Design, synthesis and biological evaluation of nitrogen-containing macrocyclic bisbibenzyl derivatives as potent anticancer agents by targeting the lysosome

Article abstract of DOI:10.1016/j.ejmech.2017.05.050

A series of novel nitrogen-containing macrocyclic bisbibenzyl derivatives was designed, synthesized, and evaluated for antiproliferative activity against three anthropic cancer cell lines. Among these novel molecules, the tri-O-alkylated compound 18a disp

Full text of DOI:10.1016/j.ejmech.2017.05.050

European Journal of Medicinal Chemistry 136 (2017) 603e618
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Design, synthesis and biological evaluation of nitrogen-containing
macrocyclic bisbibenzyl derivatives as potent anticancer agents by
targeting the lysosome
,
b
,
,
Bin Sun a 1, Jun Liu b 1, Yun Gao b, Hong-bo Zheng b, Lin Li b, Qing-wen Hu b,
Hui-qing Yuan c, Hong-xiang Lou a
, b, *
a National Glycoengineering Research Center, Shandong University, Jinan, 250012, PR China
b Key Laboratory of Natural Products & Chemical Biology, Ministry of Education, Shandong University, Jinan, 250012, PR China
c School of Medicine, Shandong University, Jinan, 250012, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel nitrogen-containing macrocyclic bisbibenzyl derivatives was designed, synthesized, and
evaluated for antiproliferative activity against three anthropic cancer cell lines. Among these novel
molecules, the tri-O-alkylated compound 18a displayed the most potent anticancer activity against the
Received 24 March 2017
Received in revised form
15 May 2017
Accepted 24 May 2017
Available online 25 May 2017
A549, MCF-7, and k562 cancer cell lines, with IC50 values of 0.51, 0.23, and 0.19 mM, respectively, which
were obviously superior to those of the parent compound riccardin D, and were 3e10-fold better than
those of the clinical used drug ADR. The bis-Mannich derivative 11b also exhibited significantly enhanced
antiproliferative potency, with submicromolar IC50 values. Structure-activity relationship analyses of
these newly synthesized compounds were also performed. Mechanistic studies indicated that these
compounds could target the lysosome to induce lysosomal membrane permeabilization, and could also
induce cell death that displayed features characteristic of both apoptosis and necrosis.
© 2017 Elsevier Masson SAS. All rights reserved.
Keywords:
Bisbibenzyls
Anticancer activity
Nitrogen-containing derivatives
Lysosome
1. Introduction
lysosomal swelling and rupture that leads to lysosomal membrane
permeabilization (LMP) [7]. LMP usually causes lysosomal pro-
Lysosomes are acidic intracellular organelles that are involved in
several cellular processes, including receptor degradation, auto-
phagy, apoptosis, post-translational protein maturation, and the
extracellular release of active enzymes [1e3]. They control the
recycling of large amounts of cellular organelles and macromole-
cules through the actions of more than 50 acid hydrolytic enzymes.
Interestingly, lysosomes in cancer cells are larger, less stable, more
numerous, and exhibit greater cathepsin activity than those in
normal cells [3,4]. Additionally, weakly basic drugs have been found
to accumulate specifically in lysosomes, after which they can no
longer easily diffuse out of the vesicles because they become pro-
tonated [5,6]. This accumulation of weakly basic drugs could lead to
changes in osmolality within the lysosomes, which results in
teases to leak into the cytosol and trigger initiation of either the
apoptotic or necrotic cell death pathways [8,9]. Therefore, the
lysosome is a critical target for anticancer therapy [10,11], and
agents that interact with lysosomes show promise for the devel-
opment of novel and potent anticancer drugs.
Macrocyclic bisbibenzyls are a series of phenolic natural prod-
ucts that are mainly found in liverworts [12]. These natural prod-
ucts exhibit versatile biological activities, including antifungal,
antibacterial, antiviral, anti-mitotic, antioxidant, cytotoxic, muscle-
relaxing, LXR-modulating, and NOS-inhibiting activities [13e17].
Therefore, bisbibenzyls are of great interest to natural product re-
searchers because of their potential applications as pharmacolog-
ical agents. Riccardin D, a macrocyclic bisbibenzyl isolated from
Marchantia polymorpha L., has been shown to exhibit robust anti-
cancer activity [18,19]. In our previous study, we prepared two
Mannich base derivatives of riccardin DdRDN-1 and RDN-2
(Fig. 1)dthat exhibited markedly improved anticancer activity
compared with the parent riccardin D. Interestingly, both mole-
cules were found to accumulate in the lysosomes. Mechanistic
* Corresponding author. National Glycoengineering Research Center, Key Labo-
ratory of Natural Products & Chemical Biology, Ministry of Education, Shandong
University, Jinan, 250012, PR China.
E-mail address: louhongxiang@sdu.edu.cn (H.-x. Lou).
These authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2017.05.050
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

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B. Sun et al. / European Journal of Medicinal Chemistry 136 (2017) 603e618
Fig. 1. The chemical structures of riccardin D, RDN-1 and RDN-2.
studies indicated that both compounds could induce LMP and
cathepsin release from the lysosomes into the cytosol, and
furthermore could induce cell death that displayed features char-
acteristic to both apoptosis and necrosis. We then predicted that
the introduction of basic groups to riccardin D might improve its
anticancer activity by promoting lysosome targeting [20,21].
Based on the findings described above, a series of riccardin D
derivatives with weakly basic groups, especially groups that con-
tained nitrogen atoms, was designed. Our modification strategy for
riccardin D is shown in Fig. 2. We first modified the Mannich side
chains of RDN-1 and RDN-2, and changed the methylamino groups
to either ethylamino, pyrrolidinyl, piperidinyl, morpholino, thio-
morpholino, 4-hydroxypiperidinyl, 4-(4-methoxyphenyl)piper-
azinyl, 4-methylpiperazinyl, bis((dibutylamino)methyl) amino, or
bis(2-methoxyethyl)amino groups to evaluate how the Mannich
side chains influence anticancer activities. Second, we directly
introduced a nitrogen atom into the arene ring of riccardin D and
prepared some derivatives based on the structure of aniline to
determine the effect of these substitutions on the cytotoxic activity
of riccardin D. Finally, we focused on the modification of phenolic
groups of riccardin D by introducing O-alkylation of different
nitrogen-containing electrophiles, and the resulting mono- and tri-
O-alkylated derivatives were obtained for structure-activity rela-
tionship (SAR) studies.
potent anticancer agents that target lysosomes.
2. Results and discussion
2.1. Chemistry
Riccardin D was prepared in 11 steps with a satisfactory yield
following a procedure reported previously [22], and was used as a
staring material for further structural modifications. The general
procedure is outlined in Scheme 1. Mannich reactions of riccardin D
were carried out using different secondary amines [23]. The reac-
tion of riccardin D, amine, and formaldehyde at a molar ratio of
1:1.5:1.5 in ethanol yielded the mono-Mannich derivatives
10ae10m as the main product at a moderate yield. The position of
the Mannich side chain was then determined based on a NOE
spectrum. As shown in Fig. 3, correlations between H2-130 and H-50
were determined for representative compound 10l (Figs. S25 and
S26), which indicated that mono-Mannich derivatives have Man-
nich side-chains on the 40-position of arene B. The substitution
pattern in the mono-Mannich derivatives was consistent with that
of RDN-1, which might occur because of the stereo-hindrance effect
was reduced (compared with 2-position on arene D) and a higher
electron density (compared with 13-position on arene C) was
present at the 40-position of arene B. When the quantity of the
secondary amine and formaldehyde were increased to 3 and 3
equivalents, respectively, the bis-Mannich derivatives 11ae11m
were obtained as the main products. Basic groups were introduced
at the 40-position on arene B and 13-position on arene C, which was
confirmed by the NOE spectrum of the representative derivative
11l. As shown in Fig. 3, correlations of H2-130 with H-50, and H2-170
with H-14 were determined (Figs. S53 and S54, respectively).
Herein, we describe the synthesis of a series of novel nitrogen-
containing riccardin D derivatives and the evaluation of these
compounds for antiproliferative activities using the MTT assay. The
anticancer mechanisms of two representative derivatives were also
investigated using flow cytometry and confocal microscopy.
Together, these findings indicated that these novel weakly basic
derivatives of macrocyclic bisbibenzyl represent a novel class of
Fig. 2. Modification strategy for riccardin D derivatives.

B. Sun et al. / European Journal of Medicinal Chemistry 136 (2017) 603e618
605
Scheme 1. Synthesis of riccardin D. Reagents and conditions: (a) i. CuO, K2CO3, Py, reflux, 80%; ii. LiAlH4, THF, ꢀ40 ꢁC to r.t., 92%; iii. PPh3HBr, MeCN, reflux, 92%; (b) Pd(PPh3)4,
toluene, EtOH, Na2CO3, reflux, 75%; (c) K2CO3, 18-crown-6, DCM, reflux, 90%; (d) i. Pd/C (10%), H2, Et3N, EtOAc, r.t., 95%; ii. HCl/THF (1:1), r.t., 93%; iii. PPh3HBr, MeCN, reflux, 97%; (e)
NaOMe, DCM, r.t., 89%; (f) i. Pd/C (10%), H2, EtOAc, r.t., 92%; ii. BBr3, DCM, ꢀ40 ꢁC to r.t., 86%.
Fig. 3. Key NOESY correlations (dashed blue arrows) for compounds 10l and 11l. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
Derivative 12 with a nitro group at 13-position was prepared
from riccardin D and nitric acid at a moderate yield. The nitro group
of compound 12 was reduced by hydrogen over Pd/C to yield
compound 13, which was underwent reductive methylation to
generate derivative 14 [24]. Compound 13 was acetylated by acetyl
chloride to give derivative 15, and the selective deacetylation of

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B. Sun et al. / European Journal of Medicinal Chemistry 136 (2017) 603e618
compound 15 by sodium hydroxide provided derivative 16 [25,26].
The alkylation of riccardin D with selected amino-alkyl chloride
electrophiles [e.g., 2-chloro-N,N-dimethylethanamine hydrochlo-
ride] at a molar ratio of 1:1.5 in acetone gave the mono-alkylated
derivatives 17ae17h as the main product in the presence of po-
tassium carbonate [27]. The structural assignment of representative
compound 17a was determined based on the NOE spectrum. As
shown in Fig. 4, correlations of H2-130 with H-40, and H3-150 with H-
2 were determined for compound 17a (Fig. S69), which indicated
that the monoalkylated derivatives have basic side chains on arene
B. When the quantity of amino-alkyl chloride electrophiles and
potassium carbonate were increased to 5 and 10 equivalents,
respectively, the tri-alkylated derivatives 18ae18h were obtained
at a good yield.
excellent cytotoxic activity against the A549, MCF-7, and k562 cell
lines, with IC50 values of 0.99, 0.43, and 0.80
which were comparable to those of ADR.
mM, respectively,
The SARs of these novel riccardin D analogues were analyzed
and are summarized below.
2.2.1.1. Effects of the Mannich side chains. As shown in Scheme 2,
for the first step we introduced different Mannich side chains to the
40-position of riccardin D, and then prepared 13 mono-Mannich
derivatives, 10ae10m. The anticancer activities of these com-
pounds are shown in Table 1. Compounds 10ae10c, 10f, 10h and 10j
were substituted with less bulky Mannich side chains, such as
ethylamino, pyrrolidinyl, piperidinyl, 4-hydroxypiperidinyl, and 4-
methylpiperazinyl groups, and exhibited improved anticancer ac-
tivities compared with riccardin D and RDN-1, with IC50 values
ranging from 2.26 to 9.19 mM. By contrast, changing the piperidinyl
2.2. Biological evaluations
group to a morpholino or thiomorpholino group led to diminished
cytotoxic activity (e.g., 10d and 10e). To our surprise, when the
more sterically bulky Mannich side chains, such as 4-(4-
2.2.1. In vitro antiproliferative activity and SAR analysis
The cytotoxic activity of the synthesized analogues (10ae10m,
11ae11m, 12e16, 17ae17h, and 18ae18h) was evaluated in parallel
in three human cancer cell lines (A549, MCF-7 and k562) using
adriamycin (ADR), riccardin D (RD), RDN-1, and RDN-2 as reference
compounds. Cells were treated with each compound for 48 h, and
viability was assessed using the MTT method. Results are summa-
rized in Table 1.
Among these novel molecules, the tri-O-alkylated derivative 18a
exhibited the most potent anticancer activity against the A549,
MCF-7, and k562 cell lines, with IC50 values of 0.51, 0.23, and
methoxyphenyl)piperazinyl,
bis((dibutylamino)methyl)amino,
and bis(2-methoxyethyl)amino groups, were introduced into ric-
cardin D, the resulting derivatives 10g, 10i and 10ke10m showed
reduced antiproliferative potency, with IC50 values greater than
20
mM. We hypothesized that the receptor binding site for the
Mannich side chains was relatively small in size so that it could
barely accommodate large substituents.
In addition, 13 bis-Mannich derivatives, 11ae11m, were then
prepared and evaluated for anticancer activities. As shown in
Table 1, the bis-Mannich derivatives with less bulky substituents
displayed obviously enhanced cytotoxic activity compared with
mono-Mannich derivatives (11a vs. 10a, 11b vs. 10b, 11c vs. 10c, 11e
vs. 10e, 11f vs.10f, 11h vs. 10h, and 11j vs.10j). Moreover, compound
11b had two pyrrolidinyl groups and was the most potent com-
pound among all of the Mannich derivatives that we tested. How-
ever, the introduction of bulky basic groups led to bis-Mannich
derivatives 11g, 11i, and 11ke11m that did not have anticancer
activity.
0.19
mM, respectively, which was clearly superior to those of the
parent compound riccardin D (IC50 ¼ 28.14, 18.31, and 17.56
mM,
respectively). Surprisingly, these values were three- to ten-fold
better than those of the clinically used drug ADR (IC50 ¼ 1.45,
2.30, and 0.61
mM, respectively). Additionally, three other tri-O-
alkylated derivatives, 18b, 18d and 18e, also exhibited significant
antiproliferative activities, with IC50 values ranging from 0.24 to
0.97
mM. Moreover, the bis-Mannich derivative 11b displayed
We then found the following cytotoxic activity trend based
on the small Mannich side chains: pyrrolidinyl > piperidinyl ¼
ethylamino > 4-methylpiperazinyl > 4-hydroxypiperidinyl > (2-
hydroxyethyl)(methyl)amino
> thiomorpholino > morpholino.
Further increasing the size of the basic side chains resulted in total
loss of activity, which suggested the existence of a relatively small
binding pocket for the receptor around the Mannich base moiety,
with a pyrrolidinyl group representing the optimal size. These
findings indicated that the presence of a less bulky Mannich base at
the 40-position of riccardin D is critical for anticancer activity, and
that the introduction of a second Mannich side chain could
dramatically enhance the activity, perhaps because of the increased
total basicity of the molecule. These data are consistent with our
prediction mentioned above.
2.2.1.2. Effects of the aniline substituents. To obtain more SAR in-
formation, the nitrogen atom was directly introduced into the arene
ring of riccardin D and, as shown in Scheme 3, a series of derivatives
based on aniline were obtained (compounds 12e16). However, all
of these derivatives displayed reduced anticancer activity
compared with riccardin D, with an IC50 value greater than 20
mM.
This trend might occur because of p- conjugate interactions be-
p
tween the nitrogen atom and arene ring, which led to a subtle effect
on the pKa of aniline and a reduction in basicity.
2.2.1.3. Effects of basic ether side chains. After confirming the ef-
fects of Mannich side chains and an aniline moiety on anticancer
activity, we then focused on modifying riccardin D by O-alkylation
Fig. 4. Key NOESY correlations (dashed blue arrows) for compound 17a. (For inter-
pretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)

