Synthesis and biological evaluation of 4-(2-fluorophenoxy)-3,3′-bipyridine derivatives as potential c-met inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.04.062

Six series of novel 4-(2-fluorophenoxy)-3,3′-bipyridine derivatives conjugated with aza-aryl formamide/amine scaffords were designed and synthesized through a structure-based molecular hybridization approach. The target compounds were evaluated for c-Met kinase inhibitory activities and cytotoxicity against four cancer cell lines (HT-29, A549, MKN-45 and MDA-MB-231) in vitro. Most compounds exhibited moderate to excellent potency, and the most promising candidate 26c (c-Met kinase IC₅₀ = 8.2 nM) showed a 4.7-fold increase in cytotoxicity against c-Met-addicted MKN-45 cell line in vitro (IC₅₀ = 3 nM), superior to that of Foretinib (IC₅₀ = 23 nM). The preliminary structure-activity relationship indicated that a 1H-benzo [e] [1,3,4]thiadiazine-3-carboxamide-4,4-dioxide moiety as linker contributed to the antitumor potency.

Full text of DOI:10.1016/j.ejmech.2016.04.062

Accepted Manuscript
Synthesis and biological evaluation of 4-(2-fluorophenoxy)-3,3′-bipyridine derivatives
as potential c-met inhibitors
Sijia Zhao, Yu Zhang, Hongyang Zhou, Shuancheng Xi, Bin Zou, Guanglong Bao,
Limei Wang, Jiao Wang, Tianfang Zeng, Ping Gong, Xin Zhai
PII:
S0223-5234(16)30355-5
10.1016/j.ejmech.2016.04.062
EJMECH 8577
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 January 2016
Revised Date: 20 April 2016
Accepted Date: 23 April 2016
Please cite this article as: S. Zhao, Y. Zhang, H. Zhou, S. Xi, B. Zou, G. Bao, L. Wang, J. Wang, T.
Zeng, P. Gong, X. Zhai, Synthesis and biological evaluation of 4-(2-fluorophenoxy)-3,3′-bipyridine
derivatives as potential c-met inhibitors, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.04.062.
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ACCEPTED MANUSCRIPT
Graphical Abstract
F
Six series of 5-atom linkers
N
S
N
H
F
N
F
linker
R
A
B
O
O
O
O
O
N
N
26c
N
N
IC50 (nM)
Compd.
26c
A549
HT-29
MKN-45
MDA-MB-231
c-Met
8.2
430
900
3
10
Six series of novel 4-(2-fluorophenoxy)-3,3'-bipyridine derivatives conjugated with aza-aryl
formamide/amine scaffords were synthesized and evaluated for their cytotoxicity and c-Met kinase activity
in vitro. Compound 26c was identified as a lead compound for further structural optimization and antitumor
activity screening purpose.

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of 4-(2-fluorophenoxy)-3,3'-bipyridine
derivatives as potential c-met inhibitors
Sijia Zhao, Yu Zhang, Hongyang Zhou, Shuancheng Xi, Bin Zou, Guanglong Bao, Limei Wang,
Jiao Wang, Tianfang Zeng, Ping Gong, Xin Zhai
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical
University, Shenyang 110016, PR China
Abstract
Six series of novel 4-(2-fluorophenoxy)-3,3'-bipyridine derivatives conjugated with aza-aryl
formamide/amine scaffords were designed and synthesized through a structure-based molecular
hybridization approach. The target compounds were evaluated for c-Met kinase inhibitory
activities and cytotoxicity against four cancer cell lines (HT-29, A549, MKN-45 and
MDA-MB-231) in vitro. Most compounds exhibited moderate to excellent potency, and the most
promising candidate 26c (c-Met kinase IC50 = 8.2 nM) showed a 4.7-fold increase in cytotoxicity
against c-Met-addicted MKN-45 cell line in vitro (IC50 = 3 nM), superior to that of Foretinib (IC50
=
23 nM). The preliminary structure–activity relationship indicated that
a
1H-benzo[e][1,3,4]thiadiazine-3-carboxamide-4,4-dioxide moiety as linker contributed to the
antitumor potency.
Keywords: Synthesis; Bipyridine derivatives; Aza-aryl formamides; c-Met; Cytotoxicity
1. Introduction
The c-mesenchymal-epithelia transition factor (c-Met), known as a hepatocyte growth factor
receptor (HGFR), belongs to a prototype member of the receptor tyrosine kinases (RTKs)
subfamily [1, 2]. c-Met plays a key role in several cellular processes [3], while abnormal c-Met
stimulates signaling pathways responsible for proliferation, invasion, migration, angiogenesis,
survival, metastasis, and drug resistance [4-6]. Therefore, deregulation of the c-Met/HGF
signaling axis has been identified as a key contributing factor for cancer treatment [7]. To date,
significant progress has been made on the development of c-Met inhibitors, resulting in the
marketing of Foretinib (GSK1363089) [6], Cabozantinib (XL-184) [8], and more than ten
candidates currently under clinical trials, such as MGCD265 [9], BMS-777607 [10] and E-7050
[11] (Fig.1). Besides that, varieties of hit-like molecules are reported recently [12-14].
(Fig. 1 should be listed here)
BMS-777607, a pyridine-based selective ATP-competitive c-Met kinase inhibitor which
primarily targets c-Met (IC50 = 3.9 nM) and several Met family members, including Axl (IC50
=
1.1 nM), Ron (IC50 = 1.8 nM), and Tyro3 (IC50 = 4.3 nM), is currently undergoing phase I clinical
trials for various cancers [15, 16]. Due to its excellent efficacy in vivo, favorable pharmacokinetic
and preclinical safety profiles, our attention was drown to the optimization of BMS-777607 as an
extension of our work on the development of novel potent c-Met inhibitors. Application of
bioisosterism theory, the aminopyridine moiety in BMS-777607 was replaced with 3, 3'-bipyridine
Corresponding author. Tel. / fax: +86 24 2398 6429.
E-mail address:zhaixin_syphu@126.com (X. Zhai).
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ring to give a new parent structure, which led to the design of the target compounds in this paper.
Further modification mainly focused on the linkers between moiety A and moiety B (Fig. 2),
which was characterized important for gaining favorable enzyme activities and cellular potencies.
According to our previous work, two structural characteristics of the linkers were interpreted
[17-21]. One is “five-atom regulation”, which demonstrates a distance of six chemical bonds
should be existed between moiety A and moiety B, and the other characteristic is that the linkers
should have “both hydrogen-bond donor and acceptor” as well. Thus, in order to explore
more varieties of linkers according to the “five-atom regulation”, we designed six different cyclic
moieties. Differ from other aza-aryl formamide linkers, a pyrimidine-4, 6-diamine framework was
introduced to further enrich the “five-atom regulation”. Additionally, on the molecular design
level, various substituents were introduced to the terminal phenyl ring (moiety B) aiming at
exploring the influence of substituents on anticancer activity by regulating the electronic and steric
effects. Accordingly, six series of novel 4-(2-fluorophenoxy)-3, 3'-bipyridine derivatives
conjugated with aza-aryl formamide/amine scaffords (23a-f, 24a-d, 25a-e, 26a-f, 27a-b and 28a-b)
were designed and synthesized (Fig. 2).
(Fig. 2 should be listed here)
In this paper, all target compounds were evaluated for their cytotoxic activity against four cell
lines (HT-29, A549, MKN-45 and MDA-MB-231) in vitro. Moreover, the enzymatic assays
against c-Met of most compounds were also performed.
2. Chemistry
A series of 4-phenoxy-3,3'-pyridine derivatives and their intermediates were synthesized
according to the pathways described in Scheme 1-6.
As shown in Scheme 1, the general synthesis of compounds 23a-g involved six steps.
Compound 1 was obtained from bromination reaction of commercially available 4-aminopyridine
with N-bromosuccinimide (NBS) [22]. Treatment of 1 with sodium nitrite in sulfuric acid aqueous
solution afforded the diazotization intermediate, which was subsequently hydrolysis with sodium
hydroxide, without further purification, gave rise to 3-bromopyridin-4-ol 2 [23]. 2 was O-arylated
with 3, 4-difluoronitrobenzene in the presence of potassium carbonate in N, N-dimethylformamide
(DMF) to provide 4-phenoxypyridine intermediate 3. Suzuki coupling reaction of intermediate 3
with 3-pyridinylborate, which was afforded by treatment of 3-bromopyridine with
bis(pinacolato)diboron using trans-dichlorobis(triphenyl-phosphine)Palladium(II) and potassium
carbonate as catalyst, to furnish compound 4 [24]. The key intermediates 5 were obtained via
reduction of compound 4 with iron powder and catalytic amounts of ammonium chloride in
ethanol at reflux [25]. Finally, the target compounds 23a-g were successfully obtained by the
nucleophilic displacement of 5 with 6a-g in the presence of N, N-diisopropylethylamine (DIPEA)
in ethanol for 15h at reflux. Herein, intermediates 6a-g were available via the N-arylation of 4,
6-dichloropyrimidine with substituted anilines under alkaline conditions in an acceptable yield
(30.0% - 45.1%).
(Scheme 1 should be listed here)
The target compounds 24a-f were synthesized according to the method summarized in Scheme
2. With one-pot strategy, treatment of commercially available substituted anilines with sodium
nitrite in concentrated hydrochloric acid provided corresponding substituted phenylhydrazine
intermediates, which then underwent an electrophilic substitution reaction with
2

