Investigation on the 1,6-naphthyridine motif: Discovery and SAR study of 1H-imidazo[4,5-h][1,6]naphthyridin-2(3H)-one-based c-Met kinase inhibitors

Article abstract of DOI:10.1039/c2ob26710a

The 1,6-naphthyridine motif is a multivalent scaffold in medicinal chemistry presenting various bioactivities when properly substituted. By incorporating a cyclic urea pharmacophore into the 1,6-naphthyridine framework through conformationally constraining the 7,8-positions, the resulting 1H-imidazo[4,5-h][1,6]naphthyridin-2(3H)-one was identified as a new class of c-Met kinase inhibitor. A comprehensive SAR study indicated that an N-1 alkyl substituent bearing a terminal free amino group, a hydrophobic substituted benzyl group at the N-3 position and the tricyclic core were essential for retaining effective Met inhibition of the 1H-imidazo[4,5-h][1,6]naphthyridin- 2(3H)-one chemotype. Further introduction of a 4′-carboxamide phenoxy group at the C-5 position significantly improved the potency. The best c-Met kinase inhibitory activity was exemplified by 2t with an IC₅₀ = 2.6 μM, which also displayed effective inhibition against TPR-Met phosphorylation and the proliferation of the BaF3-TPR-Met cells at low micromolar concentrations.

Full text of DOI:10.1039/c2ob26710a

Organic &
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Investigation on the 1,6-naphthyridine motif: discovery
and SAR study of 1H-imidazo[4,5-h][1,6]naphthyridin-
2(3H)-one-based c-Met kinase inhibitors
Cite this: Org. Biomol. Chem., 2013, 11,
1545
Yong Wang,† Zhong-Liang Xu,† Jing Ai,† Xia Peng, Jian-Ping Lin, Yin-Chun Ji,
Mei-Yu Geng* and Ya-Qiu Long*
The 1,6-naphthyridine motif is a multivalent scaffold in medicinal chemistry presenting various bioactiv-
ities when properly substituted. By incorporating a cyclic urea pharmacophore into the 1,6-naphthyridine
framework through conformationally constraining the 7,8-positions, the resulting 1H-imidazo[4,5-h][1,6]-
naphthyridin-2(3H)-one was identified as a new class of c-Met kinase inhibitor. A comprehensive SAR
study indicated that an N-1 alkyl substituent bearing a terminal free amino group, a hydrophobic substi-
tuted benzyl group at the N-3 position and the tricyclic core were essential for retaining effective Met
inhibition of the 1H-imidazo[4,5-h][1,6]naphthyridin-2(3H)-one chemotype. Further introduction of a
4’-carboxamide phenoxy group at the C-5 position significantly improved the potency. The best c-Met
kinase inhibitory activity was exemplified by 2t with an IC50 = 2.6 μM, which also displayed effective inhi-
bition against TPR-Met phosphorylation and the proliferation of the BaF3-TPR-Met cells at low micro-
molar concentrations.
Received 30th August 2012,
Accepted 31st October 2012
DOI: 10.1039/c2ob26710a
www.rsc.org/obc
inhibitors constitute the largest effort within the pharma-
ceutical industry towards c-Met-based therapies.
Introduction
Met is a receptor tyrosine kinase which is activated by its
Small molecule kinase inhibitors targeting the intracellular
specific natural ligand hepatocyte growth factor (HGF, also kinase domain are predicted to inhibit c-Met activity irrespec-
known as scatter factor). Met signaling plays an important tive of the activation mechanism, while most of the other
physiological role in embryogenesis and early development.1 intervention strategies prevent ligand-mediated activation of
However, the dysregulation of c-Met in mature adult tissues the receptor, such as HGF/c-Met biological antagonists and
through overexpression, gene amplification, mutation,
a
antibodies. Therefore, small-molecule c-Met inhibitors could
ligand-dependent autocrine/paracrine loop or interaction with benefit a different, and potentially larger, cancer patient popu-
other receptor families (heterodimerization/cross-talk) can lation. Numerous c-Met kinase inhibitors have been reported
lead to oncogenic transformation.1–4 In fact, aberrant HGF/ in the literature,8–10 which have basically been categorized into
c-Met signaling is found in a variety of human cancers and two classes based on their chemotype and predicted binding
typically associated with poor prognosis, aggressive disease, mode in the c-Met kinase domain.11 Class I inhibitors bind in
increased metastasis and shortened patient survival.1–4 Fur- a U-shaped conformation to the ATP-binding site at the
thermore, constitutive c-Met activation due to MET amplifica- entrance of the kinase pocket and wrap around Met1211,
tion was found to be linked to acquired resistance of lung while class II inhibitors bind to c-Met with an extended confor-
cancers to epidermal growth factor receptor (EGFR) inhibi- mation that stretches from the ATP-binding site, delineated by
tors.5,6 Therefore, targeting the HGF/c-Met signaling pathway the kinase hinge to the deep hydrophobic Ile 1145 pocket near
as a means of cancer therapy has become increasingly popular the C-helix region. Some representative chemical structures
with a number of different therapeutic approaches undergoing from each class are shown in Fig. 1. In general, class I inhibi-
clinical trials,2,4,7 among which small molecule c-Met tors block c-Met kinase activity with high selectivity against
other kinases, whereas a majority of class II molecules are
multikinase inhibitors, bearing the general theme of
a
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,
thiourea/urea or cyclized derivatives of the urea. Recent studies
have suggested that certain mutations near the active site of
c-Met may lead to resistance to class I inhibitors.12,13 In this
regard, class II inhibitors might be more effective against the
Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, P. R. China.
E-mail: yqlong@mail.shcnc.ac.cn, mygeng@mail.shcnc.ac.cn; Fax: +86-21-50806876,
+86-21-50806072; Tel: +86-21-50806876, +86-21-50806072
†These authors equally contributed to this work.
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the pharmacophore urea group into the core structure by con-
straining the 7,8-positions of 1,6-naphthyridine into an imida-
zolidin-2-one ring (Scheme 1). Encouragingly, the resulting
1,3,5-trisubstituted-1H-imidazo[4,5-h][1,6]naphthyridin-2(3H)-
ones effectively inhibit the c-Met kinase as well as the phos-
phorylation of c-Met in TPR-Met-transformed BaF3 cell lines
which stably express a constitutively active, ligand-indepen-
dent, oncogenic form of c-Met. Furthermore, these new
scaffold c-Met inhibitors significantly inhibit the proliferation
of BaF3-TPR-Met cells at low micromolar concentrations.
Herein we report the synthesis, structure–activity relationship
study and biological evaluation of this new class of c-Met
kinase inhibitors, which were predicted to function as class II
inhibitors by molecular modeling. Based on this promising
new chemotype c-Met inhibitor and its SAR information dis-
closed here, further effort to improve the potency and selectiv-
ity over other protein kinases is under way.
Fig. 1 Examples of class I and class II c-Met kinase inhibitors.
mutations that disrupt the class I binding mode since their
binding interactions extend the entrance of c-Met active site.
Moreover, the selectivity profiles of class II c-Met inhibitors
can be significantly improved by structure-based optimization
to avoid off-target side effects.14
Chemistry
Our interest in the 1,6-naphthyridine-7-carboxamide motif
was initiated by the HIV-1 integrase inhibitor L-870,810,15 The synthesis of 5,7-substituted-1,6-naphthyridine-7-carboxa-
which entered clinical trials as an antiviral drug with favorable mides (1a–m) and the conformationally constrained 1,3,5-sub-
pharmacological and pharmaceutical properties but was termi- stituted-1H-imidazo[4,5-h] [1,6]naphthyridin-2(3H)-one derivatives
nated due to liver and kidney toxicity observed in dogs.15,16 2a–v is depicted in Schemes 2 and 3. The key precursor, 5-
The drug repurposing study on L-870,810 with an amino sub- halo-8-hydroxy-1,6-naphthyridine-7-carboxylate (4a–b) was pre-
stituent at the 8-position and proper benzyl substitution at the pared conveniently according to our modified methodology,17
7-position resulted in the discovery of novel antitumor from which 7-various amido substituted-1,6-naphthyridines
agents.17 The replacement of the hydroxyl group with a (5a–l) and 8-various amino substituted-1,6-naphthyridines
diamino structure at the 8-position was the key to the activity (9a–d) were prepared via amidation and aromatic nucleophilic
switch from HIV-1 inhibition to tumor cytotoxicity. During the substitution, respectively (Scheme 2). The introduction of a
target identification, a kinase profiling assay indicated that the
5,8-substituted-1,6-naphthyridine-7-carboxamide
derivatives
(lead compound LNZ-57, Scheme 1) have inhibitory activity on
multiple kinases, including Lck, IGF-1R, Abl (T315I), FGFR,
Met and ALK.17 Furthermore, the 1,6-naphthyridines were
reported to inhibit FGFR-1 and VEGFR-2 kinases when substi-
tuted at positions 2 and 7 by amino and 3 by aryl groups
suggesting that various substitutions on this active scaffold
could confer diverse biological activities.18,19 Therefore, we
were intrigued to develop novel class II c-Met inhibitors based
on the 1,6-naphthyrdine-7-carboxamide framework by varying
the substitution at the 5,8-position and further introducing
Scheme 2 Reagents and conditions: (a) NHR2, toluene, reflux; (b) TsCl, TEA,
CH2Cl2, reflux; (c) tert-butyl ((1r,4r)-4-aminocyclohexyl)carbamate, or NHR3, THF,
reflux; (d) TFA, CH2Cl2, rt, or TFA (trace H2O), CH2Cl2, 30 °C (for the synthesis of
1m); (e) methyl propiolate, Pd(PPh3)2Cl2, CuI, K2CO3, THF, reflux; (f) LiOH, THF–
H2O, rt; (g) toluene, DPPA, TEA, reflux; (h) R2Br, K2CO3, DMF, rt (for synthesis of
2b–e) (note: condition d for 2a; h, d for 2f–l); (i) R2 substituted sulfonyl chloride,
TEA, THF, reflux.
Scheme 1 Design of 1H-imidazo[4,5-h][1,6]naphthyridin-2(3H)-one-based
c-Met inhibitors starting from the 1,6-naphthyridine-7-carboxamide scaffold.
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Scheme 4 Reagents and conditions: (a) 3,4-dichlorobenzyl bromide, NaH, THF,
rt; (b) methyl 4-hydroxybenzoate, NaH, DMF, 80 °C; (c) NiCl2, NaBH4, 0 °C; (d)
BTC, cat. DMF, THF, rt; (e) LiOH, THF–H2O, rt; (f) NHR1, EDCI, HOBt, CH2Cl2, rt.
Scheme 3 Reagents and conditions: (a) 3,4-dichlorobenzyl bromide, NaH, THF,
rt; (b) ROH, K2CO3, DMF, 110 °C; (c) TFA, CH2Cl2, rt; (d) NaBH4, THF, rt for 2 h; (e)
LiOH, THF–H2O, rt; (f) R’NH2, BOP, DIEA, DCM, rt.
Scheme 5 Reagents and conditions: (a) trans-1,4-diaminocyclohexane, TEA,
MeCN, 40 °C; (b) (Boc)2O, TEA, MeCN, 50 °C; (c) methyl 4-hydroxybenzoate,
NaH, DMF, 80 °C; (d) NiCl2, NaBH4, 0 °C; (e) BTC, catalytic DMF, THF, rt; (f) 3,4-
dichlorobenzyl bromide, NaH, THF, rt; (g) LiOH, THF–H2O, rt; (h) NHR1, EDCI,
HOBt, CH2Cl2, rt; (i) TFA, CH2Cl2, rt.
propiolate group at position 5 (i.e. compound 1l) was achieved
by Sonogashira coupling of the 8-amino-5-iodo-1,6-naphthyri-
dine-7-carboxamide (7l) with methyl propiolate. Treatment
with trace water containing TFA at 30 °C easily converted the 5-
propiolate 1,6-naphthyridine into the 5-β-keto propanoate
counterpart (1m).
Since the 1,3,5-substituted-1H-imidazo[4,5-h][1,6]naphthyri-
din-2(3H)-one represents a new chemotype to inhibit c-Met
kinase, an extensive SAR study was conducted to explore the
pharmacophores and guide further structural optimization.
The simplified core structure, 1H-imidazo[4,5-b]pyridin-2(3H)-
one was designed and three substitution patterns were exam-
ined while the privileged structure determined from 1H-
imidazo[4,5-h][1,6]naphthyridin-2(3H)-one series was installed
at the C-5 position (Schemes 4 and 5, Compounds 3a–o).
As depicted in Schemes 4 and 5, the 1H-imidazo[4,5-b]-
pyridin-2(3H)-one nucleus was constructed from commercially
available 2-amino-6-chloro-3-nitropyridine or 3,6-dichloro-2-
nitropyridine through triphosgene-mediated tandem reaction.
Briefly, the nitro group on the pyridine ring was reduced into
an amino group by NiCl2–NaBH4 in high yield. The resulting
primary amine was treated with triphosgene [bis(trichloro-
methyl)carbonate, BTC) and a catalytic amount of DMF in THF
to form the isocyanate followed by an intramolecular nucleo-
philic substitution to furnish the cyclic urea structure (18a–b
and 23). The 1,3,5-substituent was introduced by nucleophilic
For the construction of the 1H-imidazo[4,5-h][1,6]naphthyri-
din-2(3H)-one nucleus, we established an efficient methodo-
logy to constrain the 7-carboxyl and 8-amino groups at the 1,6-
naphthyridine ring into 1H-imidazol-2(3H)-one as a cyclic urea
derivative (Scheme 2). By employing
a tandem Curtius
rearrangement and intramolecular nucleophilic substitution
strategy, the 8-amino substituted-1,6-naphthyridine-7-car-
boxylic acid (9a–d) was transformed into acyl azide by reacting
with diphenylphosphoryl azide (DPPA) under basic conditions
in refluxing toluene, which immediately underwent Curtius
rearrangement to generate an isocyanate in situ followed by an
attack of the neighboring 8-amino group to form the cyclic
urea functionality (10a–d). Various substituents can be intro-
duced into the 3-position on the 1H-imidazo[4,5-h][1,6]-
naphthyridin-2(3H)-one scaffold by nucleophilic substitution
(2a–l) or sulfonamidation reaction (2m–n).