B. Sun et al. / European Journal of Medicinal Chemistry 136 (2017) 603e618
607
Table 1
In Vitro cytotoxicity of riccardin D derivatives against three cancer cell lines.
Cpd.
IC50
(m
M)a
Cpd.
IC50 (m
M)a
A549
MCF-7
k562
A549
MCF-7
k562
10a
10b
10c
10d
10e
10f
10g
10h
10i
5.74 1.01
2.26 0.36
7.12 1.22
>20
16.28 3.25
5.74 1.07
>20
9.07 2.31
>20
5.59 0.98
>20
7.59 1.69
2.41 0.55
5.07 1.52
>20
15.77 3.98
9.05 2.15
>20
8.28 2.00
>20
6.02 1.74
>20
7.86 2.01
4.82 1.60
5.88 1.24
>20
13.44 2.96
7.07 1.13
>20
9.19 2.07
>20
3.31 0.58
>20
12
13
14
15
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
16
>20
17a
17b
17c
17d
17e
17f
17g
17h
18a
18b
18c
18d
18e
18f
18g
18h
RDb
RDN-1
RDN-2
ADR
1.30 0.21
1.87 0.34
2.02 0.41
2.13 0.52
2.98 0.67
7.49 1.58
1.99 0.29
4.53 1.20
0.51 0.11
0.78 0.14
1.04 0.26
0.97 0.14
0.53 0.11
5.23 1.23
1.44 0.32
1.67 0.35
28.14 5.11
13.25 2.35
4.81 0.92
1.45 0.27
2.38 0.34
2.73 0.25
2.42 0.19
2.49 0.46
3.03 0.50
5.02 1.02
3.25 0.38
3.68 0.50
0.23 0.06
0.56 0.16
0.88 0.25
0.24 0.02
0.66 0.15
4.19 0.59
3.05 0.50
2.12 0.44
18.31 3.14
12.54 2.01
4.22 0.96
2.30 0.44
1.56 0.33
2.23 0.40
2.22 0.31
2.31 0.44
2.73 0.28
10.05 1.25
2.61 0.38
2.94 0.49
0.19 0.02
0.65 0.11
1.79 0.44
0.56 0.12
0.26 0.04
3.43 0.58
2.50 0.41
2.13 0.37
17.56 3.20
10.14 1.65
2.84 0.39
0.61 0.20
10j
10k
10l
>20
>20
>20
>20
>20
>20
10m
11a
11b
11c
11d
11e
11f
11g
11h
11i
3.78 0.72
0.99 0.24
4.85 0.96
>20
10.21 1.71
3.26 0.62
>20
5.21 1.02
>20
2.16 0.38
>20
1.05 0.16
0.43 0.09
2.04 0.54
>20
9.25 2.00
3.95 0.75
>20
6.00 1.57
>20
2.89 0.49
>20
1.86 0.37
0.80 0.20
0.66 0.17
>20
8.35 1.39
2.56 0.35
>20
5.67 1.17
>20
3.32 0.55
>20
11j
11k
11l
>20
>20
>20
11m
>20
>20
>20
a
The IC50 values (mM) indicate the concentrations corresponding to 50% inhibition of the growth of the indicated cell line growth; Mean values are based on three in-
dependent experiments.
b
RD represent riccardin D.
Scheme 2. Synthesis of aminomethylation derivatives of riccardin D (10a-10m, 11a-11m). Reagents and conditions: (a) HNR1R2 (1.5 eq), 37% HCHO (1.5 eq), EtOH, reflux, 62%e75%;
(b) HNR1R2 (3 eq), 37% HCHO (3 eq), EtOH, reflux, 51%e70%.
of the hydroxyl group with a series of nitrogen-containing elec-
trophiles to evaluate the effects of these aminoalkyl side chains. As
shown in Scheme 4, 16 mono- and tri-O-alkylated derivatives were
prepared; the anticancer activities of these compounds are pre-
sented in Table 1. Surprisingly, when the modification pattern was
changed from aminomethylation to O-alkylation, the “effect of
large size” disappeared, and all of the O-alkylated derivatives dis-
played potent cytotoxic activity. Introduction of aminoalkyl side
chains to the C-30 position on arene B yielded the mono-alkylated
derivatives 17ae17h, which exhibited markedly improved anti-
cancer activity compared with riccardin D, with IC50 values ranging
ethyl group, was found to be the most potent agent among the
mono-alkylated series, with IC50 values of 1.30 M (for A549),
2.38 M (for MCF-7) and 1.56 M (for K562), respectively. When the
m
m
m
(diethylamino)ethyl, five-membered (pyrrolidin-1-yl)ethyl, six-
membered (piperidin-1-yl)ethyl, or seven-membered (azepan-1-
yl)ethyl groups were introduced to the C-30 position on arene B,
the anticancer activities of the resulting compounds 17b, 17c, 17d,
and 17e, respectively, were slightly diminished compared with 17a.
Unexpectedly, the cytotoxic activity of compound 17f, which had a
six-membered (morpholino-1-yl)ethyl group at position C-30 of
arene B, was markedly reduced compared with compound 17d
(which contained a cyclic six-membered aminoalkyl side chain).
from 1.30 to 10.05 mM. Compound 17a, with the (dimethylamino)

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B. Sun et al. / European Journal of Medicinal Chemistry 136 (2017) 603e618
Scheme 3. Synthesis of amination derivatives of riccardin D 12e16. Reagents and conditions: (a) HNO3, AcOH, DCM, 24 h, 65%; (b) H2, Pd/C (10%), MeOH, r.t., 8 h, 80%; (c) HCHO,
NaBH4, THF, 2 h, 54%; (d) AcCl, Et3N, DCM, 0 ꢁC to r.t., 92%; (e) 1 N NaOH, 0 ꢁC, 30 min, 64%.
Scheme 4. Synthesis of alkylation derivatives of riccardin D (17a-17h, and 18a-18h). Reagents and conditions: (a) RCl$HCl (1.5 eq), K2CO3 (5 eq), acetone, 60 ꢁC, 66%e78%; (b) RCl$HCl
(5 eq), K2CO3 (10 eq), acetone, 60 ꢁC, 83%e90%.
Additionally, when the ethyl linkage in the aminoalkyl side chain of
compounds 17a and 17c was expanded to three carbon atoms, the
resulting compounds 17g and 17h also displayed reduced activity.
Further introduction of aminoalkyl side chains resulted in tri-O-
alkylated derivatives 18ae18h. Among these derivatives, com-
pound 18a, bearing three (dimethylamino)ethyl groups, was found
improved pKa values of the molecules.
The antiproliferative activity of the most potent compounds 11b
and 18a was representative of the Mannich and O-alkylated series,
respectively. These compounds were further assessed in A549 cells
using the xCELLigence system; hydroxycholoroquine (HCQ) was
used as a positive control. As shown in Fig. 5, compound 11b
inhibited A549 cell proliferation within 5 h and markedly reduced
the number of cells within 20 h, which is similar to the actions of
HCQ. After treatment for ~60 h, the effects of 11b were similar to
those of HCQ. The cell growth curve shows that compound 11b
inhibited A549 cell proliferation and, subsequently, induced cell
death.
to be the most potent agent with IC50 values of 0.51
mM (for A549),
0.23 M (for MCF-7) and 0.19 M (for K562), respectively. Inter-
m
m
estingly, when the dimethylamino groups of 18a were changed to
larger diethylamino or pyrrolidin-1-yl groups, the resulting com-
pounds 18b and 18c displayed diminished activity. However,
further increasing the size to piperidin-1-yl and azepan-1-yl groups
reversed this trend in activity, and led to derivatives 18d and 18e
that exhibited more potent anticancer activity compared with 18b
and 18c. The introduction of morpholino-1-yl groups or extending
the ethyl linkage in the aminoalkyl side chains also resulted in
decreased activity (18f vs. 18d, 18g vs. 18a, and 18h vs. 18c), which is
consistent with our observations of the mono-alkylated series
described above. Notably, all of the tri-O-alkylated derivatives
displayed more potent antiproliferative activity than the corre-
sponding mono-O-alkylated derivatives (18a vs.17a,18b vs.17b,18c
vs. 17c, 18d vs. 17d, 18e vs. 17e, 18f vs. 17f, 18g vs. 17g, and 18h vs.
17h), perhaps because the loss of acidic phenol groups and the
increase in basic centers occurred simultaneously, resulting in
2.2.2. Riccardin D derivatives induce lysosomal membrane
permeabilization (LMP)
After the evaluating the antiproliferative activities, we
extended our work to a mechanistic and phenotypic study of the
effects of riccardin
D derivatives on lysosomal stability by
applying confocal microscopy and flow cytometry. Two lysoso-
motropic fluorescence probes, AO and LysoTracker Red, were
used to determine the effect of compounds 11b and 18a on the
lysosomal stability of A549 cancer cells. AO, a metachromatic
fluorophore, is retained in its charged form (AOHþ) by proton
trapping inside of lysosomes and emits red fluorescence.

B. Sun et al. / European Journal of Medicinal Chemistry 136 (2017) 603e618
609
Fig. 5. A549 cells were treated with compound 11b and 18a as indicated. Cells were then plated 2000 cells/well into E-plate 16 and analyzed using a xCELLigence RTCA DP in-
strument. The results are representative of three independent experiments.
Lysosomal rupture in these AO-loaded cells results in diminished
red fluorescence. LysoTracker Red, a fluorescent weak base, could
accumulate in acidic organelles and successfully stain lysosomes
in living cells. The LysoTracker Red and AO-uptake in lysosomes
results were monitored by confocal microscopy and flow
cytometry. As shown in Fig. 6, after treatment with compounds
11b or 18a for 6 h, lysosomal rupture could be observed, as
indicated by increased numbers of “pale cells,” i.e., cells with
reduced numbers of LysoTracker Red- and AO-accumulating ly-
sosomes. Our data demonstrated that both of the riccardin de-
rivatives 11b and 18a could induce lysosomal membrane
permeabilization in A549 cancer cells.
both Mannich and O-alkylated derivatives could target the lyso-
some and induce lysosomal membrane permeabilization, which
could lead to cell death that displayed features characteristic to
both apoptosis and necrosis. Together, these data indicated that the
introduction of nitrogen-containing groups to riccardin D, which
increased the total basicity of the molecule, is critical for the
improved cytotoxicity. This activity might occur because the basic
derivatives accumulate in acidic lysosomes, which further results in
the death of cancer cells. In conclusion, this present study discov-
ered several potent nitrogen-containing bisbibenzyl derivatives,
and our systematic research has yielded novel molecular scaffolds
for the further development of antitumor agents that target
lysosomes.
2.2.3. Riccardin D derivatives trigger apoptosis and necrosis in
A549 cells
4. Experimental section
The significant antiproliferative activity of riccardin D de-
rivatives and their obvious lysosomal rupture properties encour-
aged us to future investigate the underlying biological mechanism.
We examined the effect of compounds 11b and 18a on apoptosis
and necrosis in A549 cells using microscopy and flow cytometry. As
shown in Fig. 7A, after treatment with compounds 11b or 18a,
morphological changes in A549 cells were observed using DAPI
staining. Both derivatives induced marked morphological alter-
ations characteristic of apoptosis, including cell shrinkage and
granular apoptotic body formation. Additionally, A549 cells treated
with specified doses of 11b and 18a were stained with Annexin V-
FITC and PI, respectively, and then were analyzed by flow cytometry
to assess whether both derivatives induced cell death in cancer
cells via apoptosis or necrosis. Annexin V-FITCþ/PI- represents a
hallmark of early apoptosis. Annexin V-FITCþ/PIþ staining repre-
sents the end-stage of apoptosis or necrosis. As shown in Fig. 7B,
treatment with compounds 11b or 18a caused significant increases
in the fraction of apoptotic and necrotic A549 cancer cells,
respectively.
4.1. Chemistry
Chemicals were commercially available and used as received
without further purification. Solvents (THF, Acetone, MeCN and
DCM) were dried and freshly distilled before use according to
procedures reported in the literature. Reactions were monitored by
thin-layer chromatography using Merck plates with fluorescent
indicator. Column chromatography was carried out on silica gel or
alumina (200e300 mesh). Melting points were determined in
capillary tubes using a Mel-Temp apparatus and are not corrected.
Infrared spectra were obtained as films on salt plates using CHCl3 as
the solvent except where otherwise specified, using a Perkin-Elmer
Spectrum One FT-IR spectrometer, and are baseline-corrected. The
NMR spectra were recorded on a Bruker Spectrospin spectrometer
at 600 MHz (13C NMR at 150 MHz) using TMS as an internal stan-
dard. The chemical shifts are reported in parts per million (ppm d)
referenced to the residual 1H resonance of the solvent (CDCl3,
7.26 ppm). Abbreviations used in the splitting pattern were as
follows: s ¼ singlet, d ¼ doublet, t ¼ triplet, quin ¼ quintet,
m ¼ multiplet, and br ¼ broad. Mass spectral analyses were per-
formed on SHIMASZU LCMS 2020 at the Shandong University-
Chemical Analysis Center. All HRMS spectra (ESI) were obtained
on a LTQ Orbitrap mass spectrometer.
3. Conclusions
In this present study, a series of novel riccardin D derivatives
was designed, synthesized and evaluated for antiproliferative ac-
tivity against three anthropic cancer cell lines, resulting in the
discovery of several novel and potent antitumor agents. Among
these molecules, the tri-O-alkylated compound 18a displayed the
most potent anticancer activity, with IC50 values that ranged from
4.1.1. General procedure 1 (GP 1) for the Mannich reaction
A mixture of riccardin D, secondary amine and aqueous form-
aldehyde (37%) in ethanol was stirred under reflux for 24 h. The
solvent was then removed in vacuo and the residue was purified by
flash column chromatography, eluting with a gradient mixture of
EtOAc/MeOH/ aqueous ammonia (93:5:2 to 80:18:2, V/V), to yield
the pure product (compounds 10a-10m and 11a-11m) as a solid in
moderate yield.
0.19 to 0.51
mM. This activity was obviously superior to that of
parent riccardin D, and was 3e10-fold better than the clinically
used drug ADR. The bis-Mannich derivative 11b also exhibited
significantly improved antiproliferative potency, with an IC50 value
in the submicromolar range. Our mechanistic studies indicated that

610
B. Sun et al. / European Journal of Medicinal Chemistry 136 (2017) 603e618
Fig. 6. Compounds 11b and 18a induce lysosomal membrane permeabilization (LMP) in cancer cells. (A) Lysosomal rupture assayed based on LysoTracker Red- and AO-uptake
methods. A549 cells were treated with 11b and 18a for 6 h, and then were exposed to LysoTracker Red and AO as described in the experimental section. Cells were measured
by confocal microscopy. (B) A549 cells were treated with 11b and 18a for 6 h, exposed to LysoTracker Red and AO, and then finally measured by flow cytometry.
4.1.2. General procedure 2 (GP 2) for the alkylation of phenol
A mixture of riccardin D, alkyl chloride hydrochloride and po-
tassium carbonate in acetone was stirred under reflux for 24 h. The
insoluble material was then filtered off and the filtrate was
concentrated to provide the residue that was purified by flash
column chromatography, eluting with a gradient mixture of MeOH/
EtOAc/aqueous ammonia (93:5:2 to 80:18:2, V/V), to yield the pure
product (compounds 17a-17f and 18a-18f) as a solid in good yield.
(t, J ¼ 7.9 Hz, 1H), 7.08 (dd, J ¼ 7.7, 1.1 Hz, 1H), 6.97e6.89 (m, 3H),
6.86e6.83 (m, 3H), 6.78 (d, J ¼ 8.0 Hz, 1H), 6.75 (dd, J ¼ 8.1, 1.9 Hz,
1H), 6.53 (d, J ¼ 1.4 Hz, 1H), 6.32 (dd, J ¼ 7.7, 1.5 Hz, 1H), 5.60 (s, 1H),
5.42 (d, J ¼ 1.9 Hz, 1H), 4.87 (s, 1H), 4.77 (s, 1H), 3.04e2.96 (m, 2H),
2.87e2.92 (m, 1H), 2.83e2.72 (m, 2H), 2.69e2.56 (m, 3H); MS (ESI)
425 (MþH)þ.
4.1.4. 40-((Diethylamino)methyl)-riccardin D (10a)
This compound was prepared from riccardin
D (60 mg,
4.1.3. Riccardin D
0.142 mmol), diethylamine (15 mg, 0.212 mmol), and aqueous
formaldehyde (18 mg, 0.212 mmol) by following GP 1 in 66% yield.
White solid; mp 110e1110 ꢁC. IR (film) 3368, 1588, 1521, 1463, 1365,
Riccardin D (5 g) was prepared from compounds 1, 2, 4, and 5 in
11 steps with satisfied yield by following the procedure reported
previously [22]. White solid. mp 152e153 ꢁC; 1H NMR (CDCl3)
d
7.36
1150, 956 cmꢀ1
;
1H NMR (CDCl3)
d7.08 (d, J ¼ 7.8 Hz, 1H), 6.96 (d,

