Design and synthesis of novel furoquinoline based inhibitors of multiple targets in the PI3K/Akt-mTOR pathway

Article abstract of DOI:10.1016/j.bmcl.2008.04.078

We herein report the design and synthesis of furoquinoline based novel molecules (16-36) and their in vitro multiple targeted inhibitory potency against PI3K/Akt phosphorylation and mTOR using cell based and cell-free kinase assay. In particular, compound

Full text of DOI:10.1016/j.bmcl.2008.04.078

Bioorganic & Medicinal Chemistry Letters 18 (2008) 3603–3606
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of novel furoquinoline based inhibitors
of multiple targets in the PI3K/Akt-mTOR pathway
Manoj V. Lohar a, Ramswaroop Mundada a, Mandar Bhonde b, Amol Padgaonkar b,
Vijaykumar Deore a, Nilambari Yewalkar a, Dimple Bhatia b, Maggie Rathos b,
Kalpana Joshi b, Ram A. Vishwakarma a, Sanjay Kumar a,
*
a Department of Medicinal Chemistry, Nicholas Piramal Research Centre, Goregaon (East), Mumbai 400 063, India
b Department of Pharmacology, Nicholas Piramal Research Centre, Goregaon (East), Mumbai 400 063, India
a r t i c l e i n f o
a b s t r a c t
Article history:
We herein report the design and synthesis of furoquinoline based novel molecules (16–36) and their
in vitro multiple targeted inhibitory potency against PI3K/Akt phosphorylation and mTOR using cell
based and cell-free kinase assay. In particular, compound 23 in addition to PI3K-mTOR inhibitory
potency, it has shown potent inhibition of hypoxia-induced accumulation of HIF-1a protein in U251-
HRE cell line. The inhibitory activities of compound 23 were confirmed by Western blot analysis, using
human non-small cell lung carcinoma H-460 cell line and glioblastoma U251 cell lines.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 16 November 2007
Revised 11 April 2008
Accepted 30 April 2008
Available online 4 May 2008
Phosphatidylinositol-3-kinases (PI3Ks) are members of a family
of lipid kinases that regulate cellular metabolism and growth by
phosphorylation of the 3-position of phosphatidylinositol diphos-
phate (PIP2) to phosphatidylinositol triphosphate (PIP3). The PI3K
signalling pathway is negatively regulated by PTEN. Which is most
frequently mutated in human cancer, leading to amplification of
signalling and as such is a promising target for small molecule inhi-
bition, with potential therapeutic target for anti-cancer drug devel-
opment.1–5 Based on the primary structure and mechanism of
action PI3Ks are divided into two major classes viz. class I and class
II.6,7 The class I PI3Ks are further divided into class IA enzymes:
p110a, p110b and p110d which are activated by tyrosine kinase
receptors whereas the only member of class IB enzyme p110c, is
activated by G-protein-coupled receptor. The class II PI3Ks C2a,
C2b and C2c are characterized by the presence of C2 domain at
C-terminus. The recent studies with isoform-specific small-mole-
cule inhibitors helped to elucidate the distinct cellular function
of different class I isoforms (p110a, p110b, p110d and p110c). It
has been reported that inhibition of p110a is essential to affect
growth suppression in malignant cell lines.8 The other class I iso-
forms have their therapeutic potential in other disease areas viz.
inflammation, autoimmune disease (p110d and p110c) and throm-
bosis (p110b).1,3,9–11
factor signalling cascade that generate proliferative and antiapo-
ptotic responses. The PI3K/Akt-mTOR pathway is also responsible
for the regulation of hypoxia-inducible factor-1. HIF-1 is a hetero-
dimer consisting of a and b subunit. The HIF-1a subunit is de-
graded rapidly in normoxic conditions and stabilized under
hypoxic conditions, while HIF-1b is constitutively expressed. In
general, the availability and activity of HIF-1a protein determine
the bioactivity of HIF-1.14 HIF-1a is master regulator of transcrip-
tional response to oxygen deficiency. It is upregulated in response
to hypoxia and growth factor stimuli. In addition to HIF-1’s associ-
ation in tumour progression, HIF-1 has been implicated in the reg-
ulation of genes involved in angiogenesis, for example, vascular
endothelial growth factor (VEGF), inducible nitric oxide synthase
and anaerobic metabolism (glycolytic enzymes).15 Overexpression
of HIF-1a has been demonstrated in many common human cancers
such as pancreatic carcinoma, lung carcinoma, colorectal carci-
noma and glioblastoma, to name a few.16 Moreover, a link with
HIF-1 signalling pathway has been suggested from the studies with
various cell types.17,18 Recently, several inhibitors have been iden-
tified to inhibit PI3K including wortmannin and Ly-294002.19,20
The example of HIF-1a inhibitors includes YC-1, PX-478 and topo-
tecan.21–23
As a part of our ongoing research in the identification of novel
multiple target inhibitors for anti-cancer drug development, we
herein report the design, synthesis and therapeutic evaluation of
natural product based molecules, which can significantly inhibit
multiple targets of same pathway (PI3K-Akt-mTOR-HIF) in order
to enhance the potency towards various cancer cell types relative
to drugs that address only a single target of the particular pathway.
The role of PI3K/Akt, mTOR and HIF-1a is well known in inflamma-
It has been reported that the inhibition of multiple target in the
PI3K/Akt pathway (PI3K/Akt, mTOR, HIF-1a, VEGF) may lead to
greater therapeutic potential.12,13 Akt, which is also named as pro-
tein kinase B (PKB), is an important downstream effector of growth
* Corresponding author. Tel.: +91 22 30818317; fax: +91 22 30818334.
E-mail address: sanjay.kumar@piramal.com (S. Kumar).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.078

