Identification of novel 7-amino-5-methyl-1,6-naphthyridin-2(1H)-one derivatives as potent PI3K/mTOR dual inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.12.112

Inhibition of the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway is one of the most intensively studied approaches to cancer therapy. Rational design led to the identification of novel 7-amino-5-methyl-1,6-naph

Full text of DOI:10.1016/j.bmcl.2013.12.112

Bioorganic & Medicinal Chemistry Letters 24 (2014) 790–793
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of novel 7-amino-5-methyl-1,6-naphthyridin-2(1H)
-one derivatives as potent PI3K/mTOR dual inhibitors
⇑
⇑
Songwen Lin , Fangbin Han , Peng Liu, Jing Tao, Xuechao Zhong, Xiujie Liu, Chongqin Yi , Heng Xu
PKUCare Pharmaceutical R&D Center, A106-109, Biotech Innovation Works, No. 29 Life Science Park Road, Changping District, Beijing 102206, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibition of the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signal-
ing pathway is one of the most intensively studied approaches to cancer therapy. Rational design led to
the identification of novel 7-amino-5-methyl-1,6-naphthyridin-2(1H)-one derivatives as potent PI3K/
mTOR dual inhibitors. Design, synthesis and structure activity relationship are reported.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 29 October 2013
Revised 23 December 2013
Accepted 24 December 2013
Available online 3 January 2014
Keywords:
Phosphoinositide 3-kinase
Mammalian target of rapamycin
Dual inhibitor
Anti-tumor activity
The phosphoinositide 3-kinase (PI3K)/AKT/mammalian target
of rapamycin (mTOR) signaling pathway is a critical regulator of
many essential cellular functions including cell growth and prolif-
eration and is perhaps the most commonly activated signaling
pathway in human cancer.1,2 Inhibition of this pathway by
targeting PI3K, AKT and mTOR with small molecules individually
or jointly is expected to have a substantial therapeutic effect and
has therefore become one of the most intensively studied ap-
proaches to cancer therapy.3 Notably along this pathway, mTOR
is in the PI3K superfamily and bears considerable structural
Me
Me
Me
Me
Br
Br
I
Br
N
N
N
a
c
Br
b
N
N
N
N
N
NH
R1
H2N
2
3
1
4
Me
Me
O
Me
d
N
N
OEt
N
f
e
N
NH
R1
N
N
O
H2N
N
O
R1
R1
5
6
7
Me
Br
O
Ar
O
h
g
N
H2N
N
H2N
N
R1
R1
8
9
Scheme 1. Synthesis of compounds 9. Reagents and conditions: (a) 2,5-Hexanedione, TsOH, PhMe, reflux; (b) LDA, I2, À78 °C; (c) R1NH2, DIEA, NMP, 160 °C; (d) ethyl acrylate,
Pd(PPh3)4, TEA, 130 °C; (e) t-BuOK, DBU, DMA, 150 °C; (f) NH2OH–HCl, EtOH/H2O, reflux; (g) NBS, DMF, rt; (h) aryl borate or boronic acid, Palladium catalyst, K2CO3, DMF/H2O,
100 °C.
⇑
Corresponding authors. Tel.: +86-10-80712871 (H. Xu).
E-mail addresses: xuheng@pkucare-pharm.com (H. Xu), yichongqin@founder.
com (C. Yi).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.112

S. Lin et al. / Bioorg. Med. Chem. Lett. 24 (2014) 790–793
791
Table 1
PI3K/mTOR inhibiting activities of 9a–k
Ar
O
N
H2N
X
N
R1
9a-k
Compound
X
R1
Ar
PI3K
IC50
a
mTOR
IC50
(nM)15 (nM)16
N
N
OMe
OMe
PF-04691502
N
C
8.33
7.90
O
O
HO
HO
9a
35.41
11.19
Figure 1. Predicted binding mode for 9k (yellow) with PI3K
c
(PDB ID: 3ML9).
Hydrogen bonding interactions are shown in red dashed lines to the hinge region
(Val 882) and the catalytic lysine (Lys833). Images generated using PyMol.
N
N
OMe
OMe
OMe
9b
9c
9d
9e
9f
N
C
C
C
C
C
C
C
2.80
13.60
6.45
30.09
39.04
36.54
159.96
12.25
82.12
13.91
171.72
Table 2
PI3K/mTOR inhibiting activities of 9l–u
N
OMe
NH
N
N
N
O
S
O
H2N
N
O
R2
N
OMe
F
5.95
9l-u
IC50 (nM)15
H
N
Compound
R2
PI3K
a
mTOR IC50 (nM)16
62.64
N
2.45
9l
Me
23.14
69.11
9m
9n
9o
9p
9q
9r
140.9
19.2
N
N
N
9g
9h
9i
15.20
2.90
116.75
174.28
14.27
MeO
NC
F3C
F
65.64
24.69
78.20
35.02
H
N
N
H
N
N
N
174.88
72.59
6.00
N
OMe
O
O
S
9s
60.51
13.88
9j
C
C
12.03
2.42
33.92
8.55
N
H
F
F
F
F
N
OMe
O
9t
37.33
31.16
NDa
8.49
O
S
F
N
9k
H
9u
F
Cl
O
S
HO
a
ND = not determined.
4-Methylpyridopyrimidinone (MPP) has proven to be a potent
similarity to class I PI3Ks.2,4,5 This similarity presents an opportu-
nity to generate dual-specificity compounds, targeting PI3K/mTOR
simultaneously in one pathway. These dual inhibitors could, in
principle, effectively block the signal transduction and overcome
feedback loops.6 Another advantage derived from dual inhibitors
is anticipated to be the largely reduced possibility of drug resis-
tance. In recent years, a number of dual PI3K/mTOR inhibitors have
crowded into the clinical trials to generate clinical efficacy and are
on the way towards regulatory approvals.7–13
chemical scaffold for dual PI3K/mTOR inhibitors, demonstrating
excellent anti-tumor activities in both cell proliferation assays
and xenograft models.8,9 PF-04691502, with such a scaffold, en-
tered into Phase I/II clinical trials in patients with early breast can-
cer, further elucidating its anti-tumor effectiveness.14
The key interactions between MPP and PI3K have been vali-
dated through determination of co-crystal structure of PF-
04691502 bound in PI3Kc (PDB ID: 3ML9). The NH2 functionality
together with the N atom adjacent to the methyl group at the 4

