Synthesis and in vitro antitumor activity of asperphenamate derivatives as autophagy inducer

Article abstract of DOI:10.1016/j.bmcl.2012.01.101

In an effort to improve the aqueous solubility and the antitumor activity of natural product asperphenamate, we have designed and synthesized three series of asperphenamate derivatives, including series I (simplifying molecular skeleton series), series II (introducing a hydroxyl group to A-phenyl ring series) and series III (disrupting molecular planarity series). All derivatives have displayed a significantly increased solubility compared with asperphenamate. Their growth inhibitory activities in vitro were screened by the standard MTT method in MCF-7, HeLa, and BEL-7402 cell lines. With the exception of the derivatives in series I, most of derivatives in series II and series III showed growth inhibitory activity. Among all derivatives, IM23b in series III showed the greatest potency in human breast cancer MCF-7 cells. The cellular potency of IM23b was approximately 1.5-fold more potent than that of cisplatin. The mechanism of cell death induced by IM23b in human breast cancer MCF-7 cells was further investigated. We concluded that the cell death was induced by autophagy instead of apoptosis or cell cycle arrest.

Full text of DOI:10.1016/j.bmcl.2012.01.101

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2216–2220
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro antitumor activity of asperphenamate derivatives
as autophagy inducer
Lei Yuan a, Yanchun Li b, Chunyang Zou c, Chao Wang a, Jian Gao a, Caixia Miao b, Enlong Ma b,
,
⇑
Tiemin Sun a,
⇑
a School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, PR China
b Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, PR China
c Department of Pharmacy, Liaoning College of Health Vocation and Technology, Shenyang 110101, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
In an effort to improve the aqueous solubility and the antitumor activity of natural product asperphena-
mate, we have designed and synthesized three series of asperphenamate derivatives, including series I
(simplifying molecular skeleton series), series II (introducing a hydroxyl group to A-phenyl ring series) and ser-
ies III (disrupting molecular planarity series). All derivatives have displayed a significantly increased solu-
bility compared with asperphenamate. Their growth inhibitory activities in vitro were screened by the
standard MTT method in MCF-7, HeLa, and BEL-7402 cell lines. With the exception of the derivatives
in series I, most of derivatives in series II and series III showed growth inhibitory activity. Among all
derivatives, IM23b in series III showed the greatest potency in human breast cancer MCF-7 cells. The cel-
lular potency of IM23b was approximately 1.5-fold more potent than that of cisplatin. The mechanism of
cell death induced by IM23b in human breast cancer MCF-7 cells was further investigated. We concluded
that the cell death was induced by autophagy instead of apoptosis or cell cycle arrest.
Received 3 November 2011
Revised 11 January 2012
Accepted 25 January 2012
Available online 4 February 2012
Keywords:
Asperphenamate
Autophagy
Aqueous solubility
Structure–activity relationships
Inducer
Ó 2012 Elsevier Ltd. All rights reserved.
Autophagy is an important mechanism to maintain cell homeo-
stasis and promote cell survival during starvation.1,2 But persistent
stress or prolonged starvation can result in autophagic cell death, or
type II programmed cell death, which is distinct from apoptotic cell
death (type I programmed cell death).3,4 More recently, autophagy
had emerged as potential mechanism for effective cancer thera-
pies.5 Several anticancer agents have been documented to trigger
the cellular autophagic process.6–11 However, whether autophagy
in response to cancer therapies is pro-death or pro-survival is still
controversial. Therefore, the discovery and development of novel
autophagy modulator have important significances in both
clinical applications and the further understanding of autophagy
mechanisms.
To improve aqueous solubility of asperphenamate, we have
designed and synthesized three series of derivatives, including ser-
ies I: simplifying the skeleton of asperphenamate; series II: intro-
ducing a hydroxyl group into A-phenyl ring; and series III:
disrupting molecular planarity. Thermodynamic aqueous solubility
assay was used to confirm the aqueous solubility of all deriva-
tives.14,15 As expected, all derivatives have displayed more potent
in solubility than asperphenamate. The solubility values were
listed in Table S1 (see Supplementary data).
Asperphenamate, 1a, was first isolated by Clark from Aspergillus
flavus (Fig. 1).12 1a was also isolated by our group from raw malt,
a traditional medicine for the treatment of hyperplasia of mam-
mary glands. In our previous work, we have reported the synthesis
of four stereoisomers of asperphenamate, and the preliminary bio-
logical evaluation showed that the (R, S) stereoisomer exhibited
more potent bioactivity in inhibiting the growth of breast cancer
T47D and MDA-MB-231 cell lines.13 However, the further studies
were impeded due to its poor aqueous solubility.
⇑
Corresponding authors. Tel./fax: +86 24 23986398 (T.S.), tel./fax: +86 24
23986302 (E.M.).
E-mail addresses: enlongma@hotmail.com (E. Ma), suntiemin@126.com (T. Sun).
Figure 1. The structure of asperphenamate and IM23b.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.101

