Identification of novel bivalent mimetics of annonaceous acetogenins via a scaffold-hopping strategy

Article abstract of DOI:10.1016/j.bmcl.2014.02.072

A series of novel bivalent mimetics of annonaceous acetogenins have been designed, synthesized, and evaluated. Among these, compound 7 bearing a homopiperazine ring in the middle region exhibited more potent growth inhibitory activity and higher selectivity against cancer cells over normal cells by comparison with AA005. This work indicates that modification of the middle piperazine ring is a useful optimizing tool for the simplified acetogenin mimetics.

Full text of DOI:10.1016/j.bmcl.2014.02.072

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1650–1653
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of novel bivalent mimetics of annonaceous
acetogenins via a scaffold-hopping strategy
a,
Yanghan Liu a, Yongqiang Liu b, Zhen Li c, Guang-Biao Zhou b, Zhu-Jun Yao d, , Sheng Jiang
⇑
⇑
a Laboratory of Medicinal Chemistry, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
b Division of Molecular Carcinogenesis and Targeted Therapy for Cancer, State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology,
Chinese Academy of Sciences, Beijing 100101, China
c Department of Translational Imaging, The Houston Methodist Research Institute, Houston, TX 77030, United States
d State Key Laboratory of Coordination Chemistry, Nanjing National Laboratory of Microstructures, School of Chemistry and Chemical Engineering, Nanjing University,
22 Hankou Road, Nanjing 210093, 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 bivalent mimetics of annonaceous acetogenins have been designed, synthesized, and
evaluated. Among these, compound 7 bearing a homopiperazine ring in the middle region exhibited more
potent growth inhibitory activity and higher selectivity against cancer cells over normal cells by compar-
ison with AA005. This work indicates that modification of the middle piperazine ring is a useful optimiz-
ing tool for the simplified acetogenin mimetics.
Received 11 November 2013
Revised 2 February 2014
Accepted 27 February 2014
Available online 12 March 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Annonaceous acetogenins
Homopiperazine
Bivalent analogues
Cytotoxicity
Selectivity
Annonaceous acetogenins are a class of natural polyketides iso-
lated from Annonaceous plants, and more than 400 members have
been found and characterized in the past three decades.1–3 They
were found to show a broad range of biological activities, such as
anticancer, antimalarial, anthelmintic, antiviral, and antimicrobial
effects. It is generally accepted that annonaceous acetogenins serve
as the blockers of complex I (NADH-ubiquinone oxidoreductase) in
mitochondria and reduce the production of ATP.4,5 Annonaceous
acetogenins have been attracting worldwide attention for a long
period because of their unique chemical structures and attractive
biological activities especially for their anticancer activities.1,2,6
During our efforts of simplifying natural annonaceous acetogenins
into the corresponding mimetics,7–15 we successfully invented a
simple analogue AA005 by replacement of the bis-THF rings of nat-
ural bullatacin with an linear ethylene glycol ether functionality.
AA005 exhibited significant potency of inhibiting the proliferation
of several human cancer cell lines and selective action between
human cancerous and healthy cells.8–10 Subsequently, we further
developed an improved mimicking compound having a biphenyl
moiety in the left hydrocarbon chain, showing more potent
inhibitory activity against cancer cell proliferation and higher cell
selectivity.16
Discovery of bivalent ligand inhibitors has proven to be a useful
protocol in medicinal chemistry and gained worldwide attention
due to the potential high functional affinities through bivalent li-
gand–receptor interactions.17 We recently applied this concept
into synthesizing a new series of linear dimeric analogues mimick-
ing natural annonaceous acetogenins.18 Unfortunately, these sim-
pler dimers were identified to exhibit 8–100 times less potency
than AA005 against the growth of SGC7901 cells. In this study,
we want to report a new series of bivalent analogues by a scaf-
fold-hopping strategy, which have simple structures and potent
growth inhibitory activity.
As shown in Figure 1, the newly designed mimetics are com-
posed of three functionalities: diverse analogues of ubiquinone at
the terminal(s) (P), hydrophilic piperazine ring in the middle and
hydrophobic hydrocarbon chain(s) as the linkers. The common
c-lactone moiety in the natural acetogenins is suggested to be
essential for binding at the quinone binding site of mitochondria
complex I. Previously, several groups including us found that
replacement of the c-lactone moiety of natural acetogenins with
the quinone portion of ubiquinone could maintain or increase
the potency of complex I inhibition.18–21 These results indicated
that the quinone portion of ubiquinone is a good functional
⇑
Corresponding authors. Fax: +86 20 32015318 (S.J.).
E-mail addresses: yaoz@nju.edu.cn (Z.-J. Yao), jiang_sheng@gibh.ac.cn (S. Jiang).
http://dx.doi.org/10.1016/j.bmcl.2014.02.072
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