The 5-substituted 1H-imidazo[4,5-h][1,6]naphthyridin-2-
(3H)-one derivatives (2o–v) were synthesized from the key inter-
mediate 10a by Williamson etherification reaction (Scheme 3).
The 5-bromo tricyclic urea 2b was heated in DMF with substi-
tuted phenol and K2CO3 to afford 5-phenyloxy substituted 1H-
imidazo[4,5-h][1,6]naphthyridin-2(3H)-ones (11o–s). Then Boc
cleavage of 1-(4-aminocyclohexyl) by TFA delivered the 5-sub-
stituted products 2o–r. It is noteworthy that the 5-hydroxy sub-
stituted analog 11o was accidentally obtained by the hydrolysis
of intermediate 2b under the Williamson ether synthesis con-
ditions. Further transformation was performed to introduce
more structural variation on the 5-phenoxyl ring to further
explore the SAR of the 5-position, e.g. the reduction of the alde-
hyde (2s) and the hydrolysis of the ester followed by amidation
affording the desired products (2t–v).
substitution and Williamson etherification reaction in
a
different sequence dependent on the substitution pattern. For
example, 3,5-substituted 1H-imidazo[4,5-b]pyridin-2(3H)-ones
(18a) was synthesized by a sequential nucleophilic displace-
ment of the 2-amino-6-chloro-3-nitropyridine, Williamson
ether synthesis and the formation of the cyclic urea moiety,
whereas 1,5-substituted 1H-imidazo[4,5-b]pyridin-2(3H)-ones
(18b) required Williamson ether synthesis prior to the nucleo-
philic substitution then the similar construction of the 1H-imi-
dazol-2(3H)-one ring by reacting with triphosgene. Further
saponification and coupling with different amines delivered
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various 1,3,5-substituted-1H-imidazo[4,5-b]pyridin-2(3H)-one scaffold to achieve effective Met kinase inhibition. Consistent
derivatives (3a–j). with the SAR established in the 1,6-naphthyridine-7-carboxa-
For the synthesis of 4-(1-(4-aminocyclohexyl)-3-(3,4-dichloro- mide series, a dramatic increase in enzyme potency was
benzyl)-2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-5-yloxy)-N- observed upon installation of the hydrophobic substituent at
substituted benzamide series (3k–o), starting from the 3,6- the 2, 3 or 4-position of the phenyl ring (Table 2, 2h–l com-
dichloro-2-nitropyridine, nucleophilic displacement of the 3- pared to 2g). Consistently, the replacement of the benzyl
chloro group by trans-1,4-diaminocyclohexane gave the 3-ami- moiety by a more polar sulfonyl substituent at the N-3 position
nocyclohexyl substituted analog 20, which underwent similar impaired the Met inhibitory activity (2m–n), among which the
transformations as 13 to generate the target products relatively hydrophobic 3,4-dichlorobenzenesulfonyl group
(Scheme 5).
partly restored the activity (2n). Thus, the optimal 3,4-dichloro-
benzyl moiety at the N-3 position was held constant as a
further structural variation on the C-5 substitution was investi-
gated to improve the potency.
Results and discussion
SAR and lead generation
Gratifyingly, most of the 5-aryloxy substituted 1H-imidazo-
[4,5-h][1,6]naphthyridin-2(3H)-one
derivatives
gave
an
Initial efforts were directed at exploring the SAR of the improved Met kinase inhibitory activity (2p–v), especially an
8-amino substituted-1,6-naphthyridine scaffold with structural additional introduction of the benzylamino or aliphatic cyclic
variation on the 7-carboxamide portion. Encouragingly, these amino group such as piperidin-1-yl or morpholino on the phe-
analogs were active in inhibiting the Met kinase in low to mid noxyl ring by a carboxamide bridge greatly advanced the
micromolar concentration range, representing a new chemo- enzyme inhibition (2t, IC50 = 2.6 μM; 2u, IC50 = 2.8 μM; 2v,
type of c-Met inhibitors. The variation on the C-7 substituent IC50 = 6.1 μM). The hydrophobic structure attached to the
remarkably impacted the enzymatic activity, with a hydro- 4′-carboxamide site of the 5-phenoxyl ring seemed beneficial
phobic substituent being more favorable on the phenyl ring for the activity. However, a formyl group substituted at the phe-
(exemplified by compound 1f, IC50 = 6.1 μM, Table 1). A polar noxyl ring was unfavorable for the c-Met inhibition (2q, 2r).
substituent such as an amino group was disfavored at the 4′- Another unusual activity change was observed on the simple
position of the 7-benzyl ring (1g). Accordingly, the replacement 5-hydroxy analog which displayed an equally potent inhibition
of the 7-phenyl ring by a heterocycle such as a pyridine, thio- against c-Met kinase (2o, IC50 = 3.5 μM) compared to the
phene or indole ring resulted in a big loss of the Met inhibi- 5-phenoxyl substituted derivatives (2t–v). We proposed that the
tory activity (1h–j), while the hydrophobic naphthalene two structures might adopt a different conformation when
displacement maintained a moderate inhibition effect (1k). binding to the ATP binding pocket since they bear different
Introduction of the propiolate group, as a Michael reaction substitution.
acceptor, at the 5-position had no potentiating effect (1l), but
Inspired by this observation, we envisioned that the bulky
its hydrolysis derivative β-ketoester showed an improved tricyclic core structure might be truncated into a bicyclic struc-
potency (1m, IC50 = 6.6 μM), indicating that substitution at the ture to gain deep access to the ATP binding pocket. On the
5-position had a subtle effect on the c-Met inhibition thus this other hand, for this new scaffold Met inhibitor, we need more
was an optimizable site.
SAR information to reveal the structural requirements. By
In order to improve the c-Met potency of the 1,6-naphthyri- installing the privileged structures as constant substituents at
dine based inhibitors, we incorporated the urea pharmaco- positions N-1, N-3 and C-5, three substitution patterns were
phore, typically carried by the class II inhibitors, into the new examined. However, when 1H-imidazo[4,5-h][1,6]naphthyridin-
scaffold. Interestingly, the conformationally constrained C-7 2(3H)-one was truncated into 1H-imidazo[4,5-b]pyridin-2(3H)-
and C-8 analogs with a cyclic urea moiety turned out superior one, the Met inhibitory activity was generally sacrificed regard-
to the 8-amino-1,6-naphthyridine-7-carboxamides in inhibiting less of the substitution pattern, only the 1-(4-aminocyclo-
Met kinase and proliferation of BaF3-TPR-Met cells that hexyl)-3-(3,4-dichlorobenzyl)-substituted series stably exhibited
contain an overexpression level of Met gene (Table 2, 2f vs. 1c). a moderate level of enzyme inhibition at a concentration of
Structural variation at the N-1 position of the 1H-imidazo[4,5-h]- 10 μM (Table 3). The negative results indicated that the 1H-
[1,6]naphthyridin-2(3H)-one core by replacing the 4-aminocyclo- imidazo[4,5-h][1,6]naphthyridin-2(3H)-one framework played
hexyl group with a cyclohexyl, benzyl, cyclopropyl or 4-(tert- an important role in the binding to the c-Met kinase, probably
butoxycarbonylamino)cyclohexyl group all led to inactivation being involved in the interaction with the residues of the
(2b–e), indicating the critical role of the free amino substituent kinase protein and orienting the essential substituents into an
at the N-1 position in the Met kinase inhibition. Therefore, the appropriate region in the binding pocket.
4-aminocyclohexyl moiety at the N-1 position was held constant
as the substituent at the N-3 or C-5 position was explored to
In vitro cell assays
achieve a comprehensive SAR study and enhanced potency.
Some of the more potent compounds that were identified in
The N-3 unsubstituted analog 2a was inactive in the enzy- the enzyme assay were further tested for their ability to inhibit
matic assay suggesting that the hydrophobic benzyl group was c-Met signaling in cellular settings. The TPR-Met-transformed
essential for the 1H-imidazo[4,5-h][1,6]naphthyridin-2(3H)-one BaF3 cells were selected for this purpose, which stably express
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Table 1 c-Met enzymatic activity of 5,7-substituted-1,6-naphthyridine-7-carboxamidesa
c-Met inhibition
% at 10 μM
19.6
Compd.
R2
R1
Br
IC50 (μM)b
1a
>10
1b
1c
1d
Br
Br
Br
24.0
36.6
45.4
>10
>10
>10
1e
1f
Br
Br
58.4
50.6
NDc
6.1 0.51
1g
1h
Br
Br
25.6
9.4
>10
>10
1i
1j
Br
Br
24.9
10.6
>10
>10
1k
Br
42.8
>10
1l
34.6
54.6
>10
1m
6.6 1.9
a See the Experimental section for assay details. b IC50’s were calculated by the Logit method from the results of at least two independent tests
with six concentrations each and expressed as means SD. c Not determined.
a constitutively active, ligand-independent, oncogenic form of proliferation, we first evaluated the inhibitory effect of these
c-Met.20,21 Since activation of c-Met ultimately results in cell c-Met kinase inhibitors on the growth of BaF3-TPR-Met cell
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Table 2 c-Met enzymatic activity of 1,3,5-substituted-1H-imidazo[4,5-h][1,6]naphthyridin-2(3H)-one derivativesa
c-Met inhibition
Compd.
R3
R2
H
R1
Br
% at 10 μM
IC50 (μM)b
2a
0.0
—
2b
2c
2d
2e
2f
Br
Br
Br
Br
Br
0.0
0
—
—
0
—
0
—
55.6
8.7 0.1
2g
2h
Br
Br
0.0
—
23.0
>10
2i
2j
Br
Br
21.7
16.1
>10
>10
2k
2l
Br
Br
Br
52.3
30.7
6.5
NDc
>10
—
2m
2n
2o
Br
40.6
82.4
>10
OH
3.5 2.2
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Table 2 (Contd.)
c-Met inhibition
% at 10 μM
54.3
Compd.
R3
R2
R1
IC50 (μM)b
2p
12.7 2.9
2q
2r
2s
2t
30.3
20.4
65
>10
>10
7.7 1.5
2.6 0.5
64.2
2u
2v
74.7
57
2.8 1.1
6.1 1.2
a See the Experimental section for assay details. b IC50’s were calculated by the Logit method from the results of at least two independent tests
with six concentrations each and expressed as means SD. c Not determined.
lines. As shown in Table 4, the 1,3,5-trisubstituted-1H-imidazo- activity and almost consistent with the antiproliferative activity
[4,5-h][1,6]naphthyridin-2(3H)-one derivatives (2o–p, 2s–v) sig- in BaF3 cells. However, the 2-methylbenzyl analog 2v displayed
nificantly inhibited the cell growth at 10 μM, while only a marginal inhibitory effect on TPR-Met phosphorylation. As a
compound 2t retained good inhibitory effect at 1 μM concen- comparison, the open frame 5,7,8-substituted 1,6-naphthyri-
tration. Of note, the trends observed for enzyme potency for dine-7-carboxamide derivatives 1m and 1f lacked activity
these compounds correlated with those that were observed for against the phosphorylation of c-Met protein even at a concen-
their cellular activities. Obviously, the urea-containing 1H- tration of 10 μM, in agreement with the cell growth assay. Con-
imidazo[4,5-h][1,6]naphthyridin-2(3H)-one, especially bearing sequently, compound 2t stood out as a promising lead for
a 5-phenoxy substituent, is superior to the 1,6-naphthyridine- further structural optimization to gain improved potency and
7-carboxamide in cellular potency.
selectivity.
Then some selected compounds with representative struc-
tures (1m, 1f, 2t, 2v) were further assessed for their ability to
inhibit the phosphorylation of c-Met receptor in TPR-Met-
transformed BaF3 cells. The phosphorylation levels of the Met
protein determined by Western blotting are illustrated in
Fig. 2. Significantly, the 1,3,5-trisubstituted 1H-imidazo[4,5-h]-
[1,6]naphthyridin-2(3H)-one derivative 2t effectively inhibited
the TPR-Met phosphorylation in BaF3-TPR-Met cells in a dose-
dependent manner, consistent with the enzyme inhibitory
Kinase selectivity of the 1H-imidazo[4,5-h][1,6]naphthyridin-2-
(3H)-one scaffold
Since our initial investigation on the 1,6-naphthyridine-7-car-
boxamide structure disclosed its multiple kinase inhibitory
property, we were interested in examining the kinase selectivity
of the newly developed 1H-imidazo[4,5-h][1,6]naphthyridin-2-
(3H)-one scaffold. The lead compound 2t was selected for
kinase profiling against c-Met family member, Ron, along with
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Table 3 c-Met enzymatic activity of 1,3,5-substituted-1H-imidazo[4,5-b]pyridin-2(3H)-one derivativesa
Compd.
R3
H
R2
R1
c-Met inhibition % at 10 μM
3a
27.7
3b
3c
3d
3e
H
H
H
H
0
0
21.9
17.2
3f
H
13.6
3g
3h
3i
H
H
H
H
8.7
22.3
4.3
3j
11
3k
3l
OMe
40.0
33.6
26.8
23.0
3m
3n
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Table 3 (Contd.)
Compd.
R3
R2
R1
c-Met inhibition % at 10 μM
3o
29.8
a See the Experimental section for assay details.
