Design, Synthesis and Structure-Activity Relationship Studies of Glycosylated Derivatives of Marine Natural Product Lamellarin D

Article abstract of DOI:10.1016/j.ejmech.2021.113226

Lamellarin D, a marine natural product, acts as a potent inhibitor of DNA topoisomerase I (Topo I). To modify its physicochemical property and biological activity, a series of mono- and di-glycosylated derivatives were designed and synthesized through 22–26 multi-steps. Their inhibition of human Topo I was evaluated, and most of the glycosylated derivatives exhibited high potency in inhibiting Topo I activity as well as lamellarin D. All the 15 target compounds were evaluated for their cytotoxic activities against five human cancer cell lines. The typical lamellarin derivative ZL?3 exhibited the best activity with IC₅₀ values of 3 nM, 10 nM, and 15 nM against human lung cancer A549 cells, human colon cancer HCT116 cells and human hepatocellular carcinoma HepG2 cells. Compound ZL?1 exhibited anti-cancer activity with IC₅₀ of 14 nM and 24 nM against human colon cancer HCT116 cells and human hepatocellular carcinoma HepG2 cells, respectively. Cell cycle analysis in MDA-MB-231 suggested ZL?3 inhibited cell growth through arresting cells at the G2/M phase of the cell cycle. Further tests showed a significant improvement in aqueous solubility of ZL?1 and ZL?7. This study suggested that glycosylation could be utilized as a useful strategy to optimize lamellarin D derivatives as Topo I inhibitors and anticancer agents.

Full text of DOI:10.1016/j.ejmech.2021.113226

European Journal of Medicinal Chemistry 214 (2021) 113226
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, Synthesis and Structure-Activity Relationship Studies of
Glycosylated Derivatives of Marine Natural Product Lamellarin D
Liuliu Zheng a, Tingting Gao a, Zhiwei Ge b, Zhongjun Ma a, Jinzhong Xu a, Wanjing Ding a,
,
*
Li Shen a
a Ocean College, Zhejiang University, Zhoushan, 316021, China
b Analysis Center of Agrobiology and Environmental Sciences, Zhejiang University, Hangzhou, 310058, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Lamellarin D, a marine natural product, acts as a potent inhibitor of DNA topoisomerase I (Topo I). To
modify its physicochemical property and biological activity, a series of mono- and di-glycosylated de-
rivatives were designed and synthesized through 22e26 multi-steps. Their inhibition of human Topo I
was evaluated, and most of the glycosylated derivatives exhibited high potency in inhibiting Topo I
activity as well as lamellarin D. All the 15 target compounds were evaluated for their cytotoxic activities
against five human cancer cell lines. The typical lamellarin derivative ZLꢀ3 exhibited the best activity
with IC50 values of 3 nM, 10 nM, and 15 nM against human lung cancer A549 cells, human colon cancer
HCT116 cells and human hepatocellular carcinoma HepG2 cells. Compound ZLꢀ1 exhibited anti-cancer
activity with IC50 of 14 nM and 24 nM against human colon cancer HCT116 cells and human hepato-
cellular carcinoma HepG2 cells, respectively. Cell cycle analysis in MDA-MB-231 suggested ZLꢀ3
inhibited cell growth through arresting cells at the G2/M phase of the cell cycle. Further tests showed a
significant improvement in aqueous solubility of ZLꢀ1 and ZLꢀ7. This study suggested that glycosylation
could be utilized as a useful strategy to optimize lamellarin D derivatives as Topo I inhibitors and
anticancer agents.
