Anticancer Effects of Honokiol via Mitochondrial Dysfunction Are Strongly Enhanced by the Mitochondria-Targeting Carrier Berberine

Article abstract of DOI:10.1021/acs.jmedchem.0c00881

Mitochondrion is a favorable therapeutic target in cancer, given its regulation of bioenergetics and cell death. Honokiol exhibits antiproliferative effects through mitochondria-mediated death signaling. To enhance its anticancer potential and selectivity, we conjugated honokiol to berberine, a mitochondria-targeting carrier. All designed derivatives displayed 1 order of magnitude increased cytotoxicity compared with the parent compounds, especially with massive cytoplasmic vacuoles. Biological evaluation demonstrated the representative compound 6b localized within the mitochondria, and mitochondrial dilation resulted in vacuolization. 6b induced vacuolation-associated cell death and apoptosis with obvious mitochondrial dysfunction, as demonstrated by booming reactive oxygen species generation, opening mitochondrial permeability transition pore, and reducing mitochondrial membrane potential. The targeting property also conferred 6b with selectivity for tumor cells compared to normal cells. 6b inhibited cancer cell proliferation in the zebrafish xenograft model. These results demonstrate that berberine-linked honokiol derivatives open up a direction for novel mitochondrial-targeting antitumor agents.

Full text of DOI:10.1021/acs.jmedchem.0c00881

Subscriber access provided by Stony Brook University | University Libraries
Article
Anti-cancer effects of honokiol via mitochondrial dysfunction are
strongly enhanced by the mitochondria-targeting carrier berberine
xiaojia Shi, Tao Zhang, Hongxiang Lou, Huina Song, Changhao Li, and Peihong Fan
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00881 • Publication Date (Web): 30 Sep 2020
Downloaded from pubs.acs.org on September 30, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Anti-cancer effects of honokiol via mitochondrial
dysfunction are strongly enhanced by the
mitochondria-targeting carrier berberine
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
†
‡
†
†
†
*,†
Xiaojia Shi , Tao Zhang , Hongxiang Lou , Huina Song , Changhao Li , Peihong Fan
†Department of Natural Product Chemistry, Key Lab of Chemical Biology of Ministry of
Education, School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong
University, Jinan 250012, P. R. China.
‡Shandong Provincial Key Laboratory of Neuroprotective Drugs, Zibo 255400, China.
ABSTRACT. Mitochondrion is a favorable therapeutic target in cancer given its regulation of
bioenergetics and cell death. Honokiol exhibits antiproliferative effects through mitochondria-
mediated death signaling. To enhance its anticancer potential and selectivity, we conjugated
honokiol to berberine, a mitochondria-targeting carrier. All designed derivatives displayed one
order of magnitude increased cytotoxicity compared with the parent compounds, especially with
massive cytoplasmic vacuoles. Biological evaluation demonstrated the representative compound
6b localized within the mitochondria and mitochondrial dilation resulted in vacuolization. 6b
induced vacuolation-associated cell death and apoptosis with obvious mitochondrial dysfunction
ACS Paragon Plus Environment
1

Journal of Medicinal Chemistry
Page 2 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
as demonstrated by booming reactive oxygen species generation, opening mitochondrial
permeability transition pore and reducing mitochondrial membrane potential. The targeting
property also conferred 6b selectivity for tumor cells compared to normal cells. 6b inhibited cancer
cell proliferation in zebrafish xenograft model. These results demonstrate that berberine-linked
honokiol derivatives open up a direction for novel mitochondrial targeting antitumor agents.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
KEYWORDS. Honokiol, Berberine, Mitochondria-targeting derivatives, Vacuoles,
Mitochondrial dysfunction.
INTRODUCTION
Cancer cells have developed various biological capabilities to adapt to extraordinary
microenvironments, sustain survival and evade cell death. Among these hallmarks of cancer,
resisting cell death remains a great challenge to chemotherapy by reducing the accumulation of
1
-2
cellular drugs and escaping apoptotic execution . Mitochondria are pivotal regulators in the
processes of programmed cell death, including apoptosis, mitochondrial permeability transition
2-4
(
MPT)-driven necrosis, and paraptosis etc . Direct targeting of therapeutic agents to mitochondria
improves antiproliferative effects and cell selectivity and decreases cancer invasion in vitro and
5
-7
vivo .
Honokiol (HN), a biphenyl component extracted from Magnolia officinalis, exhibits multiple
antineoplastic activities by regulating mitochondria-mediated death signaling. HN treatment (from
0 to 60 μM) inhibits the growth and proliferation of cancer cells via caspase-dependent and -
2
independent apoptosis8-10, paraptosis with swelling of endoplasmic reticulum and/or
mitochondria11 or cyclophilin D-dependent mitochondrial permeability transition pore-driven
3
necrosis . To improve the anticancer effect and cell selectivity of honokiol, we attempted to design
ACS Paragon Plus Environment
2

Page 3 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
mitochondria-targeted honokiol derivatives. Currently, the most common mitochondria-targeting
delivery system is delocalized lipophilic cations (DLCs), such as triphenylphosphonium,
rhodamine and pyridinium, which selectively accumulate in mitochondria of cancer cells based on
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
the increased mitochondrial membrane potential (Δψ ) of cancer cells compared with normal
m
1
2
cells . The natural isoquinoline alkaloid berberine (BBR) also serves as a DLC and exhibits
several advantages, such as antiproliferative effects, safety and fluorescent properties13-17. In this
work, we synthesized ten mitochondria-targeted derivatives by conjugating honokiol to a berberine
moiety and studied the anti-cancer activity and mechanisms of action using compound 6b as a
representative agent.
RESULTS
Design and synthesis. Ten berberine-linked honokiol derivatives were designed and synthesized
according to the Scheme 1. Briefly, the key intermediate compound 3 was synthesized from
condensation of BBR (1) with acetone, followed by reaction with various iodides to give
compounds 4a ~ 4c, which are berberine analogues bearing linkers of different length in the C-13
1
8
position . Etherification of hydroxyl groups of HN (2a) with iodoalkanes yielded compounds 2b
2e. Finally, in the present of excessive K CO , reactions of compounds 4a/4b/4c with 2a in
~
2
3
refluxing MeCN yielded 5a ~ 5c and 6a ~ 6c after isolation using column chromatography. The
condensation of compound 4b with 2b ~ 2e produced 5d, 6d, 5e and 6e. The structure of these
1
13
novel derivatives were confirmed via H-NMR, C-NMR and HR-MS with the aid of HSQC and
HMBC. The purity of key compounds for biological evaluation was established as ≥95% by high
performance liquid chromatography (HPLC).
Scheme 1. Synthesis of target derivatives Series 5 and 6a.
ACS Paragon Plus Environment
3