Table 4 Inhibitory effect of selected compounds on BaF3 cell proliferationa
Table 5 Effect of 2t on a panel of RTKs at 10 μM concentrationa
Inhibition rate (%)
RTK
Inhibition rate (%)
Compd
1m
1f
10 μM
1 μM
0.1 μM
Ron
Tyro3
Mer
IGF1R
PDGFR-α
PDGFR-β
FGFR1
ErbB4
Flt-1
KDR
c-Kit
RET
EGFR
ABL
77.3
57.2
29.5
24.5
81.6
54.2
79.6
74.4
72.8
84.0
75.7
83.7
63.3
63.2
86.4
20.7
22.6
10.1
8.0
5.7
2.6
2.3
−0.5
1.9
6.6
3.6
2.8
−0.2
19.3
0.4
−4.6
−4.0
−3.8
−3.4
−3.2
−0.8
−5.0
−2.2
−8.5
−6.4
−6.0
−2.6
2f
−1.3
12.1
82.9
86.4
70.5
69.8
90.5
90.2
85.9
85.7
78.5
79.5
67.4
68.3
0.4
2o
−4.9
−5.5
−2.9
−2.7
0.8
2p
2s
4.2
2t
63.1
64.8
−1.5
−6.2
1.2
EPH-A2
a The kinase profiling assay was conducted as described in the
Experimental section by using ELISA kinase assay. Each reference
compound and its inhibition rate for every kinase at a concentration of
1 μM was indicated below: PF0234106 for RON (91.9%); XL880 for
Tyro3 (80.8%) and Mer (59.1%); CCA9 for IGF1R (90.8%); Su11248 for
2u
2v
5.8
a The inhibition ratio (%) of cell proliferation was calculated using the PDGFR-α (98.1%), PDGFR-β (96.4%), Flt-1 (83.2%), KDR (88.1%), c-Kit
following equation: [1 − (A570/A570 control)] × 100%.
(96.5%) and RET (78.9%); BIBW2992 for ErbB4 (96.8%) and EGFR
(97.5%); Dasatinib for ABL (89.4%) and EPH-A2 (96.7%).
RET, EGFR, ABL, and EPH-A2 (Table 5). As shown in Table 5,
besides being an effective c-Met inhibitor, 2t also displayed
more than 70% inhibition for RON, PDGFR-α, FGFR1, ErbB4,
Flt-1, KDR, c-Kit, RET, and EPH-A2 kinase at a concentration
of 10 μM. This result demonstrated the 1H-imidazo[4,5-h][1,6]-
naphthyridin-2(3H)-one scaffold was a multi-target kinase
inhibitor, which is consistent with the observation that 2t
exhibited a more potent antiproliferative effect with a sub-
micromolar IC50 value than its c-Met kinase enzyme as well as
Met phosphorylation inhibitory activity. However, 2t still dis-
played good selectivity over some other kinases such as Mer
and IGF1R; moreover, its kinase profile has changed distinctly
compared to 1,6-naphthyridine-7-carboxamide chemotype,17
Fig. 2 Effects of selected compounds on TPR-Met phosphorylation in BaF3-
TPR-Met cells.
14 other oncogenic tyrosine kinases including Tyro3, Mer,
IGF1R, PDGFR-α, PDGFR-β, FGFR1, ErbB4, Flt-1, KDR, c-Kit,
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point for lead optimization and further development of new
structure targeted cancer therapeutics.
Experimental section
Chemistry
Unless otherwise noted, all reagents and solvents were
obtained from commercial suppliers and used without further
purification. Dry solvents were purified and stored according
to standard procedures. TLC was performed on F254 plates of
0.25 mm thickness. Products were visualised under UV light
(254 nM) and/or stained with iodine, potassium permanganate
or phosphomolybdic acid solutions followed by heating. Stan-
dard silica gel (SiO2) column chromatography was employed as
a method of purification using the indicated solvent mixtures.
1H NMR spectra were recorded at Varian Mercury-400 or
300 MHz and 13C NMR spectra at Varian Mercury-100 MHz.
Mass spectra were recorded using a Finnigan MAT-95 instru-
ment for EI and Finnigan LCQ Deca for ESI. Elemental analy-
sis were performed on a CE 1106 elemental analyzer.
Fig. 3 A proposed binding mode of the lead compound 2t to the Met kinase
domain, based on the X-ray co-crystal structure of c-Met in complex with a small
molecule inhibitor.22
implying the great potential and promise of the lead structure
to be evolved into selective c-Met kinase inhibitors by further
structural optimization.
Binding mode predicted by molecular modeling
Based on the X-ray co-crystal structure of the class II c-Met
inhibitor bound to the Met kinase domain (PDB: 3C1X),22 we
established the binding mode of lead compound 2t in the Met
kinase domain by autodocking. As demonstrated by Fig. 3, the
1H-imidazo[4,5-h][1,6]naphthyridin-2(3H)-one based c-Met
inhibitor 2t adopted an extended conformation in the ATP-
binding site of the enzyme with the activation loop in an inac-
tive, DFG-out conformation. However, the heterotricyclic core
resides in the middle of the channel between the hinge region
and the DFG motif, due to the bulky size, thus losing the key
hydrogen bonding interactions with Met1160. That might be
the major reason why the kinase inhibitory activity of this che-
motype fell in the low micromolar range. However, the carbo-
nyl oxygen in the 1H-imidazole ring formed a hydrogen bond
with the NH of Asp1222, and the 3-(3,4-dichlorobenzyl) and
1-(4-aminocyclohexyl) groups were favorably positioned into
the hydrophobic pocket and solvent area, respectively. More-
over, the 5-(4-(piperidine-1-carbonyl)phenoxy) moiety engages
in the hydrophobic interaction within the hinge region.
GENERAL PROCEDURE FOR THE PREPARATION OF 8-AMINO-5-BROMO-1,6-
NAPHTHYRIDINE-7-CARBOXAMIDE
COMPOUNDS
(1A–1K).
METHOD
A. Taking the synthesis of 1d as an example. A solution of (4-
chloro-3-(trifluoromethyl))methanamine (86 mg, 0.41 mmol)
and methyl 5-bromo-8-hydroxy-1,6-naphthyridine-7-carboxylate
(4a) (97 mg, 0.34 mmol) in toluene (2 mL) was heated to reflux
for 20 h. Upon cooling to room temperature, the precipitate
was collected by filtration and washed with small amount of
toluene to afford 5d as a white solid (116 mg, yield 74.1%),
which was used for the next step directly.
The intermediate 5d (116 mg, 0.25 mmol) was dissolved in
dry DCM (5 mL) under nitrogen. Methylbenzenesulfonyl chlor-
ide (95 mg, 0.5 mmol) and TEA (0.1 mL) were added. The
resulting solution was heated to 40 °C and stirred for 12 h.
The reaction mixture was then partitioned between water and
dichloromethane. The aqueous layer was then extracted with
dichloromethane (20 mL × 3). The combined organic extracts
were washed with saturated NH4Cl solution (20 mL × 2) and
saturated NaCl solution (20 mL × 1). The organic layer was
dried over Na2SO4 and evaporated. The residue was purified by
Conclusions
Starting from the active scaffold of 1,6-naphthyridine, the 1H- column chromatography on silica gel eluting with PE : EtOAc
imidazo[4,5-h][1,6]naphthyridin-2(3H)-one based c-Met inhibi- (1 : 1) to give 6d as a white solid (75 mg, yield 48.5%): 1H NMR
tors were designed by incorporation of the urea pharmaco- (300 MHz, CDCl3): δ 9.01 (d, J = 4.2 Hz, 1H), 8.57 (d, J = 8.1 Hz,
phore.
A systematic SAR study revealed the privileged 1H), 8.09 (t, J = 6.3 Hz, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.67–7.7
structures at the 1,3,5-positions and emphasized the impor- (m, 2H), 7.47–7.56 (m, 2H), 7.31 (d, 2H), 4.68 (d, J = 6.3 Hz,
tant role of the tricyclic core structure. This new chemotype 2H), 2.46 (s, 3H).
effectively inhibits the c-Met kinase as well as the phosphoryl-
The precursor 6d (75 mg, 0.12 mmol) was dissolved in dry
ation of TPR-Met in the TPR-Met-transformed BaF3 cell line THF (10 mL) under nitrogen. (1r,4r)-Cyclohexane-1,4-diamine
which contains an amplified MET gene locus in a dose-depen- (65 mg, 0.57 mmol) and TEA (0.1 mL) were added, and the
dent manner. More significantly, these new scaffold c-Met reaction mixture was heated to 70 °C and stirred for 12 h. After
inhibitors effectively inhibit the proliferation of BaF3-TPR-Met the reaction was completed, the reaction solution was concen-
cells at low to sub-micromolar concentrations. Molecular mod- trated. The residue was diluted with dichloromethane (20 mL).
eling predicted the 1H-imidazo[4,5-h][1,6]naphthyridin-2(3H)- Usual work-up with the organic phase and purification by
one based Met inhibitors functioned as class II inhibitor with column chromatography on silica gel eluting with PE : EtOAc
multiple kinase inhibition, providing a promising starting (1 : 1) afforded target compound 1d as a light yellow solid
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1
(40 mg, yield 59%): H NMR (300 MHz, CDCl3): δ 9.54 (d, J = prepared analogously using general method A, giving a light
8.1 Hz, 1H), 8.95 (d, J = 3.9 Hz, 1H), 8.41 (m, 2H), 7.68 (s, 1H), yellow solid, yield 30.5%: 1H NMR (300 MHz, CDCl3): δ 9.69 (d,
7.6 (dd, J = 8.1, 3.9 Hz, 1H), 7.48 (m, 2H), 4.97 (m, 1H), 4.64 (d, J = 8.1 Hz, 1H), 8.95 (d, J = 4.2 Hz, 1H), 8.4 (d, J = 8.7 Hz, 1H),
J = 6.9 Hz, 2H), 2.74 (m, 1H), 2.17 (d, J = 14.4 Hz, 2H), 1.9 (d, 8.29 (t, J = 6 Hz, 1H), 8.12 (d, J = 7.5 Hz, 1H), 7.82–7.91 (m,
J = 12 Hz, 2H), 1.26–1.43 (m, 4H); MS-EI m/z: 555 (M)+, 557 2H), 7.44–7.6 (m, 5H), 5.10 (d, J = 6 Hz, 2H), 4.98 (m, 1H), 2.82
(M + 2)+; Anal. calcd for C23H22BrClF3N5O·1/4CH3OH: C 49.44, (m, 1H), 2.21 (d, J = 12 Hz, 2H), 1.96 (d, J = 11.4 Hz, 2H),
H 4.10, N 12.40, found: C 49.74, H 4.32, N 12.06%.
1.29–1.52 (m, 4H); MS-EI m/z: 503 (M)+, 505 (M + 2)+; Anal.
Compounds 1a, 1b, 1g, 1h and 1i were prepared according Calcd for C26H26BrN5O: C 61.91, H 5.20, N 13.88, found:
to the same procedure reported previously.17
C 61.80, H 5.55, N 13.73%.
8-(((1R,4R)-4-AMINOCYCLOHEXYL)AMINO)-5-BROMO-N-(3,4-DICHLOROBENZYL)-
1,6-NAPHTHYRIDINE-7-CARBOXAMIDE (1C). 1c was prepared analogously
GENERAL PROCEDURE FOR THE PREPARATION OF 5-PROPIOLATE ESTER OR
β-KETO
ESTER-1,6-NAPHTHYRIDINE-7-CARBOXAMIDE
COMPOUNDS
(1L–
using general method A, giving a light yellow solid, yield 1M). Method B. 7l was prepared analogously following Method
1
1
73.4%: H NMR (300 MHz, CDCl3): δ 9.56 (d, 1H, J = 8.1 Hz), A, giving a light yellow solid, yield 74.2%: H NMR (300 MHz,
8.94 (d, 1H, J = 3.9 Hz), 8.42–8.35 (m, 3H), 7.59 (dd, 1H, J = 4.2, CDCl3): δ 9.58 (d, J = 8.1 Hz, 1H), 8.88 (d, J = 4.8 Hz, 1H), 8.41
8.7 Hz), 7.44–7.38 (m, 2H), 7.20 (m, 1H), 4.96 (m, 1H), 4.57 (d, (t, J = 6.6 Hz, 1H), 8.25 (d, J = 9.9 Hz, 1H), 7.68 (s, 1H), 7.58
2H, J = 6.0 Hz), 2.76 (m, 1H), 2.18 (m, 2H), 1.91 (m, 2H), 1.37 (dd, J = 4.8, 9.9 Hz, 1H), 7.46–7.52 (m, 2H), 4.96 (m, 1H), 4.65
(m, 4H); EI-MS m/z: 521 (M)+, 523 (M + 2)+.
