Novel indole α-methylene-γ-lactones as potent inhibitors for AKT-mTOR signaling pathway kinases

Article abstract of DOI:10.1016/j.bmcl.2005.07.050

In an effort to generate novel anticancer agents, a series of hybrids of α-methylene-γ-lactones and 2-phenyl indoles has been synthesized and evaluated for inhibition activities on the phosphorylation of AKT, mTOR, p70S6 kinase, and 4E-BP1. The results in

Full text of DOI:10.1016/j.bmcl.2005.07.050

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4799–4802
Novel indole a-methylene-c-lactones as potent inhibitors for
AKT-mTOR signaling pathway kinases
Huasheng Ding, Chao Zhang, Xihan Wu, Chunhao Yang,* Xiongwen Zhang,
Jian Ding and Yuyuan Xie
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Graduate School of the CAS, 555 Zuchongzhi Road, Shanghai 201203, PR China
Received 16 May 2005; revised 4 July 2005; accepted 18 July 2005
Available online 6 September 2005
Abstract—In an effort to generate novel anticancer agents, a series of hybrids of a-methylene-c-lactones and 2-phenyl indoles has
been synthesized and evaluated for inhibition activities on the phosphorylation of AKT, mTOR, p70S6 kinase, and 4E-BP1. The
results indicate that substitutes on the c-position of lactones have a rather significant influence on inhibition activities.
Ó 2005 Elsevier Ltd. All rights reserved.
In recent years, great efforts have been made to im-
prove the selectivity of anti-cancer agents by targeting
the cancer-specific proteins or signaling pathways.
Some small molecule compounds, such as gleevec,1
against these targets have been developed and ap-
proved for use in clinical practice. On the other hand,
these successes also reversely promote further studies
on these new targets.
p70S6K. The p70S6K is recognized as the regulation
kinase of the multiple phosphorylation of 40S ribo-
somal protein S6 in vivo so as to control the tran-
scription of a class of mRNAs that contain an
oligopyrimidine tract at the transcriptional start site.4
eIF4E is negatively regulated by 4E-BP1. mTOR-med-
iated phosphorylation of 4E-BP1 dissociated the 4E-
4E-BP1 complex, freeing 4E for its primary function
of binding to the cap structure of mRNA as part of
the translation initiation complex.5 eIF4E exerts a
mitogenic and oncogenic effect through activation of
the Ras signaling pathway.6
AKT-mTOR signaling pathway kinases, important
components of intracellular signal systems, have been
receiving increasing interest for their significant effect
upon cell growth and oncogenesis. Mammalian target
of rapamycin (mTOR) is considered a member of the
PIK-related kinase family, with its C-terminus sharing
strong homology to the catalytic domain of PI3-kinase
(PI3K). PI3K and potentially protein kinase B (PKB;
Akt) lie upstream of mTOR acting as apoptotic regu-
lators, which are activated in many cancers and may
promote drug resistance in vitro.2 Ribosomal p70S6
kinase (p70S6K) and eukaryotic initiation factor-4E
(eIF-4E) binding protein isoforms (4EBP1-3) are typi-
cal downstream mTOR effector molecules.3 mTOR
activates p70S6K by phosphorylating the Thr 389 of
Wortmannin (Fig. 1), isolated from the culture broth of
Talaromyces wortmannii KY12420, has shown strong
inhibitory activities for mTOR (IC50 = 40 nm).7 Its
activity may come from a near planar structure and an
electrophilic exocyclic double bond conjugated to the
carbonyl group of the d-lactone ring.8 Furthermore, in-
dole rings, especially those substituted by alkyloxy or
O
OAc
MeO
O
H
Keywords: Indole a-methylene-c-lactones; AKT-mTOR signaling
pathway kinase; Inhibitors.
O
O
O
*
Corresponding author. Tel./fax: +86 21 50806770; e-mail:
chyang@mail.shcnc.ac.cn
Figure 1. Wortmannin.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.050

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H. Ding et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4799–4802
R1
R1
R1
R2
R2
i
ii
R2
N
N
N
H
O
O
OH
3a-c
1a-c
2a-c
R1
R2
iii
a: R1=H, R2=H;
N
b: R1=OCH3, R2=H;
c: R1=MOMO, R2=H;
O
H
4a-c
i: BrCH COOEt, NaH, DMF, rt., 8h;
ii: LiAlH4, THF, rt., 3h;
2
iii: DCC, Py, TFA, DMSO,Benzene, rt., 30h
Scheme 1.
R1
R1
R1
R2
R2
i
R2
ii
N
N
N
H
O
O
O
OH
1a-e
5a-e
2a-e
R1
R2
iii
a: R1=H, R2=H; b: R1=OCH3, R2=H;
N
1a-e
c: R1=MOMO, R2=H; d: R1=H, R2=OCH3;
e: R1=H, R2=MOMO;
+
O
6a-e
i: BrCH2COOEt, NaH, DMF, rt., 8h;
iii: CH3Li, Et2O,rt., 2h;
ii: NaOH, EtOH, 80 °C, 30min;
Scheme 2.
R1
R2
O
N
Zn,THF
HCl
OEt
Br
4a-c, or 6a-e
+
R3
O
7: R1=H, R2=H, R3=H; 8: R1=OCH3, R2=H, R3=H;
O
9: R1=MOMO, R2=H, R3=H; 10: R1=H, R2=H, R3=CH3;
7-14
11: R1=OCH3, R2=H, R3=CH3; 12: R1=MOMO, R2=H, R3=CH3;
13: R1=H, R2=OCH3, R3=CH3; 14: R1=H, R2=MOMO, R3=CH3
Scheme 3.
R1
R1
hydroxy groups, are found in many natural products
with anticancer activities.9 Here, we report the prepara-
tion and biological evaluation of a series of novel conju-
gates that combine indoles and a-methylene-c-lactones
keeping functional groups of the d-lactone ring, tending
to find some small molecule compounds showing inhib-
itive activities to the AKT-mTOR signaling pathway
kinases.
R2
R2
N
N
HCl
R3
R3
O
O
O
O
15, 16
12, 14
15: R1=HO, R2=H, R3=CH3;
16: R1=H, R2=HO, R3=CH3.
Ethyl 2-(bromomethyl)acrylate was prepared as report-
ed in the literature,10,11 and the indoles were obtained
by the Fischer method. Schemes 1 and 2 illustrate the
Scheme 4.

