsic apoptosis pathway. (a,b) Antiproliferative effects of tetrandrine (1) and RMS1-RMS10

determined by CellTiter-Blue^®cell viability assay after exposure of VCR-R CEM cells to the compounds for 48 h. (a) Relative proliferation at 10

M (One-Way ANOVA followed by

Dunnett’s multiple comparison test, relative proliferation was compared with that of tetrandrine, *P < 0.05, ***P < 0.001, ****P < 0.0001) and (b) IC₅₀values are shown. (c)

Immunoblotting of VCR-R CEM cell lysates after exposure to tetrandrine (1) and RMS1-RMS10 (5 M, 24 h) with antibodies against p-Akt (Ser473), total Akt (t-Akt), PARP and the

m

anti-apoptotic proteins Bcl-xL and Mcl-1. The stain-free technology was used as loading control (ctrl). (d,e) Mitochondrial membrane potential was measured with the ﬂuorescent

probe JC-1 after treatment with tetrandrine (1) and analogues for 24 h. A shift towards JC-1 green ﬂuorescence serves indicator for reduction of the mitochondrial membrane

potential DJm. (d) Gating strategies are exemplarily shown for RMS5 and RMS10. (e) Statistical evaluation of data from (d). Bar graphs indicate means SEM of at least three

independent experiments (One-Way ANOVA followed by Dunnett’s multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001). The mitochondrial uncoupler CCCP (100 mM, 1 h)

served as positive control. -: solvent control, Rel.: relative, tet: tetrandrine (1). (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the Web

version of this article.)

Fig. 6. Toxicity of tetrandrine (1), RMS3 and RMS5 to other primary cells. (a) Viability of human umbilical vein endothelial cells (HUVECs) after a 24 h exposure (1, 5 and 10 mM) to

the compounds was assessed by CellTiter-Blue^®cell viability assay. Fluorescence intensities were normalized to vehicle control. Bar graph indicates means SEM of three inde-

pendent 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.)

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. More-

over, based on these encouraging data and facilitated by our recent

work on effective total synthesis of the bisbenzylisoquinoline al-

kaloids tetrandrine and isotetrandrine [44], further investigations

8

R. Schütz et al. / European Journal of Medicinal Chemistry 207 (2020) 112810

regarding structure-activity relationships in this chemical class

should be part of future medicinal chemistry projects.

Reverse Transcription Kit, Applied Biosystems, Waltham, USA) and

quantitative real-time PCR (ABI 7300 Real-Time PCR System,

Applied Biosystems) with human actin serving as housekeeping

gene were performed as described before [58]. Primers were pur-

chased from Metabion. Primer sequences for detecting CYP3A4

mRNA expression were taken from the work of Nozaki and co-

workers [59] (FW primer: 5⁰-GTATGGAAAAGTGTGGGGCT-3⁰, RV

primer: 5⁰-GACCATCTCCTTGAGTTTTCCA-3⁰).

4. Experimental section

4.1. Biological assays

4.1.1. Cell lines and culture

HepaRG™ cells were purchased from Life Technologies (Wal-

tham, USA) and they were plated and maintained in Williams’

medium E supplemented with GlutaMAX and HepaRG Thaw, Plate

& General Purpose Medium Supplement (Thaw, Plate, & General

Purpose Working Medium) (all purchased from Life Technologies).

Vincristine-resistant (VCR-R) CEM cells were a kind gift from Prof.

Maria Kavallaris (University of New South Wales, Australia) and

cultivated with RPMI 1640 (PAN Biotech, Aidenbach, Germany),

supplemented with 10% fetal calf serum (FCS) (PAA Laboratories,

Toronto, Canada). HepG2 cells were obtained from German

Research Centre of Biological Material (DSMZ) and cultivated with

DMEM (PAN Biotech), supplemented with 10% fetal calf serum

(FCS) (PAA Laboratories). Human umbilical vein endothelial cells

(HUVECs) were purchased from Promocell and cultivated with

ECGM Kit enhanced (PELO Biotech, Planegg, Germany) supple-

mented with 10% (FCS) (PAA Laboratories) and 1% penicillin/

streptomycin/amphotericin B (all purchased from PAN Biotech).

HUVECs were cultured for a maximum of six passages. All cells

were cultured at 37 ^ꢀC with 5% CO₂with constant humidity are

proven to be mycoplasma-free on a quarterly basis.

4.1.5. 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^TMcells without transfection: Hep-

aRG™ cells were differentiated in Williams’ medium E supple-

mented with GlutaMAX and HepaRG Maintenance/Metabolism

Medium Supplement (Maintenance/Metabolism Working Me-

dium) (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^TMcells 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

4.1.2. Molecular cloning

The cDNA template was generated by isolation of mRNA from

HepaRG™ progenitor cells (QIAGEN RNeasy Mini Kit) and subse-

quent reverse transcription (High Capacity cDNA Reverse Tran-

scription Kit, Applied Biosystems, Waltham, USA). cDNA was

ampliﬁed by PCR (Thermo Scientiﬁc Phusion Green Hot Start II

High-Fidelity Polymerase, Thermo Fisher, Waltham, USA) (CYP3A4-

FW: 5⁰-ATATATGGTACCGCCACCATGGCTCTCATCCCA-3⁰, CYP3A4-

RV: 5⁰-ATCTCGAGGGCTCCACTTACGGTGCCA-3⁰). The obtained PCR

product was cloned into the pcDNA3-EGFP vector using FastDigest

KpnI, FastDigest XhoI and T4 DNA Ligase (all purchased from

Thermo Fisher) as indicated by the manufacturer. pcDNA3-EGFP

was a gift from Doug Golenbock (Addgene plasmid #13031;

http://n2t.net/addgene:13031; RRID:Addgene_13031). Correct

insertion of the insert was conﬁrmed by PCR, restriction digestion

and Sanger sequencing. Sequencing services were provided by

Euroﬁns Genomics (Munich, Germany). Primers were purchased

from Metabion (Planegg, Germany).

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 % ꢂ 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) trans-

fection 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: 5⁰-

TACATCAATGGGCGTGGATAG-3⁰, pcDNA3-reverse: 5⁰- AGGAAGG-

GAAGAAAGCGAAAG-3⁰). Primers were purchased from metabion.

HepG2 cells stably expressing either CYP3A4-EGFP or pcDNA3-

EGFP were seeded at a density of 0.1 ꢁ 10⁶cells per well of a 24-

well 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).

4.1.3. 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 ꢁ 10⁶cells 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

PBMCs: PBMCs were isolated from anticoagulated whole blood

from healthy donors by density gradient centrifugation using

Ficoll-Paque PLUS density gradient medium (GE Healthcare, Chi-

cago, 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

(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.

4.1.4. Quantitative real-time PCR

Isolation of mRNA (RNeasy Mini Kit, QIAGEN, Venlo,

Netherlands), synthesis of cDNA templates (High Capacity cDNA

analyzed by propidium iodide (Carl Roth, Karlsruhe, Germany; 5

mL in PBS) staining and ﬂow cytometry using a BD FACS Canto II

mg/

R. Schütz et al. / European Journal of Medicinal Chemistry 207 (2020) 112810

9

(BD Biosciences). For evaluation, cell debris was excluded and

propidium iodide positive cells were determined using FlowJo 7.6

(BD Biosciences).

free technology (Bio-Rad, Hercules, USA) [61] was used to conﬁrm

equal protein loading using Image Lab 6.0.1 software (Bio-Rad).

HUVECs: HUVECs were seeded at a density of 0.125 ꢁ 10³cells

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.

4.1.11. 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 0.1 ꢁ 10⁶cells per well of a 24-well plate and allowed to

adhere overnight. VCR-R CEM cells were seeded at a density of

0.125 ꢁ 10⁶cells per well of a 24-well plate 4 h before treatment.

Subsequently, cells were treated for 24 h or 48 h with DMSO or

4.1.6. Cell proliferation assays

VCR-R CEM cells were seeded at a density of 0.02 ꢁ 10⁶cells per

well of a 96-well plate, incubated for 4 h before stimulation with

the indicated concentrations of compounds for 48 h. 4 h after

seeding, initial metabolic activity was determined and used as zero

value. 2 h before the end of stimulation time, CellTiter-Blue^®re-

agent (Promega) was added and ﬂuorescence at 590 nm was

€

5

(1

m

M of the respective compounds. Cells were incubated with JC-1

g/mL) for 1 h before cells were harvested (for staining of VCR-R

CEM cells, the P-gp inhibitor elacridar (5 M; Sellekchem Chem-

icals, Houston, USA) was present to prevent efﬂux of the mito-

chondrial uncoupler CCCP (100 M)), washed with PBS and

m

detected with

a Sunrise ELISA reader (Tecan, Mannedorf,

Switzerland). Half-maximal inhibitory concentrations (IC₅₀) values

were calculated by nonlinear regression using GraphPad Prism

8.4.0 software.

m

resuspended in PBS for ﬂow cytometric analysis on a BD FACS Canto

II (BD Biosciences). In parallel, compensation samples were pre-

pared using Anti-Mouse Ig,

Particles Set (BD Biosciences) and a BD PE Mouse IgG1,

Control (BD Biosciences #555749) and a BD Alexa Fluor^®488 Mouse

IgG1 Isotype Control (BD Biosciences #557721) as described by

k

/Negative Control (FBS) Compensation

4.1.7. Calcein-AM retention assay

The calcein-AM retention assay was performed as described

previously [42].

k/Isotype

k

the manufacturer. Prior to analysis of cellular samples, compensa-

tion of spectral overlap was performed on a BD FACS Canto II (BD

Biosciences) according to the manufacturer’s instructions. The

percentage of Alexa-Fluor^®-488-A positive populations was deter-

mined using FlowJo 7.6 (BD Biosciences).

4.1.8. P-gp cell surface expression

2 ꢁ 10⁴VCR-R CEM cells were incubated in presence of 1

mM of

the respective compounds at 37 ^ꢀC for 1.5 h, then cells were

transferred to pre-cooled FACS tubes (BD Biosciences), centrifuged

(400 g, 5 min, 4 ^ꢀC) and washed with ice-cold 1% BSA in PBS. After

aspiration, pelleted cells were resuspended in 20

Anti-Human P-gp antibody (BD Biosciences #557002), diluted with

80

l PBS per test. Cells were incubated for 30 min at 4 ^ꢀC in the

dark. To determine unspeciﬁc antibody binding, FITC Mouse IgG2b

Isotype Control (BD #58555742) was used (20 l per test, diluted

in 80 l PBS). Thereafter, cells were washed once with ice-cold 1%

BSA (Anprotec, Bruckberg, Germany) in PBS and twice with 0.5% (v/

v) Tween 20 (Carl Roth, Karlsruhe, Germany) in PBS and resus-

pended in ice-cold PBS. Subsequently, cells were analyzed by ﬂow

cytometry on a BD FACS Canto II (BD Biosciences).

m

l of FITC Mouse

4.2. Chemistry

m

4.2.1. 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 ¹H and 101 MHz for ¹³C), 500 MHz

(500 MHz for ¹H and 126 MHz for ¹³C) or 800 MHz Bruker Biospin

Avance spectrometer (800 MHz for ¹H and 201 MHz for ¹³C). 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

(CDCl₃: d_H¼ 7.26 ppm, d_C¼ 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 (Tcl₂

[100 ^ꢀC]: d_H¼ 5.92 ppm, d_C¼ 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 Fin-

nigan 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^LCMS 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 Agi-

lent 1100 system equipped with an Agilent 1100/1200 Diode Array

k

m

4.1.9. Apoptosis assay

VCR-R CEM cells were seeded at a density of 0.125 ꢁ 10⁶cells per

well of a 24-well plate and incubated for 4 h. Treatment was per-

formed 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).

4.1.10. Western blot analysis and antibodies

HepG2 were seeded at a density of 0.5 ꢁ 10⁶cells per well of a 6-

well plate and allowed to adhere overnight. VCR-R CEM cells were

seeded at a density of 2 ꢁ 10⁶cells 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 (radio-

immunoprecipiation) 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 anti-

bodies and detection of chemiluminescence on a Chemidoc™

Touch Imaging System (Bio-Rad) were performed as described

previously. The following antibodies were used: phospho-Akt

(Ser473) (1:1000, Cell Signaling Technologies (CST), Danvers, USA,

#9271), total Akt (1:1000, CST, #9272), Bcl-xL (1:1000, CST, #2762),

PARP (1:1000, CST, #9542), Mcl-1 (1:1000, CST, #4572). The stain

Detector and an Agilent Zorbax Eclipse Plus C18-column (5.0 mm,

150 ꢁ 4.6 mm) using following method: ﬂow 0.8 mL/min; tem-

perature 50 ^ꢀC; eluent system for compounds RMS1-10, 13c-d and

14c-e: 80% MeOH, 20% water, NaOH buffer pH 9; for compounds

10

R. Schütz et al. / European Journal of Medicinal Chemistry 207 (2020) 112810

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%.

3H, 2⁰-OCH₃). ¹³C NMR, DEPT, HMQC, HMBC (101 MHz, CDCl₃):

d

4), 126.8 (C-6), 122.5 (C-3), 104.5 (C-1⁰), 61.1 (2⁰-OCH₃). IR (ATR): ṽ

[cm^ꢂ1] ¼ 3063, 2932, 2833, 1726, 1569, 1474, 1427, 1206, 1070, 782.

HRMS (EI): m/z calcd for [C₉H⁷₉⁹BrO]^ꢃþ211.9831, found: 211.9829.

[ppm] ¼ 149.2 (C-2⁰), 138.1 (C-1), 131.0 (C-2), 129.8 (C-5), 128.7 (C-

4.2.2. 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

4.2.4. (E/Z)-3-Bromo-1-(2-methoxyvinyl)-4-(triﬂuoromethoxy)

benzene (12b)

^ꢀC under nitrogen atmosphere. A solution of lithium diisopro-

Prepared from 3-bromo-4-(triﬂuoromethoxy)benzaldehyde

(300 mg, 1.12 mmol) following General Procedure 1 (Wittig oleﬁ-

nation). Puriﬁcation was accomplished by ﬂash column chroma-

tography (2.5% ethyl acetate in hexanes, R_f¼ 0.30) to give the title

compound as a light yellow oil (266 mg, 0.895 mmol, 80%, E,Z-

isomer ratio 1:0.71, estimated by NMR integrals). NMR data of the

pylamide (1.4 equiv., 2.0 M solution in THF) was added dropwise

and the resulting mixture stirred for 45 min. A solution of the ar-

omatic 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 ace-

tate. The combined organic phases were washed with brine, dried

over anhydrous Na₂SO₄and concentrated in vacuo to afford the

crude product which was puriﬁed by column chromatography.

General procedure 2: N-Acyl Pictet-Spengler condensation: A so-

lution 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 tem-

perature and stirred for 6e12 h. A saturated NaHCO₃solution was

then added for neutralization and the mixture extracted 3x with

dichloromethane. The combined organic phases were dried over

anhydrous Na₂SO₄and concentrated in vacuo to afford the crude

product which was puriﬁed by column chromatography.

General procedure 3: Ullmann-type CeO coupling reaction: Ac-

cording to a modiﬁed procedure of Wang et al. [62], bromoarene

(1.0e1.2 equiv.), phenol (1.0e2.0 equiv.), CuBr$Me₂S (1.0 equiv.) and

Cs₂CO₃(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 concentra-

tion 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 col-

umn chromatography.

major E-isomer: ¹H NMR, COSY (500 MHz, CDCl₃):

d

[ppm] ¼ 7.48

(d, J ¼ 2.1 Hz, 1H, 2-H), 7.19 (dd, J ¼ 8.4, 1.4 Hz, 1H, 5-H), 7.15 (dd,

J ¼ 8.5, 2.1 Hz, 1H, 6-H), 7.03 (d, J ¼ 13.0 Hz, 1H, 2⁰-H), 5.71 (d,

J ¼ 12.9 Hz, 1H, 1⁰-H), 3.70 (s, 3H, 2⁰-OCH₃). ¹³C NMR, DEPT, HMQC,

HMBC (126 MHz, CDCl₃):

d

[ppm] ¼ 150.6 (C-2⁰), 144.2 (d, J ¼ 2.0 Hz,

C-4), 137.2 (C-1), 130.1 (C-2), 125.1 (C-6), 122.6 (C-5), 120.7 (q,

J ¼ 258.7 Hz, OCF₃), 116.5 (C-3), 102.9 (C-1⁰), 56.9 (2⁰-OCH₃). IR

(ATR): ṽ [cm^ꢂ1] ¼ 2940, 2838, 1641, 1492, 1247, 1156, 1096, 932, 833.

79

HRMS (EI): m/z calcd for [C

H

BrF₃O₂]^ꢃþ295.9654, found:

10

8

295.9651.

4.2.5. (E/Z)-4-Bromo-2-(2-methoxyvinyl)thiophene (12c)

Prepared from 4-bromothiophene-2-carboxaldehyde

(0.500 mg, 2.62 mmol) following General Procedure 1 (Wittig

oleﬁnation). Puriﬁcation was accomplished by ﬂash column chro-

matography (hexanes, R_f¼ 0.28) to give the title compound as a

light yellow oil (471 mg, 2.15 mmol, 82%, E,Z-isomer ratio 1:1.13,

estimated by NMR integrals). NMR data of the E-isomer: ¹H NMR,

COSY (400 MHz, CDCl₃):

d

[ppm] ¼ 6.97 (d, J ¼ 12.9 Hz, 1H, 2⁰-H),

6.89 (d, J ¼ 1.4 Hz, 1H, 5-H), 6.70e6.68 (m, 1H, 3-H), 5.87 (d,

J ¼ 12.8 Hz, 1H, 1⁰-H), 3.67 (s, 3H, OCH₃). ¹³C NMR, DEPT, HMQC,

HMBC (101 MHz, CDCl₃):

d

[ppm] ¼ 149.9 (C-2⁰), 141.4 (C-2), 125.0

(C-3), 118.5 (C-5), 109.8 (C-4), 98.6 (C-1⁰), 57.0 (OCH₃). IR (ATR): ṽ

[cm^ꢂ1] ¼ 3110, 2933, 2849, 2053, 1727, 1637, 1221, 1093, 819, 722.

HRMS (EI): m/z calcd for [C₇H⁷₇⁹BrOS]^ꢃþ217.9395, found: 217.9395.

4.2.6. (E/Z)-3-Bromo-4-chloro-1-(2-methoxyvinyl)benzene (12d)

Prepared from 3-bromo-4-chlorobenzaldehyde (500 mg,

2.28 mmol) following General Procedure 1 (Wittig oleﬁnation).