L. Yuan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2216–2220
2217
Meanwhile, the anti-proliferative activities of all derivatives
in vitro were examined by the standard MTT method in MCF-7,
HeLa, and BEL-7402 cell lines. Among all derivatives, IM23b in ser-
ies III displayed most potent growth inhibitory activity in MCF-7
IM20 and IM23 in series III). The synthesis of the two series of deriv-
atives was outlined in Scheme 2. Benzyloxy substituted carboxylic
acids IM10–14 were prepared by reacting benzyl bromide with var-
ious hydroxyl-substituted benzoic or phenylacetic acid in the pres-
ence of potassium carbonate. Treatment IM10–14 with thionyl
chloride gave IM15–19. Starting from IM6, IM20–24 were prepared
using a strategy similar to that described in the synthesis of com-
pounds 1–5. Compound 6–10 were synthesized via hydrogenolysis
using ammonium formate and 10% Pd/C in MeOH.
cells with an IC50 value of 6.06 lM (Fig. 1).
The cell death mechanisms IM23b-induced were further
explored in human breast cancer MCF-7 cells. Flow cytometric
analysis indicated that IM23b did not induce apoptosis and cell cy-
cle arrest. Similar with apoptosis, autophagy is also believed to be a
fundamental programmed cell death mechanism. Thus, inducing
autophagy for IM23b in MCF-7 cell lines was further studied by
observing the morphological changes after fluorescent staining
with AO and MDC and the conversion of LC3-I to LC3-II using wes-
tern blotting analysis. The results showed that IM23b caused cell
death by the induction of autophagy.
Biological activity and SAR
All of these analogs including asperphenamate were screened
in vitro for antitumor activities against MCF-7, HeLa and BEL-
7402 cells using the standard MTT method, and cisplatin was used
as the positive control.19 The results were presented in Table S1.
In series I, only 4a, 4c, 5a and 5c displayed very weak growth
inhibitory activity. Other compounds were inactive with an aver-
Chemistry
Because the rigid structure of asperphenamate may lead to poor
aqueous solubility, we first tried to simplify its skeleton by eliminat-
ing A–D phenyl group, respectively (compounds 1–5 in series I). The
synthesis of compounds in series I was carried out according to
Scheme 1. IM1–2 were prepared according to the method developed
by McKennon et al.16 The amino group of IM1–2 was acylated to
give IM3–5. Condensation of optically pure N-BOC-phenylalanine
or N-BOC-alanine with IM3–5 was promoted by 1,10-carbonyldiim-
idazole (CDI) to afford key intermediate IM6–9. After the removal of
the BOC group, 1–5 were obtained through acylation reaction.
The strategy of introducing hydrophilic group into the molecule
is widely used for increasing aqueous solubility.17 Therefore, a hy-
droxyl group was introduced to the A-phenyl ring of asperphena-
mate. (compounds 6–10 in series II); Otherwise, molecular
planarity is known to influence the solubility.18 Thus, the large
group, such as benzyloxy substituent, was introduced into ortho-po-
sition of A-phenyl ring to disrupt molecular planarity (compounds
age IC50 value greater than 100 lM. In series II and series III, with
the exception of compounds 7a, 7d, 8a, 9a, 10c, 10d, IM20a and
IM23a, most of the compounds showed growth inhibitory effect.
In series II, the most active compound was ortho-hydroxyl benzoyl
substituted derivative 6c with (R, S) configuration, which exhibited
slightly more potency than cisplatin against BEL-7402 cells. The
antitumor activities of its stereoisomers were approximately
two- to fivefold less potent than that of 6c. In meta-hydroxyl 7
and para-hydroxyl 8 benzoyl substituted derivatives, apart from
7a, 7d and 8a, other analogues displayed a similar growth inhibi-
tory activity and were significantly less active than 6c. The activity
assay in vitro of ortho-hydroxyl 9 and para-hydroxyl 10 phenylace-
tyl substituted derivatives showed that the growth inhibitory
activity against MCF-7 and HeLa cell lines for 9b was superior to
other analogues in 9 and 10. Compound 9b showed comparable
activity to cisplatin against HeLa cell lines. However, 9b showed
a one- to twofold reduction in activity compared with that of 6c.
Scheme 1. Synthesis of compounds in series I. Reagents and conditions: (i) H2SO4, NaBH4, I2, THF, reflux, 98%; (ii) IM3 and IM5: benzoyl chloride, K2CO3, MeOH, rt, 95%; IM4:
(CH3CO)2O, K2CO3, MeOH, rt, 92%; (iii) N-BOC-( or )-phenylalanine or N-BOC-( or )-alanine, CDI, CHCl3, rt, 65%; (iv) (a) 1.5 N HCl/AcOEt, rt, 76%; (b) 1–3, 5: benzoyl
chloride, pyridine, rt,70–90%; 4: (CH3CO)2O, pyridine, rt, 84–90%.
L
D
L
D