B. Sun et al. / European Journal of Medicinal Chemistry 136 (2017) 603e618
611
Fig. 7. Analysis of apoptosis and necrosis in compound 11b- and 18a-treated A549 cancer cell lines. (A) A549 cells were treated with various concentrations of 11b or 18a for 8 h,
stained with DAPI, and finally measured by microscopy. (B) A549 cells were treated with various concentrations of 11b or 18a for 8 h. Cells were collected and stained with FITC-
conjugated Annexin V and PI and analyzed using flow cytometry. The mean percentages of Annexin V/(PIþ plus PIꢀ) cells obtained from three independent experiments with the
indicated cell lines SD are shown.
J ¼ 7.8 Hz, 1H), 6.93e6.86 (m, 4H), 6.85e6.77 (m, 2H), 6.74 (dd,
J ¼ 8.1, 2.0 Hz, 1H), 6.54 (dd, J ¼ 7.8, 1.8 Hz, 1H), 6.29 (d, J ¼ 1.7 Hz,
1H), 5.38 (d, J ¼ 2.0 Hz, 1H), 3.87 (q, J ¼ 14.3 Hz, 2H), 3.01e2.89 (m,
3H), 2.86e2.76 (m, 3H), 2.69 (q, J ¼ 7.2 Hz, 4H), 2.65e2.50 (m, 2H),
4.1.7. 40-(Morpholinomethyl)-riccardin D (10d)
This compound was prepared from riccardin
D (60 mg,
0.142 mmol), morpholine (18 mg, 0.212 mmol), and aqueous
formaldehyde (18 mg, 0.212 mmol) by following GP 1 in 65% yield.
White solid; mp 130e131 ꢁC. IR (film) 3359, 1580, 1527, 1424, 1351,
1.11 (t, J ¼ 7.2 Hz, 6H); 13C NMR (CDCl3)
d 155.2, 153.3, 152.8, 146.7,
143.6, 143.3, 142.1, 140.4, 133.3, 132.1, 129.7, 128.9, 128.5, 124.5,
122.5, 122.0, 121.8, 121.6, 120.9, 119.4, 118.7, 116.2, 114.9, 56.4, 46.2,
38.1, 37.7, 36.8, 34.5, 10.7; MS (ESI) 510 (MþH)þ; HRMS (ESI) calcd
1139, 941 cmꢀ1
;
1H NMR (CDCl3)
d
7.14 (d, J ¼ 8.2 Hz, 1H), 6.99 (d,
J ¼ 7.8 Hz,1H), 6.91 (d, J ¼ 8.1 Hz, 2H), 6.89e6.85 (m, 2H), 6.83e6.77
(m, 2H), 6.74 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.51 (dd, J ¼ 7.8, 1.8 Hz, 1H),
6.32 (d, J ¼ 1.7 Hz, 1H), 5.38 (d, J ¼ 2.0 Hz, 1H), 3.83e3.68 (m, 6H),
for C33H36NO4 510.2639; found 510.2633 (MþH) þ
.
2.98e2.75 (m, 7H), 2.70e2.51 (m, 5H); 13C NMR (CDCl3)
d 156.6,
154.3, 153.7, 153.0, 152.8, 146.7, 146.0, 143.3, 140.3, 133.2, 132.0,
129.6, 129.0, 124.1, 122.5, 122.3, 122.1, 121.2, 118.8, 116.1, 114.9, 66.2,
60.4, 52.7, 38.0, 37.7, 36.8, 34.6; MS (ESI) 524 (MþH)þ; HRMS (ESI)
4.1.5. 40-((Pyrrolidin-1-yl)methyl)-riccardin D (10b)
This compound was prepared from riccardin D (60 mg,
0.142 mmol), pyrrolidine (15 mg, 0.212 mmol), and aqueous
formaldehyde (18 mg, 0.212 mmol) by following GP 1 in 69% yield.
White solid; mp 125e126 ꢁC. IR (film) 3351, 1571, 1510, 1449, 1360,
calcd for C33H34NO5 524.2431; found 524.2438 (MþH) þ
.
1141, 950 cmꢀ1; 1H NMR (CDCl3)
d
7.20 (d, J ¼ 7.8 Hz, 1H), 6.98 (d,
4.1.8. 40-(Thiomorpholinomethyl)-riccardin D (10e)
J ¼ 7.8 Hz, 1H), 6.91 (d, J ¼ 6.7 Hz, 1H), 6.89 (d, J ¼ 7.3 Hz, 2H), 6.86
(d, J ¼ 7.7 Hz, 1H), 6.80 (d, J ¼ 7.8 Hz, 2H), 6.73 (dd, J ¼ 8.1, 2.0 Hz,
1H), 6.50 (dd, J ¼ 7.8, 1.7 Hz, 1H), 6.35 (d, J ¼ 1.7 Hz, 1H), 5.37 (d,
J ¼ 2.0 Hz, 1H), 4.09 (d, J ¼ 13.6 Hz, 1H), 3.92 (d, J ¼ 13.6 Hz, 1H),
2.99e2.74 (m, 9H), 2.73e2.47 (m, 3H), 1.91e1.89 (m, 4H); 13C NMR
This compound was prepared from riccardin D (60 mg,
0.142 mmol), thiomorpholine (20 mg, 0.212 mmol), and aqueous
formaldehyde (18 mg, 0.212 mmol) by following GP 1 in 63% yield.
White solid; mp 142e143 ꢁC. IR (film) 3357, 1571, 1525, 1467, 1354,
1138, 935 cmꢀ1 1H NMR (600 MHz, CDCl3)
; d 7.16 (s, 1H), 7.00 (d,
(CDCl3)
d
154.3, 153.3, 152.8, 146.7, 144.3, 143.4, 142.6, 140.3, 133.2,
J ¼ 7.8 Hz, 1H), 6.91 (d, J ¼ 8.1 Hz, 1H), 6.91e6.87 (m, 2H), 6.86 (d,
J ¼ 7.7 Hz, 1H), 6.81 (d, J ¼ 8.1 Hz, 2H), 6.74 (dd, J ¼ 8.1, 2.0 Hz, 1H),
6.54e6.45 (m, 1H), 6.34 (s, 1H), 5.37 (d, J ¼ 1.9 Hz, 1H), 3.99e3.73
(m, 2H), 3.12e2.90 (m, 5H), 2.90e2.74 (m, 6H), 2.75e2.47 (m, 5H);
132.0, 129.7, 129.1, 128.9, 124.5, 122.5, 122.2, 122.1, 120.5, 119.1,
116.1, 114.9, 61.6, 53.3, 38.0, 37.7, 36.8, 34.7, 23.5; MS (ESI) 508
(MþH)þ; HRMS (ESI) calcd for C33H34NO4 508.2482; found
þ
508.2488 (MþH)
.
13C NMR (CDCl3)
d 154.3, 153.0, 152.7, 146.6, 143.3, 140.3, 133.2,
131.9, 129.5, 129.0, 124.3, 122.5, 122.1, 122.0, 121.3, 118.7, 116.1, 114.9,
60.4, 54.2, 37.9, 37.7, 36.7, 34.6; MS (ESI) 540 (MþH)þ; HRMS (ESI)
4.1.6. 40-((Piperidin-1-yl)methyl)-riccardin D (10c)
calcd for C33H34NO4S 540.2203; found 540.2207 (MþH) þ
.
This compound was prepared from riccardin
D (60 mg,
0.142 mmol), piperidine (17 mg, 0.212 mmol), and aqueous form-
aldehyde (18 mg, 0.212 mmol) by following GP 1 in 62% yield.
White solid; mp 134e1350 ꢁC. IR (film) 3374,1590,1522,1469,1363,
4.1.9. 40-((4-Hydroxypiperidin-1-yl)methyl)-riccardin D (10f)
This compound was prepared from riccardin (60 mg,
0.142 mmol), piperidin-4-ol (22 mg, 0.212 mmol), and aqueous
formaldehyde (18 mg, 0.212 mmol) by following GP 1 in 62% yield.
White solid; mp 177e178 ꢁC. IR (film) 3360, 1578, 1531, 1473, 1356,
D
1159, 910 cmꢀ1
;
1H NMR (CDCl3)
d
7.22 (s, 1H), 6.99 (d, J ¼ 7.9 Hz,
1H), 6.91 (d, J ¼ 7.9 Hz, 1H), 6.89 (d, J ¼ 8.0 Hz, 2H), 6.85 (d,
J ¼ 7.7 Hz, 1H), 6.82e6.78 (m, 2H), 6.73 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.49
(dd, J ¼ 7.7, 1.7 Hz, 1H), 6.37 (s, 1H), 5.37 (d, J ¼ 1.9 Hz, 1H),
3.93e3.87 (m, 2H), 3.21e2.89 (m, 4H), 2.89e2.81 (m, 2H),
2.80e2.76 (m, 2H), 2.70e2.29 (m, 4H), 1.90e1.30 (m, 6H); 13C NMR
1149, 976 cmꢀ1; 1H NMR (CD3OD)
d
7.07 (d, J ¼ 7.8 Hz, 1H), 6.93 (d,
J ¼ 7.8 Hz, 2H), 6.86 (d, J ¼ 7.9 Hz, 1H), 6.79 (d, J ¼ 7.9 Hz, 2H),
6.77e6.73 (m, 2H), 6.68 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.31 (dd, J ¼ 7.8,
1.7 Hz, 1H), 6.24 (d, J ¼ 1.6 Hz, 1H), 5.44 (d, J ¼ 2.0 Hz, 1H), 3.84 (s,
2H), 3.73e3.68 (m, 1H), 3.00e2.85 (m, 5H), 2.80e2.55 (m, 5H),
2.49e2.38 (m, 2H), 1.97e1.79 (m, 2H), 1.62e1.53 (m, 2H); 13C NMR
(CDCl3)
d 154.3, 153.2, 152.7, 149.1, 146.7, 143.3, 142.8, 140.3, 133.2,
131.9, 129.6, 128.9, 124.7, 122.6, 122.4, 122.1, 122.0, 121.2, 119.0, 116.1,
114.9, 60.4, 53.4, 37.9, 37.7, 36.7, 34.7, 24.4, 23.1, 14.2; MS (ESI) 522
(MþH)þ; HRMS þ(ESI) calcd for C34H36NO4 522.2639; found
(CD3OD)
d 156.3, 155.4, 154.8, 148.9, 145.3, 145.0, 142.9, 141.7, 134.3,
522.2645 (MþH)
.
133.9,130.6,130.1,129.6,127.0,123.5,123.1,122.9,122.2,122.1,121.0,

612
B. Sun et al. / European Journal of Medicinal Chemistry 136 (2017) 603e618
119.4, 118.2, 118.0, 116.9, 61.5, 51.2, 49.5, 49.3, 38.9, 38.0, 35.9, 34.6;
1.7 Hz, 1H), 6.29 (d, J ¼ 1.6 Hz, 1H), 5.36 (d, J ¼ 1.9 Hz, 1H), 3.92 (d,
J ¼ 14.0 Hz, 1H), 3.73 (d, J ¼ 13.9 Hz, 1H), 3.16e2.89 (m, 5H),
2.89e2.81 (m, 4H), 2.79 (d, J ¼ 10.6 Hz, 3H), 2.68e2.49 (m, 4H), 2.45
MS (ESI) 538 (MþH)þ; HRMS (ESI) calcd for C34H36NO5 538.2588;
found 538.2582 (MþH) þ
.
(s, 3H); 13C NMR (CDCl3)
d 154.4, 152.9, 152.7, 146.6, 144.2, 143.3,
4.1.10. 40-((4-(4-Methoxyphenyl)piperazin-1-yl)methyl)-riccardin
D (10g)
142.3, 140.3, 133.2, 132.0, 129.5, 129.0, 129.1, 129.0, 124.0, 122.5,
122.3, 122.2, 122.0, 121.0, 118.6, 117.7, 116.1, 114.9, 65.3, 60.9, 60.4,
42.0, 38.0, 37.7, 36.8, 34.5; MS (ESI) 537 (MþH)þ; HRMS (ESI) calcd
This compound was prepared from riccardin
D (60 mg,
0.142 mmol), 1-(4-methoxyphenyl)- piperazine (41 mg,
0.212 mmol), and aqueous formaldehyde (18 mg, 0.212 mmol) by
following GP 1 in 63% yield. White solid; mp 147e148 ꢁC. IR (film)
for C34H37N2O4 537.2748; found 537.2743 (MþH) þ
.
4.1.14. 40-((Bis(2-methoxyethyl)amino)methyl)-riccardin D (10l)
This compound was prepared from riccardin (60 mg,
3358, 1577, 1531, 1459, 1345, 1140, 979 cmꢀ1
;
1H NMR (600 MHz,
D
CDCl3)
d
7.19 (s, 1H), 7.01 (d, J ¼ 7.8 Hz, 1H), 6.96e6.84 (m, 6H),
0.142 mmol), bis(2-methoxyethyl)amine (29 mg, 0.212 mmol), and
aqueous formaldehyde (18 mg, 0.212 mmol) by following GP 1 in
75% yield. Colorless oil; IR (film) 3371, 1599, 1521, 1403, 1371,
6.84e6.76 (m, 4H), 6.73 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.51 (dd, J ¼ 7.7,
1.7 Hz, 1H), 6.34 (s, 1H), 5.38 (d, J ¼ 2.0 Hz, 1H), 3.99 (d, J ¼ 13.6 Hz,
1H), 3.85e3.78 (m, 1H), 3.75 (s, 3H), 3.38e3.02 (m, 4H), 3.02e2.69
1250 cmꢀ1
;
1H NMR (CDCl3)
d
7.08 (d, J ¼ 7.8 Hz, 1H), 6.96 (d,
(m, 9H), 2.69e2.30 (m, 3H); 13C NMR (CDCl3)
d
154.4, 153.1, 152.8,
J ¼ 7.8 Hz, 1H), 6.92 (d, J ¼ 7.8 Hz, 2H), 6.88 (d, J ¼ 7.8 Hz, 2H),
6.82e6.79 (m, 2H), 6.73 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.56 (dd, J ¼ 7.8,
1.7 Hz, 1H), 6.27 (d, J ¼ 1.7 Hz, 1H), 5.37 (d, J ¼ 2.0 Hz, 1H), 4.08 (d,
J ¼ 14.0 Hz, 1H), 3.86 (d, J ¼ 14.0 Hz, 1H), 3.61e3.50 (m, 4H), 3.30 (s,
6H), 3.02e2.74 (m, 10H), 2.62e2.50 (m, 2H); 13C NMR (CDCl3)
146.7, 143.4, 142.5, 140.3, 133.2, 132.0, 129.6, 129.0, 124.3, 122.5,
122.4, 122.1, 122.0, 121.2, 119.0, 118.8, 116.1, 114.9, 114.6, 114.5, 60.1,
55.5, 52.4, 50.1, 38.0, 37.7, 36.7, 34.6; MS (ESI) 629 (MþH)þ; HRMS
(ESI) calcd for C40H41N2O5 629.3010; found 629.3015 (MþH) þ
.
d
154.4, 153.4, 152.7, 146.7, 143.8, 143.3, 141.9, 140.4, 133.3, 132.0,
4.1.11. 40-{[(2-Hydroxyethyl)(methyl)amino]methyl}-riccardin D
(10h)
129.8, 128.7, 124.9, 122.5, 122.1, 121.9, 121.6, 120.7, 119.9, 119.0, 116.2,
114.8, 69.8, 58.8, 58.4, 53.4, 38.0, 37.7, 36.7, 34.3; MS (ESI) 570
(MþH)þ; HRMS þ(ESI) calcd for C35H40NO6 570.2850; found
This compound was prepared from riccardin
D (60 mg,
0.142 mmol), 2-(methylamino)ethanol (16 mg, 0.212 mmol), and
aqueous formaldehyde (18 mg, 0.212 mmol) by following GP 1 in
63% yield. White solid; mp 126e127 ꢁC. IR (film) 3360, 1581, 1527,
570.2855 (MþH)
.
4.1.15. 40-((4-(Tert-butoxycarbonyl)piperazin-1-yl)methyl)-
riccardin D (10m)
1440, 1325, 1100, 907 cmꢀ1; 1H NMR (CD3OD)
d
7.02 (d, J ¼ 7.8 Hz,
1H), 6.88 (d, J ¼ 7.8 Hz, 2H), 6.81 (d, J ¼ 7.8 Hz, 1H), 6.76 (d,
J ¼ 3.1 Hz, 1H), 6.74 (d, J ¼ 2.7 Hz, 1H), 6.71 (d, J ¼ 8.5 Hz, 2H), 6.63
(dd, J ¼ 8.1, 2.0 Hz, 1H), 6.26 (dd, J ¼ 7.7, 1.7 Hz, 1H), 6.20 (d,
J ¼ 1.6 Hz,1H), 5.39 (d, J ¼ 2.0 Hz,1H), 3.77 (s, 2H), 3.65 (t, J ¼ 5.7 Hz,
This compound was prepared from riccardin D (60 mg,
0.142 mmol), tert-butyl piperazine-1- carboxylate (40 mg,
0.212 mmol), and aqueous formaldehyde (18 mg, 0.212 mmol) by
following GP 1 in 66% yield. White solid; mp 158e159 ꢁC. IR (film)
2H), 2.94e2.44 (m, 10H), 2.34 (s, 3H); 13C NMR (CD3OD)
d
156.4,
3362, 1578, 1522, 1478, 1360, 1151, 946 cmꢀ1 1H NMR (600 MHz,
;
155.3, 154.7, 148.9, 145.2, 144.9, 142.8, 141.7, 134.3, 134.0, 130.6,
130.1, 129.5, 127.1, 123.5, 123.1, 122.9, 122.4, 122.1, 121.0, 120.1, 118.2,
116.8, 61.9, 59.7, 59.5, 41.7, 38.9, 38.8, 38.0, 35.9; MS (ESI) 512
(MþH)þ; HRMS (ESI) calcd for C32H33NO5 512.2431; found
CDCl3)
d
7.07 (d, J ¼ 7.8 Hz, 1H), 6.98 (d, J ¼ 7.8 Hz, 1H), 6.91 (d,
J ¼ 8.1 Hz, 2H), 6.89e6.84 (m, 2H), 6.83e6.77 (m, 2H), 6.73 (dd,
J ¼ 8.1, 2.0 Hz, 1H), 6.52 (dd, J ¼ 7.7, 1.7 Hz, 1H), 6.30 (d, J ¼ 1.7 Hz,
1H), 5.37 (d, J ¼ 1.9 Hz, 1H), 3.89 (d, J ¼ 14.0 Hz, 1H), 3.66 (d,
J ¼ 13.9 Hz, 1H), 3.57e3.05 (m, 4H), 2.97e2.78 (m, 6H), 2.67e2.35
512.2436 (MþH) þ
.
(m, 6H), 1.44 (s, 9H); 13C NMR (CDCl3)
d 154.5, 154.4, 152.9, 152.7,
4.1.12. 40-((4-Benzylpiperazin-1-yl)methyl)-riccardin D (10i)
This compound was prepared from riccardin (60 mg,
146.7, 144.0, 143.3, 142.3, 140.3, 133.3, 132.0, 129.6, 129.0, 128.9,
128.2, 124.0, 122.5, 122.2, 122.1, 121.0, 118.7, 118.1, 116.1, 114.9, 80.1,
61.6, 52.3, 38.1, 37.8, 36.8, 34.4, 28.4; MS (ESI) 623 (MþH)þ; HRMS
D
0.142 mmol), 1-benzylpiperazine (36 mg, 0.212 mmol), and
aqueous formaldehyde (18 mg, 0.212 mmol) by following GP 1 in
69% yield. White solid; mp 140e141 ꢁC. IR (film) 3375, 1566, 1535,
(ESI) calcd for C38H43N2O6 623.3116; found 623.3120 (MþH) þ
4.1.16. 13, 40-Bis((diethylamino)methyl)-riccardin D (11a)
.
1444, 1325, 1110, 906 cmꢀ1 1H NMR (CDCl3)
; d 7.41e7.25 (m, 5H),
7.07 (d, J ¼ 7.8 Hz,1H), 6.97 (d, J ¼ 7.8 Hz,1H), 6.91 (d, J ¼ 8.1 Hz,1H),
6.90e6.87 (m, 1H), 6.85 (dd, J ¼ 8.2, 2.3 Hz, 1H), 6.83e6.76 (m, 2H),
6.74 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.52 (dd, J ¼ 7.7, 1.7 Hz, 1H), 6.29 (d,
J ¼ 1.7 Hz, 1H), 5.36 (d, J ¼ 2.0 Hz, 1H), 3.89 (d, J ¼ 14.0 Hz, 1H), 3.70
(d, J ¼ 14.1 Hz,1H), 3.61e3.50 (m, 2H), 3.11e2.86 (m, 4H), 2.86e2.75
This compound was prepared from riccardin D (60 mg,
0.142 mmol), diethylamine (30 mg, 0.424 mmol), and aqueous
formaldehyde (36 mg, 0.424 mmol) by following GP 1 in 70% yield.
White solid; mp 132e133 ꢁC. IR (film) 3369, 1587, 1520, 1464, 1366,
1140, 961 cmꢀ1
;
1H NMR (CDCl3)
d
7.05 (d, J ¼ 7.7 Hz, 1H), 6.94 (d,
(m, 4H), 2.74e2.14 (m, 8H); 13C NMR (CDCl3)
d
154.5, 153.0, 152.7,
J ¼ 7.8 Hz, 1H), 6.90 (d, J ¼ 7.6 Hz, 1H), 6.89e6.86 (m, 1H), 6.84 (d,
J ¼ 8.3 Hz, 3H), 6.53 (dd, J ¼ 7.8, 1.8 Hz, 1H), 6.52 (s, 1H), 6.32 (d,
J ¼ 1.6 Hz, 1H), 5.33 (d, J ¼ 1.9 Hz, 1H), 3.89 (dd, J ¼ 14.2, 4.8 Hz, 2H),
3.82 (dd, J ¼ 14.2, 9.5 Hz, 2H), 2.98e2.87 (m, 3H), 2.83e2.70 (m,
7H), 2.66 (q, J ¼ 7.2 Hz, 4H), 2.60e2.55 (m, 1H), 2.51 (dd, J ¼ 12.2,
10.6 Hz, 1H), 1.20 (t, J ¼ 7.2 Hz, 6H), 1.09 (t, J ¼ 7.2 Hz, 6H); 13C NMR
146.7, 143.8, 143.3, 142.2, 140.3, 133.2, 132.0, 129.6, 129.4, 128.9,
128.8, 128.4, 128.3, 124.1, 122.5, 122.1, 122.0, 121.0, 118.9, 116.1, 114.9,
62.5, 61.1, 60.4, 52.3, 38.1, 37.7, 36.8, 34.4; MS (ESI) 613 (MþH)þ;
HRMS (ESI) calcd for C40H41N2O4 613.3061; found 613.3067 (MþH)
þ
.
(CDCl3)
d 155.3, 153.5, 153.2, 147.8, 144.9, 143.6, 142.0, 139.7, 132.1,
4.1.13. 40-((4-Methylpiperazin-1-yl)methyl)-riccardin D (10j)
131.6, 129.4, 128.6, 128.3, 124.3, 122.8, 122.2, 121.7, 121.6, 121.3,
120.8, 119.1, 118.8, 116.1, 65.3, 56.5, 46.3, 46.1, 38.1, 37.7, 36.7, 34.5,
10.8; MS (ESI) 595 (MþH)þ; HRMS (ESI) calcd for C38H47N2O4
This compound was prepared from riccardin
D (60 mg,
0.142 mmol), 1-methylpiperazine (22 mg, 0.212 mmol), and
aqueous formaldehyde (18 mg, 0.212 mmol) by following GP 1 in
73% yield. White solid; mp 144e145 ꢁC. IR (film) 3359, 1578, 1524,
595.3530; found 595.3537 (MþH) þ
.
1460, 1265, 1210, 977 cmꢀ1
;
1H NMR (CDCl3)
d
7.07 (d, J ¼ 7.8 Hz,
4.1.17. 13, 40-Bis((pyrrolidin-1-yl)methyl)-riccardin D (11b)
This compound was prepared from riccardin
0.142 mmol), pyrrolidine (30 mg, 0.424 mmol), and aqueous
1H), 6.98 (d, J ¼ 7.8 Hz, 1H), 6.91e6.89 (m, 2H), 6.88e6.84 (m, 2H),
D (60 mg,
6.82e6.76 (m, 2H), 6.72 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.50 (dd, J ¼ 7.8,

