Novel 1,6-naphthyridin-2(1H)-ones as potential anticancer agents targeting Hsp90

Article abstract of DOI:10.1016/j.ejmech.2016.04.050

Hsp90 is an ATP-dependent chaperone known to be overexpressed in many cancers. This way, Hsp90 is an important target for drug discovery. Novobiocin, an aminocoumarin antibiotic, was reported to inhibit Hsp90 targeting C-terminal domain, and showed anti-proliferative properties, leading to the development of new and more active compounds. Consequently, a new set of novobiocin analogs derived from 1,6-naphthyridin-2(1H)-one scaffold was designed, synthesized and evaluated against two breast cancer cell lines. Subsequently, cell cycle progression and apoptosis were conducted on best candidates, finally Western Blot analysis was performed to measure their ability to induce degradation of Hsp90 client proteins.

Full text of DOI:10.1016/j.ejmech.2016.04.050

European Journal of Medicinal Chemistry 119 (2016) 17e33
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel 1,6-naphthyridin-2(1H)-ones as potential anticancer agents
targeting Hsp90
a
b
a
b
a
ꢀ
David Montoir , Sophie Barille-Nion , Alain Tonnerre , Philippe Juin , Muriel Duflos ,
Marc-Antoine Bazin a
,
*
a
ꢀ
ꢀ
ꢀ
ꢀ
Universite de Nantes, Nantes Atlantique Universites, Laboratoire de Chimie Therapeutique, Cibles et Medicaments des Infections et du Cancer, IICiMed
UPRES EA 1155, UFR de Sciences Pharmaceutiques et Biologiques, 1 rue Gaston Veil, 44035 Nantes, France
b
ꢀ
ꢀ
UMR 892 INSERM/6299 CNRS/Universite de Nantes, Team 8 ‘Cell Survival and Tumor Escape in Breast Cancer’, Institut de Recherche Therapeutique de
ꢀ
l'Universite de Nantes, 8 quai Moncousu, BP 70721, 44007 Nantes Cedex 1, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Hsp90 is an ATP-dependent chaperone known to be overexpressed in many cancers. This way, Hsp90 is
an important target for drug discovery. Novobiocin, an aminocoumarin antibiotic, was reported to inhibit
Hsp90 targeting C-terminal domain, and showed anti-proliferative properties, leading to the develop-
ment of new and more active compounds. Consequently, a new set of novobiocin analogs derived from
1,6-naphthyridin-2(1H)-one scaffold was designed, synthesized and evaluated against two breast cancer
cell lines. Subsequently, cell cycle progression and apoptosis were conducted on best candidates, finally
Western Blot analysis was performed to measure their ability to induce degradation of Hsp90 client
proteins.
Received 3 February 2016
Received in revised form
18 April 2016
Accepted 19 April 2016
Available online 22 April 2016
Keywords:
Heat shock protein 90
Hsp90 inhibitors
© 2016 Elsevier Masson SAS. All rights reserved.
Novobiocin analogs
1,6-Naphthyridin-2(1H)-ones
Breast cancer
1. Introduction
to induce client protein degradation, which highlights the impor-
tance of Hsp90 as a promising cancer therapeutic target. Unfortu-
The 90 kDa heat shock protein (Hsp90) is a highly conserved and
specialized chaperone responsible for the maturation of a select
clientele of proteins. Many of clients (Raf-1, Her2, Akt, etc.) are
involved in multiple oncogenic pathways and associated with
cancer cell survival, making Hsp90 an attractive target for cancer
chemotherapy [1,2]. In terms of structure, Hsp90 exists as a
homodimer with each monomer consisting of three domains; a N-
terminal nucleotide binding domain, a middle domain with high
affinity for co-chaperones and client proteins, and a C-terminal
dimerization domain. In recent years, considerable effort in the
discovery of Hsp90 inhibitors was performed. 17-AAG (tanes-
pimycin), a semi-synthetic derivative of geldanamycin, was the first
Hsp90 inhibitor to be evaluated in clinical trials and has shown
significant anti-tumor activity at tolerable doses. In addition, 16
other molecules specifically targeting the N-terminal ATP-binding
site have entered clinical trials for evaluation against various can-
cers [3]. These N-terminal inhibitors have demonstrated an ability
nately, induction of a pro-survival heat shock response (HSR) at the
same concentration needed to promote Hsp90 client protein
degradation has compromised their future in therapeutic. This HSR
expands the chaperone buffering capacity to contribute in the
maturation of mutated and oncogenic clients leading to drug
resistance and tumor metastasis [4]. In contrast to N-terminal in-
hibitors, recent studies have demonstrated that inhibitors of the C-
terminal region do not induce the HSR and therefore provide an
attractive alternative for the development of new Hsp90 modula-
tors that exhibit better activities [5,6].
Novobiocin, an aminocoumarin member of the antibiotic family
acting through inhibition of DNA gyrase, was the first natural
product identified as Hsp90 C-terminal inhibitor that does not
compete with other inhibitors for binding to the N-terminal
domain [7]. Unfortunately, novobiocin displays poor anti-
proliferative activity (IC50 ~ 700 mM against SKBr3 cells and 260 mM
against MCF-7 cell line) and consequently has limited its thera-
peutic use. Although the binding mode of the N-terminal Hsp90
inhibitors is well established, the structural moieties required for
interaction with the C-terminal domain are very poorly
* Corresponding author.
E-mail address: marc-antoine.bazin@univ-nantes.fr (M.-A. Bazin).
http://dx.doi.org/10.1016/j.ejmech.2016.04.050
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

18
D. Montoir et al. / European Journal of Medicinal Chemistry 119 (2016) 17e33
characterized [8]. Since to date, no cocrystal structure of Hsp90
bound to C-terminal inhibitors has been yet reported. Preliminary
investigations on novobiocin analogs revealed structural key ele-
ments for elucidation of the Hsp90 C-terminal binding pocket and
subsequent structureeactivity relationships led to identify prom-
ising synthetic compounds that exhibit potent antiproliferative
activity against a large panel of cancer cell lines [9e23]. The syn-
thesis of these libraries has helped to highlight the following
structureeactivity relationships (SARs): (1) the lactone present in
the coumarin core is not essential for Hsp90 inhibition while 4-
hydroxy lowers the activity; (2) replacement of the noviose at the
7-position with hydrophilic amines residues is tolerated without
compromising inhibitory activity (3) the aromatic side chain and
the amide linker are critical for antiproliferative activity (Fig. 1).
On the basis of these SARs, we started a new medicinal chem-
istry program to design and synthesize novel novobiocin analogs as
potent antiproliferative agents targeting the Hsp90 chaperone
machinery.
methylamine provided monochloro compound 4 in 96% yield.
Finally, a reduction/oxidation sequence afforded the desired alde-
hyde 5. The key amine intermediate 6 was then obtained in a good
yield from 5 and the condensation with ethyl glycinate.
The target compounds were obtained in two steps from the 1,6-
naphthyridin-2-(1H)-one 6. Amides 7e18 were synthesized by re-
action of amine 6 with functionalized acyl chlorides in anhydrous
pyridine [27]. Introduction of various carboxamides (alkyle, cin-
namoyle, aryles and heterocycles) in the 3-position sometimes
required the preliminary synthesis of corresponding acyl chlorides.
Finally, aromatic nucleophilic substitutions with aliphatic amines
afforded analogs of novobiocin 19e45 in moderate to very good
yields [28]. The structure elucidation of synthesized compounds
was accomplished using extensive 1D and 2D NMR spectroscopic
studies supplemented with MS analysis.
2.2. Biological evaluation
First, we suggested that the coumarin core be replaced with a
1,6-naphthyridin-2(1H)-one scaffold. As the lactone is considered
as non-essential for biological activity, bioisosteric exchange of the
pyran-2-one ring with a pyridin-2-one core was realized. A pyri-
dine ring is attached to complete the scaffold and to form the bi-
cycle. To overcome problems with regard to solubility or reactivity
of free NeH-containing 1,6-naphthyridones, we decided to inves-
tigate N-methylated 1,6-naphthyridones [24]. Then, a large variety
of amides was introduced to the 3-position of a key amine inter-
mediate, the 3-amino-7-chloro-1-methyl-1,6-naphthyridin-2(1H)-
one, which was functionalized at the 7-position with hydrophilic
amines.
2.2.1. Antiproliferative activity (MTT)
To follow up on the synthesis of our naphthyridinones, an
evaluation of their antiproliferative activity against MCF-7 (estro-
gen receptor positive breast cancer cells) and MDA-MB-468 (triple
negative, estrogen and progesterone receptors negative, no HER2
expression) cell lines was conducted (Table 1).
The IC50 values of 17-AAG (N-terminal inhibitor) and novobiocin
were consistent with those found in the literature.
Nine molecules (19, 20, 21, 23, 25, 27, 28, 32 and 44) had no
significant effect at a concentration greater than 100 mM on two cell
lines tested, and three (34, 40, 42) exhibited antiproliferative effect
only against MCF-7 cell line. Four analogs (36, 37, 39, 41) displayed
an activity less than 10 mM to both cell lines, but none is as active as
17-AAG. The p53 status of these cell lines, p53-wild type for MCF-7
and p53-mutant for MDA-MB-468, could generate different cellular
responses. In view of these heterogeneous activities, we chose to
undergo further biological assays to obtain more information
regarding the antiproliferative mechanism of action of the most
promising compounds.
2. Results and discussion
2.1. Chemistry
In previous works, we developed efficient one-pot syntheses of
3,7-disubstituted 1,6-naphthyridin-2(1H)-ones through regiose-
lective palladium-catalyzed cross-coupling reactions [25,26]. In
this paper, the same procedure was used to synthesize in multi-
gram quantities an ortho-amino nicotinaldehyde (Scheme 1).
First, condensation of a commercial solution of diethyl 3-
oxopentanedioate 1 with triethyl orthoformate led to an interme-
diate which was cyclized with aqueous ammonia to the ester 2.
Subsequent chlorination reaction was carried out with phosphor-
us(V) oxychloride and allowed isolation of the compound 3.
Regioselective nucleophilic substitution in the 4-position with
2.2.2. Apoptosis detection
Seven compounds that have shown
a significant anti-
proliferative activity on MCF-7 and MDA-MB-468 cell lines were
selected for evaluation. For this purpose, the compounds 30, 41 and
45 were chosen because they exhibited low IC50 values. All biphenyl
derivatives 36e39 appeared promising and were also selected.
Having demonstrated antiproliferative activity of these com-
pounds, we investigated whether this effect could be the conse-
quence of an induced cell death caused by these molecules.
Analyzes were performed on MDA-MB-468 cells and at con-
centrations corresponding to the IC50 of the compounds (Fig. 2).
The Annexin V/Propidium iodide (PI) protocol is a commonly
used approach for studying the cell death and discriminate
apoptotic from necrotic cells through differences in plasma mem-
brane integrity and permeability. Three cell populations can be
distinguished: viable cells (AV-, PI-), cells in early apoptosis (AVþ,
PI-) and cells in late apoptosis or necrosis (AVþ, PIþ). Among the
seven analyzed molecules, only 30 and 38 have shown significant
cytotoxicity with respective mortalities of 40 and 80% greater than
that obtained with the 17-AAG. Molecules 36 and 39 did not induce
cell death. Finally, compounds 37, 41 and 45 presented a low level
compared to 17-AAG. The antiproliferative activity of novobiocin on
the MDA-MB-468 line is known to be low, so it was not included in
cell death assays.
2.2.3. Cell cycle analysis
Fig. 1. SARs established from previous studies related to novobiocin analogs.
To complete these apoptosis assays, analysis of the cell cycle

D. Montoir et al. / European Journal of Medicinal Chemistry 119 (2016) 17e33
19
CO2Et
OH
CO2Et
Cl
CO2Et
NH
O
1
N
N
N
a
b
c
EtO2C
CO2Et
HO
Cl
Cl
2
3
4
H
NH2
O
CHO
NH
N
R
N
N
N
d
e
f
O
Cl
Cl
N
O
Cl
N
5
6
7-18
H
N
R
N
g
O
R''R'N
N
O
19-45
Scheme 1. Synthesis of new analogs of novobiocin derived from 1,6-naphthyridin-2(1H)-one scaffold. Reagents and conditions: (a) HC(OEt)3, Ac2O, 120 ꢀC, 1.5 h, then NH3 (30%w),
ꢀC, 70%; (b) POCl3, 110 ꢀC, 2 h, 95%; (c) CH3NH2 (40%w), CH3CN, 0 ꢀC, 30 min, then rt, 3 h, 96%; (d) LiAlH4, THF, -78 ꢀC, 3 h, then MnO2, CH2Cl2, rt, 2 h, 84%; (e) Ethyl glycinate, K2CO3,
anh. DMF, 90 ꢀC, 5 h, 92%; (f) ClCOR, anh. Pyridine, 1e4 h, 33e84%; (g) R0R00NH2, -wave, 130e170 ꢀC, 1e8 h, 19e95%.
0
m
distribution of treated cancer cells was determined for each of the
selected compounds. Cells were stained with PI and the proportion
of cells in different phases of the cell cycle was recorded. MDA-MB-
468 cells control (DMSO) presented a profile of cellular cycle
conventionally observed in cancer cell lines in optimal growth
conditions. With regard to the treated cells, a gradual accumulation
of MDA-MB-468 cells and a blockage in G2/M phase for 17-AAG was
observed after 48 h of incubation, which is consistent with the data
from the literature [30]. Compounds 36, 39, 41, 45 induced a sig-
nificant increase in G2/M but not 37 (Fig. 3). No major change in the
cell cycle for molecules 30 and 38 was observed, however a subG1
peak characteristic of apoptotic cells population was detected in
agreement with previous apoptotic assays.
3. Conclusion
In summary, a library of 1,6-naphthyridinones derivatives based
on novobiocin structure was synthesized and evaluated for their
antiproliferative activities on MCF-7 and MDA-MB-468 breast
cancer cell lines. Compounds having the biphenyl structure were
the most convenient and all exhibited good activity on both cancer
cell lines, suggesting the importance of a bulky benzamide group.
Moreover, the addition of alkylamino substituents at the 7-position
ensured an increased solubility for bioassays and this motif might
be involved in the interaction with the target. Some compounds
showed promising results in different biological assays (cell death,
cell cycle distribution, expression of Hsp90 client proteins).
Particularly, Western Blot analyses suggest these compounds
manifest antiproliferative activity through Hsp90 inhibition. In-
vestigations are currently underway to further develop this scaffold
and the biological evaluation is still under progress to consider
relevant SARs.
2.2.4. Western blot analysis
In order to ensure the antiproliferative activity through Hsp90
inhibition, Western Blot analyses of two Hsp90 client protein levels
in MCF-7 and one in MDA-MB-468 were performed. As shown in
Fig. 4, the Hsp90-dependent client proteins Raf-1 and ER
a
(only
4. Experimental protocols
express in MCF-7 cells) were completely degraded in MCF-7 cell
line upon treatment with 17-AAG. In addition, induction of a pro-
survival heat shock response (HSR) was observed as described
with other N-terminal inhibitors. These results were less pro-
nounced in MDA-MB-468 cells. The heat shock response was also
visible in MCF-7 cell line for 37 and 38 but to a lesser extent. All
compounds induced degradation of client proteins in two cell lines,
except for 45. The analogue 39 seemed to be the most promising
compound, exhibited important client proteins degradation in two
cell lines, and was better than 17-AAG in MDA-MB-468 cells. The
non-Hsp90-dependent protein, actin, was not altered upon
administration of these compounds, indicating selective degrada-
tion of Hsp90-dependent proteins. In addition, Hsp90 levels
remained unchanged except for 37 and 38, which is consistent with
other known Hsp90 C-terminal inhibitors [31].
4.1. Chemistry
All commercial reagents were used without further purification.
All solvents were reagent or HPLC grade. THF was distilled on Na/
benzophenone system prior to use. Analytical TLC was performed
on silica gel 60 F254 plates. Flash column chromatography was
performed on silica gel 60 (70e230 mesh ASTM). Yields refer to
chromatographically and spectroscopically pure compounds.
Melting points were determined on an Electrothermal melting
point apparatus. 1H NMR and 13C NMR spectra were recorded in
DMSO-d6 or CDCl3 on a 400 MHz spectrometer. Chemical shifts are
reported as
d values in parts per million (ppm) relative to tetra-
methylsilane as internal standard and coupling constants (J) are
given in hertz. Multiplicities are reported as follows: s ¼ singlet,
d ¼ doublet, dd ¼ doublet of doublets, ddd ¼ doublet of doublet of
doublets, t ¼ triplet, m ¼ multiplet, q ¼ quartet, br s ¼ broad singlet.
Low resolution mass spectra were recorded using an Electrospray
Ionization (ESI) Method with Waters ZQ 2000 spectrometer. UPLC
column used was an Acquity UPLC® BEH Phenyl (2.1 mm i.d.,
A few brief comments on SARs reveals that all the seven tested
compounds displaying the best activities bear an apolar core in the
3-position (i.e. benzothienyle, biphenyle, chlorophenyle and
methoxyphenyle). Four of them are biphenyle derivatives but only
compound 39 seems to be the most potent due to client proteins
Raf-1 and ER-
a
inhibition on the two breast cancer cell lines. One
50 mm length, 1.7 mm particle size) from Waters. A linear mobile
might wonder why the protonatable nitrogen atom or the steric
hindrance on polar chains like in compounds 36e38 are useful.
Other pharmacomodulations should confirm this hypothesis.
phase gradient was used with a mobile phase A as 100% of aceto-
nitrile in water (at 2%) and mobile phase B as 100% acetonitrile. The
gradient table was: 0e0.5 min, 0% B; 0.5e4.0 min 0 / 100% B;