Puriﬁcation was accomplished by ﬂash column chromatography

(2.5% ethyl acetate in hexanes, R_f¼ 0.36) to give the title compound

as a light yellow oil (490 mg, 1.98 mmol, 87%, E,Z-isomer ratio

1.08:1, estimated by NMR integrals). NMR data of the major E-iso-

General procedure 4: Carbamate reduction: Lithium alanate (12

equiv.) was suspended in 1 mL anhydrous THF under nitrogen at-

mosphere. 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 Na₂SO₄and concentrated in

vacuo to afford the crude product which was puriﬁed by column

chromatography.

mer: ¹H NMR, COSY (500 MHz, CDCl₃):

1H, 2-H), 7.30 (d, J ¼ 7.2 Hz, 1H, 5-H), 7.08 (dd, J ¼ 8.3, 2.1 Hz, 1H, 6-

H), 7.03 (d, J ¼ 13.0 Hz, 1H, 2⁰-H), 5.69 (d, J ¼ 13.0 Hz, 1H, 1⁰-H), 3.69

(s, 3H, OCH₃). ¹³C NMR, DEPT, HMQC, HMBC (126 MHz, CDCl₃):

d

[ppm] ¼ 7.47 (d, J ¼ 2.1 Hz,

d

[ppm] ¼ 150.3 (C-2⁰), 137.0 (C-1), 131.0 (C-4), 130.4 (C-5), 130.1 (C-

2), 125.1 (C-6), 122.7 (C-3) 103.1 (C-1⁰), 56.9 (OCH₃). IR (ATR): ṽ

[cm^ꢂ1] ¼ 2934, 2833, 1701, 1640, 1467, 1192, 1120, 1023, 822. HRMS

(EI): m/z calcd for [C₉H₈⁷⁹Br³⁵ClO]^ꢃþ245.9442, found: 245.9444.

4.2.3. (E/Z)-3-Bromo-1-(2-methoxyvinyl)benzene (12a)

Prepared from 3-bromobenzaldehyde (1.00 g, 5.41 mmol)

following General Procedure 1 (Wittig oleﬁnation). Puriﬁcation was

accomplished by ﬂash column chromatography (2.5% ethyl acetate

in hexanes, R_f¼ 0.25) to give the title compound as a colourless oil

(940 mg, 4.41 mmol, 82%, E,Z-isomer ratio 0.92:1, estimated by

NMR integrals). NMR data of the major Z-isomer: ¹H NMR, COSY

4.2.7. 5-Bromo-1-pentanal (12e)

Pyridinium chlorochromate (503 mg, 2.33 mmol, 1.3 equiv.) was

suspended in 10 mL anhydrous dichloromethane under nitrogen

atmosphere. Then 5-bromo-1-pentanol (300 mg, 1.80 mmol) was

added and the mixture was stirred for 6 h at ambient temperature.

The volatiles were removed in vacuo and puriﬁcation of the residue

by ﬂash column chromatography (25% diethyl ether in hexanes,

R_f¼ 0.43) gave the product as a colourless oil (191 mg, 1.16 mmol,

(400 MHz, CDCl₃):

J ¼ 7.8, 1.4 Hz, 1H, 6-H), 7.28e7.23 (m, 1H, 4-H), 7.16e7.09 (m, 1H, 5-

d

[ppm] ¼ 7.77 (t, J ¼ 1.8 Hz, 1H, 2-H), 7.43 (dt,

H), 6.17 (d, J ¼ 7.0 Hz, 1H, 2⁰-H), 5.15 (d, J ¼ 7.0 Hz, 1H, 1⁰-H), 3.80 (s,

64%). ¹H NMR, COSY (400 MHz, CDCl₃):

d

[ppm] ¼ 9.78 (t, J ¼ 1.5 Hz,

R. Schütz et al. / European Journal of Medicinal Chemistry 207 (2020) 112810

11

1H, 1-H), 3.42 (t, J ¼ 6.5 Hz, 2H, 5-H), 2.49 (td, J ¼ 7.1, 1.5 Hz, 2H, 2-

H), 1.95e1.86 (m, 2H, 4-H), 1.84e1.75 (m, 2H, 3-H). ¹³C NMR, DEPT,

11⁰, C-13⁰), 114.3 (C-8⁰), 113.3 (C-5⁰), 109.8 (C-5), 61.2 (OCH₂CH₃,

‘OCH₂CH₃), 60.7 (7-OCH₃), 56.6 (6’-OCH₃, 6-OCH₃), 56.1 (C-1’), 52.0

HMQC, HMBC (126 MHz, CDCl₃):

d

[ppm] ¼ 201.7 (C-1), 42.9 (C-2),

(C-1), 42.2 (C-a’), 40.2 (C-a), 38.8 (C-3’), 37.8 (C-3), 28.3 (C-4’), 28.1

33.0 (C-5), 31.9 (C-4), 20.6 (C-3). IR (ATR): ṽ [cm^ꢂ1] ¼ 2954, 2865,

2840, 1725, 1437, 1358, 1122, 1058. HRMS (EI): m/z calcd for

[C₅H⁷₉⁹BrO]^ꢃþ162.9753, found: 162.9752.

(C-4), 14.6 (‘OCH₂CH₃), 14.3 (OCH₂CH₃). The resonances of C-8 and

C-4a could not be identiﬁed. IR (ATR): ṽ [cm^ꢂ1] ¼ 2935, 1689, 1512,

1426, 1332, 1239, 1112, 1023, 769. Purity (HPLC) ¼ 92% ( ¼ 210 nm).

l

79

HRMS (ESI): m/z calcd for [C

H

BrN₂O₉þ H]^þ789.2381, found:

41 45

789.2390.

4.2.8. ( )-8-((1-(4-Hydroxybenzyl)-N-(ethoxycarbonyl)-6-

methoxy-1,2,3,4-tetrahydroisoquinolin-7-yl)oxy)-N-

ethoxycarbonyl-6,7-dimethoxy-1-(3⁰-bromobenzyl)-1,2,3,4-

tetrahydroisoquinoline (13a; separable mixture of racemic

diastereomers)

4.2.9. ( )-8-((1-(4-Hydroxybenzyl)-N-(ethoxycarbonyl)-6-

methoxy-1,2,3,4-tetrahydroisoquinolin-7-yl)oxy)-N-

ethoxycarbonyl-6,7-dimethoxy-1-((3’-bromo-4⁰-triﬂuoromethoxy)

benzyl)-1,2,3,4-tetrahydroisoquinoline (13b.; separable mixture of

racemic diastereomers)

Carbamate 11 (250 mg, 0.411 mmol) and enol ether 12a (160 mg,

0.751 mmol) were condensed following General Procedure 2. The

reaction was completed after 4 h. Puriﬁcation by ﬂash column

chromatography (20% ethyl acetate in dichloromethane, R_f¼ 0.13

(1R,1⁰R)/(1S,1⁰S) isomers and 0.23 (1R,1⁰S)/(1S,1⁰R) isomers) gave the

title compounds as white solids.

Carbamate 11 (250 mg, 0.411 mmol) and enol ether 12b (146 mg,

0.493 mmol) were condensed following General Procedure 2. The

reaction was completed after 12 h. Puriﬁcation by ﬂash column

chromatography (15% ethyl acetate in dichloromethane, R_f¼ 0.10

(1R,1⁰R)/(1S,1⁰S) isomers and 0.15 (1R,1⁰S)/(1S,1⁰R) isomers) gave the

title compounds as white solids.

(1R,1⁰R)/(1S,1⁰S) isomers: yield: 111 mg, 0.141 mmol, 34%. mp:

112.0e113.0 ^ꢀC. ¹H NMR, COSY (400 MHz, Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 7.30e7.22 (m, 1H, 12-H), 7.18 (t, J ¼ 1.8 Hz, 1H, 10-H), 7.03

(1R,1⁰R)/(1S,1⁰S) isomers: yield: 134 mg, 0.153 mmol, 37%. mp:

(t, J ¼ 7.7 Hz, 1H, 13-H), 6.95 (d, J ¼ 7.6 Hz, 1H, 14-H), 6.81e6.76 (m,

2H, 10⁰-H, 14⁰-H), 6.70 (s, 1H, 5⁰-H), 6.58e6.51 (m, 3H, 5-H, 11⁰-H,

13⁰-H), 6.20 (s, 1H, 8⁰-H), 5.34 (d, J ¼ 9.6 Hz, 1H, 1-H), 5.02 (t,

J ¼ 6.5 Hz, 1H, 1⁰-H), 4.68 (s, 1H, OH), 4.16e4.05 (m, 1H, 3-H), 4.00

(q, J ¼ 7.1 Hz, 2H, ‘OCH₂CH₃), 3.96e3.85 (m,1H, 3⁰-H), 3.90 (s, 3H, 6⁰-

OCH₃), 3.85 (s, 3H, 6-OCH₃), 3.88e3.70 (m, 2H, OCH₂CH₃), 3.63 (s,

3H, 7-OCH₃), 3.33 (ddd, J ¼ 13.2, 10.4, 5.0 Hz, 1H, 3-H), 3.18 (ddd,

91.5e92.0 ^ꢀC. ¹H NMR, COSY (400 MHz, Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 7.31

(d, J ¼ 2.0 Hz, 1H, 10-H), 7.10 (dd, J ¼ 8.4, 1.6 Hz, 1H, 13-H), 6.99 (dd,

J ¼ 8.4, 2.1 Hz,1H, 14-H), 6.81e6.76 (m, 2H, 10⁰-H,14⁰-H), 6.70 (s, 1H,

5⁰-H), 6.57e6.51 (m, 2H, 11⁰-H, 13⁰-H), 6.54 (s, 1H, 5-H), 6.19 (s, 1H,

8⁰-H), 5.29 (s, 1H, 1-H), 5.02 (t, J ¼ 6.5 Hz, 1H, 1⁰-H), 4.64 (s, 1H, OH),

4.15e4.03 (m, 1H, 3-H), 4.00 (q, J ¼ 7.0 Hz, 2H, ‘OCH₂CH₃), 3.90 (s,

3H, 6⁰-OCH₃), 3.85 (s, 3H, 6-OCH₃), 3.97e3.71 (m, 3H, OCH₂CH₃, 3⁰-

H), 3.62 (s, 3H, 7-OCH₃), 3.39e3.29 (m, 1H, 3-H), 3.24e3.13 (m, 1H,

J ¼ 13.3, 9.5, 4.4 Hz, 1H, 3⁰-H), 3.10 (dd, J ¼ 13.9, 3.6 Hz, 1H,

a-H),

2.93e2.85 (m, 1H, 4-H), 2.84 (t, J ¼ 6.8 Hz, 2H,

a

⁰-H), 2.81e2.72 (m,

3⁰-H), 3.11 (dd, J ¼ 13.9, 3.7 Hz, 1H,

a-H), 2.91e2.72 (m, 5H, a -

-H, a⁰

2H,

a

-H, 4⁰-H), 2.60 (d, J ¼ 16.4 Hz,1H, 4-H), 2.54 (dt, J ¼ 16.0, 4.6 Hz,

H, 4⁰-H, 4-H), 2.62 (d, J ¼ 15.4 Hz, 1H, 4-H), 2.54 (dt, J ¼ 16.0, 4.6 Hz,

1H, 4⁰-H), 1.14 (t, J ¼ 6.9 Hz, 3H, ’ OCH₂CH₃), 0.88 (br s, 3H,

OCH₂CH₃). ¹³C NMR, HSQC, HMBC (101 MHz, Tcl₂, 100 ^ꢀC)

1H, 4⁰-H),1.14 (t, J ¼ 7.0 Hz, 3H, ‘OCH₂CH₃), 0.90 (br s, 3H, OCH₂CH₃).

¹³C NMR, HSQC, HMBC (101 MHz, Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 155.5 (‘C]

O), 155.3 (C]O), 154.2 (C-12⁰), 152.7 (C-6), 148.2 (C-6⁰), 145.9 (C-7⁰),

141.6 (C-9), 140.9 (C-7), 132.5 (C-10), 130.5 (C-10⁰, C-14⁰), 130.5 (C-

9⁰), 130.0 (C-8a⁰), 129.4 (C-13), 129.2 (C-12), 128.5 (C-4a⁰), 128.2 (C-

14), 123.8 (C-8a), 122.1 (C-11), 115.4 (C-11⁰, C-13⁰), 114.1 (C-8⁰), 113.4

(C-5⁰), 110.1 (C-5), 61.2 (‘OCH₂CH₃), 61.1 (OCH₂CH₃), 60.7 (7-OCH₃),

56.7 (6⁰-OCH₃or 6-OCH₃), 56.6 (6⁰-OCH₃or 6-OCH₃), 55.9 (C-1⁰),

d

[ppm] ¼ 155.4 (‘C]O), 155.2 (C]O), 154.2 (C-12⁰), 152.8 (C-6),

148.1 (C-6⁰), 145.8 (C-7⁰), 145.0 (C-12), 140.9 (C-7), 139.9 (C-9), 134.8

(C-10), 130.5 (C-10⁰, C-14⁰), 130.4 (C-9⁰), 129.6 (C-14), 129.3 (C-8a⁰),

123.5 (C-8a), 121.5 (C-13), 115.6 (C-11), 115.4 (C-11⁰, C-13⁰), 114.1 (C-

8⁰), 113.4 (C-5⁰), 110.2 (C-5), 61.2 (‘OCH₂CH₃), 61.1 (OCH₂CH₃), 60.7

(7-OCH₃), 56.7, 56.6 (6⁰-OCH₃, 6-OCH₃), 55.9 (C-1⁰), 51.9 (C-1), 42.1

51.9 (C-1), 42.1 (C-

a

⁰), 40.1 (C-

a

), 38.8 (C-3⁰), 37.4 (C-3), 28.3 (C-4⁰),

(C-a a

⁰), 39.6 (C- ), 38.8 (C-3⁰), 38.2 (C-3), 28.3 (C-4⁰), 27.9 (C-4), 14.6

27.9 (C-4), 14.6 (‘OCH₂CH₃), 14.2 (OCH₂CH₃). The resonances of C-8

(‘OCH₂CH₃), 14.1 (OCH₂CH₃).The resonances of C-8, C-4a, C-4a⁰and

OCF₃could not be identiﬁed. IR (ATR): ṽ [cm^ꢂ1] ¼ 2931, 1668, 1612,

and C-4a could not be identiﬁed. IR (ATR): ṽ [cm^ꢂ1] ¼ 2930, 1693,

1513, 1426, 1332, 1199, 1112, 1021, 835, 765. Purity (HPLC) ¼ 90%

1514, 1426, 1332, 1247, 1168, 1119, 1023, 763. Purity (HPLC) ¼ 85%

79

(

l

¼ 210 nm). HRMS (ESI): m/z calcd for [C₄₁

H

BrN₂O₉þ H]^þ

(l

¼ 210 nm). HRMS (ESI): m/z calcd for [C₄₂

H

BrF₃N₂O₁₀þ H]^þ

45

44

789.2381, found: 789.2383.

873.2204, found: 873.2217.

(1R,1⁰S)/(1S,1⁰R) isomers: yield: 95.0 mg, 0.121 mmol, 29%. mp:

99.0e101.5 ^ꢀC. ¹H NMR, COSY (400 MHz, Tcl₂,100 ^ꢀC)

[ppm] ¼ 7.28

(1R,1⁰S)/(1S,1⁰R) isomers: yield: 91 mg, 0.104 mmol, 25%. mp:

93.5e95.0 ^ꢀC. ¹H NMR, COSY (400 MHz, Tcl₂, 100 ^ꢀC)

[ppm] ¼ 7.37

d

(dt, J ¼ 7.8, 1.6 Hz, 1H, 12-H), 7.24 (s, 1H, 10-H), 7.05 (t, J ¼ 7.7 Hz, 1H,

13-H), 6.99 (d, J ¼ 7.6 Hz, 1H, 14-H), 6.81e6.74 (m, 2H, 10⁰-H, 14⁰-H),

6.70 (s, 1H, 5⁰-H), 6.60e6.53 (m, 2H, 11⁰-H, 13⁰-H), 6.52 (s, 1H, 5-H),

6.11 (s, 1H, 8⁰-H), 5.32 (s, 1H, 1-H), 5.18 (s, 1H, OH), 4.99 (t, J ¼ 6.5 Hz,

1H, 1⁰-H), 4.15e4.06 (m, 1H, 3-H), 4.01 (q, J ¼ 7.3 Hz, 2H, ‘OCH₂CH₃),

3.96e3.87 (m, 1H, 3⁰-H), 3.89 (s, 3H, 6⁰-OCH₃), 3.83 (s, 3H, 6-OCH₃),

3.86e3.76 (m, 2H, OCH₂CH₃), 3.60 (s, 3H, 7-OCH₃), 3.36 (ddd,

(d, J ¼ 2.0 Hz, 1H, 10-H), 7.12 (dd, J ¼ 8.4, 1.6 Hz, 1H, 13-H), 7.03 (dd,

J ¼ 8.4, 2.1 Hz,1H, 14-H), 6.81e6.72 (m, 2H,10⁰-H,14⁰-H), 6.70 (s, 1H,

5⁰-H), 6.59e6.53 (m, 2H, 11⁰-H, 13⁰-H), 6.52 (s, 1H, 5-H), 6.09 (s, 1H,

8⁰-H), 5.29 (d, J ¼ 5.6 Hz, 1H, 1-H), 4.98 (t, J ¼ 6.4 Hz, 1H, 1⁰-H),

4.10e4.01 (m, 1H, 3-H), 4.02 (q, J ¼ 7.2 Hz, 2H, ‘OCH₂CH₃), 3.88 (s,

3H, 6⁰-OCH₃), 3.96e3.76 (m, 3H, OCH₂CH₃, 3⁰-H), 3.83 (s, 3H, 6-

OCH₃), 3.58 (s, 3H, 7-OCH₃), 3.37 (ddd, J ¼ 13.3, 10.0, 4.8 Hz, 1H,

J ¼ 13.1, 10.0, 4.8 Hz, 1H, 3-H), 3.23 (dd, J ¼ 14.0, 4.1 Hz, 2H,

a

-H, 3⁰-

⁰-H, 4⁰-H),

3-H), 3.23 (dd, J ¼ 13.8, 4.0 Hz, 2H,

a a-

-H, 3⁰-H), 2.95e2.81 (m, 3H,

H), 2.97e2.83 (m, 3H,

a

-H,

a

⁰-H, 4-H), 2.82e2.72 (m, 2H,

a

H, a a

⁰-H, 4-H), 2.81e2.72 (m, 2H, ⁰-H, 4⁰-H), 2.64e2.54 (m, 2H, 4⁰-H,

2.57 (dt, J ¼ 16.0, 4.5 Hz, 2H, 4⁰-H, 4-H), 1.15 (t, J ¼ 7.1 Hz, 3H,

4-H), 1.16 (t, J ¼ 7.1 Hz, 3H, ‘OCH₂CH₃), 0.96 (br s, 3H, OCH₂CH₃). ¹³

C

‘OCH₂CH₃), 0.96 (s, 3H, OCH₂CH₃). ¹³C NMR, HSQC, HMBC (101 MHz,

NMR, HSQC, HMBC (101 MHz, Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 155.5 (C]O,

Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 155.5 (C]O, ‘C]O), 154.4 (C-12⁰), 152.6 (C-

‘C]O), 154.3 (C-12⁰), 154.2 (C-6), 148.2 (C-6⁰), 145.6 (C-7⁰), 145.2 (C-

12), 143.1 (C-7), 139.9 (C-9), 135.0 (C-10), 130.6 (C-10⁰, C-14⁰), 130.4

(C-9⁰), 130.0 (C-8a⁰), 129.7 (C-14), 123.0 (C-8a), 121.9 (C-13), 115.3

(C-11), 115.3 (C-11⁰, C-13⁰), 114.4 (C-8⁰), 113.3 (C-5⁰), 109.8 (C-5), 61.3

6), 148.2 (C-6⁰), 145.7 (C-7⁰), 141.6 (C-9), 140.7 (C-7), 132.6 (C-10),

130.5 (C-10⁰, C-14⁰), 130.3 (C-9⁰), 129.4 (C-13), 129.2 (C-12),129.2 (C-

8a⁰), 128.4 (C-4a⁰), 128.3 (C-14), 123.4 (C-8a), 122.1 (C-11), 115.3 (C-

12

R. Schütz et al. / European Journal of Medicinal Chemistry 207 (2020) 112810

(‘OCH₂CH₃), 61.2 (OCH₂CH₃), 60.7 (7-OCH₃), 56.6, 56.5 (6⁰-OCH₃, 6-

1690, 1511, 1421, 1221, 1099, 1022, 819,764. Purity (HPLC) ¼ 95%

79

OCH₃), 56.5 (C-1⁰), 52.0 (C-1), 42.2 (C-

a

⁰), 39.7 (C-

a

), 38.9 (C-3⁰),

(l

¼ 210 nm). HRMS (ESI): m/z calcd for [C₃₉

H

BrN₂O₉SeH]^-

43

38.0 (C-3), 28.3 (C-4⁰), 28.1 (C-4), 14.6 (‘OCH₂CH₃), 14.3 (OCH₂CH₃).