Received 9 December 2020
Received in revised form
14 January 2021
Accepted 22 January 2021
Available online 28 January 2021
Keywords:
Lamellarin D
Topoisomerase I
Cytotoxicity activity
Aqueous solubility
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
multidrug resistance activity [15,16], and kinase inhibition activity
[17,18]. In addition, some lamellarins even displayed immunomodu-
Lamellarins were firstly isolated by Faulkner and coworkers from
the prosobranch mollusk Lamellaria sp. on Ngarol Island, which
contain a common 14-phenyl-6H-benzopyrano[40,30:4,5]pyrrolo[2,1-
a]isoquinoline scaffold [1]. In the past 30 years, more than 70 lamel-
larins and theiranalogues have been foundmainly but notexclusively
in a variety of marine mollusks, sponges, and ascidians [1e4]. They
can be classified into two categories according to their structural
features[5e7], including lamellarintypeI: pentacyclicskeletonwitha
pyrrolo [2,1-a]isoquinoline core, and lamellarin type II: monocycle
skeleton with an unfused 3,4-diarylpyrrole core. The lamellarins
exhibit broad anticancer activity [8,9], and the majority of Structure-
Activity Relationship (SAR) studies indicated that they are multi-
target anticancer agents, for instance, they have Topo I inhibition
activity [10,11], mitochondrial apoptotic activity [12e14], reversal of
lating activity [19,20], HIV integrase inhibitory activity [19,21], anti-
oxidant, and antibacterial [22] activities et al.
One of the most notable lamellarins is lamellarin D, which has
been proved to be a potent inhibitor of Topo I [10,23,24]. Bailly and
coworkers proposed a ternary complex model of DNA-Topo I-
lamellarin D by docking simulation studies [25], which demon-
strated that the hydroxy groups at C8 and C20 are hydrogen-bonded
with Asn722 and Glu356, and the carbonyl of the lactone ring is
hydrogen-bonded with Arg364 [25]. Besides, the SAR study indi-
cated the C5]C6 double bond ensures the planarity, which is
essential for Topo I inhibition of lamellarin D [26e28].
Despite the potent biological activities of lamellarin D in vitro,
further progression was limited due to its poor aqueous solubility
[29], which is caused by its unique pentacyclic skeleton. For
improving the solubility, intensive researches have been developed
on lamellarin D, for instance, introducing nuclear location signal
peptide [25], amino acid residues [30], PEG conjugates [29] or
Mannich side chains [31].
* Corresponding author.
E-mail address: shenli@zju.edu.cn (L. Shen).
https://doi.org/10.1016/j.ejmech.2021.113226
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

L. Zheng, T. Gao, Z. Ge et al.
European Journal of Medicinal Chemistry 214 (2021) 113226
Fig. 1. Novel glycosylated derivative of marine natural product lamellarin D as a cytotoxic agent towards cancer cells.
Glycosylation is an effective method to improve the solubility of
lead compounds [32e34]. The use of glycosyl moieties generally
improves biological activity [35] and targeting effect [36]. Otto
Warburg and coworkers demonstrated that cancer cells exhibit an
increased dependence on glycolytic pathways even in the presence
of sufficient oxygen [37], which indicated that cancer cells would
consume more glucose during their high-speed growth than
normal cells. Such theory was proved to be related to the over-
expression of membrane glucose transporters GLUT in cancer
cells [35,38,39]. Further studies showed a variety of anticancer
drugs, such as doxorubicin and paclitaxel, could be delivered spe-
cifically to cancer cells by covalently binding to carbohydrates
[35,40,41]. Hence, novel glycosylated optimization on C-8, 14, or
20-OH of lamellarine D could be an effective strategy for improving
the physicochemical bioactive properties which has not been
applied in the research of lamellarin D yet.
via attaching different glycosyl groups on hydroxy groups at C-8, 14
or 20 position of lamellarin D, to improve the solubility and cyto-
toxic activity of lamellarin D (Fig.1). In this study, fifteen mono- and
di-glycosylated analogues were designed and synthesized with
glucose [35], mannose, galactose [34,42] or ribose [43] as glycosyl
donors, and all these glycosylated derivatives were tested for their
cytotoxicity towards cancer cells and Topo I inhibition, in parallel
with the structureecytotoxicity analysis.
2. Results and discussion
2.1. Chemistry
The formation of pyrrole ring through a modified N-ylide method
[44e46] has been adopted as the key step in our route, which has
been shown in Scheme 1 for the retrosynthesis. The glycosylated
lamellarin D could be started from the lamellarin D derivative with C-
8, 14 and 20-OH selective exposure. The lactone ring D was
Taking all the above-mentioned aspects into consideration, we
initiated our work to generate a new class of antineoplastic agents
Scheme 1. Retrosynthetic analysis for monoglycosylated lamellarin D.
2

Scheme 2. Synthesis of hydroxyl selectively protected lamellarin D.