H. Ding et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4799–4802
4801
Figure 2. Western blot analysis was performed as follows. Rh30 cells were starved in serum-free RPMI-1640 culture medium for 24 h and then
treated with 50 lM compounds for 1.5 h at 37 °C. Kinase phosphorylation was stimulated by 100 ng/ml EGF for 10 min. Cells (106) were collected by
centrifugation at 3000 rpm for 3 min, washed with pre-cooled PBS for two times, and then resuspended in lysis buffer on ice for an hour. The lysates
were centrifuged at 13,000 rpm for 15 min at 4 °C. Equivalent amounts of proteins were analyzed by 10% or 15% SDS–PAGE. After electrophoresis,
the proteins were transferred onto nitrocellulose membrane (Millipore). After blocking the non-specific binding in blocking solution (5% non-fat milk
in TBS/Tween 20), membranes were incubated with appropriate antibodies to: phosphorylated-AKT (1:1,000), phosphorylated-p70S6K (1:1,000),
and phosphorylated-4EBP1 (1:1000), b-actin (1:1,000) at 4 °C overnight. Proteins were visualized with a peroxidase-coupled secondary antibody,
using ECL-plus for detection. b-Actin served as loading control.
synthesis of N-indolyl-ketones or aldehydes 4 and 6. We
first used ethyl a-bromoacetate as the alkylation agent
to obtain indole derivatives 2. Compounds 2a–c could
then be reduced by LiAlH4 to alcohols, which were sub-
sequently oxidized to the corresponding aldehydes 4 via
the Moffat reaction (Scheme 1). 2 could also be hydro-
lyzed to their corresponding carboxylic acids, and treat-
ed with methyl lithium, and then the ketones 6 were
obtained with a lesser amount of indoles 2 (Scheme 2).
on the phosphorylation of AKT, mTOR, p70S6
kinase, and 4E-BP1 at 50 lM than other methyl-con-
taining compounds 10-16, and the former compoundsÕ
activity is nearly equal to that of wortmannin in Fig-
ure 1. The good inhibition activity of compounds 7, 8,
and 9 implies that less substitution on the lactones is
favorable. However, compared to the apparent effect
of space hindrance, the nature of substituents on in-
doles influences the inhibition ability slightly.
Interestingly, we found that when indole carboxylic
acids were treated with alkyl lithium in dry Et2O,
the products were obtained readily. But when the
reaction was carried out in a solution of dry THF,
the corresponding indoles were obtained, while no ke-
tones were produced. The desired compounds 7-14
were then synthesized by reacting intermediates 4 or
6 with ethyl 2-(bromomethyl)acrylate and zinc powder
in dry THF (Reformatsky-type condensation) just as
It can also be seen from Figure 1 that compounds 13,
14, and 16 with substituents on 2-phenyl ring show a
better inhibition ability than their counterparts 11, 12,
and 15 with substituents on the indole ring. Moreover,
substitution with alkoxy group seems to be not
advantageous as appearing in compounds 11, 12,
and 13.
The present work not only gives the chemical synthesis
of novel a-methylene-c-lactone-indoles hybrids but also
their inhibition ability on the phosphorylation of AKT,
mTOR, p70S6 kinase, and 4E-BP1. Initial results indi-
cate that keeping the c-position of the lactone not
substituted is crucial for the inhibition activity. Besides,
a methoxy substituent on the phenyl is more favorable
than on the indole ring.
12
¨
Ohler et al. had reported (Scheme 3). Compounds
15 and 16 with a hydroxy substitute were prepared,
respectively, by hydrolysis of the corresponding com-
pounds 12 and 14 in dilute hydrochloride solution,
as shown in Scheme 4.
The impact of compounds 7–16 on the phosphorylation
of AKT, mTOR, p70S6K, and 4E-BP1 in RH30 cell line
is shown in Figure 2. To exclude the interference of mul-
tiple growth factors in serum, we starved cells overnight
and used 100 ng/ml EGF to stimulate unspecific reaction
on many signal transduction kinases. Western blot re-
sults illustrated that four kinases we investigated were
in a hypophosphorylated state after starvation and
showed rather good identity when treated with target
compounds.
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