The resonances of C-8, C-4a, C-4a⁰and OCF₃could not be identiﬁed.

IR (ATR): ṽ [cm^ꢂ1] ¼ 2922, 1668, 1613, 1514, 1455, 1333, 1253, 1170,

793.1800, found: 793.1829.

4.2.11. ( )-8-((1-(4-Hydroxybenzyl)-N-(ethoxycarbonyl)-6-

methoxy-1,2,3,4-tetrahydroisoquinolin-7-yl)oxy)-N-

ethoxycarbonyl-6,7-dimethoxy-1-(3⁰-bromo-4⁰-chlorobenzyl)-

1,2,3,4-tetrahydroisoquinoline (13d; separable mixture of racemic

diastereomers)

1120, 1024, 764. Purity (HPLC) ¼ 90% ( ¼ 210 nm). HRMS (ESI): m/z

l

79

calcd for [C

H

BrF₃N₂O₁₀þ H]^þ873.2204, found: 873.2217.

42 44

4.2.10. ( )-8-((1-(4-Hydroxybenzyl)-N-(ethoxycarbonyl)-6-

methoxy-1,2,3,4-tetrahydroisoquinolin-7-yl)oxy)-N-

ethoxycarbonyl-6,7-dimethoxy-1-((4⁰-bromothiophen-2⁰-yl)

methyl)-1,2,3,4-tetrahydroisoquinoline (13c; separable mixture of

racemic diastereomers)

Carbamate 11 (350 mg, 0.575 mmol) and enol ether 12d

(171 mg, 0.695 mmol) were condensed following General Proced-

ure 2. The reaction was completed after 12 h. Puriﬁcation by ﬂash

column chromatography (15% ethyl acetate in dichloromethane,

R_f¼ 0.09 (1R,1⁰R)/(1S,1⁰S) isomers and 0.13 (1R,1⁰S)/(1S,1⁰R) iso-

mers) gave the title compound as a white solid.

Carbamate 11 (300 mg, 0.493 mmol) and enol ether 12c

(130 mg, 0.591 mmol) were condensed following General Proced-

ure 2. The reaction was completed after 12 h. Puriﬁcation by ﬂash

column chromatography (15% ethyl acetate in dichloromethane,

R_f¼ 0.10 (1R,1⁰R)/(1S,1⁰S) isomers and 0.15 (1R,1⁰S)/(1S,1⁰R) isomers)

gave the title compounds as white solids.

(1R,1⁰R)/(1S,1⁰S) isomers: yield: 128 mg, 0.155 mmol, 27%. mp:

84.0e88.0 ^ꢀC. ¹H NMR, COSY (400 MHz, Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 7.28

(d, J ¼ 2.0 Hz, 1H, 10-H), 7.24 (d, J ¼ 8.2 Hz, 1H, 13-H), 6.91 (dd,

J ¼ 8.1, 2.0 Hz,1H, 14-H), 6.81e6.76 (m, 2H, 14⁰-H and 10⁰-H), 6.70 (s,

1H, 5⁰-H), 6.57e6.52 (m, 2H, 13⁰-H and 11⁰-H), 6.54 (s, 1H, 5-H), 6.19

(s, 1H, 8⁰-H), 5.34e5.27 (m, 1H, 1-H), 5.02 (t, J ¼ 6.3 Hz, 1H, 1⁰-H),

4.66 (s, 1H, OH), 4.15e4.04 (m, 1H, 3-H), 4.00 (q, J ¼ 6.9 Hz, 2H,

‘OCH₂CH₃), 3.89 (s, 3H, 6⁰-OCH₃), 3.85 (s, 3H, 6-OCH₃), 3.93e3.74

(m, 3H, OCH₂CH₃, 3⁰-H), 3.62 (s, 3H, 7-OCH₃), 3.37e3.26 (m, 1H, 3-

(1R,1⁰R)/(1S,1⁰S) isomers: yield: 137 mg, 0.172 mmol, 35%. mp:

108.5e114.5 ^ꢀC. ¹H NMR, COSY (400 MHz, Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 6.96 (d, J ¼ 1.4 Hz, 1H, 12-H), 6.81e6.77 (m, 2H, 14⁰-H and

10⁰-H), 6.68 (s, 1H, 5⁰-H), 6.58 (s, 1H, 10-H), 6.57e6.54 (m, 2H, 13⁰-H

and 11⁰-H), 6.52 (s,1H, 5-H), 6.22 (s,1H, 8⁰-H), 5.33 (d, J ¼ 9.5 Hz,1H,

1-H), 5.02 (t, J ¼ 6.4 Hz, 1H, 1⁰-H), 4.58 (s, 1H, OH), 4.11e4.05 (m, 1H,

3-H), 4.01 (q, J ¼ 7.0 Hz, 2H, ‘OCH₂CH₃), 3.94e3.81 (m, 3H, 3⁰-H,

OCH₂CH₃), 3.87 (s, 3H, 6⁰-OCH₃), 3.85 (s, 3H, 6-OCH₃), 3.61 (s, 3H, 7-

H), 3.24e3.13 (m, 2H, 3⁰-H), 3.07 (dd, J ¼ 14.0, 3.8 Hz, 1H,

a-H),

2.92e2.73 (m, 5H, 4-H,

a

-H, 4⁰-H,

a

⁰-H), 2.61 (d, J ¼ 14.5 Hz, 1H, 4-

H), 2.54 (dt, J ¼ 16.1, 4.5 Hz, 1H, 4⁰-H), 1.14 (t, J ¼ 7.0 Hz, 3H,

OCH₃), 3.31 (dd, J ¼ 15.2, 3.7 Hz, 1H,

a

-H), 3.29e3.12 (m, 2H, 3-H, 3⁰-

‘OCH₂CH₃), 0.90 (br s, 3H, OCH₂CH₃). ¹³C NMR, HSQC, HMBC

H), 3.01 (dd, J ¼ 15.1, 9.3 Hz,1H,

a

-H), 2.84 (dd, J ¼ 9.1, 6.5 Hz,1H, a⁰

-

(101 MHz, Tcl₂,100 ^ꢀC)

d

[ppm] ¼ 155.4 (C]O or C]O⁰), 155.3 (C]O

H), 2.91e2.72 (m, 3H, 4-H, 4⁰-H,

a

⁰-H), 2.56 (ddd, J ¼ 20.0, 11.4,

or C]O⁰), 154.2 (C-12⁰), 152.7 (C-6), 148.2 (C-6⁰), 145.8 (C-7⁰), 140.9

(C-7), 139.7 (C-9), 134.6 (C-10), 132.1 (C-12), 130.5 (C-14⁰and C-10⁰),

130.5 (C-9⁰), 129.7 (C-13 or C-14), 129.6 (C-13 or C-14), 129.2 (C-8a⁰),

128.6 (C-4a⁰),123.5 (C-8a),121.8 (C-11),115.4 (C-13⁰and C-11⁰),114.1

(C-8⁰), 113.4 (C-5⁰), 110.1 (C-5), 61.2 (OCH₂CH₃and ‘OCH₂CH₃), 60.7

(7-OCH₃), 56.7 (6-OCH₃), 56.6 (6⁰-OCH₃), 55.9 (C-1⁰), 51.9 (C-1), 42.1

3.9 Hz, 2H, 4-H, 4⁰-H), 1.14 (t, J ¼ 7.0 Hz, 3H, ‘OCH₂CH₃), 1.01 (br s,

3H, OCH₂CH₃). ¹³C NMR, HSQC, HMBC (101 MHz, Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 155.5 (C]O or C]O⁰), 155.4 (C]O or C]O⁰), 154.2 (C-

12⁰), 152.8 (C-6), 148.4 (C-6⁰), 146.0 (C-7⁰), 142.6 (C-9), 140.8 (C-7),

130.6 (C-14⁰and C-10⁰), 130.4 (C-9⁰), 129.3 (C-8a⁰), 128.6 (C-10),

123.1 (C-8a), 121.3 (C-12), 115.4 (C-13⁰and C-11⁰), 114.4 (C-8⁰), 113.5

(C-5⁰), 110.0 (C-5), 108.9 (C-11), 61.3 (OCH₂CH₃), 61.2 (‘OCH₂CH₃),

60.7 (7-OCH₃), 56.6 (6-OCH₃or 6⁰-OCH₃), 56.5 (6-OCH₃or 6⁰-OCH₃),

(C-a a

⁰), 39.6 (C- ), 38.8 (C-3⁰), 37.5 (C-3), 28.3 (C-4⁰), 27.9 (C-4), 14.6

(‘OCH₂CH₃), 14.2 (OCH₂CH₃). The resonances of C-8 and C-4a could

not be identiﬁed. IR (ATR): ṽ [cm^ꢂ1] ¼ 3174, 1689, 1509, 1419, 1201,

55.9 (C-1⁰), 51.9 (C-1), 42.1 (C-

a

⁰), 38.7 (C-3⁰), 37.6 (C-3), 34.6 (C-

a

),

1097, 1021, 762. Purity (HPLC) ¼ 89% ( ¼ 210 nm). HRMS (ESI): m/z

l

79

28.3 (C-4⁰), 27.9 (C-4), 14.6 (‘OCH₂CH₃), 14.3 (OCH₂CH₃). The reso-

nances of C-8, C-4a and C-4a⁰could not be identiﬁed. IR (ATR): ṽ

[cm^ꢂ1] ¼ 2927, 2843, 1667, 1513, 1422, 1331, 1222, 1109, 1023, 821,

calcd for [C H

Br³⁵ClN₂O₉e H]^-821.1846, found: 821.1851.

41 44

(1R,1⁰S)/(1S,1⁰R) isomers: yield: 122 mg, 0.150 mmol, 26%. mp:

106.5e108.5 ^ꢀC. ¹H NMR, COSY (400 MHz, Tcl₂, 100 ^ꢀC)

764. Purity (HPLC) ¼ 93% (

l

¼ 210 nm). HRMS (ESI): m/z calcd for

d

[ppm] ¼ 7.34 (d, J ¼ 2.0 Hz, 1H, 10-H), 7.25 (d, J ¼ 8.1 Hz, 1H, 13-H),

79

[C

H

BrN₂O₉SeH]^-793.1800, found: 793.1829.

6.95 (dd, J ¼ 8.2, 2.0 Hz, 1H, 14-H), 6.80e6.75 (m, 2H, 14⁰-H and 10⁰-

H), 6.69 (s,1H, 5⁰-H), 6.58e6.53 (m, 2H,13⁰-H and 11⁰-H), 6.51 (s, 1H,

5-H), 6.08 (s, 1H, 8⁰-H), 5.28 (d, J ¼ 8.5 Hz, 1H, 1-H), 4.98 (t,

J ¼ 6.7 Hz, 1H, 1⁰-H), 4.02 (q, J ¼ 6.9 Hz, 3H, 3⁰-H, ‘OCH₂CH₃), 3.88 (s,

3H, 6⁰-OCH₃), 3.87 (ddd, J ¼ 25.0, 16.3, 5.2 Hz, 3H, 3-H, OCH₂CH₃),

3.83 (s, 3H, 6-OCH₃), 3.58 (s, 3H, 7-OCH₃), 3.36 (ddd, J ¼ 13.2, 9.9,

4.7 Hz, 1H, 3⁰-H), 3.28e3.22 (m, 1H, 3-H), 3.20 (dd, J ¼ 13.8, 4.1 Hz,

39 43

(1R,1⁰S)/(1S,1⁰R) isomers: yield: 130 mg, 0.163 mmol, 33%. mp:

110.0e119.5 ^ꢀC. ¹H NMR, COSY (400 MHz, Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 6.98 (d, J ¼ 1.4 Hz,1H,12-H), 6.80e6.76 (m, 2H,14⁰-H,10⁰-

H), 6.68 (s, 1H, 5⁰-H), 6.62 (s, 1H, 10-H), 6.59e6.54 (m, 2H, 13⁰-H and

11⁰-H), 6.50 (s, 1H, 5-H), 6.11 (s, 1H, 8⁰-H), 5.29 (d, J ¼ 7.0 Hz, 1H, 1-

H), 4.99 (t, J ¼ 6.6 Hz, 1H, 1⁰-H), 4.02 (q, J ¼ 7.2 Hz, 3H, 3⁰-H,

‘OCH₂CH₃), 4.00e3.88 (m, 3H, 3-H, OCH₂CH₃), 3.86 (s, 3H, 6⁰-OCH₃),

3.83 (s, 3H, 6-OCH₃), 3.58 (s, 3H, 7-OCH₃), 3.45 (dd, J ¼ 15.2, 3.9 Hz,

1H, a-H), 2.93e2.83 (m, 3H, a a

-H, 4⁰-H, ⁰-H), 2.82e2.73 (m, 2H, 4-H,

a

⁰-H), 2.63e2.54 (m, 2H, 4-H, 4⁰-H),1.15 (t, J ¼ 7.1 Hz, 3H, ‘OCH₂CH₃),

1H,

a

-H), 3.35e3.20 (m, 2H, 3-H, 3⁰-H), 3.08 (dd, J ¼ 15.1, 8.9 Hz, 1H,

0.99 (br s, 3H, OCH₂CH₃). ¹³C NMR, HSQC, HMBC (101 MHz, Tcl₂,

-H), 2.93e2.81 (m, 2H, 4⁰-H,

a

⁰-H), 2.77 (dd, J ¼ 13.9, 7.0 Hz, 2H, 4-

100 ^ꢀC)

d

[ppm] ¼ 155.5 (C]O), 155.5 (C]O⁰), 154.4 (C-12⁰), 152.7

H,

a

⁰-H), 2.63e2.51 (m, 2H, 4-H, 4⁰-H), 1.16 (t, J ¼ 7.1 Hz, 3H,

(C-6), 148.2 (C-6⁰), 145.6 (C-7⁰), 140.7 (C-7), 139.7 (C-9), 134.7 (C-10),

132.1 (C-12), 130.6 (C-14⁰and C-10⁰),130.4 (C-9⁰), 129.7 (C-14),129.7

(C-13), 129.2 (C-8a⁰), 128.5 (C-4a⁰), 123.1 (C-8a), 121.8 (C-11), 115.3

(C-13⁰and C-11⁰), 114.4 (C-8⁰), 113.3 (C-5⁰), 109.8 (C-5), 61.3

(OCH₂CH₃), 61.2 (‘OCH₂CH₃), 60.7 (7-OCH₃), 56.6 (6-OCH₃and 6⁰-

‘OCH₂CH₃), 1.07 (br s, 3H, OCH₂CH₃). ¹³C NMR, HSQC, HMBC

(101 MHz, Tcl₂,100 ^ꢀC)

d

[ppm] ¼ 155.7 (C]O or C]O⁰),155.5 (C]O

or C]O⁰), 154.3 (C-12⁰), 152.8 (C-6), 148.3 (C-6⁰), 145.7 (C-7⁰), 142.6

(C-9), 140.7 (C-7), 130.6 (C-14⁰and C-10⁰), 130.4 (C-9⁰), 129.2 (C-8a⁰),

128.7 (C-10), 122.6 (C-8a), 121.3 (C-12), 115.3 (C-13⁰and C-11⁰), 114.6

(C-8⁰), 113.4 (C-5⁰), 109.7 (C-5), 108.9 (C-11), 61.4 (OCH₂CH₃), 61.2

(‘OCH₂CH₃), 60.7 (7-OCH₃), 56.5 (6-OCH₃and 6⁰-OCH₃), 56.1 (C-1⁰),

OCH₃), 56.1 (C-1⁰), 52.0 (C-1), 42.2 (C- ⁰), 39.8 (C-

a a), 38.8 (C-3), 38.0

(C-3⁰), 28.3 (C-4⁰), 28.1 (C-4), 14.6 (‘OCH₂CH₃), 14.3 (OCH₂CH₃). The

resonances of C-8 and C-4a could not be identiﬁed. IR (ATR): ṽ

[cm^ꢂ1] ¼ 3355, 2841, 1689, 1668, 1514, 1424, 1331, 1203, 1110, 1023,

52.0 (C-1), 42.2 (C-a a

⁰), 38.9 (C-3), 38.0 (C-3⁰), 34.6 (C- ), 28.3 (C-4⁰),

28.0 (C-4), 14.6 (‘OCH₂CH₃), 14.5 (OCH₂CH₃). The resonances of C-8,

765. Purity (HPLC) ¼ 95% ( ¼ 210 nm). HRMS (ESI): m/z calcd for

l

C-4a and C-4a⁰could not be identiﬁed. IR (ATR): ṽ [cm^ꢂ1] ¼ 2936,

[C

H

Br³⁵ClN₂O₉e H]^-821.1846, found: 821.1852.