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ethyl(2-cyanoacetyl)carbamate to generate compounds 7a-f. Intramolecular cyclization of 7a-f
with glyoxal in the presence of sodium acetate furnished 8a-f, which were methylated with
iodomethane and sodium hydride afforded 9a-f. Subsequently, the cyano group of 9a-f was
hydrolyzed with glyoxal and hydrochloric acid gave rise to carboxylic acid derivatives 10a-f [26,
27]. N-acylation reaction of 10a-f with thionyl chloride produced the corresponding acyl chlorides,
which were further treatment with intermediate 5 in the presence of DIPEA in dichloromethane to
obtain target compounds 24a-f.
(Scheme 2 should be listed here)
Scheme 3 depicts the sequence of reactions that led to the preparation of compounds 25a–e.
Acylation of substituted phenylhydrazine 12a-e with O-formyl carboxylic acid 11 gave access to
dihydrophthalazinecarboxylic acid 13a-e [28]. Herein, intermediate 11 was prepared by oxidation
of naphthalene with potassium permanganate under basic condition. Meanwhile, treatment of
substituted anilines with sodium nitrite in sulfuric acid aqueous solution to give the corresponding
diazotization intermediates, which were subjected to reduction reaction with sodium sulfite to
obtain substituted phenylhydrazine 12a-e [29]. Subsequently, the resultant carboxylates 13a-e
were converted to the desired acyl chlorides on exposure to thionyl chloride in toluene, which
underwent a N-acylation reaction with intermediate 5 to provide target compounds 25a-e.
(Scheme 3 should be listed here)
The general strategy to synthesize compounds 26a-c were outlined in Scheme 4. According to
this approach，the side chains 17a–c were prepared from 2-fluorobenzenethiol by a four-step
procedure. 2-Fluorobenzenethiol was substituted by ethyl chloroacetate led to 14 [30]. Oxidation
reaction of 14 with 30% hydrogen peroxide aqueous in glacial acetic acid to obtain the sulfone 15
successfully [31], which was further treated with various substituted phenylhydrazines to generate
compounds 16a-c. An intramolecular N-arylation reaction of 16a-c with potassium carbonate
catalyst gave a good yield of benzothiadiazine intermediate，which were hydrolyzed in the
presence of sodium hydroxide to give the key intermediates 17a-c. Finally, the target compounds
26a-c were synthesized as previously described reactions shown in Scheme 2.
(Scheme 4 should be listed here)
The synthesis of compounds 27a-b was illustrated in Scheme 5. Treatment of intermediates
12d-e with glyoxal in 40% acetic acid aqueous solution provided Schiff base 18a-b, respectively.
Knoevenagel reaction of 18a-b with meldrum’s acid on exposure to pyridine in toluene furnished
methylene condensation compounds 19a-b, which underwent an intramolecular nucleophilic
substitution reaction with sodium methoxide to afford pyridazinone acids 20a-b [32]. Ultimately,
the synthesis of 27a-b was progressed as previously described N-acylation reaction in Scheme 2.
(Scheme 5 should be listed here)
The target compounds 28a-b were prepared according to the method as depicted in Scheme 6.
Treatment of substituted phenylacetic acid with phosphorus oxychloride in DMF led to the
formation of Vinamidinium salt (1,5-diazapentadiene salt) 21a-b, which were widely used in the
synthesis of the aromatic ring and a variety of aza-aromatic scaffolds by virtue of the constructing
of three carbon atoms skeleton [33]. Compounds 21a-b were treated with diethyl malonate in the
presence of potassium t-butoxide in tetrahydrofuran gave the quatemary ammonium intermediates,
which were passed into methylamine gas, without separation, to obtain pyridonecarboxylic acids
22a-b. Similarly, the desired compounds 28a-b were accomplished using the same method as
preparing 24a-f.
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(Scheme 6 should be listed here)
3. Result and discussion
The prepared twenty-five target compounds (23a-g, 24a-f, 25a-e, 26a-c, 27a-b and 28a-b) were
evaluated for their cytotoxicity in vitro against c-Met-addicted cancer cell lines including HT-29
(human colon cancer), A549 (human lung adenocarcinoma), MKN-45 (human gastric cancer) and
a c-Met less sensitive MDA-MB-231 (human breast cancer) by the 3-(4,5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide (MTT) assay, taking Foretinib as positive control. The results
expressed as half-maximal inhibitory concentration (IC50) values and are presented in Table 1, as
mean values of experiments performed in triplicate.
As illustrated in Table 1, most target compounds showed moderate to significant cytotoxic
activities against one or more tested cancer cells with potencies in the single digit micromole
range, which suggested that the introduction of six new moiety as “five-atom linker” to
4-(2-fluorophenoxy)-3, 3'-bipyridine framework maintained the potent cytotoxic activity. Four
compounds (23e, 25b, 26a and 26c) exhibited promising cytotoxicity with IC50 values ranging
from 0.003 to 1.75 µM, which was comparable to that of Foretinib. Notably, the most prominent
candidate 26c displayed prominent activity with IC50 value of 0.43 µM, 0.9 µM, 0.003 µM and
0.01 µM against HT-29, A549, MKN-45 and MDA-MB-231 cells, which was 4.7- and 54-fold
more active than that of Foretinib against c-Met-addicted MKN-45 cell (0.003 µM vs. 0.023 µM)
and c-Met less sensitive MDA-MB-231 cell (0.01 µM vs. 0.54 µM). Moreover, three compounds
(23c, 25c and 26b) showed antitumor potency either, whose IC50 value less than 0.1 µM. As a
general trend, the target compounds were more potent on MKN-45 cell than on other three cells,
suggesting the higher selectivity for gastric cancer.
Fourteen new compounds were further determined for c-Met kinase activity using homogenous
time-resolved fluorescence (HTRF) assays. As shown in Table 1, the tested compounds exhibited
moderate c-Met enzymatic potency with IC50 values ranging from 8.2 to 106.4 nM, suggesting
that the inhibition of c-Met may be a main mechanism for the antitumor activity of the prepared
compounds. Though compound 26c demonstrated the best c-Met enzymatic potency (IC50 = 8.2
nM) superior to others, whose biological activity was slightly lower than that of the positive
control Foretinib (8.2 nM vs. 1.1 nM).
The structure-activity relationship (SAR) was commenced by the introduction of “five-atom
linker” that contains different kinds of nitrogen aromatic heterocyclic between moiety A and
moiety B. The pharmacological activity data indicated that the different linker affected the activity
dramatically. A clear preference for activity in compounds 25a-e and 26a-c whose linker bears
4-oxo-3,4-dihydrophthalazine-1-carboxamide and 1H-benzo[e][1,3,4]thiadiazine-3-carboxamide-4,
4-dioxide fragment respectively, indicating that a benzoheterocyle moiety of the linkers
contributed positively to the antitumor potency. Compounds 24a-f, 27a-b and 28a-b containing
4-methyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxamide,
3-oxo-2,3-dihydropyridazine-4-carboxamide
and
1-methyl-2-oxo-1,2-dihydropyridine-3-carboxamide linkers respectively, by contrast, revealed a
weak antitumor activity. Furthermore, 26a-c displayed higher potency than that of 25a-e, which
probably due to the existence of sulfonyl group that was favor of hydrogen-bond interaction
between molecules with target proteins. Interestingly, compounds 23a-g bearing
pyrimidine-4,6-diamine skeleton showed moderate activity as well, disclosing that the acylamino
4