20
D. Montoir et al. / European Journal of Medicinal Chemistry 119 (2016) 17e33
Table 1
Antiproliferative activity of 1,6-naphthyridinones, novobiocin analogs.
H
N
R
N
O
R''R'N
N
O
Compound
R
R’R00N
MCF-7 (IC50
,
m
M)a
MDA-MB-468 (IC50
1.57 1.42
, m
M)a
17-AAG
e
e
0.029 1.54
(tanespimycin)
Novobiocin
(Lit: 5 nM) [29]
293.3 2.60
e
e
338.40 9.05
(Lit: 260 mM) [23]
19
CH3
>100
>100
O
N
N
N
N
N
H
20
21
CH3
>100
>100
>100
>100
N
N
H
CH ¼ CHPh
O
N
H
22
23
CH ¼ CHPh
27.41 1.68
12.05 1.81
N
N
H
Ph
>100
>100
O
N
H
24
25
Ph
35.09 1.24
18.27 1.51
N
N
H
Ph-(p)NH2
>100
>100
O
N
H
26
27
Ph-(p)NH2
Ph-(p)OH
55.77 1.91
20.41 1.50
N
N
H
>100
>100
O
N
N
N
H
28
Ph-(p)OCH3
>100
>100
O
N
H
29
30
Ph-(p)OCH3
Ph-(p)OCH3
37.26 1.58
8.59 1.43
15.71 1.56
10.47 3.30
N
N
H
N
N
HO
31
32
Ph-(p)OCH3
Ph-(m)OCH3
35.37 0.33
12.55 1.57
N
N
O
>100
>100
N
N
H

D. Montoir et al. / European Journal of Medicinal Chemistry 119 (2016) 17e33
21
Table 1 (continued )
Compound
33
R
R’R00N
MCF-7 (IC50
,
m
M)a
MDA-MB-468 (IC50
17.44 1.72
, m
M)a
Ph-(m)OCH3
43.25 1.38
N
N
H
34
Ph-(m,m’,p)OCH3
48.56 1.78
>100
O
N
N
H
35
36
Ph-(m,m’,p)OCH3
Ph-(m)Ph
21.47 1.40
4.85 1.31
15.59 1.92
1.96 1.83
N
N
H
O
N
N
H
37
38
Ph-(m)Ph
Ph-(m)Ph
1.93 1.55
1,68 1,67
N
N
H
15.20 1.31
14.67 1.70
N
N
HO
O
39
40
Ph-(m)Ph
Ph-(p)Cl
6.00 1.34
6.35 2.04
1.87 1.59
N
H
>100
N
N
N
N
H
41
42
Ph-(p)Cl
3.09 2.04
4.47 4.12
1.64 1.56
N
N
H
4-pyridyl
>100
O
N
H
43
44
4-pyridyl
36.56 2.48
8.36 2.68
N
N
H
2-benzothienyl
>100
>100
O
N
H
45
2-benzothienyl
12.03 1.40
3.57 1.76
N
N
H
a
Values represent mean standard deviation for at least three separate experiments performed in triplicate.
4.0e5.5% 100% B; 5.5e5.7 min 100 / 0% B; 5.7e7.5 min 0% B. at
flow rate 0.5 mL minꢁ1 and column temperature 35 ꢀC. High res-
olution mass spectra were obtained by ESI/TOF. Microwave re-
actions were carried out in a CEM Discover microwave reactor in
sealed vessels (monowave, maximum power 300 W, temperature
control via IR-sensor, fixed temperature).
5
8
4
NH2
3
4a
8a
N
7
O
Cl
N
Compounds 2e5 were synthesized following the procedure
previously described [25].
To a mixture of 5 (2.00 g, 11.7 mmol) in anhydrous DMF (30 mL)
were added ethyl glycinate (1.80 g, 12.9 mmol, 1.10 equiv.) and
K2CO3 (3.23 g, 23.4 mmol, 2.0 equiv.). The reaction mixture was
stirred at 90 ꢀC for 5 h. After cooling, water was added, and the
mixture was extracted with CH2Cl2. The organic layer was washed
with brine, dried over Na2SO4, filtered, and concentrated under
reduced pressure. Trituration with diisopropyl ether afforded 6
4.1.1. 3-Amino-7-chloro-1-methyl-1,6-naphthyridin-2(1H)-one (6)

22
D. Montoir et al. / European Journal of Medicinal Chemistry 119 (2016) 17e33
100
80
60
40
20
0
AnxV+/PI+
AnxV+/PI-
100
80
60
40
20
0
G1
S
G2/M
polyploid
SubG1
Fig. 3. Cell cycle distribution in MDA-MB-468 cells treated for 48 h with selected
compounds.
Fig. 2. Percentage of annexin V-positive cells in MDA-MB-468 cells treated for 48 h
with selected compounds.
4.1.2.2. (2E)-N-(7-Chloro-1-methyl-2-oxo-1,2-dihydro-1,6-
naphthyridin-3-yl)-3-phenylprop-2-enamide (8).
(2.25 g, 92% yield) as a pale yellow powder. Mp: 260e261 ꢀC; IR,
(cmꢁ1): 3445 and 3325 (
NeH); 1622 ( C]O); 1481, 1423 ( C]
C); 777 ( 8.48 (s, 1H, H5),
CeCl); 1H NMR (400 MHz, DMSO-d6)
7.48 (s, 1H, H8), 6.80 (s, 1H, H4), 5.94 (s, 2H, NH2), 3.68 (s, 3H, NCH3);
13C NMR (100 MHz, DMSO-d6)
157.96 (C2), 145.72 (C5), 144.47
(C8a), 138.24 (C7 or C3), 138.19 (C3 or C7), 118.13 (C4a), 107.98 (C8),
101.58 (C4), 29.71 (NCH3); MS (ESI) m/z (%): 210.1 (100) [M þ H]þ,
212.1 (40) [M þ H þ 2]þ; UPLC purity ¼ 96%, Rt 1.86 min; HRMS
(ESI): calcd. for C9H8ClN3O [M þ H]þ 210.0429, found: 210.0421.
n
e
n
n
n
f
d
n
d
a
H
N
5
8
4
4a
8a
3
c
N
d
b
O
7
N
O
Cl
The reaction was carried out using amine 6 (400 mg, 1.91 mmol)
and (2E)-3-phenylprop-2-enoyl chloride (1.15 g, 6.30 mmol) and
4.1.2. General procedure for the synthesis of carboxamides 7e18
Compound 6 (400 mg, 1.91 mmol) was dissolved in dry pyridine
(10 mL). The corresponding acyl chloride (6.30 mmol, 3.3 equiv.)
was added and the reaction mixture was refluxed for 1e5 h. After
cooling, water was added and the organic layer was extracted with
CH2Cl2. The organic layer was washed with 1 M HCl, brine, dried
over Na2SO4, filtered, and concentrated under reduced pressure.
The crude product was then purified by silica gel chromatography
to provide 7e18.
4.1.2.1. N-(7-Chloro-1-methyl-2-oxo-1,2-dihydro-1,6-naphthyridin-
3-yl)acetamide (7).
H
N
5
8
4
4a
8a
3
N
O
7
O
Cl
N
The reaction was carried out using amine 6 (400 mg, 1.91 mmol)
and acetyl chloride (448 L, 6.30 mmol) and refluxed in pyridine for
m
3 h. Purification by silica gel chromatography (dichloromethane/
ethyl acetate, 90:10) afforded 7 (302 mg, 63% yield) as a white
powder. Mp: 264e265 ꢀC; IR,
n
(cmꢁ1): 3361 (
C ¼ C); 682 (
9.70 (s, 1H, NH), 8.76 (s, 1H, H5), 8.72 (s, 1H,
n
NeH); 2986, 2970
n
(
n
CeHal); 1771, 1645 (
n
d
C ¼ O); 1498 (
n
CeCl); 1H NMR
(400 MHz, DMSO-d6)
H4), 7.64 (s,1H, H8), 3.70 (s, 3H, NCH3), 2.23 (s, 3H, COCH3); 13C NMR
(100 MHz, DMSO-d6)
d 170.69 (NHeC]O), 157.29 (C3eC]O),
149.21 (C5), 148.48 (C8a), 142.33 (C7), 129.13 (C3), 116.68 (C4), 116.00
(C4a), 108.40 (C8), 30.07 (NCH3), 24.11 (COCH3); MS (ESI) m/z (%):
252.1 (100) [M þ H]þ, 254.1 (40) [M þ H þ 2]þ; UPLC purity ¼ 98%,
Rt 1.92 min; HRMS (ESI): calcd. for C11H10ClN3O2 [M þ H]þ
252.0534, found: 252.0525.
Fig. 4. Western Blot analyses of MCF-7 and MDA-MB-468 cell lysates for Hsp90 client
protein degradation after 48 h of incubation. All compounds are tested at concentra-
tions of 1.5 times their IC50 values (
mM). 17-AAG (0.5 mM) and DMSO were employed,
respectively, as positive and negative controls.