79

41 44

R. Schütz et al. / European Journal of Medicinal Chemistry 207 (2020) 112810

13

4.2.12. ( )-8-((1-(4-Hydroxybenzyl)-N-(ethoxycarbonyl)-6-

methoxy-1,2,3,4-tetrahydroisoquinolin-7-yl)oxy)-N-

ethoxycarbonyl-6,7-dimethoxy-1-(4-bromobutyl)-1,2,3,4-

tetrahydroisoquinoline (13e; separable mixture of racemic

diastereomers)

Carbamate 11 (300 mg, 0.493 mmol) and aldehyde 12e (98 mg,

0.591 mmol) were condensed following General Procedure 2. The

reaction was completed after 12 h. Puriﬁcation by ﬂash column

chromatography (15% ethyl acetate in dichloromethane, R_f¼ 0.10

(precursor of RMS10) and 0.16 (precursor of RMS9)) gave the title

compound as a white solid.

13a (50.0 mg, 0.0633 mmol of each diastereomer) were reacted

following General Procedure 3. The reactions were completed after

42 h. Puriﬁcation was accomplished by ﬂash column chromatog-

raphy (30% acetone in hexanes, R_f¼ 0.18) and the products obtained

as beige solids.

(1R,1⁰R)/(1S,1⁰S) isomers: yield: 34.6 mg, 0.0488 mmol, 77%. mp:

83.0e83.5 ^ꢀC. ¹H NMR, COSY (400 MHz, Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 7.39

(d, J ¼ 8.4 Hz, 1H, 10⁰-H or 14⁰-H), 7.14 (t, J ¼ 7.8 Hz, 1H, 13-H), 7.10

(dd, J ¼ 8.1, 2.5 Hz, 1H, 11⁰-H or 13⁰-H), 6.97 (dd, J ¼ 8.1, 2.5 Hz, 1H,

12-H), 6.64 (s, 1H, 5⁰-H), 6.70e6.60 (m, 2H, 11⁰-H or 13⁰-H, 14-H),

6.48 (t, J ¼ 1.9 Hz, 1H, 10-H), 6.33 (s, 1H, 5-H), 6.22e6.16 (m, 1H, 10⁰-

H or 14⁰-H), 6.18 (s, 1H, 8⁰-H), 5.31 (dd, J ¼ 8.1, 3.6 Hz, 1H, 1-H), 5.05

(d, J ¼ 10.4 Hz, 1H, 1⁰-H), 4.36e4.21 (m, 3H, ‘OCH₂CH₃, 3-H), 3.99

(ddd, J ¼ 12.2, 5.7, 3.5 Hz, 1H, 3⁰-H), 3.86e3.76 (m, 2H, OCH₂CH₃),

Precursor of RMS10: yield: 133 mg, 0.178 mmol, 36%. mp:

77.5e78.0 ^ꢀC. ¹H NMR, COSY (400 MHz, Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 6.80e6.75 (m, 2H, 14⁰-H and 10⁰-H), 6.65 (s, 1H, 5⁰-H),

6.62e6.58 (m, 2H, 13⁰-H and 11⁰-H), 6.52 (s, 1H, 5-H), 6.19 (s, 1H, 8⁰-

H), 5.12 (s, 1H, 1-H), 5.02 (t, J ¼ 6.5 Hz, 1H, 1⁰-H), 4.14e3.94 (m, 6H,

3-H, 3⁰-H, ‘OCH₂CH₃, OCH₂CH₃), 3.86 (s, 3H, 6⁰-OCH₃), 3.82 (s, 3H, 6-

OCH₃), 3.58 (s, 3H, 7-OCH₃), 3.27 (t, J ¼ 6.9 Hz, 2H, 4⁰⁰-H), 3.26e3.20

(m, 1H, 3-H), 3.16 (ddd, J ¼ 13.5, 9.7, 4.4 Hz, 1H, 3⁰-H), 2.93e2.84 (m,

3.74 (s, 3H, 6-OCH₃), 3.49 (dd, J ¼ 13.5, 5.0 Hz, 1H,

a

⁰-H), 3.41 (td,

J ¼ 11.7, 4.8 Hz, 2H, 3⁰-H, 3-H), 3.34 (s, 3H, 6⁰-OCH₃), 3.26 (s, 3H, 7-

OCH₃), 3.19e3.08 (m, 1H, 4⁰-H), 2.95e2.80 (m, 2H, 4⁰-H, 4-H),

2.80e2.72 (m, 2H,

a-H), 2.71e2.59 (m, 2H, a

⁰-H, 4-H), 1.36 (t,

J ¼ 6.8 Hz, 3H, ‘OCH₂CH₃), 0.89 (br s, 3H, OCH₂CH₃). ¹³C NMR, HSQC,

1H, 4-H), 2.82 (t, J ¼ 5.5 Hz, 1H,

a

⁰-H), 2.78e2.72 (m, 1H, 4⁰-H), 2.68

HMBC (101 MHz, Tcl₂,100 ^ꢀC)

d

[ppm] ¼ 160.3 (C-11),155.9 (C]O or

(dt, J ¼ 16.5, 4.5 Hz, 1H, 4-H), 2.51 (dt, J ¼ 16.0, 4.6 Hz, 1H, 4⁰-H),

‘C]O), 155.6 (C]O or ‘C]O), 154.2 (C-12⁰), 152.1 (C-6), 149.1 (C-6⁰),

147.0 (C-8), 144.9 (C-7⁰), 142.0 (C-9), 138.9 (C-7), 134.6 (C-9⁰), 132.2

(C-10⁰or C-14⁰), 130.1 (C-10⁰or C-14⁰), 129.1 (C-13), 128.8 (C-8a⁰),

128.2 (C-4a⁰), 122.9 (C-8a), 122.2 (C-14), 122.0 (C-11⁰or C-13⁰), 121.6

(C-11⁰or C-13⁰), 119.7 (C-8⁰), 115.4 (C-10, C-12),114.0 (C-5⁰),107.3 (C-

5), 61.4 (‘OCH₂CH₃), 60.7 (OCH₂CH₃), 60.3 (7-OCH₃), 57.7 (C-1⁰), 57.0

1.86e1.68 (m, 3H, 1⁰⁰-H, 3⁰⁰-H), 1.68e1.56 (m, 1H, 1⁰⁰-H), 1.47e1.38

(m, 2H, 2⁰⁰-H), 1.13 (t, J ¼ 7.0 Hz, 6H, ‘OCH₂CH₃and OCH₂CH₃). ¹³

C

NMR, HSQC, HMBC (101 MHz, Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 155.7 (‘C]O),

155.4 (C]O), 154.5 (C-12⁰), 152.4 (C-6), 148.2 (C-6⁰), 146.3 (C-7⁰),

145.1 (C-8), 140.6 (C-7), 130.4 (C-14⁰and C-10⁰), 130.1 (C-9⁰), 129.6

(C-8a⁰), 129.3 (C-4a), 128.3 (C-4a⁰), 125.1 (C-8a), 115.4 (C-13⁰and C-

11⁰), 114.2 (C-8⁰), 113.6 (C-5⁰), 110.2 (C-5), 61.2 (‘OCH₂CH₃or

OCH₂CH₃), 61.1 (‘OCH₂CH₃or OCH₂CH₃), 60.6 (7-OCH₃), 56.7 (6-

OCH₃or 6⁰-OCH₃), 56.6 (6-OCH₃or 6⁰-OCH₃), 55.8 (C-1⁰), 50.1 (C-

(6⁰-OCH₃), 56.3 (6-OCH₃), 53.5 (C-1), 42.0 (C- ⁰), 3⁰, 41.6 (C-

a a), 36.6

(C-3), 28.0 (C-4), 27.9 (C-4⁰), 14.8 (OCH₂CH₃, ‘OCH₂CH₃). The reso-

nance of C-4a could not be identiﬁed. IR (ATR): ṽ [cm^ꢂ1] ¼ 2925,

2854, 1691, 1586, 1506, 1417, 1332, 1278, 1211, 1107, 1025, 839, 765.

1), 42.1 (C-

a

⁰), 38.6 (C-3⁰), 37.4 (C-3), 33.7 (C-1⁰⁰), 33.6 (C-4⁰⁰), 32.3

Purity (HPLC) ¼ 100% ( ¼ 210 nm). HRMS (ESI): m/z calcd for

l

(C-3⁰⁰), 28.2 (C-4⁰), 27.8 (C-4), 24.9 (C-2⁰⁰), 14.6 (‘OCH₂CH₃), 14.5

(OCH₂CH₃). IR (ATR): ṽ [cm^ꢂ1] ¼ 2928, 2854, 2605, 2498,1692,1513,

[C₄₁H₄₄N₂O₉þ H]^þ709.3120, found: 709.3125.

(1R,1⁰S)/(1S,1⁰R) isomers: yield: 18 mg, 0.0254 mmol, 40%. mp:

1426, 1331, 1234, 1100, 1026, 767. Purity (HPLC) ¼ 95% (

l

¼ 210 nm).

92.0e93.5 ^ꢀC. ¹H NMR, COSY (400 MHz, Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 7.36

79

HRMS (ESI): m/z calcd for [C

H

BrN₂O₉þ H]^þ755.2538, found:

(d, J ¼ 8.2 Hz, 1H, 10⁰-H or 14⁰-H), 7.11 (t, J ¼ 7.8 Hz, 1H, 13-H), 7.05

(d, J ¼ 7.2 Hz, 1H, 11⁰-H or 13⁰-H), 6.94 (dd, J ¼ 8.2, 2.5 Hz, 1H, 12-H),

6.64 (s, 1H, 5⁰-H), 6.66e6.60 (m, 2H, 11⁰-H or 13⁰-H, 14-H),

6.40e6.32 (m, 2H, 10-H, 10⁰-H or 14⁰-H), 6.28 (s, 1H, 5-H), 6.20 (s,

1H, 8⁰-H), 5.29 (d, J ¼ 9.4 Hz,1H,1-H), 5.11 (dd, J ¼ 9.8, 6.5 Hz,1H,1⁰-

H), 4.29e4.15 (m, 3H, ‘OCH₂CH₃, 3-H), 3.97e3.89 (m, 1H, 3⁰-H),

3.88e3.76 (m, 2H, OCH₂CH₃), 3.73 (s, 3H, 6-OCH₃), 3.60 (s, 3H, 6⁰-

38 47

755.2548.

Precursor of RMS9: yield: 85.5 mg, 0.113 mmol, 23%. mp:

200.5 ^ꢀC. ¹H NMR, COSY (400 MHz, Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 6.81e6.75 (m, 2H, 14⁰-H and 10⁰-H), 6.66 (s, 1H, 5⁰-H),

6.60e6.55 (m, 2H, 13⁰-H and 11⁰-H), 6.49 (s, 1H, 5-H), 6.10 (s, 1H, 8⁰-

H), 5.15e5.07 (m, 1H, 1-H), 4.97 (t, J ¼ 6.6 Hz, 1H, 1⁰-H), 4.16e3.97

(m, 6H, 3-H, 3⁰-H, ‘OCH₂CH₃, OCH₂CH₃), 3.85 (s, 3H, 6⁰-OCH₃), 3.81

(s, 3H, 6-OCH₃), 3.56 (s, 3H, 7-OCH₃), 3.33 (t, J ¼ 6.9 Hz, 2H, 4⁰⁰-H),

OCH₃), 3.57e3.51 (m, 1H, a

⁰-H), 3.49e3.41 (m, 1H, 3⁰-H), 3.37e3.27

(m, 1H, 3-H), 3.21e3.14 (m, 2H, a

-H, 4⁰-H), 3.16 (s, 3H, 7-OCH₃),

3.30e3.20 (m, 2H, 3-H, 3⁰-H), 2.96e2.85 (m, 2H, 4-H,

a

⁰-H),

2.88e2.76 (m, 2H, 4⁰-H, 4-H), 2.71 (dd, J ¼ 12.8, 10.5 Hz, 1H,

a

⁰-H),

2.84e2.74 (m, 2H, 4⁰-H,

a

⁰-H), 2.69 (dt, J ¼ 16.2, 4.4 Hz, 1H, 4-H),

2.62 (dd, J ¼ 13.7, 9.6 Hz, 1H,

a-H), 2.53e2.42 (m, 1H, 4-H), 1.33 (t,

2.57 (dt, J ¼ 15.5, 4.4 Hz, 1H, 4⁰-H), 1.91e1.78 (m, 3H, 1⁰⁰-H, 3⁰⁰-H),

1.73e1.61 (m, 1H, 1⁰⁰-H), 1.51e1.40 (m, 2H, 2⁰⁰-H), 1.21e1.12 (m, 6H,

‘OCH₂CH₃and OCH₂CH₃). ¹³C NMR, HSQC, HMBC (101 MHz, Tcl₂,

J ¼ 7.1 Hz, 3H, ‘OCH₂CH₃), 0.96 (br s, 3H, OCH₂CH₃). ¹³C NMR, HSQC,

HMBC (101 MHz, Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 160.4 (C-11), 155.8, 155.4

(‘C]O, C]O), 154.3 (C-12⁰), 152.5 (C-6), 150.1 (C-6⁰), 144.4 (C-7⁰),

142.4 (C-9), 137.9 (C-7), 134.7 (C-9⁰), 131.5 (C-10⁰or C-14⁰), 130.5 (C-

4a⁰), 130.0 (C-10⁰or C-14⁰), 128.9 (C-13), 128.3 (C-8a⁰), 122.4 (C-14),

122.0 (C-11⁰, C-13⁰), 120.6 (C-8a), 119.6 (C-8⁰), 115.8 (C-10), 115.4 (C-

12), 111.8 (C-5⁰), 106.8 (C-5), 61.3 (‘OCH₂CH₃), 60.9 (OCH₂CH₃), 60.5

(7-OCH₃), 57.0 (C-1⁰), 56.3 (6-OCH₃), 56.1 (6⁰-OCH₃), 54.2 (C-1), 41.8

100 ^ꢀC)

d

[ppm] ¼ 155.9 (C]O or C]O⁰), 155.5 (C]O or C]O⁰),

154.4 (C-12⁰),152.4 (C-6),148.4 (C-6⁰),146.1 (C-7⁰),145.3 (C-8),140.5

(C-7), 130.6 (C-14⁰and C-10⁰), 130.4 (C-9⁰), 129.6 (C-8a⁰), 124.7 (C-

8a), 115.3 (C-13⁰and C-11⁰), 114.7 (C-8⁰), 113.5 (C-5⁰), 109.8 (C-5),

61.3 (‘OCH₂CH₃or OCH₂CH₃), 61.2 (‘OCH₂CH₃or OCH₂CH₃), 60.6 (7-

OCH₃), 56.7 (6-OCH₃or 6⁰-OCH₃), 56.5 (6-OCH₃or 6⁰-OCH₃), 56.1

(C-a a

⁰), 41.5 (C-3⁰), 39.6 (C- ), 36.8 (C-3), 28.2 (C-4), 28.1 (C-4⁰), 14.8

(C-1⁰), 50.3 (C-1), 42.2 (C-

a

⁰), 38.9 (C-3⁰), 37.8 (C-3), 33.9 (C-1⁰⁰), 33.6

(‘OCH₂CH₃), 14.2 (OCH₂CH₃). The resonances of C-4a and C-8 could

not be identiﬁed. IR (ATR): ṽ [cm^ꢂ1] ¼ 2924, 1693, 1584, 1507, 1418,

1330, 1276, 1208, 1099, 1022, 837, 769. Purity (HPLC) ¼ 88%

(C-4⁰⁰), 32.5 (C-3⁰⁰), 28.3 (C-4⁰), 28.1 (C-4), 25.0 (C-2⁰⁰), 14.6

(‘OCH₂CH₃and OCH₂CH₃). The resonances of C-4a and C-4a⁰could

not be identiﬁed. IR (ATR): ṽ [cm^ꢂ1] ¼ 2927, 2851, 1690, 1611, 1513,

(

l

¼ 210 nm). HRMS (ESI): m/z calcd for [C₄₁H₄₄N₂O₉þ H]^þ

1427, 1332, 1233, 1100, 1023, 806, 761. Purity (HPLC) ¼ 93%

709.3120, found: 709.3121.

79

(l

¼ 210 nm). HRMS (ESI): m/z calcd for [C₃₈

H

BrN₂O₉þ H]^þ

47

755.2538, found: 755.2558.

4.2.14. ( )-N,N⁰-Bis-(ethoxycarbonyl)-12-desmethoxy-12-

triﬂuoromethoxy-bisnortetrandrine and eisotetrandrine (14b)

Previously separated diastereomers of bisbenzylisoquinoline

14b (60.0 mg, 0.0687 mmol of each diastereomer) were reacted

following General Procedure 3. The reactions were completed after

4.2.13. ( )-N,N⁰-Bis-(ethoxycarbonyl)-12-desmethoxy-

bisnortetrandrine and eisotetrandrine (14a)

Previously separated diastereomers of bisbenzylisoquinoline

14

R. Schütz et al. / European Journal of Medicinal Chemistry 207 (2020) 112810

30 h. Puriﬁcation was accomplished by ﬂash column chromatog-

raphy (25% acetone in hexanes, R_f¼ 0.23) and the products ob-

tained as beige solids.