Journal of Medicinal Chemistry
Page 4 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Reagents and conditions: (a) 5 N NaOH, acetone, rt, stir 30 min. (b) MeCN, N , various iodides,
2
avoid light, reflux 5 ~ 8 h. (c) K CO , DMF, various iodides, rt, stir overnight. (d) K CO , MeCN,
2
3
2
3
reflux 2 ~ 6 h.
In vitro biological activity. MTT assays were employed to test the cytotoxicity of target
derivatives in six human cancer cell lines and one normal hepatocyte cell line HL7702.
Doxorubicin was selected as the positive control drug. The biological activity results are listed in
Table 1 and graphs of MTT data with concentration range against all the cell lines are shown in
Figure S1. The antiproliferative activity of compounds 5a ~ 5e and 6a ~ 6e increased by one order
of magnitude in the tested cancer cell lines compared with HN or BBR (Table 1). Structure-activity
analysis suggested that the length of the linker between the moiety of BBR and HN influences
cytotoxicity. In general, a longer linker exhibits a lower IC value. For example, as the length of
5
0
the linker increased from 3C to 8C, the IC value of 5a ~ 5c against A2780 ranged from 3.03 to
50
1.97 μ M. To study the potency of bare hydroxyls in HN, we synthesized the etherification
products of 5b and 6b bearing methyl and propyl groups, i.e., compound 5d and 5e as well as 6d
ACS Paragon Plus Environment
4

Page 5 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
and 6e, respectively. These ethers exhibited slightly increased cytotoxic effects compared with 5b
and 6b, and the type of and position of alkyl group had no obvious effect on antiproliferative
activity. 50% inhibitory concentration of intermediate 2b ~ 2e against cancer cells decreased
slightly compared with parent compound HN, but their cytotoxic effects are still much inferior to
series 5 and 6 and their cell selectivity are not obvious.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 1. The antiproliferative activity of tested derivatives against various cell linesa.
IC50 (μM)
Compound
A2780
> 50
A549
> 50
HepG2
> 50
HT29
> 50
PANC1
> 50
PC3
HL7702
> 50
BBR
HN
5a
31.72±0.51
31.58±4.20 38.04±0.08
23.85±2.06
31.58±4.20 31.24±4.10
29.03±6.98 25.49±1.87
3.03±0.23b
3.71±0.68b
2.26±0.54b
5.21±0.89b
> 5
> 5
> 5
1.59±0.18b
2.90±0.24b
2.32±0.17b
3.24±0.37b
1.59±0.04b
3.04±1.05b
5
b
> 5
1.97±0.61b
2.09±0.08b
1.27±0.22b
2.54±1.07b
2.48±0.39b
2.00±0.09b
5.77±0.27b
5
c
1.36±0.48b
1.68±0.35b
1.56±0.33b
2.22±0.76b
1.71±0.03b 1.04±0.23b,c, 1.56±0.63b
2.96±0.48b
2.56±0.62b
6.02±1.24b
3.34±0.62b
1.12±0.35b
2.97±0.30b
5
d
1.66±0.23b
3.25±1.04b
3.00±0.93b
0.92±0.12b,c 2.39±1.40b
1.43±0.47b 3.62±0.02b
5
e
2.19±0.16b
1.63±0.33b
1.17±0.23b
2.25±0.03b
3.56±0.42b
4.61±0.07b
6
a
> 5
> 5
6b
6c
1.52±0.41b
1.84±0.83b
0.77±0.04b
2.89±1.08b
2.09±0.62b
1.74±0.50b
4.82±0.02b
1.17±0.03b
1.33±0.37b
1.05±0.15b,d
1.64±0.80b
1.73±0.33b,d
0.99±0.50b
3.43±0.34b
6
d
1.41±0.23b 1.25±0.11b,d 0.95±0.17b,e 1.95±0.64b 1.69±0.10b,e 1.22±0.23b
3.05±0.18b
6
e
2
b
14.67±2.56 21.97±2.03
24.66±3.90 > 30
17.47±3.58
29.65±0.66
19.11±2.90
15.48±2.45
21.07±0.51 16.73±0.08
35.37±4.86 16.42±0.09
26.47±0.52 17.79±0.52
26.41±0.50 15.90±0.02
17.87±0.03 23.94±2.10
24.84±0.66 35.27±2.37
21.08±0.55 27.83±3.08
17.01±0.78 22.35±0.83
2
c
2
d
14.73±0.08 20.97±0.56
14.69±0.64 14.72±0.09
2
e
ACS Paragon Plus Environment
5

Journal of Medicinal Chemistry
Page 6 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
doxorubicinf
0.71±0.08
3.08±0.15
0.67±0.24
5.92±1.57
1.73±0.25
0.93±0.01
2.24±0.39
aThe values were presented as the mean±standard deviations (SD), and cell viability was assessed
b
c
d
e
after incubation for 48 h. P < 0.0001 vs. HN. P < 0.0001 vs. 5b. P < 0.05 vs. 6b. P < 0.01 vs.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
f
6
b. Doxorubicin was used as a positive control.
Morphologically, we found a quick and impressive accumulation of cytoplasmic vacuoles in
series 5/6 treated cells as assessed by optical microscopy (Figure S2, S3), especially compound
6b. It is an interesting and extraordinary event in cell death. We need to explore the vacuolization
deeply. The parent agent BBR had no effect on vacuolization at the tested dose (40 μM), and
greater than 30 μM HN induced obvious masssive vacuoles (Figure S4), which indicated the HN
moiety was important for vacuolization.
For subsequent investigation, a representative compound and cell line need to be selected.
Among all cancer cell lines tested, the hepatoma cell line HepG2 was relatively more sensitive to
these compounds regarding cytotoxicity or vacuolization (Figure S3). In terms of compound,
since any variation in chemical structure might alter the mechanism of action, we tended to choose
a derivative bearing similar structure with parent agents, so that their mechanism could be
compared. Compound 6b was chosen as a representative compound given its structural similarity,
higher vacuole inducing potency, relatively good cytotoxicity and cell selectivity. It possesses a
moderate-length linker and a bare hydroxyl structurally, suppressed HepG2 cell viability with an
IC50,48h of 1.63 μM, 14.6-fold reduced compared with HN, and initiated striking cytoplasmic
vacuoles in a time- and dose-dependent manner (Figure 1A, 1B). Moreover, 6b exhibited some
preferable cell selectivity between HepG2 cells and the normal cell line HL7702, the IC ratio in
5
0
HL7702 to HepG2 cells is 2.8, while there is no selectivity for HN (Table 1). Thus, the subsequent
experiments were performed using 6b in HepG2 cells.
ACS Paragon Plus Environment
6

Page 7 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. Effects of 6b on cell viability and vacuolation. (A) Cells were treated with increasing
doses of 6b for indicated time. The values were determined using MTT assays. (B) Vacuolization
induced by 6b. Cells were treated with 4 μ M 6b for indicated time (upper images) or with
increasing concentrations for 10 h (lower images), and morphological changes were imaged by
optical microscopy. Scar bar 10 μm.
To study the importance of the whole skeleton of BBR-linked HN derivatives on their
biological performance and the contribution of the synergy effects of HN and BBR moiety, we
further synthesized some structural fragments of 6b such as compound 2f and 4d (Figure 2A，
synthetic method see supporting Scheme S1 ), and then designed some drug combinations of HN
moiety with BBR moiety to detect their antiproliferative effects and compare with 6b. The degree
of drug interaction in terms of synergism and antagonism were analyzed by CompuSyn software.
In all cases where combination index (CI) can be determined, following diagnostic rule apply: CI
<
1 indicates synergism, CI=1 indicates additive effect and CI > 1 indicates antagonism. Fa-CI
point, a plot of CI on y-axis as a function of effect level (Fa) on the x-axis, exhibited the drug
1
9
interaction visually . As shown in Figure 2B, for combination effect, all data points yielded from
combination of BBR with HN are above the CI=1 horizontal line (Table S1), indicating an
ACS Paragon Plus Environment
7