2218
L. Yuan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2216–2220
Scheme 2. Synthesis of compounds in series II and series III. Reagents and conditions: (i) PhCH2Br, K2CO3, acetone, reflux, 86–90%; (ii) SOCl2, reflux, 75–90%; (iii) (a)1.5 N HCl/
AcOEt, rt, 76%; (b) IM15–19, pyridine, rt, 76–82%; (iv) HCOONH4, Pd/C, MeOH, reflux, 70–76%.
In series III, IM20c with (R, S) configuration was most active ana-
logue against tested cell lines in ortho-benzyloxy benzoyl substi-
tuted derivatives IM20. Nevertheless, IM20c showed significantly
decreased activity as compared with that of its corresponding
ortho-hydroxyl benzoyl analogue 6c. While ortho-benzyloxy phen-
ylacetyl substituted derivative IM23b exhibited a similar activity
to IM20c and IM23c against HeLa and BEL-7402 cell lines, IM23b
showed the greatest potency among reported compounds against
MCF-7 cell line. The cellular potency of IM23b was approximately
1.5-fold more potent than that of cisplatin and twofold higher than
that of 6c. It is interesting that IM23b against MCF-7 cells showed
fivefold selectivity over HeLa and BEL-7402 cells.
Taken together, the biological activity results indicated that (a)
the molecular configuration had an impact on antitumor activity;
(b) Introduction of a hydroxyl or benzyloxy substituent on the
A-phenyl was beneficial for activity; (c) In general, the ortho-hydro-
xyl substituted derivatives showed more potent activity than meta-
and para-hydroxyl substituted derivatives; (d) The substitution
Figure 2. Flow cytometric analysis of cell cycle distribution in MCF-7 cells treated with 10 lM IM23b for 24, 48 and 72 h. The 100 lM of H2O2 treating for 12 h was used as a
positive control.