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M. V. Lohar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3603–3606
Table 1
Table 1 (continued)
In vitro inhibition of PI3K/Akt-mTOR activity in cell-based assay for compounds 16–
Compound
x
y
n
R
PI3Kb
NI
mTOR.b
NI
36a
CN
R
34
H
OCH3
0
x
n
S
S
y
O
COOH
N
35
36
H
H
OCH3
OCH3
0
0
NI
NI
Compound
x
y
n
R
PI3Kb
NI
mTOR.b
47
NO2
NO2
NO2
16
O
O
1
1
1
0
0
1
1
0
0
0
1
0
0
0
NI
40
NI
S
O
O
S
Ly-294002
45
17
18
19
20
21
22
23
24
25
26
27
28
29
O
O
NI
NI
67
43
45
58
48
43
64
57
64
58
58
44
51
NI
NI
NI
NI
49
NI
NI
NI
NI
NI
NI
a
Values are means of three experiments.
%Inhibition at 10 lM in H460 cell line. Ly-294002 was evaluated at 30 lM to get
significant inhibition; NI, No inhibition observed up to 30 lM.
b
N
N
O
O
O
tion and cancer. Based on the various isoforms of the PI3K (p110 a,
b, c and d) and their homology, we thought to take advantage of
this information and selected an anti-inflammatory natural prod-
uct, which on suitable modification could yield novel molecules
with anti-cancer properties. A sizable number of natural products
are known in the literature with potent anti-inflammatory activity.
One of them being Evolitrine, an alkaloid, which has been isolated
from several plants sources and has been reported for its antifee-
dant, anti-inflammatory and anti-cancer activity.24,25 Though it
shows significant anti-inflammatory property its anti-cancer prop-
erties have a lot of scope for improvement. Thus we wanted to
make modifications on Evolitrine and enhance its anti-cancer
properties targeting the PI3K/Akt-mTOR/HIF-1a pathway.
The compounds (16–36) described in this Letter (Table 1) are
prepared as outlined in Scheme 1.25 In brief, the condensation of
suitably substituted anilines with diethylmalonate was carried
out by treating sodium diethylmalonate salt with chloroacetyl
chloride to get compounds 1–3, which on thermal cyclization in di-
phenyl ether yielded corresponding substituted dihydrofuran
quinoline dione (4–6). Compounds 4–6 on treatment with Aliquat
336 and POCl3 yielded substituted 4-chlorofuro-quinoline-3-one
(7–9), which on subsequent reduction with NaBH4 yielded 3-hy-
droxy compounds 10–12. The 3-hydroxy compound on dehydra-
tion with KHSO4 yielded substituted 4-chlorofuroquinoline 13–15
in quantitative yield. The target compounds 16–36 (Table 1) were
prepared by treating substituted 4-chlorofuroquinoline 13–15
with a variety of substituted phenols/thiophenols/amines/N-het-
erocycles using NaH in DMF at 150 °C for 10–15 h in 30–70% yields.
The in vitro inhibitory potency of all newly synthesized target
molecules 16–36 are evaluated against the phosphorylation of
Akt at Ser 473, which is an universally accepted read-out of PI3K
activity and phosphorylation of p70S6 kinase at Thr 389 which is
a bonafide read-out of mTOR activity by using human non-small
cell lung cancer H-460 cells as per previously described assay pro-
tocol.26 We found several compounds which inhibited either PI3K
(19–30) or mTOR (16–18, 23, 30–31) or both PI3K and mTOR (23,
30) activities (Table 1). All the compounds (16–36) were tested
at 10 lM concentration in PI3K and mTOR assay and inhibitory
activity has been reported in % inhibition. Ly-294002 was used as
a standard PI3K and mTOR inhibitor in all the experiments, which
shows 40% inhibition in PI3K and 45% inhibition in mTOR assay at
30 lM concentration (Table 1).
H
OCH3
OCH3
O
O
O
OCH3
O
N
F
O
O
CN
NO2
O
O
H
OCH3
OCH3
OCH3
O
S
O
N
H
NO2
N
H
N
NO2
NO2
O
N
N
N
OCH3
OCH3
H
OCH3
OCH3
OCH3
N
N
N
N
N
O
N
30
31
32
33
O
O
1
0
0
0
40
NI
NI
NI
45
53
NI
NI
COCH3
H
OCH3
OCH3
OCH3
N
H
NO2
Br
OCH3
H
S
S
We have synthesized a series of molecules by keeping furoquin-
oline backbone constant and doing the modification particularly at
positions 4, 6 and 7 of furoquinoline ring. The positions 6 and 7
have been explored by using 7-methoxy or 6,7-dimethoxy or 6,7-
methylenedioxo groups. The modification at positions 6 and 7 re-