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group was not essential for antitumor potency, as very meaningful for extending the structural
categories of traditional aza-aryl formamide or aliphatic acylamino moiety as linkers.
Further investigations were performed to study the effect of different substituents on the phenyl
ring (moiety B) on the cytotoxic activity. Introduction of mono-electron-withdrawing groups
(mono-EWGs) on the phenyl ring led to an obvious improvement in activity, such as compounds
26c (R = 3-fluoro, IC50 = 3.0 nM) and 26a (R = 3-trifluoromethyl, IC50 = 27.0 nM). However, the
introduction of double-electron-withdrawing groups (double-EWGs) caused negative effect
against MKN-45 cells, such as 26b (R = 3, 4-difluoro, IC50 = 90.0 nM). Another case in point is
that 24e with double-EWGs (R = 3-chloro-4-flourophenyl) nearly vanished the cytotoxicity on all
tested cell lines compared to 24a (R = 2-flouro) and 24b (R = 4-chloro) with mono-EWGs.
However, 23b (R = 4-trimethoxy) and 23g (R = 3, 5-dimethoxy) is an exception.
The data shown in Table 1 indicated that the introduction of halogen group (F, Cl or CF3) at the
phenyl ring B made a good contribution to potency. Compounds with electron-donating groups, by
contrast, lost activity significantly. Compared 4-trimethoxy analog 25a with 3-fluoro analog 25b,
a 4.7-fold drop in c-Met inhibition and approximate 3- to 145-fold decrease against tested cells
were observed. It suggested that fluoro group at meta-position of phenyl ring B is a preference for
enhanced activity.
(Table 1. should be list here)
4. Conclusions
In summary, twenty-five novel bipyridine derivatives bearing aza-aryl formamide/amine
skeleton as potential c-Met kinase inhibitors were designed and synthesized. Our preliminary
cytotoxic investigation showed that most compounds displayed moderate to excellent activity
against MKN-45, MDA-MB-231 and A549 cancer cell lines. The exploration of preliminary SAR
led to the identification of c-Met inhibitor 26c as a valuable leading molecule, which possessed
excellent c-Met kinase inhibition on a single-digital nanomolar level (IC50 = 8.2 nM). Moreover, it
displayed better cytotoxicity against MKN-45 (IC50 = 3.0 nM) and MDA-MB-231 (IC50 = 10.0 nM
nM) than against HT-29 (IC50 = 430 nM) and A549 cells (IC50 = 900 nM). The initial SARs
discovered a novel 1H-benzo[e][1,3,4]thiadiazine-3-carboxamide-4,4-dioxide scaffold as the
preferred linker for enhanced potency. Moreover, fluoro group at meta-position on the phenyl ring
(moiety B) was a best option, and compounds with mono-EWGs were more active than those with
double-EDGs or without substituents on moiety B. Further studies on structural optimization and
the mechanism of kinase selectivity are in progress and will be reported in the future.
5. Experimental protocols
5.1. Chemistry
All melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi
Labortechnik, Flawil, Switzerland) and were uncorrected. Mass spectra (MS) were taken in ESI
1
mode on Agilent 1100 LC-MS (Agilent, palo Alto, CA, USA). H NMR spectra was performed
using Bruker 300 MHz spectrometers (Bruker Bioscience, Billerica, MA, USA) with TMS as an
internal standard. Column chromatography was run on silica gel (200-300 mesh) from Qingdao
Ocean Chemicals (Qingdao, Shandong, China). Unless otherwise noted, all materials were
obtained from commercially available sources and were used without further purification.
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5.1.2. Preparation of 3-bromo-4-aminopyridine (1)
To the mixture of 4-aminopyridine (20.0 g, 0.21 mol) and acetonitrile (300.0 mL), NBS (39.8 g,
0.22 mol) was added in batches at 0°C avoiding light. Subsequently, the reaction mixture was
stirred at room temperature for 24 h. After completion of reaction as indicated by thin layer
chromatography (TLC), the mixture was then cooled and filtered, and the filtrate was concentrated
under reduced pressure. The residue was purified by flash column chromatography (silica,
dichloromethane/methanol, 20:1) to afford the desired compound 1 as a light yellow solid (33.2 g,
91.4%). MS (ESI) m/z: 172.9 [M+H]+.
5.1.3. Preparation of 3-bromo-4-hydroxypyridine (2)
Concentrated sulfuric acid (85.0 mL) and the 3-bromo-4-aminopyridine (113.0 g, 0.65 mol)
were added successively to the distilled water (550.0 mL) at 0°C, and then NaNO2 (63.1 g, 0.91
mol) aqueous solution (130.0 mL) was added drop-wise to maintain the temperature between
0-5°C. After stirring at r.t. for 1 h, the reaction mixture was heated at 95°C for a further 0.5 h.
Upon cooling to below 0°C, the mixture was alkalized to pH 6 with saturated sodium hydroxide
solution, washed with ethyl acetate (200mL×3) and brine (200 mL), dried over anhydrous Na2SO4,
filtered and concentrated to yield the title compound 2 as a yellow solid (75.1 g, 66.2%). MS (ESI)
m/z: 173.9 [M+H]+.
5.1.4. Preparation of 3-bromo-4-(2-fluoro-4-nitrohenoxyl)pyridine (3)
To a stirred solution of 3-bromo-4-hydroxypyridine (2) (17.0 g, 98.0 mmol) in DMF (80.0 mL)
was added 3, 4-difluoronitrobenzene (13.0 mL, 0.12 mol) and anhydrous K2CO3 (34.0 g, 0.25
mol). Upon the completion of addition, the reaction mixture was heated at 80°C for 2 h. The
mixture was cooled and poured into ice-water. The mixture was filtrated and washed with water,
and then dried to give 3 as a yellow solid (20.1 g, 66.1%). MS (ESI) m/z: 312.9 [M+H]+.
5.1.5. Preparation of 4-(2-fluoro-4-nitrophenoxyl)-3,3'-bipyridine (4)
A mixture of 3-bromopyridine (5.5 mL, 57.0 mmol), bis(pinacolato)diboron (18.3 g, 72.0
mmol), potassium acetate (19.9 g, 0.14 mol), bis(triphenylphosphine)palladium dichloride (1.7 g,
2.4 mmol) in dioxane (300.0 mL) was heated at 90°C for 4h under nitrogen atmosphere. Upon
cooling to room temperature, 3-bromo-4-(2-fluoro-4-nitrohenoxyl)pyridine (3) (15.0 g, 48.0
mmol), sodium carbonate (15.3 g, 0.14 mol) and H2O (30.0 mL) were added, the reaction mixture
was stirred for another 4.5 h. The solid was removed by filtration, and the filtrate was concentrated
to dryness in vacuum. The residue was purified by column chromatography (silica,
dichloromethane/methanol, 30: 1) to furnish the compound 4 as a yellow solid (8.2 g, 55.4%). MS
(ESI) m/z: 312.1[M+H]+.
5.1.6. Preparation of 4-(2-fluoro-4-aminophenoxyl)-3,3'-bipyridine (5)
To the mixture of 4-(2-fluoro-4-nitrophenoxyl)-3,3'-bipyridine (4) (22.0 g, 71.0 mmol), 2.2 mL
glacial acetic acid and 60% ethanol (150.0 mL), iron powder (11.9 g, 0.21 mol) and catalytic
amount ammonium chloride were added. Upon the completion of addition, the reaction mixture
was heated at reflux for 5 h, and then filtered immediately. Subsequently, the filtrate was
concentrated and anhydrous ether (200.0 mL) was added to the residue, stirred for 0.5h and then
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filtrated to give the compound 5 as a yellow solid (15.0 g, 73.2%). MS (ESI) m/z: 282.1[M+H]+.
5.1.7. Preparation of 6-chloro-N-(substituted phenyl)pyrimidin-4-amine (6a-g)
A mixture of 4, 6-dichloropyrimidine (3.0 g, 23.6 mmol), substituted aniline (23.6 mmol),
DIPEA (4.1 mL, 25.0 mmol) in ethanol (30.0 mL) was heated at reflux for 12 h. Upon cooling to
room temperature, the mixture was filtered and the filtrate was concentrated to give the oil,
column chromatography (CH2Cl2: MeOH = 25: 1) to give compounds (6a-g).
5.1.7.1. 6-Chloro-N-phenylpyrimidin-4-amine (6a).
Yield: 45.1%; MS (ESI) m/z: 206.1 [M+H]+.
5.1.7.2. 6-Chloro-N-(4-methoxyphenyl)pyrimidin-4-amine (6b).
Yield: 30.0%; MS (ESI) m/z: 236.1 [M+H]+.
5.1.7.3. 6-Chloro-N-(3, 4-difluorophenyl)pyrimidin-4-amine (6c).
Yield: 34.7%; MS (ESI) m/z: 242.0 [M+H]+.
5.1.7.4. 6-Chloro-N-(4-chlorophenyl)pyrimidin-4-amine (6d).
Yield: 42.2%; MS (ESI) m/z: 240.0 [M+H]+.
5.1.7.5. 6-Chloro-N-(3-chlorophenyl)pyrimidin-4-amine (6e).
Yield: 45.0%; MS (ESI) m/z: 240.0 [M+H]+.
5.1.7.6. 6-Chloro-N-(4-fluorophenyl)pyrimidin-4-amine (6f).
Yield: 36.9%; MS (ESI) m/z: 224.1 [M+H]+.
5.1.7.7. 6-Chloro-N-(3,5-dimethoxyphenyl)pyrimidin-4-amine (6g).
Yield: 38.4%; MS (ESI) m/z: 266.1 [M+H]+.
5.1.8. Preparation of ethyl (Z)-(2-cyano-2-(substituted phenyl)hydrazono)acetyl) carbamate (7a-f)
To the mixture of substituted anilines (0.08 mol), concentrated hydrochloric acid (45.0 mL) and
water (120.0 mL), aqueous solution of NaNO2 (60.0 mL, 0.11 mol) was added drop-wise under
stir at a rate that the temperature below 0°C, and the reaction mixture was stirred for 0.5 h. A
mixture of ethyl (2-cyanoacetyl)carbamate (13.7 g, 0.09 mol) and sodium acetate (24.0 g, 0.27
mol) in ethanol (400.0 mL) was added drop-wise to the resulting diazonium salt solution below
0°C and stirred for a further 2 h. The precipitate was collected by filtration and washed with water
(40.0 mL×2), dried to give compounds 7a-f.
5.1.8.1. Ethyl (Z)-(2-cyano-2-(2-(2-fluorophenyl)hydrazono)acetyl)carbamate (7a)
Orange solid; yield: 88.07%; MS (ESI) m/z: 279.1 [M+H]+.
5.1.8.2. Ethyl (Z)-(2-cyano-2-(2-(4-chlorophenyl)hydrazono)-2-cyanoacetyl)carbamate (7b)
Orange solid; yield: 89.34%; MS (ESI) m/z: 295.1 [M+H]+.
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5.1.8.3. Ethyl (Z)-(2-cyano-2-(2-(3-(trifluoromethyl)phenyl)hydrazono)acetyl)carbamate (7c)
Orange solid; yield: 84.27%; MS (ESI) m/z: 329.1 [M+H]+.
5.1.8.4. Ethyl (Z)-(2-cyano-2-(2-(2, 5-dichlorophenyl)hydrazono)acetyl)carbamate (7d)
Orange solid; yield: 85.07%; MS (ESI) m/z: 329.0 [M+H]+.
5.1.8.5. Ethyl (Z)-(2-(2-(3-chloro-4-fluorophenyl)hydrazono)-2-cyanoacetyl)carbamate (7e)
Orange solid; yield: 90.02%; MS (ESI) m/z: 313.1 [M+H]+.
5.1.8.6. Ethyl (Z)-(2-cyano-2-(2-(2, 4-dimethoxyphenyl)hydrazono)acetyl)carbamate (7f)
Orange solid; yield: 90.02%; MS (ESI) m/z: 321.2 [M+H]+.
5.1.9.
Preparation
of
2-(substituted
phenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,
4-triazine-6-carbonitrile (8a-f)
A mixture of compounds 7a-f (0.07 mol), sodium acetate (29.0 g, 0.35 mol) and glacial acetic
acid (400.0 ml) was stirred at reflux for 2 h. Subsequently, upon cooling to room temperature, the
mixture was poured into water (1.2 L), and then stirred for 0.5 h .The precipitate was collected by
filtration and washed with water (60.0 mL×2) and petroleum ether (40.0 mL×2), dried to give
compounds 8a-f.
5.1.9.1. 2-(2-Fluorophenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (8a)
Orange solid; yield: 65.21%; MS (ESI) m/z: 233.2 [M+H]+.
5.1.9.2. 2-(4-Chlorophenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (8b)
Orange solid; yield: 67.21%; MS (ESI) m/z: 249.0 [M+H]+.
5.1.9.3. 3,5-Dioxo-2-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile
(8c)
Orange solid; yield: 63.01%; MS (ESI) m/z: 283.1 [M+H]+.
5.1.9.4. 2-(2,5-Dichlorophenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (8d)
Orange solid; yield: 66.15%; MS (ESI) m/z: 282.9 [M+H]+.
5.1.9.5.
2-(3-Chloro-4-fluorophenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile
(8e)
Orange solid; yield: 68.20%; MS (ESI) m/z: 267.0 [M+H]+.
5.1.9.6. 2-(2,4-Dimethoxyphenyl)-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (8f)
Orange solid; yield: 61.89%; MS (ESI) m/z: 275.1 [M+H]+.
5.1.10.
Preparation
of
2-(substituted
phenyl)-4-methyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6- carbonitrile (9a-f).
To the solution of compounds 8a-f (36 mmol) in DMF (60.0 mL), sodium hydride was added
in portions after stirred for 0.5h in an ice bath, then the solution of iodomethane (2.35 mL, 38
8

ACCEPTED MANUSCRIPT
mmol) in DMF (10.0 mL) was added drop-wise to the reaction mixture stirred for 2 h. The
reaction mixture was poured into water (200.0 mL), the precepitate was collected by filtration and
washed with water (30.0 mL×2) and petroleum ether(20.0 mL×2), dried to give compounds 9a-f.
5.1.10.1. 2-(2-Fluorophenyl)-4-methyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile
(9a)
Orange solid; yield: 78.89%; MS (ESI) m/z: 247.06 [M+H]+.
5.1.10.2. 2-(4-Chlorophenyl)-4-methyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile
(9b)
Orange solid; yield: 75.04%; MS (ESI) m/z: 263.03 [M+H]+.
5.1.10.3.
4-methyl-3,5-dioxo-2-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,2,
4-triazine-6-carbonitrile (9c)
Orange solid; yield: 79.34%; MS (ESI) m/z: 297.06 [M+H]+.
5.1.10.4.
2-(2,5-Dimethoxyphenyl)-4-methyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-
carbonitrile (9d)
Orange solid; yield: 75.78%; MS (ESI) m/z: 289.10 [M+H]+.
5.1.10.5.
2-(3-Chloro-4-fluorophenyl)-4-methyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-
carbonitrile (9e)
Orange solid; yield: 80.08%; MS (ESI) m/z: 281.03 [M+H]+.
5.1.10.6.
2-(2,4-Dimethoxyphenyl)-4-methyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-
carbonitrile (9f)
Orange solid; yield: 79.03%; MS (ESI) m/z: 289.10 [M+H]+.
5.1.11.
Preparation
of
2-(substituted
phenyl)-4-methyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6- carboxylic acid (10a-f ).
A mixture of compounds 9a-f (0.03 mol), concentrated hydrochloric acid (72.0 mL) and
glacial acetic acid (150.0 mL) was stirred at reflux for 4 h. The solvent was evaporated in vacuo,
then water (50.0 mL) was added, the precepitate was collected by filtration. Subsequently, the
precepitate was stirred for 10min in anhydrous ether (30.0 mL), dried to give compounds 10a-f.
5.1.11.1.
2-(2-Fluorophenyl)-4-methyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxylic
acid (10a)
Pale white solid; yield: 39.03%; MS (ESI) m/z: 266.1 [M+H]+.
5.1.11.2.
acid (10b)
Pale white solid; yield: 45.05%; MS (ESI) m/z: 282.0 [M+H]+.
2-(4-Chlorophenyl)-4-methyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxylic
5.1.11.3.
4-Methyl-3,5-dioxo-2-(3-(trifluoromethyl)phenyl)-2,3,4,5-tetrahydro-1,2,4-triazine-6-
9