D. Montoir et al. / European Journal of Medicinal Chemistry 119 (2016) 17e33
23
refluxed in pyridine for 3 h. Purification by silica gel chromatog-
raphy (mixtures of dichloromethane/methanol of increasing po-
8.23 (d, 3J ¼ 8.8 Hz, 2H, Hb), 7.74 (s, 1H, H8), 3.76 (s, 3H, NCH3); 13
C
NMR (100 MHz, DMSO-d6)
d
162.27 (NHeC]O), 156.54 (C3eC]O),
larity) and recrystallization from EtOH provided 8 (381 mg, 59%
150.91 (C8a), 150.69 (C5), 150.21 (Cd), 144.96 (C7), 140.29 (Ca), 133.29
(C3), 131.50 (2Cb), 125.86 (C4a), 124.29 (2Cc), 114.41 (C4), 109.17 (C8),
30.00 (NCH3); MS (ESI) m/z (%): 359.1 (100) [M þ H]þ, 361.1 (40)
[M þ H þ 2]þ; UPLC purity ¼ 97%, Rt 3.09 min; HRMS (ESI): calcd.
for C16H11ClN4O4 [M þ H]þ 359.0542, found: 359.0527.
yield) as a brown powder. Mp: 254e255 ꢀC; IR,
n
(cmꢁ1): 3335
(
n
NeH); 3063 (
n
CeHar); 1682, 1643 (
n
C ¼ O); 1574, 1512 (
n
C ¼ C);
766 (
n
CeCl); 1H NMR (400 MHz, DMSO-d6)
d
9.94 (s, 1H, NH), 8.90
(s, 1H, H5), 8.80 (s, 1H, H4), 7.70e7.65 (m, 3H, Hb, He), 7.61 (s, 1H, H8),
7.49e7.45 (m, 4H, Ha, Hd, Hf), 3.73 (s, 3H, NCH3); 13C NMR (100 MHz,
DMSO-d6)
d
164.90 (NHeC]O), 157.36 (C3eC]O), 149.34 (C5),
4.1.2.5. N-(7-Chloro-1-methyl-2-oxo-1,2-dihydro-1,6-naphthyridin-
148.60 (C8a), 142.42 (C7), 141.13 (Cb), 134.69 (Cc), 129.94 (Cf), 129.29
(C3), 128.94 (2Ce), 127.93 (2Cd), 122.06 (Ca), 117.12 (C4), 116.05 (C4a),
108.40 (C8), 30.15 (NCH3); MS (ESI) m/z (%): 340.2 (100) [M þ H]þ,
342.2 (40) [M þ H þ 2]þ; UPLC purity ¼ 96%, Rt 3.30 min; HRMS
(ESI): calcd. for C18H14ClN3O2 [M þ H]þ 340.0847, found: 340.0835.
3-yl)-4-hydroxybenzamide (11).
c
d
OH
b
H
N
5
8
4
4a
8a
3
a
N
4.1.2.3. N-(7-Chloro-1-methyl-2-oxo-1,2-dihydro-1,6-naphthyridin-
3-yl)benzamide (9).
O
7
O
N
Cl
c
d
b
H
N
5
8
4
The reaction was carried out using amine 6 (400 mg, 1.91 mmol)
and 4-(acetyloxy)benzoyl chloride [32] (1.25 g, 6.30 mmol) and
refluxed in pyridine for 5 h. After concentration under reduced
pressure, NaOH (108 mg, 2.69 mmol, 1.4 equiv.) was added to a
stirred solution of the crude product in MeOH/water 1:1 (30 mL).
After 2 h at room temperature, the reaction mixture was acidified to
pH 5 with concentrated HCl. The precipitate was collected by
filtration and rinsed with cold EtOH to afford 11 (394 mg, 63% yield,
4a
8a
3
a
N
O
7
O
N
Cl
The reaction was carried out using amine 6 (400 mg, 1.91 mmol)
and benzoyl chloride (731 L, 6.30 mmol) and refluxed in pyridine
for 3 h. Purification by silica gel chromatography (dichloro-
2 steps) as a yellow powder. Mp: 325e326 ꢀC; IR,
NeH, OeH); 1628, 1574 ( C ¼ C); 756 (
C ¼ O); 1504 (
NMR (400 MHz, DMSO-d6)
n
(cmꢁ1): 3264
m
(n
n
n
n
n
CeCl); 1H
methane/ethyl acetate, 99.5:0.5) and recrystallization from EtOH
d
10.35 (s, 1H, OH), 9.41 (s, 1H, NH), 8.85
(s, 1H, H5), 8.81 (s, 1H, H4), 7.87 (d, 3J ¼ 8.5 Hz, 2H, Hb), 7.71 (s, 1H,
provided
225e226 ꢀC; IR,
1518 (
9
(425 mg, 71% yield) as
a
white powder. Mp:
C ¼ O); 1578,
CeCl); 1H NMR (400 MHz, DMSO-d6)
n d 9.61 (s,
H8), 6.96 (d, 3J ¼ 8.5 Hz, 2H, Hc), 3.76 (s, 3H, NCH3); 13C NMR
n
(cmꢁ1): 3380 (
n
NeH); 1672, 1649 (n
n
C ¼ C); 704 (
(100 MHz, DMSO-d6)
d 164.77 (NHeC]O), 161.33 (Cd), 157.71
1H, NH), 8.86 (s, 1H, H5), 8.83 (s, 1H, H4), 7.99 (d, 3J ¼ 7.6 Hz, 2H, Hb),
(C3eC]O), 149.43 (C5), 148.67 (C8a), 142.34 (C7), 129.38 (2Cb),
128.74 (C3), 123.90 (Ca), 116.74 (C4), 116.04 (C4a), 115.48 (2Cc), 108.67
(C8), 30.26 (NCH3); MS (ESI) m/z (%): 330.1 (100) [M þ H]þ, 332.1
(40) [M þ H þ 2]þ; UPLC purity ¼ 98%, Rt 2.52 min; HRMS (ESI):
calcd. for C16H12ClN3O3 [M þ H]þ 330.0640, found: 330.0635.
7.71 (s, 1H, H8), 7.70e7.60 (m, 3H, Hc, Hd), 3.75 (s, 3H, NCH3); 13C
NMR (100 MHz, DMSO-d6)
d 165.33 (NHeC]O), 157.64 (C3eC]O),
149.59 (C5), 148.92 (C8a), 142.57 (C7), 133.47 (Ca), 132.40 (Cd), 128.90
(2Cc), 128.54 (C3), 127.29 (2Cb), 117.75 (C4), 115.89 (C4a), 108.70 (C8),
30.29 (NCH3); MS (ESI) m/z (%): 314.1 (100) [M þ H]þ, 316.1 (40)
[M þ H þ 2]þ; UPLC purity ¼ 98%, Rt 3.07 min; HRMS (ESI): calcd.
for C16H12ClN3O2 [M þ H]þ 314.0691, found: 314.0678.
4.1.2.6. N-(7-Chloro-1-methyl-2-oxo-1,2-dihydro-1,6-naphthyridin-
3-yl)-4-methoxybenzamide (12).
c
4.1.2.4. N-(7-Chloro-1-methyl-2-oxo-1,2-dihydro-1,6-naphthyridin-
3-yl)-4-nitrobenzamide (10).
d
O
b
H
N
5
8
4
4a
8a
3
c
a
d
NO2
N
b
O
H
N
5
8
4
7
O
Cl
N
4a
8a
3
a
N
O
7
N
O
Cl
The reaction was carried out using amine 6 (400 mg, 1.91 mmol)
and 4-methoxybenzoyl chloride (1.08 g, 6.30 mmol) and refluxed in
pyridine for
(dichloromethane/ethyl acetate, 99:5) provided 12 (499 mg, 76%
yield) as a white powder. Mp: 229e230 ꢀC; IR, (cmꢁ1): 3366
C ¼ O); 1580, 1456
9.49 (s, 1H,
4 h. Purification by silica gel chromatography
The reaction was carried out using amine 6 (400 mg, 1.91 mmol)
and 4-nitrobenzoyl chloride (1.17 g, 6.30 mmol) and refluxed in
pyridine for 1.5 h. After cooling, the precipitate was collected by
filtration and rinsed with cold water to afford 10 (541 mg, 79%
n
(
(
n
n
NeH); 2914, 2837 (nCeHal); 1653, 1639 (n
C ¼ C); 682 (
n d
CeCl); 1H NMR (400 MHz, DMSO-d6)
yield) as a pale yellow powder. Mp: 348e349 ꢀC; IR,
n
(cmꢁ1): 3385
C ¼ C); 1512 (nasNO2), 1236
NH), 8.85 (s, 1H, H5), 8.81 (s, 1H, H4), 7.97 (d, 3J ¼ 8.6 Hz, 2H, Hb),
7.71 (s, 1H, H8), 7.14 (d, 3J ¼ 8.6 Hz, 2H, Hc), 3.89 (s, 3H, OCH3), 3.75
(
n
NeH); 1738, 1639 (
n
C ¼ O); 1587 (
n
(nsyNO2); 710 (
n
CeCl); 1H NMR (400 MHz, DMSO-d6)
d
10.04 (s, 1H,
(s, 3H, NCH3); 13C NMR (100 MHz, DMSO-d6)
d 164.64 (NHeC]O),
162.49 (Cd), 157.67 (C3eC]O), 149.47 (C5), 148.75 (C8a), 142.40 (C7),
NH), 8.90 (s, 1H, H5), 8.86 (s, 1H, H4), 8.43 (d, 3J ¼ 8.8 Hz, 2H, Hc),

24
D. Montoir et al. / European Journal of Medicinal Chemistry 119 (2016) 17e33
129.27 (2Cb), 128.65 (C3), 125.49 (Ca), 117.14 (C4), 115.97 (C4a), 114.11
(2Cc), 108.66 (C8), 55.50 (OCH3), 30.25 (NCH3); MS (ESI) m/z (%):
344.1 (100) [M þ H]þ, 346.1 (40) [M þ H þ 2]þ; UPLC purity ¼ 99%,
Rt 3.12 min; HRMS (ESI): calcd. for C17H14ClN3O3 [M þ H]þ
344.0796, found: 344.0796.
(ESI): calcd. for C19H18ClN3O5 [M þ H]þ 404.1008, found: 404.1014.
4.1.2.9. N-(7-Chloro-1-methyl-2-oxo-1,2-dihydro-1,6-naphthyridin-
3-yl)biphenyl-3-carboxamide (15).
e
d
c
f
4.1.2.7. N-(7-Chloro-1-methyl-2-oxo-1,2-dihydro-1,6-naphthyridin-
3-yl)-3-methoxybenzamide (13).
h
H
N
5
8
4
g
4a
8a
3
i
a
N
b
e
d
f
j
O
7
N
O
Cl
H
N
5
8
4
4a
8a
3
a
c
O
N
b
O
7
Cl
N
O
The reaction was carried out using amine 6 (400 mg, 1.91 mmol)
and biphenyl-3-carbonyl chloride [33] (1.37 g, 6.30 mmol) and
refluxed in pyridine for 1 h. Purification by silica gel chromatog-
raphy (mixtures of dichloromethane/methanol of increasing po-
larity) provided 15 (626 mg, 84% yield) as a white powder. Mp:
The reaction was carried out using amine 6 (400 mg, 1.91 mmol)
and 3-methoxybenzoyl chloride (1.10 g, 6.30 mmol) and refluxed in
pyridine for 1.5 h. Purification by silica gel chromatography
(dichloromethane/ethyl acetate, 98:2) provided 13 (365 mg, 76%
213e214 ꢀC; IR,
C ¼ O); 1578, 1489 (
DMSO-d6) 9.79 (s,1H, NH), 8.85 (s,1H, H5), 8.82 (s,1H, H4), 8.20 (br
n
(cmꢁ1): 3300 (
C ¼ C); 766 (
n
NeH); 2969 (
nCeHal); 1771, 1636
(n
n
n
CeCl); 1H NMR (400 MHz,
yield) as a pale yellow powder. Mp: 195e196 ꢀC; IR,
NeH); 2941( CeHal); 1652, 1632 (
C ¼ O); 1574, 1494 (
765 ( 9.58 (s, 1H, NH), 8.85
CeCl); 1H NMR (400 MHz, DMSO-d6)
n
(cmꢁ1): 3375
C ¼ C);
d
s, 1H, Hb), 7.97e7.95 (m, 2H, Hd, Hf), 7.80e7.70 (m, 4H, H8, He, Hh),
7.56 (appt, appJ ¼ 7.2 Hz, 2H, Hi), 7.47 (t, 3J ¼ 6.6 Hz, 1H, Hj), 3.74 (s,
(n
n
n
n
n
d
3H, NCH3); 13C NMR (100 MHz, DMSO-d6)
d 165.61 (NHeC]O),
(s, 1H, H5), 8.80 (s, 1H, H4), 7.71 (s, 1H, H8), 7.54e7.53 (m, 2H, Hb, Hf),
7.49e7.48 (m,1H, He), 7.27e7.24 (m,1H, Hd), 3.89 (s, 3H, OCH3), 3.74
157.84 (C3eC]O),149.79 (C5),149.17 (C8a), 142.83 (C7), 140.84 (Cc or
Cg), 139.45 (Cg or Cc), 134.48 (Ca), 130.75 (Cd), 129.69 (Ce), 129.24
(2Ci), 128.80 (C3), 128.14 (Cj), 127.09 (2Ch), 126.60 (Cb), 125.82 (Cf),
118.47 (C4), 116.04 (C4a), 108.85 (C8), 30.41 (NCH3); MS (ESI) m/z (%):
390.1 (100) [M þ H]þ, 392.1 (40) [M þ H þ 2]þ; UPLC purity ¼ 96%,
Rt 3.88 min; HRMS (ESI): calcd. for C22H16ClN3O2 [M þ H]þ
390.1004, found: 390.0999.
(s, 3H, NCH3); 13C NMR (100 MHz, DMSO-d6)
d 165.10 (NHeC]O),
159.14 (Cc), 157.62 (C3eC]O), 149.59 (C5), 148.94 (C8a), 142.59 (C7),
134.94 (Ca), 130.09 (Ce), 128.37 (C3), 119.24 (Cb), 117.95 (C4), 117.83
(Cd), 115.84 (C4a), 112.59 (Cf), 108.70 (C8), 55.35 (OCH3), 30.34
(NCH3); MS (ESI) m/z (%): 344.1 (100) [M þ H]þ, 346.1 (40)
[M þ H þ 2]þ; UPLC purity ¼ 96%, Rt 3.13 min; HRMS (ESI): calcd.
for C17H14ClN3O3 [M þ H]þ 344.0796, found: 344.0782.
4.1.2.10. 4-Chloro-N-(7-chloro-1-methyl-2-oxo-1,2-dihydro-1,6-
naphthyridin-3-yl)benzamide (16).
4.1.2.8. N-(7-Chloro-1-methyl-2-oxo-1,2-dihydro-1,6-naphthyridin-
3-yl)-3,4,5-trimethoxybenzamide (14).
c
d
Cl
b
O
c
H
N
5
8
4
O
O
d
4a
8a
3
b
a
N
H
N
5
8
4
4a
8a
3
O
a
N
7
O
N
Cl
O
7
O
N
Cl
The reaction was carried out using amine 6 (400 mg, 1.91 mmol)
and 4-chlorobenzoyl chloride (1.10 g, 6.30 mmol) and refluxed in
The reaction was carried out using amine 6 (400 mg, 1.91 mmol)
and 3,4,5-trimethoxybenzoyl chloride (1.45 g, 6.30 mmol) and
refluxed in pyridine for 3 h. Purification by silica gel chromatog-
raphy (mixtures of dichloromethane/ethanol of increasing polarity)
provided 14 (444 mg, 58% yield) as a white powder. Mp:
pyridine for
(dichloromethane) and trituration with acetonitrile afforded 16
(350 mg, 53% yield) as a white powder. Mp: 278e279 ꢀC; IR,
(cmꢁ1): 3379 (
NeH); 1686, 1636 (
C ¼ O); 1580, 1518, 1483
3 h. Purification by silica gel chromatography
n
n
n
249e250 ꢀC; IR,
1639 (
C ¼ O); 1584, 1489 (
(400 MHz, DMSO-d6) 9.63 (s, 1H, NH), 8.86 (s, 1H, H5), 8.77 (s, 1H,
n
(cmꢁ1): 3674 (
n
NeH); 2963, 2901 (
n
CeHal); 1717,
(n
C ¼ C); 743, 606 (
n d 9.72 (s,
CeCl); 1H NMR (400 MHz, DMSO-d6)
n
nC ¼ C); 630 (
n
CeCl); 1H NMR
1H, NH), 8.86 (s, 1H, H5), 8.81 (s, 1H, H4), 8.01 (d, 3J ¼ 8.2 Hz, 2H, Hc),
d
7.71 (s, 1H, H8), 7.68 (d, 3J ¼ 8.2 Hz, 2H, Hb), 3.75 (s, 3H, NCH3); 13
C
H4), 7.73 (s, 1H, H8), 7.29 (s, 2H, Hb), 3.92 (s, 6H, 2CceOCH3), 3.79 (s,
NMR (100 MHz, DMSO-d6) d 164.56 (NHeC]O), 157.62 (C3eC]O),
3H, CdeOCH3), 3.76 (s, 3H, NCH3); 13C NMR (100 MHz, DMSO-d6)
149.56 (C5), 149.02 (C8a), 142.72 (C7), 137.15 (Cd), 132.30 (Ca), 129.40
(2Cc), 128.87 (2Cb), 128.51 (C3), 118.42 (C4), 115.82 (C4a), 108.71 (C8),
30.24 (NCH3); MS (ESI) m/z (%): 348.0 (100) [M þ H]þ, 350.0 (65)
[M þ H þ 2]þ, 352.0 (10) [M þ H þ 4]þ; UPLC purity ¼ 98%, Rt
3.44 min; HRMS (ESI): calcd. for C16H11Cl2N3O2 [M þ H]þ 348.0301,
found: 348.0287.
d
165.07 (NHeC]O), 157.69 (C3eC]O), 152.84 (2Cc), 149.56 (C5),
148.97 (C8a), 142.69 (C7), 140.91 (Cd), 128.91 (Ca), 128.64 (C3), 118.50
(C4), 115.85 (C4a), 108.69 (C8), 104.97 (2Cb), 60.13 (CdeOCH3), 56.09
(2CceOCH3), 30.21 (NCH3); MS (ESI) m/z (%): 404.1 (100) [M þ H]þ,
406.1 (40) [M þ H þ 2]þ; UPLC purity ¼ 97%, Rt 3.02 min; HRMS

