Design and synthesis of novel 2-(4-(2-(dimethylamino)ethyl)-4H-1,2,4- triazol-3-yl)pyridines as potential antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2014.04.059

New 2-(4-(2-(dimethylamino)ethyl)-4H-1,2,4-triazol-3-yl)pyridine derivatives were synthesized and evaluated for their in vitro cytotoxicity against five cancer cell lines namely MKN-45, H460, HT-29, A549 and U87MG, as well as the normal cell line WI-38. Nearly all the compounds exhibited superior potency to sorafenib with a better selectivity towards the MKN-45, H460 and HT-29 cell lines. In addition, the enzymatic screening result demonstrated that the optimized compounds possessed potent Raf kinase inhibition as well as favorable enzyme selectivity. The most promising compound, 11f, showed high levels of cytotoxicity against MKN-45, H460 and HT-29 cells with IC₅₀ values of 51, 72 and 130 nM, respectively, which are 45.5, 30.4 and 27.8 folds higher than the corresponding IC₅₀ values for sorafenib against these cell lines. Structure-activity relationships revealed that the dimethylaminoethyl group was crucial for high activity.

Full text of DOI:10.1016/j.ejmech.2014.04.059

European Journal of Medicinal Chemistry 81 (2014) 47e58
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of novel 2-(4-(2-(dimethylamino)ethyl)-4H-
,2,4-triazol-3-yl)pyridines as potential antitumor agents
1
a
a
b
a
a
a
c
Mingze Qin , Xin Zhai , Hongbo Xie , Junjie Ma , Kuan Lu , Yu Wang , Lihui Wang ,
d
a,
*
Yucheng Gu , Ping Gong
a
Key Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical University), Ministry of Education, 103 Wenhua Road,
Shenyang 110016, PR China
College of Bioinformatics Science and Technology, Harbin Medical University, Harbin 150081, PR China
Department of Pharmacology, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China
Syngenta, Jealott’s Hill International Research Centre, Bracknell, Berkshire RG42 6EY, UK
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 December 2013
Received in revised form
New 2-(4-(2-(dimethylamino)ethyl)-4H-1,2,4-triazol-3-yl)pyridine derivatives were synthesized and
evaluated for their in vitro cytotoxicity against five cancer cell lines namely MKN-45, H460, HT-29, A549
and U87MG, as well as the normal cell line WI-38. Nearly all the compounds exhibited superior potency
to sorafenib with a better selectivity towards the MKN-45, H460 and HT-29 cell lines. In addition, the
enzymatic screening result demonstrated that the optimized compounds possessed potent Raf kinase
inhibition as well as favorable enzyme selectivity. The most promising compound, 11f, showed high
levels of cytotoxicity against MKN-45, H460 and HT-29 cells with IC50 values of 51, 72 and 130 nM,
respectively, which are 45.5, 30.4 and 27.8 folds higher than the corresponding IC50 values for sorafenib
against these cell lines. Structureeactivity relationships revealed that the dimethylaminoethyl group was
crucial for high activity.
20 March 2014
Accepted 21 April 2014
Available online 24 April 2014
Keywords:
Synthesis
4-(2-(Dimethylamino)ethyl)-4H-1,2,4-
triazole
Antitumor activity
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
patients with advanced renal and hepatocellular carcinomas [13].
In light of its satisfactory antitumor properties, as well as its proven
Cancer is a group of diseases characterized by uncontrolled cell
growth in a body, causing over 7 million deaths a year [1]. The
current chemotherapeutics are far from satisfactory due to serious
side effects and acquired drug resistance, which lead to an ongoing
need to explore safer and more efficient antitumor agents [2,3].
Recently, small molecule inhibitors have attracted increasing levels
of interest in cancer therapy, not only for their outstanding anti-
tumor activity, but also for their diverse mechanisms of action
exerted in different malignancies, which offer the possibility of
minimizing relative toxicity and circumventing drug resistance [4e
clinical utility, scientists have paid much attention to the modifi-
cation of sorafenib and have designed and synthesized a large
number of related compounds. A series of benzimidazole-based
compounds with general structure I (Fig. 1) was reported to
exhibit excellent Raf kinase inhibition (0.001e0.15 mM) and to have
favorable pharmacokinetic properties [14]. Further optimization on
the methyl amide fragment by introducing a trifluoromethyl
imidazole moiety led to the identification of RAF265, a potent in-
hibitor of Raf and VEGFR-2 kinases which is in phase I clinical trials
for the treatment of metastatic melanoma [15,16].
1
0].
The co-crystal structure of sorafenib in complex with B-Raf ki-
nase revealed that the urea and pyridine moieties interact with
amino acid residues (Asp593, Glu500 and Cys531) via hydrogen
bonds, and the trifluoromethyl phenyl ring occupied the hydro-
phobic pocket [17,18]. Therefore, with the intention of identifying
more potent analogs of novel chemotypes, we focused on modifi-
cation of the pharmacophore of sorafenib while maintaining the
interaction mode with the kinase domains.
Sorafenib is an orally available multikinase inhibitor which is
active against Raf kinase by targeting the MAPKs signaling pathway,
and shows a powerful inhibitory action on VEGFR and PDGFR
[11,12]. It demonstrates significant inhibition across a broad spec-
trum of tumor types, and is considered to be a first-line therapy in
Despite the high potency of sorafenib, its low level of bioavail-
ability persuaded us to pursue a program to identify compounds
*
Corresponding author.
E-mail address: gongpinggp@126.com (P. Gong).
http://dx.doi.org/10.1016/j.ejmech.2014.04.059
223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.
0

48
M. Qin et al. / European Journal of Medicinal Chemistry 81 (2014) 47e58
desired intermediate 4a, which then reacted with N,N-dime-
thylformamide dimethylacetal (DMF-DMA) to afford 5a. Interme-
diate 5b was synthesized from 3 in a similar manner to that
described for 5a, but employing hydrazine hydrate instead of
ammonia. The key intermediate 6a was obtained by cyclization of
5
a with methylhydrazine in glacial acetic acid [29], while 6b and 6c
were prepared by reaction of 5b with appropriate amines
methylamine, N,N-dimethylethylenediamine) under the same
(
conditions [30]. After reduction of the nitro compounds 6ae6c with
hydrazine hydrate in ethanol, the resultant amino derivatives 7ae
7c were further treated with phenyl chloroformate in acetone in
the presence of anhydrous K CO to give the corresponding in-
2
3
termediates 8ae8c in good yields. In the following step, 8ae8c
were treated with hydrazine hydrate in refluxing 1,4-dioxane
generating 9ae9c, respectively. Finally, condensation of 9ae9c
with aromatic aldehydes in isopropanol in the presence of a cata-
lytic amount of glacial acetic acid yielded compounds 10ae10c,
11ae11x and 12ae12c [31].
The chemical structures of compounds 10ae10c, 11ae11x and
1
2ae12c were confirmed by IR, NMR and mass spectra. 2D NOESY
NMR spectra were recorded in order to determine configuration of
the imino double bonds of these compounds. In the case of com-
pound 11f, a clear NOESY signal was observed between the proton
of eNHeN] (
.43 ppm, singlet), indicating that compound 11f has the E-
configuration (Fig. 3 and supplementary data). Similarly, for com-
pound 11g, the NOE interaction between eNHeN] ( 11.29 ppm,
singlet) and eN]CHe ( 8.36 ppm, singlet) confirmed that it was
d 11.33 ppm, singlet) and the proton of eN]CHe (d
Fig. 1. Structures of sorafenib and its analogs.
8
d
with high potency combined with an improved physicochemical
profile, especially higher levels of water-solubility [19e21]. As part
of our overall design strategy, we inserted an additional imine
group between the urea and phenyl ring, arguing that it should
improve structure flexibility, which might be beneficial to biolog-
ical activity and the solubility of the compounds [22,23]. Further-
more, triazole, a privileged moiety in modern drug design [24e26],
replaced the methyl amide fragment to build a more drug-like
skeleton (Fig. 2).
d
the E-isomer. The other related compounds were assigned the same
E-configuration by analogy.
3. Results and discussion
3.1. In vitro cytotoxicity and structureeactivity relationships
The cytotoxicity of compounds 10ae10c, 11ae11x and 12ae12c
We started with a study of structureeactivity relationships
around the triazole moiety. We hypothesized that enhanced solu-
bility could be achieved by introducing an ionogenic amino group
on the triazole unit, and thus compounds 10ae10c, with variations
in the position of linkage and nature of substitution were prepared.
Biological evaluation identified compound 10c, which possessed a
dimethylaminoethyl group at position-4 of triazole, as being of
particular interest (comparable potency and superior physico-
chemical profile to sorafenib), and suggested that we should look
more closely at 2-(4-(2-(dimethylamino)ethyl)-4H-1,2,4-triazol-3-
yl)pyridine derivatives. Therefore, our further research efforts
focused on optimizing the skeleton of 10c, with modifications to
the terminal aryl moiety to find a substitution pattern which would
improve the potency even further. Substituents varied in nature,
steric-hindrance as well as electron property were introduced to
yield compounds 11ae11x and 12ae12c. In this paper, we report
the newly synthesized compounds and their in vitro antitumor
potency, together with the results of a study of the mechanism of
action.
were evaluated in vitro against five cancer cell lines, namely MKN-
5 (human gastric cancer), H460 (human lung cancer), HT-29
human colorectal cancer), A549 (human non-small-cell lung can-
cer) and U87MG (human glioblastoma), by the standard MTT assay.