(d, J = 6.6 Hz, 2H), 4.39 (br s, 1H), 3.48 (m, 1H), 2.18 (d, J =
8-((1R,4R)-4-AMINOCYCLOHEXYLAMINO)-5-BROMO-N-(4-(BENZYLOXY)BENZYL)- 12.6 Hz, 2H), 2.06 (d, J = 13.8 Hz, 2H), 1.44 (s, 9H), 1.23–1.38
1,6-NAPHTHYRIDINE-7-CARBOXAMIDE (1E). 1e was prepared analogously (m, 4H).
using general method A, giving a light yellow solid, yield
7l (0.104 g, 0.15 mmol), Pd(PPh3)2Cl2 (4 mg), CuI (2 mg),
1
56.2%: H NMR (300 MHz, CDCl3): δ 9.66 (d, J = 8.1 Hz, 1H), and anhydrous potassium carbonate (50 mg, 0.36 mmol) were
8.94 (d, 1H, J = 4.2 Hz), 8.41 (d, 1H, J = 10.2 Hz), 8.25 (t, J = dissolved in dry THF (3 mL) under nitrogen. Methyl propiolate
6.3 Hz, 1H), 7.58 (dd, 1H, J = 4.2, 10.2 Hz), 7.3–7.45 (m, 9H), (60 μL) was added and the reaction mixture was heated to
6.96 (d, 2H, J = 8.7 Hz), 5.06 (s, 1H), 4.94 (m, 1H), 4.56 (d, J = 65 °C and stirred for 12 h. After the reaction was completed,
6.3 Hz, 2H), 2.76 (m, 1H), 2.17 (d, 2H, J = 12.3 Hz), 1.9 (d, 2H, the reaction solution was concentrated. The residue was dis-
J = 13.5 Hz), 1.39 (m, 4H); MS-EI m/z: 559 (M)+; HR-EIMS calcd solved in dichloromethane (20 mL). The DCM layer was
for C29H30BrN5O2: 559.1583, found: 559.1588.
washed with saturated NH4Cl solution (20 mL × 2) and satu-
8-((1R,4R)-4-AMINOCYCLOHEXYLAMINO)-5-BROMO-N-(4-(CYCLOHEXYLOXY)- rated NaCl solution (20 mL × 1). The organic layer was dried
3-(TRIFLUOROMETHYL)BENZYL)-1,6-NAPHTHYRIDINE-7-CARBOXAMIDE (1F). 1f over Na2SO4 and evaporated. The residue was purified by
was prepared analogously using general method A, giving a column chromatography on silica gel eluting with PE : EtOAc
light yellow solid, yield 61.8%: 1H NMR (300 MHz, CDCl3): δ (1 : 1) to give 5-propiolate ester compound as a light yellow solid
1
9.61 (d, J = 8.4 Hz, 1H), 8.95 (d, J = 2.7 Hz, 1H), 8.42 (d, J = 8.7 76 mg, yield 77.8%: H NMR (300 MHz, CDCl3): δ 10.35 (d, J =
Hz, 1H), 8.29 (t, J = 6.3 Hz, 1H), 7.59 (dd, J = 2.7, 8.7 Hz, 1H), 8.4 Hz, 1H), 8.93 (d, J = 4.2 Hz, 1H), 8.67 (t, J = 6.6 Hz, 1H), 8.51
7.55 (s, 1H), 7.46 (d, J = 6.3 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H), (d, J = 8.1 Hz, 1H), 7.68 (s, 1H), 7.60 (dd, J = 4.2, 8.1 Hz, 1H),
4.98 (m, 1H), 4.57 (d, J = 6.3 Hz, 2H), 4.39 (m, 1H), 2.83 (m, 7.46–7.52 (m, 2H), 5.11 (m, 1H), 4.63 (d, J = 6.6 Hz, 2H), 4.42 (br
1H), 2.19 (m, 2H), 1.86–1.97 (m, 5H), 1.51–1.54 (m, 3H), s, 1H), 3.88 (s, 3H), 3.49 (m, 1H), 2.24 (d, J = 10.2 Hz, 2H), 2.09
1.25–1.45 (m, 10H); 13C NMR (100 MHz, CDCl3): δ 167.7, 155.2, (d, J = 13.8 Hz, 2H), 1.45 (s, 9H), 1.25–1.38 (m, 4H).
150.6, 145.0, 144.9, 136.3, 132.3, 129.7, 126.7, 126.6, 124.4,
The compound obtained from the last step was dissolved in
124.3, 123.6, 114.3, 75.7, 53.7, 50.0, 42.1, 34.8, 33.2, 31.2, 25.5, dry DCM (2 mL) and cooled to 0 °C under nitrogen. TFA
23.0; MS-ESI m/z: 620 (M + H)+, 622 (M + 2 + H)+; Anal. calcd (0.2 mL) was added dropwise over 3 min. The ice bath was
for C29H33BrF3N5O2: C 56.13, H 5.36, N 11.29, found: C 55.84, removed and the reaction solution was allowed to warm slowly
H 5.37, N 11.20%.
to room temperature. After the reaction was completed, the
8-((1R,4R)-4-AMINOCYCLOHEXYLAMINO)-5-BROMO-N-((1H-INDOL-5-YL)METHYL)- reaction mixture was diluted dichloromethane (20 mL). The
1,6-NAPHTHYRIDINE-7-CARBOXAMIDE (1J). 1j was prepared analogously DCM layer was washed with saturated NaHCO3 solution
using general method A, giving a light yellow solid, yield (20 mL × 2) and saturated NaCl solution (20 mL × 2). The
1
36.3%: H NMR (300 MHz, CDCl3): δ 9.7 (d, J = 9 Hz, 1H), 8.94 organic layer was dried over Na2SO4 and evaporated. The
(d, J = 3.9 Hz, 1H), 8.41 (d, J = 7.8 Hz, 1H), 8.29 (t, J = 5.1 Hz, residue was purified by column chromatography on silica gel
1H), 8.19 (m, 1H), 7.66 (s, 1H), 7.57 (dd, J = 3.9, 7.8 Hz, 1H), eluting with CH2Cl2 : CH3OH (10 : 1) to give 1l as a light yellow
1
7.4 (d, J = 7.8 Hz, 1H), 7.23–7.26 (m, 1H), 6.55 (m, 1H), 4.97 solid 15 mg, yield: 59.7%: H NMR (300 MHz, CDCl3): δ 10.32
(m, 1H), 4.73 (d, J = 5.1 Hz, 2H), 2.8 (m, 1H), 2.2 (d, J = 14.7 (d, J = 8.4 Hz, 1H), 8.94 (d, J = 6 Hz, 1H), 8.68 (t, J = 6 Hz, 1H),
Hz, 2H), 1.95 (d, J = 12.9 Hz, 2H), 1.36–1.46 (m, 4H); MS-EI 8.52 (d, J = 6.3 Hz, 1H), 7.69 (s, 1H), 7.60 (dd, J = 6, 6.3 Hz,
m/z: 492 (M)+, 494 (M + 2)+; Anal. Calcd for C21H23BrN6O· 1H), 7.46–7.53 (m, 2H), 5.14 (m, 1H), 4.63 (d, J = 6 Hz, 2H),
2/5CF3COOH·3/4CH3OH: C 54.50, H 5.08, N 14.93, found: C 3.88 (s, 3H), 2.81 (m, 1H), 2.24 (d, J = 12.3 Hz, 2H), 1.96 (d, J =
54.56, H 4.84, N 14.66%.
8-((1R,4R)-4-AMINOCYCLOHEXYLAMINO)-5-BROMO-N-(NAPHTHALEN-1- 2)+; Anal. calcd for C27H25ClF3N5O3·1/3H2O: C 57.30, H 4.57, N
13.5 Hz, 2H), 1.33–1.51 (m, 4H); MS-EI m/z: 559 (M)+, 561 (M +
YLMETHYL)-1,6-NAPHTHYRIDINE-7-CARBOXAMIDE
(1K). 1k
was 12.37, found: C 57.55, H 4.78, N 12.07%.
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But if the deprotecting condition was changed to be stirred
Organic & Biomolecular Chemistry
1-((1R,4R)-4-AMINOCYCLOHEXYL)-5-BROMO-1H-IMIDAZO[4,5-H][1,6]-
at 30 °C, 1m was produced as a tan solid, yield 84.9%: 1H NMR NAPHTHYRIDIN-2(3H)-ONE (2A). 11a (75 mg, 0.16 mmol) was
(300 MHz, CDCl3): δ 10.72 (d, J = 8.4 Hz, 1H), 9.68 (d, J = 8.7 deprotected in 20% TFA–DCM solution (4 ml) to give 2a as a
1
Hz, 1H), 8.91 (d, J = 3.9 Hz, 1H), 8.83 (t, J = 6.9 Hz, 1H), 7.80 (s, light yellow solid 45 mg (yield: 77.6%): H NMR (300 MHz, d6-
1H), 7.56–7.63 (m, 2H), 7.48 (d, J = 8.4 Hz, 2H), 5.20 (m, 1H), DMSO): δ 9.07 (d, J = 3.0 Hz, 1H), 8.56 (d, J = 6.9 Hz, 1H), 7.54
4.66 (d, J = 6.9 Hz, 2H), 3.92 (s, 2H), 3.54 (s, 3H), 2.83 (m, 1H), (dd, J = 3.0, 6.9 Hz, 1H), 5.25 (br s, 1H), 2.85 (t, 1H), 2.66 (d, J =
2.25 (d, J = 11.1 Hz, 2H), 1.98 (d, J = 11.4 Hz, 2H), 1.38–1.49 12 Hz, 2H), 1.96 (d, J = 11.1 Hz, 2H), 1.74 (d, J = 10.2 Hz, 2H),
(m, 4H); MS-EI m/z: 577 (M)+, 579 (M + 2)+; Anal. calcd for 1.31 (m, 2H); 13C NMR (100 MHz, d6-DMSO): δ 154.1, 140.1,
C27H27ClF3N5O4·1/10CF3COOH·4/5H2O: C 54.11, H 4.79, N 135.2, 120.2, 116.7, 49.2, 34.4, 29.9; MS-ESI m/z: 362 (M + H)+;
11.60, found: C 54.32, H 5.06, N 11.24%.
HR-ESI MS calcd for C15H16BrN5O3 (M + H)+: 362.0616, found:
METHYL
5-BROMO-8-(TOSYLOXY)-1,6-NAPHTHYRIDINE-7-CARBOXYLATE 362.0620.
(8). Following the procedure as compound 6a, methylbenzene-
GENERAL PROCEDURE FOR THE PREPARATION OF 1-SUBSTITUTED-3-(3,4-
sulfonyl chloride (0.302 g, 1.58 mmol), methyl 5-bromo- DICHLOROBENZYL)-5-BROMO-1H-IMIDAZO[4,5-H][1,6]NAPHTHYRIDIN-2(3H)-
8-hydroxy-1,6-naphthyridine-7-carboxylate (4a) (0.120 g, ONE COMPOUNDS (2B–2E). Method C. 2b. 10a (91 mg, 0.2 mmol),
0.42 mmol), TEA (0.5 mL), gave compound 8a as a brown solid anhydrous K2CO3 (55 mg, 0.4 mmol) and 4-(bromomethyl)-1,2-
1
140 mg (yield: 76.2%): H NMR (300 MHz, CDCl3): δ 9.05 (d, dichlorobenzene (0.05 mL, 0.3 mmol) were added to DMF
J = 4.2 Hz, 1H), 8.59 (d, J = 7.8 Hz, 1H), 7.85 (d, J = 8.1 Hz, 2H), (2 mL). The mixture was stirred at room temperature for 12 h.
7.70 (dd, J = 4.2, 7.8 Hz, 1H), 7.34 (d, J = 8.1 Hz, 2H), 3.83 (s, After the reaction was completed, the reaction mixture was
3H), 2.47 (s, 3H).
diluted with dichloromethane (20 mL). The DCM layer was
5-BROMO-8-(((1R,4R)-4-((TERT-BUTOXYCARBONYL)AMINO)CYCLOHEXYL)AMINO)- washed with saturated NH4Cl solution (20 mL × 2) and satu-
1,6-NAPHTHYRIDINE-7-CARBOXYLIC ACID (9A). Following the procedure rated NaCl solution (20 mL × 1). The organic layer was dried
as compound 1a, tert-butyl (1r,4r)-4-aminocyclohexylcarbamate over Na2SO4 and evaporated. The residue was purified by
(0.208 g, 0.97 mmol), compound 8 (142 mg, 0.32 mmol), TEA column chromatography on silica gel eluting with PE : EtOAc
(0.15 mL), gave a light yellow solid 63 mg (yield: 41.1%): 1H (2 : 1) to give compound 2b as a white solid 113 mg (yield:
1
NMR (300 MHz, CDCl3): δ 8.96 (d, J = 3.9 Hz, 1H), 8.88 (br, 90.9%): H NMR (300 MHz, CDCl3): δ 9.02 (d, J = 3.9 Hz, 1H),
1H), 7.48 (d, J = 8.4 Hz, 1H), 7.64 (dd, J = 3.9, 8.4 Hz, 1H), 4.93 8.57 (d, J = 9.0 Hz, 1H), 7.62 (s, 1H), 7.44 (dd, J = 3.9, 9.0 Hz,
(m, 1H), 4.40 (br, 1H), 3.98 (s, 3H), 3.5 (m, 1H), 2.20 (d, J = 1H), 7.38 (s, 2H), 5.18 (s, 2H), 4.43 (br s, 1H), 3.71 (br s, 1H),
11.4 Hz, 2H), 2.08 (d, J = 13.5 Hz, 2H), 1.45 (s, 9H), 1.25–1.42 2.88 (br s, 1H), 2.17 (d, J = 12.6 Hz, 2H), 1.85 (d, J = 11.4 Hz,
(m, 4H).
The compound obtained from the last step (0.052 g,
2H), 1.47 (s, 9H), 1.21–1.40 (m, 4H); MS-ESI m/z: 622(M + H)+.