D. Montoir et al. / European Journal of Medicinal Chemistry 119 (2016) 17e33
25
4.1.2.11. N-(7-Chloro-1-methyl-2-oxo-1,2-dihydro-1,6-naphthyridin-
4.1.3. Typical procedure for the synthesis of carboxamides 19e45
3-yl)pyridine-4-carboxamide (17).
In a 10 mL vessel were introduced amide 11 (75 mg, 0.22 mmol)
and N,N-dimethylethylenediamine (0.47 mL, 4.36 mmol). The tube
was sealed, and the reaction mixture was heated under microwave
irradiation at 130 ꢀC for 6 h. After cooling, water was added, and the
mixture was extracted with CH2Cl2. The organic layer was washed
with brine, dried over Na2SO4, filtered, and concentrated under
reduced pressure. The crude product was purified by silica gel
chromatography (mixtures of dichloromethane/methanol of
increasing polarity) and trituration with diisopropyl ether provided
35 (44 mg, 50% yield) as a pale yellow powder.
c
b
N
H
N
5
8
4
4a
8a
3
a
N
O
7
O
N
Cl
4.1.3.1. N-(1-Methyl-7-{[2-(morpholin-4-yl)ethyl]amino}-2-oxo-1,2-
dihydro-1,6-naphthyridin-3-yl)acetamide (19).
The reaction was carried out using amine 6 (400 mg, 1.91 mmol)
and isonicotinoyl chloride hydrochloride (1.12 g, 6.30 mmol) and
refluxed in pyridine for 2 h. After cooling, water was added, and the
mixture was extracted with CH2Cl2. The organic layer was washed
with brine, dried over Na2SO4, filtered, and concentrated under
reduced pressure. The precipitate was collected by filtration and
rinsed with cold water to afford 17 (239 mg, 39% yield) as a yellow
H
N
5
4
d
4a
8a
3
c
O
N
a
O
N
d
7
N
O
N
c
b
8
H
powder. Mp: 308e309 ꢀC; IR,
n
(cmꢁ1): 3352 (
C ¼ O); 1582, 1518, 1491 (
n
NeH); 2961, 2901
C ¼ C); 743
d 9.95 (s, 1H, NH), 8.87 (s,
(
n
CeHal); 1717, 1638 (
n
n
(n
CeCl); 1H NMR (400 MHz, DMSO-d6)
The reaction was carried out using amide 7 (75 mg, 0.30 mmol)
and 2-(morpholin-4-yl)ethanamine (789
L, 6.00 mmol) at 170 ꢀC
1H, H5), 8.84 (d, 3J ¼ 6.0 Hz, 2H, Hc), 8.83 (s, 1H, H4), 7.88 (d,
m
3J ¼ 6.0 Hz, 2H, Hb), 7.72 (s, 1H, H8), 3.74 (s, 3H, NCH3); 13C NMR
for 1 h. Purification by silica gel chromatography (mixtures of
dichloromethane/methanol of increasing polarity) and trituration
with diisopropyl ether afforded 19 (20 mg, 19% yield) as beige
(100 MHz, DMSO-d6)
d 164.40 (NHeC]O), 157.53 (C3eC]O),
150.52 (2Cc), 149.79 (C5), 149.24 (C8a), 142.94 (C7), 140.63 (Ca),
128.29 (C3), 121.29 (2Cb), 119.37 (C4), 115.69 (C4a), 108.73 (C8), 30.23
(NCH3); MS (ESI) m/z (%): 315.1 (100) [M þ H]þ, 317.1 (40)
[M þ H þ 2]þ; UPLC purity ¼ 98%, Rt 2.03 min; HRMS (ESI): calcd.
for C15H11ClN4O2 [M þ H]þ 315.0643, found: 315.0635.
powder. Mp: 203e204 ꢀC; IR,
CeHal); 1620 (
C ¼ O); 1589, 1514 (
CDCl3)
n nNeH); 2970
(cmꢁ1): 3291, 3237 (
(n
n
n
C ¼ C); 1H NMR (400 MHz,
d
8.61 (s, 1H, H4), 8.36 (s, 1H, H5), 8.33 (s, 1H, C3eNH), 6.06 (s,
1H, H8), 5.44 (br s, 1H, C7eNH), 3.74 (t, 3J ¼ 4.4 Hz, 4H, Hd), 3.66 (s,
3H, NCH3), 3.42e3.37 (m, 2H, Ha), 2.68 (t, 3J ¼ 5.6 Hz, 2H, Hb),
2.54e2.49 (m, 4H, Hc), 2.22 (s, 3H, COCH3); 13C NMR (100 MHz,
4.1.2.12. N-(7-Chloro-1-methyl-2-oxo-1,2-dihydro-1,6-naphthyridin-
3-yl)-1-benzothiophene-2-carboxamide (18).
CDCl3)
d 168.90 (NHeC]O), 158.82 (C7), 158.53 (C3eC]O), 149.86
(C5), 143.18 (C8a), 124.03 (C3), 119.60 (C4), 109.55 (C4a), 87.66 (C8),
66.85 (2Cd), 56.87 (Cb), 53.30 (2Cc), 38.38 (Ca), 29.78 (NCH3), 24.78
d
e
c
H]þ; UPLC
f
(COCH3); MS (ESI) m/z (%): 346.1 (100) [M
purity ¼ 98%, Rt 1.15 min; HRMS (ESI): calcd. for C17H23N5O3
þ
b
H
N
5
8
4
[M þ H]þ 346.1874, found: 346.1873.
g
h
4a
8a
3
a
S
N
4.1.3.2. N-(7-{[2-(dimethylamino)ethyl]amino}-1-methyl-2-oxo-1,2-
dihydro-1,6-naphthyridin-3-yl)acetamide (20).
O
7
O
Cl
N
H
N
5
4
4a
8a
3
c
The reaction was carried out using amine 6 (400 mg, 1.91 mmol)
and benzo[b]thiophene-2-carbonyl chloride (1.24 g, 6.30 mmol)
and refluxed in pyridine for 3 h. After cooling, water was added, and
the mixture was extracted with CH2Cl2. The organic layer was
washed with brine, dried over Na2SO4, filtered, and concentrated
under reduced pressure. The precipitate was collected by filtration
and trituration with CH3CN afforded 18 (234 mg, 33% yield) as a
N
a
O
N
7
N
O
N
b
8
H
The reaction was carried out using amide 7 (75 mg, 0.30 mmol),
yellow powder. Mp: 287e288 ꢀC; IR,
1580 ( C ¼ C); 617 (
C ¼ O); 1525 (
DMSO-d6) 9.85 (s, 1H, NH), 8.87 (s, 1H, H5), 8.78 (s, 1H, H4), 8.48 (s,
1H, Hb), 8.13e8.07 (m, 2H, Hd, Hg), 7.73 (s, 1H, H8), 7.59e7.51 (m, 2H,
He, Hf), 3.77 (s, 3H, NCH3); 13C NMR (100 MHz, DMSO-d6)
160.57
n
n
(cmꢁ1): 3354 (
n
NeH); 1643,
N,N-dimethylethylenediamine (646 mL, 6.00 mmol) and CuI (28 mg,
n
n
CeCl); 1H NMR (400 MHz,
0.15 mmol) at 130 ꢀC for 5 h. Purification by silica gel chromatog-
raphy (mixtures of dichloromethane/methanol of increasing po-
larity) afforded 20 (44 mg, 48% yield) as a beige powder. Mp:
d
d
132e133 ꢀC; IR,
C ¼ O); 1587, 1504 (
n nNeH); 2970 (nCeHal); 1639
(cmꢁ1): 3292, 3227 (
(NHeC]O), 157.51 (C3eC]O), 149.67 (C5), 149.08 (C8a), 142.76 (C7),
140.71 (Ca), 139.07 (Ch), 137.92 (Cc), 128.19 (C3), 127.08 (Cb), 126.97
(Ce or Cf), 125.82 (Cd or Cg), 125.21 (Cf or Ce),122.90 (Cg or Cd), 118.75
(C4), 115.79 (C4a), 108.73 (C8), 30.27 (NCH3); MS (ESI) m/z (%): 370.1
(100) [M þ H]þ, 372.0 (40) [M þ H þ 2]þ; UPLC purity ¼ 95%, Rt
3.65 min; HRMS (ESI): calcd. for C18H12ClN3O2S [M þ H]þ 370.0412,
found: 370.0398.
(n
n
C ¼ C); 1H NMR (400 MHz, CDCl3)
d 8.60 (s,
1H, H4), 8.35 (s, 1H, H5), 8.34 (s, 1H, C3eNH), 6.10 (s,1H, H8), 5.34 (br
s, 1H, C7eNH), 3.64 (s, 3H, CONCH3), 3.45e3.41 (m, 2H, Ha), 2.64 (t,
3J ¼ 5.8 Hz, 2H, Hb), 2.33 (s, 6H, Hc), 2.22 (s, 3H, COCH3); 13C NMR
(100 MHz, CDCl3) d 168.89 (NHeC]O), 158.84 (C7), 158.58 (C3eC]
O), 149.77 (C5), 143.09 (C8a), 123.97 (C3), 119.64 (C4), 109.52 (C4a),
88.14 (C8), 57.77 (Cb), 45.02 (2Cc), 38.32 (Ca), 29.74 (CONCH3), 24.77

26
D. Montoir et al. / European Journal of Medicinal Chemistry 119 (2016) 17e33
(COCH3); MS (ESI) m/z (%): 304.2 (100) [M
þ
H]þ; UPLC
(Ca), 120.07 (C4), 109.71 (C4a), 88.55 (C8), 57.93 (Ch), 45.10 (2Ci),
39.10 (Cg), 29.79 (CONCH3); MS (ESI) m/z (%): 392.1 (100) [M þ H]þ;
UPLC purity ¼ 95%, Rt 2.46 min; HRMS (ESI): calcd. for C22H25N5O2
[M þ H]þ 392.2081, found: 392.2077.
purity ¼ 96%, Rt 1.17 min; HRMS (ESI): calcd. for C15H21N5O2
[M þ H]þ 304.1768, found: 304.1767.
4.1.3.3. (2E)-N-(1-Methyl-7-{[2-(morpholin-4-yl)ethyl]amino}-2-
oxo-1,2-dihydro-1,6-naphthyridin-3-yl)-3-phenylprop-2-enamide
(21).
4.1.3.5. N-(1-Methyl-7-{[2-(morpholin-4-yl)ethyl]amino}-2-oxo-1,2-
dihydro-1,6-naphthyridin-3-yl)benzamide (23).
e
c
f
d
d
b
j
a
H
N
H
N
h
5
8
4
5
8
4
4a
8a
3
4a
8a
3
i
g
a
c
O
N
O
N
b
g
e
O
O
N
N
7
7
O
N
O
N
N
N
h
f
H
H
The reaction was carried out using amide 8 (75 mg, 0.22 mmol)
and 2-(morpholin-4-yl)ethanamine (579
L, 4.40 mmol) at 150 ꢀC
The reaction was carried out using amide 9 (75 mg, 0.24 mmol)
and 2-(morpholin-4-yl)ethanamine (631
L, 4.80 mmol) at 170 ꢀC
m
m
for 4 h. Purification by silica gel chromatography (mixtures of
dichloromethane/methanol of increasing polarity) afforded 21
for 1 h. Purification by silica gel chromatography (mixtures of
dichloromethane/methanol of increasing polarity) afforded 23
(36 mg, 38% yield) as a yellow powder. Mp: 231e232 ꢀC; IR,
n
(91 mg, 93% yield) as a beige powder. Mp: 168e169 ꢀC; IR,
n
(cmꢁ1):
C ¼ O); 1591, 1514
d 9.20 (s, 1H, C3eNH), 8.82 (s,
(cmꢁ1): 3294 (
n
NeH); 2922 (
n
CeHal); 1622 (
n
C ¼ O); 1589, 1520
3663 (nNeH); 2986, 2970 (nC-Hal); 1632 (n
C ¼ C); 1H NMR (400 MHz, CDCl3)
d 8.77 (s, 1H, H4), 8.55 (s, 1H,
(
n
C ¼ C); 1H NMR (400 MHz, CDCl3)
(n
C3eNH), 8.38 (s, 1H, H5), 7.72 (d, 3J ¼ 15.6 Hz, 1H, Hb), 7.56 (dd,
1H, H4), 8.41 (s, 1H, H5), 7.94 (d, 3J ¼ 7.2 Hz, 2H, Hb), 7.58e7.49 (m,
3H, Hc, Hd), 6.08 (s, 1H, H8), 5.48 (t, 3J ¼ 4.8 Hz, 1H, C7eNH), 3.75 (t,
3J ¼ 4.6 Hz, 4H, Hh), 3.70 (s, 3H, NCH3), 3.43e3.39 (m, 2H, He), 2.68
3J ¼ 8.0 Hz, J ¼ 2.4 Hz, 2H, He), 7.40e7.38 (m, 3H, Hd, Hf), 6.61 (d,
4
3J ¼ 15.6 Hz, 1H, Ha), 6.06 (s, 1H, H8), 5.51 (br s, 1H, C7eNH), 3.74 (t,
3J ¼ 4.4 Hz, 4H, Hj), 3.68 (s, 3H, NCH3), 3.42e3.38 (m, 2H, Hg), 2.68
(t, J ¼ 6.0 Hz, 2H, Hf), 2.53e2.50 (m, 4H, Hg); 13C NMR (100 MHz,
3
(t, J ¼ 5.8 Hz, 2H, Hh), 2.53e2.49 (m, 4H, Hi); 13C NMR (100 MHz,
3
CDCl3)
d 165.71 (NHeC]O), 159.13 (C3eC]O), 158.64 (C7), 150.02
CDCl3)
d 164.34 (NHeC]O), 158.90 (C3eC]O), 158.59 (C7), 150.03
(C5), 143.28 (C8a), 134.32 (Ca), 132.07 (Cd), 128.83 (2Cc), 127.14 (2Cb),
124.17 (C3), 119.85 (C4), 109.72 (C4a), 87.85 (C8), 66.97 (2Ch), 56.93
(Cf), 53.38 (2Cg), 38.61 (Ce), 29.94 (NCH3); MS (ESI) m/z (%): 408.1
(100) [M þ H]þ; UPLC purity ¼ 95%, Rt 2.03 min; HRMS (ESI): calcd.
for C22H25N5O3 [M þ H]þ 408.2030, found: 408.2040.
(C5), 143.23 (C8a), 142.32 (Cb), 134.58 (Cc), 130.07 (Cf), 128.92 (2Ce),
128.05 (2Cd), 124.16 (C3), 120.71 (Ca), 120.09 (C4), 109.63 (C4a), 87.56
(C8), 66.90 (2Cj), 56.84 (Ch), 53.31 (2Ci), 38.43 (Cg), 29.83 (NCH3);
MS (ESI) m/z (%): 435.2 (100) [M þ H]þ; UPLC purity ¼ 94%, Rt
2.49 min; HRMS (ESI): calcd. for C24H27N5O3 [M þ H]þ 434.2187,
found: 434.2201.
4.1.3.6. N-(7-{[2-(dimethylamino)ethyl]amino}-1-methyl-2-oxo-1,2-
dihydro-1,6-naphthyridin-3-yl)benzamide (24).
4.1.3.4. (2E)-N-(7-{[2-(dimethylamino)ethyl]amino}-1-methyl-2-
oxo-1,2-dihydro-1,6-naphthyridin-3-yl)-3-phenylprop-2-enamide
(22).
c
d
b
a
H
N
5
8
4
4a
8a
3
e
g
N
f
d
e
O
N
a
H
N
5
8
4
7
O
N
N
4a
8a
3
i
f
c
H
N
b
g
O
N
7
O
N
N
The reaction was carried out using amide 9 (75 mg, 0.24 mmol)
and N,N-dimethylethylenediamine (517
L, 4.80 mmol) at 130 ꢀC
h
H
m
for 6 h. Purification by silica gel chromatography (mixtures of
dichloromethane/methanol of increasing polarity) afforded 24
The reaction was carried out using amide 8 (75 mg, 0.22 mmol)
and N,N-dimethylethylenediamine (474
L, 4.40 mmol) at 130 ꢀC
for 3 h. Purification by silica gel chromatography (mixtures of
dichloromethane/methanol of increasing polarity) afforded 22
m
(58 mg, 66% yield) as a yellow powder. Mp: 145e146 ꢀC; IR,
n
(cmꢁ1): 3383, 3250 (
n
NeH); 2978, 2949 (
n
CeHal); 1641 (
n
C ¼ O);
1593, 1518 (
n
C ¼ C); 1H NMR (400 MHz, CDCl3)
d 9.20 (s, 1H,
(21 mg, 24% yield) as a yellow powder. Mp: 161e162 ꢀC; IR,
n
C3eNH), 8.81 (s, 1H, H4), 8.41 (s, 1H, H5), 7.94 (d, 3J ¼ 6.8 Hz, 2H, Hb),
7.58e7.48 (m, 3H, Hc, Hd), 6.12 (s, 1H, H8), 5.50 (s, 1H, C7eNH), 3.68
(s, 3H, CONCH3), 3.44e3.40 (m, 2H, He), 2.62 (t, 3J ¼ 6.0 Hz, 2H, Hf),
(cmꢁ1): 3354 (
n
NeH); 2922 (
n
CeHal); 1649 (
n
C ¼ O); 1587, 1514
(n
C ¼ C); 1H NMR (400 MHz, CDCl3)
d 8.76 (s, 1H, H4), 8.54 (s, 1H,
C3eNH), 8.38 (s, 1H, H5), 7.73 (d, 3J ¼ 15.6 Hz, 1H, Hb), 7.56 (dd,
3J ¼ 6.0 Hz, 4J ¼ 2.4 Hz, 2H, He), 7.41e7.36 (m, 3H, Hd, Hf), 6.61 (d,
3J ¼ 15.6 Hz, 1H, Ha), 6.15 (s, 1H, H8), 5.67 (br s, 1H, C7eNH), 3.66 (s,
3H, CONCH3), 3.52e3.57 (m, 2H, Hg), 2.71 (t, 3J ¼ 5.6 Hz, 2H, Hh),
2.31 (s, 6H, Hg); 13C NMR (100 MHz, CDCl3)
d 165.62 (NHeC]O),
159.11 (C3eC]O), 158.73 (C7), 149.98 (C5), 143.16 (C8a), 134.33 (Ca),
132.05 (Cd), 128.81 (2Cc), 127.11 (2Cb), 124.04 (C3), 119.90 (C4),
109.52 (C4a), 88.04 (C8), 57.73 (Cf), 45.12 (2Cg), 39.49 (Ce), 29.76
(CONCH3); MS (ESI) m/z (%): 366.2 (100) [M þ H]þ; UPLC
purity ¼ 98%, Rt 1.93 min; HRMS (ESI): calcd. for C20H23N5O2
[M þ H]þ 366.1925, found: 366.1921.
2.38 (s, 6H, Hi); 13C NMR (100 MHz, CDCl3)
d 164.37 (NHeC]O),
158.90 (C3eC]O), 158.50 (C7), 149.91 (C5), 143.13 (C8a), 142.28 (Cb),
134.59 (Cc),130.09 (Cf),129.01 (2Ce),128.10 (2Cd),124.11 (C3),120.76