Journal of Medicinal Chemistry
Page 8 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
antagonistic effect. Other drug combinations, 2f with BBR, 4d with HN, produced partially CI <
1
(values of 0.79 to 0.98), indicating weak synergism at different doses case (Table S2, S3). The
combination of 2f with 4d yielded CI value of 0.269 to 0.921 revealed a moderate synergistic effect
Table S4). But the cytotoxicity in HepG2 of all drug combinations is lower than 6b at the same
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
dose (Figure 2C). These results suggested that the entire molecular skeleton of 6b is essential to
its cytotoxicity. For morphological changes, the effect on vacuolization of intermediate 2f is
weaker than that of its parent compound HN, while BBR derivative 4d exhibited some ability to
induce vacuoles at 40 μM as well (Figure S4). In drug combinations, more obvious vacuolation
were observed than that in single compound group (Figure S5), which displayed the synergism
relationship between BBR and HN scaffold in terms of morphological manifestation.
ACS Paragon Plus Environment
8

Page 9 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. The degree of drug combinations in HepG2. (A) Chemical structures and IC against
5
0
HepG2 of 2f, 4d and 6b. (B) Fa-CI point yielded from various drug combinations. The CI and Fa
values were calculated by CompuSyn software. (C) Growing inhibition of designed drug
combinations and 6b in HepG2 cells. Cells were treated with indicated drugs for 48 h and viability
were tested by MTT assays.
6
b mediates cytoplasmic vacuolation via dilation of mitochondria. Upon treatment with
different doses of 6b for indicated time, decreasing cell viability and increasing remarkable
cytoplasmic vacuoles are completely synchronized (Figure 1), suggesting that formation and
development of vacuoles were pivotal for 6b-induced cytotoxicity. Thus, it is important and
ACS Paragon Plus Environment
9

Journal of Medicinal Chemistry
Page 10 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
necessary to explore the formation of cytoplasmic vacuolation. The mitochondria-specific
fluorescent probe mito-tracker red CMXRos was used to study whether cytoplasmic vacuoles
originated from mitochondria. As shown in Figure 3A, mitochondria labeled with red fluorescence
in untreated cells displayed a compact, rod-like and reticulate shape, whereas appeared swollen
and turgid in 6b-treated cells. The merged red fluorescence and phase-contrast images
demonstrated that mito-tracker was incorporated into the small vacuoles, suggesting these
vacuoles originated from swelling of mitochondria. Dilated mitochondria after treatment with 6b
were observed by transmission electron microscopy as well (Figure 3B). These findings indicate
that mitochondria was demolished by 6b and it played a crucial role in vacuolation-associated cell
death.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Cytoplasmic vacuoles originated from dilation of mitochondria. (A) Cells were stained
with 150 nM mito-tracker probe for 15 min after treatment with 3 μM 6b for 12 h. Fluorescent
ACS Paragon Plus Environment
1
0

Page 11 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
and phase-contrast images were obtained by laser confocal microscopy. Scar bar, 10 μm. (B)
Transmission electron microscopy images of microstructural changes of mitochondria in HepG2
cells incubated with or without 3 μM 6b. Black arrowheads point mitochondria. Scar bar in the
original and enlarged images indicates 1.2 μm and 0.6 μm respectivily.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cellular uptake and localization of 6b. To confirm that the increasing cytotoxicity was related
to mitochondrial targeting, intracellular drug content and subcellular localization were measured
by HPLC assays and confocal microscopy, respectively. The total cellular uptake of 6b was 2.5-
fold increased compared with honokiol after treatment with 5 μM drugs for 12 h in HepG2 cells
(Figure 4A). The concentration of 6b and HN in isolated mitochondria was 66.3 μM and 17.8 μM
respectively, revealing that mitochondrial accumulation of 6b was 3.7-fold increased compared
with HN (Figure 4A). Since 6b can emit blue fluorescence according to its emission spectrum
(Figure S6), we employed laser confocal microscopy to examine its potential subcellular
distribution. As shown in Figure 4B, we observed clusters of blue fluorescence in the 6b-treated
group but not the blank control. Here, 6b (blue) colocalized with mitochondria (red) stained with
the mito-tracker probe. These data indicate that BBR facilitated HN entry into cells and locate in
mitochondria.
ACS Paragon Plus Environment
1
1

Journal of Medicinal Chemistry
Page 12 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Cellular intake and subcellular localization of 6b. (A) Standard curve of HN and HPLC
chromatograms of extracted HN from HepG2 cells (a). Standard curve of 6b and HPLC
ACS Paragon Plus Environment
1
2

Page 13 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
chromatograms of extracted 6b from HepG2 cells (b). Histogram of mitochondrial, non-
mitochondrial and total cellular accumulation of HN and 6b *P < 0.5 , *** P < 0.001(c). (B)
Colocalization of 6b with mito-tracker in HepG2 cells was imaged by confocal microscopy. Scar
bar, 10 μm.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
b induces apoptosis and vacuolation-associated cell death. Based on the mitochondrial
targeting feature of 6b and swelling of mitochondria in treated cells, we hypothesized that this
compound may induce mitochondria-driven cell death. Flow cytometry detected the number of
apoptotic cells by annexin V/PI double staining. HepG2 cells treated for 24 h revealed that 6b
induced significantly apoptosis in a dose-dependent manner and 1 μM, 2 μM and 4 μM 6b
induced apoptosis in 9.0%, 8.9% and 42.9% of cells, respectively (Figure 5A). Western blot
indicated cleaved caspase 3, 8 and 9 were upregulated, together with decreasing product of pro-
caspase 12 and increasing expression of cleaved poly (ADP-ribose) polymerase (PARP) (Figure
5B). Next, we detected the cellular localization of apoptosis-inducing factor (AIF), a protein that
20
induces apoptosis in the presence of irreversible caspase inhibitor z-VAD-fmk . The result
obtained via immunofluorescence staining showed that AIF was not translocated to the nucleus
but distributed widely in cytoplasm in the presence of 6b (Figure 5C). These demonstrate caspase-
dependent extrinsic and intrinsic apoptosis invovled in 6b-induced cell death. Pretreatment with
the pan-caspase inhibitor z-VAD-fmk slightly affected apoptosis and cell death initiated by 4 μM
6
b (Figure 5D (a, b)) , but had no obvious impact on the size and number of vacuoles (Figure 5D
(c)), indicating that alternative non-apoptotic programs may exist. Then, we examined whether
necroptosis or autophagy is involved in the process of vacuolization and cell proliferation
2
1
inhibition. Necrostatin-1 (Nec-1), a small molecule inhibitor of necroptosis , partially prevented
cell death in approximately 12 h, but no significant difference was noted between the 6b and 6b
ACS Paragon Plus Environment
1
3