Scheme 3. Synthesis of the monoglycosylated derivatives of lamellarin D.
3

L. Zheng, T. Gao, Z. Ge et al.
European Journal of Medicinal Chemistry 214 (2021) 113226
Scheme 4. Synthesis of the diglycosylated derivatives of lamellarin D.
constructedatalatestagefromN-ylidemediatedpyrrolefromthekey
precursor phenacyl bromide 5 and isoquinoline 11 which would be
achieved by multistep reactions from commercially available homo-
vanilic acid and phenylethanamine 8. Both of the phenylethanamine
8 and phenacyl bromide 5 could be synthesized starting from
isovanillin.
Based on our previous work [31] (Scheme 2), the preparation of
lamellarin D derivative with all phenols protected 17 began with
the commercially available isovanillin 1, followed by Baeyer-Villiger
oxidation, hydrolysis, Friedel-Crafts acylation [47], methanesulfo-
11 with phenacyl bromide 5 under basic condition gave the key
intermediate 12. Then the carboxylic acid 14 was afforded by a
Vilsmeier-Haack formylation 13 [44] and oxidation. Later, the acetyl
group of 14 was further hydrolyzed to give the compound 15 which
was then transformed to lactone 16 by intramolecular cyclization.
Lactone 16 was treated with DDQ [50] to provide the compound 17.
As shown in Scheme 3, the synthesis of the glycosylated ana-
logues was started with hydroxy groups selectively protected
lamellarin D 17. Treatment of 17 with different methods allowed to
obtain hydroxy groups selectively exposed lamellarin D (18, 21 or
25), which were then treated with trichloroacetimidate glycosyl
donors (glucose, mannose, galactose and ribose) to afford the
essential precursors. The remaining hydroxyl protecting groups
were removed to provide mono-glycosylated Lamellarin D ZL 1e12
as target moleculars.
nation, acetation and a-bromination to obtain phenacyl bromide 5.
Meanwhile, isovanillin was readily converted into phenylethan-
amine 8 via a three-step sequence involving hydroxyl protection,
Henry reaction [48,49] and reduction. Amide moiety 10 was ob-
tained by acetylation of phenylethylamine 8 and homovanilic acid
9, and it was subsequently submitted to a Bischler-Napieralski re-
action [47], affording isoquinoline 11. Condensation of isoquinoline
After successful synthesis of the mono-glycosylated lamellarin
D, we turned our attention towards the synthesis of di-glycosylated
4

L. Zheng, T. Gao, Z. Ge et al.
European Journal of Medicinal Chemistry 214 (2021) 113226
Fig. 2. Variable temperature NMR experiment of compound ZL-3 in DMSO‑d6 from 25 to 70 ꢁC.
lamellarin D (Scheme 4), which began with a hydroxyl selective
exposure of 19a or 22a, followed by a similar synthetic sequence
(glycosylation and deprotection of hydroxyl group), to furnish the
final di-glycosylated lamellarin D ZL 13e15. However, after many
attempts, we still failed to obtain tri-glycosylated derivatives.
Finally, fifteen target compounds were prepared using commer-
cially available isovanillin, homovanilic acid, and glycosyl donors
(glucose, mannose, galactose, and ribose) as the original material
through 22e26 multi-steps, with an overall yield of 0.6e2.8%. All the
targetcompoundswere purifiedbysilicagelcolumnchromatography
and identified by high resolution mass spectrometry (HRMS), 1H and
13C NMR spectroscopy (Supporting Information).
Interestingly, according to the effect of neighboring group
participation, a single favorable steric configuration could be ob-
tained. However, the 1H and 13C NMR spectra of most of the syn-
thesized compounds showed the presence of two sets of NMR
signals of comparable intensity, and the J-value of hydrogen on
anomeric carbon is consistent, for example, the two sets of J-values
on compound ZL-3 are 6.6 Hz. We assumed that the two sets of
NMR signals are caused by a pair of slowly interconverting con-
formers and our assumption was supported by variable tempera-
ture proton spectra for typical compound ZLꢀ3 (Fig. 2). The two
rotameric underwent coalescence at 70 ꢁC and these changes in the
NMR spectra were reversible upon cooling, which demonstrated
the presence of rotational isomers.