D. Montoir et al. / European Journal of Medicinal Chemistry 119 (2016) 17e33
27
4.1.3.7. 4-Amino-N-(1-methyl-7-{[2-(morpholin-4-yl)ethyl]amino}-
4.1.3.9. N-(1-Methyl-7-{[2-(morpholin-4-yl)ethyl]amino}-2-oxo-1,2-
2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl)benzamide (25).
dihydro-1,6-naphthyridin-3-yl)-4-hydroxybenzamide (27).
c
c
d
d
OH
NH2
b
b
a
H
N
H
h
h
5
8
4
5
8
4
a
g
N
4a
8a
3
4a
8a
3
g
O
N
O
N
e
e
O
O
N
N
7
7
N
O
O
N
N
N
f
f
H
H
The reaction was carried out using amide 11 (75 mg, 0.23 mmol)
and 2-(morpholin-4-yl)ethanamine (605
L, 4.60 mmol) at 170 ꢀC
The reaction was carried out using amide 10 (75 mg,
m
0.21 mmol) and 2-(morpholin-4-yl)ethanamine (552
mL,
for 1.5 h. The reaction mixture was adjusted to pH 7 with a 1 M HCl
solution. The precipitate was collected by filtration, rinsed with
cold water and purified by silica gel chromatography (mixtures of
dichloromethane/methanol of increasing polarity). Trituration with
diisopropyl ether afforded 27 (49 mg, 53% yield) as a yellow pow-
4.20 mmol) at 170 ꢀC for 1 h. Purification by silica gel chroma-
tography (mixtures of dichloromethane/methanol of increasing
polarity) and trituration with diisopropyl ether afforded 25
(55 mg, 62% yield) as a yellow powder. Mp: 250e251 ꢀC; IR,
n
(cmꢁ1): 3383, 3350, 3240 (
n
NeH); 2968 (
n
CeHal); 1627 (
n
C ¼ O);
der. Mp: 232e233 ꢀC; IR,
CeHal); 1591, 1578 (
C ¼ O); 1528, 1503, 1445 (
(400 MHz, DMSO-d6)
n nNeН, nΟeН); 2943
(cmꢁ1): 3229 (
1587, 1508 (
n
C ¼ C); 1H NMR (400 MHz, DMSO-d6)
d 9.04 (s, 1H,
(n
n
d
n
C ¼ C); 1H NMR
C3eNH), 8.56 (s, 1H, H4), 8.42 (s, 1H, H5), 7.66 (d, 3J ¼ 8.6 Hz, 2H,
Hb), 6.76 (t, 3J ¼ 5.2 Hz, 1H, C7eNH), 6.67 (d, 3J ¼ 8.6 Hz, 2H, Hc),
6.39 (s, 1H, H8), 5.92 (s, 2H, NH2), 3.64e3.61 (m, 7H, Hh, NCH3),
3.50e3.45 (m, 2H, He), 2.56e2.54 (m, 2H, Hf), 2.49e2.45 (m, 4H,
10.26 (s,1H, OH), 9.19 (s,1H, C3eNH), 8.56 (s,
1H, H4), 8.44 (s, 1H, H5), 7.83 (d, 3J ¼ 8.4 Hz, 2H, Hb), 6.93 (d,
3J ¼ 8.4 Hz, 2H, Hc), 6.81 (s, 1H, C7eNH), 6.40 (s, 1H, H8), 3.62e3.61
(m, 7H, Hh, NCH3), 3.48e3.46 (m, 2H, He), 3.21e3.20 (m, 2H, Hf),
Hg); 13C NMR (100 MHz, DMSO-d6)
d 164.44 (NHeC]O), 158.69
2.49e2.44 (m, 4H, Hg); 13C NMR (100 MHz, DMSO-d6)
d 164.52
(C3eC]O), 158.44 (C7), 152.57 (Cd), 149.03 (C5), 142.39 (C8a),
128.65 (2Cb), 123.35 (Ca), 119.98 (C3), 119.12 (C4), 112.90 (2Cc),
108.31 (C4a), 88.60 (C8), 66.15 (2Ch), 57.37 (Cf), 53.33 (2Cg), 38.10
(Ce), 29.43 (NCH3); MS (ESI) m/z (%): 423.2 (100) [M þ H]þ; UPLC
purity ¼ 97%, Rt 1.67 min; HRMS (ESI): calcd. for C22H26N6O3
[M þ H]þ 423.2139, found: 423.2153.
(NHeC]O), 161.14 (Cd), 159.15 (C3eC]O), 158.72 (C7), 149.53 (C5),
142.86 (C8a), 129.37 (2Cb), 124.63 (C3), 123.33 (Ca), 120.48 (C4),
115.64 (2Cc), 108.39 (C4a), 88.84 (C8), 66.40 (2Ch), 57.54 (Cf), 53.58
(2Cg), 38.29 (Ce), 29.75 (NCH3); MS (ESI) m/z (%): 425.2 (100)
[M þ H]þ; UPLC purity ¼ 95%, Rt 1.29 min; HRMS (ESI): calcd. for
C
22H25N5O4 [M þ H]þ 424.1979, found: 424.1970.
4.1.3.8. 4-Amino-N-(7-{[2-(dimethylamino)ethyl]amino}-1-methyl-
2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl)benzamide (26).
4.1.3.10. N-(1-Methyl-7-{[2-(morpholin-4-yl)ethyl]amino}-2-oxo-
1,2-dihydro-1,6-naphthyridin-3-yl)-4-methoxybenzamide (28).
c
d
c
NH2
b
d
O
b
a
H
N
5
8
4
a
g
H
N
4a
8a
3
h
5
8
4
g
4a
8a
3
N
O
N
e
O
N
e
O
7
N
O
N
N
7
f
N
O
N
H
f
H
The reaction was carried out using amide 10 (75 mg,
The reaction was carried out using amide 12 (75 mg, 0.22 mmol)
and 2-(morpholin-4-yl)ethanamine (579
L, 4.40 mmol) at 170 ꢀC
0.21 mmol) and N,N-dimethylethylenediamine (452
m
L,
m
4.20 mmol) at 130 ꢀC for 8 h. Purification by silica gel chroma-
tography (mixtures of dichloromethane/methanol of increasing
polarity) and trituration with diisopropyl ether afforded 26
for 2 h. Purification by silica gel chromatography (mixtures of
dichloromethane/methanol of increasing polarity) and trituration
with diisopropyl ether afforded 28 (39 mg, 40% yield) as a beige
(15 mg, 19% yield) as an orange powder. Mp: 180e181 ꢀC; IR,
n
powder. Mp: 195e196 ꢀC; IR,
CeHal); 1632 (
(400 MHz, CDCl3) d 9.12 (s, 1H, C3eNH), 8.80 (s, 1H, H4), 8.40 (s, 1H,
n nNeH); 2945
(cmꢁ1): 3362 (
(cmꢁ1): 3379, 3345, 3335 (
n
NeH); 2949 (
n
CeHal); 1626 (
n
C ¼ O);
(n
n
C ¼ O); 1585, 1499, 1454 (
n
C ¼ C); 1H NMR
1532, 1505 (
n
C ¼ C); 1H NMR (400 MHz, CDCl3)
d
9.07 (s, 1H,
C3eNH), 8.77 (s, 1H, H4), 8.38 (s, 1H, H5), 7.78 (d, 3J ¼ 8.4 Hz, 2H,
Hb), 6.71 (d, 3J ¼ 8.4 Hz, 2H, Hc), 6.14 (s, 1H, H8), 5.57 (br s, 1H,
C7eNH), 4.04 (s, 2H, NH2), 3.68 (s, 3H, CONCH3), 3.48e3.44 (m, 2H,
He), 2.67 (t, 3J ¼ 5.2 Hz, 2H, Hf), 2.35 (s, 6H, Hg); 13C NMR (100 MHz,
H5), 7.91 (d, 3J ¼ 8.8 Hz, 2H, Hb), 6.99 (d, 3J ¼ 8.8 Hz, 2H, Hc), 6.08 (s,
1H, H8), 5.47 (br s,1H, C7eNH), 3.88 (s, 3H, OCH3), 3.76e3.74 (m, 4H,
3
Hh), 3.70 (s, 3H, NCH3), 3.44e3.39 (m, 2H, He), 2.68 (t, J ¼ 5.5 Hz,
2H, Hf), 2.55e2.51 (m, 4H, Hg); 13C NMR (100 MHz, CDCl3)
d 165.21
CDCl3)
d
165.56 (NHeC]O), 159.67 (C7), 158.62 (C3eC]O), 150.20
(NHeC]O), 162.68 (Cd), 159.17 (C3eC]O), 158.56 (C7), 149.96 (C5),
143.16 (C8a), 129.03 (2Cb), 126.58 (Ca), 124.33 (C3), 119.49 (C4),
114.02 (2Cc), 109.69 (C4a), 87.56 (C8), 66.95 (2Ch), 56.83 (Cf), 55.50
(OCH3), 53.32 (2Cg), 38.46 (Ce), 29.80 (NCH3); MS (ESI) m/z (%):
438.2 (100) [M þ H]þ; UPLC purity ¼ 98%, Rt 2.20 min; HRMS (ESI):
calcd. for C23H27N5O4 [M þ H]þ 438.2136, found: 438.2138.
(Cd), 149.65 (C5), 143.05 (C8a), 129.17 (2Cb), 124.75 (Ca), 123.91 (C3),
119.28 (C4), 114.44 (2Cc), 110.02 (C4a), 88.57 (C8), 58.06 (Cf), 45.23
(2Cg), 39.55 (Ce), 29.87 (CONCH3); MS (ESI) m/z (%): 381.2 (100)
[M þ H]þ; UPLC purity ¼ 95%, Rt 1.64 min; HRMS (ESI): calcd. for
C
20H24N6O2 [M þ H]þ 381.2034, found: 381.2033.