Sorafenib was used as the positive control. Potent compounds were
further assessed for their safety towards the non-malignant cell
line WI-38 (human fetal lung fibroblasts). The bioactivity data are
presented as IC50 values in Tables 1 and 2. The IC50 values shown are
the mean of the data generated by at least three replicated
experiments.
The biological data presented in Table 1 suggested that the
substitution pattern on the triazole moiety was closely related to
antitumor potency and physicochemical profile. As shown in
Table 1, compound 10a that has a methyl substituent at position-1,
was almost inactive against all the tested cancer cells and possessed
a ClogP of 3.82 units. However, shifting the methyl to position-4
4
(
(10b) increased the potency significantly and decreased ClogP by
0.82 units, indicating a contribution derived from the position of
the substituent. Furthermore, replacement of the methyl group
with a dimethylaminoethyl group (10c) led to a 2.8e16.1-fold
improvement of the antitumor activity, coupled with a compara-
ble ClogP but a favorable reduction of ClogD7.4 by 1.59 units, which
probably means a better distribution of the compound in blood
serum. Given these encouraging results, we retained the promising
skeleton of 10c, and modified the terminal aryl moiety with the aim
of improving antitumor potency even further.
2
. Chemistry
The general route to synthesize the target compounds is
described in Scheme 1. The starting material picolinic acid 1 was
chlorinated with thionyl chloride, and then treated with methanol
in an ice-bath to give the methyl ester 2 as a light-yellow solid [27].
Subsequently, a nucleophilic substitution of 2 with 2-fluoro-4-
nitrophenol in refluxing chlorobenzene provided 3 in a moderate
yield [28]. Aminolysis of 3 with ammonia in acetone led to the
As shown in Table 2, most of the 2-(4-(2-(dimethylamino)ethyl)-
4H-1,2,4-triazol-3-yl)pyridine derivatives exhibited excellent ac-
tivity, similar to or even higher than that of sorafenib against one or

M. Qin et al. / European Journal of Medicinal Chemistry 81 (2014) 47e58
49
Fig. 2. Design strategy of the target compounds.
more cancer cell lines. Compounds 11ae11g, 11j, 11l, 11s and 12ae
2c displayed better efficacy than sorafenib against all the tested
cancer cell lines with IC50 values ranging from 0.051 to 13.23 M.
Compound 11h showed excellent levels of inhibition against MKN-
compounds. As shown, the 2,3-dichloro-phenyl derivative 11f
showed outstanding activity towards MKN-45 cells, with an IC50
1
m
value of 0.051
with the 2,6-dichloro-phenyl derivative 11h (IC50 ¼ 0.16
further decrease in potency was observed with the 2,4-dichloro-
M).
mM, whereas the potency was reduced by 3.2 folds
mM), and a
4
0
5, H460, HT-29 and A549 cancer cells with IC50 values of 0.16, 0.35,
.28 and 1.41 M, respectively. Also noteworthy is that the potency
m
phenyl derivative 11g (IC50 ¼ 0.75
m
of the compounds was generally more pronounced against the
MKN-45, H460, HT-29 cells than against A549 and U87MG cells.
Further analysis clearly revealed that the potency of the com-
pounds was extremely dependent on the electronic properties of
the substituent(s) on the phenyl moiety. As a matter of fact, almost
all the compounds (11ae11h, 11j, 11l) with electron-withdrawing
groups (EWGs) displayed an enhanced potency in comparison to
that of 10c (R ¼ Ph), whereas compounds (11me11s, 11ue11x) with
Incorporation of bulky moieties such as bicyclic groups (12ae
12c) maintained the potent activity, suggesting an insensitivity to
the bulk of the aryl ring to a certain degree.
It is worth mentioning that most of the tested compounds were
less toxic to human fibroblasts WI-38 in comparison to their effects
on cancer cells, suggesting that the cancer cells were more sensitive
to the newly synthesized compounds than the non-malignant cells.
In particular, compound 11f possessed a low level of toxicity to-
electron-donating groups (EDGs) showed
a
comparative or
wards WI-38 (IC50 ¼ 6.13
mM), and the selectivity indexes for MKN-
45 and H460 were 120.2 and 85.1, respectively, values which are 37
and 24.7 folds better than that of sorafenib.
decreased activity. It can be concluded that EWGs on the phenyl
ring are more effective than EDGs. However, the introduction of
EDGs at both the meta- and para-position appears to influence the
inhibitory activity against the H460 and HT-29 cells, a case in point
is that derivative 11r possesses marked inhibition towards the
3.2. In vitro enzymatic assay
H460 and HT-29 cells, exhibiting IC50 values of 0.057 and 0.12 mM,
respectively. These results could be ascribed to the degree of elec-
tron density on the phenyl ring as well as the specificity of cancer
cells.
In addition, it is interesting to note that the nature of the
halogen atoms on the phenyl ring plays an important role on
antitumor activity. Compound 11a, with a chlorine atom at the
para-position, promoted the antitumor potency towards the H460
cells by 6.2 folds as compared to 11c, which contains a fluorine at
the same region, suggesting that chlorine is better tolerated than
fluorine at the para-position. Further support for this conclusion
In order to explore the mechanism of action of the newly syn-
thesized compounds, analogs (11b, 11d, 11f, 11g, 11r, 11u and 12c)
that had structural diversity and potent antitumor potency were
screened in a panel of kinases (c-Met, VEGFR-2, B-Raf, Raf-1,
PDGFR
and 12c) exhibited significant inhibition against the two tested Raf
kinase isoforms (B-Raf and Raf-1) at a concentration of 10 M, while
a). As shown in Table 3, five compounds (11b, 11d, 11f, 11g
m
their effects on other tested kinases were much lower. These results
implied that some compounds might be potential Raf kinase in-
hibitors, and had a favorable enzyme selectivity. In addition, the
decreased affinity observed for 11r and 11u further confirmed SARs
that the hydrophobic pocket was sensitive to the electronic prop-
erties of the substituent(s) on aryl fragment, with EWGs being well
tolerated. However, we also noticed that all these analogs were less
potent than sorafenib against Raf kinase. Therefore, we are
currently carrying out more experiments to confirm the exact
can be drawn by comparing 11g (R ¼ 2,4-di-Cl-Ph; IC50 ¼ 0.13
mM)
with 11e (R ¼ 2-Cl-4-F-Ph, IC50 ¼ 0.72
m
M). Similarly, a comparison
of 11i (R ¼ 3-F-4-OH-Ph) with 11j (R ¼ 3-Br-4-OH-Ph) in cytotoxic
activity against H460 and HT-29 cells indicated that bromine is
preferred in comparison to fluorine at the meta-position. In addi-
tion, the substitution position influenced the cytotoxicity of the

50
M. Qin et al. / European Journal of Medicinal Chemistry 81 (2014) 47e58
ꢀ
ꢀ
ꢀ
Scheme 1. Reagents and conditions: (a) SOCl
2
, NaBr, chlorobenzene, 50 C, 30 min, 85 C, 20 h; (b) MeOH, toluene, 0e15 C, 1.5 h; (c) 2-fluoro-4-nitrophenol, chlorobenzene, reflux,
ꢀ
ꢀ
ꢀ
ꢀ
12 h; (d) NH
3
.H
2
O, acetone, 50 C, 3 h; (e) NH
2
2
NH .H
2
O, MeCN, 25 C, 3 h; (f) DMF-DMA, CH
2
Cl
2
, reflux, 2e3 h; (g) CH
NH .H
3
NHNH
2
, HOAc, 90 C, 3 h; (h) appropriate amine, HOAc, 90 C,
ꢀ
ꢀ
3
5
h; (i) NH
e8 h.
2
2
NH .H
2
O, EtOH, reflux, 3e6 h; (j) phenyl chloroformate, K
2
CO
3
, acetone, 0e25 C, 3 h; (k) NH
2
2
2
O, dioxane, 90 C, 3 h; (l) substituted aldehyde, isopropanol, reflux,
targets to which these compounds bind and which leads to their
interesting antitumor potency.
the binding model overlay of the binding model of compound 11f
with sorafenib. It suggests that the two compounds act in a very
similar way, with matching occupancy of the hydrophobic pocket
and similar hydrogen-bond interactions. As shown in Fig. 4 (right),
compound 11f binds well to B-Raf kinase domain via four hydrogen
bonds. The urea moiety of compound 11f formed hydrogen-
bonding interactions with residues Asp593 and Glu500 in the
catalytic region, and the pyridine nitrogen atom interacted with
residue Cys531 via a hydrogen bond. In addition, the active-site
groove of 1UWH is occupied by the dimethylaminoethyl tail,
which is surrounded by the residues Ile462, Val470 and Phe594.