5-BROMO-1-CYCLOHEXYL-3-(3,4-DICHLOROBENZYL)-1H-IMIDAZO[4,5-H]-
0.11 mmol) was dissolved in 1 N LiOH (2 mL) and THF (2 mL). [1,6]NAPHTHYRIDIN-2(3H)-ONE (2C). 2c was prepared analogously
The solution was stirred under nitrogen at rt. After the reaction with general method C, giving a light yellow solid, yield 82.3%:
was complete, the reaction mixture was concentrated. The 1H NMR (300 MHz, CDCl3): δ 8.98 (d, J = 4.2 Hz, 1H), 8.54 (d, J
residue was then partitioned between H2O (10 mL) and = 8.7 Hz, 1H), 7.62 (s, 1H), 7.42 (dd, J = 4.2, 8.7 Hz, 1H), 7. 38
dichloromethane (10 mL). The pH of the H2O phase was (d, J = 0.9 Hz, 2H), 5.17 (s, 2H), 2.64 (m, 1H), 1.89 (m, 4H), 1.71
adjusted to 3 and was extracted with DCM (10 mL × 2). The (m, 2H), 1.46 (m, 4H); 13C NMR (100 MHz, CDCl3): δ 153.7,
DCM layer was dried over Na2SO4 and evaporated to give crude 152.5, 139.0, 138.6, 137.8, 136.6, 134.5, 132.6, 131.9, 130.6,
9a as a tan solid 0.051 g (yield: 99.6%): 1H NMR (300 MHz, 130.6, 128.1, 120.6, 42.7, 31.2, 29.9, 26.1, 25.2; MS-EI m/z 505
CDCl3): δ 10.75 (br s, 1H), 8.99 (d, J = 5.7 Hz, 1H), 8.49 (d, J = (M)+; HR-EI MS calcd for C22H19BrCl2N4O (M)+: 505.2225,
10.2 Hz, 1H), 7.68 (dd, J = 5.7, 10.2 Hz, 1H), 5.03 (m, 1H), 4.40 found: 505.2221.
(br s, 1H), 3.49 (m, 1H), 2.20 (d, J = 13.8 Hz, 2H), 2.09 (d, J =
1-BENZYL-5-BROMO-3-(3,4-DICHLOROBENZYL)-1H-IMIDAZO[4,5-H][1,6]-
13.2 Hz, 2H), 1.45 (s, 9H), 1.25–1.41 (m, 4H); MS-EI m/z: 464 NAPHTHYRIDIN-2(3H)-ONE (2D). 2d was prepared similar to the
1
(M)+, 466 (M + 2)+.
general method C, giving a light yellow solid, yield 72.3%: H
TERT-BUTYL ((1R,4R)-4-(5-BROMO-2-OXO-2,3-DIHYDRO-1H-IMIDAZO[4,5-H]- NMR (300 MHz, CDCl3): δ 8.98 (d, J = 4.2 Hz, 1H), 8.52 (d, J =
[1,6]NAPHTHYRIDIN-1-YL)CYCLOHEXYL)CARBAMATE (10A). 9a (108 mg, 8.7 Hz, 1H), 7.56 (m, 3H), 7.42 (dd, J = 4.2, 8.7 Hz, 1H), 7.37
0.23 mmol), DPPA (0.06 ml, 0.25 mmol), and TEA (0.05 ml) (m, 2H), 5.78 (s, 2H), 5.21 (s, 2H); 13C NMR (100 MHz, CDCl3):
were dissolved in anhydrous toluene (2 mL). The reaction δ 154.1, 153.1, 138.7, 138.6, 137.7, 137.4, 136.4, 135.5, 134.7,
mixture was heated to 90 °C. After the reaction was completed, 132.6, 132.1, 130.6, 130.5, 128.4, 128.0, 127.7, 121.0, 120.7,
the mixture was filtered to give crude 10a as a brown–yellow 115.5, 46.3, 42.9; MS-EI m/z 513 (M)+; HR-EI MS calcd for
1
solid 89 mg (yield: 83.9%): H NMR (300 MHz, CDCl3): δ 9.05 C23H15BrCl2N4O (M)+: 513.2015, found: 513.2010.
(d, J = 3.9 Hz, 1H), 8.58 (d, J = 8.7 Hz, 1H), 7.47 (dd, J = 3.9, 8.7
5-BROMO-1-CYCLOPROPYL-3-(3,4-DICHLOROBENZYL)-1H-IMIDAZO[4,5-H]-
Hz, 1H), 4.47 (br s, 1H), 2.19 (d, J = 12.3 Hz, 2H), 1.87 (d, J = [1,6]NAPHTHYRIDIN-2(3H)-ONE (2E). 2e was prepared according to
11.7 Hz, 2H), 1.48 (s, 9H), 1.30–1.42 (m, 4H); 13C NMR the procedure described in the general method C, giving a
(100 MHz, d6-DMSO): δ 154.9, 152.8, 139.6, 137.4, 133.3, 121.3, light yellow solid, yield 86.3%: 1H NMR (300 MHz, CDCl3): δ
119.3, 115.7, 77.5, 48.3, 32.0, 28.3, 28.0; MS-EI m/z: 461 (M)+, 9.02 (d, J = 4.2 Hz, 1H), 8.57 (d, J = 8.4 Hz, 1H), 7.64 (s, 1H),
463 (M + 2)+.
7.45 (dd, J = 4.2, 8.4 Hz, 1H), 7.37 (d, J = 1.2 Hz, 2H), 5.16 (s,
1556 | Org. Biomol. Chem., 2013, 11, 1545–1562
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2H), 3.49 (m, 1 H), 1.56 (m, 2 H), 1.25 (m, 2 H); MS-EI m/z: 464 2.98 (t, J = 10.2 Hz, 1H), 2.76 (m, 2H), 2.04 (d, J = 11.7 Hz, 2H),
(M)+; HR-EI MS calcd for C19H13BrCl2N4O (M)+: 463.9645, 1.88 (d, J = 13.8 Hz, 2H), 1.36–1.48 (m, 2H); MS-EI m/z: 519
found: 463.9640.
(M)+, 521 (M + 2)+; HR-EI MS calcd for C23H21BrF3N5O (M)+:
GENERAL PROCEDURE FOR THE PREPARATION OF 1-((1R,4R)-4-AMINOCY- 519.0882, found: 519.0878; Anal. calcd for C23H21BrF3N5O·1/
CLOHEXYL)-3-(SUBSTITUTED-BENZYL)-5-BROMO-1H-IMIDAZO[4,5-H][1,6]- 6CF3COOH: C 51.96, H 3.96, N 12.98, found: C 51.98, H 3.88,
NAPHTHYRIDIN-2(3H)-ONE COMPOUNDS (2F–2L). Method D. 2f. 2b N 12.93%.
(91 mg, 0.15 mmol) was deprotected in 20% TFA–DCM solu-
1-((1R,4R)-4-AMINOCYCLOHEXYL)-3-(4-CHLOROBENZYL)-5-BROMO-1H-
tion (4 ml) to give 14b as a light yellow solid 55 mg (yield: IMIDAZO[4,5-H][1,6]NAPHTHYRIDIN-2(3H)-ONE (2K). 2k was pre-
1
70.6%): H NMR (300 MHz, CDCl3): δ 8.97 (d, J = 2.7 Hz, 1H), pared as described above in the general method D, giving a
8.56 (d, J = 10.5 Hz, 1H), 7.61 (s, 1H), 7.43 (dd, J = 2.7, 10.5 Hz, light yellow solid, yield 60%: 1H NMR (300 MHz, CDCl3): δ
1H), 7.38 (s, 2H), 5.17 (s, 2H), 2.97 (t, 1H), 2.74 (m, 2H), 2.03 8.97 (d, J = 3.9 Hz, 1H), 8.57 (d, J = 8.7 Hz, 1H), 7.49 (d, J = 8.4
(d, J = 12.0 Hz, 2H), 1.89 (d, J = 16.2 Hz, 2H), 1.35–1.48 (m, Hz, 2H), 7.44 (dd, J = 3.9, 8.7 Hz, 1H), 7.29 (d, J = 8.4 Hz, 2H),
2H); 13C NMR (100 MHz, CDCl3): δ 153.8, 152.4, 138.9, 137.9, 5.20 (s, 2H), 3.00 (t, J = 11.7 Hz, 1H), 2.75 (m, 2H), 2.05 (d, J =
136.4, 134.7, 132.5, 131.9, 130.5, 128.1, 120.7, 120.6, 49.4, 42.6, 13.2 Hz, 2H), 1.88 (d, J = 12.9 Hz, 2H), 1.38–1.50 (m, 2H);
35.5, 28.2; MS-EI m/z: 519 (M)+, 521 (M + 2)+; HR-EI MS calcd MS-ESI m/z: 488 (M + H)+; HR-ESI MS calcd for C22H21BrClN5O
for C22H20BrCl2N5O (M)+: 519.0228, found: 519.0229; Anal. (M
+
Na)+: 508.0516, found: 508.0503; Anal. calcd for
calcd for C22H21BrFN5O·1/4CF3COOH: C 49.16, H 3.71, N C22H21BrClN5O·1/6CF3COOH: C 53.03, H 4.22, N 13.85, found:
12.74, found: C 49.28, H 3.75, N 12.77%.
C 53.19, H 4.25, N 13.76%.
1-((1R,4R)-4-AMINOCYCLOHEXYL)-3-BENZYL-5-BROMO-1H-IMIDAZO[4,5-H]-
1-((1R,4R)-4-AMINOCYCLOHEXYL)-3-(2-CHLOROBENZYL)-5-BROMO-1H-
[1,6]NAPHTHYRIDIN-2(3H)-ONE (2G). 2g was prepared as described IMIDAZO[4,5-H][1,6]NAPHTHYRIDIN-2(3H)-ONE (2L). 2l was prepared
above in the general method D, giving a light yellow solid, as described above in the general method D, giving a light
1
yield 44.8%: H NMR (300 MHz, CD3OD): δ 9.06 (d, J = 3.6 Hz, yellow solid, yield 48.6%: 1H NMR (300 MHz, CDCl3): δ 9.00 (d,
1H), 8.68 (d, J = 8.4 Hz, 1H), 7.60 (m, 1H), 7.43 (d, J = 6.9 Hz, J = 2.7 Hz, 1H), 8.57 (d, J = 8.7 Hz, 1H), 7.38–7.47 (m, 2H),
2H), 7.32 (m, 3H), 5.24 (s, 2H), 2.84 (m, 2H), 2.21 (d, J = 14.7 7.13–7.23 (m, 2H), 7.06 (d, J = 6.0 Hz, 1H), 5.39 (s, 2H), 3.03 (t,
Hz, 2H), 2.02 (d, J = 8.1 Hz, 2H), 1.59–1.72 (m, 2H); MS-EI m/z: J = 12.6 Hz, 1H), 2.81 (m, 2H), 2.09 (d, J = 10.8 Hz, 2H), 1.93 (d,
451 (M)+, 453 (M + 2)+.
J = 10.5 Hz, 2H), 1.42–1.54 (m, 2H); MS-ESI m/z: 488 (M + H)+;
1-((1R,4R)-4-AMINOCYCLOHEXYL)-3-(4-FLUOROBENZYL)-5-BROMO-1H-IMIDAZO- Anal. calcd for C22H21BrClN5O·1/3CF3COOH: C 51.88, H 4.10,
[4,5-H][1,6]NAPHTHYRIDIN-2(3H)-ONE (2H). 2h was prepared as N 13.34, found: C 52.10, H 4.36, N 13.17%.
described above in the general method D, giving a light yellow
GENERAL PROCEDURE FOR THE PREPARATION OF 1-((1R,4R)-4-AMINOCY-
solid, yield 61.4%: 1H NMR (300 MHz, CDCl3): δ 8.96 (d, J = 3.9 CLOHEXYL)-3-(SUBSTITUTED-SULFONYL)-5-BROMO-1H-IMIDAZO[4,5-H][1,6]-
Hz, 1H), 8.56 (d, J = 7.5 Hz, 1H), 7.53 (t, J = 9.0 Hz, 2H), 7.42 NAPHTHYRIDIN-2(3H)-ONE COMPOUNDS (2M–2N). Method E. 2m. A
(dd, J = 3.9, 7.5 Hz, 1H), 6.99 (t, J = 8.4 Hz, 2H), 5.20 (s, 2H), solution of 10a (69 mg, 0.15 mmol), TsCl (71 mg, 0.3 mmol)
2.96 (t, 1H), 2.75 (m, 2H), 2.03 (d, J = 13.2 Hz, 2H), 1.87 (d, J = and TEA (0.05 ml) in DCM (4 mL) was refluxed for 12 h. After
9.9 Hz, 2H), 1.34–1.46 (m, 2H); MS-ESI m/z: 470 (M + H)+; Anal. the reaction was complete, the reaction mixture was diluted
calcd for C22H21BrFN5O·1/4CF3COOH: C 54.17, H 4.29, N with dichloromethane (20 mL). The DCM layer was washed
14.04, found: C 54.20, H 4.32, N 14.03%.
with saturated NH4Cl solution (20 mL × 2) and saturated NaCl
1-((1R,4R)-4-AMINOCYCLOHEXYL)-3-(3-FLUORO-4-METHOXYBENZYL)-5-BROMO- solution (20 mL × 1). The organic layer was dried over Na2SO4
1H-IMIDAZO[4,5-H][1,6]NAPHTHYRIDIN-2(3H)-ONE (2I). 2i was prepared and evaporated. The residue was purified by column chrom-
as described above in the general method D, giving a light atography on silica gel eluting with PE : EtOAc (4 : 1) to give a
yellow solid, yield 82.6%: 1H NMR (300 MHz, CDCl3): δ 8.96 (d, light yellow solid 50 mg (yield: 54.1%): 1H NMR (300 MHz,
J = 3.9 Hz, 1H), 8.55 (d, J = 8.7 Hz, 1H), 7.42 (dd, J = 3.9, 8.7 CDCl3): δ 9.02 (d, J = 3.3 Hz, 1H), 8.59 (d, J = 8.7 Hz, 1H), 8.21
Hz, 1H), 7.29 (d, J = 3.9 Hz, 1H), 7.26 (m, 1H), 6.90 (t, J = 8.7 (d, J = 8.1 Hz, 2H), 7.52 (dd, J = 3.3, 8.7 Hz, 1H), 7.36 (d, J =
Hz, 1H), 5.15 (s, 2H), 3.84 (s, 3H), 2.94 (t, J = 11.1 Hz, 1H), 2.74 8.4 Hz, 2H), 4.42 (br s, 1H), 3.64 (br s, 1H), 2.76 (m, 2H), 2.42
(m, 2H), 2.00 (d, J = 12.0 Hz, 2H), 1.86 (d, J = 11.1 Hz, 2H), (s, 3H), 2.14 (d, J = 12.9 Hz, 2H), 1.80 (d, J = 12.9 Hz, 2H), 1.46
1.31–1.43 (m, 2H); 13C NMR (100 MHz, CDCl3): δ 153.7, 153.3, (s, 9H), 1.25–1.40 (m, 2H); MS-ESI m/z: 618 (M + H)+.