Journal of Medicinal Chemistry
Page 14 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
plus Nec-1 group, and couldn’t rescue cell after treatment for 12 h. Morphologically Nec-1 didn’t
block 6b-induced vacuolization (Figure 6A). The autophagy inhibitors chloroquine (CQ) and 3-
methyladenine (3-MA) couldn't averted vacuolization or cell death as well (Figure 6B), and the
expression of LC3 II and P62, the key indicator protein of autophagy, shown no significant changes
in 6b-treated cells compared with control. Invalid response of Nec-1, CQ and 3-MA on viability
and vacuolization, with undifferentiated expression of autophagy marker, highlight necroptosis
and autophagy are not indispensable in 6b-induced antineoplastic process. These results suggest
that mitochondrial caspase-dependent apoptosis is involved in cell death but not vacuolization,
whereas the necroptosis and autophagy pathways have no effect on either. Therefore, we focused
more attention on mitochondrial dysfunction to assess the crucial role of 6b in cell death.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment
1
4

Page 15 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Compound 6b induced apoptosis. (A) Apoptosis initiated by 6b. HepG2 cells were
treated with 1, 2 and 4 μM 6b for 24 h. Apoptotic levels were assessed using annexin V/PI double
staining and flow cytometry. Related histogram for living and apoptotic cells are expressed. ** P
<
0.01 vs. control. (B) Western blot measured caspase family proteins and PARP levels in HepG2
ACS Paragon Plus Environment
1
5

Journal of Medicinal Chemistry
Page 16 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
cells treated with 0 ~ 4 μM 6b. (C) Immunofluorescence assays detected the position of AIF
following treatment with 4 μ M 6b for 24 h. Red fluorescence indicates AIF, whereas blue
represents the nucleus. Scar bar, 50 μm. (D) The effects of z-VAD-fmk on vacuolization and cell
death induced by 6b. Cells were pretreated with 20 μM z-VAD-fmk for 2 h and then incubated
with 4 μM 6b for 24 h. Flow cytometry analysis of cell distribution and quantification of the
number of surviving and apoptotic levels (a). MTT assays tested cell viability after exposure to
different doses of 6b in the presence or absence of z-VAD-fmk (b). Optical microscopy images of
HepG2 cells preincubated with or without z-VAD-fmk before treatment with 3 μM 6b (c). Scar
bar, 10 μm.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment
1
6

Page 17 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Figure 6. Cell death and cytoplasmic vacuoles induced by 6b were not associated with necrosis
and autophagy. (A) The effects of Nec-1 (30 μ M, pretreated for 2 h) on cell viability and
vacuolization in HepG2 cells treated with 3 μM 6b. MTT assays tested survival rates. The green
curve indicates viability after treatment with 6b, blue demonstrates viability in cells treated with
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
b and Nec-1. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control. Morphological changes were
imaged using optical microscopy after treatment for 24 h. Scale bar, 10 μm. (B) Cell viability
following treatment with 3 μM 6b for 24 h in the presence of CQ (10 μM) or 3-MA (1 mM).
Growing inhibition analysis examined by MTT assays, *** P < 0.001 vs. control. Phase-contrast
images of cells treated with indicated agents were performed. Scar bar, 10 μm. Western blot was
performed to detect LC3 and P62 expression.
6
b initiates mitochondrial dysfunction via interference with reactive oxygen species
homeostasis and mitochondrial permeability. To obtain a general sense of the role of 6b in
mitochondrial dysfunction, we assessed the level of reactive oxygen species (ROS), changes of
mitochondrial permeability transition pore (MPTP) and Δψ following 6b treatment. Mitochondria
m
are major contributors of endogenous ROS in tumors. ROS generated from mitochondria control
2
2
cellular redox regulation and render cells vulnerable to chemotherapeutic drugs . We detected the
ROS generation by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). ROS levels were
indeed upregulated in a dose-dependent manner (Figure 7A (a)) beginning 2 h after treatment with
2 μM 6b and persisting up to 24 h (Figure 7A (b)). Oxidants are essential for the induction of
mitochondrial permeability transition pore, resulting in mitochondrial degradation and cell death23,
24.
We then detected the status of the permeability transition pore using the probe calcein-AM and
the quencher CoCl . Calcein-AM can enter cells freely and be hydrolyzed to the fluorescent form
2
of calcein, which is only able to escape mitochondria when the permeability transition pore is open.
ACS Paragon Plus Environment
1
7

Journal of Medicinal Chemistry
Page 18 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Given that the residual calcein present in cytosol and nucleus is quenched by CoCl , the
2
2
5
fluorescence localized to mitochondria is imaged against dark background . As shown in Figure
7B, green fluorescence in mitochondria is lost following treatment with 2 μM 6b for 12 h, and
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
dispersive puncta were observed upon exposure to 4 μM 6b. The collapse of Δψ caused by 6b
m
was obtained by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1)
staining (Figure 7C). These results demonstrate that 6b disturbed redox homeostasis, promoted
MPTP opening, reduced mitochondrial membrane permeability and gradually resulted in distinct
mitochondrial dysfunction in HepG2 cells.
ACS Paragon Plus Environment
1
8

Page 19 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Figure 7. Mitochondrial dysfunction induced by 6b. (A) ROS generation in HepG2 cells exposed
to 6b. Cells were treated with 6b at indicated concentrations for 24 h or treated with 2 μM 6b for
different times. Samples were loaded with 10 μM DCFH-DA for 30 min and tested by flow
cytometry. Ros-up was the positive agent provided in the test kit, *** P < 0.001 vs. Control. (B)
Mitochondria leakage induced by 6b. Cells were exposed to 6b at indicated doses for 12 h and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
then stained with calcein-AM in the presence of CoCl . Images were captured by fluorescence
2
microscopy. (C) Changes in Δψ were analyzed using JC-1 following treatment for 24 h.
m
Histogram of JC-1 aggregates are expressed. *** P < 0.001 vs. control.
In vivo anti-tumor effects of 6b. The antiproliferative efficiency of 6b against HepG2 cells in
vivo was assessed in zebrafish xenograft model, an adopted animal model for drug screening
popularly5, 26. Generally, embryos were obtained from pairwise mathing of sexually mature
zebrafish and then microinjected of HepG2 cells labeled with cell tracker CM-Dil to enstablish
xenograft model. Zebrafish bearing hepatoma cancer cells were incubated with different
concentrations of 6b for 48 h. A group of zebrafish administered by doxorubicin seved as a positive
control. Based on the reduced red fluorescence intensity in xenotransplantation, 6b inhibited
significantly the growth of HepG2 cells in vivo compared with the control group (Figure 8).
ACS Paragon Plus Environment
1
9

Journal of Medicinal Chemistry
Page 20 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 8. Compound 6b inhibited the proliferation of HepG2 in zabrafish xenograft model. (A)
Tumor growth in every group was detected using confocal microscopy after drugs administration
for 48 h. (B) Histogram of fluorescence intensities in different groups of xenograft zebrafish. ***
P < 0.001 vs. control.
CONCLUSION AND DISCUSSION
Current literatures about the antitumor activity and mechanism of honokiol derivatives have
uncovered its potential in chemotherapy. In an attempt to improve its therapeutic profile, there has
been interest in obtaining derivatives with increased anti-cancer effects and cell selectivity and
identifying the precise mechanisms of action in cell death. The mitochondrion is a most studied
target in cancer therapy given its indispensable role in energy metabolism, redox signaling and
2
programmed cell death . Mitochondria-targeted cytotoxic conjugates formed by linking parent
drugs to mitochondria-directing moieties are major approaches in targeted therapeutics driven by
the increased mitochondrial membrane potential in cancer cells (-220 mV) compared with normal
ACS Paragon Plus Environment
2
0