74.5e76.0 ^ꢀC. ¹H NMR, COSY (400 MHz, Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 7.35

(d, J ¼ 8.2 Hz, 1H, 14⁰-H or 10⁰-H), 7.13 (dd, J ¼ 8.2, 2.5 Hz, 1H, 13⁰-H

or 11⁰-H), 6.70 (d, J ¼ 5.8 Hz, 1H, 13⁰-H or 11⁰-H), 6.60 (s, 1H, 5⁰-H),

6.41 (d, J ¼ 1.7 Hz,1H, 12-H), 6.34 (s, 1H, 5-H), 6.28 (s, 1H, 10-H), 6.19

(dd, J ¼ 8.3, 2.2 Hz, 1H, 14⁰-H or 10⁰-H), 6.01 (s, 1H, 8⁰-H), 5.45 (d,

J ¼ 6.8 Hz, 1H, 1-H), 5.00 (s, 1H, 1⁰-H), 4.30e4.19 (m, 2H, ‘OCH₂CH₃),

4.11e4.02 (m, 1H, 3-H), 4.01e3.88 (m, 3H, 3⁰-H, OCH₂CH₃), 3.73 (s,

(1R,1⁰R)/(1S,1⁰S) isomers: yield: 35.0 mg, 0.0441 mmol, 64%. mp:

77.5e78.5 ^ꢀC. ¹H NMR, COSY (400 MHz, Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 7.41

(d, J ¼ 8.4 Hz, 1H, 10⁰-H or 14⁰-H), 7.15e7.07 (m, 2H, 13-H and 11⁰-H

or 13⁰-H), 6.68e6.64 (m, 1H, 11⁰-H or 13⁰-H), 6.65 (s, 1H, 5⁰-H), 6.62

(dd, J ¼ 8.1, 2.0 Hz, 1H, 14-H), 6.59 (d, J ¼ 1.9 Hz, 1H, 10-H), 6.33 (s,

1H, 5-H), 6.22 (dd, J ¼ 8.3, 2.2 Hz, 1H, 10⁰-H or 14⁰-H), 6.17 (s, 1H, 8⁰-

H), 5.29 (dd, J ¼ 7.9, 2.8 Hz,1H, 1-H), 5.05 (s, 1H,1⁰-H), 4.37e4.27 (m,

1H, 3-H), 4.26 (q, J ¼ 7.2 Hz, 2H, ‘OCH₂CH₃), 4.00 (ddd, J ¼ 12.2, 5.7,

3.5 Hz, 1H, 3⁰-H), 3.87e3.76 (m, 2H, OCH₂CH₃), 3.74 (s, 3H, 6-OCH₃),

3H, 6-OCH₃), 3.50e3.37 (m, 3H, 3-H, 3⁰-H,

OCH₃), 3.22 (s, 3H, 7-OCH₃), 3.15e3.04 (m, 1H, 4⁰-H), 3.00 (dd,

J ¼ 15.2, 2.6 Hz,1H, -H), 2.91e2.75 (m, 3H, 4-H,

-H, 4⁰-H), 2.69 (dt,

⁰-H), 1.35 (t,

a

⁰-H), 3.32 (s, 3H, 6⁰-

a

J ¼ 16.1, 4.9 Hz, 1H, 4-H), 2.63 (d, J ¼ 11.8 Hz, 1H,

a

J ¼ 7.1 Hz, 3H, ‘OCH₂CH₃), 1.06 (br s, 3H, OCH₂CH₃). ¹³C NMR, HSQC,

3.51 (d, J ¼ 8.5 Hz, 1H,

a

⁰-H), 3.41 (td, J ¼ 11.8, 4.9 Hz, 2H, 3⁰-H, 3-H),

HMBC (101 MHz, Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 157.0 (C-12⁰), 152.3 (C-6),

3.34 (s, 3H, 6⁰-OCH₃), 3.26 (s, 3H, 7-OCH₃), 3.15 (td, J ¼ 11.3, 10.8,

149.3 (C-6⁰), 144.9 (C-7⁰), 141.5 (C-9), 139.2 (C-7), 134.4 (C-9⁰), 132.1

(C-14⁰or C-10⁰), 130.3 (C-4a⁰), 129.8 (C-14⁰or C-10⁰), 128.6 (C-8a⁰),

123.5 (C-8a), 121.0 (C-13⁰and C-11⁰), 120.2 (C-8⁰), 118.2 (C-10), 113.7

(C-5⁰), 107.3 (C-5), 101.1 (C-12), 61.4 (‘OCH₂CH₃), 61.1 (OCH₂CH₃),

60.2 (7-OCH₃), 57.7 (C-1⁰), 56.6 (6⁰-OCH₃), 56.3 (6-OCH₃), 54.2 (C-1),

5.6 Hz, 1H, 4⁰-H), 2.94e2.84 (m, 1H, 4-H), 2.84e2.72 (m, 3H, -H, 4⁰-

a

H), 2.73e2.61 (m, 2H,

a

⁰-H, 4-H), 1.36 (t, J ¼ 6.2 Hz, 3H, ‘OCH₂CH₃),

0.87 (br s, 3H, OCH₂CH₃). ¹³C NMR, HSQC, HMBC (101 MHz, Tcl₂,

100 ^ꢀC)

d

[ppm] ¼ 155.9 (‘C]O), 155.5 (C]O), 153.5 (C-12⁰), 152.3

(C-6), 152.2 (C-11), 149.1 (C-6⁰), 147.0 (C-8), 144.8 (C-7⁰), 140.8 (C-9),

138.9 (C-7), 136.6 (q, J ¼ 1.8 Hz, C-12), 135.3 (C-9⁰), 132.3, 130.3 (C-

10⁰, C-14⁰), 130.2 (C-4a⁰), 128.7 (C-8a⁰), 122.5 (C-13), 122.5 (C-8a),

122.0 (C-14), 121.9, 121.3 (C-11⁰, C-13⁰), 119.6 (C-8⁰), 117.4 (C-10),

113.9 (C-5⁰), 107.4 (C-5), 61.4 (‘OCH₂CH₃), 60.8 (OCH₂CH₃), 60.3 (7-

OCH₃), 57.6 (C-1⁰), 56.9 (6⁰-OCH₃), 56.3 (6-OCH₃), 53.4 (C-1), 42.0

41.6 (C-3⁰and C- ⁰), 38.5 (C- ), 38.2 (C-3), 28.0 (C-4), 27.9 (C-4⁰),

a a

14.8 (‘OCH₂CH₃), 14.4 (OCH₂CH₃). The resonances of ‘C]O, C]O, C-

8, C-4a and C-11 could not be identiﬁed. IR (ATR): ṽ [cm^ꢂ1] ¼ 2929,

1693, 1558, 1505, 1416, 1277, 1219, 1101, 1020, 875, 770. Purity

(HPLC)

¼

95%

(

l

¼

210 nm). HRMS (ESI): m/z calcd for

[C₃₉H₄₂N₂O₉S þ H]^þ715.2684, found: 715.2686.

(C-a

⁰, C-3⁰), 41.1 (C-

a

), 36.6 (C-3), 27.9 (C-4), 27.8 (C-4⁰), 14.8

(1R,1⁰S)/(1S,1⁰R) isomers: yield: 15.3 mg, 0.0214 mmol, 17%. mp:

116.0e119.0 ^ꢀC. ¹H NMR, COSY (400 MHz, Tcl₂, 100 ^ꢀC)

(‘OCH₂CH₃), 14.0 (OCH₂CH₃). The resonances of C-4a and OCF₃

could not be identiﬁed. IR (ATR): ṽ [cm^ꢂ1] ¼ 1693, 1505, 1423, 1281,

d

[ppm] ¼ 7.31 (dd, J ¼ 8.2, 2.2 Hz, 1H, 14⁰-H or 10⁰-H), 7.08 (dd,

1248, 1201, 1110, 1024, 842, 767. Purity (HPLC) ¼ 96% (

l

¼ 210 nm).

J ¼ 7.9, 2.2 Hz, 1H, 13⁰-H or 11⁰-H), 6.66 (dd, J ¼ 8.3, 2.6 Hz, 1H, 13⁰-H

or 11⁰-H), 6.62 (s, 1H, 5⁰-H), 6.40 (dd, J ¼ 8.2, 2.2 Hz, 1H, 14⁰-H or 10⁰-

H), 6.31 (d, J ¼ 1.7 Hz, 1H, 10-H), 6.27 (s, 1H, 5-H), 6.17 (s, 1H, 8⁰-H),

5.97 (d, J ¼ 1.7 Hz, 1H, 12-H), 5.32 (d, J ¼ 8.0 Hz, 1H, 1-H), 5.11 (t,

J ¼ 8.7 Hz, 1H, 1⁰-H), 4.29e4.19 (m, 3H, 3-H, ‘OCH₂CH₃), 4.00e3.88

(m, 3H, 3⁰-H, OCH₂CH₃), 3.72 (s, 3H, 6-OCH₃), 3.58 (d, J ¼ 6.6 Hz, 1H,

HRMS (ESI): m/z calcd for [C₄₂H₄₃F₃N₂O₁₀þ H]^þ793.2943, found:

793.2955.

(1R,1⁰S)/(1S,1⁰R) isomers: yield: 15.0 mg, 0.0189 mmol, 28%. mp:

73.5e74.5 ^ꢀC. ¹H NMR, COSY (400 MHz, Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 7.38

(d, J ¼ 8.0 Hz, 1H, 10⁰-H or 14⁰-H), 7.11e7.05 (m, 2H, 11⁰-H or 13⁰-H,

13-H), 6.64 (s, 1H, 5⁰-H), 6.62 (dt, J ¼ 8.3, 2.6 Hz, 2H, 11⁰-H or 13⁰-H,

14-H), 6.47 (s, 1H, 10-H), 6.39 (d, J ¼ 8.4 Hz, 1H, 10⁰-H or 14⁰-H), 6.29

(s, 1H, 5-H), 6.19 (s, 1H, 8⁰-H), 5.26 (d, J ¼ 9.4 Hz, 1H, 1-H), 5.12 (q,

J ¼ 7.5 Hz, 1H, 1⁰-H), 4.29e4.15 (m, 3H, ‘OCH₂CH₃, 3-H), 3.99e3.88

(m, 1H, 3⁰-H), 3.88e3.76 (m, 2H, OCH₂CH₃), 3.73 (s, 3H, 6-OCH₃),

a

⁰-H), 3.55 (s, 3H, 6⁰-OCH₃), 3.40 (td, J ¼ 11.4, 4.9 Hz, 1H, 3⁰-H), 3.32

(d, J ¼ 15.0 Hz, 1H,

a

-H), 3.27e3.13 (m, 2H, 3-H, 4⁰-H), 3.12 (s, 3H, 7-

OCH₃), 2.86e2.70 (m, 3H, 4-H, 4⁰-H,

a

⁰-H), 2.68e2.54 (m, 2H, 4-H,

a

-

H), 1.33 (t, J ¼ 7.0 Hz, 3H, ‘OCH₂CH₃), 1.09 (br s, 3H, OCH₂CH₃). ¹³

C

NMR, HSQC, HMBC (101 MHz, Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 156.8 (C-12⁰),

3.60 (s, 3H, 6⁰-OCH₃), 3.58e3.52 (m, 1H,

a

⁰-H), 3.45 (td, J ¼ 11.5,

155.8 (C]O), 152.6 (C-6), 150.1 (C-6⁰), 144.0 (C-7⁰), 141.8 (C-9), 137.8

(C-7),134.8 (C-9⁰), 131.6 (C-14⁰or C-10⁰), 129.6 (C-14⁰or C-10⁰), 128.3

(C-8a⁰), 122.1 (C-13⁰or C-11⁰), 121.3 (C-13⁰or C-11⁰), 120.3 (C-8a),

119.8 (C-8⁰), 118.0 (C-12), 111.8 (C-5⁰), 106.7 (C-5), 99.6 (C-10), 61.3

(‘OCH₂CH₃), 61.2 (OCH₂CH₃), 60.5 (7-OCH₃), 57.1 (C-1⁰), 56.3 (6-

4.9 Hz, 1H, 3⁰-H), 3.35e3.25 (m, 1H, 3-H), 3.22e3.10 (m, 2H, -H, 4⁰-

a

H), 3.16 (s, 3H, 7-OCH₃), 2.86e2.73 (m, 2H, 4⁰-H, 4-H), 2.72 (dd,

J ¼ 12.8, 10.6 Hz, 1H,

a

⁰-H), 2.61 (dd, J ¼ 13.8, 9.7 Hz, 1H,

a-H), 2.50

(d, J ¼ 16.3 Hz, 1H, 4-H), 1.34 (t, J ¼ 7.1 Hz, 3H, ‘OCH₂CH₃), 0.98 (br s,

3H, OCH₂CH₃). ¹³C NMR, HSQC, HMBC (101 MHz, Tcl₂, 100 ^ꢀC)

OCH₃), 56.0 (6⁰-OCH₃), 55.7 (C-1), 41.9 (C-3⁰), 41.2 (C-

3), 35.6 (C-

a

⁰), 37.2 (C-

a

), 28.1 (C-4), 28.0 (C-4⁰), 14.8 (‘OCH₂CH₃), 14.6

d

[ppm] ¼ 155.8, 155.3 (‘C]O, C]O), 153.8 (C-12⁰), 152.6 (C-6),

152.3 (C-11), 150.1 (C-6⁰), 144.3 (C-7⁰), 141.1 (C-9), 138.0 (C-7), 136.7

(C-12), 135.4 (C-9⁰), 131.7, 130.7 (C-10⁰, C-14⁰), 128.2 (C-8a⁰), 122.2

(C-11⁰or C-13⁰or C-13, C-14),121.7 (C-11⁰or C-13⁰or C-13),120.5 (C-

8a), 119.6 (C-8⁰), 117.9 (C-10), 111.8 (C-5⁰), 106.9 (C-5), 61.4

(‘OCH₂CH₃), 61.0 (OCH₂CH₃), 60.5 (7-OCH₃), 56.9 (C-1⁰), 56.3 (6-

(OCH₂CH₃). The resonances of ‘C]O, C-4a⁰, C-4a, C-8, and C-11

could not be identiﬁed. IR (ATR): ṽ [cm^ꢂ1] ¼ 2927, 2854, 1694, 1557,

1505, 1417, 1219, 1098, 1022, 842, 770. Purity (HPLC) ¼ 90%

(

l

¼ 210 nm). HRMS (ESI): m/z calcd for [C₃₉H₄₂N₂O₉S þ H]^þ

715.2684, found: 715.2688.

OCH₃), 56.0 (6⁰-OCH₃), 54.1 (C-1), 41.8 (C- ⁰, C-3⁰), 39.3 (C-

a a), 36.9

(C-3), 28.2 (C-4), 28.1 (C-4⁰), 14.8 (‘OCH₂CH₃), 14.2 (OCH₂CH₃). IR

(ATR): ṽ [cm^ꢂ1] ¼ 1694, 1506, 1420, 1273, 1248, 1200, 1099, 1021,

4.2.16. ( )-N,N⁰-Bis-(ethoxycarbonyl)-12-desmethoxy-12-chloro-

bisnortetrandrine and eisotetrandrine (14d)

841, 770. Purity (HPLC) ¼ 89% (

l

¼ 210 nm). HRMS (ESI): m/z calcd

Previously separated diastereomers of bisbenzylisoquinoline

13d (110 mg, 0.133 mmol of each diastereomer) were reacted

following General Procedure 3. The reactions were completed after

60 h. Puriﬁcation was accomplished by ﬂash column chromatog-

raphy (25% acetone in hexanes, R_f¼ 0.15) and the products obtained

as a white solid.

for [C₄₂H₄₃F₃N₂O₁₀þ H]^þ793.2943, found: 793.2955.

4.2.15. ( )-N,N⁰-Bis-(ethoxycarbonyl) ring C-thiophene analogues

of bisnortetrandrine and eisotetrandrine (14c)

Previously separated diastereomers of bisbenzylisoquinoline

13c (100 mg, 0.126 mmol of each diastereomer) were reacted

following General Procedure 3. The reactions were completed after

70 h. Puriﬁcation was accomplished by ﬂash column chromatog-

raphy (25% acetone in hexanes, R_f¼ 0.17) and the products obtained

as white solids.

(1R,1⁰R)/(1S,1⁰S) isomers: yield: 48.4 mg, 0.0652 mmol, 49%. mp:

50.0e56.0 ^ꢀC. ¹H NMR, COSY (400 MHz, Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 7.41

(d, J ¼ 8.2 Hz, 1H, 14⁰-H or 10⁰-H), 7.21 (d, J ¼ 7.9 Hz, 1H, 13-H), 7.13

(dd, J ¼ 8.2, 2.6 Hz, 1H, 13⁰-H or 11⁰-H), 6.70e6.64 (m, 1H, 13⁰-H or

11⁰-H), 6.64 (s, 1H, 5⁰-H), 6.58 (dd, J ¼ 8.0, 1.9 Hz, 1H, 14-H), 6.54 (d,

J ¼ 1.8 Hz, 1H, 10-H), 6.32 (s, 1H, 5-H), 6.21 (dd, J ¼ 8.3, 2.2 Hz, 1H,

(1R,1⁰R)/(1S,1⁰S) isomers: yield: 51.3 mg, 0.0718 mmol, 57%. mp:

R. Schütz et al. / European Journal of Medicinal Chemistry 207 (2020) 112810

15

14⁰-H or 10⁰-H), 6.16 (s,1H, 8⁰-H), 5.28 (d, J ¼ 6.1 Hz,1H,1-H), 5.05 (s,

1H, 1⁰-H), 4.35e4.22 (m, 3H, 3-H, ‘OCH₂CH₃), 3.99 (ddd, J ¼ 12.7, 5.7,

3.6 Hz, 1H, 3⁰-H), 3.87e3.76 (m, 2H, OCH₂CH₃), 3.73 (s, 3H, 6-OCH₃),

OCH₂CH₃), 4.18e4.08 (m, 2H, ‘OCH₂CH₃), 4.04e3.96 (m, 2H, 3-H, 4⁰⁰-

H), 3.81 (dt, J ¼ 12.4, 5.5 Hz, 1H, 3⁰-H), 3.70 (s, 3H, 6-OCH₃),

3.62e3.56 (m, 1H, 3⁰-H), 3.55 (s, 3H, 6⁰-OCH₃), 3.37 (dd, J ¼ 12.2,

3.50 (dd, J ¼ 11.9, 5.2 Hz, 1H,

a

⁰-H), 3.45e3.36 (m, 2H, 3-H, 3⁰-H),

5.2 Hz, 1H, a

⁰-H), 3.33e3.24 (m, 1H, 3-H), 3.13 (s, 3H, 7-OCH₃), 2.98

3.35 (s, 3H, 6⁰-OCH₃), 3.25 (s, 3H, 7-OCH₃), 3.14 (ddd, J ¼ 16.5, 11.2,

(ddd, J ¼ 14.9, 9.0, 5.4 Hz, 1H, 4⁰-H), 2.88e2.75 (m, 2H, 4-H, 4⁰₀-H),

5.8 Hz, 1H, 4⁰-H), 2.93e2.77 (m, 2H, 4-H, 4⁰-H), 2.76e2.72 (m, 2H,

a

-

2.64 (dt, J ¼ 16.2, 4.6 Hz, 1H, 4-H), 2.54 (t, J ¼ 11.6 Hz, 1H,

a -H),

H), 2.69e2.61 (m, 2H, 4-H,

a

⁰-H), 1.36 (t, J ¼ 7.0 Hz, 3H, ‘OCH₂CH₃),

1.91e1.80 (m, 1H, 3⁰⁰-H), 1.65e1.52 (m, 4H, 1⁰⁰-H, 2⁰⁰-H, 3⁰⁰-H),

1.49e1.39 (m, 1H, 2⁰⁰-H), 1.32 (t, J ¼ 7.1 Hz, 3H, OCH₂CH₃), 1.25 (t,

J ¼ 7.1 Hz, 3H, ‘OCH₂CH₃). ¹³C NMR, HSQC, HMBC (101 MHz, Tcl₂,

0.88 (br s, 3H, OCH₂CH₃). ¹³C NMR, HSQC, HMBC (101 MHz, Tcl₂,

100 ^ꢀC)

d

[ppm] ¼ 155.9 (C]O or C]O⁰), 155.7 (C-11), 155.5 (C]O

or C]O⁰), 153.8 (C-12⁰), 152.1 (C-6), 149.1 (C-6⁰), 147.0 (C-8), 144.8

(C-7⁰), 140.6 (C-9), 138.9 (C-7), 135.2 (C-9⁰), 132.3 (C-14⁰or C-10⁰),

130.2 (C-14⁰or C-10⁰), 130.2 (C-4a⁰), 129.7 (C-13), 128.7 (C-8a⁰),

128.2 (C-4a), 122.7 (C-14), 122.5 (C-8a), 121.9 (C-13⁰or C-11⁰), 121.4

(C-13⁰or C-11⁰), 120.3 (C-12), 119.6 (C-8⁰), 117.0 (C-10), 113.9 (C-5⁰),

107.4 (C-5), 61.4 (‘OCH₂CH₃), 60.8 (OCH₂CH₃), 60.3 (7-OCH₃), 57.6

(C-1⁰), 56.9 (6⁰-OCH₃), 56.3 (6-OCH₃), 53.4 (C-1), 41.9 (C-3⁰and C-

100 ^ꢀC)

d

[ppm] ¼ 158.0 (C-12⁰), 155.8 (‘C]O), 155.7 (C]O), 152.3

(C-6), 149.6 (C-6⁰), 145.0 (C-7⁰), 138.2 (C-7), 131.7 (C-14⁰or C-10⁰),

129.6 (C-4a⁰), 129.5 (C-14⁰or C-10⁰), 128.1 (C-8a⁰), 123.3 (C-8a),120.1

(C-8⁰), 116.9 (C-13⁰and C-11⁰), 113.2 (C-5⁰), 107.2 (C-5), 68.9 (C-4⁰⁰),

61.3 (OCH₂CH₃), 61.0 (‘OCH₂CH₃), 60.1 (7-OCH₃), 57.4 (C-1⁰), 56.8

(6⁰-OCH₃), 56.2 (6-OCH₃), 50.6 (C-1), 41.6 (C- ⁰), 40.9 (C-3⁰), 37.7 (C-

a

3), 34.1 (C-1⁰⁰), 28.6 (C-3⁰⁰), 28.0 (C-4 and C-4⁰), 22.0 (C-2⁰⁰), 14.8

(OCH₂CH₃), 14.7 (‘OCH₂CH₃). The resonances of C-9⁰, C-4a and C-8

could not be identiﬁed. IR (ATR): ṽ [cm^ꢂ1] ¼ 2924, 2840, 1689, 1509,

1415, 1273, 1207, 1099, 1023, 879, 769. Purity (HPLC) ¼ 99%

a

⁰), 41.1 (C- ), 36.7 (C-3), 27.9 (C-4 and C-4⁰), 14.8 (‘OCH₂CH₃), 14.1

a

(OCH₂CH₃). IR (ATR): ṽ [cm^ꢂ1] ¼ 2927, 2853, 1692, 1505, 1417, 1277,

1205,1105, 1023, 840, 765. Purity (HPLC) ¼ 95% ( ¼ 210 nm). HRMS

l

35

(ESI): m/z calcd for [C

H

ClN₂O₉þ H]^þ743.2730, found: 743.2735.