B. Sun et al. / European Journal of Medicinal Chemistry 136 (2017) 603e618
613
formaldehyde (36 mg, 0.424 mmol) by following GP 1 in 53% yield.
White solid; mp 133e134 ꢁC. IR (film) 3360, 1578, 1525, 1461, 1360,
1158, 936 cmꢀ1 1H NMR (CDCl3)
4.1.21. 13, 40-Bis((4-hydroxypiperidin-1-yl)methyl)-riccardin D
(11f)
This compound was prepared from riccardin
0.142 mmol), piperidin-4-ol (44 mg, 0.424 mmol), and aqueous
formaldehyde (36 mg, 0.424 mmol) by following GP 1 in 65% yield.
White solid; mp 157e158 ꢁC. IR (film) 3362, 1580, 1520, 1469, 1364,
;
d
7.12 (d, J ¼ 7.7 Hz, 1H), 6.96 (d,
D
(60 mg,
J ¼ 7.8 Hz, 1H), 6.89 (d, J ¼ 7.7 Hz, 2H), 6.86e6.81 (m, 3H), 6.74 (s,
1H), 6.53 (dd, J ¼ 7.7, 1.7 Hz, 1H), 6.27 (d, J ¼ 1.7 Hz, 1H), 5.38 (d,
J ¼ 1.9 Hz, 1H), 4.15e4.03 (m, 3H), 3.82 (d, J ¼ 13.8 Hz, 1H),
3.03e2.90 (m, 6H), 2.85e2.77 (m, 3H), 2.74 (s, 4H), 2.58e2.30 (m,
1155, 965 cmꢀ1; 1H NMR (CD3OD)
d
7.07 (d, J ¼ 7.8 Hz, 1H), 6.95 (d,
3H), 2.03e1.90 (m, 4H), 1.87e1.75 (m, 4H); 13C NMR (CDCl3)
d
154.8,
J ¼ 8.1 Hz, 1H), 6.93 (d, J ¼ 8.1 Hz, 1H), 6.87 (d, J ¼ 8.2 Hz, 1H), 6.79
(d, J ¼ 7.7 Hz, 1H), 6.77e6.70 (m, 2H), 6.61 (d, J ¼ 1.9 Hz, 1H), 6.31
(dd, J ¼ 7.7, 1.6 Hz, 1H), 6.24 (d, J ¼ 1.6 Hz, 1H), 5.40 (d, J ¼ 1.8 Hz,
1H), 3.85 (s, 2H), 3.80 (s, 2H), 3.77e3.63 (m, 2H), 3.11e2.81 (m, 7H),
2.81e2.27 (m, 9H), 2.01e1.87 (m, 4H), 1.74e1.45 (m, 4H); 13C NMR
153.2, 147.6, 143.9, 143.7, 142.0, 140.8, 140.1, 132.5, 132.2, 129.6,
128.7, 128.3, 124.4, 122.7, 122.1, 122.0, 121.4, 120.8, 119.0, 118.9, 117.0,
64.8, 58.0, 53.3, 53.2, 38.1, 37.7, 36.7, 34.5, 23.6, 23.5; MS (ESI) 591
(MþH)þ; HRMS (ESI) calcd for C38H43N2O4 591.3217; found
591.3222 (MþH) þ
.
(CD3OD) d 156.3, 155.4, 154.6, 148.9, 145.4, 144.9, 142.8, 141.8, 134.0,
133.6, 130.6, 130.2, 129.6, 127.0, 123.5, 123.4, 123.1, 122.2, 121.0,
118.0, 117.7, 61.6, 60.3, 51.4, 51.1, 50.6, 49.6, 49.2, 48.9, 38.9, 38.8,
38.0, 35.9; MS (ESI) 651 (MþH)þ; HRMS (ESI) calcd for C40H47N2O6
4.1.18. 13, 40-Bis((piperidin-1-yl)methyl)-riccardin D (11c)
This compound was prepared from riccardin
D
(60 mg,
651.3429; found 651.3432 (MþH) þ
.
0.142 mmol), piperidine (34 mg, 0.424 mmol), and aqueous form-
aldehyde (36 mg, 0.424 mmol) by following GP 1 in 57% yield.
White solid; mp 156e157 ꢁC. IR (film) 3366, 1584, 1529, 1464, 1369,
4.1.22. 13, 40-Bis((4-(4-methoxyphenyl)piperazin-1-yl)methyl)-
riccardin D (11g)
1159, 959 cmꢀ1
;
1H NMR (CDCl3)
d
7.05 (d, J ¼ 7.8 Hz, 1H), 6.94 (d,
This compound was prepared from riccardin
D (60 mg,
J ¼ 7.8 Hz, 1H), 6.90 (d, J ¼ 7.7 Hz, 1H), 6.88 (dd, J ¼ 7.4, 2.3 Hz, 1H),
6.86e6.79 (m, 3H), 6.54 (dd, J ¼ 7.7, 1.8 Hz, 2H), 6.32 (d, J ¼ 1.6 Hz,
1H), 5.33 (d, J ¼ 1.9 Hz, 1H), 3.83 (d, J ¼ 14.1 Hz, 2H), 3.67 (d,
J ¼ 14.1 Hz, 2H), 3.02e2.85 (m, 4H), 2.85e2.74 (m, 4H), 2.73e2.61
(m, 2H), 2.58 (t, J ¼ 11.4 Hz, 2H), 2.52 (t, J ¼ 11.4 Hz, 2H), 2.46e2.16
(m, 2H), 1.83e1.67 (m, 4H), 1.59e1.35 (m, 8H); 13C NMR (CDCl3)
0.142 mmol), 1-(4-methoxyphenyl)- piperazine (82 mg,
0.424 mmol), and aqueous formaldehyde (36 mg, 0.424 mmol) by
following GP 1 in 54% yield. White solid; mp 149e150 ꢁC. IR (film)
3368, 1589, 1522, 1464, 1365, 1151, 951 cmꢀ1 1H NMR (600 MHz,
;
CDCl3)
d
7.11 (d, J ¼ 7.8 Hz,1H), 6.99 (d, J ¼ 7.8 Hz,1H), 6.96e6.88 (m,
4H), 6.88e6.73 (m, 9H), 6.60e6.53 (m, 2H), 6.34 (d, J ¼ 1.7 Hz, 1H),
5.38 (d, J ¼ 1.9 Hz, 1H), 3.98e3.83 (m, 3H), 3.77 (s, 3H), 3.75 (s, 3H),
3.74 (s, 1H), 3.22 (s, 4H), 3.09 (br, 4H), 3.00e2.73 (m, 13H),
d
155.0, 153.4, 153.1, 147.7, 144.6, 143.6, 142.1, 139.7, 132.1, 131.7,
129.4, 128.7, 128.5, 124.2, 122.7, 122.2, 121.8, 121.5, 120.8, 119.0,
118.4, 116.2, 61.8, 61.7, 53.7, 38.1, 37.7, 36.7, 34.4, 25.4, 25.3, 23.8,
23.7, 14.2; MS (ESI) 619 (MþH)þ; HRMS (ESI) calcd for C40H47N2O4
2.70e2.45 (m, 3H); 13C NMR (CDCl3)
d 154.6, 154.3, 153.4, 153.1,
619.3530; found 619.3536 (MþH) þ
.
147.9,145.2,145.1,144.4,144.1,142.2,139.8,132.1,132.0,129.4,128.9,
128.8, 124.1, 122.8, 122.3, 122.2, 121.3, 120.9, 118.7, 118.1, 116.4, 114.5,
114.4, 61.3, 55.6, 55.5, 52.6, 50.5, 38.1, 37.7, 36.7, 34.5; MS (ESI) 833
(MþH)þ; HRMS (ESI) calcd for C52H57N4O6 833.4273; found
4.1.19. 13, 40-Bis(morpholinomethyl)-riccardin D (11d)
833.4270 (MþH) þ
.
This compound was prepared from riccardin
D (60 mg,
0.142 mmol), morpholine (36 mg, 0.424 mmol), and aqueous
formaldehyde (36 mg, 0.424 mmol) by following GP 1 in 60% yield.
White solid; mp 138e139 ꢁC. IR (film) 3360, 1581, 1532, 1456, 1377,
4.1.23. 13, 40-Bis{[(2-hydroxyethyl)(methyl)amino]methyl}-
riccardin D (11h)
1159, 969 cmꢀ1
;
1H NMR (CDCl3)
d
7.10 (d, J ¼ 7.8 Hz, 1H), 6.98 (d,
This compound was prepared from riccardin
D (60 mg,
J ¼ 7.8 Hz, 1H), 6.89 (d, J ¼ 7.8 Hz, 2H), 6.87e6.78 (m, 3H), 6.59 (s,
1H), 6.52 (dd, J ¼ 7.7, 1.7 Hz, 1H), 6.32 (d, J ¼ 1.7 Hz, 1H), 5.37 (d,
J ¼ 2.0 Hz, 1H), 3.93e3.82 (m, 6H), 3.82e3.58 (m, 6H), 3.06e2.68
0.142 mmol), 2-(methylamino)ethanol (32 mg, 0.424 mmol), and
aqueous formaldehyde (36 mg, 0.424 mmol) by following GP 1 in
59% yield. White solid; mp 132e133 ꢁC. IR (film) 3368, 1581, 1527,
(m, 10H), 2.68e2.43 (m, 6H); 13C NMR (CDCl3)
d 154.4, 153.3, 153.0,
1464, 1361, 1154, 954 cmꢀ1
;
1H NMR (CD3OD)
d
7.06 (d, J ¼ 7.8 Hz,
147.8, 144.2, 144.1, 142.2, 139.8,132.3,132.1, 129.4,129.1, 128.8,124.1,
122.7, 122.3, 121.1, 121.0, 118.6, 117.6, 116.6, 66.4, 66.3, 61.6, 60.4,
52.8, 52.7, 38.1, 37.6, 36.7, 34.5; MS (ESI) 623 (MþH)þ; HRMS (ESI)
1H), 6.91 (d, J ¼ 7.8 Hz, 2H), 6.85 (d, J ¼ 7.8 Hz, 1H), 6.78 (d,
J ¼ 7.7 Hz, 1H), 6.74 (d, J ¼ 7.9 Hz, 2H), 6.58 (d, J ¼ 2.0 Hz, 1H), 6.28
(dd, J ¼ 7.7, 1.7 Hz, 1H), 6.23 (d, J ¼ 1.6 Hz, 1H), 5.38 (d, J ¼ 1.9 Hz,
1H), 3.82 (s, 2H), 3.80 (s, 2H), 3.76 (t, J ¼ 5.8 Hz, 2H), 3.68 (t,
J ¼ 5.7 Hz, 2H), 2.94e2.84 (m, 3H), 2.77e2.52 (m, 9H), 2.41 (s, 3H),
calcd for C38H43N2O6 623.3116; found 623.3121 (MþH) þ
.
4.1.20. 13, 40-Bis(thiomorpholinomethyl)-riccardin D (11e)
2.37 (s, 3H); 13C NMR (CD3OD)
d 156.5, 155.4, 154.7, 149.0, 145.4,
This compound was prepared from riccardin
D
(60 mg,
144.8, 142.7, 141.7, 134.0, 133.5, 130.6, 130.1, 129.4, 127.1, 123.7, 123.5,
123.1, 122.5, 122.0, 121.0, 120.3, 118.2, 117.6, 62.1, 60.9, 59.8, 59.7,
59.6, 58.3, 41.9, 41.7, 38.9, 38.8, 38.0, 35.9; MS (ESI) 599 (MþH)þ;
HRMS (ESI) calcd for C36H43N2O6 599.3116; found 599.3110 (MþH)
0.142 mmol), thiomorpholine (39 mg, 0.424 mmol), and aqueous
formaldehyde (36 mg, 0.424 mmol) by following GP 1 in 53% yield.
White solid; mp 159e160 ꢁC. IR (film) 3361, 1580, 1531, 1474, 1360,
1142, 955 cmꢀ1; 1H NMR (600 MHz, CDCl3)
d
7.10 (t, J ¼ 7.8 Hz, 1H),
.
þ
6.98 (d, J ¼ 7.7 Hz, 1H), 6.91 (t, J ¼ 7.2 Hz, 1H), 6.89e6.87 (m, 1H),
6.87e6.82 (m, 2H), 6.82e6.77 (m, 1H), 6.73 (dd, J ¼ 8.1, 1.9 Hz, 1H),
6.52 (dt, J ¼ 8.1, 1.9 Hz, 1H), 6.31 (dd, J ¼ 5.0, 1.6 Hz, 1H), 5.37 (d,
J ¼ 2.4 Hz,1H), 3.91e3.87 (m, 2H), 3.76e3.72 (m, 2H), 3.57e2.99 (m,
5H), 2.98e2.86 (m, 8H), 2.85e2.77 (m, 4H), 2.77e2.62 (m, 5H),
4.1.24. 13, 40-Bis((4-benzylpiperazin-1-yl)methyl)-riccardin D (11i)
This compound was prepared from riccardin
D (60 mg,
0.142 mmol), 1-benzylpiperazine (72 mg, 0.424 mmol), and
aqueous formaldehyde (36 mg, 0.424 mmol) by following GP 1 in
60% yield. White solid; mp 148e149 ꢁC. IR (film) 3362, 1578, 1532,
2.62e2.43 (m, 2H); 13C NMR (CDCl3)
d 154.5, 152.9, 152.7, 147.7,
146.6, 143.3, 142.3, 142.2, 140.3, 133.2, 132.1, 132.0, 129.5, 128.9,
124.1, 122.6, 122.5, 122.2, 122.1, 121.1, 120.9, 118.8, 118.7, 116.1, 114.9,
61.9, 60.4, 54.3, 54.2, 45.3, 38.0, 37.7, 36.7, 34.4; MS (ESI) 655
(MþH)þ; HRMS (ESI) calcd for C38H43N2O4S2 655.2659; found
1460, 1366, 1154, 946 cmꢀ1 1H NMR (CDCl3)
; d 7.37e7.27 (m, 9H),
7.25e7.22 (m, 1H), 7.06 (d, J ¼ 7.9 Hz, 1H), 6.95 (d, J ¼ 7.8 Hz, 1H),
6.89 (d, J ¼ 7.7 Hz, 1H), 6.87 (d, J ¼ 2.1 Hz, 1H), 6.83e6.81 (m, 3H),
6.52 (dd, J ¼ 7.7, 1.7 Hz, 1H), 6.48 (d, J ¼ 2.1 Hz, 1H), 6.32 (d,
J ¼ 1.6 Hz, 1H), 5.33 (d, J ¼ 1.9 Hz, 1H), 3.87 (d, J ¼ 14.1 Hz, 1H), 3.82
655.2652 (MþH) þ
.