M. V. Lohar et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3603–3606
3605
Cl
O
O
O
O
x
y
C2H5O
O
x
x
x
c
a
b
n
n
n
NH2
y
y
O
y
O
N
H
N
N
H
O
(7-9)
(1-3)
(4-6)
Cl
N
R
N
Cl
N
OH
x
x
n
y
x
n
y
d
e
f
n
y
O
O
O
(13-15)
(16-36)
(10-12)
1, 4, 7, 10, 13: x = H, y = -OCH3, n = 0; 2, 5, 8, 11, 14: x = y = -OCH3, n = 0; 3, 6, 9, 12, 15: x = y = -O-, n = 1
Scheme 1. Reagent and conditions: (a) NaH, chloroacetyl chloride, TEA, diethyl malonate, THF, 24 h, 45–65%; (b) diphenyl ether, 250 °C, 30 min, 70–80%; (c) Aliquat 336,
POCl3, rt, 48 h, 65–70%; (d) NaBH4, MeOH, 1 h, 90–92%; (e) KHSO4, dioxane, reflux, 2–3 h, 65–75%; (f) substituted phenol/thiophenol/amine/N-heterocyles, NaH, DMF, 150 °C,
10–15 h, 30–70%.
veal that 7-methoxy was more suitable modification for getting an
active entity. We explored position 4 by substituting with variety
of R groups (Table 1) viz. phenoxy, thiophenoxy, N-phenyl, pyri-
doxy, imidazo, piperazine, etc. While evaluating the activity in
PI3K and mTOR several molecules (19–30) were found active in
PI3k assay. Upon screening this subset of molecules for PI3K and
mTOR dual activity only two molecules (23 and 30) exhibited sig-
nificant activity. Then these two molecules were subjected for HIF-
1a assay and compound 23 was found active thus demonstrating
multiple target inhibitor profile. To perform a SAR on compound
23 the para nitro group was substituted with a electron donating
group like amine (data not shown), electronically neutral group
(halogen) but neither of these molecules exhibited any significant
activity. Thus other EWG (34, 35, and 36) were tried at this position
but as shown in Table 1 none of them demonstrate any activity.
Compound 23 turns out to be the most active multi targeted
(PI3K/Akt-mTOR-HIF-1a) inhibitor of this furoquinoline series.
Upon screening Evolitrine for similar assay it exhibited no activity
in PI3K, mTOR or HIF-1a up to 30 lM concentration.
Figure 1. (a) H460 cells were either left untreated (lane 1), or were treated with 20
To address question whether the inhibition of Akt phosphoryla-
tion was a result of targeting the PI3 kinase inhibition or due to di-
rect inhibition of Akt phosphorylation, we performed cell-free
kinase assay with PI3K inhibitors (19–30) using recombinant PI3Ka
(p110a). The read-out of this assay was the formation of PI3P
(phosphatidylinositol-3-phosphate) from PI (phosphatidylinositol)
in presence of PI3Ka as per published procedure.8 Surprisingly, we
found that only compound 23 showed significant inhibition in PI3
kinase (p110a) assay with IC50 4 lM (data not shown). The com-
pounds, which inhibited Akt phosphorylation in cell-based assay,
but showed no inhibition of PI3 kinase activity, might be directly
inhibiting Akt without affecting the PI3 kinase (p110a). The PI3K-