3.3. Binding model analysis
To elucidate the binding mode of this series of compounds with
Raf kinase and to guide further SARs, a detailed docking analysis
was performed on the most promising compound 11f. The analysis
was conducted using the AutoDock 4.0 protocol, and the crystal
structure of wild type B-Raf kinase with sorafenib (PDB code:
1
UWH) was obtained from the Protein Data Bank. Fig. 4 (left) shows

M. Qin et al. / European Journal of Medicinal Chemistry 81 (2014) 47e58
51
ꢀ
8
5 C and stirred for another 20 h. The completion of reaction was
determined by TLC, at which point the solvent was removed under
reduced pressure to give a brown oil, which was immediately dis-
solved in toluene (300 mL). Then anhydrous methanol (80 mL) was
ꢀ
added dropwise in an ice-bath. The mixture was stirred at 15 C for
1
(
K
.5 h. The precipitate was filtered out and dissolved in CH
500 mL). The organic solution was washed with saturated aqueous
CO (300 mL), dried over Na SO and evaporated to give 2 as a
light-yellow solid (158.5 g, yield 76.0%). M.p. 67e68 C; H NMR
300 MHz, DMSO-d
8.65 (d, J ¼ 5.2 Hz, 1H), 8.02 (d, J ¼ 1.7 Hz,
H), 7.78 (dd, J ¼ 5.1, 1.6 Hz, 1H), 3.85 (s, 3H); ESI-MS m/z: 194.0
2 2
Cl
2
3
2
4
ꢀ
1
(
1
6
) d
þ
[MþNa] .
5.1.2. Methyl 4-(2-fluoro-4-nitrophenoxy)picolinate (3)
A solution of 2 (20 g, 0.117 mol) and 2-fluoro-4-nitrophenol
(27.5 g, 0.175 mol) in chlorobenzene (400 mL) was stirred under
reflux for 12 h. After cooling to room temperature, the mixture was
concentrated under vacuum. The residue was dissolved in CH
2
Cl
CO
2
(
(
500 mL), and then washed with saturated aqueous K
2
3
3 ꢁ 250 mL), followed by brine (200 mL). The organic phase were
SO and concentrated to yield a gray solid, which was
recrystallized from absolute ethanol to generate 3 as a pale-yellow
dried over Na
2
4
Fig. 3. NOESY effects of compounds 11f and 11g.
ꢀ
1
3
solid (15.5 g, yield 45.4%). M.p. 86e88 C; H NMR (300 MHz, CDCl )
d
8.70 (d, J ¼ 5.5 Hz, 1H), 8.19e8.17 (m, 1H), 8.16 (t, J ¼ 3.0 Hz, 1H),
4
. Conclusion
7
1
.69 (d, J ¼ 2.4 Hz, 1H), 7.40e7.34 (m, 1H), 7.11 (dd, J ¼ 5.5, 2.5 Hz,
þ
H), 4.01 (s, 3H); ESI-MS m/z: 315.0 [MþNa] .
In this study, we have developed an interesting series of 2-(4-(2-
dimethylamino)ethyl)-4H-1,2,4-triazol-3-yl)pyridine derivatives.
(
5
.1.3. 4-(2-Fluoro-4-nitrophenoxy)picolinamide (4a)
Most compounds exhibited excellent cytotoxic activity towards
MKN-45, H460 and HT-29 cancer cells, while showed much lower
toxic effects towards human fibroblasts WI-38. Furthermore, some
of the target compounds were evaluated as potential Raf kinase
inhibitors with a satisfactory enzyme selectivity. An analysis of
SARs revealed that the dimethylaminoethyl substituent on the
triazole moiety is crucial for the observed activity, and also im-
proves the physicochemical profile of the compounds. In addition,
EWGs on the terminal aryl fragment can improve antitumor po-
tency even further, and maintain favorable Raf kinase affinity.
Taking the biological data together, compound 11f has emerged as a
promising candidate for further development of more selective Raf
kinase inhibitors.
Aqueous ammonia (30 mL) was added to a solution of 3 (2.92 g,
0
.01 mol) in acetone (30 mL) at room temperature. The mixture was
ꢀ
stirred at 50 C for 3 h. The completion of the reaction was proved
by TLC, and then poured into water. After stirring for 30 min, the
precipitate was collected by filtration, and dried to provide 4a as a
ꢀ
white solid (2.09 g, yield 75.6%). M.p. 107e109 C; ESI-MS m/z:
00.1 [MþNa] .
þ
3
5
.1.4. N-((dimethylamino)methylene)-4-(2-fluoro-4-nitrophenoxy)
picolinamide (5a)
A mixture of 4a (2.77 g, 0.01 mol) and DMF-DMA (3.58 g,
2 2
.03 mol) in CH Cl (20 mL) was stirred under reflux for 2 h. The
0
solvent was removed under reduced pressure. The residue was
washed with water, filtered, and dried to give 5a as a light-yellow
5
. Experimental
ꢀ
solid (2.84 g, yield 85.6%). M.p. 128e131 C; ESI-MS m/z: 333.3
þ
[
MþH] .
5.1. Chemistry
Unless otherwise noted, all materials were obtained from
5.1.5. 4-(2-Fluoro-4-nitrophenoxy)-2-(1-methyl-1H-1,2,4-triazol-
5-yl)pyridine (6a)
commercially available sources and used without further purifica-
tion. All melting points were determined on a Büchi Melting Point
B-540 apparatus (Büchi Labortechnik, Flawil, Switzerland) and
A mixture of 5a (2 g, 6 mmol) and methylhydrazine (1.1 g,
ꢀ
24 mmol) in glacial acetic acid (20 mL) was heated to 90 C and
1
13
were uncorrected. H NMR (1D and 2D) and C NMR spectroscopy
were performed using Bruker ARX-300 or ARX-600 spectrometers
stirred for 3 h. The mixture was concentrated and then poured into
ice water. After stirring for 30 min, the precipitate was collected by
filtration, and then washed with water, subsequently dried to give
(
Bruker Bioscience, Billerica, MA, USA) with TMS as an internal
ꢀ
standard. Mass spectra (MS) were determined in ESI mode on an
Agilent 1100 LC-MS (Agilent, Palo Alto, CA, USA). Elemental analysis
was determined on a Carlo-Erba 1106 Elemental analysis instru-
ment (Carlo Erba, Milan, Italy). The IR spectra were recorded by
6a as a light-yellow solid (0.98 g, yield 52.1%). M.p. 139e141 C; ESI-
MS m/z: 316.2 [MþH] .
þ
5.1.6. 4-(2-Fluoro-4-nitrophenoxy)picolinohydrazide (4b)
means of the KBr pellet technique on
spectrometer.
a
Bruker FTS 135
A mixture of 3 (14.6 g, 0.05 mol) and 80% hydrazine hydrate
(70 mL) in MeCN (100 mL) was stirred at room temperature. TLC
indicated that the reaction had gone to completion after 3 h. The
5.1.1. Methyl 4-chloropicolinate (2)
mixture was diluted with CH
2 2
Cl (300 mL), and then washed with
To a solution of picolinic acid (150 g, 1.22 mol) and sodium
brine (3 ꢁ 100 mL). The organics were evaporated to furnish 4b as a
ꢀ
1
bromide (20.1 g, 0.197 mol) in chlorobenzene (200 mL), thionyl
chloride (355 mL, 4.88 mol) was added slowly at room temperature.
pale-yellow solid (9.97 g, yield 68.3%). M.p. 98e100 C; H NMR
(300 MHz, DMSO-d
10.01 (s, 1H), 8.60 (d, J ¼ 5.6 Hz, 1H), 8.47
(dd, J ¼ 10.5, 2.7 Hz,1H), 8.23 (m,1H), 7.69 (t, J ¼ 8.5 Hz,1H), 7.53 (d,
6
) d
ꢀ
The reaction mixture was stirred at 50 C for 30 min, and heated to

52
M. Qin et al. / European Journal of Medicinal Chemistry 81 (2014) 47e58
Table 1
In vitro cytotoxicity, ClogP and ClogD values of compounds 10ae10c.
Compd.
R
1
IC50 (
m
M)
ClogPa
ClogDb
MKN-45
H460
HT-29
A549
NA
U87MG
NA
1
1
1
0a
0b
0c
NA
52.23 ꢂ 3.79
30.17 ꢂ 2.35
3.82
3.00
3.82
3.00
7.61 ꢂ 1.91
15.73 ꢂ 1.47
8.79 ꢂ 0.69
7.56 ꢂ 1.01
36.32 ꢂ 4.39
2.73 ꢂ 0.76
2.32 ꢂ 0.25
1.78 ꢂ 0.23
2.19 ꢂ 0.11
1.12 ꢂ 0.27
3.61 ꢂ 0.36
1.86 ꢂ 0.86
1.92 ꢂ 0.12
2.25 ꢂ 0.75
3.02
4.34
1.41
4.34
Sorafenib
NA: compound showing IC50 value > 100
15.57 ꢂ 1.78
m
M.
a
ClogP values, calculated using JChem_For_Excel_6.0.0.865.