Page 21 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
cells (-160 mV)27, 28. Recent studies repurposed isoquinoline alkaloid BBR as a mitochondria-
targeting carrier based on its delocalized positive charge and amphiphilicity14, 17. Our efforts to
improve cell selectivity and enhance the cytotoxicity of the parent drug HN focused on developing
mitochondria-targeting BBR-linked HN derivatives and assessing its overall mechanisms of action
in cell death.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In the present work, we designed and synthesized series of novel derivatives based on the
natural product honokiol conjugated with BBR, a natural DLC that serves as a mitochondria-
targeting carrier. Compound 6b was chosen as a representative for deep study based on its potential
inducing vacuoles, as well as its increased anticancer effect and selectivity compared with its
parent compounds HN and BBR. For increasing cytotoxicity and cell selectivity of these novel
derivatives, enhanced endocytosis and specific mitochondrial targeting feature could illustrate
(Figure 4). Researches that study alkyl-substituted BBR derivatives summarized the enhanced
lipophilicy and improving tissue penetration29, 30, which allowed BBR moiety mediate HN to
penetrate biomembranes. In addition, cytoplasmaic membrane potential enhanced endocytosis of
cations dependent on potential difference outside and inside the cell membrane. Nernst equation
[
Δψ =61.5*log (C /C )] describes cellular uptake of cationic drugs. C and C are the
10 in out in out
concentration of ions in the inner and outer of the biomembrane, such as cytoplasmic and
mitochondrial membrane. The equation indicated that every 61.5 mV membrane potential will
1
2
induce 10 times increasing of concentration in inner membrane space . In the process of drugs
accumulation, cytoplasmaic membrane potential of -30~40 mV typically leads to a 3~4-fold
increasing in the cytosolic domain compared with extracellular area in principle. In fact, the total
cellular accumulation of 6b was about 2.5-fold increasing than that of HN (Figure 4A). Next,
cationic 6b loaded in cytoplasm can be concentrated in mitochondria with the help of higher
ACS Paragon Plus Environment
2
1

Journal of Medicinal Chemistry
Page 22 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
mitochondrial membrane potential in cancer cells (Figure 4). Thus, more BBR-linked HN
derivatives penetrated cell membrane and targeted selectively to mitochondria to exert the
anticancer efficiency though these force.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The detail mechanisms of action of 6b was explored in this study. Here, 6b induced massive
dilation of mitochondria and marked mitochondria-mediated cell death, including vacuolation-
associated cell death and apoptosis. ROS play a dual role in survival. Elevated ROS production
contributes to oncogenic transformation, but excessive toxic levels increased oxidative stress and
2
2
induced tumor cell death . In our mechanistic study, we found that the ROS balance was rapidly
disrupted in treated cells approximately 2 h after treatment (Figure 7A) and followed by massive
cytoplasmic vacuoles that formed from dilated mitochondria (Figure 3). Oxidant stress elicits an
abrupt increase in inner mitochondrial membrane permeability via induction of the mitochondria
permeability transition pore, which results in mitochondrial depolarization.23, 24In 6b-treated cells,
the MPTP began to open in succession with reducing of Δψ (Figure 7B, 7C). Here, 6b disrupts
m
the integrity of the mitochondrial inner and outer membranes upon entry into mitochondria
possibly via ROS-driven formation of the permeability transition pore.
In this context, BBR serves as a mitochondrial-targeting carrier, and the BBR-linked HN
derivative 6b exhibits some differences in its ability to induce cell death, compared with its parent
compound HN. Although exposure of HN resulted in ROS-mediated apoptosis and autophagy in
human osteosarcoma cells or hepatocellular carcinoma9, 31, the cell death initiated by 6b was
associated with vacuolization as early as apoptosis, without autophagy and necroptosis in HepG2
cells. Consistently, conjugating the “war head” of HN to the mitochondrial-targeting carrier BBR
contributes to the alternative mechanisms of action and increased antitumor effects. In addition 6b
ACS Paragon Plus Environment
2
2

Page 23 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
treatment inhibited the growth of HepG2 cells in xenograft zebrafish, that confirming anti-tumor
efficiency in vivo of 6b (Figure 8).
Taken together, we demonstrate that BBR serves as an effective mitochondrial-targeting
cation carrier, and the antitumor efficacy and potency of honokiol can be increased upon
conjugation to BBR.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EXPERIMENTAL SECTION
General Procedures. NMR spectra were obtained using a Bruker Avance DRX-600 spectrometer
1
13
operating at 600 ( H) and 150 ( C) MHz. The chemical shifts are reported as δ (ppm), and the
coupling constants (J) are given in Hz for NMR. HR-ESI-MS was conducted using an LTQ
Orbitrap XL instrument. Column chromatography was performed with silica gel (200~300 mesh;
Qingdao Haiyang Chemical Co. Ltd., Qingdao, China). Thin-layer chromatography (TLC) was
performed with precoated silica gel GF-254 glass plates (Qingdao Haiyang Chemical Co. Ltd.,
Qingdao, China) and polyamide plates (Sinopharm Chemical Regent Co. Ltd., Taizhou, China).
For biological evaluation, the purity of key compounds was established as ≥95% by HPLC using
an Agilent 1100 series with a ZORBAX SB-C18 column (250 mm × 4.6 mm, 5 μm). The eluent
used for HPLC was methanol-0.1% acetic acid aqueous solution (gradient elution, 0 ~ 30 min ,
7
0% ~ 100% methanol), and the detection wavelength was 265 nm. Berberine chloride and
honokiol possessed a purity of at least 95% were purchased from Dibo Bio-tech Co. Ltd., Shanghai,
China and Ailan Chemical Technology Co. Ltd., Shanghai, China, respectively.
Preparation of intermediate 2b ~ 2f, 3 and 4a ~ 4d. To a solution of honokiol (2a) (2 mmol) and
excessive K CO (12 mmol) in DMF, various iodides (4 mmol) was added dropwise. After
2
3
stiring overnight at room temperature and monitored by TLC, the mixture was taken into 10-fold
ACS Paragon Plus Environment
2
3