mTOR inhibitory potency of compound 23 was further confirmed
by Western blot analysis using H-460 cell line for phosphorylation
of Akt (PI3K) and 4E-BP1 (mTOR) at concentrations of 3 and 10 lM.
We observed a concentration dependent inhibition of phosphory-
lation of PI3K and mTOR targets (Fig. 1a). The dual (PI3K and
mTOR) inhibitory potency of compound 23 encouraged us to see
the effect of these inhibitors in downstream signalling pathway.
For the same we decided to evaluate the inhibitory potency of
the target molecules 16–36 against hypoxia-induced HIF-1 activa-
tion in U251-HRE glioblastoma cell line. The inhibitory potencies of
HIF-1 activation were obtained using a HRE mediated cell based re-
porter gene assay under hypoxic conditions (1% O2, 94% N2 and 5%
CO2) as well as DFX induced hypoxic condition similar to published
protocol.15 In this assay out of all reported compounds 16–36, only
compound 23 has showed significant inhibition of hypoxia-in-
duced HIF-1 activation, with IC50 3 lM (data not shown). YC-1 a
known HIF-1 inhibitor used as standard (IC50 14.8 lM).
% FCS (lane 2) in the presence of 3 lM compound 23 (lane 3) or 10 lM compound 23
(lane 4). (b) U251-HRE-Luc cells were either left untreated (lane 1), or were treated
for 6 h with DFX (lane 2), in the presence of 0.5 lM Topotecan (lane 3) or 10 lM
compound 23 (lane 4).
To confirm the HIF-1 inhibitory activity, compound 23 was
evaluated by Western blot analysis for the expression of HIF-1a
protein in U251-HRE cell line (Fig. 1b).
These results suggest that compound 30 is a dual inhibitor of
PI3K and mTOR activities, and compound 23 is a multiple inhibitor
of PI3K, mTOR and HIF-1a. However, further studies will unravel
the mechanism of action of these inhibitors in the context of
PI3K isoforms (p110a, p110b, p110c, p110d) as well as antiprolifer-
ative and in vivo efficacy.
In conclusion, we have designed and synthesized variety of
furoquinoline based compounds. The primary screening shows
that 7-methoxy-4-(4-nitrophenylthio)furo[2,3-b]quinoline (23) is
a multi-target inhibitor of PI3K/Akt-mTOR-HIF-1a and identified
as most potent inhibitor in this series. Further investigations are
warranted to establish more detailed mechanism of these
molecules.
Acknowledgments
Authors are grateful to Dr. Giovanni Melillo, Development Ther-
apeutics Program-Tumor Hypoxia Laboratory, National Cancer
Institute at Frederick, Maryland USA. We are also thankful to
department of analytical chemistry for providing nmr, mass,
microanalysis and hplc for characterization of all novel molecules

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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.04.078.
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