28
D. Montoir et al. / European Journal of Medicinal Chemistry 119 (2016) 17e33
4.1.3.11. N-(7-{[2-(dimethylamino)ethyl]amino}-1-methyl-2-oxo-
4.1.3.13. N-(1-Methyl-7-(4-methylpiperazin-1-yl)-2-oxo-1,2-
1,2-dihydro-1,6-naphthyridin-3-yl)-4-methoxybenzamide (29).
dihydro-1,6-naphthyridin-3-yl)-4-methoxybenzamide (31).
c
c
d
O
d
O
b
b
a
H
N
H
N
5
8
4
a
5
8
4
g
4a
8a
3
4a
8a
3
N
N
e
e
O
O
N
f
7
7
O
N
N
N
O
N
f
H
N
g
The reaction was carried out using amide 12 (75 mg, 0.22 mmol)
and N,N-dimethylethylenediamine (474
L, 4.40 mmol) at 130 ꢀC
m
for 2.5 h. Purification by silica gel chromatography (mixtures of
dichloromethane/methanol of increasing polarity) and trituration
with diisopropyl ether afforded 29 (46 mg, 53% yield) as a beige
The reaction was carried out using amide 12 (75 mg, 0.22 mmol)
and 1-methylpiperazine (488
L, 4.40 mmol) at 150 ꢀC for 1 h.
Purification by silica gel chromatography (mixtures of dichloro-
methane/methanol of increasing polarity) and trituration with
diisopropyl ether afforded 31 (46 mg, 51% yield) as a pale yellow
m
powder. Mp: 146e147 ꢀC; IR,
C ¼ O); 1593, 1497, 1462 (
n nNeH); 1641
(cmꢁ1): 3397, 3240 (
(
d
n
n
C ¼ C); 1H NMR (400 MHz, CDCl3)
9.12 (s, 1H, C3eNH), 8.78 (s, 1H, H4), 8.39 (s, 1H, H5), 7.91 (d,
powder. Mp: 248e249 ꢀC; IR,
CeHal); 1639 (
(400 MHz, CDCl3)
n nNeH); 2936
(cmꢁ1): 3368 (
3J ¼ 8.8 Hz, 2H, Hb), 6.98 (d, 3J ¼ 8.8 Hz, 2H, Hc), 6.15 (s, 1H, H8), 5.63
(br s, 1H, C7eNH), 3.88 (s, 3H, OCH3), 3.68 (s, 3H, CONCH3),
3.48e3.46 (m, 2H, He), 2.69 (t, 3J ¼ 5.6 Hz, 2H, Hf), 2.36 (s, 6H, Hg);
(n
n
d
C ¼ O); 1593, 1528, 1503 (
n
C ¼ C); 1H NMR
9.15 (s, 1H, NH), 8.81 (s, 1H, H4), 8.47 (s, 1H, H5),
7.91 (d, 3J ¼ 8.4 Hz, 2H, Hb), 6.99 (d, 3J ¼ 8.4 Hz, 2H, Hc), 6.30 (s, 1H,
H8), 3.88 (s, 3H, OCH3), 3.74e3.72 (m, 7H, He, CONCH3), 2.69e2.63
13C NMR (100 MHz, CDCl3)
d 165.20 (NHeC]O), 162.66 (Cd), 159.17
(C7), 158.49 (C3eC]O), 149.76 (C5), 143.05 (C8a), 129.02 (2Cb),
126.61 (Ca), 124.30 (C3), 119.47 (C4), 114.01 (2Cc), 109.73 (C4a), 88.45
(C8), 57.83 (Cf), 55.49 (OCH3), 44.97 (2Cg), 39.16 (Ce), 29.78
(CONCH3); MS (ESI) m/z (%): 396.3 (100) [M þ H]þ; UPLC
purity ¼ 96%, Rt 1.90 min; HRMS (ESI): calcd. for C21H25N5O3
[M þ H]þ 396.2030, found: 396.2036.
(m, 4H, Hf), 2.44 (s, 3H, Hg); 13C NMR (100 MHz, CDCl3)
d 165.26
(NHeC]O), 162.72 (Cd), 159.14 (C7), 158.91 (C3eC]O), 149.30 (C5),
143.07 (C8a), 129.05 (2Cb), 126.51 (Ca), 124.97 (C3), 119.03 (C4),
114.03 (2Cc), 109.94 (C4a), 88.69 (C8), 55.50 (OCH3), 54.56 (2Cf),
45.84 (Cg), 44.98 (2Ce), 29.77 (CONCH3); MS (ESI) m/z (%): 408.3
(100) [M þ H]þ; UPLC purity ¼ 96%, Rt 2.01 min; HRMS (ESI): calcd.
for C22H25N5O3 [M þ H]þ 408.2030, found: 408.2026.
4.1.3.12. N-7-[4-(2-Hydroxyethyl)piperazin-1-yl]-1-methyl-2-oxo-
1,2-dihydro-1,6-naphthyridin-3-yl)-4-methoxybenzamide (30).
4.1.3.14. N-(1-Methyl-7-{[2-(morpholin-4-yl)ethyl]amino}-2-oxo-
1,2-dihydro-1,6-naphthyridin-3-yl)-3-methoxybenzamide (32).
c
d
O
b
e
H
N
d
f
5
8
4
a
4a
8a
3
j
H
N
5
8
4
N
4a
8a
3
e
i
c
a
O
O
N
O
f
b
7
g
N
O
N
O
N
h
7
O
N
N
N
h
H
HO
g
The reaction was carried out using amide 12 (75 mg, 0.22 mmol)
and 1-(2-hydroxyethyl)piperazine (540
L, 4.40 mmol) at 130 ꢀC for
h. Purification by silica gel chromatography (mixtures of
dichloromethane/methanol of increasing polarity) afforded 30
(67 mg, 70% yield) as a beige powder. Mp: 238e239 ꢀC; IR, (cmꢁ1):
C ¼ O); 1591, 1501, 1437
9.31 (s, 1H, NH), 8.61 (s,
The reaction was carried out using amide 13 (75 mg, 0.22 mmol)
and 2-(morpholin-4-yl)ethanamine (579
L, 4.40 mmol) at 170 ꢀC
for 1 h. Trituration with diisopropyl ether afforded 32 (83 mg, 86%
m
m
1
yield) as a beige powder. Mp: 183e184 ꢀC; IR,
NeH); 2967 ( CeHal); 1641 (
C ¼ O); 1587, 1504 (
NMR (400 MHz, CDCl3) d 9.17 (s, 1H, C3eNH), 8.80 (s, 1H, H4), 8.40
n
(cmꢁ1): 3304, 3227
n
(n
n
n
n
C ¼ C); 1H
3385 (
nNeH); 2920, 2849 (
n
CeHal); 1641 (
n
(n
C ¼ C); 1H NMR (400 MHz, DMSO-d6)
d
(s, 1H, H5), 7.49e7.46 (m, 2H, Hb, Hf), 7.40 (appt, appJ ¼ 8.0 Hz, 1H,
He), 7.09 (ddd, 3J ¼ 8.0 Hz, 4J ¼ 2.0 Hz, 4J ¼ 2.0 Hz, 1H, Hd), 6.09 (s,
1H, H8), 5.50 (br s, 1H, C7eNH), 3.88 (s, 3H, OCH3), 3.75 (t,
3J ¼ 4.8 Hz, 4H, Hj), 3.69 (s, 3H, NCH3), 3.44e3.40 (m, 2H, Hg), 2.69
1H, H4), 8.56 (s, 1H, H5), 7.94 (d, 3J ¼ 9.0 Hz, 2H, Hb), 7.13 (d,
3J ¼ 9.0 Hz, 2H, Hc), 6.62 (s, 1H, H8), 4.50 (m, 1H, OH), 3.88 (s, 3H,
OCH3), 3.69 (s, 3H, NCH3), 3.67e3.65 (m, 4H, He), 3.61e3.57 (m, 2H,
Hh), 2.59e2.56 (m, 4H, Hf), 2.49 (t, 3J ¼ 6.2 Hz, 2H, Hg); 13C NMR
3
(t, J ¼ 6.0 Hz, 2H, Hh), 2.54e2.51 (m, 4H, Hi); 13C NMR (100 MHz,
(100 MHz, CDCl3)
d
165.40 (NHeC]O), 162.86 (Cd), 159.30 (C3eC]
CDCl3) d 165.54 (NHeC]O), 162.02 (Cd), 159.08 (C7), 158.56
O), 159.20 (C7), 149.46 (C5), 143.21 (C8a), 129.19 (2Cb), 126.67 (Ca),
125.04 (C3), 119.23 (C4), 114.18 (2Cc), 109.98 (C4a), 88.69 (C8), 59.52
(Ch), 57.94 (Cg), 55.65 (OCH3), 52.77 (2Cf), 45.57 (2Ce), 29.89 (NCH3);
MS (ESI) m/z (%): 438.3 (100) [M þ H]þ; UPLC purity ¼ 97%, Rt
2.01 min; HRMS (ESI): calcd. for C23H27N5O4 [M þ H]þ 438.2136,
found: 438.2149.
(C3eC]O), 149.97 (C5), 143.27 (C8a), 135.76 (Ca), 129.82 (Ce), 124.16
(C3), 119.87 (C4), 118.91 (Cb), 118.37 (Cd), 112.34 (Cf), 109.63 (C4a),
87.82 (C8), 66.73 (2Cj), 56.93 (Ch), 55.49 (OCH3), 53.29 (2Ci), 38.28
(Cg), 29.82 (NCH3); MS (ESI) m/z (%): 439.2 (100) [M þ H]þ; UPLC
purity ¼ 95%, Rt 2.18 min; HRMS (ESI): calcd. for C23H27N5O4
[M þ H]þ 438.2136, found: 438.2154.

D. Montoir et al. / European Journal of Medicinal Chemistry 119 (2016) 17e33
29
4.1.3.15. N-(7-{[2-(dimethylamino)ethyl]amino}-1-methyl-2-oxo-
4.1.3.17. N-(7-{[2-(dimethylamino)ethyl]amino}-1-methyl-2-oxo-
1,2-dihydro-1,6-naphthyridin-3-yl)-3-methoxybenzamide (33).
1,2-dihydro-1,6-naphthyridin-3-yl)-3,4,5-trimethoxybenzamide (35).
e
f
d
O
H
N
5
8
4
c
d
i
O
O
4a
8a
3
b
a
c
a
O
N
b
H
N
g
5
8
4
g
4a
8a
3
O
N
7
N
O
N
N
h
e
H
O
N
7
O
N
N
f
H
The reaction was carried out using amide 13 (75 mg, 0.22 mmol)
and N,N-dimethylethylenediamine (474
L, 4.40 mmol) at 130 ꢀC
for 6 h. Purification by silica gel chromatography (mixtures of
dichloromethane/methanol of increasing polarity) and trituration
with diisopropyl ether afforded 33 (44 mg, 50% yield) as a pale
m
The reaction was carried out using amide 14 (75 mg, 0.19 mmol)
and N,N-dimethylethylenediamine (409
L, 3.80 mmol) at 130 ꢀC
for 3.5 h. Purification by silica gel chromatography (mixtures of
dichloromethane/methanol of increasing polarity) afforded 35
m
yellow powder. Mp: 154e155 ꢀC; IR,
2951 ( CeHal); 1717, 1645 (
NMR (400 MHz, CDCl3)
n
(cmꢁ1): 3395 (
C ¼ O); 1589, 1518, 1456 (
nNeH); 2980,
n
(52 mg, 60% yield) as a beige powder. Mp: 188e189 ꢀC; IR,
n
n
(cmꢁ1):
C ¼ C);
9.11 (s, 1H, C3eNH), 8.77 (s, 1H, H4),
n
n
C ¼ C); 1H
3240 (
n
NeH); 2938 (
n
CeHal); 1636 (
n
C ¼ O); 1586, 1495 (
d
9.18 (s, 1H, C3eNH), 8.78 (s, 1H, H4), 8.38
1H NMR (400 MHz, CDCl3)
d
(s, 1H, H5), 7.49e7.48 (m, 2H, Hb, Hf), 7.40 (appt, appJ ¼ 8.0 Hz, 1H,
He), 7.09 (ddd, 3J ¼ 8.0 Hz, 4J ¼ 2.0 Hz, 4J ¼ 2.0 Hz, 1H, Hd), 6.22 (s,
1H, H8), 5.86 (br s, 1H, C7eNH), 3.88 (s, 3H, OCH3), 3.67 (s, 3H,
CONCH3), 3.59e3.55 (m, 2H, Hg), 2.82 (t, 3J ¼ 5.4 Hz, 2H, Hh), 2.47 (s,
8.39 (s, 1H, H5), 7.15 (s, 2H, Hb), 6.16 (s, 1H, H8), 5.66 (t, 3J ¼ 5.6 Hz,
1H, C7eNH), 3.95 (s, 6H, 2Cc-OCH3), 3.91 (s, 3H, CdeOCH3), 3.68 (s,
3H, CONCH3), 3.50e3.46 (m, 2H, He), 2.70 (t, 3J ¼ 5.6 Hz, 2H, Hf),
2.37 (s, 6H, Hg); 13C NMR (100 MHz, CDCl3)
d 165.30 (NHeC]O),
6H, Hi); 13C NMR (100 MHz, CDCl3)
d 165.53 (NHeC]O),160.01 (Cc),
159.13 (C3eC]O),158.58 (C7), 153.35 (2Cc), 149.90 (C5), 143.10 (C8a),
141.41 (Cd), 129.70 (Ca), 124.05 (C3), 119.88 (C4), 109.62 (C4a), 104.40
(2Cb), 88.45 (C8), 60.98 (CdeOCH3), 57.83 (Cf), 56.35 (2Cc-OCH3),
44.96 (2Cg), 39.13 (Ce), 29.79 (CONCH3); MS (ESI) m/z (%): 456.3
(100) [M þ H]þ; UPLC purity ¼ 97%, Rt 1.44 min; HRMS (ESI): calcd.
for C23H29N5O5 [M þ H]þ 456.2241, found: 456.2257.
159.04 (C7), 158.15 (C3eC]O), 149.53 (C5), 143.12 (C8a), 135.75 (Ca),
129.81 (Ce), 124.29 (C3), 119.67 (C4), 118.91 (Cb), 118.37 (Cd), 112.33
(Cf), 109.88 (C4a), 89.48 (C8), 58.13 (Ch), 55.48 (OCH3), 44.60 (2Ci),
38.30 (Cg), 29.86 (CONCH3); MS (ESI) m/z (%): 396.3 (100) [M þ H]þ;
UPLC purity ¼ 98%, Rt 1.84 min; HRMS (ESI): calcd. for C21H25N5O3
[M þ H]þ 396.2030, found: 396.2022.
4.1.3.18. N-(1-Methyl-7-{[2-(morpholin-4-yl)ethyl]amino}-2-oxo-
4.1.3.16. N-(1-Methyl-7-{[2-(morpholin-4-yl)ethyl]amino}-2-oxo-
1,2-dihydro-1,6-naphthyridin-3-yl)-3,4,5-trimethoxybenzamide (34).
1,2-dihydro-1,6-naphthyridin-3-yl)biphenyl-3-carboxamide
(36).
e
O
d
c
f
c
h
d
H
N
O
n
5
8
4
b
4a
8a
3
m
i
a
O
N
H
N
g
h
a
5
8
4
b
g
4a
8a
3
k
j
O
N
O
O
N
7
O
N
N
e
l
H
O
N
7
O
N
N
f
H
The reaction was carried out using amide 15 (75 mg, 0.19 mmol)
and 2-(morpholin-4-yl)ethanamine (500
L, 3.80 mmol) at 170 ꢀC
m
The reaction was carried out using amide 14 (75 mg, 0.19 mmol)
and 2-(morpholin-4-yl)ethanamine (500
L, 3.80 mmol) at 170 ꢀC
for 1 h. Purification by silica gel chromatography (mixtures of
dichloromethane/methanol of increasing polarity) afforded 34
for 1 h. Purification by silica gel chromatography (mixtures of
dichloromethane/methanol of increasing polarity) and trituration
with diisopropyl ether afforded 36 (65 mg, 71% yield) as a white
m
powder. Mp: 171e172 ꢀC; IR,
n
(cmꢁ1): 3231 (
n
NeH); 1639 (
n
C ¼ O);
(52 mg, 55% yield) as a beige powder. Mp: 197e198 ꢀC; IR,
n
n
(cmꢁ1):
C ¼ C);
9.11 (s, 1H, C3eNH), 8.79 (s, 1H, H4),
1591, 1512, 1466 (
n
C ¼ C); 1H NMR (400 MHz, CDCl3)
d 9.25 (s, 1H,
3238 (
n
NeH); 2922 (
n
CeHal); 1634 (
n
C ¼ O); 1587, 1495 (
C3eNH), 8.84 (s, 1H, H4), 8.42 (s, 1H, H5), 8.15 (s, 1H, Hb), 7.90 (d,
3J ¼ 7.6 Hz, 1H, Hf), 7.78 (d, 3J ¼ 8.0 Hz, 1H, Hd), 7.65 (d, 3J ¼ 7.6 Hz,
2H, Hh), 7.57 (appt, appJ ¼ 7.6 Hz, 1H, He), 7.48 (appt, appJ ¼ 7.4 Hz,
2H, Hi), 7.39 (t, 3J ¼ 7.2 Hz, 1H, Hj), 6.09 (s, 1H, H8), 5.50 (br s, 1H,
C7eNH), 3.75 (t, 3J ¼ 4.0 Hz, 4H, Hn), 3.70 (s, 3H, NCH3), 3.44e3.39
1H NMR (400 MHz, CDCl3)
d
8.41 (s, 1H, H5), 7.15 (s, 2H, Hb), 6.09 (s, 1H, H8), 5.51 (t, 3J ¼ 5.2 Hz,
1H, C7eNH), 3.95 (s, 6H, 2Cc-OCH3), 3.92 (s, 3H, CdeOCH3), 3.75 (t,
3J ¼ 4.6 Hz, 4H, Hh), 3.70 (s, 3H, NCH3), 3.43e3.39 (m, 2H, He), 2.69
(t, 3J ¼ 5.8 Hz, 2H, Hf), 2.53e2.51 (m, 4H, Hg); 13C NMR (100 MHz,
(m, 2H, Hk), 2.69 (t, 3J ¼ 5.2 Hz, 2H, Hl), 2.54e2.50 (m, 4H, Hm); 13
C
CDCl3)
d
165.32 (NHeC]O), 159.14 (C3eC]O), 158.66 (C7), 153.36
NMR (100 MHz, CDCl3) d 165.63 (NHeC]O), 159.11 (C7), 158.66
(2Cc), 150.08 (C5), 143.22 (C8a), 141.44 (Cd), 129.68 (Ca), 124.08 (C3),
119.90 (C4), 109.57 (C4a), 104.41 (2Cb), 87.57 (C8), 66.93 (2Ch), 60.98
(CdeOCH3), 56.81 (2Cc-OCH3), 56.36 (Cf), 53.31 (2Cg), 38.43 (Ce),
29.81 (NCH3); MS (ESI) m/z (%): 498.3 (100) [M þ H]þ; UPLC
purity ¼ 99%, Rt 1.53 min; HRMS (ESI): calcd. for C25H31N5O6
[M þ H]þ 498.2347, found: 498.2364.
(C3eC]O), 150.09 (C5), 143.27 (C8a), 142.01 (Cc or Cg), 140.08 (Cg or
Cc), 134.91 (Ca), 130.74 (Cd), 129.28 (Ce), 128.96 (2Ci), 127.87 (Cj),
127.26 (2Ch), 125.93 (Cb), 125.80 (Cf), 124.13 (C3), 119.96 (C4), 109.58
(C4a), 87.59 (C8), 66.92 (2Cn), 56.83 (Cl), 53.31 (2Cm), 38.42 (Ck),
29.81 (NCH3); MS (ESI) m/z (%): 484.3 (100) [M þ H]þ; UPLC
purity ¼ 96%, Rt 2.79 min; HRMS (ESI): calcd. for C28H29N5O3