above. The cytotoxic activities of ZLꢀ1 (IC50HCT116 ¼ 14 nM;
IC50HepG2
¼
24 nM) and ZLꢀ3 (IC50HCT116
¼
10 nM;
IC50HepG2 ¼ 15 nM) against HCT116 and HepG2 were even better
than lamellarin D (IC50HCT116 ¼ 25 nM; IC50HepG2 ¼ 88 nM). ZLꢀ4,
ZLꢀ6, ZLꢀ10, ZLꢀ11 and ZLꢀ12 showed moderate cytotoxic activity
against the five cancer cells with IC50 < 5 mM. However, cytotox-
icities of lamellarin D bearing glycosyl group at C-14 OH (ZLꢀ2,
ZLꢀ5, ZLꢀ8, ZLꢀ13 or ZLꢀ15) were decreased in the cultures of all
the five cell lines.
According to the IC50 values shown in Table 1, the cytotoxic
activity was related to the monosaccharide types, the connecting
position and amounts on the lamellarin D. In most case, the tested
compounds with glycosyl group at C-8 or 20-OH position showed
much better anti-proliferation activities than the test compounds
with glycosyl group at C-14-OH position. The test compounds with
glucose and galactose substitution generally resulted in higher
anti-proliferation activities than mannose or ribose substitution. In
addition, when introducing the same glycosyl group, mono-
glycosylated derivatives exhibited better anticancer activities than
the di-glycosylated derivatives.
To investigate the selectivity of glycosylated derivatives toward
Topo I, we used in vitro Topo I relaxation inhibition assays. As
illustrated in Fig. 3, the supercoiled DNA entirely changed to
relaxed DNA in the presence of Topo I, and the positive control
lamellarin D inhibited the relaxation, as indicated by the increase of
the supercoiled DNA, which was because lamellarin D stabilizes
Topo I-DNA cleavable complexes to inhibit Topo I activity. Based on
the result of Topo I inhibitory assays, most of glycosylated de-
rivatives showed potent Topo I inhibitory activity which were
similar to lamellarin D. Unexpectedly, ZLꢀ2, ZLꢀ5, ZLꢀ8, ZLꢀ13,
and ZLꢀ15 retained the Topo I inhibitory activity, but their anti-
proliferative activities were decreased compared with lamellarin
D. There might be other targets or mechanisms of the glycosylated
derivatives for the anti-proliferative activities in different levels.
The effects on the cell cycle distribution of compound ZLꢀ3 in
MDA-MB-231 cells were examined by flow cytometry after staining
of the cells with propidium iodide (Fig. 4A). MDA-MB-231 cells
2.2. Biological evaluation
All of the lamellarin D analogues ZL 1e15 were assayed for their
anticancer activities against five human cell lines, including human
breast cancer cells (MDA-MB-231), human gastric carcinoma cells
(HGC27), human lung cancer cells (A549), human colon cancer cells
(HCT116) and human hepatoma cells (HepG2). The cytotoxic ac-
tivities of the target compounds were evaluated by MTT assay and
their IC50 values are shown in Table 1. Among the glycosylated
derivatives, ZLꢀ1, ZLꢀ3, ZLꢀ7, ZLꢀ9 and ZLꢀ14 showed potent
anti-proliferation activities against the five cell lines mentioned
5

L. Zheng, T. Gao, Z. Ge et al.
European Journal of Medicinal Chemistry 214 (2021) 113226
Table 1
Structures and in vitro cytotoxicity of glycosylated lamellarin D.
Comp.