614
B. Sun et al. / European Journal of Medicinal Chemistry 136 (2017) 603e618
(d, J ¼ 13.8 Hz, 1H), 3.75 (d, J ¼ 13.9 Hz, 1H), 3.69 (d, J ¼ 14.1 Hz, 1H),
3.59 (s, 2H), 3.49 (s, 2H), 3.02e2.89 (m, 4H), 2.89e2.73 (m, 8H),
4.1.28. 13, 40-Bis ((4-(tert-butoxycarbonyl)piperazin-1-yl)methyl)-
riccardin D (11m)
2.72e2.39 (m, 12H); 13C NMR (CDCl3)
d
154.7, 153.4, 153.0, 147.8,
This compound was prepared from riccardin D (60 mg,
144.5, 143.9, 142.2, 139.6, 132.1, 131.7, 129.4, 129.3, 129.2, 128.7,
128.4, 128.3, 127.4, 127.3, 124.0, 122.8, 122.3, 122.0, 121.3, 120.9,
118.8, 118.2, 116.2, 62.7, 61.3, 60.9, 60.4, 52.6, 52.4, 52.3, 52.2, 38.1,
37.6, 36.7, 34.5; MS (ESI) 801 (MþH)þ; HRMS (ESI) calcd for
0.142 mmol), tert-butyl piperazine-1- carboxylate (79 mg,
0.424 mmol), and aqueous formaldehyde (36 mg, 0.424 mmol) by
following GP 1 in 59% yield. White solid; mp 125e126 ꢁC. IR (film)
3369, 1582, 1521, 1461, 1366, 1157, 947 cmꢀ1 1H NMR (600 MHz,
;
C
52H57N4O4 801.4374; found 801.4377 (MþH) þ
.
CDCl3)
d
7.06 (d, J ¼ 7.8 Hz, 1H), 6.97 (d, J ¼ 7.8 Hz, 1H), 6.89 (d,
J ¼ 7.7 Hz, 1H), 6.87e6.83 (m, 4H), 6.52 (dd, J ¼ 7.8, 1.7 Hz, 1H), 6.46
(s, 1H), 6.33 (d, J ¼ 1.7 Hz, 1H), 5.35 (d, J ¼ 2.1 Hz, 1H), 3.89 (d,
J ¼ 14.0 Hz, 1H), 3.79 (d, J ¼ 13.8 Hz, 1H), 3.73 (d, J ¼ 13.7 Hz, 1H),
3.67 (d, J ¼ 14.0 Hz, 1H), 3.59e3.34 (m, 7H), 2.97e2.71 (m, 7H),
2.68e2.44 (m, 10H), 1.47 (s, 9H), 1.44 (s, 9H); 13C NMR (CDCl3)
4.1.25. 13, 40-Bis((4-methylpiperazin-1-yl)methyl)-riccardin D (11j)
This compound was prepared from riccardin (60 mg,
D
0.142 mmol), 1-methylpiperazine (44 mg, 0.424 mmol), and
aqueous formaldehyde (36 mg, 0.424 mmol) by following GP 1 in
51% yield. White solid; mp 139e140 ꢁC. IR (film) 3360, 1588, 1527,
d
154.5, 154.4, 153.0, 147.8, 144.2, 144.1, 142.3, 140.4, 139.8, 132.1,
1466, 1368, 1157, 950 cmꢀ1
;
1H NMR (CDCl3)
d
7.06 (d, J ¼ 7.8 Hz,
131.8, 129.4, 128.8, 127.4, 122.3, 121.0, 120.9, 118.6, 117.9, 114.9, 113.7,
80.1, 65.4, 52.3, 42.0, 38.0, 37.6, 36.7, 34.5, 30.2, 29.1, 28.4, 28.3; MS
(ESI) 821 (MþH)þ; HRMS (ESI) calcd for C48H61N4O8 821.4484;
1H), 6.96 (d, J ¼ 7.8 Hz, 1H), 6.92e6.86 (M, 2H), 6.85e6.78 (m, 3H),
6.52 (dd, J ¼ 7.8, 1.7 Hz, 1H), 6.47 (d, J ¼ 2.0 Hz, 1H), 6.32 (d,
J ¼ 1.7 Hz, 1H), 5.34 (d, J ¼ 2.0 Hz, 1H), 3.89 (d, J ¼ 13.9 Hz, 1H), 3.81
(d, J ¼ 13.8 Hz, 1H), 3.76 (d, J ¼ 13.9 Hz, 1H), 3.70 (d, J ¼ 14.0 Hz, 1H),
3.17e2.85 (m, 5H), 2.85e2.72 (m, 7H), 2.72e2.57 (m, 6H), 2.57e2.45
(m, 4H), 2.42 (s, 3H), 2.40e2.34 (m, 1H), 2.31 (d, J ¼ 2.0 Hz, 3H),
found 821.4489 (MþH) þ
.
4.1.29. 13-Nitro-riccardin D (12)
To a solution of riccardin D (50 mg, 0.12 mmol) in DCM (0.7 mL)
was added the mixture of nitric acid in acetic acid (5.3 mL HNO3 in
2.29e2.15 (m, 1H); 13C NMR (CDCl3)
d 154.0, 153.4, 153.0, 147.8,
0.5 mL acetic acid) at 0 ꢁC. The reaction was stirred at room tem-
perature for 24 h and poured into ice-water. The organic phase was
isolated and the aqueous solution was extract with DCM. The
combined organic phase then washed with brine, dried, and
evaporated. The residue was purified by silica gel column chro-
matography to give compound 12 as yellow solid, yield 65%; m.p.
88e89 ꢁC. IR (film) 3388, 1596, 1529, 1478, 1360, 1150, 949 cmꢀ1; 1H
144.3, 144.0, 142.2, 139.7, 132.1, 131.9, 130.9, 129.3, 128.8, 124.0,
122.7, 122.3, 122.1, 121.4, 121.3, 120.9, 118.7, 118.1, 116.3, 65.0, 61.2,
54.7, 54.4, 51.9, 51.8, 45.5, 45.3, 38.1, 37.7, 36.7, 34.5; MS (ESI) 649
(MþH)þ; HRMS (ESI) calcd for C40H49N4O4 649.3748; found
649.3742 (MþH) þ
.
4.1.26. 13, 40-Bis((dibutylamino)methyl)-riccardin D (11k)
This compound was prepared from riccardin
NMR (CDCl3)
d
10.72 (s, 1H), 7.63 (s, 1H), 7.38 (t, J ¼ 7.8 Hz, 1H), 7.28
D
(60 mg,
(s, 1H), 7.10 (d, J ¼ 7.7 Hz, 1H), 6.96 (d, J ¼ 8.1 Hz, 1H), 6.93 (d,
J ¼ 8.0 Hz,1H), 6.88 (m, 3H), 6.84 (d, J ¼ 8.1 Hz,1H), 6.56 (s,1H), 6.38
(d, J ¼ 7.6 Hz, 1H), 5.81 (s, 1H), 4.96 (s, 1H), 4.94 (s, 1H), 3.06e2.57
0.142 mmol), 1-methylpiperazine (44 mg, 0.424 mmol), and
aqueous formaldehyde (55 mg, 0.424 mmol) by following GP 1 in
68% yield. Colorless oil. IR (film) 3367, 1589, 1523, 1464, 1368, 1157,
(m, 8H); 13C NMR (CDCl3)
d 153.5, 153.2, 152.3, 149.7, 144.3, 143.7,
956 cmꢀ1; 1H NMR (600 MHz, CDCl3)
d
6.93 (d, J ¼ 8.1 Hz, 2H), 6.88
143.0, 140.6, 134.0, 132.3, 132.0, 130.5, 129.7, 129.5, 123.1, 122.9,
122.4, 122.3, 122.0, 121.2, 117.7, 117.1, 116.5, 113.6, 39.8, 37.9, 37.3,
36.7, 35.0; MS (ESI) 492 (MþNa)þ; HRMS (ESI) calcd for
(d, J ¼ 8.0 Hz,1H), 6.86e6.82 (m, 2H), 6.66 (t, J ¼ 2.0 Hz,1H), 6.62 (d,
J ¼ 8.2 Hz, 2H), 6.47 (dd, J ¼ 8.2, 2.7 Hz, 1H), 6.21 (d, J ¼ 7.5 Hz, 1H),
5.44 (d, J ¼ 1.9 Hz, 1H), 3.80e3.75 (m, 3H), 3.00 (s, 4H), 2.75 (q,
J ¼ 8.1, 7.6 Hz, 4H), 2.56 (t, J ¼ 7.6 Hz, 3H), 2.48 (dd, J ¼ 9.2, 6.4 Hz,
4H), 1.67e1.51 (m, 4H), 1.51e1.40 (m, 5H), 1.34 (q, J ¼ 7.5 Hz, 4H),
C
28H23NNaO6 492.1418, found: 492.1414 [MþNa]þ.
4.1.30. 13-Amino-riccardin D (13)
1.30e1.20 (m, 5H), 0.91 (t, J ¼ 7.3 Hz, 6H), 0.86 (t, J ¼ 7.3 Hz, 6H); 13
C
Compound 12 (98 mg, 0.21 mmol) and Pd/C (10 mg) was dis-
solved in MeOH (3 mL) and hydrogen gas was bubbled through the
mixture for 8 h. DCM was added and the catalyst was removed by
filtration, The filtrate was evaporated and the residue was puried
with silica gel column chromatography to give compound 13
(92 mg, yield 80%) as red solid; m.p. 84e85 ꢁC. IR (film) 3392, 1592,
NMR (CDCl3)
d 157.8, 153.5, 150.8, 147.1, 144.9, 142.0, 139.9, 138.5,
135.7, 129.4, 127.7, 124.4, 122.3, 121.6, 120.8, 119.8, 115.7, 115.5, 112.1,
57.9, 53.2, 36.6, 36.0, 34.8, 30.2, 28.3, 26.9, 20.7, 20.6, 14.0, 13.9; MS
(ESI) 707 (MþH)þ; HRMS (ESI) calcd for C46H63N2O4 707.4782;
found 707.4788 (MþH) þ
.
1515, 1462, 1354, 1151, 966 cmꢀ1 1H NMR (CDCl3)
; d 7.34 (t,
4.1.27. 13, 40-Bis((Bis(2-methoxyethyl)amino)methyl)-riccardin D
(11l)
J ¼ 7.9 Hz, 1H), 7.07 (d, J ¼ 7.7 Hz, 1H), 6.91 (d, J ¼ 7.8 Hz, 2H), 6.82
(m, 3H), 6.76 (d, J ¼ 7.9 Hz, 1H), 6.49 (s, 1H), 6.34 (d, J ¼ 7.6 Hz, 1H),
6.28 (s, 1H), 4.85 (s, 1H), 3.06e2.82 (m, 3H), 2.76e2.65 (m, 2H), 2.60
This compound was prepared from riccardin
D (60 mg,
0.142 mmol), bis(2-methoxyethyl)amine (60 mg, 0.424 mmol), and
aqueous formaldehyde (36 mg, 0.424 mmol) by following GP 1 in
70% yield. Colorless oil. IR (film) 3371, 1582, 1514, 1459, 1359, 1148,
(m, 3H); 13C NMR (CDCl3)
d 153.6, 153.0, 152.8, 146.8, 144.0, 143.9,
140.2, 134.4, 132.6, 131.4, 131.1, 130.2, 129.2, 129.1, 122.8, 122.6,
122.2, 121.7, 117.2, 117.0, 113.3, 109.8, 107.2, 46.4, 37.8, 37.6, 36.9,
35.0; MS (ESI) 440 (MþH)þ; HRMS (ESI) calcd for C28H26NO4
440.1856, found: 440.1851 [MþH]þ.
957 cmꢀ1
;
1H NMR (CDCl3)
d
7.04 (d, J ¼ 7.8 Hz, 1H), 6.94 (d,
J ¼ 7.8 Hz, 1H), 6.88 (d, J ¼ 7.8 Hz, 2H), 6.856.83 (m, 3H), 6.56 (dd,
J ¼ 7.8, 1.7 Hz, 1H), 6.48 (d, J ¼ 1.9 Hz, 1H), 6.28 (d, J ¼ 1.7 Hz, 1H),
5.33 (d, J ¼ 1.9 Hz, 1H), 4.03 (d, J ¼ 14.0 Hz, 1H), 3.97 (d, J ¼ 13.9 Hz,
1H), 3.87 (d, J ¼ 13.9 Hz, 1H), 3.81 (d, J ¼ 14.1 Hz, 1H), 3.60 (t,
J ¼ 5.5 Hz, 4H), 3.56e3.45 (m, 4H), 3.36 (s, 6H), 3.29 (s, 6H),
2.95e2.84 (m, 9H), 2.84e2.71 (m, 5H), 2.60e2.37 (m, 2H); 13C NMR
4.1.31. 60-Dimethylamino-riccardin D (14)
A suspension of compound 13 (30 mg, 0.07 mmol) and NaBH4 in
THF was added slowly to a mixture of 3 M H2SO4 and formaldehyde
at 0 ꢁC. The reaction was allowed to stirred for 1 h and quenched
with 1 mL H2O. KOH then added and stirred for 0.5 h, and solution
was extract ether. The organic phase was dried and evaporated for
silica gel column chromatography to give compound 14 (17 mg,
54%) as white solid; m.p. 99e100 ꢁC. IR (film) 3352, 1581,1511, 1463,
(CDCl3)
d 154.6, 153.7, 153.5, 148.1, 144.6, 143.8, 141.9, 139.7, 132.1,
131.7, 129.5, 128.5, 124.8, 123.0, 122.2, 122.0, 121.8, 121.4, 120.6,
119.7, 119.4, 116.4, 70.4, 70.1, 58.8, 58.7, 58.2, 53.5, 53.4, 38.1, 37.7,
36.7, 34.4; MS (ESI) 715 (MþH)þ; HRMS (ESI) calcd for C45H55N2O8
715.3953; found 715.3957 (MþH) þ
.
1360 cmꢀ1
;
1H NMR (CDCl3)
d
7.35 (t, J ¼ 7.9 Hz, 1H), 7.07 (d,

