Syntheses and structure-activity relationship studies of N-substituted-β-d-glucosaminides as selective cytotoxic agents

Article abstract of DOI:10.1016/j.bmcl.2012.09.075

Twenty-four diosgenyl saponins bearing cinnamoyl, carbamido and thiosemicarbazone groups were synthesized concisely. The cytotoxicities of the synthetic compounds on six human caner cell lines were evaluated employing MTT method. Structure-activity relationship could be observed, and two of the synthesized compounds (5c and 5f) exhibited selective inhibition on HeLa and MCF-7 cells, while three of them (5d, 5f and 5h) showed strong inhibition against HT1080.

Full text of DOI:10.1016/j.bmcl.2012.09.075

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7110–7113
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Syntheses and structure–activity relationship studies
of N-substituted-b-D-glucosaminides as selective cytotoxic agents
⇑
Bo Wang, Yang Liu, Yanshi Wang, Xin Liu, Mao-Sheng Cheng
Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University,
Shenyang 110016, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 July 2012
Revised 4 September 2012
Accepted 21 September 2012
Available online 28 September 2012
Twenty-four diosgenyl saponins bearing cinnamoyl, carbamido and thiosemicarbazone groups were syn-
thesized concisely. The cytotoxicities of the synthetic compounds on six human caner cell lines were
evaluated employing MTT method. Structure–activity relationship could be observed, and two of the syn-
thesized compounds (5c and 5f) exhibited selective inhibition on HeLa and MCF-7 cells, while three of
them (5d, 5f and 5h) showed strong inhibition against HT1080.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Glucosaminides
Cinnamoyl
Urea
Thiosemicarbazone
Selective inhibition
Saponins which are composed of a saccharide moiety and the
aglycone have attracted much attention due to their cytotoxic
activity. Among the saponins isolated from nature, either steroidal
or triterpenoidal, the presenting saccharides attached on the
structures. Since trans-Cinnamic acid has been demonstrated to
possess a variety of biological activities including anti-cancer and
used to induce a reversal of malignant properties of several human
tumor cells in vitro.9–12 While both carbamido and thiosemicarba-
zone groups are quite common substructures in the anti-cancer
compounds. Especially for thiosemicarbazone, it has been
researched for decades, and believed that the antitumor activity
seems to be due to an inhibition of DNA synthesis.13–15 Based on
these researches, three series of diosgenyl glucosaminide with sub-
stituents of cinnamoyl, carbamido and thiosemicarbazone on their
amino groups were designed and synthesized to test their in vitro
cytotoxicities against different cancer cell lines and find the SARs of
these compounds (Fig. 1).
aglycon are usually b-D-glucopyranose, a-L-rhamnopyranose, and
b- -galactopyranose. Though it is quite common as a constituent
D
of many natural poly- or oligosaccharides, glucosamine rarely
emerged in natural saponin, however they are presenting strong
in vitro cytotoxicity towards different tumor cell lines.1,2 The potent
cytotoxic property indicates that the sugar chains of glucosamine
dramatically enhance the bioactivity of their aglycons. Therefore,
it is a quite rational design that some modifications are performed
on the glucosamine. Up to date, most researches are focused on the
isolation of glucosaminides, fewer works are done with the struc-
tural modification of those saponins.3,4 The earliest reference could
As parts of our research on saponins, the representative agly-
cons of steroidal saponins diosgenin was selected as scaffolds to
date back to the year of 2003 in which 2-amino-2-deoxy-b-
D
-
prepare the N-substituted-b-D-glucosaminide derivatives (Scheme
glucopyranoside hydrochloride was synthesized, and in combina-
tion with cladribine it can increase the number of apoptotic B cells
isolated from B-CLL patients.5,6 Later in 2008 and 2009, modifica-
tions on the amino group of diosgenyl 2-amino-2-deoxy-b-
1). 2,2,2-Trichloroethoxycarbonyl group (Troc) was used to protect
amino group in glycosyl imidate, considering the toleration and
deprotection conditions of the protecting group.16 The synthetic
route began with the glycosylation between diosgenin and glycosyl
imidate catalyzed by TMSOTf as showing in Scheme 1. The config-
uration of newly formed glucosidic bond was b (demonstrated by
the 8.2 Hz coupling constant of J1-2 in 1H NMR), due to Troc group’s
neighboring group participating capacity. Intermediate 2 was pre-
pared by following known procedures.7,8 After deprotection of the
Troc group, the amine 3 was condensed with substituted cinnam-
oyl chloride, which were prepared via Knoevenagel reaction from
commercially available substituted benzaldehydes and malonic
acid in the presence of pyridine to afford intermediates 4a–4n,17
D-glucopyranoside were initially reported, which showed that the
substitutents on amino groups had great influences on their
cytotoxicity against several tumor cell lines.7,8
Inspired by the previous results, we assumed that the introduc-
tion of some cytotoxic pharmaphores onto glucosamine saponin
might be
a rational method to produce more potent lead
⇑
Corresponding author. Tel.: +86 24 23986413; fax: +86 24 23995043.
E-mail address: mscheng@syphu.edu.cn (M.-S. Cheng).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.075

B. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7110–7113
7111
Figure 1. Structures of N-substituted-b-D-glucosaminide.
Scheme 1. Reagents and conditions: (a) TMSOTf, CH2Cl2, 0 °C, 84%; (b) Zinc dust, AcOH, overnight; (c) substituted cinnamoyl chloride, pyridine, CH2Cl2, 5 h; (d) R-NH2, DIEA,
DMSO, 70 °C; (e) thiocarbonyl chloride, CaCO3, CH2Cl2, H2O, rt, then 80% hydrazine hydrate, ethanol, rt, 52.4% in two steps; (f) substituted benzaldehydes, THF, reflux, 1 h;
(g) NH3, MeOH, overnight, yields and compound details of cinnamoyl series see Table 1; 7a: R5 = benzyl, 62.7%; 7b: R5 = 4-flurobenzyl, 55.4%; 7c: R5 = 4-methoxybenzyl,
68.3%; 7d: R5 = cyclopropyl, 71.2%; 7e: R5 = t-butyl, 69.4%; 7f: R5 = heptyl, 54.6%; 7g: R5 = piperidyl, 75.1%; 10a: R6 = R7 = H, 48.4%; 10b: R6 = H, R7 = OMe, 52.5%; 10c: R6 = CF3,
R
7 = H, 53.0%.
followed by a fully deprotection in NH3-MeOH to provide the
N-cinnamoyl-b- -glucosaminide 5a–5n. All the synthetic com-
pounds’ structural details and yields were summerized in Table
1. For the synthesis of urea series, the key intermediate 2 was
directly condensed with commercially available amines in the
presence of DIEA in DMSO at 70 °C as shown in Scheme 1, then
D