R1
R2
R3
IC50（mM）
MDA-MB-231
HGC27
A549
HCT116
HepG2
ZLꢀ1
Glu
H
H
Man
H
H
Gal
H
H
Rib
H
H
H
Glu
H
H
Man
H
H
Gal
H
H
Rib
H
H
H
Glu
H
H
Man
H
H
Gal
H
H
Rib
H
Glu
Glu
H
0.032
>50
0.034
1.991
>50
0.316
0.018
>50
0.025
5.081
2.113
2.693
13.257
0.036
7.373
0.031
0.114
>50
0.047
>50
0.003
3.239
>50
0.374
0.013
>50
0.020
4.121
0.659
2.856
13.707
0.026
16.363
0.004
0.014
26.236
0.010
1.424
>50
0.206
0.037
13.735
0.018
1.283
0.614
1.156
5.283
0.040
6.771
0.025
0.024
42.922
0.015
3.881
>50
0.570
0.048
>50
0.058
0.937
0.332
0.627
40.101
0.118
28.771
0.088
ZLꢀ2
ZLꢀ3
0.162
0.219
31.347
0.137
0.114
16.594
0.090
1.238
0.224
0.862
27.080
0.074
7.218
0.236
ZLꢀ4
ZLꢀ5
ZLꢀ6
ZLꢀ7
ZLꢀ8
ZLꢀ9
ZLꢀ10
ZLꢀ11
ZLꢀ12
ZLꢀ13
ZLꢀ14
ZLꢀ15
Lam-D
Glu
Glu
H
Glu
H
Glu
H
H
(IC50：the half-maximal inhibitory concentration; Lam-D: lamellarin D; MDA-MB-231: human breast cancer cells; HGC27: human gastric carcinoma cells; A549: human lung
cancer cells; HCT116: human colon cancer cells; HepG2: human hepatocellular carcinoma cells.).
were exposed to ZLꢀ3 (10 nM, 20 nM and 50 nM) for 24 h and
lamellarin D (20 nM and 50 nM) as the positive control. As shown in
Fig. 4A and B, both ZLꢀ3 and lamellarin D induced an increase of
cells in the G2/M phase arrests in a concentration-dependent
manner. Notably, ZLꢀ3 caused more accumulation of cells in G2/
M phase, compared to lamellarin D as the positive control. And
apoptosis significantly occurred in MDA-MB-231 cells after treat-
ment with a higher concentration of ZLꢀ3 (Fig. 4A and C).
To evaluate the binding mode of glycosylated derivatives, mo-
lecular docking studies were performed in the active sites of Topo I
(PDB code: 1k4t) by AutoDock 4.0. To confirm that our top docking
pose was reasonable, lamellarin D was docking into the binding site
of the crystal structure contain ligand camptothecin (CPT), and the
fact confirmed that the binding poses of the lamellarin D were
successfully docked in the correct binding site in the DNA-Topo I
complex.
Top binding poses of lamellarin D (Fig. 5A) showed that the hy-
droxygroupsatC8, C14, C20 andthecarbonyloxygenofthelactonering
were hydrogen-bonded with Glu356, Asn352, Asn722 and Arg364.
ZLꢀ1 (Fig. 5B, ꢀ10.1 kcal/mol), ZLꢀ3 (Fig. 5D, ꢀ10.27 kcal/mol) dis-
played a better docking energy for DNA-Topo I complex than ZLꢀ2
(Fig. 5C, ꢀ9.46 kcal/mol), which was in consistent with the experi-
mental in vitro anti-cancer activity observed. The hydroxy groups at
C14 and C20 of ZLꢀ1 established hydrogen bonds with Asn352,
Asn722, and the 6-OH of glycosyl formed hydrogen bonds with Glu
356. As for ZLꢀ2, the absence of the C-14 hydroxyl reduced the sta-
bility of the ternary complex, only the carbonyl oxygen of the lactone
ring bound to Arg364, and the hydroxy group of glycosyl bound to
Asn352, Tyr426. However, due to the change of glycosyl configura-
tion, the hydrogen bonds between glycosyl and enzyme were not
fixed. In the binding of the ZLꢀ3-DNA-Topo I complex, the hydroxy
groups at C8 and C14 and the carbonyl oxygen of the lactone ring were
engaged in hydrogen bonds with Asn722, Asn352, and Arg364, and
the 6-OH of glycosyl was hydrogen-bonded with Glu356.
glycosylated lamellarin D displayed lower binding affinities for
DNA-Topo I complex. The key interactions of the core of lamellarin
D with DNA-Topo I complex were significantly disturbed when
more glycosyl groups were placed on lamellarin D, such as in
Fig. 6A, only the oxygen atom of the glycosyl group interacted with
Asp533. In Fig. 6B, the carbonyl oxygen and oxygen atom of the
lactone ring established hydrogen bonds with Arg364, the oxygen
atoms of the glycosyl groups interacted with Asp533, Tyr426, and
Thr718 residues through H-bonds. For ZLꢀ15, the hydroxy group at
C8 was interacting with Met428 and Asn352. As for the tri-
glycosylated lamellarin D, it engaged the glycosyl groups with
hydrogen bonds to the Arg364, Asn722, Met428, and Tyr426.
