Simplified aminocoumarin analogues as anticancer agents: Amino isosteric replacement in the noviose moiety resulted in substantial enhancement of antiproliferative activity

Article abstract of DOI:10.1016/j.cclet.2013.04.046

Simplified aminocoumarin analogues, either noviosylated or simple basic heterocycle attached 3-amido-coumarin compounds, are known to be promising anticancer agents targeting the C-terminal ATP-binding site of Hsp90. In this study, 3′-amino isosteric replacement in the noviose moiety of two known noviose containing Hsp90 C-terminal inhibitors was synthetically realized for the first time. In vitro evaluation of these compounds suggested that the introduction of a basic amino group into the noviose subunit resulted in significant improvement of their cytotoxicities.

Full text of DOI:10.1016/j.cclet.2013.04.046

Chinese Chemical Letters 24 (2013) 719–722
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Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Simplified aminocoumarin analogues as anticancer agents: Amino isosteric
replacement in the noviose moiety resulted in substantial enhancement of
antiproliferative activity
*
*
Ting Zhang, Zheng Yan, Yun-Feng Li , Nan Wang
State Key Laboratory of Bioactive Substance and Function of Natural Medicine, Beijing Key Laboratory of Active Substance Discovery and Drugability Evaluation, Institute of Materia
Medica, Peking Union Medical College & Chinese Academy of Medical Sciences, Beijing 100050, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Simplified aminocoumarin analogues, either noviosylated or simple basic heterocycle attached 3-amido-
coumarin compounds, are known to be promising anticancer agents targeting the C-terminal ATP-
binding site of Hsp90. In this study, 30-amino isosteric replacement in the noviose moiety of two known
noviose containing Hsp90 C-terminal inhibitors was synthetically realized for the first time. In vitro
evaluation of these compounds suggested that the introduction of a basic amino group into the noviose
subunit resulted in significant improvement of their cytotoxicities.
Received 5 March 2013
Received in revised form 12 April 2013
Accepted 18 April 2013
Available online 31 May 2013
Keywords:
ß 2013 Yun-Feng Li and Nan Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All
rights reserved.
3-Amino-3-deoxy-L-noviose
Aminocoumarin analogues
Cytotoxicity
Hsp90 inhibitors
1. Introduction
complex noviose moiety could be replaced by a simple piperidine
appendage without deterring in vitro anticancer activity [6].
Aminocoumarin antibiotics are coumarin 7-O-
L
-noviosides
Undoubtedly, the data disclosed so far are encouraging, but in
terms of the potency of the screened compounds, the mission to a
clinically useful Hsp90 C-terminal targeting aminocoumarin
analogue is still far from success. To this end, we report herein
the synthesis and in vitro cytotoxicities of a newly designed series
of aminocoumarin analogues that could be structurally general-
ized by 7 (Fig. 1).
produced by different species of Streptomyces [1]. Novobiocin
(1) and clorobiocin (2) (Fig. 1), two typical members of this natural
product family, are historically known as potent antibacterial
agents that target bacterial DNA gyrase B [2]. However, Neckers
and coworkers revealed in 2000 that both 1 and 2 bound, although
in low potency, to the previously unknown C-terminal ATP-
binding site of heat shock protein 90 (Hsp90) and therefore might
represent a distinct class of Hsp90 inhibitors [3].
As Hsp90 is a growingly recognized anticancer drug target [4],
the findings of Neckers provoked pioneering chemical explorations
by Blagg et al., leading to the identification of the drastically
simplified, but biologically improved, novobiocin analogue 3 [5].
Unfortunately, this compound was found lacking cytotoxicity
against cancer cells in spite of its low micro-molar affinity to the
Hsp90 C-terminal ATP binding site [6]. Studies were therefore
continued by modifying the coumarin 3-N-acyl group, which
resulted in the discovery of the prototype cytotoxic compound 4
and the more potent analogue 5 [7]. Moreover, the recently
reported compound 6 (Fig. 1) suggested that the structurally
2. Experimental
2.1. Chemistry
Our synthesis of the target molecules started with the
construction of N-Cbz-3-amino-3-deoxy-
ing a previously reported approach to the N-benzoyl variant
(Scheme 1) [8]. The seven-step sequence starting from dimethyl
L-noviose (8) by follow-
L-
tartrate was reproduced to give the chiral homoallylic amine 9,
which was then treated with benzyl chloroformate to afford amide
10. After oxidative cleavage of the terminal C55C double bond in 10,
the resulting hemiketal 11 was dehydrated with TsCl in pyridine
under reflux, giving the dihydropyrane derivative 12 in excellent
yield [9]. The substrate controlled stereo-selective epoxidation of
12 with m-CPBA and subsequent in situ hydrolysis of the resulting
epoxide helped furnish the preparation of 8.
* Corresponding authors.
E-mail addresses: liyunfeng@imm.ac.cn (Y.-F. Li), wangnan@imm.ac.cn
(N. Wang).
1001-8417/$ – see front matter ß 2013 Yun-Feng Li and Nan Wang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2013.04.046