ClogD values, predicted at PH ¼ 7.4.
b
J ¼ 2.5 Hz,1H), 7.34 (dd, J ¼ 5.6, 2.6 Hz,1H), 4.72 (s, 2H); ESI-MS m/z:
1.5 mmol) was added 80% hydrazine hydrate aqueous solution
(6.25 mL, 0.1 mol) with vigorous agitation. Upon completion of
addition, the mixture was stirred under reflux for 3e6 h until TLC
showed the completion of the reaction. The reaction mixture was
þ
3
15.1 [MþNa] .
0
.1.7. N -(4-(2-fluoro-4-nitrophenoxy)picolinoyl)-N,N-
5
dimethylformohydrazonamide (5b)
2 2
concentrated under vacuum, which was then dissolved in CH Cl ,
At room temperature, DMF-DMA (14.3 g, 0.12 mol) was added to
washed with brine, and evaporated to yield the solids 7ae7c.
4
2 2
b (11.7 g, 0.04 mol) in CH Cl (150 mL), the mixture was refluxed
for 2 h until TLC showed the completion of the reaction. The
mixture was evaporated under reduced pressure, and then washed
with diethyl ether, subsequently dried to yield 5b as an orange solid
5
.1.9.1. 3-Fluoro-4-(2-(1-methyl-1H-1,2,4-triazol-5-yl)pyridin-4-
ꢀ
1
yloxy)aniline (7a). A gray solid; Yield: 63%; M.p. 150e153 C;
NMR (300 MHz, DMSO-d
H
6
)
d
8.61 (d, J ¼ 5.7 Hz,1H), 7.99 (s,1H), 7.48
ꢀ
1
(
9.84 g, yield 70.9%). M.p. 153e155 C; H NMR (300 MHz, DMSO-
11.00 (s, 1H), 8.60 (d, J ¼ 5.6 Hz, 1H), 8.47 (dd, J ¼ 10.5, 2.6 Hz,
(
6
3
d, J ¼ 2.4 Hz, 1H), 7.14e7.01 (m, 2H), 6.56 (dd, J ¼ 13.2, 2.4 Hz, 1H),
6
d ) d
1
(
.47 (dd, J ¼ 8.7, 2.1 Hz, 1H), 5.56 (s, 2H), 4.27 (s, 3H); ESI-MS m/z:
H), 8.23 (d, J ¼ 9.1 Hz, 1H), 8.06 (s, 1H), 7.69 (t, J ¼ 8.6 Hz, 1H), 7.54
þ
08.3 [MþNa] .
d, J ¼ 2.5 Hz, 1H), 7.33 (dd, J ¼ 5.5, 2.6 Hz, 1H), 2.83 (s, 6H); ESI-MS
þ
m/z: 348.3 [MþH] .
5
.1.9.2. 3-Fluoro-4-(2-(4-methyl-4H-1,2,4-triazol-3-yl)pyridin-4-
yloxy)aniline (7b). A light-yellow solid; Yield: 61%; M.p. 173e
5
.1.8. General procedure for preparation of compounds (6b and 6c)
Appropriate amine (0.06 mol) was added slowly to a stirred
ꢀ
1
1
75 C; H NMR (300 MHz, DMSO-d ) d 8.62 (s, 1H), 8.58 (d,
6
J ¼ 5.7 Hz, 1H), 7.49 (d, J ¼ 2.2 Hz, 1H), 7.11e7.01 (m, 2H), 6.56 (dd,
solution of 5b (5.2 g, 0.015 mol) in glacial acetic acid (30 mL) at
room temperature. The mixture was stirred at 90 C for 3 h, and
then concentrated under vacuum. The residue was poured into
J ¼ 13.3, 2.3 Hz,1H), 6.47 (dd, J ¼ 8.8, 1.9 Hz,1H), 5.68 (d, J ¼ 56.8 Hz,
ꢀ
þ
2
H), 4.00 (s, 3H); ESI-MS m/z: 308.3 [MþNa] .
water, and extracted with CH
organic layers were dried over Na
reduced pressure to give the crude residue, which was purified by
silica gel column chromatography using CH Cl /MeOH (100/1 to
2
Cl
2
(3 ꢁ 100 mL). The combined
5
.1.9.3. 4-(2-(4-(2-(Dimethylamino)ethyl)-4H-1,2,4-triazol-3-yl)pyr-
2
SO , and evaporated under
4
idin-4-yloxy)-3-fluoroaniline (7c). A light-yellow solid; Yield: 52%;
M.p. 103e105 C. H NMR (300 MHz, DMSO-d ) d 8.63 (s, 1H), 8.56
6
ꢀ
1
2
2
(
6
2
d, J ¼ 5.7 Hz, 1H), 7.49 (d, J ¼ 2.4 Hz, 1H), 7.08 (m, 1H), 7.04 (m, 1H),
100/10) as eluent to obtain the solids 6b and 6c.
.55 (dd, J ¼ 13.2, 2.5 Hz, 1H), 6.46 (dd, J ¼ 8.9, 2.4 Hz,1H), 5.54 (s,
H) 4.61 (t, J ¼ 6.3 Hz, 2H), 2.57 (t, J ¼ 6.2 Hz, 2H), 2.13 (s, 6H); ESI-
5
3
.1.8.1. 4-(2-Fluoro-4-nitrophenoxy)-2-(4-methyl-4H-1,2,4-triazol-
þ
MS m/z: 343.4 [M þ H] .
-yl)pyridine (6b). A light-yellow solid; Yield: 57%; M.p. 142e
ꢀ
þ
143 C; ESI-MS m/z: 316.2 [MþH] .
5
.1.10. General procedure for preparation of compounds (8ae8c)
ꢀ
5
.1.8.2. 2-(3-(4-(2-Fluoro-4-nitrophenoxy)pyridin-2-yl)-4H-1,2,4-
To a stirred, cooled to 0 C solution of 7ae7c (4 mmol) and
K CO (0.83 g, 6 mmol) in dry acetone (20 mL) was added phenyl
chloroformate (0.81 g, 5.2 mmol). Upon completion of the addition,
the reaction mixture was heated to room temperature and stirred
for 3 h. The reaction mixture was quenched by addition of water
triazol-4-yl)-N,N-dimethylethanamine (6c). A yellow solid; Yield:
2
3
ꢀ
þ
4
2%; M.p. 117e119 C; ESI-MS m/z: 373.3 [MþH] .
5.1.9. General procedure for preparation of compounds (7ae7c)
At room temperature, to a suspension of nitro derivatives 6ae6c
2 2
and extracted with CH Cl , the combined organics were washed
(
3 2
10 mmol), active carbon (0.036 g, 3 mmol) and FeCl .6H O (0.41 g,
with brine, evaporated to give compounds 8ae8c.

M. Qin et al. / European Journal of Medicinal Chemistry 81 (2014) 47e58
53
Table 2
In vitro cytotoxicity, ClogP and ClogD values of 2-(4-(2-(dimethylamino)ethyl)-4H-1,2,4-triazol-3-yl)pyridine derivatives.
Compd.