(

l

¼ 210 nm). HRMS (ESI): m/z calcd for [C₃₈H₄₆N₂O₉þ H]^þ

41 43

(1R,1⁰S)/(1S,1⁰R) isomers: yield: 54.3 mg, 0.0732 mmol, 55%. mp:

113.5e118.0 ^ꢀC. ¹H NMR, COSY (400 MHz, Tcl₂, 100 ^ꢀC)

675.3276, found: 675.3288.

Precursor of RMS9: yield: 21.9 mg, 0.0325 mmol, 29%. mp:

d

[ppm] ¼ 7.39 (d, J ¼ 8.2 Hz, 1H, 14⁰-H or 10⁰-H), 7.18 (d, J ¼ 7.9 Hz,

178.0 ^ꢀC. ¹H NMR, COSY (400 MHz, Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 7.20 (d,

1H,13-H), 7.09 (dd, J ¼ 8.2, 2.5 Hz,1H,13⁰-H or 11⁰-H), 6.65 (s,1H, 5⁰-

H), 6.62 (dd, J ¼ 8.4, 2.2 Hz, 2H, 13⁰-H or 11⁰-H), 6.58 (dd, J ¼ 8.0,

1.8 Hz, 1H, 14-H), 6.43e6.37 (m, 2H, 10-H, 14⁰-H or 10⁰-H), 6.28 (s,

1H, 5-H), 6.18 (s, 1H, 8⁰-H), 5.25 (d, J ¼ 8.2 Hz, 1H, 1-H), 5.11 (dd,

J ¼ 10.4, 6.2 Hz,1H,1⁰-H), 4.31e4.11 (m, 3H, 3, ‘OCH₂CH₃), 3.99e3.88

(m, 1H, 3⁰-H), 3.87e3.77 (m, 2H, OCH₂CH₃), 3.73 (s, 3H, 6-OCH₃),

J ¼ 6.5 Hz,1H,14⁰-H or 10⁰-H), 6.96 (d, J ¼ 6.7 Hz,1H,13⁰-H or 11⁰-H),

6.60 (s, 1H, 5⁰-H), 6.43 (d, J ¼ 5.8 Hz, 1H, 13⁰-H or 11⁰-H), 6.28 (s, 1H,

5-H), 6.23 (d, J ¼ 7.4 Hz, 1H, 14⁰-H or 10⁰-H), 5.90 (s, 1H, 8⁰-H), 5.16

(dd, J ¼ 9.5, 2.0 Hz, 1H, 1-H), 4.98 (dd, J ¼ 10.3, 5.6 Hz, 1H, 1⁰-H),

4.27e4.11 (m, 4H, ‘OCH₂CH₃, OCH₂CH₃), 4.11e4.00 (m, 3H, 3-H, 4⁰⁰-

H), 3.87e3.78 (m, 1H, 3⁰-H), 3.72 (s, 3H, 6-OCH₃), 3.58 (s, 3H, 6⁰-

3.60 (s, 3H, 6⁰-OCH₃), 3.55 (dd, J ¼ 11.8, 5.7 Hz, 1H,

a

⁰-H), 3.46 (td,

OCH₃), 3.55e3.46 (m, 1H, 3⁰-H), 3.43 (dd, J ¼ 12.7, 6.0 Hz, 1H,

a

⁰-H),

J ¼ 11.5, 4.9 Hz, 1H, 3⁰-H), 3.30 (td, J ¼ 13.8, 12.9, 4.5 Hz, 1H, 3-H),

3.30e3.20 (m, 1H, 3-H), 3.15 (s, 3H, 7-OCH₃), 3.06 (ddd, J ¼ 15.5, 9.8,

3.16 (s, 3H, 7-OCH₃), 3.19e3.08 (m, 2H,

a

-H, 4⁰-H), 2.82 (dt, J ¼ 15.7,

5.5 Hz, 1H, 4⁰-H), 2.88e2.74 (m, 2H, 4-H, 4⁰-H), 2.67e2.55 (m, 2H, 4-

4.6 Hz, 1H, 4⁰-H), 2.80e2.74 (m, 1H, 4-H), 2.72 (dd, J ¼ 12.8, 10.6 Hz,

H, a

⁰-H), 1.81e1.70 (m, 2H, 1⁰⁰-H, 3⁰⁰-H), 1.69e1.59 (m, 1H, 1⁰⁰-H),

1H,

a

⁰-H), 2.63e2.54 (m, 1H,

a

-H), 2.47 (d, J ¼ 16.8 Hz, 1H, 4-H), 1.33

1.58e1.49 (m, 1H, 3⁰⁰-H), 1.48e1.38 (m, 1H, 2⁰⁰-H), 1.36e1.24 (m, 7H,

2⁰⁰-H, ‘OCH₂CH₃, OCH₂CH₃). ¹³C NMR, HSQC, HMBC (101 MHz, Tcl₂,

(t, J ¼ 7.1 Hz, 3H, ‘OCH₂CH₃), 0.98 (br s, 3H, OCH₂CH₃). ¹³C NMR,

HSQC, HMBC (101 MHz, Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 155.8 (C-11), 155.4

100 ^ꢀC)

d

[ppm] ¼ 156.4 (C-12⁰), 155.9 (C]O or C]O⁰), 155.7 (C]O

(C]O and C]O⁰), 153.8 (C-12⁰), 152.6 (C-6), 150.1 (C-6⁰), 144.0 (C-

7⁰), 140.8 (C-9), 137.9 (C-7), 135.3 (C-9⁰), 131.6 (C-14⁰or C-10⁰), 130.6

(C-4a⁰), 130.2 (C-14⁰or C-10⁰), 129.6 (C-4a), 129.4 (C-13), 128.3 (C-

8a⁰), 123.0 (C-14), 121.9 (C-13⁰and C-11⁰), 120.2 (C-8a and C-12),

119.6 (C-8⁰), 117.3 (C-10), 111.9 (C-5⁰), 106.9 (C-5), 61.4 (‘OCH₂CH₃),

61.0 (OCH₂CH₃), 60.5 (7-OCH₃), 57.0 (C-1⁰), 56.2 (6-OCH₃), 56.1 (6⁰-

or C]O⁰),152.2 (C-6),149.9 (C-6⁰),144.7 (C-7⁰),138.2 (C-7),130.9 (C-

14⁰or C-10⁰), 130.1 (C-4a⁰), 129.4 (C-14⁰or C-10⁰), 128.6 (C-8a⁰), 121.8

(C-8a), 119.7 (C-8⁰), 112.3 (C-5⁰), 107.1 (C-5), 68.7 (C-4⁰⁰), 61.3

(‘OCH₂CH₃or OCH₂CH₃), 61.2 (‘OCH₂CH₃or OCH₂CH₃), 60.4 (7-

OCH₃), 57.1 (C-1⁰), 56.4 (6⁰-OCH₃), 56.3 (6-OCH₃), 50.3 (C-1), 41.4

(C-a

⁰), 41.3 (C-3⁰), 37.7 (C-3), 33.5 (C-1⁰⁰), 28.3 (C-4), 28.1 (C-4⁰), 27.4

OCH₃), 54.1 (C-1), 41.7 (C-3⁰), 41.5 (C-

a

⁰), 39.2 (C-

a), 37.3 (C-3), 28.2

(C-3⁰⁰), 21.4 (C-2⁰⁰), 14.8 (OCH₂CH₃), 14.8 (‘OCH₂CH₃). The reso-

nances of C-9⁰, C-4a and C-8 could not be identiﬁed. IR (ATR): ṽ

[cm^ꢂ1] ¼ 2927, 2860, 1690, 1508, 1414, 1275, 1203, 1098, 1023, 768.

(C-4), 28.1 (C-4⁰), 14.8 (‘OCH₂CH₃), 14.2 (OCH₂CH₃). The resonance

of C-8 could not be identiﬁed. IR (ATR): ṽ [cm^ꢂ1] ¼ 2936, 2828,

1692, 1506, 1416, 1276, 1204, 1099, 1022, 840, 768. Purity

Purity (HPLC) ¼ 83% (

l

¼ 210 nm). HRMS (ESI): m/z calcd for

(HPLC)

¼

99%

(

l

¼

210 nm). HRMS (ESI): m/z calcd for

[C₃₈H₄₆N₂O₉þ H]^þ675.3276, found: 675.3274.

35

[C H

41 43

ClN₂O₉þ Na]^þ765.2549, found: 765.2550.

4.2.18. ( )-12-Desmethoxytetrandrine and eisotetrandrine (RMS1-

2)

4.2.17. ( )-N,N⁰-Bis-(ethoxycarbonyl) ring C-propylidene analogues

of bisnortetrandrine and eisotetrandrine (14e)

RMS1: (1R,1⁰S)/(1S,1⁰R) isomers of carbamate 14a (17.0 mg,

0.024 mmol) were reduced following General Procedure 4. The

reaction was completed after 12 h. Puriﬁcation by ﬂash column

chromatography (ethyl acetate / 1.0% triethylamine and 7.5%

methanol in ethyl acetate, R_f¼ 0.14) affording the product as a beige

solid (13 mg, 0.0219 mmol, 91%). mp: 196.5e198.0 ^ꢀC. ¹H NMR,

Previously separated diastereomers of bisbenzylisoquinoline

14e (85 mg, 0.112 mmol of each diastereomer), potassium iodide

(3.70 mg, 0.0225 mmol, 0.2 equiv.) and potassium carbonate

(37.2 mg, 0.225 mmol, 2.0 equiv.) were dissolved in 6.0 mL of

anhydrous DMF. The mixture was stirred for 48 h at 105 ^ꢀC. Puri-

ﬁcation was accomplished by ﬂash column chromatography (25%

acetone in hexanes, R_f¼ 0.25) and the products obtained as white

solids.

COSY (500 MHz, CDCl₃):

d

[ppm] ¼ 7.28 (dd, J ¼ 8.3, 2.1 Hz,1H,10⁰-H

or 14⁰-H), 7.18 (t, J ¼ 7.8 Hz, 1H, 13-H), 7.07 (dd, J ¼ 8.1, 2.5 Hz, 1H,

11⁰-H or H-13⁰), 6.96 (dd, J ¼ 8.1, 2.5 Hz,1H,12-H), 6.85 (d, J ¼ 7.4 Hz,

1H,14-H), 6.62 (dd, J ¼ 8.2,1.7 Hz,1H, 11⁰-H or 13⁰-H), 6.53 (s, 1H, 5⁰-

H), 6.45e6.37 (m, 2H,10-H, 10⁰-H or 14⁰-H), 6.28 (s,1H, 5-H), 5.99 (s,

1H, 8⁰-H), 3.92e3.82 (m, 2H, 1-H, 1⁰-H), 3.75 (s, 3H, 6-OCH₃), 3.60 (s,

Precursor of RMS10: yield: 45.3 mg, 0.0672 mmol, 60%. mp:

103.5e104.5 ^ꢀC. ¹H NMR, COSY (400 MHz, Tcl₂, 100 ^ꢀC)

d

[ppm] ¼ 7.24 (br s, 1H, 14⁰-H or 10⁰-H), 6.86 (br s, 1H, 13⁰-H or 11⁰-

H), 6.65 (br s, 1H, 13⁰-H or 11⁰-H), 6.60 (s, 1H, 5⁰-H), 6.28 (s, 1H, 5-H),

6.20 (br s, 1H, 14⁰-H or 10⁰-H), 5.66 (s, 1H, 8⁰-H), 5.26 (d, J ¼ 4.5 Hz,

1H,1-H), 4.93 (dd, J ¼ 10.0, 4.1 Hz,1H,1⁰-H), 4.28e4.18 (m, 3H, 4⁰⁰-H,

3H, 6⁰-OCH₃), 3.45e3.36 (m, 1H, 3⁰-H), 3.33e3.21 (m, 2H,

3.12 (s, 3H, 7-OCH₃), 3.07 (d, J ¼ 13.9 Hz,1H, -H), 2.96e2.88 (m, 2H,

⁰-H, 4⁰-H), 2.86e2.75 (m, 4H, 3-H, 3⁰-H, 4-H, 4⁰-H), 2.68e2.61 (m,

a

⁰-H, 3-H),

a

16

R. Schütz et al. / European Journal of Medicinal Chemistry 207 (2020) 112810

1H,

a

-H), 2.58 (s, 3H, 2⁰-NCH₃), 2.43e2.34 (m, 1H, 4-H), 2.25 (s, 3H,

132.4 (C-10⁰or C-14⁰), 130.5 (C-10⁰or C-14⁰), 127.8 (C-8a⁰), 123.3 (C-

14), 122.5 (C-13), 121.8 (C-11⁰or C-13⁰), 121.5 (C-11⁰or C-13⁰), 120.4

(C-8a), 119.9 (C-8⁰), 117.2 (C-10), 111.3 (C-5⁰), 105.6 (C-5), 63.8 (C-1⁰),

62.2 (C-1), 60.7 (7-OCH₃), 55.9 (6-OCH₃), 55.7 (6⁰-OCH₃), 46.1 (C-3⁰),

2-NCH₃). ¹³C NMR, DEPT, HMQC, HMBC (126 MHz, CDCl₃):

d

[ppm] ¼ 160.4 (C-11), 154.3 (C-12⁰), 152.0 (C-6), 150.0 (C-6⁰), 144.5

(C-9),143.7 (C-7⁰),137.2 (C-7),135.4 (C-9⁰),132.3 (C-10⁰or 14⁰),130.3

(C-10⁰or 14⁰),129.1 (C-13),127.9 (C-8a⁰),123.4 (C-14),122.0 (C-11⁰or

13⁰), 121.5 (C-11⁰or 13⁰), 121.0 (C-8a), 120.0 (C-8⁰), 115.3 (C-10), 115.1

(C-12), 111.3 (C-5⁰), 105.6 (C-5), 63.9 (C-1⁰), 62.1 (C-1), 60.7 (7-

OCH₃), 55.9 (6-OCH₃), 55.7 (6⁰-OCH₃), 46.2 (C-3⁰), 45.4 (C-3), 43.0

45.3 (C-3), 42.9 (2⁰-NCH₃), 42.7 (2-NCH₃), 39.2 (C- ⁰),

a), 37.7 (C-a

25.9 (C-4⁰), 22.8 (C-4). The resonances of C-8, C-4a, C-4a⁰and OCF₃

could not be identiﬁed. IR (ATR): ṽ [cm^ꢂ1] ¼ 2933, 2843, 1593, 1505,

1416, 1198, 1164, 1112, 1008, 838, 729. Purity (HPLC) ¼ 98%, d.r. 95:5

(2⁰-NCH₃), 42.9 (2-NCH₃), 39.4 (C-

a

), 37.9 (C-

a

⁰), 25.9 (C-4⁰), 23.7 (C-

(

l

¼ 210 nm). HRMS (ESI): m/z calcd for [C₃₈H₃₉F₃N₂O₆þ H]^þ

4). The resonances of C-8, C-4a and C-4a⁰could not be identiﬁed. IR

(ATR): ṽ [cm^ꢂ1] ¼ 2923, 2853, 1740, 1606, 1506, 1462, 1255, 1114,

677.2833, found: 677.2838.

RMS 4: (1R,1⁰R)/(1S,1⁰S) isomers of carbamate 14b (32.0 mg,

0.0404 mmol) were reduced following General Procedure 4. The

reaction was completed after 12 h. Puriﬁcation by ﬂash column

chromatography (ethyl acetate / 1.5% triethylamine and 7.5%

methanol in ethyl acetate, R_f¼ 0.11) affording the product as a

white solid (20.2 mg, 0.0298 mmol, 74%). mp: 174.0e176.0 ^ꢀC. ¹H

1018, 841, 698. Purity (HPLC) ¼ 99%, d.r. > 99:1 ( ¼ 210 nm). HRMS

l

(ESI): m/z calcd for [C₃₇H₄₀N₂O₅þ H]^þ593.3010, found: 593.3011.