ACCEPTED MANUSCRIPT
carboxylic acid (10c）
Pale white solid; yield: 41.76%; MS (ESI) m/z: 316.1 [M+H]+.
5.1.11.4.
2-(2,5-Dichlorophenyl)-4-methyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxylic
acid
(10d)
Pale white solid; yield: 45.66%; MS (ESI) m/z: 315.9 [M+H]+.
5.1.11.5.
2-(3-Chloro-4-fluorophenyl)-4-methyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-
carboxylic acid (10e)
Pale white solid; yield: 43.15%; MS (ESI) m/z: 300.0 [M+H]+.
5.1.11.6.
2-(2,6-Dimethoxyphenyl)-4-methyl-3,5-dioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carboxylic acid
(10f)
Pale white solid; yield: 44.05%; MS (ESI) m/z: 308.1 [M+H]+.
5.1.12. Preparation of 2-carboxyethl formyl benzoic acid (11)
Potassium permanganate (132.5 g, 0.84 mol) in H2O (937.0 mL) was added drop-wise to the
vigorously stirred liquated solution of naphthalene (20.0 g, 0.16 mol) in sodium hydroxide
solution (312.0 mL, 0.5 mol). After completion of reaction as indicated by TLC, ethanol (30.0 mL)
was added and stirred for another 15 min. The mixture was then cooled and filtered, and the
filtrate was acidified by concentrated hydrochloric acid (93.8 mL) and evaporated to dryness to
give the compound 11 as a white solid (14.2 g, 45.3%). MS (ESI) m/z: 195.1 [M+H]+.
5.1.13. General procedure for the preparation of substituted phenyl hydrazine hydrochoride
(12a-e)
Substituted aniline (0.17 mol) and aqueous solution of NaNO2 (71.0 mL, 1.24 mol) were
successively added to aqueous hydrochloric acid (115mL) maintaining the temperature between
0-5°C. The reaction mixture was stirred for 2 h, alkalized to pH 6-7 with 12% w/v sodium
carbonate solution, and the resulting diazonium salt solution was added drop-wise to sodium
sulfite solution (443.0 mL, 1.9 mol) below 0°C. And then the mixture was stirred at room
temperature for a future 1 h, concentrated hydrochloric acid (277.0 mL) was added slowly, and
heated to 100°C for 3h. The mixture was then cooled and filtered, and dried to give compounds
12a-e.
5.1.13.1. (4-Methoxyphenyl)hydrazine (12a)
White solid; yield: 70.05%; MS (ESI) m/z: 138.08 [M+H]+.
5.1.13.2. (3-Fluorophenyl)hydrazine (12b)
Yellow solid; yield: 74.81%; MS (ESI) m/z: 127.06 [M+H]+.
5.1.13.3. Phenylhydrazine (12c)
Red solid; yield: 78.74%; MS (ESI) m/z: 109.07 [M+H]+.
10

ACCEPTED MANUSCRIPT
5.1.13.4. (4-Fluorophenyl)hydrazine (12d)
Yellow solid; yield: 78.15%; MS (ESI) m/z: 127.06 [M+H]+.
5.1.13.5. (4-Chlorophenyl)hydrazine (12e)
Yellow solid; yield: 70.67%; MS (ESI) m/z: 143.03 [M+H]+.
5.1.14.
General
procedure
for
the
preparation
of
3-substituted
phenyl-4-oxo-3,4-dihydrophthalazine- 1-carboxylic acid (13a-e)
A mixture of compounds 11 (15.7 g, 81 mmol) and 12a-e (88 mmol) in ethanol (25.5 mL) and
water (62.7 mL) was stirred at room temperature for 12 h. The precipitate was collected by
filtration, and then dissolved in 5% potassium hydroxide solution, stirred for 1h. The reaction
mixture was filtrated, washed with dichloromethane, and then acidified to pH 3-4 by concentrated
hydrochloric acid. The target compounds 13a-e was collected by filtration.
5.1.14.1. 3-(4-Methoxyphenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxylic acid (13a)
White solid; yield: 36.20%; MS (ESI) m/z: 297.1 [M+H]+.
5.1.14.2. 3-(3-Fluorophenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxylic acid (13b)
Yellow solid; yield: 29.00%; MS (ESI) m/z: 285.2 [M+H]+.
5.1.14.3. 4-Oxo-3-phenyl-3,4-dihydrophthalazine-1-carboxylic acid (13c)
Red solid; yield: 39.81%; MS (ESI) m/z: 267.1 [M+H]+.
5.1.14.4. 3-(4-Fluorophenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxylic acid (13d)
Yellow solid; yield: 45.10%; MS (ESI) m/z: 285.1 [M+H]+.
5.1.14.5. 3-(4-Chlorophenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxylic acid(13e)
Yellow solid; yield: 30.62%; MS (ESI) m/z: 301.0 [M+H]+.
5.1.15. Preparation of 2-(2-fluorobenzenethio)ethyl acetate (14)
To the stirred mixture of 2-fluorobenzeneththiol (17.0 mL, 0.16 mol), anhydrous potassium
carbonate (43.0 g, 0.31 mol) and acetone (120.0 mL), ethyl chloroacetate (18 mL, 0.168 mol) was
added drop-wise below 25°C. After completion of reaction as indicated by TLC, the reaction
mixture was filtrated, and the filtrate was concentrated to give the compound 14 as a pale yellow
oil (28.11 g, 82.1%), MS (ESI) m/z: 215.1 [M+H]+.
5.1.16. Preparation of 2-(2-fluorobenzenesulfonyl)ethyl acetate (15)
To the mixture of compound 14 (30.0 g, 0.14 mol) and glacial acetic acid (120.0 mL), 30%
aqueous hydrogen peroxide (50.0 mL, 0.5 mol) was added drop-wise. After completion of reaction
as indicated by TLC, the mixture was cooled and poured into 300.0 mL water, then extracted with
dichloromethane (60.0 mL×3) and the organic phase was washed with 10% aqueous sodium
bicarbonate (40.0 mL×2), then washed to neutral by water. The organic phase was dried over
anhydrous sodium sulfate and filtered, the filtrate was concentrated to yield the title compound 15
11

ACCEPTED MANUSCRIPT
as a white solid (25.9 g, 74.9%), MS (ESI) m/z: 247.0 [M+H]+.
5.1.17. General procedure for the preparation of (E)-2-(2-fluorobenzenesulfonyl)-2-(2-substituted
phenyl hydrazone) ethyl acetate (16a-c)
To the mixture of substituted aniline (0.04 mol), concentrated hydrochloric acid (13.0 mL) and
water (20.0 mL), aqueous solution of NaNO2 (40.0 mL, 0.08 mol) was added drop-wise at a rate
that the temperature below 0°C, and the reaction mixture was stirred for 0.5 h. A mixture of 15 (5.0
g, 0.02 mol) and sodium acetate (6.6 g, 0.08 mol) in methanol (150.0 mL) was added drop-wise to
the resulting diazonium salt solution and stirred for a further 2 h. The precipitate was collected by
filtration and washed with water (30.0 mL×2), dried to give the compounds16a-c.
5.1.17.1.
(E)-ethyl-2-((2-fluorophenyl)sulfonyl)-2-(2-(2-(trifluoromethyl)phenyl)hydrazono)acetate (16a)
White solid; yield 42.21%; MS (ESI) m/z: 419.1 [M+H]+.
5.1.17.3. (E)-ethyl-2-(2-(3,4-difluorophenyl)hydrazono)-2-((2-fluorophenyl)sulfonyl)acetate (16b)
White solid; yield 47.10%; MS (ESI) m/z: 369.2 [M+H]+.
5.1.17.2. (E)-ethyl-2-(2-(3-fluorophenyl)hydrazono)-2-((2-fluorophenyl)sulfonyl)acetate (16c)
White solid; yield 49.80%; MS (ESI) m/z: 387.2 [M+H]+.
5.1.18.
General
procedure
for
the
preparation
of
1-substituted
phenyl-1H-4,1,2-benzothiadiazine-3- carboxylic acid-4,4-dioxide (17a-c)
A mixture of 16a-c (2.0 mmol), anhydrous potassium carbonate (0.33 g, 2.4 mmol) in DMF
(10.0 mL) was stirred at room temperature for 4h, then the reaction mixture was poured into water
(50.0 mL) and stirred for another 0.5 h. The solids was collected by filtration and washed with
water (30.0 mL×2), dried to give compounds 17a-c.
5.1.18.1.
(E)-ethyl-2-((2-fluorophenyl)sulfonyl)-2-(2-(2-(trifluoromethyl)phenyl)hydrazono)acetate (17a)
Off-white solid; yield 80.10%; MS (ESI) m/z: 419.1 [M+H]+.
5.1.18.2. (E)-ethyl-2-(2-(3,4-difluorophenyl)hydrazono)-2-((2-fluorophenyl)sulfonyl)acetate (17b)
Off-white solid; yield 76.20%; MS (ESI) m/z: 387.4 [M+H]+.
5.1.18.3. (E)-ethyl-2-(2-(3-fluorophenyl)hydrazono)-2-((2-fluorophenyl)sulfonyl)acetate (17c)
Off-white solid; yield 85.10%; MS (ESI) m/z: 369.3 [M+H]+.
5.1.19. Preparation of (E)-2-(2-substituted phenylhydrazono)acetaldehyde (18a-b)
To the mixture of water (25mL) and glacial acetic acid (25mL), substituted phenylhydrazine (8g,
0.049mol) and 40% aqueous glyoxal solution (23mL, 0.2mol) were added drop-wise successively.
Upon the completion of addition, the reaction mixture was stirred at room temperature for 4h, then
the precipitate was collected by filtration and washed with water (30mL×2), dried to give
compounds 18a-b.
12

ACCEPTED MANUSCRIPT
5.1.19.1. (E)-2-(2-(4-chlorophenyl)hydrazono)acetaldehyde (18a)
White solid; yield 80.20%; MS (ESI) m/z: 183.0 [M+H]+.
5.1.19.2. (E)-2-(2-(4-fluorophenyl)hydrazono)acetaldehyde (18b)
White solid; yield 77.68%; MS (ESI) m/z: 167.3 [M+H]+.
5.1.20.
Preparation
of
(E)-5-(2-Substituted
phenyl
hydrazono)ethylidene)-2,2-dimethyl-1,3-dioxane- 4,6-dione (19a-b)
A mixture of 18a or 18b (35 mmol), meldrum’s acid (5.0 g, 35 mmol), glacial acetic acid (0.5
mL) and piperidine (0.5 mL) intoluene (50.0 mL) was stirred at r.t. for 24 h. The solid was
obtained by filtration and washed with petroleum ether, dried to give compounds 19a or 19b.
5.1.20.1. (Z)-5-((E)-2-(2-(4-chlorophenyl)hydrazono)ethylidene)-2,2-dimethyl-1,3-dioxan-4-one
(19a)
White solid; yield 75.32%; MS (ESI) m/z: 295.1 [M+H]+.
5.1.20.2. (Z)-5-((E)-2-(2-(4-fluorophenyl)hydrazono)ethylidene)-2,2-dimethyl-1,3-dioxan-4-one
(19b)
White solid; yield 74.80%; MS (ESI) m/z: 279.1 [M+H]+.
5.1.21. Preparation of 2-substituted phenyl-3-oxo-2,3-dihydropyridazine-4-carboxylic acid
(20a-b)
To the solution of sodium methoxide (0.65 g, 12 mmol) in methanol (30.0 mL), compound 19a
or 19b (0.01 mol) was added, and heated at reflux for 12 h. Subsequently, the mixture was poured
into ice-water (150.0 mL) slowly, acidified to pH 1 by aqueous 10% HCl solution, then extracted
with dichloromethane (60.0 mL×3), dried over Na2SO4, filtered and concentrated to yield
compounds 20a or 20b.
5.1.21.1. 2-(4-Chlorophenyl)-3-oxo-2,3-dihydropyridazine-4-carboxylic acid (20a)
White solid; yield 57.98%; MS (ESI) m/z: 251.0 [M+H]+.
5.1.21.2. 2-(4-Fluorophenyl)-3-oxo-2,3-dihydropyridazine-4-carboxylic acid (20b)
White solid; yield 58.37%; MS (ESI) m/z: 235.1 [M+H]+.
5.1.22. Preparation of N-(3-(dimethylamino)-2-substituted phenylallylidene)-N-methyl ammonium
hexafluorophosphate (21a-b)
A solution of substituted phenylacetic acid (5.0 g, 32 mmol) in DMF (30.0 mL) was heated at
70°C for 20 min under nitrogen atmosphere. Then, phosphorus oxychloride (6.1 mL) was added
drop-wise to the mixture, and stirred at 75°C for another 1 h. Upon cooling to room temperature, a
brown solution was obtained. During this process, hexafluorophosphoric acid (3.1 mL, 35 mmol)
and aqueous sodium hydroxide solution (9.7 mL, 48 mmol) were added to water (20.0 mL) and
stirred for 0.5 h. Subsequently, the former brown solution and aqueous sodium hydroxide solution
(13.1 mL, 65 mmol) were added drop-wise to maintaining the temperature below 10°C and stirred
13