R
IC50
(
mM)
ClogPa
ClogDb
MKN-45
H460
HT-29
A549
U87MG
WI-38
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0c
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
1r
Ph
4-Cl-Ph
3-Br-Ph
4-F-Ph
2,4-di-F-Ph
2-Cl-4-F-Ph
2,3-di-Cl-Ph
2,4-di-Cl-Ph
2,6-di-Cl-Ph
3-F-4-OH-Ph
3-Br-4-OH-Ph
3,5-di-Br-4-OH-Ph
2-NO
3-OH-Ph
4-OH-Ph
2,4-dieOHePh
3,4-dieOHePh
3-OH-4-OCH
4-OH-3-OCH
4-OH-3,5-dieCH
3,5-di-tert-butyl-2-OH-Ph
2,4-dieOCH
2,5-dieOCH
2.73 ꢂ 0.76
0.57 ꢂ 0.01
0.45 ꢂ 0.03
1.03 ꢂ 0.12
0.81 ꢂ 0.03
0.87 ꢂ 0.07
0.051 ꢂ 0.022
0.75 ꢂ 0.06
0.16 ꢂ 0.03
1.97 ꢂ 0.27
1.86 ꢂ 0.27
4.32 ꢂ 0.52
1.02 ꢂ 0.06
1.37 ꢂ 0.32
0.82 ꢂ 0.19
2.86 ꢂ 0.67
3.24 ꢂ 0.13
2.51 ꢂ 0.12
3.78 ꢂ 0.32
1.51 ꢂ 0.52
3.30 ꢂ 0.23
1.68 ꢂ 0.37
1.51 ꢂ 0.28
2.02 ꢂ 0.17
2.90 ꢂ 0.10
1.78 ꢂ 0.23
0.32 ꢂ 0.03
0.52 ꢂ 0.07
1.98 ꢂ 0.08
1.93 ꢂ 0.08
0.72 ꢂ 0.12
0.072 ꢂ 0.016
0.13 ꢂ 0.07
0.35 ꢂ 0.15
2.62 ꢂ 0.15
0.43 ꢂ 0.17
3.76 ꢂ 0.67
1.27 ꢂ 0.13
0.78 ꢂ 0.07
1.78 ꢂ 0.09
11.32 ꢂ 0.93
1.93 ꢂ 0.13
0.62 ꢂ 0.11
0.057 ꢂ 0.012
2.01 ꢂ 0.06
0.59 ꢂ 0.08
1.57 ꢂ 0.058
2.93 ꢂ 0.12
1.79 ꢂ 0.068
2.77 ꢂ 0.32
1.12 ꢂ 0.27
0.53 ꢂ 0.02
0.38 ꢂ 0.06
1.37 ꢂ 0.23
0.96 ꢂ 0.13
0.51 ꢂ 0.06
0.13 ꢂ 0.09
0.39 ꢂ 0.16
0.28 ꢂ 0.09
3.31 ꢂ 0.93
0.45 ꢂ 0.22
2.67 ꢂ 0.85
0.95 ꢂ 0.21
0.33 ꢂ 0.16
1.92 ꢂ 0.32
52.63 ꢂ 5.27
1.89 ꢂ 0.07
0.96 ꢂ 0.12
0.12 ꢂ 0.02
1.22 ꢂ 0.13
0.68 ꢂ 0.15
1.78 ꢂ 0.36
2.07 ꢂ 0.079
3.97 ꢂ 0.72
3.62 ꢂ 0.32
1.86 ꢂ 0.86
0.78 ꢂ 0.15
0.97 ꢂ 0.11
1.79 ꢂ 0.31
1.87 ꢂ 0.17
1.12 ꢂ 0.04
1.09 ꢂ 0.12
0.81 ꢂ 0.15
1.41 ꢂ 0.13
13.37 ꢂ 1.26
1.37 ꢂ 0.11
NA
2.25 ꢂ 0.75
1.37 ꢂ 0.07
0.83 ꢂ 0.13
2.31 ꢂ 0.09
1.90 ꢂ 0.37
1.21 ꢂ 0.12
13.23 ꢂ 1.67
2.33 ꢂ 0.98
NA
ND
3.02
3.62
3.78
3.16
3.30
3.76
4.22
4.22
4.22
1.75
2.25
2.93
2.96
2.20
2.17
1.60
1.81
2.16
2.16
3.38
5.41
2.70
2.70
2.54
2.54
1.41
2.01
2.18
1.56
1.69
2.15
2.62
2.62
2.62
1.32
1.97
2.93
1.35
1.11
1.11
0.82
0.81
0.95
0.95
2.13
4.20
1.09
1.09
0.93
0.93
1.87 ꢂ 0.63
4.23 ꢂ 0.32
3.98 ꢂ 1.17
5.03 ꢂ 0.45
2.09 ꢂ 0.33
6.13 ꢂ 0.21
4.76 ꢂ 0.55
3.32 ꢂ 0.27
ND
NA
12.38 ꢂ 0.69
5.56 ꢂ 0.72
NA
ND
3
-Ph
1.20 ꢂ 0.12
7.15 ꢂ 0.71
NA
7.75 ꢂ 0.85
15.43 ꢂ 1.26
NA
6.86 ꢂ 0.53
ND
ND
ND
ND
NA
3.56 ꢂ 0.81
12.71 ꢂ 2.70
NA
NA
NA
1.64 ꢂ 0.25
0.98 ꢂ 0.09
2.82 ꢂ 0.12
1.95 ꢂ 0.25
6.68 ꢂ 0.83
NA
3
-Ph
-Ph
NA
NA
ND
3
5.32 ꢂ 0.35
3.61 ꢂ 0.68
2.35 ꢂ 0.23
6.63 ꢂ 1.17
ND
1s
1t
1u
1v
1w
1x
3
ePh
7.71 ꢂ 0.68
0.52 ꢂ 0.07
12.63 ꢂ 0.52
6.40 ꢂ 0.73
7.65 ꢂ 0.82
22.47 ꢂ 3.32
3
ePh
ePh
3
2,3,4-trieOCH
3,4,5-trieOCH
3
ePh
ePh
ND
ND
3
1
2a
0.79 ꢂ 0.02
2.02 ꢂ 0.12
1.63 ꢂ 0.37
0.89 ꢂ 0.11
1.30 ꢂ 0.12
0.67 ꢂ 0.06
0.82 ꢂ 0.22
0.88 ꢂ 0.11
0.69 ꢂ 0.034
0.62 ꢂ 0.06
3.62 ꢂ 0.37
3.25 ꢂ 1.55
3.11
2.89
1.51
2.11
1
1
2b
2c
1.97 ꢂ 0.23
2.32 ꢂ 0.25
1.17 ꢂ 0.15
2.19 ꢂ 0.11
0.47 ꢂ 0.09
3.61 ꢂ 0.36
1.83 ꢂ 0.13
1.92 ꢂ 0.12
0.72 ꢂ 0.08
4.27 ꢂ 0.69
7.54 ꢂ 0.78
2.64
4.34
1.03
4.34
Sorafenib
15.57 ꢂ 1.78
NA: compound showing IC50 value > 100 mM.
ND: not determined.
a
ClogP values, calculated using JChem_For_Excel_6.0.0.865.
ClogD values, predicted at PH ¼ 7.4.
b
5.1.10.1. Phenyl 3-fluoro-4-(2-(1-methyl-1H-1,2,4-triazol-5-yl)pyr-
5.1.10.3. Phenyl 4-(2-(4-(2-(dimethylamino)ethyl)-4H-1,2,4-triazol-
idin-4-yloxy)phenylcarbamate (8a). A white solid; Yield: 59%; M.p.
3-yl)pyridin-4-yloxy)-3-fluorophenylcarbamate (8c). A white solid;
Yield: 83%; M.p. 121e123 C; ESI-MS m/z: 463.3 [MþH] .
ꢀ
1
ꢀ
þ
1
64e166 C; H NMR (300 MHz, DMSO-d
6
)
d
10.66 (s, 1H), 8.66 (d,
J ¼ 5.7 Hz, 1H), 8.00 (s, 1H), 7.68 (dd, J ¼ 12.8, 2.0 Hz, 1H), 7.51 (d,
J ¼ 2.5 Hz, 1H), 7.48 (d, J ¼ 4.2 Hz, 1H), 7.47e7.43 (m, 3H), 7.27 (m
5.1.11. General procedure for preparation of compounds (9ae9c)
3
H), 7.18 (dd, J ¼ 5.7, 2.6 Hz, 1H), 4.27 (s, 3H); ESI-MS m/z: 406.2
To a solution of 8ae8c (3 mmol) in dioxane, was added 80%
þ
[MþH] .
hydrazine hydrate (0.38 mL, 6 mmol). After 3 h of vigorous stirring
ꢀ
at 90 C, the mixture was cooled to room temperature. The pre-
cipitate was collected by filtration, and dried to provide compounds
5
.1.10.2. Phenyl 3-fluoro-4-(2-(4-methyl-4H-1,2,4-triazol-3-yl)pyr-
9ae9c.
idin-4-yloxy)phenylcarbamate (8b). A white solid; Yield: 77%; M.p.
ꢀ
1
198e200 C; H NMR (300 MHz, DMSO-d ) d 10.66 (s, 1H), 8.63 (m
6
2
H), 7.68 (d, J ¼ 12.5 Hz, 1H), 7.52 (m, 1H), 7.49e7.40 (m, 4H), 7.29
5.1.11.1. N-(3-Fluoro-4-(2-(1-methyl-1H-1,2,4-triazol-5-yl)pyridin-4-
(
[
m, 3H), 7.16 (dd, J ¼ 5.6, 2.6 Hz, 1H), 4.00 (s, 3H); ESI-MS m/z: 428.2
yloxy)phenyl)hydrazinecarboxamide (9a). A white solid; Yield: 71%;
þ
ꢀ
1
MþNa] .
M.p. 210e213 C; H NMR (300 MHz, DMSO-d ) d 9.05 (s, 1H), 8.58
6

54
M. Qin et al. / European Journal of Medicinal Chemistry 81 (2014) 47e58
Table 3
C
22 7 2
H18FN O (%): C, 61.25; H, 4.21; N, 22.73. Found (%): C, 61.27; H,
Enzymatic screening of compounds 11b, 11d, 11f, 11g, 11r, 11u and 12c in vitro.
4.19; N, 22.77.
Compd.