B. Sun et al. / European Journal of Medicinal Chemistry 136 (2017) 603e618
615
J ¼ 7.7 Hz,1H), 6.91 (d, J ¼ 8.0 Hz, 2H), 6.82 (m, 4H), 6.59 (s,1H), 6.53
(s, 1H), 6.36 (d, J ¼ 7.6 Hz, 1H), 5.22 (s, 1H), 3.00e2.86 (m, 3H), 2.78
(s, 6H), 2.77e2.71 (m, 2H), 2.68e2.58 (m, 3H); 13C NMR (CDCl3)
0.142 mmol), 2-chloro-N,N- diethylethanamine hydrochloride
(37 mg, 0.212 mmol), and potassium carbonate (98 mg, 0.71 mmol)
by following GP 2 in 78% yield. White solid; mp 198e199 ꢁC. IR
(film) 3370, 1581, 1524, 1464, 1360, 1151, 959 cmꢀ1; 1H NMR (CDCl3)
d
153.6, 153.1, 152.9, 146.8, 143.9, 140.9, 139.9, 137.5, 131.9, 131.5,
130.2, 129.2, 129.1, 122.9, 122.7, 122.5, 122.3, 121.5, 117.1, 117.1, 113.þ3,
d
7.38 (t, J ¼ 7.9 Hz, 1H), 7.16 (d, J ¼ 7.7 Hz, 1H), 6.99 (d, J ¼ 7.3 Hz,
112.8, 112.2, 44.4, 37.8, 37.5, 37.0, 35.0; MS (ESI) 468 (MþH)
;
1H), 6.93 (d, J ¼ 7.9 Hz, 2H), 6.94e6.82 (m, 4H), 6.74 (d, J ¼ 7.5 Hz,
1H), 6.51 (d, J ¼ 6.1 Hz, 1H), 6.28 (s, 1H), 5.40 (s, 1H), 4.30e4.20 (m,
2H), 2.98e2.90 (m, 4H), 2.82 (d, J ¼ 10.5 Hz, 2H), 2.70 (s, 4H), 2.63 (t,
J ¼ 11.1 Hz,1H), 2.50 (t, J ¼ 11.1 Hz,1H),1.27 (s, 6H); 13C NMR (CDCl3)
HRMS (ESI) calcd for
C30H30NO4 468.2169, found: 468.2166
[MþH]þ.
4.1.32. N-Acetamide-acetyl-riccardin D (15)
Acetyl chloride (33.7 L) was added dropwise to a solution of
compound 13 (47 mg, 0.107 mmol) and Et3N (104 L) in DCM (2 mL)
d 155.8, 153.8, 152.7, 146.8, 144.4, 143.4, 141.8, 140.5, 133.4, 131.7,
m
130.0,129.0,128.8,124.1, 122.7, 122.0,121.9,120.9,116.1, 114.8,109.6,
m
64.5, 50.9, 46.8, 37.6, 36.9, 34.5, 9.4; MS (ESI) 524 (MþH)þ; HRMS
at 0 ꢁC. The reaction was allowed to room temperature for 1 h and
dilute HCl was added. The organic phase was isolated and the
aqueous solution was extract with DCM. The combined organic
phase then washed with brine, dried, and evaporated. The residue
was purified with silica gel column chromatography to give com-
pound 15 (60 mg, yield 80%) as white solid; m.p. 114e115 ꢁC. IR
(film) 1759, 1591, 1505, 1457, 1194, 1150, 956 cmꢀ1; 1H NMR (CDCl3)
(ESI) calcd for C34H38NO4 524.2795; found 524.2798 (MþH) þ
.
4.1.36. 30-(2-(Pyrrolidin-1-yl)ethoxy)-riccardin D (17c)
This compound was prepared from riccardin
D (60 mg,
0.142 mmol), 1-(2-chloroethyl)pyrrolidine hydrochloride (37 mg,
0.212 mmol), and potassium carbonate (98 mg, 0.71 mmol) by
following GP 2 in 70% yield. White solid; mp 224e225 ꢁC. IR (film)
d
7.82 (s, 1H), 7.42 (t, J ¼ 7.8 Hz, 1H), 7.35 (d, J ¼ 7.8 Hz, 1H), 7.22 (s,
3369, 1585, 1524, 1463,1366,1150, 951 cmꢀ1; 1H NMR (CDCl3)
d 7.33
1H), 6.97 (d, J ¼ 7.9 Hz, 1H), 6.90 (m, 2H), 6.83 (d, J ¼ 7.9 Hz, 1H),
6.73 (m, 2H), 6.60 (d, J ¼ 9.5 Hz, 2H), 2.92 (m, 3H), 2.77 (m, 5H), 2.40
(s, 3H), 2.22 (s, 3H), 2.04 (s, 3H), 1.83 (s, 3H). 13C NMR (CDCl3)
(t, J ¼ 7.9 Hz, 1H), 7.10 (dd, J ¼ 7.8, 1.1 Hz, 1H), 7.00 (dd, J ¼ 7.8, 1.1 Hz,
1H), 6.91 (dd, J ¼ 7.8, 1.1 Hz, 1H), 6.90 (d, J ¼ 7.8, 1H), 6.84 (d,
J ¼ 7.6 Hz, 1H), 6.83e6.74 (m, 3H), 6.72 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.57
(dd, J ¼ 7.7, 1.7 Hz, 1H), 6.20 (d, J ¼ 1.7 Hz, 1H), 5.38 (d, J ¼ 2.0 Hz,
1H), 4.24e4.03 (m, 2H), 3.01e2.75 (m, 8H), 2.72e2.47 (m, 5H),
d
169.7, 168.5, 168.3, 168.1, 152.9, 150.7, 148.7, 147.6, 143.7, 141.6,
140.2, 139.4, 132.0, 131.2, 129.8, 129.3, 129.2, 129.0, 128.1, 127.6,
125.9, 124.9, 123.3, 122.4, 122.1, 119.9, 114.4, 113.1, 37.7, 37.4, 37.0,
34.6, 24.9, 21.0, 20.7, 20.5; MS (ESI) 630 (MþNa) þ; HRMS (ESI) calcd
for C36H37N2O8 625.2544 found: 625.2552 [MþNH4]þ.
2.46e2.33 (m, 1H), 1.90e1.75 (m, 4H).13C NMR (CDCl3)
d 156.0,
154.6, 152.7, 146.9, 144.0, 143.4, 141.5, 140.6, 133.5, 131.6, 130.3,
128.7, 128.5, 127.6, 123.6, 122.7, 121.9, 121.6, 120.9, 116.1, 114.8, 109.1,
65.9, 54.6, 54.1, 53.4, 50.8, 37.8, 37.6, 37.0, 34.4, 23.3; MS (ESI) 522
(MþH)þ; HRMS (ESI) calcd for C34H36NO4 522.2639; found
4.1.33. N-riccardin -acetamide (16)
D
522.2635 (MþH) þ
.
The solution of 1 N NaOH (0.5 mL) was added to a suspension of
compound 15 (50 mg, 0.1 mmol) in MeOH (0.5 mL) at 0 ꢁC. The
solution was quenched after stirring for 0.5 h with hydrochloric
acid. Then DCM was added and isolated. The organic phase was
dried and evaporated for silica gel column chromatography to give
compound 16 (25 mg, 64%) as white solid, m.p. 117e118 ꢁC. IR (film)
4.1.37. 30-(2-(Piperidin-1-yl)ethoxy)-riccardin D (17d)
This compound was prepared from riccardin
D (60 mg,
0.142 mmol), 1-(2-chloroethyl)piperidine hydrochloride (42 mg,
0.212 mmol), and potassium carbonate (98 mg, 0.71 mmol) by
following GP 2 in 76% yield. White solid; mp 220e221 ꢁC. IR (film)
3368, 1760, 1588, 1515, 1453, 1360, 1151 cmꢀ1
;
1H NMR (600 MHz,
(CD3)2CO) 8.96 (s, 1H), 8.65 (s, 1H), 7.73 (s, 1H), 7.20 (m, 3H), 6.97
d
3369, 1585, 1524, 1465, 1364, 1151, 950 cmꢀ1; 1H NMR (CDCl3)
d 7.36
(m, 2H), 6.87 (d, J ¼ 6.9 Hz, 1H), 6.80 (d, J ¼ 7.5 Hz, 1H), 6.73 (m, 3H),
6.28 (m, 2H), 5.18 (s, 1H), 2.92e2.83 (m, 3H), 2.73 (m, 1H), 2.60 (m,
(t, J ¼ 8.0 Hz, 1H), 7.13 (d, J ¼ 7.8 Hz, 1H), 7.02 (d, J ¼ 7.4 Hz, 1H), 6.93
(d, J ¼ 8.0 Hz, 2H), 6.84 (d, J ¼ 7.6 Hz, 1H), 6.83e6.78 (m, 3H), 6.74
(dd, J ¼ 8.1, 1.5 Hz, 1H), 6.55 (d, J ¼ 7.6 Hz, 1H), 6.29 (s, 1H), 5.40 (d,
J ¼ 1.5 Hz, 1H), 4.30e4.25 (m, 1H), 4.15e4.12 (m, 1H), 3.03e2.71 (m,
8H), 2.65e2.40 (m, 6H), 1.75e1.62 (m, 4H), 1.50e1.42 (m, 2H); 13C
5H), 2.21 (s, 3H). 13C NMR (151 MHz, (CD3)2CO)
d 170.2, 155.5, 155.0,
154.3, 149.5, 144.9, 142.5, 141.3, 136.0, 133.7, 133.6, 132.9, 130.2,
123.0,129.3,128.4, 126.0, 123.0, 122.8,122.5, 122.4, 121.1, 117.7, 115.1,
113.7, 38.4, 38.3, 35.6, 23.9; MS (ESI) 504 (MþNa)þ; HRMS (ESI)
calcd for C30H27NNaO5 504.1781, found: 504.1775 [MþNa]þ.
NMR (CDCl3)
d 155.9, 154.2, 152.7, 146.9, 144.1, 143.4, 141.7, 140.6,
133.5, 131.7, 130.2,128.8, 128.7, 127.2, 123.9, 122.7, 121.7, 120.9, 116.0,
114.8,109.4, 64.4, 57.4, 54.5, 37.8, 37.6, 37.0, 34.4, 29.7, 24.3; MS (ESI)
536 (MþH)þ; HRMS (ESI) calcd for C35H38NO4 536.2795; found
4.1.34. 30-(2-(Dimethylamino)ethoxy)-riccardin D (17a)
This compound was prepared from riccardin
D (60 mg,
536.2790 (MþH) þ
.
0.142 mmol), 2-chloro-N,N- dimethylethanamine hydrochloride
(31 mg, 0.212 mmol), and potassium carbonate (98 mg, 0.71 mmol)
by following GP 2 in 66% yield. White solid; mp 162e163 ꢁC. IR
4.1.38. 30-(2-(Azepan-1-yl)ethoxy)-riccardin D (17e)
(film) 3368, 1589, 1520, 1465, 1360, 1142, 955 cmꢀ1
;
1H NMR
This compound was prepared from riccardin D (60 mg,
(CDCl3)
d
7.35 (t, J ¼ 7.9 Hz, 1H), 7.12 (d, J ¼ 7.8 Hz, 1H), 7.02 (d,
0.142 mmol), 1-(2-chloroethyl)azepane hydrochloride (42 mg,
0.212 mmol), and potassium carbonate (98 mg, 0.71 mmol) by
following GP 2 in 72% yield. White solid; mp 189e190 ꢁC. IR (film)
J ¼ 7.9 Hz,1H), 6.96e6.93 (m, 2H), 6.89 (d, J ¼ 7.6 Hz,1H), 6.86e6.77
(m, 3H), 6.75 (dd, J ¼ 8.1, 1.6 Hz, 1H), 6.65 (d, J ¼ 7.6 Hz, 1H), 6.21 (s,
1H), 5.38 (d, J ¼ 1.6 Hz, 1H), 4.17e4.02 (m, 2H), 3.07e2.96 (m, 2H),
2.96e2.80 (m, 4H), 2.70e2.61 (m, 1H), 2.61e2.53 (m, 2H), 2.42 (t,
3364,1582,1520,1464,1365,1155, 957 cmꢀ1; 1H NMR (CDCl3)
d 7.34
(t, J ¼ 7.9 Hz, 1H), 7.11 (d, J ¼ 7.5 Hz, 1H), 6.98 (d, J ¼ 7.2 Hz, 1H), 6.92
(d, J ¼ 8.0 Hz, 2H), 6.87e6.76 (m, 4H), 6.73 (dd, J ¼ 8.1, 2.0 Hz, 1H),
6.56 (d, J ¼ 7.9 Hz, 1H), 6.23 (s, 1H), 5.38 (s, 1H), 4.23e4.02 (m, 2H),
3.02e2.79 (m, 8H), 2.71 (s, 4H), 2.63e2.40 (m, 2H), 1.64e1.58 (m,
J ¼ 12.0 Hz, 1H), 2.29 (s, 6H); 13C NMR (CDCl3)
d 156.1, 154.7, 152.8,
146.8, 143.8, 143.4, 141.4, 140.6, 133.5, 131.5, 130.5, 128.5, 128.3,
127.8, 123.6, 123.3, 122.8, 122.0, 121.5, 121.1, 116.3, 114.8, 108.9, 65.1,
58.0, 45.1, 37.8, 37.6, 37.1, 34.2; MS (ESI) 496 (MþH)þ; HRMS (ESI)
8H); 13C NMR (CDCl3)
d 155.9, 154.3, 152.7, 146.8, 144.1, 143.4, 141.6,
calcd for C32H34NO4 496.2482; found 496.2480 (MþH) þ
4.1.35. 30-(2-(Diethylamino)ethoxy)-riccardin D (17b)
.
140.5,133.5, 131.7, 130.2, 128.7, 128.6,127.1, 123.8, 122.8,122.0,121.7,
121.6, 120.9, 116.2, 114.8, 65.1, 55.6, 55.4, 37.8, 37.7, 37.0, 34.4, 27.0,
26.9; MS (ESI) 550 (MþH)þ; HRMS (ESI) calcd for C36H40NO4
This compound was prepared from riccardin
D
(60 mg,
550.2952; found 550.2946 (MþH) þ
.