Y. Liu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1650–1653
1651
Figure 1. Design of new bivalent analogues of annonaceous acetogenins.
equivalent for the
c
-lactone of natural acetogenins. Therefore,
Syntheses of bivalent analogues 2–11 were shown in Scheme 2.
Parallel treatment of piperazine or homopiperazine with corre-
sponding quinones 27–32, in the presence of catalytic amount of
tetrabutylammonium iodide in acetonitrile under refluxing
conditions, afforded the corresponding analogues 2–11.
diverse analogues of ubiquinone were designed as an essential
functionality for our bivalent mimicking. Hydrophilic piperazine
ring was introduced in the middle because of its structural similar-
ities with the bis-THF rings of natural acetogenins. We envisioned
that modification of the conformational property of piperazine ring
would affect inhibitory activity. Accordingly, a series of homopip-
erazine derivatives were also designed for comparison. The hydro-
phobic hydrocarbon chains of natural annonaceous acetogenins
retain the amphiphilic nature of these acetogenin mimetics.
Syntheses of the ubiquinone analogues were shown in
Scheme 1. Oxidation of 2,3,5-trimethylphenol 18 got 19 in 90%
yield.22 Treatment of 2,5-dimethyl-p-benzoquinone with acetic
anhydride and boron trifluoride-etherate provided 21 in 92% yield.
Then, compound 21 was treated with sodium hydroxide and di-
methyl sulfate in methanol to afford 22 in 80% yield. Subsequent
oxidation of compound 22 with phenyliodine diacetate (PIDA) pro-
vided 23 in 60% yield.22 Finally, compounds 27–32 were synthe-
sized in parallel via radical alkylation of the substituted quinones
19 and 23–26 in 20–56% yields,23 via decarboxylation of the bro-
mo-acid under silver nitrate and ammonium persulfate at 75 °C.
A
number of monovalent analogues 12–17 were also
synthesized (Scheme 3). Coupling of 4-biphenylcarboxylic acid
with benzyl piperazine-1-carboxylate followed by hydrogenation
of 37 afforded compound 39 in 60% yield. Compound 40 was
prepared by the same procedure starting from benzyl 1,4-diaze-
pane-1-carboxylate. Parallel reactions of compound 39 or 40 with
quinones 27–31, respectively, in the presence of catalytic amount
of tetrabutylammonium iodide in acetonitrile under refluxing con-
ditions, afforded monovalent analogues 12–17.
All the synthesized compounds 1–17 were evaluated with MTT
assays for their inhibitory activity against the proliferation of hu-
man gastric cancer cell line (SGC7901), colorectal carcinoma cell
line (HCT-116), human lung fibroblasts (HLF) and human bronchial
epithelial cell line (16HBE). The results are summarized in Table 1.
Among the compounds 2–5 featuring a common piperazine ring in
the middle region, compound 2 bearing natural ubiquinone ring at