Enzyme inhibition (%)a,b
5
4
.1.12.3. (E)-2-benzylidene-N-(4-(2-(4-(2-(dimethylamino)ethyl)-
H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-fluorophenyl)hydrazine-
c-Met
VEGFR-2
B-Raf
Raf-1
PDGFRa
1
1
1
1
1
1
1
1b
1d
1f
1g
1r
1u
2c
7.8
1.3
2.2
3.1
10.2
6.1
5.8
e
1.1
21.7
12.8
9.2
33.2
19.6
7.3
72.3
77.2
82.6
67.5
56.6
42.7
70.5
95.7
e
66.1
59.7
71.1
73.7
52.3
37.9
61.6
97.3
e
13.2
16.6
5.9
11.2
3.7
22.3
12.3
e
carboxamide (10c). A light-yellow solid; Yield: 63%; M.p. 170e
ꢀ
ꢃ1
171 C; IR (KBr, cm ): 3437.7, 2919.3, 2850.5, 1699.9, 1593.9,
1
1508.3; H NMR (300 MHz, DMSO-d
8
2
6
)
d
11.26 (s, 1H) 9.55 (s, 1H),
.64 (s, 1H), 8.60 (d, J ¼ 5.8 Hz, 1H), 8.01 (s, 1H), 7.91 (dd, J ¼ 13.5,
.7 Hz, 1H), 7.85 (d, J ¼ 6.8 Hz, 2H), 7.62 (d, J ¼ 9.3 Hz, 1H), 7.55 (d,
J ¼ 2.4 Hz, 1H), 7.47e7.37 (m, 4H), 7.13 (dd, J ¼ 5.7, 2.1 Hz, 1H), 4.61
Sorafenib
Foretinib
e
13
97.5
99.6
98.7
(t, J ¼ 6.1 Hz, 2H), 2.57 (t, J ¼ 6.3 Hz, 2H), 2.13 (s, 6H); C NMR
(600 MHz, DMSO-d 165.34, 156.62, 155.01, 153.47, 152.58, 151.73,
150.62, 150.06, 147.47, 141.89, 139.35, 137.67, 134.87, 129.93, 129.08,
a
6
) d
Values were the average of two independent experiments, SD < 10%.
Compounds tested at a concentration of 10 mM.
b
1
27.51, 124.05, 116.84, 112.23, 109.23, 108.74, 59.25, 45.54 (2C),
þ
43.85; ESI-MS m/z: 489.2 [MþH] ; Anal. calcd. for C25
8 2
H25FN O (%):
(
7
(
d, J ¼ 5.7 Hz, 1H), 7.93 (s, 1H), 7.79 (d, J ¼ 13.6 Hz, 1H), 7.63 (s, 1H),
C, 61.47; H, 5.16; N, 22.94. Found (%): C, 61.41; H, 5.19; N, 23.03.
.42 (m, 2H), 7.26 (t, J ¼ 8.8 Hz,1H), 7.10 (dd, J ¼ 6.6, 3.0 Hz,1H), 4.39
þ
s, 2H), 4.21 (s, 3H); ESI-MS m/z: 366.1 [MþNa] .
5.1.12.4. (E)-2-(4-chlorobenzylidene)-N-(4-(2-(4-(2-(dimethyla-
mino)ethyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-fluorophenyl)
hydrazinecarboxamide (11a). A white solid; Yield: 47%; M.p. 135e
5
4
6
8
1
4
.1.11.2. N-(3-Fluoro-4-(2-(4-methyl-4H-1,2,4-triazol-3-yl)pyridin-
ꢀ
ꢃ1
-yloxy)phenyl)hydrazinecarboxamide (9b). A white solid; Yield:
137 C; IR (KBr, cm ): 3385.7, 2920.1, 2851.5, 1696.9, 1594.9,
ꢀ
1
1508.1; H NMR (300 MHz, DMSO-d )
1
6
d
11.00 (s, 1H), 9.28 (s, 1H),
8.64 (s, 1H), 8.61 (d, J ¼ 5.7 Hz, 1H), 7.97 (s, 1H), 7.94e7.88 (m, 3H),
.64 (d, J ¼ 8.5 Hz, 1H), 7.56 (d, J ¼ 2.2 Hz, 1H), 7.50 (d, J ¼ 8.4 Hz,
2H), 7.42 (t, J ¼ 9.1 Hz, 1H), 7.15 (dd, J ¼ 5.7, 2.5 Hz, 1H), 4.62 (t,
2%; M.p. 196e197 C; H NMR (300 MHz, DMSO-d
6
)
d
9.08 (s, 1H),
.61 (m, 2H), 7.85 (d, J ¼ 13.5 Hz, 1H), 7.65 (s, 1H), 7.52 (d, J ¼ 2.2 Hz,
7
H), 7.45 (s, 1H), 7.32 (t, J ¼ 9.0 Hz, 1H), 7.12 (dd, J ¼ 5.7, 2.5 Hz, 1H),
þ
.54 (s, 2H), 4.00 (s, 3H); ESI-MS m/z: 344.2 [MþH] .
13
J ¼ 6.0 Hz, 2H), 2.59 (t, J ¼ 6.2 Hz, 2H), 2.14 (s, 6H); C NMR
600 MHz, DMSO-d 165.29, 154.56, 153.41, 152.94, 151.71, 150.57,
50.02, 147.50, 140.52, 139.04, 134.53, 134.41, 133.70, 129.26 (2C),
(
6
) d
5.1.11.3. N-(4-(2-(4-(2-(dimethylamino)ethyl)-4H-1,2,4-triazol-3-yl)
1
pyridin-4-yloxy)-3-fluorophenyl)hydrazinecarboxamide
(9c). A
white solid; Yield: 81%; M.p. 207e210 C; H NMR (300 MHz,
DMSO-d
ꢀ
1
129.11 (2C), 124.04, 117.16, 112.28, 109.09, 59.15, 45.45 (2C), 43.73.
þ
ESI-MS m/z: 523.1 [MþH] ; Anal. calcd. for C25
8 2
H24ClFN O (%): C,
6
)
d
9.05 (s, 1H), 8.64 (s, 1H), 8.59 (d, J ¼ 5.7 Hz, 1H), 7.85 (d,
5
7.42; H, 4.63; N, 21.43. Found (%): C, 57.46; H, 4.57; N, 21.50.
J ¼ 13.6 Hz, 1H), 7.64 (s, 1H), 7.52 (d, J ¼ 2.5, 1H), 7.45 (s, 1H), 7.33 (t,
J ¼ 9.1 Hz, 1H), 7.12 (dd, J ¼ 2.6, 5.8 Hz, 1H), 4.62 (t, J ¼ 6.0 Hz, 2H),
5
.1.12.5. (E)-2-(3-Bromobenzylidene)-N-(4-(2-(4-(2-(dimethyla-
4
.52 (s, 2H) 2.59 (t, J ¼ 6.1 Hz, 2H), 2.14 (s, 6H); ESI-MS m/z: 423.2
þ
mino)ethyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-fluorophenyl)
hydrazinecarboxamide (11b). A white solid; Yield: 52%; M.p. 161e
[MþNa] .
ꢀ
ꢃ1
1
63 C; IR (KBr, cm ): 3384.1, 2919.7, 2851.0, 1695.2, 1593.7, 1507.7;
5.1.12. General procedure for the preparation of the target
1
H NMR (300 MHz, DMSO-d
6
) d 11.09 (s, 1H), 9.38 (s, 1H), 8.65 (s,
compounds (10ae10c, 11ae11x and 12ae12c)
1
H), 8.60 (d, J ¼ 6.0 Hz, 1H), 8.19 (s, 1H), 7.99e7.85 (m, 2H), 7.78 (d,
A mixture of 9ae9c (1 mmol), appropriate aromatic aldehyde
J ¼ 7.5 Hz, 1H), 7.62e7.56 (m, 3H), 7.41 (m, 2H), 7.14 (s, 1H), 4.63 (s,
(
1.2 mmol) and a catalytic amount of glacial acetic acid was refluxed
13
2
d
H), 2.60 (s, 2H), 2.15 (s, 6H); C NMR (600 MHz, DMSO-d
165.30, 154.97, 153.41, 152.54, 151.72, 150.61, 150.07, 147.47, 140.25,
39.04, 137.22, 134.68, 132.54, 131.18, 129.41, 127.05, 124.02, 122.71,
6
)
in isopropanol (5 mL) for 5e8 h until TLC showed the completion of
the reaction. After cooling to room temperature, the precipitate was
filtered, and purified by silica gel column chromatography to afford
compounds 10ae10c, 11ae11x and 12ae12c.
1
1
17.37, 112.27, 109.19, 59.25, 45.53 (2C), 43.85. ESI-MS m/z: 567.1
þ
[MþH] ; Anal. calcd. for C25
8 2
H24BrFN O (%): C, 52.92; H, 4.26; N,
19.75. Found (%): C, 53.01; H, 4.22; N, 19.81.
5
.1.12.1. (E)-2-benzylidene-N-(3-fluoro-4-(2-(1-methyl-1H-1,2,4-
triazol-5-yl)pyridin-4-yloxy)phenyl)hydrazinecarboxamide (10a).