616
B. Sun et al. / European Journal of Medicinal Chemistry 136 (2017) 603e618
4.1.39. 30-(2-Morpholinoethoxy)-riccardin D (17f)
143.8, 141.3, 140.1, 134.9, 132.9, 130.9, 129.2, 128.8, 128.2, 127.9,
123.2, 122.6, 122.3, 121.5, 121.1, 117.1, 114.7, 112.8, 109.6, 67.9, 67.2,
66.9, 58.3, 58.2, 57.9, 46.0, 45.9, 45.8, 37.8, 37.6, 36.9, 35.2; MS (ESI)
638 (MþH)þ; HRMS (ESI) calcd for C40H52N3O4 638.3952; found
This compound was prepared from riccardin
D (60 mg,
0.142 mmol), 4-(2-chloroethyl)- morpholine hydrochloride (40 mg,
0.212 mmol), and potassium carbonate (98 mg, 0.71 mmol) by
following GP 2 in 70% yield. White solid; mp 160e161 ꢁC. IR (film)
638.3959 (MþH) þ
.
3367, 1587, 1515, 1460, 1355, 1157, 950 cmꢀ1; 1H NMR (CDCl3)
d 7.36
(t, J ¼ 8.0 Hz,1H), 7.14 (d, J ¼ 7.7 Hz,1H), 6.97 (dd, J ¼ 8.1, 2.0 Hz,1H),
6.92 (d, J ¼ 8.0 Hz, 2H), 6.84e6.78 (m, 3H), 6.73 (dd, J ¼ 8.1, 2.0 Hz,
1H), 6.53 (d, J ¼ 7.8 Hz, 1H), 6.26 (s, 1H), 5.65 (s, 1H), 5.36 (d,
J ¼ 1.8 Hz, 1H), 4.52e4.05 (m, 2H), 3.75 (s, 4H), 3.06e2.74 (m, 7H),
4.1.43. 3, 12, 30-Tri(2-(diethylamino)ethoxy)-riccardin D (18b)
This compound was prepared from riccardin (60 mg,
0.142 mmol), 2-chloro-N,N- diethylethanamine hydrochloride
(122 mg, 0.71 mmol), and potassium carbonate (200 mg,
1.42 mmol) by following GP 2 in 86% yield. White solid; mp
89e90 ꢁC. IR (film) 1587, 1522, 1465, 1361, 1150, 962 cmꢀ1; 1H NMR
D
2.73e2.14 (m, 7H); 13C NMR (CDCl3)
d 156.1, 155.6, 153.6, 152.7,
146.8, 143.4, 141.9, 140.5, 133.2, 131.8, 130.0, 128.9, 128.8, 124.3,
122.7, 122.0, 121.9, 121.0, 116.0, 114.9, 64.5, 61.1, 57.4, 53.5, 37.7, 37.6,
37.0, 34.5; MS (ESI) 538 (MþH)þ; HRMS (ESI) calcd for C34H36NO5
(CDCl3)
d
7.23 (t, J ¼ 8.0 Hz, 1H), 6.98 (d, J ¼ 7.8 Hz, 1H), 6.84 (d,
J ¼ 8.2 Hz, 2H), 6.77 (d, J ¼ 6.6 Hz, 1H), 6.712e6.68 (m, 5H), 6.36 (s,
1H), 6.20 (d, J ¼ 7.6 Hz,1H), 5.42 (d, J ¼ 1.7 Hz,1H), 4.24 (t, J ¼ 6.0 Hz,
2H), 4.07e3.97 (m, 4H), 3.13e3.06 (m, 2H), 2.96e2.84 (m, 7H), 2.81
(q, J ¼ 7.1 Hz, 4H), 2.78e2.71 (m, 1H), 2.72e2.65 (m, 1H), 2.61 (q,
J ¼ 7.1 Hz, 4H), 2.58e2.52 (m, 2H), 2.49 (q, J ¼ 7.1 Hz, 4H), 2.44e2.41
(m, 1H), 1.12 (t, J ¼ 7.2 Hz, 6H), 0.92 (t, J ¼ 7.2 Hz, 6H), 0.88 (t,
538.2588; found 538.2582 (MþH) þ
.
4.1.40. 30-(3-(Diethylamino)propoxy)-riccardin D (17g)
This compound was prepared from riccardin
D (60 mg,
0.142 mmol), 3-chloro-N,N- dimethylpropan-1-amine hydrochlo-
ride (35 mg, 0.212 mmol), and potassium carbonate (98 mg,
0.71 mmol) by following GP 2 in 71% yield. White solid; mp
J ¼ 7.2 Hz, 6H); 13C NMR (CDCl3)
d 155.8, 155.5, 153.0, 149.3, 145.7,
144.0,141.7,140.1,134.8,132.7, 129.5,129.1,128.5,127.3, 123.0,122.8,
122.1, 121.7, 121.6, 117.0, 114.3, 112.2, 109.4, 66.6, 65.3, 65.2, 51.3,
51.0, 50.8, 47.6, 47.5, 47.4, 37.7, 37.4, 36.6, 35.1, 10.8, 10.1, 10.0; MS
(ESI) 722 (MþH)þ; HRMS (ESI) calcd for C46H64N3O4 722.4891;
186e187 ꢁC. IR (film) 3365, 1588, 1523, 1465, 1365, 1156, 949 cmꢀ1
;
1H NMR (CDCl3)
d
7.36 (t, J ¼ 7.9 Hz, 1H), 7.12 (dd, J ¼ 7.8, 1.1 Hz, 1H),
6.94e6.87 (m, 4H), 6.84 (dd, J ¼ 8.3, 1.2 Hz, 1H), 6.79 (d, J ¼ 7.9 Hz,
2H), 6.72 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.43 (dd, J ¼ 7.8, 1.7 Hz, 1H), 6.27
(d, J ¼ 1.7 Hz, 1H), 5.40 (d, J ¼ 2.0 Hz, 1H), 4.02e3.97 (m, 1H),
3.90e3.86 (m, 1H), 2.98e2.85 (m, 4H), 2.82e2.71 (m, 3H),
2.70e2.48 (m, 3H), 2.21 (s, 6H), 1.85e1.74 (m, 2H); 13C NMR (CDCl3)
found 722.4898 (MþH) þ
.
4.1.44. 3, 12, 30- Tri (2-(pyrrolidin-1-yl)ethoxy)-riccardin D (18c)
This compound was prepared from riccardin (60 mg,
D
d
156.2, 153.2, 152.8, 146.8, 144.5, 143.5, 141.8, 140.4, 133.2, 132.0,
0.142 mmol), 1-(2-chloroethyl)pyrrolidine hydrochloride (120 mg,
0.71 mmol), and potassium carbonate (200 mg, 1.42 mmol) by
following GP 2 in 90% yield. White solid; mp 82e83 ꢁC. IR (film)
129.6, 129.3, 126.0, 123.8, 122.6, 122.0, 121.3, 120.6, 118.0, 116.1,
115.0, 110.3, 66.6, 55.8, 44.7, 37.8, 37.7, 36.8, 34.6, 26.9; MS (ESI) 510
(MþH)þ; HRMS (ESI) calcd for C33H36NO4 510.2639; found
1586, 1525, 1465, 1364, 1151, 954 cmꢀ1 1H NMR (CDCl3)
; d 7.27 (t,
510.2646 (MþH) þ
.
J ¼ 7.7 Hz, 1H), 7.03 (d, J ¼ 7.7 Hz, 1H), 6.91 (d, J ¼ 8.2 Hz, 1H),
6.89e6.86 (m, 2H), 6.80 (d, J ¼ 8.2 Hz, 1H), 6.78e6.73 (m, 3H), 6.71
(d, J ¼ 8.2 Hz, 1H), 6.32 (s, 1H), 6.30 (dd, J ¼ 8.5, 2.0 Hz, 1H), 5.50 (d,
J ¼ 2.0 Hz,1H), 4.39 (t, J ¼ 5.6 Hz, 2H), 4.32e4.08 (m, 4H), 3.40e3.26
(m, 2H), 3.24 (t, J ¼ 5.6 Hz, 2H), 3.11e3.02 (m, 6H), 2.94e2.76 (m,
8H), 2.72e2.51 (m, 8H), 1.97e1.87 (m, 4H), 1.87e1.78 (m, 4H),
4.1.41. 30-(3-(Pyrrolidin-1-yl)propoxy)-riccardin D (17h)
This compound was prepared from riccardin D (60 mg,
0.142 mmol), 1-(3-chloropropyl)- pyrrolidine hydrochloride
(39 mg, 0.212 mmol), and potassium carbonate (98 mg,
0.71 mmol) by following GP 2 in 73% yield. White solid; mp
1.77e1.60 (m, 4H); 13C NMR (CDCl3)
d 155.5, 155.2, 153.1, 149.4,
197e198 ꢁC. IR (film) 3368, 1588, 1528, 1464, 1365, 1153, 954 cmꢀ1
;
1H NMR (CDCl3)
d
7.38 (t, J ¼ 8.0 Hz, 1H), 7.15 (dd, J ¼ 7.8, 1.1 Hz,
145.7, 144.0, 141.8, 140.1, 135.1, 132.8, 129.5, 129.2, 128.6, 127.1, 123.1,
123.0, 122.2, 121.8, 121.6, 117.3, 115.1, 112.1, 109.0, 67.1, 64.8, 64.6,
54.5, 54.4, 54.3, 54.2, 54.1, 54.0, 37.6, 37.3, 36.7, 35.2, 23.3, 23.1, 23.0.
MS (ESI) 716 (MþH)þ; HRMS (ESI) calcd for C46H58N3O4 716.4422;
1H), 6.94e6.88 (m, 3H), 6.84 (dd, J ¼ 8.2, 1.1 Hz, 1H), 6.82e6.77 (m,
3H), 6.72 (dd, J ¼ 8.1, 2.0 Hz, 1H), 6.42 (dd, J ¼ 7.7, 1.7 Hz, 1H), 6.29
(d, J ¼ 1.7 Hz, 1H), 5.40 (d, J ¼ 2.0 Hz, 1H), 4.09e4.05 (m, 1H),
3.92e3.87 (m, 1H), 3.00e2.84 (m, 6H), 2.79e2.61 (m, 6H),
found 716.4416 (MþH) þ
.
2.58e2.48 (m, 2H), 2.00e1.88 (m, 6H); 13C NMR (CDCl3)
d 155.9,
153.3, 152.6, 146.8, 144.6, 143.5, 141.8, 140.5, 133.1, 132.2, 129.5,
129.0, 125.8, 124.2, 122.6, 122.2, 122.0, 120.4, 117.3, 116.0, 114.9,
110.7, 65.9, 53.7, 52.6, 37.8, 37.7, 36.9, 34.7, 26.9, 23.4; MS (ESI) 536
4.1.45. 3, 12, 30- Tri (2-(piperidin-1-yl)ethoxy)-riccardin D (18d)
This compound was prepared from riccardin (60 mg,
D
0.142 mmol), 1-(2-chloroethyl)piperidine hydrochloride (130 mg,
0.71 mmol), and potassium carbonate (200 mg, 1.42 mmol) by
following GP 2 in 88% yield. White solid; mp 95e96 ꢁC. IR (film)
(MþH)þ; HRMS (ESI) calcd for C35H38NO4 536.2795; found
þ
536.2790 (MþH)
.
1586, 1525, 1464, 1365, 1150, 951 cmꢀ1 1H NMR (CDCl3)
; d 7.21 (t,
4.1.42. 3, 12, 30-Tri(2-(dimethylamino)ethoxy)-riccardin D (18a)
This compound was prepared from riccardin (60 mg,
J ¼ 8.0 Hz, 1H), 6.96 (d, J ¼ 7.7 Hz, 1H), 6.83 (d, J ¼ 8.2 Hz, 2H), 6.77
(d, J ¼ 7.4 Hz, 1H), 6.74 (d, J ¼ 7.6 Hz, 1H), 6.69 (d, J ¼ 7.9 Hz, 2H),
6.66e6.65 (m, 2H), 6.30 (s, 1H), 6.24 (d, J ¼ 7.6 Hz, 1H), 5.41 (d,
J ¼ 1.7 Hz, 1H), 4.18 (t, J ¼ 6.3 Hz, 2H), 3.95e3.76 (m, 4H), 2.87e2.80
(m, 5H), 2.75e2.68 (m, 1H), 2.66e2.56 (m, 4H), 2.56e2.48 (m, 8H),
2.28 (s, 4H), 2.16 (s, 4H), 1.61e1.52 (m, 4H), 1.45 (dt, J ¼ 11.1, 5.6 Hz,
4H), 1.38 (dt, J ¼ 11.4, 5.6 Hz, 4H), 1.36e1.22 (m, 6H); 13C NMR
D
0.142 mmol), 2-chloro-N,N- dimethylethanamine hydrochloride
(102 mg, 0.71 mmol), and potassium carbonate (200 mg,
1.42 mmol) by following GP 2 in 83% yield. White solid; mp
77e78 ꢁC. IR (film) 1587, 1521, 1465, 1361, 1142, 959 cmꢀ1; 1H NMR
(CDCl3)
d
7.28 (t, J ¼ 7.9 Hz, 1H), 7.03 (dd, J ¼ 7.7, 1.1 Hz, 1H), 6.90 (d,
J ¼ 8.0 Hz, 2H), 6.81 (d, J ¼ 8.0, 2H), 6.78e6.74 (m, 2H), 6.73e6.68
(m, 2H), 6.37 (d, J ¼ 1.6 Hz, 1H), 6.28 (dd, J ¼ 7.6, 1.6 Hz, 1H), 5.47 (d,
J ¼ 2.0 Hz, 1H), 4.20 (t, J ¼ 6.3 Hz, 2H), 3.97e3.76 (m, 4H), 2.99e2.84
(m, 3H), 2.82 (t, J ¼ 6.2 Hz, 2H), 2.80e2.56 (m, 5H), 2.52 (q,
J ¼ 6.5 Hz, 2H), 2.44 (td, J ¼ 6.0, 2.7 Hz, 2H), 2.37 (s, 6H), 2.17 (s, 6H),
(CDCl3) d 156.4, 156.0, 153.0, 149.5, 146.1, 143.9, 141.4, 140.2, 134.7,
132.8, 129.4, 129.3, 128.2, 127.8, 123.2, 122.6, 122.3, 121.5, 121.0,
117.0, 114.2, 112.7, 109.4, 67.1, 66.6, 66.4, 57.9, 57.8, 57.5, 55.0, 54.9,
54.5, 37.9, 37.6, 36.9, 35.2, 25.8, 25.7, 25.6, 24.1, 23.9, 23.8. MS (ESI)
758 (MþH)þ; HRMS (ESI) calcd for C49H64N3O4 758.4891; found
2.08 (s, 6H); 13C NMR (CDCl3)
d
156.5, 156.0, 153.1, 149.8, 146.2,
758.4898 (MþH) þ
.