The solubilities of lamellarin D, mono- and di-glycosylated
lamellarin D derivatives were tested in distilled water with HPLC-
HRMS method. As shown in Table 2, the solubility of lamellarin D
was 0.563 nM, and some of the glycosylated derivatives possessed
significantly improved water solubility than that of lamellarin D. It
should be noted that ZLꢀ1, ZLꢀ7 and ZLꢀ15 had the best water
solubility of 257.78 nM, 235.82 nM and 257.55 nM, which were
almost up to 500-fold higher than that of lamellarin D. However,
the water solubilities of ZLꢀ2, ZLꢀ4, ZLꢀ5, ZLꢀ8 and ZLꢀ9 were
even not as good as that of lamellarin D. The water solubilities of
ZLꢀ10, ZLꢀ11 and ZLꢀ12 were lower than the detection limits.
As shown in Fig. 7, ZLꢀ1 and ZLꢀ7 bearing glucosyl or galactosyl
group at C-8-OH position exhibited remarkable water solubility.
However, introducing a mannosyl or ribosyl at C-8-OH position of
lamellarin D led to ZLꢀ4 and ZLꢀ10, which showed poor water
solubility. The water solubility decreased after introducing glycosyl
into the C-14 or 20-OH position of lamellarin D. Compared with the
mono-glycosylation derivatives, there was no significant improve-
ment in the solubility of di-glycosylation derivatives. These results
suggested that the aqueous solubility of glycosylated lamelllarin D
was related to the types of monosaccharide and the connecting
position on lamellarin D. The water solubility and cytotoxic activity
has been compared, it was found that the compounds with good
As shown in Fig. 6, all three di-glycosylated lamellarin D and tri-
6

L. Zheng, T. Gao, Z. Ge et al.
European Journal of Medicinal Chemistry 214 (2021) 113226
Fig. 3. Topo I inhibitory activity was evaluated by agarose gel electrophoresis. Inhibition of Topo I relaxation activity at 10 mM. Lamellarin D was a positive control. Reaction samples
were incubated at 37 ꢁC for 30 min and separated by electrophoresis on a 1% agarose gel, then stained with 4s green, and photographed under the irradiation of UV light. Lane 1 is
pBR322 DNA only; Lane 2 is the mixture of pBR322 DNA and Topo I; Lines 3e17 are the mixture of pBR322 DNA, Topo I and test compounds. Line 18 is the mixture of pBR322 DNA,
Topo I and positive control lamellarin D.
water solubility often showed potent antiproliferative activity such
as ZLꢀ1, ZLꢀ7 and ZLꢀ14. These data indicated that the water
solubility of glycosylated derivatives could affect their anti-
proliferative potencies. Interestingly, ZLꢀ3 and ZLꢀ9 exhibited
remarkable antiproliferative potencies, albeit their poor water
soluble which meant that their antiproliferative activity did not
entirely depend on the water solubility.
residue at the C-14-OH position lowered the cytotoxicity, which
was not consistent with their Topo I inhibitory activity, so there
might be other targets of the glycosylated derivatives for anti-
cancer activity. The docking study confirmed that C-8 or 20-OH
glycosylated lamellarin D-Topo I-DNA ternary complex was stable
than C-14-OH glycosylated lamellarin D-Topo I-DNA ternary com-
plex, and with the increase of glycosyl groups, the compounds led
to a lower binding affinity to the Topo I-DNA complex. In addition,
when the MDA-MB-231 cells were exposed to ZLꢀ3, the number of
cells in G0/G1 phase decreased and those in G2/M phase increased
compared with the control, and the population of cells in G2/M
phase increased in a dose-dependent manner. The investigation
suggested that ZLꢀ3 induced cell-cycle arrest in the G2/M phase
and increased cell apoptosis. The water-soluble study indicated that
the introduction of glucosyl or galactosyl to the C-8-OH position of
lamellarin D increased their aqueous solubility and their anti-
cancer activity. The synthesis and study on tri-glycosylated de-
rivatives or other derivatives are currently in progress.