L. Yuan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2216–2220
2219
Figure 3. IM23b induced autophagy in MCF-7 cells. (A) Autophagosomes observation. MCF-7 cells treated with IM23b (5 and 10
with AO and observed by fluorescence microscope. (B) Autophagosomes observation. MCF-7 cells treated with IM23b (5 and 10
l
M) for 72 h, and then the cells were stained
lM) for 72 h, and then the cells were stained
with MDC and observed by fluorescence microscope. (C) Autophagic cells detection. MCF-7 cells treated with IM23b (5 and 10
lM) for 72 h, and then the cells were stained
with MDC and subjected to flow cytometric analysis. (D) Western blotting analysis for autophagy. MCF-7 cells were treated with IM23b (5 and 10 lM) for 48 h, and then the
total proteins were extracted and detected by western blot analysis.
type along with the length of the carbonyl linker between
A-ring and nitrogen atom played an important role in activity of
the derivatives substituted in the ortho-position of A-phenyl ring.
The preference of substitution pattern is ortho-hydroxyl benzoyl
and ortho-benzyloxy phenylacetyl substitution.
Among the tested compounds, IM23b showed the potent inhib-
itory activity, and hence, the mechanism of cell death induced by
IM23b in human breast cancer MCF-7 cells was further investigated.
IM23b inducing autophagy
Autophagy is a process of self-degradation of cellar compo-
nents. Autophagy, type II programmed cell death, is a cell death
pathway distinct from apoptosis, and it has been observed in
response to several anticancer reagents. Because IM23b did not
induce apoptosis and cell cycle arrest in MCF-7 cells, we deter-
mined whether IM23b could induce autophagy. The formation of
acidic vesicular organelles (AVOs) is a characteristic of autophagy.
To assess the occurrence of AVOs, MCF-7 cells were treated with
Flow cytometric analysis for apoptosis of IM23b treated MCF-7
cells
IM23b (5 and 10 lM) for 72 h. After stained with AO and MDC,
the morphological changes were observed through fluorescence
microcopy. As shown in Figure 3A and B, MCF-7 cells treated with
IM23b showed an increase in the number of cytoplasmic AVOs
compared with control cells by the method of AO and MDC stain-
ing. MDC fluorescent intensity was also measured to determine
AVOs by flow cytometry. As shown in Figure 3C, after treatment
MCF-7 cells were treated with 10 lM IM23b for 24, 48 and 72 h,
and then examined by flow cytometry analysis after PI staining.
Compared to the positive control (H2O2), IM23b did not influence
the cell cycle progression and the characteristic hypodiploid DNA
content peak (subG1) which represents cells undergoing apoptosis
(Fig. 2). The result suggested that IM23b did not induce apoptosis
or influence the distribution of cell cycle in MCF-7 cells.
5 and 10
lM of IM23b for 72 h, MCF-7 cells showed higher MDC-
associated fluorescence than that of the control cells.

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L. Yuan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2216–2220
2. Meijer, A. J.; Codogno, P. Int. J. Biochem. Cell Biol. 2004, 36, 2445.
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LC3-II was investigated. It was known that LC3-I was involved in
the formation of autophagosome and was incorporated into the
autophagic vacuolemembrane. LC3-I must be conjugated to phos-
phatidylethanolamine (PE). The PE-conjugated LC3-I is designated
as LC3-II. The LC3-II devise is a potential marker of activation of
the autophagic pathway. Cellular extracts were prepared from cells
incubated with or without IM23b for a period of 48 h and the level of
both forms of LC3 was assessed by western blot analysis. As shown
in Figure 3D, the conversion of LC3-I to LC3-II was observed. These
results indicated that IM23b was capable of inducing autophagy.
In summary, three series of asperphenamate derivatives with
improved solubility were synthesized. The growth inhibitory activ-
ity in vitro for all derivatives was evaluated. Among the compounds
synthesized, compound IM23b in series III showed the strongest
inhibitory activity in human breast cancer MCF-7 cells. The mecha-
nism of cell death induced by IM23b in human breast cancer MCF-7
cells was further investigated. The results showed that inhibition of
growth was attributed to the autophagy induction instead of apop-
tosis or cell cycle arrest. At present, studies are underway to eluci-
date the autophagy signal pathways IM23b affected.
11. Opipari, A. W.; Tan, L. J.; Boitano, A. E.; Sorenson, D. R.; Aurora, A.; Liu, J. R.
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14. Thermodynamic solubility determination was based on the method of Fritsche
J.15 Briefly, to 1 mg of a test compound was added to 2 mL of an equal volume
of a mixture of 1:15 M phosphate buffer (pH 7.4) and EtOH. The mixture was
sonicated for 10 min in a bath sonifier and then shaken for 30 h at 25 °C. After
centrifugation, an aliquot was filtered through
(0.45 M). The filtrate was diluted in methanol. An aliquot of this solution
was subjected to HPLC analysis and UV detection at 220 nm. Separation was
achieved on a Waters Symmetry C18 analytical column (5 m, 4.6 mm (ID)
a Millipore DIMEX-13
l
l
ꢀ250 mm). The concentration of the sample solution was calculated using a
previously determined calibration curve, corrected for the dilution factor of the
sample.
Supplementary data
15. Fritsche, A.; Elfringhoff, A. S.; Fabian, J.; Lehr, M. Bioorg. Med. Chem. 2008, 16,
3489.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2012.01.101.
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