A white solid; Yield: 57%; M.p. 152e155 C; IR (KBr, cm ): 3389.7,
5
3
.1.12.6. (E)-N-(4-(2-(4-(2-(Dimethylamino)ethyl)-4H-1,2,4-triazol-
ꢀ
ꢃ1
-yl)pyridin-4-yloxy)-3-fluorophenyl)-2-(4-fluorobenzylidene)hydra-
1
2
920.2, 2850.6, 1686.1, 1593.8, 1510.7, 1491.2; H NMR (300 MHz,
DMSO-d
10.94 (s, 1H), 9.24 (s, 1H), 8.66 (d, J ¼ 5.7 Hz,1H), 7.99 (s,
H), 7.87 (d, J ¼ 6.4 Hz, 3H), 7.65 (d, J ¼ 10.0 Hz, 1H), 7.53 (d,
J ¼ 2.4 Hz, 1H), 7.43 (m, 4H), 7.18 (dd, J ¼ 5.8, 2.5 Hz, 1H), 4.27 (s,
ꢀ
zinecarboxamide (11c). A white solid; Yield: 35%; M.p. 122e123 C;
6
)
d
1
6
H NMR (300 MHz, DMSO-d ) d 10.89 (s, 1H), 9.20 (s, 1H), 8.59 (s,
2
1
1
2
H), 8.56 (d, J ¼ 5.8 Hz, 1H), 7.94e7.83 (m, 4H), 7.59 (d, J ¼ 9.0 Hz,
H), 7.51 (d, J ¼ 2.4 Hz, 1H), 7.37 (t, J ¼ 9.0 Hz, 1H), 7.23 (t, J ¼ 8.8 Hz,
13
3
H); C NMR (600 MHz, DMSO-d
6
) d 165.43, 154.98, 153.49, 152.54,
H), 7.10 (dd, J ¼ 5.7, 2.4 Hz, 1H), 4.58 (t, J ¼ 6.1 Hz, 2H), 2.55 (t,
151.79, 151.10, 150.53, 149.89, 141.92, 139.13, 134.72, 134.55, 130.03,
13
J ¼ 6.1 Hz, 2H), 2.10 (s, 6H); C NMR (600 MHz, DMSO-d
6
) d 165.31,
129.06, 127.61, 124.01, 117.09, 112.44, 109.71, 109.04, 108.81, 25.95;
162.08, 154.98, 153.48, 152.56, 151.73, 150.60, 150.05, 147.49, 140.72,
þ
ESI-MS m/z: 432.3 [MþH] ; Anal. calcd. for C22
7 2
H18FN O (%): C,
1
39.12, 134.58, 131.39, 129.82, 129.74, 124.04, 117.10, 116.19, 115.97,
61.25; H, 4.21; N, 22.73. Found (%): C, 61.30; H, 4.27; N, 22.70.
þ
1
12.29, 109.15, 59.20, 45.50 (2C), 43.79; ESI-MS m/z: 507.3 [MþH] ;
Anal. calcd. for C25 (%): C, 59.28; H, 4.78; N, 22.12. Found
(%): C, 59.32; H, 4.81; N, 22.09.
24 2 8 2
H F N O
5
.1.12.2. (E)-2-Benzylidene-N-(3-fluoro-4-(2-(4-methyl-4H-1,2,4-
(10b).
A white solid; Yield: 62%; M.p. 147e149 C; 1H NMR (300 MHz,
DMSO-d 10.99 (s, 1H), 9.29 (s, 1H), 8.65 (s, 2H), 8.01 (s, 1H), 7.88
triazol-3-yl)pyridin-4-yloxy)phenyl)hydrazinecarboxamide
ꢀ
5.1.12.7. (E)-2-(2,4-difluorobenzylidene)-N-(4-(2-(4-(2-(dimethyla-
mino)ethyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-fluorophenyl)
hydrazinecarboxamide (11d). A white solid; Yield: 42%; M.p. 117e
6
) d
(
4
m, 3H), 7.67 (d, J ¼ 7.9 Hz, 1H), 7.57 (s, 1H), 7.43 (m, 4H), 7.16 (s, 1H),
þ
ꢀ
1
.02 (s, 3H); ESI-MS m/z: 432.2 [MþH] ; Anal. calcd. for
119 C; H NMR (300 MHz, DMSO-d ) d 11.02 (s, 1H), 9.25 (s, 1H),
6

M. Qin et al. / European Journal of Medicinal Chemistry 81 (2014) 47e58
55
Fig. 4. Docking model of compound 11f with wild-type B-Raf kinase (PDB code: 1UWH). Left: a representation of the overlap of docking model of 11f with sorafenib in protein
surface. Right: a representation of the kinase with selected residues in stick model. The inhibitors were colored by atom type (grey ¼ carbon/hydrogen, red ¼ oxygen,
blue ¼ nitrogen, green ¼ chlorine, cyan ¼ fluorine). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
8
8
.59 (s, 1H), 8.56 (d, J ¼ 5.8 Hz, 1H), 8.36 (dd, J ¼ 15.7, 8.8 Hz, 1H),
.10 (s, 1H), 7.84 (dd, J ¼ 13.2, 2.4 Hz, 1H), 7.58 (d, J ¼ 8.2 Hz, 1H),
1H), 7.91 (dd, J ¼ 13.3, 2.3 Hz, 1H), 7.69 (d, J ¼ 2.0 Hz, 1H), 7.65 (d,
J ¼ 8.7 Hz, 1H), 7.57 (d, J ¼ 2.5 Hz, 1H), 7.52 (dd, J ¼ 8.6, 1.8 Hz, 1H),
7.43 (t, J ¼ 9.0 Hz,1H), 7.16 (dd, J ¼ 5.7, 2.6 Hz,1H), 4.64 (t, J ¼ 6.2 Hz,
7
.51 (d, J ¼ 2.5 Hz, 1H), 7.37 (m, 1H), 7.32e7.25 (m, 1H), 7.17 (m, 1H),
13
7
.10 (dd, J ¼ 5.7, 2.5 Hz,1H), 4.58 (t, J ¼ 6.2 Hz, 2H), 2.55 (t, J ¼ 6.0 Hz,
H), 2.11 (s, 6H); 13C NMR (600 MHz, DMSO-d
165.30, 154.99,
53.32, 152.56, 151.69, 150.60, 150.05, 147.46, 139.01, 134.70, 133.48,
28.94, 124.04, 119.15, 117.18, 112.85, 112.64, 112.28, 109.16, 108.92,
2H), 2.63 (t, J ¼ 6.1 Hz, 2H), 2.17 (s, 6H); C NMR (600 MHz, DMSO-
2
1
1
6
)
d
6
d ) d 165.27, 154.55, 153.20, 152.93, 151.72, 150.56, 149.99, 147.51,
139.00, 136.64, 134.95, 134.62, 133.75, 131.05, 129.61, 129.21, 128.08,
124.06, 117.24, 112.28, 109.10, 59.07, 45.38 (2C), 43.68; ESI-MS m/z:
þ
þ
1
04.64, 59.16, 45.43 (2C), 43.76. ESI-MS m/z: 525.2 [MþH] ; Anal.
557.3 [MþH] ; Anal. calcd. for C25
2 8 2
H23Cl FN O (%): C, 53.87; H, 4.16;
calcd. for C25 (%): C, 57.25; H, 4.42; N, 21.36. Found (%): C,
H F
23 3
N
8
O
2
N, 20.10. Found (%): C, 53.92; H, 4.11; N, 20.16.
57.17; H, 4.48; N, 21.40.
5
.1.12.11. (E)-2-(2,6-dichlorobenzylidene)-N-(4-(2-(4-(2-(dimethyla-
5
.1.12.8. (E)-2-(2-Chloro-4-fluorobenzylidene)-N-(4-(2-(4-(2-(dime-
mino)ethyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-fluorophenyl)
hydrazinecarboxamide (11h). A white solid; Yield: 47%; M.p. 138e
thylamino)ethyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-
fluorophenyl)hydrazinecarboxamide (11e). A white solid; Yield: 51%;
M.p. 150e152 C; IR (KBr, cm ): 3392.5, 2919.3, 2850.8, 1695.6,
ꢀ
1
140 C; H NMR (300 MHz, DMSO-d ) d 11.33 (s, 1H), 9.18 (s, 1H),
6
ꢀ
1
ꢃ1
8
1
2
.66 (s, 1H), 8.60 (d, J ¼ 5.1 Hz, 1H), 8.20 (s, 1H), 7.88 (d, J ¼ 13.5 Hz,
1595.9, 1507.7; H NMR (300 MHz, DMSO-d
6
)
d
11.10 (s, 1H), 9.28 (s,
H), 8.59 (s, 1H), 8.56 (d, J ¼ 5.7 Hz, 1H), 8.46e8.38 (m, 1H), 8.28 (s,
H), 7.84 (dd, J ¼ 13.4, 1.8 Hz, 1H), 7.58 (d, J ¼ 8.5 Hz, 1H), 7.53e7.45
H), 7.58 (m, 4H), 7.48e7.36 (m, 2H), 7.13 (s, 1H), 4.63 (t, J ¼ 6.1 Hz,
1
1
þ
H), 2.61 (t, J ¼ 6.2 Hz, 2H), 2.16 (s, 6H); ESI-MS m/z: 557.2 [MþH] ;
2 8 2
Anal. calcd. for C25H23Cl FN O (%): C, 53.87; H, 4.16; N, 20.10.