B. Sun et al. / European Journal of Medicinal Chemistry 136 (2017) 603e618
617
4.1.46. 3, 120, 30-Tri(2-(azepan-1-yl)ethoxy)-riccardin D (18e)
This compound was prepared from riccardin (60 mg,
6.79e6.72 (m, 2H), 6.65 (s, 1H), 6.39 (d, J ¼ 7.5 Hz, 1H), 6.59 (d,
J ¼ 2.1 Hz, 1H), 5.40 (d, J ¼ 1.9 Hz, 1H), 4.20 (t, J ¼ 5.5 Hz, 2H),
4.06e3.96 (m, 1H), 3.94e3.91 (m, 2H), 3.81e3.76 (m, 2H),
3.69e3.65 (m, 2H), 3.58e3.47 (m, 2H), 3.42e3.36 (m, 3H),
3.10e3.04 (m, 2H), 2.95e2.81 (m, 5H), 2.77e2.49 (m, 11H),
2.45e2.40 (m, 2H), 2.22e2.11 (m, 4H), 2.09e1.86 (m, 13H); 13C NMR
D
0.142 mmol), 1-(2-chloroethyl)azepane hydrochloride (140 mg,
0.71 mmol), and potassium carbonate (200 mg, 1.42 mmol) by
following GP 2 in 84% yield. White solid; mp 88e89 ꢁC. IR (film)
1585, 1522, 1463, 1365, 1152, 956 cmꢀ1 1H NMR (CDCl3)
; d 7.28 (t,
J ¼ 7.8, 1H), 7.02 (dd, J ¼ 7.8, 1.0 Hz, 1H), 6.91 (d, J ¼ 8.2 Hz, 2H),
6.84e6.80 (m, 2H), 6.77e6.72 (m, 4H), 6.37 (d, J ¼ 1.5 Hz, 1H), 6.30
(dd, J ¼ 7.6,1.5 Hz,1H), 5.47 (d, J ¼ 2.1 Hz,1H), 4.21 (t, J ¼ 6.6 Hz, 2H),
4.01e3.76 (m, 4H), 3.05 (t, J ¼ 6.6 Hz, 2H), 2.97e2.86 (m, 3H),
2.85e2.79 (m, 4H), 2.78e2.72 (m, 3H), 2.70e2.65 (m, 4H),
2.66e2.59 (m, 2H), 2.58e2.57 (m, 4H), 2.47e2.45 (m, 4H),1.69e1.66
(m, 4H), 1.62e1.57 (m, 4H), 1.52 (s, 8H), 1.46 (s, 8H); 13C NMR
(CDCl3) d 156.0, 155.6, 152.9, 149.5, 145.6, 143.7, 141.5, 140.0, 135.1,
132.4, 129.5, 128.6, 128.2, 123.9,123.1, 122.2,121.9, 121.0, 117.3, 115.7,
113.2, 111.5, 67.2, 66.5, 65.1, 53.8, 53.7, 53.5, 53.4, 53.3, 53.2, 53.1,
53.0, 37.9, 37.6, 37.1, 34.9, 26.0, 25.8, 25.7, 23.4, 23.3, 23.2, 22.4; MS
(ESI) 758 (MþH)þ; HRMS (ESI) calcd for C49H64N3O4 758.4891;
found 758.4898 (MþH) þ
.
(CDCl3)
d
156.6, 156.1, 153.1, 149.6, 146.2, 143.8, 141.3, 140.1, 134.6,
4.1.50. Antiproliferative studies
132.8,129.3,128.1,127.8,123.2,122.4,122.3,121.4,120.9,117.0,114.2,
112.7, 109.3, 67.9, 67.0, 66.8, 56.9, 56.6, 56.4, 56.0, 55.9, 55.5, 37.9,
37.6, 36.9, 35.2, 28.0, 27.9, 27.8, 27.0, 26.9; MS (ESI) 800 (MþH)þ;
HRMS (ESI) calcd for C52H70N3O4 800.5361; found 800.5354 (MþH)
Human non-small cell lung cancer A549 cell lines and human
breast adenocarcinoma cell line MCF-7 were purchased from the
Shanghai Institute for Biological Sciences (SIBS), China Academy of
Sciences (China). Human myelogenous leukemia k562 cell line was
purchased from the Department of Pharmacology, Institute of He-
matology of the Chinese Academy of Medical Sciences, Tianjin
(China). The cells were cultured in RPMI-1640 (HyClone) medium
containing 10% FBS (Sijiqing Company, Ltd.), 100 units/mL of peni-
þ
.
4.1.47. 3, 12, 30-Tri(2-morpholinoethoxy)-riccardin D (18f)
This compound was prepared from riccardin
D (60 mg,
0.142 mmol), 4-(2-chloroethyl)- morpholine hydrochloride
(130 mg, 0.71 mmol), and potassium carbonate (200 mg,
1.42 mmol) by following GP 2 in 80% yield. White solid; mp
80e81 ꢁC. IR (film) 1588, 1515, 1461, 1356, 1157, 951 cmꢀ1; 1H NMR
cillin G, and 100 mg/mL of streptomycin in a stable environment
with 37 ꢁC and 5% CO2. The antiproliferative activity of the riccardin
D derivatives on the three tumor cell lines was measured by the
MTT method. Briefly, after treatment with the candidate drug for
48 h, the absorbance of the soluble MTT product was measured at
570 nm. All experiments were performed at least three times.
(CDCl3)
d
7.29 (t, J ¼ 7.9 Hz, 1H), 7.04 (dd, J ¼ 7.8, 1.1 Hz, 1H), 6.92 (d,
J ¼ 8.2 Hz, 2H), 6.85 (d, J ¼ 7.5 Hz, 1H), 6.81 (d, J ¼ 7.6 Hz, 1H),
6.77e6.73 (m, 4H), 6.38 (d, J ¼ 1.5 Hz, 1H), 6.30 (dd, J ¼ 7.6, 1.5 Hz,
1H), 5.48 (d, J ¼ 2.1 Hz, 1H), 4.30 (s, 2H), 4.03 (s, 4H), 3.78 (t,
J ¼ 4.6 Hz, 4H), 3.64 (s, 4H), 3.56 (s, 4H), 3.05e2.86 (m, 5H),
2.82e2.55 (m, 11H), 2.45 (s, 4H), 2.29 (s, 4H); 13C NMR (CDCl3)
4.1.51. AO and LysoTracker uptake
We used LysoTracker (50 nm, Molecular Probes) or AO (5 mg/mL,
Sigma-Aldrich) for lysosomal integrity assays. Flow cytometry was
done on a FACScan cytometry (FACSCalibur, Becton Dickinson,
USA). Data were analyzed using CELLQUEST software (Verity Soft-
ware House, Topsham, Maine, USA).
d
153.1, 149.7, 146.0, 144.0, 141.9, 141.6, 140.2, 125.1, 132.8, 129.5,
128.5, 127.6, 124.9, 123.3, 122.9, 122.2, 121.7, 121.4, 117.1, 115.1, 112.5,
109.3, 67.3, 66.6, 66.5, 66.2, 66.0, 57.6, 57.5, 57.2, 54.0, 53.8, 53.5,
37.8, 37.5, 36.9, 35.2. MS (ESI) 764 (MþH)þ; HRMS (ESI) calcd for
C
46H58N3O7 764.4269; found 764.4262 (MþH) þ
.
4.1.52. DAPI staining
We put 10-mm round glass cover slips in 24-well plates. A549
cells were seeded on slips at a density of 5 ꢂ 104/mL. After incu-
bation for 24 h, cells were treated with compounds 11b and 18a for
8 h. After the cells were washed with the cold PBS, cold-methyl
alcohol/acetone (1:1) was used to fix cells. Then washed by PBS
4.1.48. 3, 12, 30- Tri (3-(diethylamino)propoxy)-riccardin D (18g)
This compound was prepared from riccardin (60 mg,
D
0.142 mmol), 3-chloro-N,N- dimethylpropan-1-amine hydrochlo-
ride (113 mg, 0.71 mmol), and potassium carbonate (200 mg,
1.42 mmol) by following GP 2 in 87% yield. White solid; mp
93e94 ꢁC. IR (film) 1588, 1520, 1465, 1366, 1156, 950 cmꢀ1; 1H NMR
and stained with DAPI (4
Cover slips containing the cells were then washed with PBS-TX
L 10% Tritonx-100) three times and mounted us-
mg/mL) for 10 min at room temperature.
(CDCl3)
d
7.32 (t, J ¼ 7.9 Hz, 1H), 7.10 (dd, J ¼ 7.8, 1.1 Hz, 1H), 6.91 (d,
(10 mL PBSþ10
m
J ¼ 8.1 Hz, 1H), 6.89e6.77 (m, 4H), 6.76 (dd, J ¼ 8.2, 2.0 Hz, 2H),
6.71e6.61 (m, 1H), 6.41 (dd, J ¼ 7.6, 1.5 Hz, 1H), 6.29 (d, J ¼ 1.5 Hz,
1H), 5.43 (d, J ¼ 2.0 Hz, 1H), 4.20 (t, J ¼ 5.9 Hz, 2H), 4.09e3.98 (m,
1H), 3.92 (t, J ¼ 5.4 Hz, 2H), 3.76e3.70 (m, 1H), 3.18e3.02 (m, 2H),
3.00e2.78 (m, 5H), 2.75e2.68 (m, 3H), 2.66 (s, 6H), 2.63e2.59 (m,
1H), 2.57 (s, 6H), 2.47 (s, 6H), 2.35e2.11 (m, 3H), 2.09e1.84 (m, 6H);
ing mounting medium (PBS:Glycerol ¼ 1:1 [v/v]) and analyzed by
fluorescence microscopy.
4.1.53. Apoptosis detection
Surface exposure of phosphatidylserine in apoptotic cells was
quantitatively detected using Annexin V/FITC and PI apoptosis
detection kit (Becton Dickinson, USA). Briefly, cells (5 ꢂ 105 per
well) were seeded into 6-well plates and then treated with varying
concentrations of compound 11b and 18a, respectively. After 8 h,
the cells were harvested and washed twice with ice-cold PBS
(0.01 M, pH7.2). After 5 min of centrifuging at 200 g, Annexin V/FITC
and PI double-staining were performed according to manufac-
turer's instruction. Cell apoptosis was analyzed on a FACScan flow
cytometry (Becton Dickinson, USA). Annexin V-positive, PI-
negative cells were scored as apoptotic. Double-stained cells were
considered either as necrotic or as late apoptotic.
13C NMR (CDCl3)
d 156.1, 155.6,153.0, 149.6, 145.8,143.7, 141.6, 140.1,
135.1, 132.4, 129.4, 128.6, 128.1, 123.7, 123.0, 122.2, 121.9, 121.0, 117.3,
115.6, 113.1, 111.0, 67.4, 66.2, 65.0, 55.9, 54.9, 54.8, 43.7, 43.0, 42.8,
37.8, 37.6, 37.1, 34.9, 25.5, 24.8, 24.6. MS (ESI) 680 (MþH)þ; HRMS
(ESI) calcd for C43H58N3O4 680.4422; found 680.4430 (MþH) þ
.
4.1.49. 3, 12, 30- Tri (3-(pyrrolidin-1-yl)propoxy)-riccardin D (18h)
This compound was prepared from riccardin (60 mg,
D
0.142 mmol), 1-(3-chloropropyl)- pyrrolidine hydrochloride
(131 mg, 0.71 mmol), and potassium carbonate (200 mg,
1.42 mmol) by following GP 2 in 90% yield. White solid; mp
86e87 ꢁC. IR (film) 1587, 1528, 1464, 1364, 1155, 954 cmꢀ1; 1H NMR
Acknowledgment
(CDCl3)
d
7.31 (t, J ¼ 7.9 Hz, 1H), 7.10 (d, J ¼ 7.7 Hz, 1H), 6.90 (d,
J ¼ 8.2 Hz, 1H), 6.85 (d, J ¼ 8.5 Hz, 2H), 6.84e6.81 (m, 2H),
This work was supported by grant from the National Natural

618
B. Sun et al. / European Journal of Medicinal Chemistry 136 (2017) 603e618
cathepsin B activity, Eur. J. Med. Chem. 79 (2014) 24e33.
[11] J. Marino, M.C.G. Vior, L.E. Dicelio, L.P. Roguin, J. Awruch, Photodynamic effects
of isosteric water-soluble phthalocyanines on human nasopharynx KB carci-
noma cells, Eur. J. Med. Chem. 45 (2010) 4129e4139.
Science Foundation (NNSF) of China (No. 81473107, No. 81630093
and No. 81102319), and Shandong Provincial Natural Science Foun-
dation, China (No. ZR2011HQ024).
[12] D.C. Harrowven, S.L. Kostiuk, Macrocylic bisbibenzyl natural products and
their chemical synthesis, Nat. Prod. Rep. 29 (2012) 223e242.
[13] B. Sun, M. Zhang, Y. Li, Q.W. Hu, H.B. Zheng, W.Q. Chang, H.X. Lou, Synthesis of
riccardin D derivatives as potent antimicrobial agents, Bioorg. Med. Chem.
Lett. 26 (2016) 3617e3620.
[14] B. Sun, L. Li, Q.W. Hu, F. Xie, H.B. Zheng, H.M. Niu, H.Q. Yuan, H.X. Lou, Design,
synthesis and biological evaluation of novel macrocyclic bisbibenzyl ana-
logues as tubulin polymerization inhibitors, Eur. J. Med. Chem. 121 (2016)
484e499.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2017.05.050.
References
[15] H. Sawada, K. Onoda, D. Morita, E. Ishitsubo, K. Matsuno, H. Tokiwa, T. Kuroda,
H. Miyachi, Structure-anti-MRSA activity relationship of macrocyclic bis(bi-
benzyl) derivatives, Bioorg. Med. Chem. Lett. 23 (2013) 6563e6568.
[16] A. Speicher, M. Groh, M. Hennrich, A.M. Huynh, Syntheses of macrocyclic
bis(bibenzyl) compounds derived from Perrottetin E, Eur. J. Org. Chem. (2010)
6760e6778.
[17] C.F. Xie, H.X. Lou, Chemical constituents from the Chinese bryophytes and
their reversal of fungal resistance, Curr. Org. Chem. 12 (2008) 619e628.
[18] Y.Y. Wang, Y. Ji, Z.Y. Hu, H.M. Jiang, F.H. Zhu, H.Q. Yuan, H.X. Lou, Riccardin D
induces cell death by activation of apoptosis and autophagy in osteosarcoma
cells, Toxicol. Vitro 27 (2013) 1928e1936.
[19] C.C. Sun, Y.S. Zhang, X. Xue, Y.N. Cheng, H.P. Liu, C.R. Zhao, H.X. Lou, X.J. Qu,
Inhibition of angiogenesis involves in anticancer activity of riccardin D, a
macrocyclic bisbibenzyl, in human lung carcinoma, Eur. J. Med. Chem. 667
(2011) 136e143.
[20] Y.Y. Wang, L.N. Wang, Z.Y. Hu, Y. Ji, Z.M. Lin, H.Q. Yuan, M. Ji, H.X. Lou, A novel
derivative of riccardin D induces cell death through lysosomal rupture in vitro
and inhibits tumor growth in vivo, Cancer Lett. 329 (2013) 207e216.
[21] L. Li, H.M. Niu, B. Sun, Y.N. Xiao, W. Li, H.Q. Yuan, H.X. Lou, Riccardin D-N
induces lysosomal membrane permeabilization by inhibiting acid sphingo-
myelinase and interfering with sphingomyelin metabolism in vivo, Toxicol.
Appl. Pharm. 310 (2016) 175e184.
[22] J. Jiang, B. Sun, Y.Y. Wang, M. Cui, L. Zhang, C.Z. Cui, Y.F. Wang, X.G. Liu,
H.X. Lou, Synthesis of macrocyclic bisbibenzyl derivatives and their anticancer
effects as anti-tubulin agents, Bioorg. Med. Chem. 20 (2012) 2382e2391.
[23] B.P. Bandgar, S.A. Patil, J.V. Totre, B.L. Korbad, R.N. Gacche, B.S. Hote, S.S. Jalde,
H.V. Chavan, Synthesis and biological evaluation of nitrogen-containing
benzophenone analogues as TNF-alpha and IL-6 inhibitors with antioxidant
activity, Bioorg. Med. Chem. 20 (2010) 2292e2296.
[24] L.E. Kiss, H.S. Ferreira, L. Torrao, M.J. Bonifacio, P.N. Palma, P. Soares-da-Silva,
D.A. Learmonth, Discovery of a long-acting, Peripherally selective inhibitor of
catechol-O-methyltransferase, J. Med. Chem. 53 (2010) 3396e3411.
[25] S.J.A. Grove, J. Kaur, A.W. Muir, E. Pow, G.J. Tarver, M.Q. Zhang, Oxyaniliniums
as acetylcholinesterase inhibitors for the reversal of neuromuscular block,
Bioorg. Med. Chem. Lett. 12 (2002) 193e196.
[26] D.A. Learmonth, A.P. Freitas, Chemical synthesis and characterization of con-
jugates of a novel catechol-O-methyltransferase inhibitor, Bioconjugate Chem.
13 (2002) 1112e1118.
[27] Z. Chen, Y.R. Wu, Y. Liu, S.J. Yang, Y.J. Chen, L.H. Lai, Discovery of dual target
inhibitors against cyclooxygenases and leukotriene A(4) hydrolyase, J. Med.
Chem. 54 (2011) 3650e3660.
[1] S. Fakih, M. Podinovskaia, X. Kong, H.L. Collins, U.E. Schaible, R.C. Hider, Tar-
geting the lysosome: fluorescent iron(III) chelators to selectively monitor
endosomal/lysosomal labile iron pools, J. Med. Chem. 51 (2008) 4539e4552.
[2] J.P. Falgueyret, S. Desmarais, R. Oballa, W.C. Black, W. Cromlish, K. Khougaz,
S. Lamontagne, F. Masse, D. Riendeau, S. Toulmond, M.D. Percival, Lysoso-
motropism of basic cathepsin K inhibitors contributes to increased cellular
potencies against off-target cathepsins and reduced functional selectivity,
J. Med. Chem. 48 (2005) 7535e7543.
[3] R. Gigli, G.J.S. Pereira, F. Antunes, A. Bechara, D.M. Garcia, D.G. Spindola,
M.G. Jasiulionis, A.C.F. Caires, S.S. Smaili, C. Bincoletto, The biphosphinic pal-
adacycle complex induces melanoma cell death through lysosomal-
mitochondrial axis modulation and impaired autophagy, Eur. J. Med. Chem.
107 (2016) 245e254.
[4] Z. Chen, X. Liang, H.Y. Zhang, H. Xie, J.W. Liu, Y.F. Xu, W.P. Zhu, Y. Wang,
X. Wang, S.Y. Tan, D. Kuang, X.H. Qian, A new class of naphthalimide-based
antitumor agents that inhibit topoisomerase II and induce lysosomal mem-
brane permeabilization and apoptosis, J. Med. Chem. 53 (2010) 2589e2600.
[5] Q.Q. Yu, Y.A. Liu, L. Xu, C.P. Zheng, F.L. Le, X.Y. Qin, Y.Y. Liu, J. Liu, Ruthenium(II)
polypyridyl complexes: cellular uptake, cell image and apoptosis of HeLa
cancer cells induced by double targets, Eur. J. Med. Chem. 82 (2014) 82e95.
[6] S. Grasso, G.J.S. Pereira, C. Palmeira-dos-Santos, A.K. Calgarotto, I. Martinez-
Lacaci, J.A. Ferragut, S.S. Smaili, C. Bincoletto, Autophagy regulates Selumetinib
(AZD6244) induced-apoptosis in colorectal cancer cells, Eur. J. Med. Chem. 122
(2016) 611e618.
[7] Z. Al-Eisawi, C. Stefani, P.J. Jansson, A. Arvind, P.C. Sharpe, M.T. Basha,
G.M. Iskander, N. Kumar, Z. Kovacevic, D.J.R. Lane, S. Sahni, P.V. Bernhardt,
D.R. Richardson, D.S. Kalinowski, Novel mechanism of cytotoxicity for the
selective selenosemicarbazone, 2-acetylpyridine 4,4-Dimethyl-3-selenosemi-
carbazone (Ap44mSe): lysosomal membrane permeabilization, J. Med. Chem.
59 (2016) 294e312.
[8] G.A. Gauna, J. Marino, M.C.G. Vior, L.P. Roguin, J. Awruch, Synthesis and
comparative photodynamic properties of two isosteric alkyl substituted zin-
c(II) phthalocyanines, Eur. J. Med. Chem. 46 (2011) 5532e5539.
[9] R. Ruiz-Gonzalez, P. Acedo, D. Sanchez-Garcia, S. Nonell, M. Canete,
J.C. Stockert, A. Villanueva, Efficient induction of apoptosis in HeLa cells by a
novel cationic porphycene photosensitizer, Eur. J. Med. Chem. 63 (2013)
401e414.
[10] A. Bechara, C.M.V. Barbosa, E.J. Paredes-Gamero, D.M. Garcia, L.S. Silva,
A.L. Matsuo, F.D. Nascimento, E.G. Rodrigues, A.C.F. Caires, S.S. Smaili,
C. Bincoletto, Palladacycle (BPC) antitumour activity against resistant and
metastatic cell lines: the relationship with cytosolic calcium mobilisation and