3. Conclusion
In this study, a new class of glycosylated derivatives were
designed and synthesized based on the skeleton of lamellarin D, the
corresponding cytotoxic activities, and Topo I inhibitions were
investigated. The result demonstrated that most of the glycosylated
derivatives kept the potent Topo I inhibitory activity which were
similar to lamellarin D. The cytotoxicity was improved after
glycosylation with glucose or galactose at the C-8 and 20-OH po-
sitions of lamellarin D. In contrast, the presence of a glycosyl
Fig. 4. (A) Cell cycle analysis: MDA-MB-231 cells were treated with lamellarin D (20 nM and 50 nM) or ZLꢀ3 (10 nM, 20 nM and 50 nM) for 24 h and PI staining was used to analyze
the cell cycle distribution. (B) The percentages of MDA-MB-231 cells in the different phases of cell cycle were presented. (C) The percentages of MDA-MB-231 cells in the different
phases of cell cycle and sub-G1 were presented.
7

L. Zheng, T. Gao, Z. Ge et al.
European Journal of Medicinal Chemistry 214 (2021) 113226
Fig. 5. Calculated binding poses of ligand to the DNA-Topo I complex (PDB ID: 1k4t (http://www.rcsb.org/structure/1K4T); cyan), key hydrogen-bonding interactions are indicated
with yellow dashes. (A) Lam D (white)/CPT (magenta)-DNA-Topo I complex, the docking energy of lamellarin D-DNA-Topo I complex was estimated to be ꢀ10.87 kcal/mol (B) ZLꢀ1
(white)-DNA-Topo I complex, the docking energy was estimated to be ꢀ10.10 kcal/mol (C) ZLꢀ2 (white)-DNA-Topo I complex, the docking energy was estimated to be ꢀ9.46 kcal/
mol; (D) ZLꢀ3 (white)-DNA-Topo I complex, the docking energy was estimated to be ꢀ10.27 kcal/mol.
3.1. Experimental section
cells were then incubated for 4 h. The medium was removed and
100 L of DMSO was added into the wells to dissolve the formazan
m
3.1.1. General procedures
crystals and the absorption at 540 nm was recorded by a microplate
reader (labsystem multiscan). Cell proliferation rates for each well
were calculated as follows: A540treated/A540control ꢂ 100%. IC50
values were determined as the in vitro concentration of compound
required to inhibit cell proliferation by 50%.
Proton NMR and carbon NMR were performed on JNM-
ECZ600R/S1 (1H, 600 MHz; 13C, 150 MHz). Waterless operation
was performed on Unilab Pro Sp (1500/780). Low temperature re-
action was performed on PSL2000. The organic solvents were dried
and purified by MB-SPS-5. Water Solubility was performed on AB
Sciex Triple TOF 5600þ. Flash column chromatography was per-
formed using silica gel (200e300 mesh) and sephadex LH-20.
Visualization was achieved under a UV lamp (254 nm). Flow
cytometry assay was performed on FACS Calibur. The organic sol-
vents were dried and purified before use. Substrates 2e18, 21, 24
and 25 were prepared according to our previous work [31].
Experimental procedures and copies of 1H, 13C NMR spectra and
HRMS spectra can be found in the Supporting Information.
3.1.3. Topo I inhibition assay
The reaction mixture (20
I Buffer (2 L), supercoiled DNA (pBR322, 250 ng, TaKaRa Bio), Topo
I (2 U), and indicated drug concentrations were incubated at 37 ꢁC
for 30 min. Reactions were terminated by the addition of 2 L 10%
SDS, digested with 2 L proteinase K for 30 min. The reaction
mL) contained 10X DNA Topoisomerase
m
m
m
product was subjected to 1% agarose gel electrophoresis in the
absence of 4s Green. After electrophoresis, the 4s Green-free gel
were stained with 0.5
photodocumentation.