Journal of Medicinal Chemistry
Page 24 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
water and extracted with ethyl acetate three times. The layer of ethyl acetate was distilled under
reduced pressure and the residue was purified via column chromatography on silica gel (petroleum
ether/ethly acetate, 50/1 ~ 30/1, v/v) to obtain compound 2b ~ 2f. Berberine chloride (1) (5.0 g,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3.5 mmol) was dissolved in 5 N NaOH (25 mL) and acetone (5 mL) was added dropwise while
stirring. After stirring for 30 min at room temperature, the reaction mixture was filtered and washed
with 80% MeOH to give intermediate 3, directly used for next reaction without purify by
chromatography. Treatment of compound 3 (5.08 mmol) with various iodide (25.5 mmol) in
acetonitrile at 80℃ for 6 ~ 10 h. Intermediates 4a ~ 4d were yielded by column chromatography
on silica gel (DCM/MeOH, 100/1 ~ 50/1, v/v).
Synthesis of target compounds of series 5 and 6. Compound 2a (1 mmol) and K CO (10 mmol)
2
3
was dissolved in 10 mL MeCN and stirred until the mixture turned yellow. Then, the intermediate
a/4b/4c (1 mmol) diluted in 5 mL acetonitrile was added dropwise and stirred at 80℃ for 2 ~ 6 h
4
until the reaction was complete according to the TLC detection using polyamide plates. The
reaction mixture was filtered to remove excessive K CO and distilled under reduced pressure.
2
3
The residue was purified by column chromatography with silica gel using DCM/MeOH 120/1 as
the eluent to give compounds 5a/6a and 5b/6b and 5c/6c as yellow solids. Products 5d, 6d, 5e and
6
e were obtained via the reaction of intermediate 4b (0.3 mmol) with 2b ~ 2e (0.5 mmol)
respectively, in the presence of excessive K CO (2 mmol) in refluxing acetonitrile.
2
3
1
3
',5-diallyl-4'-methoxy-[1,1'-biphenyl]-2-ol (2b). Light yellowish oil. 41% yield. H NMR (600
MHz, CDCl ) δ 7.27 (dd, J = 8.3, 1.9 Hz, 1H, H-6'), 7.23 (d, J = 2.4 Hz, 1H, H-2'), 7.03 (dd, J =
3
6
5
.5, 2.3 Hz, 2H, H-4, H-6), 6.92 (d, J = 8.4 Hz, 1H, H-5'), 6.87 (d, J = 9.0 Hz, 1H, H-3), 6.04 -
.91 (m, 2H, H-8, H-8'), 5.10 - 5.02 (m, 4H, H-9, H-9'), 3.84 (s, 3H, OCH -1''), 3.42 (d, J = 6.7
3
1
3
Hz, 2H, H-7'), 3.33 (d, J = 6.7 Hz, 2H, H-7). C NMR (150 MHz, CDCl ) δ 157.1, 150.9, 137.9,
3
ACS Paragon Plus Environment
2
4

Page 25 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
3
36.6, 132.2, 130.6, 130.3, 129.8, 129.2, 128.8, 128.0, 128.0, 115.9, 115.7, 115.6, 111.0 , 55.6,
-
-
9.5, 34.4 . ESI-MS m/z calculated for C H O [M-H] 279.1391, found 279.1391.
1
9
19
2
1
3,5'-diallyl-2'-methoxy-[1,1'-biphenyl]-4-ol (2c). Light yellowish oil. 24% yield. H NMR (600
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
MHz, CDCl ) δ 7.19 ( 2H, H-2', H-6'), 7.01 (s, 1H, H-6), 6.98 (d, J = 8.1 Hz, 1H, H-4), 6.80 - 6.77
3
(m, 1H, H-3), 6.68 (d, J = 7.9 Hz, 1H, H-5'), 5.97 - 5.84 (m, 2H, H-8, H-8'), 5.12 - 4.94 (m, 4H,
H-9, H-9'), 3.68 (s, 3H, OCH -1''), 3.33 (d, J = 5.7 Hz, 2H, H-7'), 3.26 (d, J = 6.1 Hz, 2H, H-7).
3
1
3
C NMR (150 MHz, CDCl ) δ 153.8, 152.2, 136.8, 135.5, 131.3, 130.4, 130.1, 129.9, 129.3,
3
1
27.9, 126.9, 124.0, 115.37, 114.52, 114.35, 110.39, 54.69, 38.34, 34.09. ESI-MS m/z calculated
-
-
for C H O [M-H] 279.1391, found 279.1378.
19 19
2
1
3',5-diallyl-4'-propoxy-[1,1'-biphenyl]-2-ol (2d). Light yellowish oil. 32% yield. H NMR (600
MHz, CDCl ) δ 7.24 (dd, J = 8.1, 2.3 Hz, 1H, H-6'), 7.23 (d, J = 2.5 Hz, 1H, H-2'), 7.05 - 7.00 (m,
3
2
H, H-4, H-6), 6.91 (d, J = 8.3 Hz, 1H, H-5'), 6.88 (d, J = 8.8 Hz, 1H, H-3), 5.98 (m, 2H, H-8,
H-8'), 5.13-5.01 (m, 4H, H-9, H-9'), 3.96 (t, J = 6.4 Hz, 2H, OCH -1''), 3.44 (d, J = 7.3 Hz, 2H, H-
2
13
7
'), 3.33 (d, J = 6.9 Hz, 2H, H-7), 1.84 (h, J = 7.0 Hz, 2H, CH -2''), 1.09-1.04 (t, 3H, CH -3''). C
2
3
NMR (150 MHz, CDCl ) δ 156.5, 150.9, 137.8, 136.7, 132.1, 130.5, 130.2, 129.9, 128.8, 128.7,
3
1
27.94, 127.86, 115.8, 115.6, 115.5, 111.7, 69.6, 39.4, 34.5, 22.7, 10.7. ESI-MS m/z calculated
-
-
for C H O [M-H] 307.1704, found 307.1687.
2
1
23
2
1
3
,5'-diallyl-2'-propoxy-[1,1'-biphenyl]-4-ol (2e). Light yellowish oil. 21% yield. H NMR (600
MHz, CDCl ) δ 7.34 (d, J = 2.3 Hz, 1H, H-2'), 7.30 (dd, J = 8.2, 2.3 Hz, 1H, H-6'), 7.12 (d, J = 2.4
3
Hz, 1H, H-6), 7.05 (dd, J = 8.3, 2.4 Hz, 1H, H-4), 6.87 (d, J = 8.3 Hz, 1H, H-3), 6.79 (d, J = 8.3
Hz, 1H, H-5'), 6.00 (dddt, J = 40.3, 16.8, 10.0, 6.6 Hz, 2H, H-8, H-8'), 5.20 - 5.11 (m, 4H, H-9, H-
9
'), 3.87 (t, J = 6.5 Hz, 2H, OCH -1''), 3.43 (dt, J = 6.7, 1.6 Hz, 2H, H-7'), 3.35 (d, J = 6.9 Hz, 2H,
2
ACS Paragon Plus Environment
2
5