[
T. Zhang et al. / Chinese Chemical Letters 24 (2013) 719–722
Coumarin
OH
H
N
R1
OH
H
N
H
N
4
O
N
H
3
O
O
O
Noviose
5'
O
R2
O
O
O
O
O
MeO
7
O
O
O
8
R1
Me
HO
O
MeO
OH
O
4'
MeO
HO
3' 2'
OH
O
OH
R1
R2
H
5
R2
O
3
4
Me
Ph
Me
R1
R2
NH
OH
Novobiocin 1
Clorobiocin 2
Me
2
H
N
H
R1
N
Cl
N
H
O
Me
O
O
O
O
O
O
O
R2
O
Me
MeO
H2N
OH
HN
7
6
values were reported against MCF-7 cancer cells, 4: 20
50
[(Scheme_1)TD$FIG] Fig. 1. Aminocoumarin natural products and anticancer analogues (IC
m
mol/L, 5: 0.5
mmol/L, 6: 0.8
mmol/L).
OH NHCbz
OH
NH2
OH
7 steps
COOMe
a
OH
OH
b
O
O
MeOOC
d
O
c
MeO
CbzHN
MeO
CbzHN
MeO
CbzHN
OMe
OMe
OH
OH
9
10
11
12
8
Scheme 1. Reagents and conditions: (a) CbzCl, 10% aq. Na2CO3, Et2O, 0 8C–r.t., 72%; (b) NaIO4, OsO4, 2,6-lutidine, 1:1 dioxane/H2O, 86%; (c) TsCl, pyridine, reflux, 89%; (d) m-
CPBA, 1:1 Et2O/H2O, 0 8C–r.t., 83%.
To accomplish the final assembly of the target molecules,
various substituted 7-hydroxy-coumarins (13a–f, Scheme 2) were
prepared according to the previous work [10], and then coupled
with the key building-block (8) under Mitsunobu conditions [11].
Due to the difficulties in the separation of the coupling products
from triphenylphosphine oxide generated in the reactions, the
mixtures were directly subjected to the hydrogenolysis catalyzed
by palladium on charcoal in methanol to remove the N-Cbz group
Hangzhou, China) and 100 units/mL of both penicillin and
streptomycin, in a humidified 5% CO2/air atmosphere at 37 8C. A
MTT assay was used to determine the cytotoxicity of the six
compounds prepared, with compound 4 and 6 (synthesized
Table 1
Yields of the final stage assembly and cytotoxicities of 7a–f.
Compounds R1
R2
Yielda IC50
A2780
60.58
(
m
mol/L)
HeLa
38.95
and delivered
a-configured 7a–f in acceptable yields (Table 1). In
fact, the -isomers that may be generated in the coupling reactions
b
Ketr-3
26.34
MCF-7
25.78b
6.92b
>100
>100
were not obtained in the above experiments. The structures and
the configurations of these target molecules were confirmed by 1H-
NMR, 13C NMR, FAB/ESI-MS and IR spectra.
4
–
–
–
H
–
–
6
–
42.41
>100
2.78
>100
2.53
>100
>100
89.34 >100
7a
7b
7c
7d
7e
7f
Me
32%
30%
Me Cl
>100
>100
>100
>100
Me Me 33%
2.2. Biological assay
Ph
Ph
Ph
H
39%
38%
17.00
15.76
12.45
14.60
2.14
59.81
Cl
2.83
7.52
5.35
2.89
13.84
8.12
The human cancer cell lines A2780 (ovarian cancer), HeLa
(cervical cancer), Ketr-3 (kidney cancer), and MCF-7 (breast
cancer) were maintained in RPMI 1640 medium, supplemented
with 10% heat inactivated fetal bovine serum (Sijiqing Biomaterial,
Me 43%
a
Purified yield of
a
-isomer (the last 2 steps).
The statistical difference between the tested and reported IC50s for compound 4
and 6 may reflect the different assay conditions.
b
[(Scheme_2)TD$FIG]
H
N
1
R
H
N
1
R
O
OH
O
a, b
O
O
O
MeO
CbzHN
O
+
2
HO
O
O
R
O
OH
MeO
2
R
H N
2
OH
7
8
13
13a R1 = Me, R2 = H 13b R1 = Me, R2 = Cl
13c R1 = Me, R2 = Me 13d R1 = Ph, R2 =H
13e R1 = Ph, R2 = Cl 13f R1 = Ph, R2 = Me
7a R = Me, R2 = H 7b R1 = Me, R2 = Cl
1
7c R = Me, R2 = Me 7d R1 = Ph, R2 = H
1
7e R = Ph, R2 = Cl 7f R1 = Ph, R2 = Me
1
Scheme 2. Reagents and conditions: (a) PPh3, DEAD, THF, 0 8C–r.t.; (b) H2, Pd-C (10%), MeOH, 30%–45% (2 steps).