RMS2: (1R,1⁰R)/(1S,1⁰S) isomers of carbamate 14a (47.0 mg,

0.0663 mmol) were reduced following General Procedure 4. The

reaction was completed after 12 h. Puriﬁcation by ﬂash column

chromatography (ethyl acetate / 1.0% triethylamine and 7.5%

methanol in ethyl acetate, R_f¼ 0.14) affording the product as a

white solid (37 mg, 0.0624 mmol, 94%). mp: 206.0e206.5 ^ꢀC. ¹H

NMR, COSY (400 MHz, CDCl₃):

d

[ppm] ¼ 7.36 (dd, J ¼ 8.2, 2.2 Hz,

1H, 10⁰-H or 14⁰-H), 7.17 (d, J ¼ 8.1 Hz, 1H, 13-H), 7.12 (dd, J ¼ 8.2,

2.6 Hz, 1H, 11⁰-H or 13⁰-H), 6.94 (d, J ¼ 8.2 Hz, 1H, 14-H), 6.76 (dd,

J ¼ 8.3, 2.6 Hz, 1H, 11⁰-H or 13⁰-H), 6.63 (d, J ¼ 1.9 Hz, 1H, 10-H), 6.51

(s, 1H, 5⁰-H), 6.31 (s, 1H, 5-H), 6.32e6.28 (m, 1H, 10⁰-H or 14⁰-H),

5.98 (s, 1H, 8⁰-H), 3.89 (dd, J ¼ 10.9, 5.7 Hz, 1H, 1⁰-H), 3.75 (s, 3H, 6-

OCH₃), 3.72 (s,1H,1-H), 3.55e3.43 (m, 2H, 3-H, 3⁰-H), 3.38 (s, 3H, 6⁰-

NMR, COSY (500 MHz, CDCl₃):

d

[ppm] ¼ 7.35 (dd, J ¼ 8.2, 2.2 Hz,

1H, 10⁰-H or 14⁰-H), 7.23 (t, J ¼ 7.8 Hz, 1H, 13-H), 7.11 (dd, J ¼ 8.1,

2.6 Hz,1H,11⁰-H or 13⁰-H), 6.99 (dd, J ¼ 7.8, 2.3 Hz,1H,12-H), 6.97 (s,

1H, 14-H), 6.77 (dd, J ¼ 8.3, 2.6 Hz, 1H, 11⁰H or 13⁰-H), 6.55 (t,

J ¼ 1.9 Hz, 1H, 10-H), 6.51 (s, 1H, 5⁰-H), 6.31 (s, 1H, 5-H), 6.30e6.28

(m, 1H, 10⁰-H or 14⁰-H), 5.99 (s, 1H, 8⁰-H), 3.92e3.86 (m, 1H, 1⁰-H),

3.78 (d, J ¼ 8.7 Hz, 1H, 1-H), 3.75 (s, 3H, 6-OCH₃), 3.56e3.48 (m, 1H,

3-H), 3.49e3.42 (m, 1H, 3⁰-H), 3.37 (s, 3H, 6⁰-OCH₃), 3.32e3.25 (m,

OCH₃), 3.31e3.24 (m, 1H, a

⁰-H), 3.18 (s, 3H, 7-OCH₃), 3.00e2.85 (m,

4H, 3-H, 3⁰-H, 4-H, 4⁰-H), 2.83 (d, J ¼ 11.7 Hz, 1H,

a

⁰-H), 2.79e2.70

(m, 2H,

a

-H, 4⁰-H), 2.63 (s, 3H, 2⁰-NCH₃), 2.54 (d, J ¼ 13.6 Hz, 1H,

a-

H), 2.41 (dd, J ¼ 11.3, 5.2 Hz, 1H, 4-H), 2.34 (s, 3H, 2-NCH₃). ¹³C NMR,

1H,

H), 2.80 (t, J ¼ 11.7 Hz, 1H,

(s, 3H, 2⁰-NCH₃), 2.59e2.52 (m, 1H,

a

⁰-H), 3.18 (s, 3H, 7-OCH₃), 2.99e2.87 (m, 4H, 3-H, 3⁰-H, 4-H, 4⁰-

⁰-H), 2.77e2.72 (m, 2H, -H, 4⁰-H), 2.63

-H), 2.49e2.38 (m, 1H, 4-H),

DEPT, HMQC, HMBC (126 MHz, CDCl₃):

d

[ppm] ¼ 153.6 (C-12⁰),

a

152.2 (C-11), 151.7 (C-6), 148.8 (C-6⁰), 148.6 (C-8), 143.9 (C-7⁰), 142.6

(C-9), 138.0 (C-7), 136.2 (C-12), 135.7 (C-9⁰), 133.0 (C-10⁰or 14⁰),

130.4 (C-10⁰or 14⁰), 128.3 (C-4a⁰), 128.2 (C-4a), 127.9 (C-8a⁰), 123.2

(C-14), 122.7 (C-8a and 13), 121.8 (C-11⁰or C-13⁰), 121.7 (C-11⁰or C-

13⁰), 121.0 (q, J ¼ 257.2 Hz, OCF₃), 120.3 (C-8⁰), 117.7 (C-10), 112.7 (C-

5⁰), 106.0 (C-5), 64.1 (C-1⁰), 61.3 (C-1), 60.4 (7-OCH₃), 55.9 (6⁰-OCH₃

a

2.34 (s, 3H, 2-NCH₃). ¹³C NMR, DEPT, HMQC, HMBC (126 MHz,

CDCl₃):

d

[ppm] ¼ 160.2 (C-11), 154.1 (C-12⁰), 151.6 (C-6), 148.8 (C-

6⁰), 144.0 (C-7⁰), 143.9 (C-9), 138.0 (C-7), 135.1 (C-9⁰), 132.8 (C-10⁰or

C-14⁰), 130.3 (C-10⁰or 14⁰), 129.2 (C-13), 128.0 (C-8a⁰), 123.3 (C-14),

123.1 (C-8a), 122.0 (C-11⁰or C-13⁰), 121.8 (C-11⁰or C-13⁰), 120.4 (C-

8⁰), 115.6 (C-10), 115.2 (C-12), 112.8 (C-5⁰), 105.9 (C-5), 64.1 (C-1⁰),

61.5 (C-1), 60.4 (7-OCH₃), 56.0 (6⁰-OCH₃or 6-OCH₃), 55.9 (6⁰-OCH₃

and 6-OCH₃), 45.4 (C-3⁰), 44.1 (C-3), 42.7 (2⁰-NCH₃), 42.3 (C-

a), 42.3

(2-NCH₃), 38.3 (C-a ṽ

⁰), 25.3 (C-4⁰), 21.9 (C-4). IR (ATR):

[cm^ꢂ1] ¼ 2926, 2846, 1596, 1504, 1250, 1200, 1163, 1113, 1020, 840.

or 6-OCH₃), 45.4 (C-3⁰), 44.2 (C-3), 42.8 (C-

a

), 42.7 (2⁰-NCH₃), 42.4

Purity (HPLC) ¼ 99%, d.r. 94:6 ( ¼ 210 nm). HRMS (ESI): m/z calcd

l

(2-NCH₃), 38.5 (C-

a

⁰), 25.2 (C-4⁰), 22.2 (C-4). The resonances of C-8,

for [C₃₈H₃₉F₃N₂O₆þ H]^þ677.2833, found: 677.2839.

C-4a and C-4a⁰could not be identiﬁed. IR (ATR): ṽ [cm^ꢂ1] ¼ 2926,

2841, 1586, 1505, 1268, 1211, 1111, 1023, 841, 785. Purity

4.2.20. ( )-Ring C thiophene analogues of tetrandrine and

eisotetrandrine (RMS5-6)

(HPLC) ¼ 99%, d.r. 95:5 ( ¼ 210 nm). HRMS (ESI): m/z calcd for

l

[C₃₇H₄₀N₂O₅þ H]^þ593.3010, found: 593.3011.

RMS5: (1R,1⁰S)/(1S,1⁰R) isomers of carbamate 14c (10.0 mg,

0.014 mmol) were reduced following General Procedure 4. The

reaction was completed after 4 h. Puriﬁcation by ﬂash column

chromatography (ethyl acetate / 2.0% trimethylamine in ethyl

acetate, R_f¼ 0.08) affording the product as a beige solid (5.2 mg,

0.00868 mmol, 62%). mp: 149.0e151.0 ^ꢀC. ¹H NMR, COSY (500 MHz,

4.2.19. ( )-12-Desmethoxy-12-triﬂuoromethoxytetrandrine and

eisotetrandrine (RMS3-4)

RMS3: (1R,1⁰S)/(1S,1⁰R) isomers of carbamate 14b (13.0 mg,

0.0164 mmol) were reduced following General Procedure 4. The

reaction was completed after 12 h. Puriﬁcation by ﬂash column

chromatography (ethyl acetate / 1.5% triethylamine and 7.5%

methanol in ethyl acetate, R_f¼ 0.11) affording the product as a

white solid (9.7 mg, 0.0143 mmol, 87%). mp: 111.0e112.5 ^ꢀC. ¹H

CDCl₃):

d

[ppm] ¼ 7.27e7.24 (m, 1H, 14⁰-H or 10⁰-H), 7.10 (dd, J ¼ 8.1,

2.6 Hz, 1H, 13⁰-H or 11⁰-H), 6.64 (dd, J ¼ 8.3, 2.6 Hz, 1H, 13⁰-H or 11⁰-

H), 6.48 (s, 1H, 5⁰-H), 6.48e6.43 (m, 1H, 14⁰-H or 10⁰-H), 6.35 (d,

J ¼ 1.7 Hz, 1H, 12-H), 6.26 (s, 1H, 5-H), 6.02 (s, 2H, 10-H, 8⁰-H), 3.94

(d, J ¼ 7.6 Hz, 1H, 1-H), 3.91e3.85 (m, 1H, 1⁰-H), 3.74 (s, 3H, 6-OCH₃),

3.52 (s, 3H, 6⁰- OCH₃), 3.50e3.43 (m, 1H, 3⁰-H), 3.28 (dd, J ¼ 12.9,

NMR, COSY (400 MHz, CDCl₃):

d

[ppm] ¼ 7.30 (dd, J ¼ 8.2, 2.1 Hz,1H,

10⁰-H or 14⁰-H), 7.12 (d, J ¼ 8.3 Hz, 1H, 13-H), 7.08 (d, J ¼ 8.5 Hz, 1H,

11⁰-H or 13⁰-H), 6.83 (dd, J ¼ 8.2,1.5 Hz,1H,14-H), 6.64e6.57 (m, 2H,

10-H, 11⁰-H or 13⁰-H), 6.53 (s, 1H, 5⁰-H), 6.45 (br s, 1H, 10⁰-H or 14⁰-

H), 6.27 (s, 1H, 5-H), 5.97 (s, 1H, 8⁰-H), 3.90e3.84 (m, 1H, 1⁰-H), 3.82

(d, J ¼ 9.6 Hz, 1H, 1-H), 3.75 (s, 3H, 6-OCH₃), 3.60 (s, 3H, 6⁰-OCH₃),

7.0 Hz, 1H, a a-H), 3.09 (s, 3H, 7- OCH₃),

⁰-H), 3.17e3.11 (m, 2H, 3-H,

2.96 (t, J ¼ 11.0 Hz, 1H,

a a

⁰-H), 2.93e2.73 (m, 6H, 3-H, 4-H, -H, 4⁰-H,

3⁰-H), 2.56 (s, 3H, 2⁰-NCH₃), 2.49 (d, J ¼ 19.4 Hz,1H, 4-H), 2.37 (s, 3H,

2-NCH₃). ¹³C NMR, DEPT, HMQC, HMBC (126 MHz, CDCl₃):

3.45e3.35 (m, 1H, 3⁰-H), 3.25 (dd, J ¼ 12.9, 6.3 Hz, 2H,

a

⁰-H, 3-H),

d

[ppm] ¼ 157.3 (C-11), 156.6 (C-12⁰), 152.0 (C-6), 149.8 (C-6⁰), 144.5

3.13 (s, 3H, 7-OCH₃), 3.05 (d, J ¼ 13.8 Hz, 1H,

a

-H), 2.96e2.87 (m, 1H,

(C-9), 143.2 (C-7⁰), 136.8 (C-7), 135.4 (C-9⁰), 132.1 (C-14⁰or C-10⁰),

130.1 (C-14⁰or C-10⁰), 129.4 (C-4a), 129.1 (C-4a⁰), 127.5 (C-8a⁰), 121.9

(C-13⁰or C-11⁰), 121.0 (C-13⁰or C-11⁰), 120.7 (C-8a), 120.0 (C-8⁰),

117.6 (C-10), 111.3 (C-5⁰), 105.2 (C-5), 99.7 (C-12), 64.1 (C-1⁰), 63.2

(C-1), 60.7 (7-OCH₃), 55.9 (6-OCH₃), 55.6 (6⁰-OCH₃), 46.2 (C-3⁰),

a

⁰-H, 4⁰-H), 2.87e2.75 (m, 4H, 3⁰-H, 3-H, 4-H, 4⁰-H), 2.63 (dd,

J ¼ 10.2, 3.6 Hz, 1H,

a

-H), 2.58 (s, 3H, 2⁰-NCH₃), 2.42e2.31 (m, 1H, 4-

H), 2.26 (s, 3H, 2-NCH₃). ¹³C NMR, DEPT, HMQC, HMBC (126 MHz,

CDCl₃):

d

[ppm] ¼ 153.7 (C-12⁰), 152.3 (C-11), 152.1 (C-6), 149.9 (C-

6⁰), 143.6 (C-7⁰), 143.4 (C-9), 137.2 (C-7), 136.1 (C-12), 136.0 (C-9⁰),

44.8 (C-3), 43.1 (2⁰-NCH₃), 43.0 (2-NCH₃), 38.1 (C- ⁰), 33.8 (C-

a a),

R. Schütz et al. / European Journal of Medicinal Chemistry 207 (2020) 112810

17

25.5 (C-4⁰), 24.8 (C-4). The resonance of C-8 could not be identiﬁed.

IR (ATR): ṽ [cm^ꢂ1] ¼ 2924, 2853, 1736, 1556, 1507, 1451, 1261, 1106,

11⁰), 120.6 (C-8a), 119.9 (C-8⁰), 119.5 (C-12), 116.5 (C-10), 111.4 (C-5⁰),

105.7 (C-5), 63.7 (C-1⁰), 62.1 (C-1), 60.6 (7-OCH₃), 55.9 (6-OCH₃),

1017, 798. Purity (HPLC) ¼ 98%, d.r. >99:1 (

l

¼ 210 nm). HRMS (ESI):

55.7 (6⁰-OCH₃), 46.1 (C-3⁰), 42.8 (2-NCH₃and 2⁰-NCH₃), 39.1 (C-

a),

m/z calcd for [C₃₅H₃₈N₂O₅S þ H]^þ599.2574, found: 599.2574.

RMS6: (1R,1⁰R)/(1S,1⁰S) isomers of carbamate 14c (20.0 mg,

0.028 mmol) were reduced following General Procedure 4. The

reaction was completed after 2 h. Puriﬁcation by ﬂash column

chromatography (ethyl acetate / 2.0% trimethylamine in ethyl

acetate, R_f¼ 0.08) affording the product as a beige solid (12.9 mg,

0.0215 mmol, 77%). mp: 251.5e252.5 ^ꢀC. ¹H NMR, COSY (400 MHz,

37.9 (C-a

⁰), 25.8 (C-4⁰).The resonances of C-4a, C-8, C-9 and C-9⁰

could not be identiﬁed. IR (ATR): ṽ [cm^ꢂ1] ¼ 2925, 2799, 1579, 1505,

1412, 1262, 1205, 1113, 1021, 817. Purity (HPLC) ¼ 96%, d.r. >99:1

35

(

l

¼ 210 nm). HRMS (ESI): m/z calcd for [C₃₇

H

ClN₂O₅þ H]^þ

39

627.2620, found: 627.2621.

RMS8: The (1R,1⁰R)/(1S,1⁰S) isomers of carbamate 14d (40.0 mg,

0.0538 mmol) were reacted and puriﬁed as described above for

RMS7 to afford the desired product as a white solid (10.0 mg,

0.0159 mmol, 30%). mp: 148.0e150.0 ^ꢀC. ¹H NMR, COSY (400 MHz,

CDCl₃):

d

[ppm] ¼ 7.31 (dd, J ¼ 8.2, 2.2 Hz, 1H, 14⁰-H or 10⁰-H), 7.13

(dd, J ¼ 8.1, 2.6 Hz, 1H, 13⁰-H or 11⁰-H), 6.78 (dd, J ¼ 8.3, 2.6 Hz, 1H,

13⁰-H or 11⁰-H), 6.50 (s, 1H, 5⁰-H), 6.44 (d, J ¼ 1.7 Hz, 1H, 12-H), 6.29

(s, 1H, 5-H), 6.26 (dd, J ¼ 8.3, 2.2 Hz, 1H, 14⁰-H or 10⁰-H), 6.24 (d,

J ¼ 1.7 Hz, 1H, 10-H), 5.90 (s, 1H, 8⁰-H), 3.93 (d, J ¼ 7.2 Hz, 1H, 1-H),

3.79 (dd, J ¼ 11.2, 5.5 Hz, 1H, 1⁰-H), 3.74 (s, 3H, 6-OCH₃), 3.52e3.43

CDCl₃):

d

[ppm] ¼ 7.36 (dd, J ¼ 8.2, 2.2 Hz, 1H, 14⁰-H or 10⁰-H), 7.28

(d, J ¼ 8.0 Hz, 1H, 13-H), 7.14 (dd, J ¼ 8.2, 2.6 Hz, 1H, 13⁰-H or 11⁰-H),

6.90 (dd, J ¼ 8.1, 1.9 Hz, 1H, 14-H), 6.76 (dd, J ¼ 8.3, 2.6 Hz, 1H, 13⁰-H

or 11⁰-H), 6.59 (d, J ¼ 1.8 Hz, 1H, 10-H), 6.51 (s, 1H, 5⁰-H), 6.30 (s, 1H,

5-H), 6.30 (dd, J ¼ 8.6, 2.1 Hz, 2H, 14⁰-H or 10⁰-H), 5.98 (s, 1H, 8⁰-H),

3.88 (dd, J ¼ 10.9, 5.6 Hz, 1H, 1⁰-H), 3.75 (s, 3H, 6-OCH₃), 3.71 (d,

J ¼ 9.9 Hz, 1H, 1-H), 3.51e3.42 (m, 2H, 3-H, 3⁰-H), 3.37 (s, 3H, 6⁰-

(m, 1H, 3⁰-H), 3.36 (s, 3H, 6⁰-OCH₃), 3.31e3.21 (m, 2H, 3-H,

a

⁰-H),

-H, 4⁰-H, 3⁰-H),

a

⁰-H), 2.76e2.67 (m, 1H, 4⁰-H), 2.58 (s, 3H,

3.13 (s, 3H, 7-OCH₃), 3.00e2.82 (m, 5H, 3-H, 4-H,

2.83e2.72 (m, 2H, -H,

a

2⁰-NCH₃), 2.46 (s, 3H, 2-NCH₃), 2.46e2.35 (m, 1H, 4-H). ¹³C NMR,

OCH₃), 3.27 (dd, J ¼ 12.3, 5.7 Hz, 1H,

a

⁰-H), 3.18 (s, 3H, 7-OCH₃), 2.94

DEPT, HMQC, HMBC (101 MHz, CDCl₃):

d

[ppm] ¼ 156.9 (C-12⁰),

(qd, J ¼ 19.6, 17.5, 7.8 Hz, 4H, 3-H, 4-H, 4⁰-H, 3⁰-H), 2.81 (t,

156.8 (C-11), 151.8 (C-6), 148.9 (C-6⁰), 148.9 (C-8), 144.3 (C-7⁰), 143.7

(C-9), 138.2 (C-7), 135.1 (C-9⁰), 132.7 (C-14⁰or C-10⁰), 129.9 (C-14⁰or

C-10⁰), 128.5 (C-4a⁰), 128.0 (C-8a⁰), 123.7 (C-8a), 121.6 (C-13⁰or C-

11⁰), 121.0 (C-13⁰or C-11⁰), 120.7 (C-8⁰), 118.5 (C-10), 112.9 (C-5⁰),

106.0 (C-5), 100.9 (C-12), 64.5 (C-1⁰), 61.8 (C-1), 60.3 (7-OCH₃), 55.9

(6-OCH₃), 55.9 (6⁰-OCH₃), 45.4 (C-3⁰), 44.5 (C-3), 42.8 (2⁰-NCH₃),

J ¼ 11.7 Hz, 1H,

a a

⁰-H), 2.77e2.68 (m, 2H, -H, 4⁰-H), 2.62 (s, 3H, 2⁰-

NCH₃), 2.53 (d, J ¼ 13.6 Hz, 1H,

a-H), 2.44e2.37 (m, 1H, 4-H), 2.32 (s,

3H, 2-NCH₃). ¹³C NMR, DEPT, HMQC, HMBC (101 MHz, CDCl₃):

d

[ppm] ¼ 155.4 (C-11), 153.5 (C-12⁰), 151.5 (C-6), 148.7 (C-6⁰), 143.8

(C-7⁰), 142.3 (C-9), 137.9 (C-7), 135.6 (C-9⁰), 132.8 (C-14⁰or C-10⁰),

130.3 (C-14⁰or C-10⁰),129.5 (C-13),128.2 (C-4a⁰), 127.9 (C-8a⁰),123.9

(C-14), 122.6 (C-8a), 121.7 (C-13⁰and C-11⁰), 120.2 (C-8⁰), 119.6 (C-

12), 117.1 (C-10), 112.7 (C-5⁰), 105.8 (C-5), 63.9 (C-1⁰), 61.3 (C-1), 60.3

(7-OCH₃), 55.8 (6-OCH₃and 6⁰-OCH₃), 45.2 (C-3⁰), 44.0 (C-3), 42.6

42.5 (2-NCH₃), 39.0 (C-a a

⁰), 37.5 (C- ), 24.9 (C-4⁰), 23.2 (C-4). The

resonance of C-4a could not be identiﬁed. IR (ATR):