1652
Y. Liu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1650–1653
O
C
OH
OH
a or b
O
c
N
N
Cbz
O
COOH
a
m
37: m = 1
38: m = 2
36
O
R3
R1
R2
19
O
18
C
N
NH
6
d
O
O
N
N
O
OMe
m
OAc
OMe
27-31
OMe
d
39: m = 1
40: m = 2
b
m
OAc
c
O
O
O
OMe
22
OAc
21
12-17
12: m = 2, R1 = R2 = OMe, R3 = Me; 13: m = 2, R1 = OMe, R2 = R3 = Me;
23
20
O
O
O
14: m = 2, R1 = R2 = R3 = Me;
16: m = 2, R1, R2 = Ph , R3 = H;
15: m = 2, R1, R2 = Ph, R3 = Me;
17: m = 1, R1 = OMe, R2 = R3 = Me
R1
R3
R1
R2
R3
e or f
Br
R2
n
Scheme 3. Reagents and conditions: (a) benzyl piperazine-1-carboxylate, EDCI,
HOBt, DCM, 82%; (b) benzyl 1,4-diazepane-1-carboxylate, EDCI, HOBt, DCM, 75%;
(c) 10% Pd/C, H2, MeOH, rt; (d) TBAI, CH3CN, reflux, 50–70%.
O
24: R1 = R2 = OMe, R3 = Me
23: R1 = OMe, R2 = R3 = Me
19: R1 = R2 = R3 = Me
27: n = 6, R1 = R2 = OMe, R3 = Me
28: n = 6, R1 = OMe, R2 = R3 = Me
29: n = 6, R1 = R2 = R3 = Me
Table 1
Antiproliferative activity of AA005 (1) and new mimetics 2–17a–c
25: R1, R2 = Ph, R3 = Me
26: R1, R2 = Ph, R3 = H
23: R1 = OMe, R2 = R3 = Me
30: n = 6, R1, R2 = Ph, R3 = Me
31: n = 6, R1, R2 = Ph, R3 = H
32: n = 1, R1 = OMe, R2 = R3 = Me
Compd
GI50
HCT116
(lM)
SGC7901
HLF
16HBE
1 (AA005)
2
3
4
5
6
7
8
0.16
9.77
0.14
>20
>20
8.69
0.04
0.14
0.12
>20
9.76
5
1.56
1.39
5.57
>20
5.27
0.03
1.95
0.18
0.07
0.26
1.98
0.04
0.3
9.715
>20
>20
>20
>20
>20
>20
0.36
4.95
>20
>20
>20
>20
>20
>20
>20
>20
3.34
2.63
>20
3.85
3.52
1.14
>20
3.32
2.7
>20
>20
10.31
3.15
4
Scheme 1. Reagents and conditions: (a) I2, H2O2, H2SO4, MeOH, 23 °C, 90%; (b)
Ac2O, BF3ꢀEt2O, 40 °C, 92%; (c) Me2SO4, aq NaOH, MeOH, 23 °C, 80%; (d) PIDA, 9:1
H2O/MeOH, 60%; (e) 11-bromoundecanoic acid, AgNO3, (NH4)2S2O8, 1:1 CH3CN/
H2O, 75 °C, 20–56%. (f) 7-Bromoheptanoic acid, AgNO3, (NH4)2S2O8, 1:1 CH3CN/H2O,
75 °C, 20%.
9
0.09
>20
10
11
12
13
14
15
16
17
O
1.48
1.36
0.89
1.78
0.97
>20
R3
R1
R2
O
H
N
n
n
R1
R2
R3
2.85
>20
3.19
N
N
O
O
a
Br
m
m
N
H
n
2.29
O
R2
R1
a
AA005 was used as a positive control.
Inhibition of cell growth by the listed compounds was determined by using
MTT assay.
33: m = 1
34: m = 2
b
27-32
R3
2-10
c
O
Standard error of the GI50 was generally less than 10%.
2: m = 1, n = 6, R1 = R2 = OMe, R3 = Me; 3: m = 1, n = 6, R1 = OMe, R2 = R3 = Me;
4: m = 1, n = 6, R1 R2 R3 = Me; 5: m = 1, n = 6, R1, R2 = Ph, R3 = Me;
6: m = 2, n = 6, R1 = R2 = OMe, R3 = Me; 7: m = 2, n = 6, R1 = OMe, R2 = R3 = Me;
=
=
methoxy group of ubiquinone ring was replaced by methyl group,
is 10 and 70 times more potent than 2 against SGC7901 and HCT-
116 cell lines, respectively. Compound 4, in which two methoxy
groups of ubiquinone ring were replaced by methyl group, is 28
times more potent than 2 against HCT-116 cell lines, but it lost
its antiproliferative activity against SGC7901 cell lines. Similarly,
compound 5 bearing benzo-quinone ring at the terminal lost its
antiproliferative activity against SGC7901 cell lines. These results
indicate that replacement of the methoxy groups of ubiquinone
ring by methyl groups may be an effective strategy for activity
improvement. Replacement of piperazine of compounds 2–5 with
homopiperazine resulted in compounds 6–9. They were found to
exhibit more potent inhibition of cell growth than the correspond-
ing compounds 2–5 against SGC7901 and HCT-116 cell lines,
respectively. For instance, compound 9 exhibits 2 and 167 times
8: m = 2, n = 6, R1
=
R2
=
R3 = Me;
9: m = 2, n = 6, R1, R2 = Ph, R3 = Me;
10: m = 2, n = 1, R1 = OMe, R2 = R3 = Me;
O
O
O
H
O
N
6
N
N
O
O
b
Br
6
N
H
O
6
35
28
O
11
O
Scheme 2. Reagents and conditions: (a) TBAI, CH3CN, reflux, 50–70%; (b) TBAI,
CH3CN, reflux, 52%.
more potency than the corresponding compound
5 against
SGC7901 and HCT-116 cell lines, respectively. We are pleased to
find that compound 7 bearing a homopiperazine ring and 3-meth-
oxy-2,5-dimethyl quinone was identified as the best compound in
the terminal showed 61–65 times less potency than AA005 in
inhibition of the cell growth (SGC7901 and HCT-116), and no inhi-
bition was found against HLF cell lines. Compound 3, in which one