7112
B. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7110–7113
Table 1
Whereas introducing carbamido and thiosemicarbazone groups
neither provided any competitive compounds comparing to the
positive control, nor generated obvious structure–activity relation-
ships and inhibition selectivity.
Structural details and yields of the cinnamoyl series compounds
Entry
R1
R2
R3
R4
Yielda (%)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
H
H
H
OMe
H
H
H
H
H
OMe
H
OMe
H
H
F
Me
H
H
OMe
H
OMe
H
OMe
OMe
H
H
H
H
H
H
H
H
H
OMe
H
OMe
H
H
H
48.8
69.2
59.0
61.4
62.8
65.7
58.6
60.0
49.5
45.4
59.5
63.3
45.3
47.9
As shown in Table 2 (from entries 1 to 15), some compounds in
cinnamoyl series showed selective inhibitions against several can-
cer cell lines. This selectivity referred to two kinds of diversities,
including different sensitivities of varied cancer cell lines to the
same samples in transverse comparison and influences of different
samples with tiny modifications in their structures on the cytotox-
icity against the same cancer cell lines in longitudinal compraison.
For most of the synthetic saponins belonging to the cinnamoyl ser-
ies, they generally showed more active in HeLa and MCF-7 than in
A549, HepG2 and HCT116, which matched the selectivity tendency
of positive control 1. The cytotoxicity diversities were significantly
relied on the types of cinnamoyl groups introduced. For instances,
cinnamoyl series derivatives 5a showed a modest increases of
cytotoxicities comparing to the positive control 1 against all the
cancer cell lines tested, however 5c, 5f which was slightly modified
from 5a showed excellent activity against HeLa and MCF-7 than 5a
and positive control 1, while changes of their cytotoxicities against
A549, HepG2 and HCT116 were not obvious. Most of the samples
showed remarkable activity increases against HT1080 comparing
to the positive control 1, nevertheless tiny modifications on their
structures still could significantly affect their cytotoxicities. The
detailed structure–activity relationships were discussed as follows.
A diverse electron withdrawing groups and electron donating
groups were introduced to the different position of phenyl ring
in 5a for investigation of the structure–activity relationships
(SAR). As shown in Table 2, compound 5c and 5f bearing methyl
and methoxy groups at para-position were significantly more ac-
tive in HeLa and MCF-7 than 5a and 5b, which contains no or elec-
tron withdrawing groups at the same positions. The similar
tendency could be observed between derivatives bearing
H
OMe
OMe
OMe
H
H
H
H
OMe
OMe
Cl
Br
H
H
NO2
H
H
a
Yields based on intermediate 2
the condensation products 6a–6g were deacetylated in NH3-MeOH
solution to give the urea-based products 7a–7g in 54.6% to 75.1%
yields. To synthesize the thiosemicarbazone series, the free amino
group in intermediate 3 was firstly converted to isosulfocyanide,
which was then reacted with hydrazine affording 8 in ethanol.
After condensation with substituted benzaldehydes in THF, all
the protective acetyl groups were cleaved to yield 10a–10c.
With all the synthetic glucosaminides with diversified substitu-
ents on their amino groups in hand, the cytotoxic effect against
HeLa, MCF-7, A549, HepG2, HCT116 and HT1080 were determined
employing MTT assay, and the IC50s were illustrated in Table 2.
Compound 1 with acetyl on the amino group and 5-fluorouracil
were chosed as positive controls. The preliminary in vitro screen-
ing indicated that some saponins belonging to the cinnamoyl series
showed moderate to excellent antiproliferative activities against
cell lines tested comparing to the positive control 1 and 5-FU.
Table 2
The in vitro cytotoxicities of three series saponins
a,b
Entry
compound
IC50
(lM)
HeLa
MCF-7
A549
HepG2
HCT116
HT1080
1
2
3
4
5
6
7
8
5a
5b
5c
5d
5e
5f
5g
5h
5i
12.8 2.2
16.0 1.3
21.2 2.0
5.3 0.7
23.1 3.2
24.1 4.4
0.5 0.2
44.4 3.7
26.7 3.3
>50
31.3 2.4
>50
>50
>50
>50
23.9 1.8
>50
>50
>50
26.8 2.9
>50
>50
48.2 4.0
>50
41.8 0.9
33.8 1.2
41.6 2.4
23.1 1.3
26.9 0.9
24.4 4.2
43.4 0.7
27.1 3.1
47.8 0.8
30.3 2.7
>50
>50
>50
>50
28.5 1.5
>50
36.3 1.3
39.8 2.7
35.5 0.9
29.5 1.2
>50
30.7 2.5
45.6 3.7
41.3 4.0
31.7 0.8
25.9 1.7
>50
>50
>50
>50
>50
>50
>50
>50
>50
39.1 3.7
35.0 2.5
40.4 1.7
38.9 2.6
>50
29.5 2.2
47.4 1.3
42.9 3.5
35.6 4.7
29.0 0.6
>50
>50
>50
>50
>50
>50
>50
>50
>50
29.8 3.2
34.9 2.5
23.5 1.5
4.3 0.3
26.6 1.5
3.5 0.8
21.2 2.1
5.8 0.1
>50
>50
>50
>50
>50
26.9 1.1
6.5 1.1
27.5 5.5
27.0 3.0
6.1 1.2
36.2 0.2
22.3 8.1
32.2 2.7
26.1 1.8
>50
>50
>50
>50
27.0 3.0
>50
9
10
11
12
13
15
16
17
18
19
20
21
22
23
24
25
26
27
5j
5k
5l
5m
5n
7a
7b
7c
7d
7e
7f
7g
10a
10b
10c
1
36.5 0.5
>50
42.0 2.2
>50
>50
>50
>50
>50
>50
43.5 3.7
27.6 6.0
>50
>50
42.3 2.3
43.0 3.0
35.1 1.4
>50
>50
>50
>50
>50
29.5 3.9
28.2 2.6
>50
>50
>50
40.0 3.4
38.2 1.8
>50
>50
>50
36.2 9.3
34.8 9.0
>50
49.7 0.1
37.3 2.2
>50
>50
88.9
>50
21.5 1.1c
63.4
18.3 0.6c
78.6
>50
51.5
>50
36.4
>50
15.2
5-FU
a
Concentration inhibiting fifty percent of cell growth for 48 h exposure period of tested samples. Data represent mean values standard deviation for independent
experiments.
b
IC50s more than 50
IC50s reported in Ref.
l
M are not shown their exact value.
c
8

B. Wang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7110–7113
7113
substituents with different electron properties at the same position
Supplementary data
(5d comparing with 5n, and 5e comparing with 5l and 5m).
Accordingly, we can conclude that electron donating groups in
the cinnamoyl groups were preferred. Meanwhile comparing the
cytotoxicities against HeLa and MCF-7 of those saponins bearing
methoxy groups (entries 4–11), it is clear that mono-substituted
at the para-position is much more active, while introduction of
methoxy groups at ortho- or meta-position leads to a decline of
cytotoxicity.
The selective inhibition against HT1080 of cinnamoyl series can
also be found, and the selectivity relies on the types and position of
the substituents. As shown in Table 2 (HT1080 datas in Entries 4, 6
and 8), methoxy group is exclusive and any introduction of other
groups leads to the decline of activity. Ortho- and para-position is
preferred, and introduction of the methoxy group at meta-position
leads to a significant decline of activity.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.09.
075.
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In summary, a series of N-substituted-b-D-glucosaminides were
synthesized from commercially available starting materials in high
yields. Cytotoxic effects of all the synthetic saponins were evalu-
ated against six human cancer cell lines. Preliminary SAR research
showed that diosgenyl saponins bearing an electron-donating sub-
stituent at parasite of cinnamoyl group is preferable against HeLa
and MCF-7, while diosgenyl saponins bearing methoxy groups at
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HT1080. Further study based on the selective inhibition on HeLa,
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The authors would like to thank New Drug Research& Develop-
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