Journal of Medicinal Chemistry
Page 26 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
H-7), 1.76 - 1.65 (m, 2H, CH -2''), 0.95 (t, J = 7.5 Hz, 3H, CH -3''). C NMR (150 MHz, CDCl )
2
3
3
δ 154.4, 153.0, 137.9, 136.6, 132.2, 131.7, 131.3, 130.9, 130.5, 128.9, 127.9, 124.7, 116.3, 115.5,
-
-
115.3, 112.7, 70.2, 39.4, 35.1 , 22.6, 10.8. ESI-MS m/z calculated for C H O [M-H] 307.1704,
2
1
23
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
found 307.1700.
1
3
',5-diallyl-4'-(pentyloxy)-[1,1'-biphenyl]-2-ol (2f). Light yellowish oil. 43 % yield. H NMR
(
600 MHz, CDCl ) δ 7.25 (dd, J = 8.3, 2.4 Hz, 1H), 7.22 (d, J = 2.2 Hz, 1H), 7.06 - 7.00 (m,
3
2
2
1
H), 6.92 (d, J = 8.3 Hz, 1H), 6.89 (d, J = 8.0 Hz, 1H), 5.98 (dddt, J = 22.1, 16.8, 10.0, 6.7 Hz,
H, H-8, H-8'), 5.13 - 5.01 (m, 4H, H-9, H-9'), 4.00 (t, J = 6.4 Hz, 2H, OCH -1''), 3.43 (dt, J = 6.8,
2
.5 Hz, 2H, H-7'), 3.34 (dt, J = 6.7, 1.6 Hz, 2H, H-7), 1.82 (m, 2H, CH -2''/3''/4'), 1.48 (m, 2H ,
2
1
3
CH -2''/3''/4'), 1.40 (m, 2H , CH -2''/3''/4''), 0.95 (t, J = 7.3 Hz, 3H, CH -5''). C NMR (150 MHz,
2
2
3
CDCl ) δ 156.6, 150.9, 137.9, 136.8, 132.2, 130.5, 130.3, 129.98, 128.83, 128.81, 128.02, 127.94,
3
1
15.9, 115.65, 115.62, 111.8, 68.2, 39.5, 34.6, 29.1, 28.4, 22.6, 14.2. ESI-MS m/z calculated for
-
-
C H O [M-H] 335.2017, found 335.2017.
2
3
27
2
1
3-(3-iodopropyl)-9,10-dimethoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquinolino[3,2-
1
a]isoquinolin-7-ium iodide (4a). Yellow powder. 47% yield. H NMR (600 MHz, CD OD) δ 9.81
3
(
s, 1H, H-8), 8.22 (d, J = 9.4 Hz, 1H, H-11/12), 8.16 (d, J = 9.4 Hz, 1H, H-11/12), 7.34 (s, 1H,
H-1), 7.04 (s, 1H, H-4), 6.12 (s, 2H, OCH O-15), 4.79 (t, J = 5.8 Hz, 2H, NH -6), 4.22 (s, 3H,
2
2
OCH -16), 4.13 (s, 3H, OCH -17), 3.66-3.63 (m, 2H, linker-CH ), 3.17-3.11 (m, 2H, H-5), 2.28
3
3
2
1
3
(
p, J = 6.9 Hz, 2H, linker-CH ). C NMR (150 MHz, CD OD) δ 151.9, 151.6, 149.0, 146.5, 145.6,
2 3
138.4, 135.3, 134.50, 134.47, 127.2, 123.2, 122.1, 110.8, 109.4, 103.7, 62.6, 58.8, 57.5, 49.6, 35.1,
+
+
3
1.5, 29.2, 4.9. ESI-MS m/z calculated for C H INO [M-I] 504.0666, found 504.0657.
23 23 4
ACS Paragon Plus Environment
2
6

Page 27 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
3-(5-iodopentyl)-9,10-dimethoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquinolino[3,2-
1
a]isoquinolin-7-ium iodide (4b). Yellow powder. 45% yield. H NMR (600 MHz, CD OD) δ 9.80
3
(s, 1H, H-8), 8.19 (d, J = 9.4 Hz, 1H, H-11/12), 8.16 (d, J = 9.2 Hz, 1H, H-11/12), 7.30 (d, J = 5.7
Hz, 1H, H-1), 7.04 (s, 1H, H-4), 6.13 (d, J = 2.8 Hz, OCH O-15), 4.80 (t, J = 5.7 Hz, 2H, NH -6),
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
4
.21 (s, 3H, OCH -16), 4.13 (s, 2H, OCH -17), 3.56 (t, J = 6.5 Hz, 1H, H-5'), 3.47 (q, J = 8.0, 7.0
3 3
Hz, 2H, H-1'), 3.21 (t, J = 6.9 Hz, 1H, H-5'), 3.14 (q, J = 5.7 Hz, 2H, H-5), 1.88 (p, J = 7.3 Hz,
1
3
2
H, linker -CH -), 1.81 (h, J = 7.0 Hz, 2H, linker -CH -), 1.56(m, 2H, linker -CH -). C NMR
2 2 2
(150 MHz, CD OD) δ 151.8, 151.4, 146.4, 145.3, 136.1, 135.2, 134.4, 127.2, 123.2, 122.3, 121.8,
3
1
10.5, 109.4, 103.7, 62.7, 58.9, 57.5, 45.5, 33.9, 33.1, 31.2, 30.8, 30.5, 29.3, 27.5, 6.5. ESI-MS
+
+
m/z calculated for C H INO [M-I] 532.0979, found 532.0981.
2
5
27
4
1
3-(8-iodooctyl)-9,10-dimethoxy-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquinolino[3,2-
1
a]isoquinolin-7-ium iodide (4c). Yellow powder. 41% yield. H NMR (600 MHz, CD OD) δ 9.79
3
(s, 1H, H-8), 8.19 (d, J = 9.5 Hz, 1H, H-11/12), 8.15 (d, J = 9.4 Hz, 1H, H-11/12), 7.30 (s, 1H,
H-1), 7.05 (s, 1H, H-4), 6.14 (s, 2H, OCH O-15), 4.80 (t, J = 5.8 Hz, 2H, NH -6), 4.21 (s, 3H,
2
2
OCH -16), 4.13 (s, 3H, OCH -17), 3.44 (t, J = 8.0 Hz, 2H, H-1'), 3.23 (t, J = 6.9 Hz, 2H, H-8'),
3
3
3
.13 (t, J = 5.8 Hz, 2H, H-5), 1.88( 2H, linker-CH -), 1.82-1.76 (m, 2H, linker-CH -), 1.47 (m, 2H,
2 2
1
3
linker-CH -), 1.42-1.32 (m, 6H, linker-CH -). C NMR (150 MHz, CDCl ) δ 150.6, 149.9, 147.4,
2
2
3
1
5
46.3, 145.6, 136.2, 134.5, 133.8, 133.3, 125.5, 122.3, 120.5, 120.4, 109.3, 108.7, 102.3, 63.3,
8.0, 57.1, 33.5, 31.2, 30.5, 30.1, 29.6, 29.0, 28.8, 28.5, 7.6. ESI-MS m/z calculated for
+
+
C H INO [M-I] 574.1449, found 574.1444.
2
8
33
4
9
,10-dimethoxy-13-pentyl-5,6-dihydro-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-7-
1
1
ium iodide (4d). Yellow solid. 20% yield. H NMR (600 MHz, CD OD). H NMR (600 MHz,
3
CD OD) δ 9.78 (s, 1H, H-8), 8.18 (d, J = 9.5 Hz, 1H, H-11/12), 8.15 (d, J = 9.4 Hz, 1H, H-11/12),
3
ACS Paragon Plus Environment
2
7