ACCEPTED MANUSCRIPT
for 1 h. The precipitate was collected by filtration and washed with water (30.0 mL×2), dried to
give compounds 21a or 21b.
5.1.22.1. N-(3-(dimethylamino)phenyl-allylidene)-N-methyl ammonium hexafluorophosphate
(21a)
White solid; yield 54.81%; MS (ESI) m/z: 349.1 [M+H]+.
5.1.22.2.
(E)-N-(3-(dimethylamino)-2-(4-fluorophenyl)allylidene)-N-methylmethanaminium
hexafluorophosphate (21b)
White solid; yield 50.21%; MS (ESI) m/z: 367.3 [M+H]+.
5.1.23. Preparation of 1-methyl-2-oxo-5-substituted phenyl-1,2-dihydropyridine-3-carboxylic acid
(22a-b)
To the solution of diethyl malonate (1.5 g, 9.4 mmol) in tetrahydrofuran (40.0 mL), potassium
t-butoxide (1.27 g, 11.3 mmol) was added in batches, keeping the temperature below 0oC, and
then the reaction mixture was stirred at room temperature for 50 min. Subsequently, compounds
21a or 22b (11.3 mmol) was added to the mixture and heated at 45°C for a further 3h. The reaction
mixture was concentrated to dryness to give a yellow solid, which was dissolved in DMF (30.0
mL), heated at 70°C for 1.5 h under methylamine atmosphere. Upon cooling to .t., water (100.0
mL) was added and then extracted with dichloromethane (30.0 mL×3), evaporated the organic
phase to dryness and dissolved in ethanol (30.0 mL). Subsequently, aqueous sodium hydroxide
solution (30.0 mL, 18.0 mmol) was added drop-wise to the solution and stirred at room
temperature for 4 h. The reaction mixture was washed with ethyl acetate (30.0 mL×3), and then
acidified to pH 3 by concentrated hydrochloric acid. The precipitate was collected by filtration and
dried to give compounds 22a or 22b.
5.1.23.1. 1-methyl-2-oxo-5-phenyl-1,2-dihydropyridine-3-carboxylic acid (22a)
White solid; yield 41.20%; MS (ESI) m/z: 230.2 [M+H]+.
5.1.23.2. 5-(4-fluorophenyl)-1-methyl-2-oxo-1,2-dihydropyridine-3-carboxylic acid (22b)
White solid; yield: 38.23%; MS (ESI) m/z: 248.1 [M+H]+.
5.1.24. General procedure for the preparation of compounds (23a-g)
A mixture of 5 (0.1 g, 0.3 mmol), N-substitutedphenyl-6-chloropyrimidine-4-amine (0.3 mmol)
and DIPEA (0.1 mL) in ethanol (10.0 mL) was heated at reflux for 15 h. Upon cooling to room
temperature, the reaction mixture was filtrated and washed with ethanol (10.0 mL×2), dried to
obtain compounds 23a-g.
5.1.24.1. N4-(4-(3,3'-bipyridin-4-yloxy)-3-fluorophenyl)-N6-phenylpyrimidine-4,6-diamine (23a).
White solid; yield:71.6%; m.p.: 172-175°C; MS (ESI) m/z: 451.1 [M+H]+; 1H-NMR (400 MHz,
DMSO-d6) δ (ppm): 9.98 (s, 1H), 9.47 (s, 1H), 8.86 (s, 1H), 8.50 (dd, J = 4.4, 1.3 Hz,1H), 8.37 (s,
1H), 8.18 (s, 1H), 8.13–8.02 (m, 2H), 7.89 (d, J = 7.6 Hz, 1H), 7.61 (m, 3H), 7.52 (dd, J = 9.0,
1.9Hz, 1H), 7.41 (dd, J = 7.9, 4.6 Hz, 1H), 7.31 (t, J = 7.9 Hz, 2H), 6.99 (t, J=7.4 Hz, 1H), 6.39 (d,
J = 7.6 Hz, 1H), 6.34 (s, 1H); Anal. calcd. for C26H19FN6O (%): C, 69.32; H, 4.25; N, 18.66.
14

ACCEPTED MANUSCRIPT
Found (%): C, 69.23; H, 4.21; N, 18.57.
5.1.24.2.
N4-(4-(3,3'-bipyridin-4-yloxy)-3-fluorophenyl)-N6-(4-methoxyphenyl)pyrimidine-4,6-diamine
(23b).
Gray solid; yield: 70.2%; m.p.: 142-144°C; MS (ESI) m/z: 481.5 [M+H]+, 503.5 [M+Na]+.
1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.61 (s, 1H), 9.05 (s, 1H), 8.85 (s, 1H), 8.50 (dd, J=3.3,
1.2 Hz, 1H), 8.30 (s, 1H), 8.15 (s, 1H), 8.10 (m, 1H), 8.02 (dd, J = 13.7, 2.1 Hz, 1H), 7.86 (d, J =
7.7 Hz, 1H), 7.61 (t, 1H), 7.47–7.35 (m, 4H), 6.92 (d, J = 8.9 Hz, 2H), 6.38 (d, J = 7.7 Hz, 1H),
6.09 (s, 1H), 3.74(s, 3H); Anal. calcd. for C27H21FN6O (%): C, 67.49; H, 4.41; N, 17.49. Found
(%): C, 67.41; H, 4.45; N, 17.47.
5.1.24.3.
N4-(4-(3,3'-bipyridin-4-yloxy)-3-fluorophenyl)-N6-(3,4-difluorophenyl)pyrimidine-4,6-diamine
(23c).
White solid; yield:68.2%; m.p.: 242-244°C; MS (ESI) m/z: 487.5 [M+H]+, 509.5 [M+Na]+,
1H-NMR (400 MHz, DMSO-d6) δ (ppm): 10.01(s, 1H), 9.76(s, 1H), 8.91(s, 1H), 8.54(d, J = 4.1 Hz,
1H), 8.42(s, 1H), 8.22(dd, J = 10.0, 4.6 Hz, 2H), 8.07(dd, J = 13.8, 2.0 Hz, 1H), 7.97-7.87(m, 2H),
7.65(t, J = 8.9 Hz, 1H), 7.54-7.47(m, 2H), 7.41-7.32(m, 2H), 6.41(d, J = 7.6 Hz, 1H), 6.32(s, 1H); Anal.
calcd. for C26H17F3N6O (%): C, 64.20; H, 3.52; N, 17.28. Found (%): C, 64.12; H, 3.56; N, 17.24.
5.1.24.4.
N4-(4-(3,3'-bipyridin-4-yloxy)-3-fluorophenyl)-N6-(4-chlorophenyl)pyrimidine-4,6-diamine (23d).
White solid; yield: 75.0%; m.p.: 178-180°C; MS (ESI) m/z: 485.6 [M+H]+, 507.5 [M+Na]+.
1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.91 (s, 1H), 9.58 (s, 1H),8.86 (s, 1H), 8.50 (dd, J = 4.4,
1.7 Hz ,1H), 8.39 (s, 1H), 8.16 (s, 1H), 8.10 (m, 1H), 8.04 (dd, J = 13.9, 1.8 Hz, 1H), 7.87 (d, J =
7.6 Hz, 1H), 7.67 (d,J = 8.8 Hz, 2H), 7.62 (t, 1H), 7.50 (dd, J = 9.6, 1.8Hz, 1H), 7.40 (dd, J = 8.1,
4.5 Hz, 1H), 7.34 (d, J = 8.8 Hz, 2H), 6.39 (d, J = 7.6 Hz, 1H), 6.31 (s, 1H); Anal. calcd. for
C26H18ClFN6O (%): C, 64.40; H, 3.74; N, 17.33. Found (%): C, 64.42; H, 3.76; N, 17.29.
5.1.24.5.
N4-(4-(3,3'-bipyridin-4-yloxy)-3-fluorophenyl)-N6-(3-chlorophenyl)pyrimidine-4,6-diamine (23e).
White solid; yield: 75.7%; m.p.: 180-182°C; MS (ESI) m/z: 485.5 [M+H]+, 507.4 [M+Na]+. IR
(KBr, cm-1): 3325.5, 3183.4, 3046.2, 3019.3, 1663.1, 1644.9, 1546.8, 1324.2, 1260.8, 1220.6,
1
1151.4, 1131.5, 1031.5, 977.1, 884.7, 758.1, 697.2, 651.3; H-NMR (400 MHz, DMSO-d6) δ
(ppm): 9.98 (s, 1H), 9.70 (s, 1H), 8.85 (s, 1H), 8.50 (dd, J = 4.0, 1.1Hz, 1H), 8.43 (s, 1H), 8.16 (s,
1H), 8.10 (m, 1H), 8.05 (dd, J = 13.7, 2.2 Hz, 1H), 7.92 (t, 1H), 7.87 (d, J=7.6 Hz, 1H), 7.64 (t,
1H), 7.52 (m, 2H), 7.40 (dd, J = 7.9, 4.6 Hz, 1H), 7.31 (t, 1H), 7.00 (m, 1H), 6.39 (d, J = 7.6
Hz,1H), 6.36 (s, 1H); Anal. calcd. for C26H18ClFN6O (%): C, 64.40; H, 3.74; N, 17.33. Found (%):
C, 64.37; H, 3.71; N, 17.26.
5.1.24.6.
N4-(4-(3,3'-bipyridin-4-yloxy)-3-fluorophenyl)-N6-(4-fluorophenyl)pyrimidine-4,6-diamine (23f).
White solid; yield: 75.4%; m.p.: 162-164°C; MS (ESI) m/z: 469.1 [M+H]+, 491.1 [M+Na]+.
15