Y. Liu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1650–1653
1653
this series and showed 4 times more potent inhibitory activity
Supplementary data
against SGC7901 cell lines than reference compound AA005 (1).
Meanwhile, compound 7 displayed higher selectivity between can-
cerous and normal cell lines than that of 1. Compound 11 bearing a
N, N0-dimethyl ethylenediamine in the middle is 244 times less
potent than compound 7 against SGC7901 cell lines, indicating that
the homopiperazine ring is a more preferred functionality. These
results indicate that modification of the conformational property
in the middle region of these mimetics could significantly enhance
the growth inhibitory activity. Compound 10 with a shorter hydro-
carbon chain lost its cytotoxicities, indicating that the moderate
distance between the middle part and terminal moiety is favorable
for the bioactivity.24 Monovalent analogues are much less potent
than the corresponding bivalent analogues (13 vs 7, 14 vs 8, and
15 vs 9), indicating that the second quinone is favorable for anti-
proliferative activities.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.02.072.
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In summary, a series of new mimetics of annonaceous acetoge-
nin have been designed, synthesized, and evaluated in this work.
Compound 7 was identified to exhibit more potent growth inhibi-
tory activity and higher selectivity against cancer cells over normal
cells by comparison with AA005. This work indicates that replace-
ment of the methoxy groups of ubiquinone ring by methyl groups
and modification of the conformational property of the piperazine
ring in the middle region may be useful optimizing tools for
improving these analogues. In addition, easier synthesis of com-
pound 7 will be advantageous for supporting more future applica-
tions and further investigations. The inhibitory study on complex I
and metastatic tumors in animal models is currently underway in
our laboratory and will be reported in due course.
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This work was supported by the National Key Program for Basic
Research (2010CB529201, 2010CB833202, 2012CB910800, and
2009CB940900), the National Natural Science Foundation (8107
1930, 81171925, and 21172220), the Special Foundation of Presi-
dent, and the Strategic Leading Science & Technology Program of
the Chinese Academy of Sciences.