mg/mL 4s Green for 50 min prior to
3.1.2. Antiproliferative assay
Cells were collected and resuspended in DMEM growth
medium þ10% fetal bovine serum (FBS). The cells were seeded on
96-well plates at the concentration of 5 000 cells/well and incubated
3.1.4. Flow cytometry assay
MDA-MB-231 (5 ꢂ 10^5) cells were seeded into six-well plates,
cultured overnight at 37 ꢁC, and treated with test compounds at
indicated concentrations (10 nM, 20 nM, 50 nM) for 24 h. After drug
treatments, the cells were collected by trypsinization, washed
twice with phosphate-buffered saline (PBS), and fixed with 70%
ethanol overnight at ꢀ20 ꢁC. Before staining, cells were washed
with PBS to remove ethanol. Fixed cells were stained with
at 37 ꢁC in 5% CO2 overnight. The following day,100
mL of compounds
with serial dilutions were added into the corresponding wells, and
the plates were further incubated at 37 ꢁC for 72 h. The cell viability
was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. The medium was
changed with a fresh medium containing 0.5 mg/mL of MTT, and the
8

L. Zheng, T. Gao, Z. Ge et al.
European Journal of Medicinal Chemistry 214 (2021) 113226
Fig. 6. Calculated binding poses of ligand to the DNA-Topo I complex (cyan), key hydrogen-bonding interactions are indicated with yellow dashes. (A) ZLꢀ13 (white)-DNA-Topo I
complex, the docking score was estimated to be ꢀ8.23 kcal/mol (B) ZLꢀ14 (white)-DNA-Topo I complex, the docking score was estimated to be ꢀ10.17 kcal/mol (C) ZLꢀ15 (white)-
DNA-Topo I complex, the docking score was estimated to be ꢀ9.27 kcal/mol. (D) Triglycosylated lamellarin D (white)-DNA-Topo I complex, the docking energy was estimated to
be ꢀ6.87 kcal/mol.
Table 2
Structures and aqueous solubility of glycosylated lamellarin D.
No.
R1
R2
R3
Aqueous solubility（nM）
ZLꢀ1
Glu
H
H
Man
H
H
Gal
H
H
Rib
H
H
H
Glu
H
H
Man
H
H
Gal
H
H
Rib
H
H
H
Glu
H
H
Man
H
H
Gal
H
H
Rib
H
Glu
Glu
H
257.78 7.903
0.41 0.090
2.00 0.288
0.23 0.000
0.14 0.006
0.37 0.085
235.82 5.451
0.49 0.033
0.12 0.001
<0.05
<0.05
<0.05
96.75 8.796
182.31 9.772
257.55 10.369
0.56 0.119
ZLꢀ2
ZLꢀ3
ZLꢀ4
ZLꢀ5
ZLꢀ6
ZLꢀ7
ZLꢀ8
ZLꢀ9
ZLꢀ10
ZLꢀ11
ZLꢀ12
ZLꢀ13
ZLꢀ14
ZLꢀ15
Lam-D
Glu
Glu
H
Glu
H
Glu
H
Fig. 7. The water solubility of lamellarin D and glycosylated derivatives. The aqueous
solubility was tested by HPLC-HRMS method. The standard curves were shown in
Supporting Information Figure. S1.
H
9

L. Zheng, T. Gao, Z. Ge et al.
European Journal of Medicinal Chemistry 214 (2021) 113226
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The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
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Acknowledgment
T. Fukuda, F. Ishibashi, L. Meijer, M. Iwao, Synthesis, resolution, and biological
evaluation of atropisomeric (aR)- and (aS)-16-methyllamellarins N: unique
effects of the axial chirality on the selectivity of protein kinases inhibition,
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Synthesis, structureeactivity relationships, and mechanism of action of anti-
This work was supported by National Natural Science Founda-
tion of China (No. 81402845 and partially No. 82073694), Natural
Science Foundation of Zhejiang Province (No. LY21H300002) and
the Science and Technology Project of Zhoushan (2018C81035). We
are grateful to Dr. Daoqiong Zheng, Dr. Pingmei Wang, Ms. Jiankun
Wu, and Ms. Yuchao Yu for Topo I Inhibition Assay and to Ms. Jinhui
Wang, Ms. Yalin Yu, and Mr. Ziyang Cheng for HPLC assay.
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