ACCEPTED MANUSCRIPT
1H-NMR (400 MHz, DMSO-d6) δ (ppm): 9.79 (s, 1H), 9.37 (s, 1H), 8.85 (d, J = 1.9 Hz, 1H), 8.50
(dd, J = 4.8, 1.4Hz, 1H), 8.36 (s, 1H), 8.16 (s, 1H), 8.10 (m, 1H), 8.03 (dd, J = 13.8, 2.1 Hz, 1H),
7.86 (d, J = 7.6 Hz, 1H), 7.64–7.56 (m, 3H), 7.47 (dd, J = 8.9, 2.0 Hz, 1H), 7.40 (dd, J = 7.8, 4.8
Hz, 1H), 7.15 (t, J = 8.9, 2H), 6.39 (d, J = 7.6Hz, 1H), 6.22 (s, 1H); Anal. calcd. for C26H18F2N6O
(%): C, 66.66; H, 3.87; N, 17.94. Found (%): C, 66.57; H, 3.76; N, 17.92.
5.1.24.7.
N4-(4-([3,3'-bipyridin]-4-yloxy)-3-fluorophenyl)-N6-(3,5-dimethoxyphenyl)pyrimidine-4,6-diamin
e (23g).
Gray solid; yield: 72.5%; m.p.: 148-150°C; MS (ESI) m/z: 511.5 [M+H]+, 533.6 [M+Na]+.
1H-NMR (400 MHz, DMSO-d6) δ (ppm)：9.61 (s, 1H), 9.05 (s, 1H), 8.85 (s, 1H), 8.50 (dd, J = 3.3,
1.2 Hz, 1H), 8.31 (s, 1H), 8.15 (s, 1H), 8.10 (m, 1H), 8.04 (dd, J = 13.7, 2.1 Hz, 1H), 7.87 (d, J =
7.7 Hz, 1H), 7.61 (t, 1H), 7.51–7.36 (m, 4H), 6.73 (s, 1H), 6.38 (d, J = 7.7 Hz, 1H), 6.09 (s, 1H),
3.74 (s, 6H); Anal. calcd. for C28H23FN6O3 (%): C, 65.87; H, 4.54; N, 16.46. Found (%): C, 65.82;
H, 4.52; N, 16.41.
5.1.25. General procedure for the preparation of compounds (24a-f, 25a-d, 26a-c, 27a-b, 28a-b)
To the solution of 10a-f (or 13a-d, 17a-c, 20a-b, 22a-b) (6.0 mmol) in toluene (20.0 mL),
thionyl chloride (10.0 mL) was added drop-wise, and then heated at reflux for 5 h. Upon cooling
to room temperature, the reaction mixture was concentrated to dryness to obtain the corresponding
acyl chloride as a powdered solid. Subsequently, the freshly prepared acyl chloride (0.75 mmol)
was added to the mixture of 5 (0.5 mmol) and DIPEA (2.5 mmol) in dichloromethane (50.0 mL) in
batches, keeping the temperature below 0°C. Then, the reaction mixture was stirred at .t. for 10 h.
Upon the completion of reaction as indicated by TLC, the mixture was washed successively with
10% aqueous potassium carbonate solution (20.0 mL×3) and brine (20.0 mL×2), the organic phase
was dried over anhydrous sodium sulfate. The solid was removed by filtration, and the filtrate was
concentrated to yield the title compounds 24a-f, 25a-d, 26a-c, 27a-b, 28a-b.
5.1.25.1.
N-(4-((3,3'-bipyridin)-4-yloxy)-3-fluorophenyl)-4-methyl-3,5-dioxo-2-phenyl-2,3,4,5-tetrahydro-1
,2,4-triazine-6-carboxamide (24a).
Yellow solid; yield: 41.2%; m.p.: 174-176°C; MS (ESI) m/z: 510.5 [M+H]+, 532.4 [M+Na]+.
1H-NMR (400 MHz, DMSO-d6) δ (ppm): 11.13(s, 1H), 8.90(s, 1H), 8.56(d, J = 4.4 Hz, 1H), 8.27(s,
1H), 8.16(d, J = 7.9 Hz, 1H), 7.98(dd, J = 9.3, 4.6 Hz, 2H), 7.83(t, J = 8.8 Hz, 1H), 7.67(dd, J = 11.9,
7.7 Hz, 3H), 7.50(dt, J = 13.4, 8.4 Hz, 3H), 6.46(d, J = 7.6 Hz, 1H), 3.36(s, 3H); Anal. calcd. for
C27H18F2N6O4 (%): C, 61.36; H, 3.43; N, 15.90. Found (%): C, 61.32; H, 3.40; N, 15.87.
5.1.25.2.
N-(4-((3,3'-bipyridin)-4-yloxy)-3-fluorophenyl)-2-(2-fluorophenyl)-4-methyl-3,5-dioxo-2,3,4,5-tet
rahydro-1,2,4-triazine-6-carboxamide (24b).
Yellow solid; yield: 43.5%; m.p.: 192-195°C; MS (ESI) m/z: 529.5 [M+H]+, 561.4 [M+Na]+.
1H-NMR (400 MHz, DMSO-d6) δ (ppm): 11.06(s, 1H), 8.86(s, 1H), 8.52(d, J = 3.9 Hz, 1H), 8.23(d,
J = 1.0 Hz, 1H), 8.13(dt, J = 8.0, 1.8 Hz, 1H), 7.93(ddd, J = 6.7, 5.8, 2.1 Hz, 2H), 7.79(t, J = 8.8 Hz,
1H), 7.63(s, 5H), 7.44(dd, J = 7.9, 4.8 Hz, 1H), 6.41(d, J = 7.6 Hz, 1H), 3.30(s, 3H); Anal. calcd. for
16

ACCEPTED MANUSCRIPT
C27H18ClFN6O4 (%): C, 59.51; H, 3.33; N, 15.42. Found (%): C, 59.54; H, 3.36; N, 15.38.
5.1.25.3.
N-(4-((3,3'-bipyridin)-4-yloxy)-3-fluorophenyl)-4-methyl-5-oxo-2-(3-(trifluoromethyl)phenyl)-2,3
,4,5-tetrahydro-1,2,4-triazine-6-carboxamide (24c).
Yellow solid; yield: 39.6%; m.p.: 193-195°C; MS (ESI) m/z: 529.5 [M+H]+, 561.4 [M+Na]+. IR
(KBr, cm-1): 3432.1, 3034.5, 2963.2, 2874.5, 1678.2, 1661.2, 1654.8, 1521.9, 1446.2, 1376.4,
1264.8, 1253.9, 1231.1, 1140.2, 1095.9, 881.3, 826.5, 755.8, 651.8; 1H-NMR (400 MHz,
DMSO-d6) δ (ppm): 11.32(s, 1H), 8.86(s, 1H), 8.51(d, J = 2.8 Hz, 1H), 8.23(s, 1H), 8.11(d, J = 7.9 Hz,
1H), 8.04-7.91(m, 4H), 7.82(dt, J = 24.6, 8.3 Hz, 3H), 7.67(d, J = 9.0 Hz, 1H), 7.42(dd, J = 7.7, 4.8 Hz,
1H), 6.42(d, J = 7.6 Hz, 1H), 3.31(s, 3H); Anal. calcd. for C28H18F4N6O4 (%): C, 58.14; H, 3.14; N,
14.53. Found (%): C, 58.11; H, 3.16; N, 14.50.
5.1.25.4.
N-(4-((3,3'-bipyridin)-4-yloxy)-3-fluorophenyl)-2-(2,5-dichlorophenyl)-4-methyl-5-oxo-2,3,4,5-tet
rahydro-1,2,4-triazine-6-carboxamide (24d).
Yellow solid; yield: 39.6%; m.p.: 183-185°C; MS (ESI) m/z: 529.5[M+H]+, 561.4 [M+Na]+.
1H-NMR (400 MHz, DMSO-d6) δ (ppm): 11.13(s, 1H), 8.85(s, 1H), 8.51(d, J = 3.7 Hz, 1H), 8.23(s,
1H), 8.11(d, J = 7.9 Hz, 1H), 7.91(dd, J = 14.4, 3.7 Hz, 2H), 7.82-7.75(m, 3H), 7.70-7.64(m, 1H),
7.59(d, J = 8.6 Hz, 1H), 7.42(dd, J = 7.8, 4.8 Hz, 1H), 6.41(d, J = 7.6 Hz, 1H), 3.37(s, 3H); Anal.
calcd. for C27H17Cl2FN6O4 (%): C, 55.97; H, 2.96; N, 14.51. Found (%): C, 55.95; H, 2.94; N,
14.54.
5.1.25.5.
N-(4-((3,3'-bipyridin)-4-yloxy)-3-fluorophenyl)-2-(3-chloro-4-fluorophenyl)-4-methyl-5-oxo-2,3,4
,5-tetrahydro-1,2,4-triazine-6-carboxamide (24e).
Yellow solid; yield: 39.6%; m.p.: 208-211°C; MS (ESI) m/z: 529.5 [M+H]+, 561.4 [M+Na]+.
1H-NMR (400 MHz, DMSO-d6) δ (ppm): 11.03(s, 1H), 8.85(s, 1H), 8.51(d, J = 3.8 Hz, 1H), 8.22(s,
1H), 8.10(dt, J = 7.9, 1.7 Hz, 1H), 7.98-7.86(m, 3H), 7.79(t, J = 8.8 Hz, 1H), 7.67-7.60(m, 3H),
7.42(dd, J = 7.8, 4.8 Hz, 1H), 6.41(d, J = 7.6 Hz, 1H), 3.30(s, 3H); Anal. calcd. for C27H17ClF2N6O4
(%): C, 57.61; H, 3.04; N, 14.93. Found (%): C, 57.60; H, 3.06; N, 14.94.
5.1.25.6.
N-(4-((3,3'-bipyridin)-4-yloxy)-3-fluorophenyl)-2-(2,4-dimethoxyphenyl)-4-methyl-3,5-dioxo-2,3,
4,5-tetrahydro-1,2,4-triazine-6-carboxamide (24f).
Yellow solid; yield: 43.5%; m.p.: 187-189°C; MS (ESI) m/z: 529.5[M+H]+, 561.4 [M+Na]+.
1H-NMR (400 MHz, DMSO-d6) δ (ppm): 11.19(s, 1H), 8.85(s, 1H), 8.51(d, J = 3.7 Hz, 1H), 8.22(s,
1H), 8.11(d, J = 7.9 Hz, 1H), 7.93(dd, J = 14.4, 3.7 Hz, 2H), 7.82-7.79(m, 3H), 7.70-7.62(m, 1H),
7.56(d, J = 8.6 Hz, 1H), 7.40(dd, J = 7.8, 4.8 Hz, 1H), 6.41(d, J = 7.6 Hz, 1H), 3.91 (s, 3H), 3.84 (s,
3H), 3.37(s, 3H); Anal. calcd. for C29H23FN6O6 (%): C, 61.05; H, 4.06; N, 14.73. Found (%): C,
61.02; H, 4.08; N, 14.74.
5.1.25.7.
N-(4-((3,3'-bipyridin)-4-yloxy)-3-fluorophenyl)-3-(4-methoxyphenyl)-4-oxo-3,4-dihydrophth
17