792
S. Lin et al. / Bioorg. Med. Chem. Lett. 24 (2014) 790–793
Table 3
Table 4
PI3K/mTOR inhibiting activities of 9j, 9v–y
PI3K/mTOR inhibiting activities of 10–14
N
OMe
O
Compound Structure
PI3K
IC50
a
mTOR
IC50
O
S
N
(nM)15
(nM)16
N
H
H2N
N
O
F
F
N
OMe
R1
9j, 9v-y
N
Compound
R1
PI3K
a
IC50 (nM)15
mTOR IC50 (nM)16
33.92
H2N
N
N
O
10
>10,000
>10,000
9j
12.03
9.90
HO
9v
28.68
13.36
N
OH
H2N
N
N
O
N
11
5551
>10,000
OMe
OMe
9w
8.60
O
HO
N
N
9x
9y
39.65
42.84
NDa
O
O
S
12
13
14
3.59
35.63
3.43
35.27
18.39
2.32
N
H
125.93
H2N
N
N
N
N
F
F
a
ND = not determined.
N
N
OMe
O
O
S
N
N
N
position of MPP forms key hydrogen bonds with the hinge residue,
Val 882. Another important interaction was identified as one addi-
tional hydrogen bonding between the ring nitrogen on the
methoxypyridine and a conserved water molecule in a selectivity
pocket.8
H
H2N
H2N
N
N
F
F
OMe
O
O
S
N
H
F
F
To search for a structurally differentiated template that could
potentially address the tolerability findings observed for PF-
04691502 in cancer clinical trials,14 we planned to explore the
MPP scaffold and discover a novel chemical series. Firstly, we no-
ticed from the co-crystal structure that there is no direct interac-
tion between the nitrogen at the 1-position of MPP and the PI3K,
indicating a good starting point to diversify the scaffold. By using
a bioisostere approach, this nitrogen was replaced with a carbon
atom. The synthetic route to prepare such analogs is illustrated
in Scheme 1: 2-amino-5-bromo-6-methylpyridine 1 was firstly
protected by 2,5-hexanedione, then followed by deprotonation
with LDA and subsequent iodination to provide 3. Iodide 3 was car-
ried out with various primary amines to afford 4, which was then
followed by Heck coupling with ethyl acrylate and cyclization to
generate 6. Deprotection of 6 by hydroxylamine afforded 7, and 7
was further brominated by NBS to yield 8. Various aryl (Ar) groups
were then attached to the 3-position of the 1,6-naphthyridin-
2(1H)-one core of 8 by Suzuki coupling to give the desired analogs
9.
Table 5
Mouse PK profile of 9k19
Dose (mg/kg)
AUCinf (h⁄ng/mL)
8087
3441
CL (mL/min/kg)
T1/2 (h)
F (%)
1.36 (IV)
6.81 (PO)
2.87
/
1.33
7.67
/
8.5
Lys833 up and inward, thereby rendering the reasonable distance
to form hydrogen bonding with both the O atom and the F atom,
which do not exist in the original MPP scaffold.17 This finding
was also observed in our SAR investigation (Table 1, 13.60 nM of
9c vs 5.95 nM of 9e in PI3K
oxyl pyridine with various indazoles also resulted in enhancement
of PI3K potency (9f, 9h, 9i vs 9c). To seek further expansion, sul-
a). Additionally, replacement of meth-
a
The compounds 9a and 9c were synthesized according to this
fonamide functionality, with its deprotonated form of NH at phys-
iological conditions to interact with Lys833,10 was placed onto the
3-position of methoxypyridine. In particular, the enzymatic po-
synthetic route and then tested in PI3K
assays.15,16 Compared to PF-04691502, however, 9a is about 4-fold
and 1.5-fold less potent in PI3K and mTOR respectively. The sim-
a and mTOR biochemical
a
tency of 9k against PI3K
a was significantly increased with an
ilar trend was also noted for 9c versus 9b. While no direct interac-
tion was derived from this nitrogen, replacement of the nitrogen
with the carbon increases the electron density of the aryl ring, con-
sequently weakening H-bonding of the NH2 and enhancing H-
bonding of the N on the ring respectively. The overall H-bonding
forming capability of the aminopyridine fragment with the hinge
is likely attributed to the potency drop.
To compensate for the decrease in potency, new interaction(s)
need to be incorporated into the chemical structure. It was previ-
ously reported that introduction of the F atom to the 3-position
of methoxypyridine was able to enhance the potency by pushing
IC50 of 2.42 nM, resulting in around a 15-fold improvement com-
pared to 9a, while its potency against mTOR was also found in-
creased (8.55 nM of 9k vs 11.19 nM of 9a). In a modeling study,
9k was docked with PI3Kc as illustrated in Figure 1, indicating that
aryl sulfonamide group fits into the space and forms additional
interactions (e.g. hydrogen bondings) with the residue, Lys833.
Meanwhile, a variety of sulfonamides with alkyl, cycloalkyl and
aryl substituents were explored (Table 2). The cyclopentanyl ring
was chosen for R1 substitution mainly due to its synthetic feasibil-
ity and enzymatic activity. Through a SAR analysis, alkyl and cyclo-
alkyl substituted sulfonamides were found to generally exhibit