150.8, 147.3, 147.2, 139.2, 138.4, 137.9, 134.6, 129.4, 129.3,
The light yellow solid (50 mg, 0.08 mmol) was deprotected
124.7, 120.6, 120.5, 116.6, 116.4, 113.2, 56.1, 49.5, 42.9, 35.8, in 20% TFA–DCM solution (2 ml) to give 2m as a light yellow
1
28.3; MS-ESI m/z: 500 (M
C23H23BrFN5O2·3/2H2O: C 52.38, H 4.97, N 13.28, found: C (d, J = 2.4 Hz, 1H), 8.58 (d, J = 8.4 Hz, 1H), 8.20 (d, J = 8.1 Hz,
52.42, H 4.73, N 13.06%. 2H), 7.52 (dd, J = 2.4, 8.4 Hz, 1H), 7.35 (d, J = 7.8 Hz, 1H), 2.97
+
H)+; Anal. calcd for solid 32 mg (yield: 77.5%): H NMR (300 MHz, CDCl3): δ 8.99
1-((1R,4R)-4-AMINOCYCLOHEXYL)-3-(3-(TRIFLUOROMETHYL)BENZYL)-5-BROMO- (t, J = 12.3 Hz, 1H), 2.66 (m, 2H), 2.05 (d, J = 10.5 Hz, 2H), 1.83
1H-IMIDAZO[4,5-H][1,6]NAPHTHYRIDIN-2(3H)-ONE (2J). 2j was prepared (d, J = 11.4 Hz, 2H), 1.35–1.48 (m, 2H); 13C NMR (100 MHz,
as described above in the general method D, giving a light CDCl3): δ 154.0, 146.0, 137.8, 135.1, 129.8, 128.6, 122.1, 49.4,
yellow solid, yield 85.4%: 1H NMR (300 MHz, CDCl3): δ 8.98 (d, 34.6, 27.6, 21.7; MS-ESI m/z: 518 (M + H)+; Anal. calcd for
J = 2.7 Hz, 1H), 8.57 (d, J = 8.7 Hz, 1H), 7.82 (s, 1H), 7.70 (d, J = C22H22BrFN5O3S·1/4CF3COOH: C 49.59, H 4.12, N 12.85,
7.5 Hz, 1H), 7.53 (d, J = 8.1 Hz, 1H), 7.46 (m, 2H), 5.28 (s, 2H), found: C 49.48, H 4.10, N 12.84%.
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1-((1R,4R)-4-AMINOCYCLOHEXYL)-3-((3,4-DICHLOROPHENYL)SULFONYL)-5- 1H), 4.94 (s, 2H), 3.00–2.80 (m, 2H), 2.30–2.16 (m, 2H),
BROMO-1H-IMIDAZO[4,5-H][1,6]NAPHTHYRIDIN-2(3H)-ONE (2N). 2n was 2.12–1.96 (m, 2H), 1.77–1.59 (m, 2H); MS-EI m/z: 561 (M)+;
prepared as described above in the general method E, giving a HR-EI MS calcd for C29H25Cl2N5O3 (M)+: 561.2353, found:
light yellow solid, yield 43.8%: 1H NMR (300 MHz, CDCl3): δ 561.2359.
9.01 (d, J = 4.2 Hz, 1H), 8.61 (d, J = 8.7 Hz, 1H), 8.46 (d, J = 2.1
4-((1-((1R,4R)-4-AMINOCYCLOHEXYL)-3-(3,4-DICHLOROBENZYL)-2-OXO-2,3-
Hz, 1H), 8.18 (d, J = 8.4 Hz, 1H), 7.65 (d, J = 8.7 Hz, 1H), 7.55 DIHYDRO-1H-IMIDAZO[4,5-H][1,6]NAPHTHYRIDIN-5-YL)OXY)-2-CHLOROBENZAL-
(dd, J = 4.2, 8.7 Hz, 1H), 2.94 (m, 1H), 2.64 (m, 2H), 2.03 (d, J = DEHYDE (2R). 2r was prepared as described above in the general
12.3 Hz, 2H), 1.88 (m, 2H), 1.33–1.45 (m, 2H); MS-ESI m/z: 572 method E, giving a light yellow solid, yield 65.5%: 1H NMR
(M
10CF3COOH: C 43.17, H 3.23, N 11.87, found: C 43.43, H 3.52, = 8.5 Hz, 1H), 8.15–8.05 (m, 1H), 7.24–7.20 (m, 1H), 7.92 (d, J =
N 11.61%. 8.4 Hz, 1H), 7.43 (d, J = 8.3 Hz, 2H), 7.30–7.27 (m, 1H),
1-((1R,4R)-4-AMINOCYCLOHEXYL)-3-(3,4-DICHLOROBENZYL)-5-HYDROXY-1H- 7.14–7.03 (m, 1H), 4.86 (s, 2H), 4.57–4.34 (m, 2H), 3.90–3.60
+
H)+; Anal. calcd for C21H18BrCl2N5O3S·2/H2O·1/ (300 MHz, CDCl3): δ 10.04 (s, 1H), 9.12–9.03 (m, 1H), 8.74 (d, J
IMIDAZO[4,5-H][1,6]NAPHTHYRIDIN-2(3H)-ONE
(2O). 2f
(90
mg, (m, 2H), 3.02–2.85 (m, 2H), 2.26–2.06 (m, 2H), 1.43–1.37 (m,
0.15 mmol) and K2CO3 (55 mg, 0.4 mmol) were added to DMF 2H); MS-EI m/z: 595 (M)+; HR-EI MS calcd for C29H24Cl3N5O3
(2 mL). The mixture was stirred at 120 °C. After the reaction (M)+: 595.1047, found: 595.1042.
was complete, the reaction mixture was diluted with dichloro-
1-((1R,4R)-4-AMINOCYCLOHEXYL)-3-(3,4-DICHLOROBENZYL)-5-(4-(HYDRO-
methane (20 mL). The DCM layer was washed with saturated XYMETHYL)PHENOXY)-1H-IMIDAZO[4,5-H][1,6]NAPHTHYRIDIN-2(3H)-ONE (2S).
NH4Cl solution (20 mL × 2) and saturated NaCl solution 2s was prepared as described above in the general method E,
(20 mL × 1). The organic layer was dried over Na2SO4 and evap- giving a light yellow solid, yield 72.6%: 1H NMR (300 MHz,
orated. The residue was purified by column chromatography CDCl3): δ 9.03 (d, J = 4.0 Hz, 1H), 8.78 (d, J = 8.5 Hz, 1H), 7.86
on silica gel to give a white solid 110 mg, then deprotected in (s, 1H), 7.50 (d, J = 7.8 Hz, 3H), 7.42–7.35 (m, 1H), 7.21 (d, J =
20% TFA–DCM solution (4 ml) to give 2o as a light yellow solid 8.1 Hz, 2H), 7.12 (d, J = 8.2 Hz, 1H), 4.88 (s, 2H), 4.70 (s, 2H),
1
39 mg (yield: 56.7% in tow steps): H NMR (300 MHz, CDCl3): 3.03–2.94 (m, 1H), 2.84–2.75 (m, 1H), 2.11–2.04 (m, 2H),
δ 8.94 (d, J = 2.5 Hz, 1H), 8.52 (d, J = 8.8 Hz, 1H), 7.52 (d, J = 1.93–1.88 (m, 2H), 1.55–1.36 (m, 4H); MS-EI m/z: 563 (M)+;
1.9 Hz, 1H), 7.41 (dd, J = 8.7, 4.2 Hz, 1H), 7.33–7.26 (m, 2H), HR-EI MS calcd for C29H27Cl2N5O3 (M)+: 563.1491, found:
5.09 (s, 2H), 3.31–3.17 (m, 1H), 2.86–2.62 (m, 2H), 2.29–2.01 563.1495.
(m, 2H), 1.95–1.79 (m, 2H), 1.72–1.47 (m, 2H); MS-EI m/z: 457
(M)+; HR-EI MS calcd for C22H21Cl2N5O2 (M)+: 457.1072, 1-CARBONYL)PHENOXY)-1H-IMIDAZO[4,5-H][1,6]NAPHTHYRIDIN-2(3H)-ONE
found: 457.1040.
(2T). 1H NMR (300 MHz, CDCl3): δ 9.12 (d, J = 5.5 Hz, 1H), 8.87
1-((1R,4R)-4-AMINOCYCLOHEXYL)-3-(3,4-DICHLOROBENZYL)-5-(4-(PIPERIDINE-
GENERAL PROCEDURE FOR THE PREPARATION OF 1,3,5-TRISUBSTITUTED- (d, J = 8.6 Hz, 1H), 7.60 (d, J = 8.5 Hz, 3H), 7.47 (d, J = 5.8 Hz,
1H-IMIDAZO[4,5-H][1,6]NAPHTHYRIDIN-2(3H)-ONE COMPOUNDS (2P–2V). 2H), 7.39 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 9.5 Hz, 1H), 4.98 (s,
Method F. 2p: A solution of 2f (91 mg, 0.15 mmol), anhydrous 2H), 3.80–3.75 (m, 1H), 3.56–3.46 (m, 1H), 3.03–2.82 (m, 3H),
K2CO3 (55 mg, 0.4 mmol) and 2-hydroxypyridine (0.05 mL, 2.31–2.22 (m, 2H), 2.11–2.00 (m, 2H), 1.84–1.68 (m, 6H),
0.3 mmol) in DMF (2 mL) was stirred at 110 °C for 12 h. After 1.68–1.50 (m, 4H), 1.40–1.26 (m, 4H); MS-EI m/z: 644 (M)+;
the reaction was complete, the reaction mixture was diluted HR-EI MS calcd for C34H34Cl2N6O3 (M)+: 644.2069, found:
with dichloromethane (20 mL). The DCM layer was washed 644.2072.
with saturated NH4Cl solution (20 mL × 2) and saturated NaCl
1-((1R,4R)-4-AMINOCYCLOHEXYL)-3-(3,4-DICHLOROBENZYL)-5-(4-(MORPHO-
solution (20 mL × 1). The organic layer was dried over Na2SO4 LINE-4-CARBONYL)PHENOXY)-1H-IMIDAZO[4,5-H][1,6]NAPHTHYRIDIN-2(3H)-
and evaporated. The residue was purified by column chrom- ONE (2U). 1H NMR (300 MHz, CDCl3): δ 8.96 (d, J = 5.7 Hz, 1H),
atography on silica gel to give a white solid 113 mg, then 8.61 (d, J = 8.2 Hz, 1H), 7.64 (s, 1H), 7.56–7.49 (m, 1H), 7.45 (d,
deprotected in 20% TFA–DCM solution (4 ml) to give 2p as a J = 8.3 Hz, 1H), 7.40–7.35 (m, 1H), 5.15 (s, 2H), 4.63 (t, J =
light yellow solid 62 mg (overall yield of 77.6% for two steps): 6.2 Hz, 2H), 3.78 (t, J = 6.3 Hz, 2H), 3.69–3.50 (m, 4H), 3.18
1H NMR (300 MHz, CDCl3): δ 9.07 (d, J = 4.2 Hz, 1H), 8.82 (d, J (dd, J = 14.6, 7.3 Hz, 4H), 2.19 (s, 1H), 2.12–2.05 (m, 2H),
= 8.6 Hz, 1H), 8.57 (d, J = 2.5 Hz, 1H), 8.53 (d, J = 4.8 Hz, 1H), 1.98–1.94 (m, 1H), 1.32–1.25 (m, 4H), 1.20–1.11 (m, 4H); MS-EI
7.78 (d, J = 7.0 Hz, 1H), 7.63–7.51 (m, 2H), 7.38 (d, J = 8.3 Hz, m/z: 646 (M)+; HR-EI MS calcd for C33H32Cl2N6O4 (M)+:
1H), 7.35 (d, J = 1.9 Hz, 1H), 7.07 (d, J = 8.3 Hz, 1H), 4.91 (s, 646.1860, found: 646.1862.
2H), 2.96–2.80 (m, 2H), 2.31–2.18 (m, 2H), 2.07–1.96 (m, 2H),
1.78–1.58 (m, 2H); MS-EI m/z: 534 (M)+; HR-EI MS calcd for 2,3-DIHYDRO-1H-IMIDAZO[4,5-H][1,6]NAPHTHYRIDIN-5-YLOXY)-N-(1-PHENYL-
C27H24Cl2N6O2 (M)+: 534.1342, found: 534.1356. ETHYL)BENZAMIDE (2V). 1H NMR (300 MHz, CDCl3): δ 8.958 (d, J =
(R)-4-((1R,4R)-4-AMINOCYCLOHEXYL)-3-(3,4-DICHLOROBENZYL)-2-OXO-
4-((1-((1R,4R)-4-AMINOCYCLOHEXYL)-3-(3,4-DICHLOROBENZYL)-2-OXO-2,3- 3.9, 1 H), 8.557 (d, J = 8.4, 1 H), 7.584 (m, 3 H), 7.425 (dd, J =
DIHYDRO-1H-IMIDAZO[4,5-H][1,6]NAPHTHYRIDIN-5-YL)OXY)BENZALDEHYDE (2Q). 3.9, 8.4, 1 H), 7.368 (m, 2 H), 7.132 (m, 1 H), 5.183 (s, 2 H),
2q was prepared as described above in the general method E, 2.142 (d, J = 7.2, 3 H); MS-EI m/z: 680 (M)+; HR-EI MS calcd for
giving a light yellow solid, yield 63.4%: 1H NMR (300 MHz, C37H34Cl2N6O3 (M)+: 680.2069, found: 680.2071.