ṽ

[cm^ꢂ1] ¼ 2925, 2853, 1561, 1505, 1357, 1262, 1205, 1102, 1015, 809,

717. Purity (HPLC) ¼ 97%, d.r. 96:4 (

l

¼ 210 nm). HRMS (ESI): m/z

(2⁰-NCH₃), 42.2 (2-NCH₃), 42.1 (C- ⁰), 25.2 (C-4⁰), 21.8 (C-

a), 38.2 (C-a

calcd for [C₃₅H₃₈N₂O₅S þ H]^þ599.2574, found: 599.2580.

4). The resonances of C-8 and C-4a could not be identiﬁed. IR (ATR):

ṽ [cm^ꢂ1] ¼ 2922, 1747, 1578, 1505, 1413, 1266, 1206, 1111, 1019, 836.

4.2.21. ( )-12-Desmethoxy-12-chlorotetrandrine and

eisotetrandrine (RMS7-8)

Purity (HPLC) ¼ 95%, d.r. >99:1 ( ¼ 210 nm). HRMS (ESI): m/z calcd

l

35

for [C

H

ClN₂O₅þ H]^þ627.2620, found: 627.2622.

37 39

RMS7: A solution of the (1R,1⁰S)/(1S,1⁰R) isomers of carbamate

14d (40.0 mg, 0.0538 mmol) in 1.0 mL anhydrous THF was cooled to

4.2.22. ( )-Ring C propylidene analogues of tetrandrine and

eisotetrandrine (RMS9-10)

0

^ꢀC and methyllithium (1.6 M solution in diethyl ether; 0.510 mL,

0.807 mmol, 15 equiv.) was added slowly. The mixture was stirred

at 0 ^ꢀC for 4 h and was then quenched with 1.0 mL deionized water.

After alkalizing to pH 12e14 with 2.0 M NaOH solution, the mixture

was extracted with ethyl acetate (3 x 20 mL). The combined organic

phases were dried over anhydrous Na₂SO₄and concentrated in

vacuo to afford the secondary amine as a yellow oil. The crude

amine was dissolved in 1.0 mL methanol, a solution of formalde-

RMS9: The ﬁrst fraction of separated diastereomers of carba-

mate 14e (20.0 mg, 0.0297 mmol) was reduced following General

Procedure 4. The reaction was completed after 5 h. Puriﬁcation by

ﬂash column chromatography (ethyl acetate / 3.0% trimethyl-

amine in ethyl acetate, R_f¼ 0.15) afforded the product as a white

solid (14.7 mg, 0.0264 mmol, 89%). mp: 92.0e94.0 ^ꢀC. ¹H NMR,

COSY (500 MHz, CDCl₃):

d

[ppm] ¼ 7.09 (dd, J ¼ 8.3, 2.2 Hz,1H,14⁰-H

hyde (37% in water; 20.0

mL, 0.269 mmol, 5 equiv.), one drop of

or 10⁰-H), 6.96 (dd, J ¼ 8.2, 2.6 Hz,1H, 13⁰-H or 11⁰-H), 6.64 (s, 1H, 5⁰-

H), 6.51 (dd, J ¼ 8.4, 2.8 Hz,1H,13⁰-H or 11⁰-H), 6.33 (s, 1H, 5-H), 6.12

(dd, J ¼ 8.4, 2.2 Hz, 1H, 14⁰-H or 10⁰-H), 5.42 (s, 1H, 8⁰-H), 4.27 (dt,

J ¼ 11.9, 4.5 Hz, 1H, 4⁰⁰-H), 4.03 (ddd, J ¼ 12.3, 9.2, 3.7 Hz, 1H, 4⁰⁰-H),

3.84 (s, 3H, 6⁰-OCH₃), 3.73 (s, 3H, 6-OCH₃), 3.56 (dd, J ¼ 10.6, 5.7 Hz,

1H, 1⁰-H), 3.41e3.33 (m, 1H, 3⁰-H), 3.30e3.21 (m, 2H, 1-H, 3-H), 3.15

acetic acid and sodium cyanoborohydride (10.7 mg, 0.161 mmol, 3

equiv.) was added. The mixture was stirred at ambient temperature

for 12 h and the volatiles removed in vacuo. Puriﬁcation by ﬂash

column chromatography (ethyl acetate / 3.0% trimethylamine in

ethyl acetate, R_f¼ 0.16) afforded the product as a white solid

(9.9 mg, 0.0159 mmol, 30%). mp: 227.5e230.0 ^ꢀC. ¹H NMR, COSY

(s, 3H, 7-OCH₃), 3.14e3.10 (m, 1H, a

⁰-H), 2.92e2.83 (m, 2H, 4-H, 4⁰-

(800 MHz, CDCl₃):

d

[ppm] ¼ 7.30 (d, J ¼ 6.3 Hz, 1H, 14⁰-H or 10⁰-H),

H), 2.83e2.77 (m, 3H, 3-H, 4⁰-H, 3⁰-H), 2.70 (dd, J ¼ 12.7, 10.6 Hz, 1H,

7.25e7.22 (m, 1H, 13-H), 7.09 (d, J ¼ 7.0 Hz, 1H, 13⁰-H or 11⁰-H), 6.79

(d, J ¼ 8.2 Hz,1H,14-H), 6.62 (s,1H,13⁰-H or 11⁰-H), 6.54 (s, 2H,10-H,

5⁰-H), 6.44 (s, 1H, 14⁰-H or 10⁰-H), 6.27 (s, 1H, 5-H), 5.97 (s, 1H, 8⁰-H),

3.87 (s, 1H, 1⁰-H), 3.80 (s, 1H, 1-H), 3.75 (s, 3H, 6-OCH₃), 3.61 (s, 3H,

a

⁰-H), 2.51 (s, 3H, 2⁰-NCH₃), 2.45 (s, 3H, 2-NCH₃), 2.44e2.38 (m, 1H,

4-H), 1.78e1.68 (m, 2H, 3⁰⁰-H, 2⁰⁰-H), 1.66e1.53 (m, 2H, 3⁰⁰-H, 1⁰⁰-H),

1.48e1.36 (m, 2H, 2⁰⁰-H, 1⁰⁰-H). ¹³C NMR, DEPT, HMQC, HMBC

(126 MHz, CDCl₃):

d

[ppm] ¼ 155.4 (C-12⁰), 152.0 (C-6), 150.5 (C-6⁰),

6⁰-OCH₃), 3.40 (s, 1H, 3⁰-H), 3.26 (s, 2H, 3-H,

OCH₃), 3.03 (d, J ¼ 13.9 Hz, 1H,

4-H, 4⁰-H, ⁰-H), 2.88e2.78 (m, 3H, 3-H, 4⁰-H, 3⁰-H), 2.66e2.54 (m,

2H,

-H), 2.58 (s, 3H, 2⁰-NCH₃), 2.44e2.30 (m, 1H, 4-H), 2.24 (s, 3H,

a

⁰-H), 3.13 (s, 3H, 7-

145.5 (C-7⁰), 138.6 (C-7), 131.9 (C-14⁰or C-10⁰), 131.1 (C-9⁰), 129.7 (C-

14⁰or C-10⁰), 129.2 (C-4a), 128.9 (C-4a⁰), 128.3 (C-8a⁰), 123.2 (C-8a),

120.5 (C-8⁰),119.0 (C-13⁰or C-11⁰),115.6 (C-13⁰or C-11⁰),112.0 (C-5⁰),

107.4 (C-5), 68.1 (C-4⁰⁰), 64.5 (C-1⁰), 60.3 (7-OCH₃), 57.9 (C-1), 56.4

(6⁰-OCH₃), 55.9 (6-OCH₃), 46.1 (C-3⁰), 44.1 (C-3), 42.8 (2⁰-NCH₃),

a

-H), 2.92 (dt, J ¼ 16.2, 6.1 Hz, 3H,

a

2-NCH₃). ¹³C NMR, DEPT, HMQC, HMBC (201 MHz, CDCl₃):

d

[ppm] ¼ 155.7 (C-11), 153.8 (C-12⁰), 152.1 (C-6), 150.0 (C-6⁰), 143.7

41.9 (2-NCH₃), 38.2 (C-a

⁰), 33.9 (C-1⁰⁰), 26.8 (C-3⁰⁰), 25.7 (C-4⁰), 22.7

(C-7⁰), 137.1 (C-7), 132.4 (C-14⁰or C-10⁰), 130.5 (C-14⁰or C-10⁰), 129.5

(C-4), 21.4 (C-2⁰⁰). The resonance of C-8 could not be identiﬁed. IR

(ATR): ṽ [cm^ꢂ1] ¼ 2925, 2850, 1607, 1508, 1451, 1266, 1216, 1010,

(C-13), 129.0 (C-4a⁰), 127.6 (C-8a⁰), 124.1 (C-14), 122.1 (C-13⁰and C-

18

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832, 750. Purity (HPLC) ¼ 96%, d.r. >99:1 ( ¼ 210 nm). HRMS (ESI):

l

m/z calcd for [C₃₄H₄₂N₂O₅þ H]^þ559.3166, found: 559.3163.

RMS10: The second fraction of separated diastereomers of

carbamate 14e (18.0 mg, 0.0267 mmol) was reduced following

General Procedure 4. The reaction was completed after 5 h. Puri-

ﬁcation by ﬂash column chromatography (ethyl acetate / 3.0%

trimethylamine in ethyl acetate, R_f¼ 0.15) afforded the product as a

white solid (10.4 mg, 0.0186 mmol, 70%). mp: 153.5 ^ꢀC. ¹H NMR,

COSY (400 MHz, CDCl₃):

d

[ppm] ¼ 7.22 (dd, J ¼ 8.3, 2.3 Hz,1H,14⁰-H

or 10⁰-H), 6.89 (dd, J ¼ 8.3, 2.7 Hz, 1H, 13⁰-H or 11⁰-H), 6.72 (dd,

J ¼ 8.3, 2.7 Hz,1H,13⁰-H or 11⁰-H), 6.48 (s,1H, 5⁰-H), 6.29 (dd, J ¼ 8.3,

2.2 Hz, 1H, 14⁰-H or 10⁰-H), 6.25 (s, 1H, 5-H), 5.53 (s, 1H, 8⁰-H), 4.29

(dt, J ¼ 9.9, 4.6 Hz, 1H, 4⁰⁰-H), 4.06 (ddd, J ¼ 11.7, 9.0, 3.2 Hz, 1H, 4⁰⁰-

H), 3.77e3.73 (m, 1H, 1⁰-H), 3.72 (s, 3H, 6-OCH₃), 3.66 (dd, J ¼ 8.2,

3.6 Hz, 1H, 1-H), 3.51 (s, 3H, 6⁰-OCH₃), 3.40e3.25 (m, 2H, 3-H, 3⁰-H),

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20190275027, Southwest Research Institute, USA, 2019.

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L. Zhang, Z.-S. Chen, D.-H. Yang, Tetrandrine interaction with ABCB1 reverses

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drugs followed by downregulation of ABCB1 expression, Molecules 24

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S. Kim, N.-P. Jung, C.-O. Lee, The bisbenzylisoquinoline alkaloids, tetrandine

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cisplatin in human drug-resistant esophageal squamous carcinoma cells by

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1681e1686.

3.22 (dd, J ¼ 12.6, 5.0 Hz, 1H,

a

⁰-H), 3.05 (s, 3H, 7-OCH₃), 3.00e2.77

⁰-H), 2.59 (s,

(m, 4H, 3-H, 4-H, 4⁰-H, 3⁰-H), 2.71e2.61 (m, 2H, 4⁰-H,

a

3H, 2⁰-NCH₃), 2.44 (s, 3H, 2-NCH₃), 2.38 (dd, J ¼ 16.3, 5.7 Hz, 1H, 4-

H),1.91e1.81 (m,1H, 3⁰⁰-H),1.73e1.56 (m, 3H, 3⁰⁰-H, 2⁰⁰-H),1.55e1.45

(m, 2H, 1⁰⁰-H). ¹³C NMR, DEPT, HMQC, HMBC (101 MHz, CDCl₃):

d

[ppm] ¼ 158.1 (C-12⁰), 151.7 (C-6), 149.1 (C-6⁰), 148.7 (C-8), 143.6

(C-7⁰), 137.3 (C-7), 132.4 (C-14⁰or C-10⁰), 131.4 (C-9⁰), 129.7 (C-14⁰or

C-10⁰), 128.4 (C-4a⁰), 128.3 (C-4a), 127.9 (C-8a⁰), 122.6 (C-8a), 121.0

(C-8⁰), 117.2 (C-13⁰or C-11⁰), 116.0 (C-13⁰or C-11⁰), 111.7 (C-5⁰), 105.8

(C-5), 68.6 (C-4⁰⁰), 64.3 (C-1⁰), 60.2 (7-OCH₃), 58.9 (C-1), 55.9 (6-

OCH₃), 55.8 (6⁰-OCH₃), 45.4 (C-3⁰), 43.9 (C-3), 42.6 (2⁰-NCH₃), 41.9

(2-NCH₃), 38.1 (C-a

⁰), 34.8 (C-1⁰⁰), 29.4 (C-3⁰⁰), 25.8 (C-4⁰), 23.5 (C-

[14] L. Fu, Y. Liang, L. Deng, Y. Ding, L. Chen, Y. Ye, X. Yang, Q. Pan, Characterization

2⁰⁰), 22.3 (C-4). IR (ATR): ṽ [cm^ꢂ1] ¼ 2926, 2854, 1656, 1578, 1507,

1352, 1262, 1203, 1114, 1012, 812. Purity (HPLC) ¼ 98%, d.r. >99:1

of tetrandrine,

a potent inhibitor of P-glycoprotein-mediated multidrug

resistance, Canc. Chemother. Pharmacol. 53 (2004) 349e356.

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kaloids from Stephania tetrandra can reverse multidrug resistance by inhib-

iting P-glycoprotein activity in multidrug resistant human cancer cells,

Phytomedicine 21 (2014) 1110e1119.

(l

¼ 210 nm). HRMS (ESI): m/z calcd for [C₃₄H₄₂N₂O₅þ H]^þ

559.3166, found: 559.3172.

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513e519.

Author contributions

MM and RMS developed the concept. RMS, MM and FG con-

ducted experiments. RMS and MM wrote the paper. AMV, FB, FG

and KB substantially revised the manuscript.

Declaration of competing interest

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

[19] M. Shamma, The Isoquinoline Alkaloids: Chemistry and Pharmacology, ﬁ rst

ed., Acad. Press, New York, 1972.

[20] Tetrandrine Tablets Used in the Treatment of COVID-19 (TT-NPC),

NCT04308317,

2020.

https://clinicaltrials.gov/ct2/show/NCT04308317?

term¼tetrandrine&draw¼2&rank¼1. (Accessed

8

September

2020).

Acknowledgments

[21] J.H. Lee, G.H. Kang, K.C. Kim, K.M. Kim, D.I. Park, B.T. Choi, H.S. Kang, Y.T. Lee,

Y.H. Choi, Tetrandrine-induced cell cycle arrest and apoptosis in A549 human

lung carcinoma cells, Int. J. Oncol. 21 (2002) 1239e1244.

[22] S.-M. Yoo, S.-H. Oh, S.-J. Lee, B.-W. Lee, W.-G. Ko, C.-K. Moon, B.-H. Lee, In-

hibition of proliferation and induction of apoptosis by tetrandrine in HepG2

cells, J. Ethnopharmacol. 81 (2002) 225e229.

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A. Watermann, M. Ulrich, D. Mayr, C. Wahl-Schott, M. Biel, A.M. Vollmar, Two-

pore channel function is crucial for the migration of invasive cancer cells,

Canc. Res. 77 (2017) 1427e1438.

This work was funded by the German Research Foundation (DFG

VO 376/19-1 and BR 1034/7-1). We thank Anna Niedrig for per-

forming the HPLC measurements as well as Bernadette Grohs and

€

Julian Fradrich for technical assistance to conduct cell-based

experiments.

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205 (2005) 275e292.

Appendix A. Supplementary data

[25] Y. Tian, J. Zheng, Metabolic activation and toxicities of bis-benzylisoquinoline

alkaloids, in: J.C. Fishbein, J.M. Heilman (Eds.), Advances in Molecular Toxi-

cology, Academic Press, Cambridge, San Diego, Oxford, London, 2017,

pp. 241e272.

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2020.112810.

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bolic activation of tetrandrine in CD-1 mice, Chem. Res. Toxicol. 24 (2011)

2142e2152.

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