ACCEPTED MANUSCRIPT
alazine-1-carboxamide (25a).
Pale yellow solid; yield: 42.1%; m.p.: 162-164°C; MS (ESI) m/z: 582.5 [M+Na]+. H-NMR
(400 MHz, DMSO-d6) δ (ppm): 11.08(d, J = 6.5 Hz, 1H), 8.85(d, J = 1.1 Hz, 1H), 8.52-8.41(m, 3H),
8.23(s, 1H), 8.13-7.91(m, 6H), 7.85-7.64(m, 4H), 7.42(dd, J = 7.8, 4.8 Hz, 1H), 7.10(d, J = 9.0 Hz, 1H),
6.42(d, J = 7.6 Hz, 1H), 3.95-3.83(m, 3H); Anal. calcd. for C32H22FN5O4 (%): C, 68.69; H, 3.96; N,
12.52. Found (%): C, 68.65; H, 3.98; N, 12.56.
1
5.1.25.8.
N-(4-((3,3'-bipyridin)-4-yloxy)-3-fluorophenyl)-3-(3-fluorophenyl)-4-oxo-3,4-dihydrophthalazine-
1-carboxamide (25b).
Gray solid; yield: 36.2%; m.p.: 193-195°C; MS (ESI) m/z: 548.1 [M+H)+, 570.1(M+Na]+. IR
(KBr, cm-1): 3412.5, 3322.5, 3183.4, 3019.3, 1670.5, 1660.9, 1644.9, 1529.9, 1262.8, 1234.6,
1145.2, 1136.2, 1095.3, 1034.7, 884.7, 798.1, 756.2, 697.4, 631.5; 1H-NMR (400 MHz, DMSO-d6)
δ (ppm): 8.85(d, J = 1.4 Hz, 1H), 8.52–8.43 (m, 3H), 8.20 (s, 1H), 8.11 (m, 1H), 8.07–7.97 (m,
3H), 7.91 (d, J=7.5 Hz, 1H), 7.78–7.69 (m, 4H), 7.60 (dd, J = 8.1, 1.2 Hz, 1H), 7.41 (dd, J = 8.2,
4.6 Hz, 1H),7.33 (m, 1H), 7.10 (d, J = 8.9 Hz, 2H), 6.41 (d, J = 7.5 Hz, 1H); Anal. calcd. for
C33H25F2N5O3 (%): C, 68.62; H, 4.36; N, 12.13. Found (%): C, 68.65; H, 4.32; N, 12.16.
5.1.25.9.
N-(4-((3,3'-bipyridin)-4-yloxy)-3-fluorophenyl)-4-oxo-3-phenyl-3,4-dihydrophthalazine-1-carbox
amide (25c).
Gray solid; yield: 37.6%; m.p.: 202-204°C; MS (ESI) m/z: 530.6 [M+H]+. 1H-NMR (400 MHz,
DMSO-d6) δ (ppm): 11.11(s, 1H), 8.86(d, J = 1.7 Hz, 1H), 8.51(dd, J = 4.8, 1.5 Hz, 1H), 8.50-8.41(m,
2H), 8.23(d, J = 0.6 Hz, 1H), 8.13-8.10(m, 1H), 8.09-7.98(m, 3H), 7.94(d, J = 7.6 Hz, 1H),
7.89-7.76(m, 3H), 7.72(dd, J = 8.8, 1.7 Hz, 1H), 7.66-7.55(m, 2H), 7.52-7.38(m, 2H), 6.42(dd, J = 7.6,
2.8 Hz, 1H); Anal. calcd. for C31H20FN5O3 (%): C, 70.32; H, 3.81; N, 13.23. Found (%): C, 70.35;
H, 3.84; N, 13.26.
5.1.25.10.
N-(4-((3,3'-bipyridin)-4-yloxy)-3-fluorophenyl)-3-(4-fluorophenyl)-4-oxo-3,4-dihydrophthalazine-
1-carboxamide (25d).
1
Gray solid; yield: 35.5%; m.p.: 168-170°C; MS (ESI) m/z: 548.1 [M+H]+, 570.1 [M+Na]+. H-
NMR (400 MHz, CDCl3) δ (ppm): 9.34 (s, 1H), 9.25 (d, J = 7.7 Hz, 1H), 8.72 (s, 1H), 8.56 (m,
2H), 8.15 (m, 1H), 8.03–7.89 (m, 3H), 7.67–7.53 (m, 3H), 7.52–7.44 (m, 2H), 7.40 (t, 1H), 7.34
(dd, J = 7.9, 5.2 Hz, 1H), 7.25 (t, 2H), 6.60 (d, J =7.7 Hz, 1H); Anal. calcd. for C31H19F2N5O3 (%):
C, 68.00; H, 3.50; N, 12.79. Found (%): C, 68.03; H, 3.54; N, 12.72.
5.1.25.11.
N-(4-([3,3'-bipyridin]-4-yloxy)-3-fluorophenyl)-3-(4-chlorophenyl)-4-oxo-3,4-dihydrophthalazine
-1-carboxamide (25e).
Gray solid; yield: 34.21%; m.p.: 178-180°C; MS (ESI) m/z: 565.0 [M+H]+, 587.1 [M+Na]+. 1H-
NMR (400 MHz, CDCl3) δ (ppm): 9.34 (s, 1H), 9.25 (d, J = 7.7 Hz, 1H), 8.72 (s, 1H), 8.56 (m,
2H), 8.15 (m, 1H), 8.04–7.90 (m, 3H), 7.67–7.64 (m, 3H), 7.48–7.39 (m, 2H), 7.40 (t, 1H), 7.34
(dd, J = 7.9, 5.2 Hz, 1H), 7.13 (t, 2H), 6.62 (d, J = 7.7 Hz, 1H); Anal. calcd. for C31H19ClFN5O3
18

ACCEPTED MANUSCRIPT
(%): C, 66.02; H, 3.40; N, 12.42. Found (%): C, 68.06; H, 3.36; N, 12.38.
5.1.25.12.
N-(4-((3,3'-bipyridin)-4-yloxy)-3-fluorophenyl)-1-(2-trifluoromethylphenyl)-1H-4,1,2-benzothiadi
azine-3-carboxamide-4,4-dioxide (26a).
Pale yellow solid; yield: 31.4%; m.p.: 186-188°C; MS (ESI) m/z: 634.0(M+H)+. 1H-NMR (400
MHz, DMSO-d6) δ (ppm): 10.53(s, 1H), 8.85(d, J = 1.4 Hz, 1H), 8.50(dd, J = 4.6, 1.2 Hz, 1H),
8.24-8.15(m, 2H), 8.08(dd, J = 16.1, 8.0 Hz, 4H), 7.92(ddd, J = 14.9, 11.7, 5.3 Hz, 3H), 7.80-7.58(m,
4H), 7.41(dd, J = 7.9, 4.8 Hz, 1H), 6.67(d, J = 8.6 Hz, 1H), 6.40(d, J = 7.6 Hz, 1 H); Anal. calcd. for
C31H19F4N5O4S (%): C, 58.77; H, 3.02; N, 11.05. Found (%): C, 58.72; H, 3.06; N, 11.02.
5.1.25.13.
N-(4-(3,3'-bipyridin-4-yloxy)-3-fluorophenyl)-1-(3,4-difluorophenyl)-1H-4,1,2-benzothiadiazine-
3-carboxamide-4,4-dioxide (26b).
Gray solid; yield: 36.1%; m.p.: 175-177°C; MS (ESI) m/z: 602.0 [M+H]+. 1H-NMR (400 MHz,
CDCl3) δ (ppm): 9.08 (s, 1H), 8.70 (s, 1H), 8.52 (d, J = 3.9 Hz, 1H), 8.16 (dd, J = 7.7, 1.4 Hz, 1H),
8.10 (m, 1H), 7.88 (dd, J = 11.7, 1.1 Hz, 1H), 7.63 (s, 1H), 7.59–7.50 (m, 2H), 7.50–7.42 (m, 3H),
7.38 (m, 2H), 7.34–7.30 (m, 2H), 6.86 (d, J = 8.5 Hz, 1H), 6.56 (d, J = 7.6 Hz, 1H); Anal. calcd.
for C30H18F3N5O4S (%): C, 59.90; H, 3.02; N, 11.64. Found (%): C, 59.86; H, 3.01; N, 11.62.
5.1.25.14.
N-(4-((3,3'-bipyridin)-4-yloxy)-3-fluorophenyl)-1-(3-fluorophenyl)-1H-4,1,2-benzothiadiazine-3-c
arboxamide-4,4-dioxide (26c).
Gray solid; yield: 38.2%; m.p.: 216-218°C; MS (ESI) m/z: 584.5 [M+H]+, 606.6 [M+Na]+. IR
(KBr, cm-1): 3423.3, 3331.9, 3182.4, 3032.5, 1678.9, 1662.1, 1653.2, 1528.4, 1479.9, 1263.4,
1252.8, 1234.6, 1152.2, 1139.4, 1136.2, 1038.3, 874.4, 758.1, 631.5;1H-NMR (400 MHz, CDCl3)
δ (ppm): 8.85 (s, 1H), 8.70 (s, 1H), 8.54 (d, J = 3.7 Hz, 1H), 8.19 (dd, J = 7.3, 2.3 Hz, 1H), 8.11
(m, 1H), 7.90 (dd, J = 12.2, 2.0 Hz, 1H), 7.62 (m, 2H), 7.55 (m, 2H), 7.47 (m, 2H), 7.38 (d, J =
8.5 Hz, 1H), 7.33 (m, 4H), 6.90 (dd, J = 7.9, 1.4 Hz, 1H), 6.57 (d, J = 7.7 Hz, 1H); Anal. calcd. for
C30H19F2N5O4S (%): C, 61.75; H, 3.28; N, 12.00. Found (%): C, 61.72; H, 3.23; N, 11.95.
5.1.25.15.
N-(4-((3,3'-bipyridin)-4-yloxy)-3-fluorophenyl)-2-(4-chlorophenyl)-3-oxo-2,3-dihydropyridazine-
4-carboxamide (27a).
Gray solid; yield: 41.2%; m.p.: 175-179°C; MS (ESI) m/z: 564.1 [M+H]+, 586.2 [M+Na]+. IR
(KBr, cm-1): 3436.2, 3312.4, 3179.2, 3029.3, 1680.9, 1663.1, 1651.9, 1525.8, 1265.1, 1254.8,
1231.1, 1156.2, 1132.7, 1097.1, 1036.7, 881.9, 798.1, 697.9, 651.2; 1H-NMR (400 MHz,
DMSO-d6) δ (ppm): 8.85 (s, 1H), 8.50 (d, J = 4.6, 1H), 8.38 (d, J = 4.0 Hz), 8.27 (d, J = 4.0 Hz),
8.21 (s, 1H), 8.11–8.02 (m, 2H), 7.91 (d, J = 7.5Hz, 1H), 7.76 (t, 1H), 7.61–7.52 (m, 3H),
7.34–7.28 (m, 3H), 6.40 (d, J = 7.6 Hz, 1H); Anal. calcd. for C27H17ClFN5O3 (%): C, 63.10; H,
3.33; N, 13.63. Found (%): C, 63.12; H, 3.28; N, 13.66.
5.1.25.16.
N-(4-((3,3'-bipyridin)-4-yloxy)-3-fluorophenyl)-2-(4-fluorophenyl)-3-oxo-2,3-dihydropyridazine-
19