CDCl3): δ 10.04 (s, 1H), 9.07 (d, J = 4.3 Hz, 1H), 8.80 (d, J =
8.7 Hz, 1H), 8.05 (d, J = 8.4 Hz, 2H), 7.90 (s, 1H), 7.57–7.53 (m, PYRIDIN-2(3H)-ONE COMPOUNDS (3A–3J). METHOD G. 6-CHLORO-N-(3,4-
1H), 7.42 (d, J = 8.8 Hz, 2H), 7.37 (s, 1H), 7.13 (d, J = 11.3 Hz, DICHLOROBENZYL)-3-NITROPYRIDIN-2-AMINE (13). To solution of
GENERAL PROCEDURE FOR THE PREPARATION OF 1H-IMIDAZO[4,5-B]-
a
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2-amino-6-chloro-3-nitropyridine (500 mg, 2.9 mmol) and 4- was quenched with aqueous NH4Cl (50 mL) and extracted with
(bromomethyl)-1,2-dichlorobenzene (0.5 mL, 3.0 mmol) in EtOAc (3 × 20 mL). The extracts were washed with H2O (20 mL)
anhydrous DMF (5 mL) was added a 60% mineral oil dis- and brine (20 mL). The organic layer was dried over Na2SO4
persion of sodium hydride (412.5 mg, 3.0 mmol) in several and evaporated. The residue was purified by column chrom-
portions. The mixture was stirred at room temperature for 6 h. atography on silica gel to give a white solid 185.3 mg (yield:
After the reaction was complete, the reaction mixture was 96%): 1H NMR (300 MHz, CDCl3) δ 8.27 (s, 1H), 8.09 (d, J =
diluted with dichloromethane (20 mL). The DCM layer was 15.0 Hz, 1H), 7.97–7.89 (m, 2H), 7.65 (d, J = 15.0 Hz, 1H),
washed with saturated NH4Cl solution (20 mL × 2) and satu- 7.40 (d, J = 3.1 Hz, 1H), 7.20 (dd, J = 15.0, 3.1 Hz, 1H),
rated NaCl solution (20 mL). The organic layer was dried over 7.11–7.04 (m, 2H), 6.58 (d, J = 15.0 Hz, 1H), 4.70 (s, 2H); EI MS
Na2SO4 and evaporated. The residue was purified by column m/z 429 [M+].
chromatography on silica gel eluting with PE : EtOAc to give
N-CYCLOPROPYL-4-((3-(3,4-DICHLOROBENZYL)-2-OXO-2,3-DIHYDRO-1H-
1
compound 13 as a light yellow solid 789 mg (yield: 82%): H IMIDAZO[4,5-B]PYRIDIN-5-YL)OXY)BENZAMIDE (3A). To a solution of
NMR (300 MHz, CDCl3) δ 7.43 (s, 1H), 7.35 (d, J = 8.2 Hz, 1H), 19a (60 mg, 0.14 mmol) and cyclopropylamine (15.9 mg,
7.18 (d, J = 8.2 Hz, 1H), 6.81 (d, J = 7.7 Hz, 1H), 6.52 (d, J = 7.7 0.28 mmol) in DCM (5 mL) was added EDCI (43.4 mg,
Hz, 1H), 4.53 (s, 2H); EI MS m/z 331 [M+].
0.28 mmol) and HOBt (137 mg, 0.28 mmol). The mixture was
METHYL 4-((6-((3,4-DICHLOROBENZYL)AMINO)-5-NITROPYRIDIN-2-YL)OXY)- stirred at rt for 4 h. After the reaction was complete, the reac-
BENZOATE (14). A solution of 13 (150 mg, 0.45 mmol), anhydrous tion mixture was diluted with EtOAc (80 mL). Usual work-up of
K2CO3 (165 mg, 1.2 mmol) and methyl 4-hydroxybenzoate the organic layer and purification of the residue by column
(137 mg, 0.9 mmol) in DMF (2 mL) was stirred at 80 °C for chromatography on silica gel gave a white solid 56.5 mg (yield:
12 h. After the reaction was completed, the reaction mixture 86%): 1H NMR (400 MHz, DMSO) δ 8.40 (d, J = 3.2 Hz, 1H),
was diluted with EtOAc (80 mL). Usual work-up with the 7.84 (d, J = 8.7 Hz, 2H), 7.55 (d, J = 8.2 Hz, 1H), 7.45 (d, J = 8.4
organic layer and purification of the residue by column chrom- Hz, 2H), 7.18 (dd, J = 7.8, 1.8 Hz, 1H), 7.05 (d, J = 8.7 Hz, 2H),
atography on silica gel gave a white solid 210.6 mg, (yield: 6.69 (d, J = 8.0 Hz, 1H), 4.86 (s, 2H), 2.87–2.78 (m, 1H), 0.69
89%): 1H NMR (300 MHz, CDCl3) δ 9.00 (s, 1H), 8.29 (d, J = 8.6 (dt, J = 7.1, 4.7 Hz, 2H), 0.56 (dt, J = 7.1, 4.7 Hz, 2H). MS-ESI
Hz, 1H), 8.01 (d, J = 8.8 Hz, 2H), 7.59–7.51 (m, 2H), 7.23 (d, J = m/z: 467.3 (M)−; HR-ESI MS calcd for C23H18Cl2N4O3Na
8.8 Hz, 2H), 7.16 (d, J = 8.6 Hz, 1H), 5.36 (s, 2H), 3.87 (s, 3H); (M + Na)+: 491.0654, found: 491.0678.
EI MS m/z 447 [M+].
METHYL
3-(3,4-DICHLOROBENZYL)-5-(4-(PIPERIDINE-1-CARBONYL)PHENOXY)-1H-
4-((5-AMINO-6-((3,4-DICHLOROBENZYL)AMINO)PYRIDIN-2-YL)- IMIDAZO[4,5-B]PYRIDIN-2(3H)-ONE (3B). White solid, yield 87.5%:
OXY)BENZOATE (15A). To a solution of 14 (200 mg, 0.45 mmol) 1H NMR (300 MHz, DMSO) δ 11.26 (s, 1H), 7.58 (d, J = 8.3 Hz,
and NiCl2·6H2O (124 mg, 0.90 mmol) in MeOH (5 mL) was 1H), 7.50 (d, J = 1.7 Hz, 1H), 7.46 (d, J = 8.1 Hz, 1H), 7.38 (d, J =
added NaBH4 (34.2 mg, 0.90 mmol) slowly at 0 °C. The 8.2 Hz, 2H), 7.19 (d, J = 8.3 Hz, 1H), 7.06 (d, J = 8.6 Hz, 1H),
mixture was stirred at room temperature for 1 h. After the reac- 6.71 (dd, J = 8.1, 0.7 Hz, 1H), 4.89 (s, 2H), 1.67–1.59 (m, 2H),
tion was completed, the reaction mixture was diluted with 1.56–1.46 (m, 3H); ESI MS m/z 497.3 [M] +; HR-ESI MS calcd for
EtOAc (80 mL). Usual work-up and purification by column C25H23Cl2N4O3 (M + H)+: 497.1674, found: 497.1682.
chromatography on silica gel afforded a white solid 171.1 mg,
3-(3,4-DICHLOROBENZYL)-5-(4-(MORPHOLINE-4-CARBONYL)PHENOXY)-1H-
1
(yield: 91%): H NMR (300 MHz, CDCl3) δ 7.94 (d, J = 8.4 Hz, IMIDAZO[4,5-B]PYRIDIN-2(3H)-ONE (3C). White solid, yield 84.7%: 1H
2H), 7.57–7.48 (m, 2H), 7.46 (s, 1H), 7.19 (d, J = 8.6 Hz, 1H), NMR (300 MHz, DMSO) δ 11.26 (s, 1H), 7.57 (d, J = 8.2 Hz, 1H),
7.06 (d, J = 8.3 Hz, 2H), 6.75 (d, J = 8.1 Hz, 1H), 4.88 (s, 2H); EI 7.49 (d, J = 1.9 Hz, 1H), 7.47–7.39 (m, 2H), 7.18 (d, J = 8.3 Hz,
MS m/z 417 [M+].
1H), 7.06 (d, J = 8.6 Hz, 1H), 6.70 (d, J = 8.1 Hz, 1H), 4.88 (s,
METHYL 4-((3-(3,4-DICHLOROBENZYL)-2-OXO-2,3-DIHYDRO-1H-IMIDAZO- 2H), 3.66–3.53 (m, 4H), 3.34–3.26 (m, 4H); ESI MS m/z 499.3
[4,5-B]PYRIDIN-5-YL)OXY)BENZOATE (18A). To
solution of 15a [M]+. HR-ESI MS calcd for C24H21Cl2N4O4 (M + H)+: 499.1801,
(200 mg, 0.48 mmol) and a catalytic amount of DMF (50 μL) in found: 499.1822.
a
THF (5 mL) was added triphosgene (60 mg, 0.20 mmol) slowly
3-(3,4-DICHLOROBENZYL)-5-(4-(PIPERAZINE-1-CARBONYL)PHENOXY)-1H-
at rt. The mixture was stirred at room temperature for 10 min. IMIDAZO[4,5-B]PYRIDIN-2(3H)-ONE (3D). White solid, yield 84.7%:
After the reaction was complete, the reaction mixture was 1H NMR (300 MHz, DMSO) δ 11.26 (s, 1H), 7.57 (d, J = 8.2 Hz,
diluted with EtOAc (80 mL). Usual work-up and purification by 1H), 7.49 (d, J = 1.9 Hz, 1H), 7.47–7.39 (m, 2H), 7.18 (d, J =
column chromatography on silica gel gave a white solid 8.3 Hz, 1H), 7.06 (d, J = 8.6 Hz, 1H), 6.70 (d, J = 8.1 Hz, 1H),
208.3 mg (yield: 98%): 1H NMR (300 MHz, CDCl3) 8.28 (s, 1H), 4.88 (s, 2H), 3.66–3.53 (m, 4H), 3.34–3.26 (m, 4H); ESI MS m/z
8.09 (d, J = 15.0 Hz, 1H), 7.81–7.71 (m, 2H), 7.65 (d, J = 498.2 [M+]. HR-ESI MS calcd for C24H22Cl2N5O3 (M + H)+:
15.0 Hz, 1H), 7.40 (d, J = 3.1 Hz, 1H), 7.20 (dd, J = 14.9, 3.0 Hz, 498.1100, found: 498.1076.
1H), 7.03–6.93 (m, 2H), 6.58 (d, J = 15.0 Hz, 1H), 4.70 (s, 2H),
3.90 (s, 3H); EI MS m/z 443 [M+].
(R)-4-((3-(3,4-DICHLOROBENZYL)-2-OXO-2,3-DIHYDRO-1H-IMIDAZO[4,5-B]-
(3E). White solid,
PYRIDIN-5-YL)OXY)-N-(1-PHENYLETHYL)BENZAMIDE
4-((3-(3,4-DICHLOROBENZYL)-2-OXO-2,3-DIHYDRO-1H-IMIDAZO[4,5-B]- yield 88.6%: 1H NMR (300 MHz, DMSO) δ 8.76 (s, 1H),
PYRIDIN-5-YL)OXY)BENZOIC ACID (19A). The solution of 18a (200 mg, 7.98–7.88 (m, 2H), 7.56–7.31 (m, 2H), 7.20 (s, 2H), 7.07 (dd, J =
0.45 mmol) and LiOH (184.5 mg, 4.5 mmol) in THF–H2O (2 : 1, 13.6, 9.2 Hz, 1H), 6.75–6.64 (m, 1H), 5.17 (t, J = 10.4 Hz, 1H),
10 mL) was stirred at rt for 12 h. Then the reaction mixture 4.86 (d, J = 9.6 Hz, 2H), 1.47 (t, J = 10.4 Hz, 3H); ESI MS m/z
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531.4 [M]−. HR-ESI MS calcd for C28H22Cl2N4O3Na (M + Na)+: 531.3 [M]−. HR-ESI MS calcd for C28H22Cl2N4O3Na (M + Na)+:
555.0967, found: 555.0972.
555.0967, found: 555.0961.
(S)-4-((3-(3,4-DICHLOROBENZYL)-2-OXO-2,3-DIHYDRO-1H-IMIDAZO[4,5-B]-
METHYL 4-((1-(4-AMINOCYCLOHEXYL)-3-(3,4-DICHLOROBENZYL)-2-OXO-
PYRIDIN-5-YL)OXY)-N-(1-PHENYLETHYL)BENZAMIDE
(3F). White solid, 2,3-DIHYDRO-1H-IMIDAZO[4,5-B]PYRIDIN-5-YL)OXY)BENZOATE (3K). White
1
1
yield 84.6%: H NMR (300 MHz, DMSO) δ 9.34 (d, J = 7.7 Hz, solid, yield 78.3%: H NMR (300 MHz, CDCl3) δ 8.01 (d, J = 8.8
1H), 8.50 (d, J = 8.8 Hz, 2H), 8.12 (d, J = 8.3 Hz, 1H), 8.02 (d, J = Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H), 7.14–7.11 (m, 1H), 7.08 (d, J =
8.2 Hz, 2H), 7.96 (d, J = 7.4 Hz, 2H), 7.89 (t, J = 7.4 Hz, 2H), 8.7 Hz, 2H), 7.03 (d, J = 8.3 Hz, 1H), 6.55 (d, J = 8.3 Hz, 1H),
7.78 (dd, J = 13.7, 7.7 Hz, 2H), 7.65 (d, J = 8.8 Hz, 2H), 7.27 (d, 4.97 (s, 2H), 4.42–4.26 (m, 1H), 3.85 (s, 3H), 3.05–2.92 (m, 1H),
J = 8.1 Hz, 1H), 5.80–5.70 (m, 1H), 5.44 (s, 2H), 2.05 (d, J = 2.46–2.26 (m, 2H), 2.20–2.05 (m, 2H), 1.90–1.77 (m, 2H),
7.0 Hz, 3H); ESI MS m/z 531.3 [M]−. HR-ESI MS calcd 1.67–1.45 (m, 2H). ESI MS m/z 541.2 [M]+. HR-ESI MS calcd for
for C28H22Cl2N4O3Na (M + Na)+: 555.0967, found: 555.0970.