30
D. Montoir et al. / European Journal of Medicinal Chemistry 119 (2016) 17e33
[M þ H]þ 484.2343, found: 484.2361.
O), 159.13 (C7), 159.10 (C3eC]O), 149.45 (C5), 143.19 (C8a), 142.03
(Cc or Cg), 140.07 (Cg or Cc), 134.86 (Ca), 130.79 (Cd), 129.30 (Ce),
128.96 (2Ci), 127.88 (Cj), 127.26 (2Ch), 125.94 (Cb), 125.80 (Cf), 124.69
(C3), 119.57 (C4), 109.72 (C4a), 88.51 (C8), 59.39 (Cn), 57.80 (Cm),
52.63 (2Cl), 45.41 (2Ck), 29.77 (NCH3); MS (ESI) m/z (%): 484.3 (100)
[M þ H]þ; UPLC purity ¼ 100%, Rt 2.59 min; HRMS (ESI): calcd. for
4.1.3.19. N-(7-{[2-(dimethylamino)ethyl]amino}-1-methyl-2-oxo-
1,2-dihydro-1,6-naphthyridin-3-yl)biphenyl-3-carboxamide
(37).
28H29N5O3 [M þ H]þ 484.2343, found: 484.2341.
e
C
d
f
H
N
h
4.1.3.21. N-[1-Methyl-2-oxo-7-n-propylamino-1,2-dihydro-1,6-
naphthyridin-3-yl]biphenyl-3-carboxamide (39).
5
8
4
c
a
4a
8a
3
m
i
g
N
b
k
j
O
N
e
7
O
N
N
d
l
f
H
h
H
N
c
5
8
4
4a
8a
3
i
a
N
g
b
The reaction was carried out using amide 15 (75 mg, 0.19 mmol)
and N,N-dimethylethylenediamine (409
L, 3.80 mmol) at 130 ꢀC
m
k
j
O
m
7
O
N
N
for 1.5 h. Purification by silica gel chromatography (mixtures of
dichloromethane/methanol of increasing polarity) and trituration
with diisopropyl ether afforded 37 (54 mg, 64% yield) as a yellow
l
H
powder. Mp: 136e137 ꢀC; IR,
CeHal); 1634 (
(400 MHz, CDCl3)
n nNeH); 2949
(cmꢁ1): 3372 (
The reaction was carried out using amide 15 (75 mg, 0.19 mmol)
and n-propylamine (312 L, 3.80 mmol) in N-methyl-2-pyrrolidone
(n
n
C ¼ O); 1593, 1518, 1474 (
n
C ¼ C); 1H NMR
m
d
9.26 (s, 1H, C3eNH), 8.82 (s, 1H, H4), 8.41 (s, 1H,
(1 mL). CuI (19 mg, 0.10 mmol, 0.5 equiv.) was added and the
mixture was heated at 130 ꢀC for 5 h under microwave irradiation.
Purification by silica gel chromatography (mixtures of dichloro-
methane/methanol of increasing polarity) and trituration with
diisopropyl ether afforded 39 (45 mg, 57% yield) as a pale yellow
H5), 8.15 (s, 1H, Hb), 7.90 (d, 3J ¼ 7.5 Hz, 1H, Hf), 7.78 (d, 3J ¼ 7.6 Hz,
1H, Hd), 7.65 (d, 3J ¼ 7.2 Hz, 2H, Hh), 7.57 (appt, appJ ¼ 7.8 Hz, 1H, He),
7.48 (appt, appJ ¼ 7.4 Hz, 2H, Hi), 7.39 (t, 3J ¼ 7.4 Hz, 1H, Hj), 6.14 (s,
1H, H8), 5.59 (br s, 1H, C7eNH), 3.68 (s, 3H, CONCH3), 3.48e3.43 (m,
2H, Hk), 2.66 (t, 3J ¼ 5.6 Hz, 2H, Hl), 2.34 (s, 6H, Hm); 13C NMR
powder. Mp: 144e145 ꢀC; IR,
n
n
(cmꢁ1): 3387, 3244 (
CeHal); 1643 (
C ¼ O); 1587, 1512
9.25 (s, 1H, C3eNH), 8.84 (s,
nNeH); 3078
(100 MHz, CDCl3)
d 165.62 (NHeC]O), 159.13 (C7), 158.66 (C3eC]
(
n
CeHar), 2961, 2936, 2876 (
n
O), 149.96 (C5), 143.18 (C8a), 142.00 (Cc or Cg), 140.09 (Cg or Cc),
134.92 (Ca), 130.72 (Cd), 129.27 (Ce), 128.95 (2Ci), 127.86 (Cj), 127.26
(2Ch), 125.93 (Cb), 125.80 (Cf), 124.08 (C3), 119.99 (C4), 109.59 (C4a),
88.28 (C8), 57.80 (Cl), 45.03 (2Cm), 39.29 (Ck), 29.79 (CONCH3); MS
(ESI) m/z (%): 442.3 (100) [M þ H]þ; UPLC purity ¼ 100%, Rt
2.73 min; HRMS (ESI): calcd. for C26H27N5O2 [M þ H]þ 442.2238,
found: 442.2232.
(n
C ¼ C); 1H NMR (400 MHz, CDCl3)
d
1H, H4), 8.40 (s, 1H, H5), 8.15 (s, 1H, Hb), 7.90 (d, 3J ¼ 8.0 Hz, 1H, Hf),
7.78 (d, 3J ¼ 8.0 Hz, 1H, Hd), 7.65 (d, 3J ¼ 7.2 Hz, 2H, Hh), 7.58 (appt,
appJ ¼ 7.8 Hz, 1H, He), 7.48 (appt, appJ ¼ 7.2 Hz, 2H, Hi), 7.40 (t,
3J ¼ 7.2 Hz, 1H, Hj), 6.05 (s, 1H, H8), 4.90e4.87 (m, 1H, C7eNH), 3.71
(s, 3H, NCH3), 3.31e3.26 (m, 2H, Hk), 1.77e1.68 (m, 2H, Hl), 1.05 (t,
3J ¼ 7.4 Hz, 3H, Hm); 13C NMR (100 MHz, CDCl3)
d 165.62 (NHeC]
O), 159.13 (C3eC]O), 158.89 (C7), 150.13 (C5), 143.35 (C8a), 142.00
(Cc or Cg), 140.09 (Cg or Cc), 134.94 (Ca), 130.72 (Cd), 129.27 (Ce),
128.95 (2Ci), 127.86 (Cj), 127.26 (2Ch), 125.93 (Cb), 125.80 (Cf), 124.03
(C3), 120.03 (C4), 109.48 (C4a), 86.86 (C8), 44.45 (Ck), 29.80 (NCH3),
22.58 (Cl), 11.62 (Cm); MS (ESI) m/z (%): 413.2 (100) [M þ H]þ; UPLC
purity ¼ 100%, Rt 3.12 min; HRMS (ESI): calcd. for C25H24N4O2
[M þ H]þ 413.1972, found: 413.1962.
4.1.3.20. N-7-[4-(2-Hydroxyethyl)piperazin-1-yl]-1-methyl-2-oxo-
1,2-dihydro-1,6-naphthyridin-3-yl)biphenyl-3-carboxamide
(38).
e
d
f
h
H
N
c
5
8
4
4a
8a
3
i
a
N
g
4.1.3.22. 4-Chloro-N-(1-methyl-7-{[2-(morpholin-4-yl)ethyl]amino}-
2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl)benzamide (40).
b
k
j
O
l
7
N
N
O
n
c
N
d
Cl
b
a
HO
m
H
N
h
5
8
4
g
4a
8a
3
O
N
e
The reaction was carried out using amide 15 (75 mg, 0.19 mmol)
and 1-(2-hydroxyethyl)piperazine (466
L, 3.80 mmol) at 130 ꢀC for
h. Purification by silica gel chromatography (mixtures of
O
N
m
7
O
N
N
f
1
H
dichloromethane/methanol of increasing polarity) and trituration
with diisopropyl ether afforded 38 (56 mg, 61% yield) as a pale
yellow powder. Mp: 188e189 ꢀC; IR,
n
n
(cmꢁ1): 3374 (
n
NeH); 2833
The reaction was carried out using amide 16 (75 mg, 0.22 mmol)
and 2-(morpholin-4-yl)ethanamine (579
L, 4.40 mmol) at 170 ꢀC
(
n
CeHal); 1638 (
n
C ¼ O); 1589, 1517 (
C ¼ C); 1H NMR (400 MHz,
m
CDCl3)
d
9.28 (s, 1H, C3eNH), 8.85 (s, 1H, H4), 8.50 (s, 1H, H5), 8.15 (s,
for 1 h. Purification by silica gel chromatography (mixtures of
dichloromethane/methanol of increasing polarity) and trituration
with diisopropyl ether afforded 40 (77 mg, 79% yield) as a yellow
1H, Hb), 7.90 (d, 3J ¼ 8.0 Hz, 1H, Hf), 7.79 (d, 3J ¼ 7.6 Hz, 1H, Hd), 7.65
(d, 3J ¼ 7.2 Hz, 2H, Hh), 7.58 (appt, appJ ¼ 7.6 Hz, 1H, He), 7.48 (appt,
appJ ¼ 7.4 Hz, 2H, Hi), 7.40 (t, 3J ¼ 7.4 Hz, 1H, Hj), 6.30 (s, 1H, H8),
3.72e3.68 (m, 9H, Hn, Hk, NCH3), 2.69 (t, 3J ¼ 5.0 Hz, 4H, Hl), 2.64 (t,
powder. Mp: 209e210 ꢀC; IR,
CeHal); 1638 (
C ¼ O); 1593, 1517, 1483 (
NMR (400 MHz, CDCl3) 9.15 (s,1H, C3eNH), 8.78 (s,1H, H4), 8.41 (s,
n
(cmꢁ1): 3383 (
C ¼ C); 613 (
n
NeH); 2978, 2951
(n
n
n
n
CeCl); 1H
3J ¼ 5.2 Hz, 2H, Hm); 13C NMR (100 MHz, CDCl3)
d
165.68 (NHeC]
d