ACCEPTED MANUSCRIPT
4-carboxamide (27b).
Gray solid; yield: 37.3%; m.p.: 204-206°C; MS (ESI) m/z: 548.2 [M+H]+, 560.2 [M+Na]+.
1H-NMR (400 MHz, DMSO-d6) δ (ppm): 8.85(s, 1H), 8.50 (d, J = 4.6, 1H), 8.38 (d, J = 4.0 Hz,
1H), 8.27(d, J = 4.0 Hz, 1H), 8.21 (s, 1H), 8.12–8.03 (m, 2H), 7.92 (d, J = 7.6 Hz, 1H), 7.78 (t,
1H), 7.71–7.62 (m, 3H), 7.45–7.39 (m, 3H), 6.40 (d, J = 7.6 Hz, 1H); Anal. calcd. for
C27H17F2N5O3 (%): C, 65.19; H, 3.44; N, 14.08. Found (%): C, 65.15; H, 3.48; N, 14.06.
5.1.25.17.
N-(4-((3,3'-bipyridin)-4-yloxy)-3-fluorophenyl)-5-phenyl-1-methyl-2-oxo-1,2-dihydropyridine-3-c
arboxamide (28a).
Pale yellow solid; yield: 48.6%; m.p.: 203-206°C; MS (ESI) m/z: 493.2 [M+H]+. IR (KBr, cm-1):
3431.7, 3031.5, 2963.6, 2882.3, 1680.8, 1665.1, 1653.0, 1529.2, 1445.1, 1377.1, 1261.6, 1252.9,
1
1233.1, 1151.7, 1135.6, 1098.1, 876.2, 821.3, 659.1; H-NMR (400 MHz, DMSO-d6) δ (ppm):
12.53(s, 1H), 8.87(s, 1H), 8.76(d, J = 2.8 Hz, 1H), 8.67(d, J = 2.6 Hz, 1H), 8.51(d, J = 3.8 Hz, 1H),
8.23(d, J = 2.0 Hz, 1H), 8.16-8.07(m, 2H), 7.99-7.89(m, 1H), 7.78(t, J = 8.7 Hz, 1H), 7.69(d, J = 7.3
Hz, 2H), 7.61(dd, J = 7.6, 1.9 Hz, 1H), 7.51(t, J = 7.7 Hz, 2H), 7.45-7.38(m, 2H), 6.41(d, J = 7.6 Hz,
1H), 3.76(s, 3H); Anal. calcd. for C29H20ClFN4O3 (%): C, 66.10; H, 3.83; N, 10.63. Found (%): C,
66.08; H, 3.77; N, 10.61.
5.1.25.18.
N-(4-((3,3'-bipyridin)-4-yloxy)-3-fluorophenyl)-5-(4-fluorophenyl)-1-methyl-2-oxo-1,2-dihydropy
ridine-3-carboxamide (28b).
Pale yellow solid; yield: 41.3%; m.p.: 193-195°C; MS (ESI) m/z: 511.2 [M+H]+. 1H-NMR (400
MHz, DMSO-d6) δ (ppm): 12.52(s, 1H), 8.97(s, 1H), 8.72(d, J = 2.8 Hz, 1H), 8.65(d, J = 2.8 Hz, 1H),
8.58(s, 1H), 8.29(d, J = 9.8 Hz, 2H), 8.11(dd, J = 13.0, 2.1 Hz, 1H), 7.99-7.93(m, 1H), 7.78(t, J = 8.8
Hz, 1H), 7.75-7.69(m, 2H), 7.59(ddd, J = 12.5, 7.8, 3.1 Hz, 2H), 7.38-7.30(m, 2H), 6.44(d, J = 7.6 Hz,
1H), 3.74(s, 3H); Anal. calcd. for C29H20F2N4O3 (%): C, 68.23; H, 3.95; N, 10.98. Found (%): C,
68.21; H, 3.92; N, 10.94.
5.2. Pharmacology
5.2.1. MTT assay in vitro
The cytotoxic activity of compounds (23a-f, 24a-d, 25a-e, 26a-f, 27a-b and 28a-b) was
evaluated with HT-29, MKN-45, A549 and MDA-MB-231 cells by the MTT assay in vitro, with
Foretinib as references. The cancer cells were cultured in minimum essential medium (MEM)
supplemented with 10% fetal bovine serum (FBS). Approximately 4×103 cells, suspended in
MEM medium, were plated onto each well of a 96-well plate and incubated in 5% CO2 at 37°C for
24 h. The test compounds were added to the culture medium at the indicated final concentrations
and the cell cultures were continued for 72 h. Fresh MTT was added to each well at a final
concentration of 5.0 µg/mL and incubated with cells at 37 °C for 4 h. The formazan crystals were
dissolved in 100.0 µL DMSO per each well, and the absorbency at 492 nm (for the absorbance of
MTT formazan) and 630 nm (for the reference wavelength) was measured with the ELISA reader.
All of the compounds were tested three times in each of the cell lines. The results expressed as
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IC50 (inhibitory concentration of 50%) were the mean ± SD and were calculated by using the
Bacus Laboratories Incorporated Slide Scanner (Bliss) software.
5.2.2. c-Met Kinase assays
The c-Met kinase activity was determined using homogenous time-resolved fluorescence
(HTRF) assays following the manufacture’s instruction, with Foretinib as positive control. Briefly,
20.0 mg/mL poly (Glu, Tyr) 4:1 (Sigma) was precoated as a substrate in 384-well plates. Then
50.0 mL of 10.0 mM ATP (Invitrogen) solution diluted in kinase reaction buffer (50.0 mM
HEPES, pH 7.0, 1.0 M DTT, 1.0 M MgCl2, 1.0 M MnCl2, 0.1% NaN3) was added to each well.
Various concentrations of compounds diluted in 10.0 mL of 1% DMSO v/v were used as the
negative control. The kinase reaction was initiated by the addition of purified tyrosine kinase
proteins duluted in 39.0 mL of kinase reaction buffer solution. Reactions were incubated for 30
min at 25°C and stopped by the addition of 5.0 mL Streptavidin-XL665 and 5.0 mL Tk Antibody
Cryptate working solution to all of wells. The plate was read using Envision (PerkinElmer) at 320
nm and 615 nm. The inhibition rate (%) was calculated using the following equation: % inhibition
= 100 - [(activity of enzyme with tested compounds - min)/(max - min)]×100 (max: the observed
enzyme activity measured in the presence of enzyme, substrates, and cofactors; min: the observed
enzyme activity in the presence of substrates, cofactors and in the absence of enzyme). IC50 values
were calculated from the inhibition curves.
Acknowledgments
The work was supported by Program for Liaoning Excellent Talents in University (LR2014030
and 2013921042) and National Natural Science Foundation of China (21002065).
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Legends
Fig. 1. The representative small-molecule c-Met kinase inhibitors.
Fig. 2. Design and structures of the target compounds.
Scheme 1. Reagents and conditions: (i) NBS, CH3CN, r.t.; (ii) NaNO2, H2SO4/H2O, NaOH 0°C to 95°C; (iii)
3,4-difluoronitrobenzene, DMF, K2CO3, 80°C; (iv) 3-bromopyridine, bis(pinacolato)diboron, KOAc, PdCl2(PPh3)2,
dioxane, 90°C to r.t.; Na2CO3, H2O, 90°C; (v) Fe, 95% EtOH, NH4Cl (cat), AcOH, reflux; (vi) DIPEA, EtOH,
reflux; (vii) DIPEA, EtOH, substituted anilines, reflux to r.t..
Scheme 2. Reagents and conditions: (i) HCl, NaNO2, AcONa, EtOH, 0°C; (ii) glyoxal, AcONa, reflux; (iii) NaH,
CH3I, DMF, 0°C (iv) glyoxal, HCl, H2O, reflux; (v) SOCl2, Tol, reflux; DIPEA, CH2Cl2, 0°C to r.t..
Scheme 3. Reagents and conditions: (i) KMnO4, NaOH, H2O, 100°C (ii) EtOH, H2O, r.t.; (iii) SOCl2, Tol, reflux;
DIPEA, CH2Cl2, 0oC to r.t.; (iv) NaNO2, HCl, H2O, 0°C to r.t.; Na2SO3, r.t..
Scheme 4. Reagents and conditions: (i) ethyl chloroacetate, K2CO3, acetone, r.t.; (ii) 30% H2O2, AcOH, 100°C; (iii)
AcONa, MeOH, 0°C; (iv) K2CO3, DMF, r.t., NaOH/H2O, r.t.;(v) SOCl2, Tol, reflux; DIPEA, CH2Cl2, 0°C to r.t..
Scheme 5. Reagents and conditions: (i) 40% glyoxal, AcOH/H2O (1:1), r.t.; (ii) meldrum’s acid, piperidine, Tol, r.t.;
(iii) MeONa, MeOH, reflux.; (v) SOCl2, Tol, reflux; DIPEA, CH2Cl2, 0°C to r.t..
Scheme 6. Reagents and conditions: (i) POCl3, DMF, HPF6, NaOH, H2O, 10°C; (ii) diethyl malonate, t-BuOK,
THF, 45°C; CH3NH2, DMF, 70°C; (iii) SOCl2, Tol, reflux; DIPEA, CH2Cl2, 0°C to r.t..
Table 1. Cytotoxicity of target compounds against A549, HT-29, MKN-45 and MDA-MB-231 cells and c-Met
kinase inhibition in vitro.
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Table 1. Cytotoxicity of target compounds against A549, HT-29, MKN-45 and MDA-MB-231 cells and c-Met
kinase inhibition in vitro.
IC50(µM) a
c-Met
Compd.
Linker
R
IC50(nM)
53.1
ND
HT-29
A549
MKN-45
6.3±0.58
MDA-MB-231
2.74±0.29
ND b
23a
23b
23c
23d
23e
23f
H
4-OCH3
3,4-di-F
4-Cl
0.88±0.07
4.11±0.39
2.32±0.15
0.98±0.10
0.69±0.06
0.33±0.03
1.13±0.12
2.10±0.18
0.44±0.04
0.51±0.055
0.78±0.082
4.78±0.49
0.89±0.092
ND
0.41±0.04
2.14±0.02
7.63±0.07
1.61±0.16
0.18±0.02
0.71±0.07
0.56±0.06
0.61±0.06
0.93±0.09
0.46±0.044
0.72±0.078
12.50±1.21
0.89±0.091
6.8±0.70
1.01±0.11
0.08±0.008
0.24±0.03
0.03±0.003
0.16±0.02
1.75±0.16
4.78±0.05
1.6±0.15
6.77±0.68
ND
37.5
ND
3-Cl
0.09±0.009
14.10±1.42
4.24±0.42
6.76±0.66
3.22±0.32
3.98±0.38
9.69±0.94
11.3±1.15
3.27±0.33
10.4±1.02
0.072±0.007
8.2±0.81
ND
24
4-F
ND
23g
24a
24b
24c
24d
24e
24f
3,5-OCH3
2-F
ND
84.6
47.5
ND
4-Cl
3-CF3
2,6-di-Cl
3-Cl,4-F
2,4-di-OCH3
4-OCH3
3-F
1.95±0.19
3.93±0.38
10.10±1.03
2.82±0.28
0.74±0.07
0.027±0.003
0.09±0.009
0.32±0.03
0.43±0.04
0.027±0.003
0.09±0.009
0.003±0.0003
4.23±0.43
56.3
103.2
ND
25a
25b
25c
25d
25e
26a
26b
26c
27a
89.3
19.0
ND
0.41±0.04
1.24±0.14
0.99±0.09
0.71±0.07
0.19±0.02
1.55±0.14
0.43±0.04
4.56±0.48
1.75±0.18
7.9±0.81
H
4-F
0.82±0.08
1.02±0.11
0.12±0.01
1.3±0.01
51.6
ND
4-Cl
ND
2-CF3
3,4-di-F
3-F
0.046±0.005
0.034±0.003
0.01±0.001
4.84±0.49
17.3
46.5
8.2
0.9±0.09
4-Cl
6.56±0.68
89.8
27b
28a
4-F
H
4.77±0.43
7.42±0.79
2.88±0.28
7.6±0.75
3.12±0.34
5.4±0.53
5.2±0.54
9.5±0.98
ND
ND
28b
4-F
11.23±1.09
0.17±0.019 c
8.3±0.81
7.1±0.68
11.9±0.18
0.54± 0.06
106.4
1.1 f
Foretinib
0.26 ± 0.026 d
0.023 ± 0.0015 e
a Data presented is the mean ± SD value of three independent determinations.
6 ND = not determined
c Reported IC50 = 29 nM.31
d Reported IC50 = 165 nM.31
e Reported IC50 = 8 nM.32
f Reported IC50 = 4 nM.31

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