S. Lin et al. / Bioorg. Med. Chem. Lett. 24 (2014) 790–793
793
moderate PI3K
a
potencies (23.14, 69.11 nM for 9l and 9m, respec-
comparable to PF-04691502 as well as acceptable cellular activity
and PK profile. Moreover, a novel AMN template differentiated
from known MPP derivatives was thus identified for further opti-
mization, yielding a broader chemical space towards developing
anti-tumor agents into clinical phases.
tively), while their mTOR potencies was decreased compared to
most of aryl substituted compounds listed in Table 2 (9l–m vs
9n, 9p, 9r–s, 9u). It was also observed that substituents on the aryl
ring play a significant part in potency. Among methoxyl, cyano, tri-
fluoro and fluoro groups introduced to the para-position of phenyl
ring (9o–r), the cyano group stands out with the highest PI3K po-
tency (e.g. IC50 = 14.27 nM). We reasoned that the size/configura-
tion of the substituent(s) that best fits into the corresponding
hydrophobic region could be dominant factor for retaining PI3K
potency. It was also noticed that the potencies of mTOR did not fol-
Acknowledgments
This research was supported in part by Returnee Scientific Inno-
vation Initiative Program of Beijing, China Postdoctoral Science
Foundation (2012M510380) and Beijing Postdoctoral Research
Foundation (2012ZZ-96).
low the same trend as those of PI3Ka among compounds 9o–r. This
could be easily attributed to the structural difference between PI3K
and mTOR binding pockets. Moreover, this indirect evidence of the
structural difference should be borne in mind particularly for
designing dual PI3K/mTOR inhibitors.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.12.
112.
Subsequently, the SAR was also explored in R1 region (Table 3).
The cyclohexyl ring attached with a hydroxyethoxyl group was re-
ported as an optimal R1 substituent in PF-04691502, helping to
achieve excellent permeability and potency.17 This R1 substituent
References and notes
also yielded the most potent compound against PI3Ka/mTOR
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among the newly synthesized compounds listed in Table 3. With
the exploration of trans-4-hydroxycyclohexyl, tetrahydro-4H-pyra-
nyl, cyclopentyl, cyclopropyl and isopropyl groups, 6-member ring
(9w) could be concluded as the most favorite, showing approxi-
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five-member ring (9j) and three-member ring (9x), respectively.
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15. See Supplementary data for the procedure of PI3Ka biochemical assay
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Bagrodia, S.; Yuan, J.; Sun, S. X.; Zhang, E.; Xiang, C.; Dalvie, D.; Rahavendran, S.
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18. See Supplementary data for the procedure of proliferation assay.
19. See Supplementary data for the procedure of PK study.
and IC50 value of 9k in this cell line was determined to be 2.1 l
M.18
A mouse PK study revealed 9k had low clearance, moderate oral
exposure and reasonable half-life (Table 5).19 The oral availability
of 9k is low (F = 8.5%) and probably caused by low absorption. Its
in vitro inhibition activities as well as PK profile support itself to
enter into a next phase of in vivo anti-tumor efficacy evaluation.
In summary, a novel series of compounds featuring 7-amino-5-
methyl-1,6-naphthyridin-2(1H)-one (AMN) moieties were discov-
ered as dual PI3K/mTOR inhibitors by rational design. Through a
systemic SAR analysis, the representative compound (e.g. 9k)
was identified, demonstrating decent PI3K/mTOR potency