[
T. Zhang et al. / Chinese Chemical Letters 24 (2013) 719–722
721
Fig. 2. The novel simplified aminocoumarin amino isosteres and the SAR from 7a–f which conforms with the known SAR.
according to the literature [6,7]) as positive controls. Briefly,
logarithmic cells described above under cell culture were plated in
assumption that 7d–f may act by targeting the C-terminus of
Hsp90, though detailed mechanistic studies are to be carried out.
However, more significant comparison is found among 7f, 4 and 6,
as it is clear that a ꢀ10-fold elevation of the compound’s anticancer
activity was achieved by simply replacing the 30-OH (4) with a 30-
NH2 (7f), and even without a fully optimized coumarin N-acyl
group, the potency of 7f has reached to the level of 6, one of the
most potent Hsp90 C-terminal inhibitors disclosed so far,
indicating that the combination of the noviose possessed lipophilic
characteristic and a basic amino group into one molecule might
constitute a useful strategy in the design of more effective
anticancer agents targeting Hsp90 C-terminus.
96-well plates at concentration of 1800/100
mL per well. Drugs at
final concentrations of 0.1–100 mol/L were added with quad-
m
ruplicates of each concentration after 24 h. The cells were
incubated further at 37 8C for 72 h, the medium was aspirated,
and 100
m
L MTT of 0.5 mg/mL in medium was added. After 4 h
L DMSO was
incubation, the medium was aspirated and 150
m
added to solubilize the formazan crystals. Absorbance of the
converted dye was measured at a wavelength of 570 nm with
background subtraction at 650 nm. The dose–response curves
were fitted with SigmaPlot and IC50s were determined.
3. Results and discussion
4. Conclusion
L
-Noviose is the signature subunit of aminocoumarins. An
In conclusion, a series of novel, simplified aminocoumarin
analogues having the noviose 3-OH replaced by an amino group
were synthesized and their cytotoxicities were evaluated in cancer
cell lines. The in vitro cytotoxicities of these compounds, particularly
that of 7f, indicated that amino isosteric replacement in the noviose
moiety of these analogues resulted in substantial enhancement of
the cytotoxicity of the compounds, and thereby may be recognized
as a useful strategy in the search of more potent anticancer
aminocoumarin analogues that target Hsp90 C-terminus.
unusual characteristic of this pseudo-sugar is the possession of a
lipophilic motif comprising the gem-dimethyl and the methoxy
groups. While this feature is known to play an important role when
1 or 2 is binding to DNA gyrase B, no evidence is available to deny
its importance to the Hsp90 affinity of 3, 4 and 5. On the other
hand, the remarkable anticancer activity of 6 may be attributed to
the basicity of the piperidine moiety. To date, the exact structure of
the Hsp90 C-terminal ATP-binding site remains elusive, but by
superimposing the two types of inhibitor substructures (Fig. 2), we
found it likely that the free hydroxyls of the noviose moiety and the
amine segment of the piperidine moiety might share common
interactive residue(s) from the protein. It is therefore reasonable to
speculate that the incorporation of a basic amino group into the
noviose moiety would possibly lead to the elevation of the
anticancer activity of the compounds. For this reason, the synthesis
and biological evaluation of compounds 7a–f are of considerable
interest.
Acknowledgments
We are grateful for the generous financial support from Youth
Science Foundation of Peking Union Medical College & Chinese
Academy of Medical Sciences (No. 521164).
Appendix A. Supplementary data
As illustrated in Table 1, compounds 7d–f, each bears 3-
benzamino substituent, exhibited relatively high potency of
growth inhibitory effects against the tested cancer cells, but all
three 3-acetoamino-substituted analogues (7a–c) proved inactive.
Moreover, the coumarin C8 substituted compounds 7e and 7f
showed better activity than unsubstituted 7d. Notably, these new
biological data are totally consistent with the known structure
activity relationships (SARs) in the molecules of simplified
aminocoumarin analogues, that both the benzamide side chain
and the coumarin C8 substitution are beneficial to Hsp90
inhibitory activities of the compounds (Fig. 2) [12]. Such SAR
fitness, as well as the close structural similarity of the newly
prepared molecules to compounds 3, 4 and 5, supported our
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.cclet.2013.04.046.
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