Journal of Medicinal Chemistry
Page 28 of 47
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
.29 (s, 1H, H-1), 7.04 (s, 1H, H-4), 6.13 (s, 2H, OCH O-15), 4.80 (t, J = 5.9 Hz, 2H, H-6), 4.21
2
(s, 3H, OCH -16), 4.13 (s, 3H, OCH -17), 3.44 – 3.39 (m, 2H, H-5/1'), 3.15 – 3.10 (m, 2H, H-
3 3
5/1'), 1.92 – 1.85 (m, 2H, CH -2'/3'/4'), 1.47 (q, J = 7.4 Hz, 2H, CH -2'/3'/4'), 1.40 (dt, J = 8.9, 7.0
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
Hz, 2H, CH -2'/3'/4'), 0.94 (t, J = 7.2 Hz, 3H, CH -3' ). C NMR (150 MHz, CD OD) δ 151.8,
2
3
3
1
1
51.3, 148.7, 146.3, 145.3, 137.8, 136.3, 135.1, 134.5, 127.1, 123.1, 122.3, 121.8, 110.4, 109.3,
+
03.8, 62.6, 58.9, 57.5, 32.7, 31.8, 30.7, 29.2, 23.6, 14.4. ESI-MS m/z calculated for C H NO
25 28
4
+
[M-I] 406.2013, found 406.2006.
1
3-(3-((3',5-diallyl-4'-hydroxy-[1,1'-biphenyl]-2-yl)oxy)propyl)-9,10-dimethoxy-5,6-
dihydro-[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-7-ium iodide (5a). Yellow solid.
1
20% yield. H NMR (600 MHz, CD OD). δ 9.68 (s, 1H, H-8), 7.90 (d, J = 9.3 Hz, 1H, H-11/12),
3
7
.79 (d, J = 9.4 Hz, 1H, H-11/12), 7.28 (s, 1H, H-1), 7.18 (d, J = 2.3 Hz, 1H, H-6'), 7.15 (dd, J =
.2, 2.3 Hz, 1H, H-6″), 7.06-7.03 (2H, H-4', H-2''), 6.91 (s, 1H, H-4), 6.87 (d, J = 8.1 Hz, 1H, H-
8
5''), 6.78 (d, J = 8.1 Hz, 1H, H-3'), 6.05 (s, 2H, OCH O-15), 5.99 (ddt, J = 16.8, 10.1, 6.7 Hz, 1H,
2
H-8'/8''), 5.86 (ddt, J = 16.8, 10.1, 6.7 Hz, 1H, H-8'/8''), 5.10-5.02 (m, 2H, H-9'/9''), 4.91 (dt, J =
1
3
7.1, 1.8 Hz, 1H, H-9'/9''), 4.84-4.80 (m, 1H, H-9'/9''), 4.70 (t, J = 5.7 Hz, 2H, NCH -6), 4.20 (s,
2
H, OCH -16), 4.12 (s, 3H, OCH -17), 3.99 (t, J = 5.0 Hz, 2H, OCH -3'''), 3.60 (t, J = 7.9 Hz, 2H,
3
3
2
H-1'''), 3.35 (d, J = 6.7 Hz, 2H, H-7'), 3.23 (d, J = 6.8 Hz, 2H, H-7''), 2.97 (t, J = 5.8 Hz, 2H, H-5),
1
3
2
1
.20 (q, J = 7.0 Hz, 2H, H-2'''). C NMR (150 MHz, CD OD) δ 155.4, 155.3, 151.6, 151.4, 148.8,
3
46.2, 145.2, 139.3, 138.3, 138.1, 135.5, 135.0, 134.3, 133.9, 132.3, 131.9, 131.4, 129.2, 128.9,
127.1, 127.0, 123.0, 122.3, 121.6, 115.7, 115.6, 115.5, 113.6, 110.5, 109.3, 103.6, 68.5, 62.6, 58.8,
+
+
5
7.5, 40.4, 35.3, 31.9, 29.2, 28.0. HR-ESI-MS m/z calculated for C H NO [M-I] 642.2850,
41 40 6
found 642.2852.
ACS Paragon Plus Environment
2
8

Page 29 of 47
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
3-(5-((3',5-diallyl-4'-hydroxy-[1,1'-biphenyl]-2-yl)oxy)pentyl)-9,10-dimethoxy-5,6-dihydro-
[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-7-ium iodide (5b). Yellow solid. 18% yield.
1
H NMR (600 MHz, CD OD) δ 9.73 (s, 1H, H-8), 8.04 (s, 2H, H-11, H-12), 7.18 (d, J = 2.3 Hz,
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
6
8
H, H-2''), 7.08 (s, 1H, H-1), 7.03 (dd, J = 8.3, 2.3 Hz, 1H, H-4'), 7.00 (d, J = 2.2 Hz, 1H, H-6'),
.98 (dd, J = 8.2, 2.3 Hz, 1H, H-6''), 6.95 (s, 1H, H-4), 6.90 (d, J = 8.3 Hz, 1H, H-3'), 6.56 (d, J =
.2 Hz, 1H, H-5''), 6.00 (s, 2H, OCH O-15), 5.99-5.93 (m, 1H, H-8'), 5.80 (ddt, J = 16.7, 10.0, 6.6
2
Hz, 1H, H-8''), 5.06-4.86 (m, 4H, H-9', 9''), 4.75 (t, J = 5.8 Hz, 2H, NCH -6), 4.23 (s, 3H, OCH -
2
3
1
6), 4.11 (s, 3H, OCH -17), 3.95 (t, J = 5.7 Hz, 2H, H-5'''), 3.33 (d, J = 7.0 Hz, 2H, H-7'), 3.27 (t,
3
J = 8.1 Hz, OCH -1'''), 3.06 (d, J = 6.6 Hz, 2H, H-7''), 3.01 (t, J = 5.9 Hz, 2H, H-5), 1.86, 1.75,
2
1
3
1.54 (each, 2H, linker-CH -2'''/3'''/4'''). C NMR (150 MHz, CD OD) δ 155.6, 154.9, 151.7, 151.3,
2
3
1
1
48.7, 146.4, 145.1, 139.4, 138.3, 137.7, 136.1, 134.9, 134.4, 133.6, 132.4, 131.9, 131.6, 131.4,
29.2, 128.8, 127.1, 126.6, 123.2, 122.2, 121.7, 115.62, 115.55, 115.2, 113.7, 110.6, 109.2, 103.6,
69.9, 62.7, 58.9, 57.6, 40.4, 35.1, 31.9, 30.7, 29.6, 29.2, 27.8. HR-ESI-MS m/z calculated for
+
+
C H NO [M-I] 670.3163, found 670.3171.
4
3
44
6
1
3-(8-((3',5-diallyl-4'-hydroxy-[1,1'-biphenyl]-2-yl)oxy)octyl)-9,10-dimethoxy-5,6-dihydro-
[1,3]dioxolo[4,5-g]isoquinolino[3,2-a]isoquinolin-7-ium iodide (5c). Yellow solid. 15% yield.
1
H NMR (600 MHz, CD OD) δ 9.75 (s, 1H, H-8), 8.11 (d, J = 9.4 Hz, 1H, H-11/12), 8.08 (d, J =
3
9
1
.4 Hz, 1H, H-11/12), 7.23 (s, 1H, H-1), 7.21 (d, J = 2.2 Hz, 1H, H-2''), 7.10 (dd, J = 8.2, 2.3 Hz,
H, H-6''), 7.02 (m, 2H, H-4', H-6'), 6.97 (s, 1H, H-4), 6.91(d, J=9.0 Hz, 1H, H-3'), 6.70 (d, J =
8.3 Hz, 1H, H-5''), 6.00 (s, 2H, OCH O-15), 5.99-5.91 (m, 2H, H-8', H-8''), 5.08-4.92 (m, 4H, H-
2
9
', H-9''), 4.75 (t, J = 5.9 Hz, 2H, NCH -6), 4.20 (s, 3H, OCH -16), 4.09 (s, 3H, OCH -17), 3.91
2 3 3
(t, J = 6.1 Hz, 2H, H-8'''), 3.36 (3H, H-1''', H-7'), 3.32 (1H, H-7'), 3.29 (dt, J = 6.5, 1.6 Hz, 2H,
H-7''), 3.07 (t, J = 5.9 Hz, 2H, H-5), 1.82 (m, 2H, linker-CH ), 1.64 (m, 2H, linker-CH ), 1.47-
2
2
ACS Paragon Plus Environment
2
9