D. Montoir et al. / European Journal of Medicinal Chemistry 119 (2016) 17e33
31
1H, H5), 7.87 (d, 3J ¼ 6.8 Hz, 2H, Hc), 7.47 (d, 3J ¼ 6.8 Hz, 2H, Hb), 6.08
(s, 1H, H8), 5.51 (br s, 1H, C7eNH), 3.75 (t, 3J ¼ 4.6 Hz, 4H, Hh), 3.69
(s, 3H, NCH3), 3.43e3.39 (m, 2H, He), 2.69 (t, 3J ¼ 5.8 Hz, 2H, Hf),
7.76 (d, 3J ¼ 6.0 Hz, 2H, Hb), 6.10 (s, 1H, H8), 5.55 (br s, 1H, C7eNH),
3.75 (t, 3J ¼ 4.4 Hz, 4H, Hg), 3.70 (s, 3H, NCH3), 3.44e3.40 (m, 2H,
Hd), 2.70 (t, 3J ¼ 6.0 Hz, 2H, He), 2.55e2.51 (m, 4H, Hf); 13C NMR
2.54e2.50 (m, 4H, Hg); 13C NMR (100 MHz, CDCl3)
d
164.51
(100 MHz, CDCl3) d 163.53 (NHeC]O), 158.96 (C7), 158.84 (C3eC]
(NHeC]O), 159.06 (C3eC]O), 158.70 (C7), 150.12 (C5), 143.29 (C8a),
138.40 (Cd), 132.66 (Ca), 129.10 (2Cc), 128.54 (2Cb), 123.90 (C3),
120.07 (C4), 109.48 (C4a), 87.58 (C8), 66.92 (2Ch), 56.82 (Cf), 53.31
(2Cg), 38.42 (Ce), 29.82 (NCH3); MS (ESI) m/z (%): 442.1 (100)
[M þ H]þ, 444.1 (40) [M þ H þ 2]þ; UPLC purity ¼ 97%, Rt 2.43 min;
HRMS (ESI): calcd. for C22H24ClN5O3 [M þ H]þ 442.1640, found:
442.1638.
O), 150.84 (2Cc), 150.29 (C5), 143.45 (C8a), 141.20 (Ca), 123.44 (C3),
120.80 (2Cb, C4), 109.30 (C4a), 87.65 (C8), 66.90 (2Cg), 56.85 (Ce),
53.35 (2Cf), 38.33 (Cd), 29.86 (NCH3); MS (ESI) m/z (%): 409.3 (100)
[M þ H]þ; UPLC purity ¼ 99%, Rt 0.98 min; HRMS (ESI): calcd. for
C
21H24N6O3 [M þ H]þ 409.1983, found: 409.1981.
4.1.3.25. N-(7-{[2-(dimethylamino)ethyl]amino}-1-methyl-2-oxo-
1,2-dihydro-1,6-naphthyridin-3-yl)pyridine-4-carboxamide
(43).
4.1.3.23. 4-Chloro-N-(7-{[2-(dimethylamino)ethyl]amino}-1-methyl-
2-oxo-1,2-dihydro-1,6-naphthyridin-3-yl)benzamide (41).
c
c
b
N
d
Cl
b
a
H
N
5
8
4
a
f
4a
8a
3
H
N
5
8
4
N
g
4a
8a
3
d
N
O
N
7
O
e
N
N
e
O
N
H
7
O
N
N
f
H
The reaction was carried out using amide 17 (75 mg, 0.24 mmol)
and N,N-dimethylethylenediamine (517
L, 4.80 mmol) at 130 ꢀC
for 3 h. Purification by silica gel chromatography (mixtures of
dichloromethane/methanol of increasing polarity) afforded 43
m
The reaction was carried out using amide 16 (75 mg, 0.22 mmol)
and N,N-dimethylethylenediamine (474
L, 4.40 mmol) at 130 ꢀC
for 5 h. Purification by silica gel chromatography (mixtures of
dichloromethane/methanol of increasing polarity) and trituration
with diisopropyl ether afforded 41 (62 mg, 70% yield) as a yellow
m
(53 mg, 60% yield) as a yellow powder. Mp: 169e170 ꢀC; IR,
n
(cmꢁ1): 3366, 3246 (
n
NeH); 2853 (
n
CeHal); 1643 (
n
C ¼ O); 1597,
C ¼ C); 1H NMR (400 MHz, CDCl3)
d 9.24 (s, 1H, C3eNH), 8.82
powder. Mp: 183e184 ꢀC; IR,
CeHal); 1634 (
C ¼ O); 1518, 1485 (
(400 MHz, CDCl3)
n
(cmꢁ1): 3375 (
nNeH); 2972
1517 (
n
(dd, 3J ¼ 4.4 Hz, 5J ¼ 1.6 Hz, 2H, Hc), 8.78 (s, 1H, H4), 8.41 (s, 1H, H5),
7.76 (dd, 3J ¼ 4.4 Hz, 5J ¼ 1.6 Hz, 2H, Hb), 6.14 (s, 1H, H8), 5.67 (br s,
1H, C7eNH), 3.68 (s, 3H, CONCH3), 3.49e3.45 (m, 2H, Hd), 2.68 (t,
3J ¼ 5.6 Hz, 2H, He), 2.36 (s, 6H, Hf); 13C NMR (100 MHz, CDCl3)
(n
n
n
C ¼ C); 613 (
n
CeCl); 1H NMR
d
9.16 (s, 1H, C3eNH), 8.77 (s, 1H, H4), 8.40 (s, 1H,
H5), 7.87 (d, 3J ¼ 8.4 Hz, 2H, Hc), 7.48 (d, 3J ¼ 8.4 Hz, 2H, Hb), 6.14 (s,
1H, H8), 5.60 (br s, 1H, C7eNH), 3.68 (s, 3H, CONCH3), 3.47e3.43 (m,
2H, He), 2.66 (t, 3J ¼ 5.6 Hz, 2H, Hf), 2.54 (s, 6H, Hg); 13C NMR
d
163.50 (NHeC]O), 158.93 (C7), 158.82 (C3eC]O), 150.88 (2Cc),
150.17 (C5),143.33 (C8a),141.24 (Ca),123.37 (C3),120.77 (2Cb),120.76
(C4), 109.29 (C4a), 88.34 (C8), 57.75 (Ce), 44.99 (2Cf), 39.17 (Cd), 29.83
(100 MHz, CDCl3) d 164.50 (NHeC]O), 159.07 (C7), 158.70 (C3eC]
O), 150.00 (C5), 143.18 (C8a), 138.38 (Cd), 132.68 (Ca), 129.09 (2Cc),
128.54 (2Cb), 123.84 (C3), 120.09 (C4), 109.48 (C4a), 88.27 (C8), 57.79
(Cf), 45.02 (2Cg), 39.27 (Ce), 29.80 (CONCH3); MS (ESI) m/z (%): 400.1
(100) [M þ H]þ, 402.1 (40) [M þ H þ 2]þ; UPLC purity ¼ 99%, Rt
2.35 min; HRMS (ESI): calcd. for C20H22ClN5O2 [M þ H]þ 400.1535,
found: 400.1524.
(CONCH3); MS (ESI) m/z (%): 367.3 (100) [M
þ
H]þ; UPLC
purity ¼ 96%, Rt 1.43 min; HRMS (ESI): calcd. for C19H22N6O2
[M þ H]þ 367.1877, found: 367.1872.
4.1.3.26. N-(1-Methyl-7-{[2-(morpholin-4-yl)ethyl]amino}-2-oxo-
1,2-dihydro-1,6-naphthyridin-3-yl)-1-benzothiophene-2-
carboxamide (44).
4.1.3.24. N-(1-Methyl-7-{[2-(morpholin-4-yl)ethyl]amino}-2-oxo-
1,2-dihydro-1,6-naphthyridin-3-yl)pyridine-4-carboxamide
(42).
d
e
c
b
f
c
H
N
l
5
8
4
k
4a
8a
3
g
b
h
N
a
S
O
N
g
H
N
5
8
4
a
i
4a
8a
3
f
O
N
O
N
7
O
N
N
j
H
d
O
N
7
O
N
N
e
H
The reaction was carried out using amide 18 (75 mg, 0.20 mmol)
and 2-(morpholin-4-yl)ethanamine (526
L, 4.00 mmol) at 170 ꢀC
m
The reaction was carried out using amide 17 (75 mg, 0.24 mmol)
and 2-(morpholin-4-yl)ethanamine (631
L, 4.80 mmol) at 170 ꢀC
for 1 h. Purification by silica gel chromatography (mixtures of
dichloromethane/methanol of increasing polarity) afforded 42
for 1 h. Purification by silica gel chromatography (mixtures of
dichloromethane/methanol of increasing polarity) afforded 44
m
(72 mg, 77% yield) as a yellow powder. Mp: 222e223 ꢀC; IR,
n
(cmꢁ1): 3320 (
n
NeH); 2845 (
n
CeHal); 1634 (
n
C ¼ O); 1585, 1530,
(44 mg, 45% yield) as a yellow powder. Mp: 235e236 ꢀC; IR,
n
1487 (
n
C ¼ C); 1H NMR (400 MHz, CDCl3)
d 9.18 (s, 1H, C3eNH), 8.76
(cmꢁ1): 3408, 3368 (
n
NeH); 2847 (
n
CeHal); 1643 (
n
C ¼ O); 1593,
(s,1H, H4), 8.40 (s,1H, H5), 7.93 (s,1H, Hb), 7.90e7.88 (m, 2H, Hg, Hd),
7.48e7.41 (m, 2H, Hf, He), 6.09 (s, 1H, H8), 5.22 (br s, 1H, C7eNH),
3.75 (t, 3J ¼ 4.6 Hz, 4H, Hl), 3.71 (s, 3H, NCH3), 3.43e3.39 (m, 2H, Hi),
1501, 1487 (
n
C ¼ C); 1H NMR (400 MHz, CDCl3)
d 9.24 (s, 1H,
C3eNH), 8.82 (d, 3J ¼ 6.0 Hz, 2H, Hc), 8.80 (s, 1H, H4), 8.42 (s, 1H, H5),

32
D. Montoir et al. / European Journal of Medicinal Chemistry 119 (2016) 17e33
2.69 (t, 3J ¼ 5.8 Hz, 2H, Hj), 2.53e2.51 (m, 4H, Hk); 13C NMR
4.2.2. Annexin V cell death detection assay
(100 MHz, CDCl3)
d
160.57 (NHeC]O), 158.90 (C7), 158.70 (C3eC]
MDA-MB-468 cells were seeded (40,000/well, 500 mL) on a 96-
O), 150.14 (C5), 143.30 (C8a), 141.39 (Ca), 139.13 (Ch), 138.64 (Cc),
126.68 (C3), 125.53 (Cb), 125.33 (Cf or Ce), 125.08 (Cg or Cd), 123.71
(Ce or Cf), 122.79 (Cd or Cg), 120.14 (C4), 109.49 (C4a), 87.58 (C8),
66.92 (2Cl), 56.81 (Cj), 53.31 (2Ck), 38.41 (Ci), 29.87 (NCH3); MS (ESI)
m/z (%): 464.4 (100) [M þ H]þ; UPLC purity ¼ 95%, Rt 2.10 min;
HRMS (ESI): calcd. for C24H25N5O3S [M þ H]þ 464.1751, found:
464.1752.
well plate and incubated in 5% CO2 incubator at 37 ꢀC for 24 h.
Compounds at varying concentrations in DMSO (1% DMSO final
concentration) were added, and cells were returned to the incu-
bator for 48 h. Floating dead cells and adherent cells after trypsin
treatment were harvested and washed once with PBS. Cells were
pelleted and re-suspended in annexin V binding buffer containing
1 mL annexin V-FITC (Miltenyi Biotec) and 1 mL propidium iodide
(SigmaeAldrich) for 15 min on ice in the dark. Subsequently, cells
were analyzed by flow cytometry, using a FACSCalibur operated by
CellQuest software and at least 10 000 events were collected per
sample. Cells were considered apoptotic if they stained positive for
Annexin V.
4.1.3.27. N-(7-{[2-(dimethylamino)ethyl]amino}-1-methyl-2-oxo-
1,2-dihydro-1,6-naphthyridin-3-yl)-1-benzothiophene-2-
carboxamide (45).
d
e
c
4.2.3. Cell cycle assay
b
f
MDA-MB-468 cells were seeded (40,000/well, 500 mL) on a 24-
H
N
5
8
4
well plate and incubated in 5% CO2 incubator at 37 ꢀC for 24 h.
Compounds at varying concentrations in DMSO (1% DMSO final
concentration) were added, and cells were returned to the incu-
bator for 48 h. Cells were harvested using trypsin and gentle
agitation, washed with PBS and fixed with 70% ethanol. Following
k
4a
8a
3
g
h
a
S
N
i
O
N
7
O
N
N
j
H
rehydration in PBS, cells were stained in PBS containing 2 mg/mL
propidium iodide. Subsequently, cells were analyzed by flow
cytometry, using a FACSCalibur operated by CellQuest software and
at least 10 000 events were collected per sample.
The reaction was carried out using amide 18 (75 mg, 0.20 mmol)
and N,N-dimethylethylenediamine (431
L, 4.00 mmol) at 130 ꢀC
for 3 h. Purification by silica gel chromatography (mixtures of
dichloromethane/methanol of increasing polarity) afforded 45
m
4.2.4. Western blot analysis
For Western Blot analysis MCF-7 and MDA-MB-468 cells were
seeded on a 6-well plate and incubated in 5% CO2 incubator at 37 ꢀC
for 24 h. Cells were treated with various concentrations of drug, 17-
AAG in DMSO (1% DMSO final concentration), or vehicle (DMSO) for
48 h. After 48 h incubation with individual compounds, cells were
washed by PBS and harvested. Cell lysates were obtained by 2 h
treatment with CHiP cell lysis buffer containing protease inhibitors
and phosphatase inhibitors on ice. After sonication, the protein
concentration in the supernatant was measured by BCA assay.
Equal amounts of protein were analyzed by SDS-PAGE, transferred
into PVDF membrane and blocked for 1 h with Western Blocking
Reagent (WBR) containing 0.05% Tween. The samples were sub-
jected to immunoblotting with specific primary antibodies over-
night at 4 ꢀC, followed by washing with TBST for 30 min. The
resulting membrane was exposed into HRP-conjugated secondary
antibody for 1 h at room temperature. After 30 min washing with
TBST, the membrane was developed by ECL prime solution and the
chemiluminescent signal was measured by ChemiDoc MP imaging
system.
(35 mg, 42% yield) as a yellow powder. Mp: 216e217 ꢀC; IR,
n
(cmꢁ1): 3244, 3360 (
n
NeH); 2970 (
n
CeHal); 1638 (
n
C ¼ O); 1593,
1522, 1503 (
n
C ¼ C); 1H NMR (400 MHz, CDCl3)
d 9.18 (s, 1H,
C3eNH), 8.75 (s, 1H, H4), 8.39 (s, 1H, H5), 7.93 (s, 1H, Hb), 7.90e7.88
(m, 2H, Hg, Hd), 7.47e7.41 (m, 2H, Hd, He), 6.15 (s, 1H, H8), 5.64 (br s,
1H, C7eNH), 3.69 (s, 3H, CONCH3), 3.49e3.45 (m, 2H, Hi), 2.67 (t,
3J ¼ 5.6 Hz, 2H, Hj), 2.35 (s, 6H, Hk); 13C NMR (100 MHz, CDCl3)
d
160.55 (NHeC]O), 158.90 (C7), 158.69 (C3eC]O), 150.00 (C5),
143.18 (C8a), 141.39 (Ca), 139.13 (Ch), 138.69 (Cc), 126.65 (C3), 125.52
(Cb), 125.32 (Cf or Ce), 125.07 (Cg or Cd), 123.65 (Ce or Cf), 122.78 (Cf
or Cg), 120.16 (C4), 109.50 (C4a), 88.36 (C8), 57.79 (Cj), 45.01 (2Ck),
39.22 (Ci), 29.84 (CONCH3); MS (ESI) m/z (%): 422.3 (100) [M þ H]þ;
UPLC purity ¼ 97%, Rt 1.96 min; HRMS (ESI): calcd. for C22H23N5O2S
[M þ H]þ 422.1645, found: 422.1634.
4.2. Biological evaluation
4.2.1. Antiproliferative assay (MTT)
Cells were grown in DMEM medium supplemented with L-
glutamine (2 mM), streptomycin (50 units/mL), penicillin (550 ng/
Acknowledgments
mL) and 5% FBS. Cells were grown to confluence in a humidified
The authors gratefully acknowledge support of this project by
atmosphere (37 ꢀC, 5% CO2), seeded (6000/well, 50
mL) in 96-well
ꢀ
the Ligue Contre le Cancer (Comite du Maine-et-Loire et de Loire-
plates, and allowed to attach for 24 h. Compounds at varying
concentrations in DMSO (1% DMSO final concentration) were
added, and cells were returned to the incubator for 48 h. At 48 h,
Atlantique). We thank Dr. Catherine Roullier for the HR MS
measurements.
50
and incubated for 3.5 h. Medium was subsequently removed from
wells and resulting formazan crystals solubilized in 100 L DMSO.
Culture plates were rocked gently for 30 min to solubilize before
optical density was measured using a microplate reader (BioRad) at
570 nm. Cells that incubated in 1% DMSO were used as 100% pro-
liferation (i.e. DMSO ¼ 100% growth) and the relative growth for
each compound concentration was compared to 1% DMSO. IC50
values were calculated from separate experiments performed in
triplicate using GraphPad Prism.
mL of MTT solution (2.5 mg/mL in PBS) was added to each well
Appendix A. Supplementary data
m
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.04.050.
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