C27H27Cl2N4O4 (M)+: 541.0363, found: 541.0371.
N-CYCLOPROPYL-4-((1-(3,4-DICHLOROBENZYL)-2-OXO-2,3-DIHYDRO-1H-
1-(4-AMINOCYCLOHEXYL)-3-(3,4-DICHLOROBENZYL)-5-(4-(PIPERIDINE-1-CAR-
IMIDAZO[4,5-B]PYRIDIN-5-YL)OXY)BENZAMIDE (3G). White solid, yield
BONYL)PHENOXY)-1H-IMIDAZO[4,5-B]PYRIDIN-2(3H)-ONE
(3L). White
87.3%: H NMR (400 MHz, DMSO) δ 8.40 (d, J = 4.1 Hz, 1H), solid, yield 82%: 1H NMR (400 MHz, CDCl3) δ 7.43–7.41 (m,
7.82 (d, J = 8.8 Hz, 2H), 7.66 (d, J = 2.0 Hz, 1H), 7.61 (d, J = 1H), 7.40 (d, J = 8.3 Hz, 1H), 7.38 (d, J = 2.0 Hz, 2H), 7.15–7.09
8.3 Hz, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.31 (dd, J = 8.3, 2.1 Hz, (m, 3H), 7.02 (d, J = 8.3 Hz, 1H), 6.58 (d, J = 8.3 Hz, 1H), 4.96
1H), 7.07 (d, J = 8.8 Hz, 2H), 6.71 (d, J = 8.2 Hz, 1H), 5.03 (s, (s, 2H), 4.35–4.21 (m, 1H), 3.66 (s, 3H), 3.44–3.30 (m, 2H),
2H), 2.86–2.78 (m, 1H), 0.68 (dt, J = 7.1, 4.7 Hz, 2H), 0.55 2.79–2.63 (m, 1H), 2.32–2.15 (m, 2H), 2.12–1.98 (m, 2H),
(dt, J = 7.1, 4.7 Hz, 2H); 13C NMR (126 MHz, DMSO) δ 166.77, 1.81–1.72 (m, 2H), 1.72–1.60 (m, 4H), 1.60–1.47 (m, 4H). ESI
157.77, 156.58, 153.62, 141.50, 137.94, 131.17, 130.90, MS m/z 594.3 [M]+. HR-ESI MS calcd for C31H34Cl2N5O3 (M)+:
130.23, 129.63, 129.61, 128.93, 127.82, 120.52, 118.96, 118.14, 594.1033, found: 541.1041.
1
104.12, 42.26, 23.00, 5.74. MS-ESI m/z: 467.2 (M)−; HR-ESI MS
1-(4-AMINOCYCLOHEXYL)-3-(3,4-DICHLOROBENZYL)-5-(4-(MORPHOLINE-4-
calcd for C23H18Cl2N4O3Na (M + Na)+: 491.0654, found: CARBONYL)PHENOXY)-1H-IMIDAZO[4,5-B]PYRIDIN-2(3H)-ONE (3M). White
491.0662.
solid, yield 91%: 1H NMR (300 MHz, CDCl3) δ 7.45 (d, J =
8.5 Hz, 2H), 7.42–7.35 (m, 2H), 7.17–7.09 (m, 3H), 7.02 (d, J =
1-(3,4-DICHLOROBENZYL)-5-(4-(PIPERIDINE-1-CARBONYL)PHENOXY)-1H-
IMIDAZO[4,5-B]PYRIDIN-2(3H)-ONE (3H). White solid, yield 92.3%: 8.3 Hz, 1H), 6.58 (d, J = 8.3 Hz, 1H), 4.96 (s, 2H), 4.35–4.18 (m,
1H NMR (400 MHz, CDCl3) δ 9.72 (s, 1H), 7.42 (t, J = 8.5 Hz, 1H), 3.79–3.62 (m, 4H), 3.49–3.35 (m, 4H), 2.76–2.60 (m, 1H),
4H), 7.18 (d, J = 8.3 Hz, 1H), 7.08 (dd, J = 8.2, 3.5 Hz, 3H), 6.60 2.32–2.12 (m, 2H), 2.09–1.92 (m, 2H), 1.84–1.67 (m, 2H),
(d, J = 8.3 Hz, 1H), 5.01 (s, 2H), 3.85–3.61 (m, 2H), 3.52–3.30 1.62–1.41 (m, 2H). ESI MS m/z 596.3 [M]+. HR-ESI MS calcd for
(m, 2H), 1.77–1.62 (m, 4H), 1.62–1.46 (m, 2H). 13C NMR C30H32Cl2N5O4 (M)+: 596.1831, found: 596.1843.
(101 MHz, CDCl3) δ 170.10, 157.82, 156.31, 153.88, 140.78,
(R)-4-((1-(4-AMINOCYCLOHEXYL)-3-(3,4-DICHLOROBENZYL)-2-OXO-2,3-
135.56, 133.08, 132.32, 131.27, 130.93, 129.35, 128.73, 126.81, DIHYDRO-1H-IMIDAZO[4,5-B]PYRIDIN-5-YL)OXY)-N-(1-PHENYLETHYL)BENZA-
119.99, 119.61, 118.12, 104.80, 48.98, 48.96, 43.54, 26.46, MIDE (3N). White solid, yield 86%: 1H NMR (300 MHz, CDCl3) δ
25.54, 24.41. ESI MS m/z 497.3 [M]+; HR-ESI MS calcd for 7.93 (d, J = 8.6 Hz, 2H), 7.67 (d, J = 7.5 Hz, 2H), 7.47–7.38 (m,
C25H23Cl2N4O3 (M + H)+: 497.1674, found: 497.1680.
4H), 7.36–7.31 (m, 2H), 7.27–7.16 (m, 1H), 7.16–7.11 (m, 2H),
1-(3,4-DICHLOROBENZYL)-5-(4-(MORPHOLINE-4-CARBONYL)PHENOXY)-1H- 7.11–7.05 (m, 1H), 6.55 (d, J = 8.2 Hz, 1H), 5.35–5.25 (m, 1H),
1
IMIDAZO[4,5-B]PYRIDIN-2(3H)-ONE (3I). White solid, yield 92.3%: H 4.98 (s, 2H), 4.40–4.26 (m, 1H), 2.82–2.70 (m, 1H), 2.44–2.26
NMR (400 MHz, CDCl3) δ 9.79 (s, 1H), 7.44–7.35 (m, 4H), 7.16 (m, 2H), 2.19–2.05 (m, 2H), 1.89–1.74 (m, 2H), 1.57 (s, 3H),
(d, J = 8.2 Hz, 1H), 7.07 (d, J = 8.4 Hz, 3H), 6.60 (d, J = 8.3 Hz, 1.54–1.47 (m, 2H). ESI MS m/z 630.3 [M]+. HR-ESI MS calcd for
1H), 4.99 (s, 2H), 3.79–3.65 (m, 4H), 3.65–3.44 (m, 4H). 13C C34H34Cl2N5O3 (M)+: 630.2039, found: 630.2008.
NMR (101 MHz, CDCl3) δ 169.97, 157.65, 156.67, 153.93,
1-(4-AMINOCYCLOHEXYL)-3-(3,4-DICHLOROBENZYL)-5-(4-(4-METHYLPIPER-
140.89, 135.60, 133.10, 132.34, 130.95, 130.49, 129.36, 129.05, AZINE-1-CARBONYL)PHENOXY)-1H-IMIDAZO[4,5-B]PYRIDIN-2(3H)-ONE (3O).
1
126.83, 120.12, 119.66, 118.05, 104.90, 66.82, 43.55. MS-ESI White solid, yield 92%: H NMR (400 MHz, CDCl3) δ 7.48 (d,
m/z: 499.3 (M)+; HR-ESI MS calcd for C24H20Cl2N4O4Na J = 8.2 Hz, 2H), 7.44–7.36 (m, 2H), 7.19–7.10 (m, 3H), 7.05 (d,
(M + Na)+: 521.0757, found: 521.0759.
J = 8.4 Hz, 1H), 6.61 (d, J = 8.3 Hz, 1H), 4.96 (s, 2H), 4.30–4.18
(R)-4-((1-(3,4-DICHLOROBENZYL)-2-OXO-2,3-DIHYDRO-1H-IMIDAZO[4,5-B]- (m, 1H), 4.02–3.67 (m, 4H), 3.20–3.02 (m, 4H), 2.73 (s, 3H),
PYRIDIN-5-YL)OXY)-N-(1-PHENYLETHYL)BENZAMIDE (3J). White solid, yield 2.69–2.63 (m, 1H), 2.26–2.12 (m, 2H), 2.09–1.99 (m, 2H),
1
88.3%: H NMR (400 MHz, CDCl3) δ 8.78 (d, J = 8.0 Hz, 1H), 1.82–1.71 (m, 2H), 1.58–1.44 (m, 2H). ESI MS m/z 609.4 [M]+.
7.91 (d, J = 8.8 Hz, 2H), 7.66 (d, J = 2.0 Hz, 1H), 7.61 (d, J = HR-ESI MS calcd for C31H35Cl2N6O3 (M)+: 609.2148, found:
8.3 Hz, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.40–7.36 (m, 2H), 609.2133.
7.35–7.28 (m, 3H), 7.21 (t, J = 7.2 Hz, 1H), 7.10 (d, J = 8.8 Hz,
Biological evaluations
2H), 6.71 (d, J = 8.2 Hz, 1H), 5.21–5.12 (m, 1H), 5.03 (s, 2H),
1.47 (d, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 164.83,
ELISA KINASE ASSAY. The kinase domain of c-Met was
157.79, 156.65, 153.62, 144.93, 141.49, 137.93, 131.18, 130.90, expressed using the Bac-to-Bac™ baculovirus expression
130.25, 129.76, 129.61, 129.17, 128.18, 127.82, 126.52, 125.98, system (Invitrogen, Carlsbad, CA, USA) and purified on Ni-NTA
120.49, 119.05, 118.15, 104.06, 48.40, 42.27, 22.24. ESI MS m/z columns (QIAGEN Inc., Valencia, CA, USA). The activity of
1560 | Org. Biomol. Chem., 2013, 11, 1545–1562
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c-Met tyrosine kinase was detected using enzyme-linked- with the spacing value equivalent to 0.375 Å, and the grid
immunosorbent assay (ELISA). Briefly, 20 μg mL−1 Poly(Glu, center is 1.5, 4.687, 50. During docking, the number of individ-
Tyr)4:1 (Sigma, St. Louis, MO) was pre-coated in 96-well ELISA uals in population, maximum number of energy evaluation,
plates as substrate. Each well was treated with 50 μL of maximum number of generations and GA-LS runs are rectified
10 μmol L−1 ATP solution which was diluted in kinase reaction to 150, 2 500 000, 27 000 and 100, respectively. Other para-
buffer (50 mM HEPES pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2, meters set as the default values implemented by the program.
0.2 mM Na3VO4, 1 mM DTT). Then 1 μL of various concen-
trations of tested compounds or reference compound dis-
solved in DMSO were added to each reaction well. Experiments
Acknowledgements
at each concentration were performed in duplicate. The reac-
tion was initiated by the addition of purified tyrosine kinase
proteins diluted in 40 μL of kinase reaction buffer. After incu-
bation at 37 °C for 60 min, the wells were washed three times
with phosphate buffered saline (PBS) containing 0.1% Tween
20 (T-PBS). Next, 100 μL anti-phosphotyrosine (PY99) antibody
(1 : 1000, Santa Cruz Biotechnology, Santa Cruz, CA) diluted in
T-PBS containing 5 mg mL−1 BSA was added and the plate was
incubated at 37 °C for 30 min. After the plate was washed
three times, 100 μL horseradish peroxidase-conjugated goat
anti-mouse IgG (1 : 2000, Calbiochem, San Diego, CA) diluted
in T-PBS containing 5 mg mL−1 BSA was added and the plate
was incubated at 37 °C for 30 min. The plate was washed, then
100 μL citrate buffer (0.1 M, pH 5.5) containing 0.03% H2O2
and 2 mg mL−1 o-phenylenediamine was added and samples
were incubated at room temperature until color emerged. The
reaction was terminated by adding 50 μL of 2 M H2SO4, and
the plate was read using a multiwell spectrophotometer (VER-
SAmax™, Molecular Devices, Sunnyvale, CA, USA) at 492 nm.
The inhibitory rate (%) was calculated with the formula: [1 −
(A492 treated/A492 control)] × 100%. IC50 values were calculated
from the inhibitory curves.
This work was supported by grants from the Natural Science
Foundation of China for Innovation Research Group
(81021062), the National Natural Science Foundation of China
(81072527, 81123004, 81102461), the National Science & Tech-
nology Major Project “Key New Drug Creation and Manufactur-
ing Program” (2012ZX09301001-007), National Program on Key
Basic Research Project of China (2012CB910704).
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