Found (%): C, 53.90; H, 4.19; N, 20.02.
(
2
d
1
m, 2H), 7.33 (m, 2H), 7.10 (dd, J ¼ 5.7, 3.1 Hz, 1H), 4.58 (t, J ¼ 6.0 Hz,
13
H), 2.57 (t, J ¼ 6.1 Hz, 2H), 2.11 (s, 6H); C NMR (600 MHz, DMSO-
165.28, 163.66, 162.00, 154.56, 153.29, 152.94, 151.71, 150.56,
50.01, 147.50, 138.95, 136.75, 134.69, 133.83, 129.79, 128.75, 124.06,
6
) d
5.1.12.12. (E)-N-(4-(2-(4-(2-(Dimethylamino)ethyl)-4H-1,2,4-triazol-
3-yl)pyridin-4-yloxy)-3-fluorophenyl)-2-(3-fluoro-4-
hydroxybenzylidene)hydrazinecarboxamide (11i). A white solid;
117.24, 115.62, 112.29, 109.08, 59.12, 45.42 (2C), 43.71; ESI-MS m/z:
þ
5
41.1 [MþH] ; Anal. calcd. for C25
2 8 2
H23ClF N O (%): C, 55.51; H, 4.29;
ꢀ
ꢃ1
N, 20.71. Found (%): C, 55.45; H, 4.33; N, 20.65.
Yield: 45%; M.p. 125e128 C; IR (KBr, cm ): 3368.9, 2919.9, 2850.7,
1
1
1
7
1
685.9, 1593.9, 1510.3; H NMR (300 MHz, DMSO-d
0.28 (s, 1H), 9.19 (s, 1H), 8.65 (s, 1H), 8.61 (d, J ¼ 5.4 Hz, 1H), 7.94e
.84 (m, 3H), 7.65 (d, J ¼ 8.5 Hz, 1H), 7.56 (s, 1H), 7.41 (t, J ¼ 9.2 Hz,
H), 7.35 (d, J ¼ 8.1 Hz, 1H), 7.15 (s, 1H), 6.98 (t, J ¼ 8.6 Hz, 1H), 4.65
6
) d 10.81 (s, 1H),
5
.1.12.9. (E)-2-(2,3-Dichlorobenzylidene)-N-(4-(2-(4-(2-(dimethyla-
mino)ethyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-fluorophenyl)
hydrazinecarboxamide (11f). A white solid; Yield: 42%; M.p. 136e
13
ꢀ
ꢃ1
(s, 2H), 2.69 (s, 2H), 2.23 (s, 6H); C NMR (600 MHz, DMSO-d
165.32, 154.98, 153.45, 153.00, 151.73, 150.60, 150.05, 147.50, 141.14,
39.19, 134.53, 134.41, 126.57, 125.20, 124.00, 118.03, 117.04, 114.24,
6
)
142 C; IR (KBr, cm ): 3442.8, 2920.8, 2851.5, 1701.7, 1596.1,
1
6
1508.3; H NMR (600 MHz, DMSO-d ) d 11.33 (s, 1H), 9.42 (s, 1H),
1
8
1
1
2
d
.67 (s, 1H), 8.62 (d, J ¼ 5.3 Hz, 1H), 8.43 (s, 1H), 8.39 (d, J ¼ 7.2 Hz,
H), 7.91 (d, J ¼ 12.8 Hz, 1H), 7.67 (dd, J ¼ 27.0, 7.5 Hz, 2H), 7.56 (s,
H), 7.49e7.40 (m, 2H), 7.16 (d, J ¼ 2.8 Hz, 1H), 4.64 (t, J ¼ 6.2 Hz,
114.04, 112.23, 109.21, 59.23, 45.52 (2C), 43.83; ESI-MS m/z: 523.1
þ
[
MþH] ; Anal. calcd. for C25
24 2 8 3
H F N O (%): C, 57.47; H, 4.63; N,
13
21.45. Found (%): C, 57.38; H, 4.55; N, 21.57.
H), 2.63 (t, J ¼ 6.3 Hz, 2H), 2.17 (s, 6H); C NMR (600 MHz, DMSO-
6
)
d
165.27, 154.56, 153.19, 152.93, 151.73, 150.56, 149.99, 147.51,
1
38.97, 137.44, 134.69, 134.39, 132.58, 131.47, 130.91, 128.61, 126.57,
5
(
.1.12.13. (E)-2-(3-Bromo-4-hydroxybenzylidene)-N-(4-(2-(4-(2-
dimethylamino)ethyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-
fluorophenyl)hydrazinecarboxamide (11j). A white solid; Yield: 37%;
124.07, 117.26, 112.29, 109.09, 59.05, 45.37 (2C), 43.66; ESI-MS m/z:
þ
5
57.3 [MþH] ; Anal. calcd. for C25
2 8 2
H23Cl FN O (%): C, 53.87; H, 4.16;
N, 20.10. Found (%): C, 53.91; H, 4.13; N, 20.16.
ꢀ
1
6
M.p. 111e113 C; H NMR (300 MHz, DMSO-d ) d 10.76 (s, 1H), 10.66
(
s, 1H), 9.22 (s, 1H), 8.69 (s, 1H), 8.62 (d, J ¼ 5.7 Hz, 1H), 8.08 (m, 1H),
5.1.12.10. (E)-2-(2,4-dichlorobenzylidene)-N-(4-(2-(4-(2-(dimethyla-
7.90 (m, 2H), 7.67e7.57 (m, 3H), 7.39 (t, J ¼ 9.0 Hz, 1H), 7.15 (dd,
mino)ethyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-fluorophenyl)
hydrazinecarboxamide (11g). A white solid; Yield: 32%; M.p. 177e
J ¼ 5.7, 2.4 Hz, 1H), 7.02 (d, J ¼ 8.4 Hz, 1H), 4.73 (t, J ¼ 6.1 Hz, 2H),
1
3
2.99 (t, J ¼ 6.0 Hz, 2H), 2.43 (s, 6H); C NMR (600 MHz, DMSO-d
6
)
ꢀ
ꢃ1
1
1
80 C; IR (KBr, cm ): 3443.5, 2918.7, 2850.4, 1688.0, 1594.3,
d 165.42, 155.80, 153.49, 152.54, 151.79, 150.58, 149.74, 147.45,
1
508.6; H NMR (600 MHz, DMSO-d
6
)
d
11.29 (s, 1H), 9.43 (s, 1H),
140.74, 139.27, 134.48, 131.51, 128.76, 127.48, 123.99, 117.18, 116.65,
8
.67 (s, 1H), 8.62 (d, J ¼ 5.7 Hz, 1H), 8.44 (d, J ¼ 8.6 Hz, 1H), 8.36 (s,
112.38, 110.47, 109.06, 108.83, 57.96, 44.38 (2C), 42.56; ESI-MS m/z:

56
M. Qin et al. / European Journal of Medicinal Chemistry 81 (2014) 47e58
þ
5
83.3 [MþH] ; Anal. calcd. for C25
H
24BrFN
8
O
3
(%): C, 51.47; H, 4.15;
129.41, 124.10, 116.19, 112.15, 109.28, 107.95, 102.96, 59.24, 45.53
þ
N, 19.21. Found (%): C, 51.39; H, 4.21; N, 19.27.
(2C), 43.82; ESI-MS m/z: 521.4 [MþH] ; Anal. calcd. for
25 8 4
C H25FN O (%): C, 57.69; H, 4.84; N, 21.53. Found (%): C, 57.62; H,
5
(
.1.12.14. (E)-2-(3,5-Dibromo-4-hydroxybenzylidene)-N-(4-(2-(4-(2-
dimethylamino)ethyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-
fluorophenyl)hydrazinecarboxamide (11k). A white solid; Yield:
4.90; N, 21.57.
5
.1.12.19. (E)-2-(3,4-Dihydroxybenzylidene)-N-(4-(2-(4-(2-(dime-
ꢀ
1
4
1%; M.p. 131e132 C; H NMR (300 MHz, DMSO-d
.33 (s, 1H), 8.68 (s, 1H), 8.62 (d, J ¼ 5.5 Hz, 1H), 8.08 (s, 2H), 7.91 (d,
J ¼ 13.2 Hz,1H), 7.84 (s, 1H), 7.64 (m, 3H), 7.41 (t, J ¼ 8.8 Hz,1H), 7.16
s, 1H), 4.71 (t, J ¼ 6.6 Hz, 2H), 2.91 (t, J ¼ 6.6 Hz, 2H), 2.38 (s, 6H);
6
) d 10.92 (s, 1H),
thylamino)ethyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-
fluorophenyl)hydrazinecarboxamide (11p). A white solid; Yield:
9
ꢀ
1
54%; M.p. 135e137 C; H NMR (300 MHz, DMSO-d
6
) d 10.57 (s, 1H),
(
9
.36 (s, 1H), 9.10 (s, 1H), 8.59 (s, 1H), 8.55 (d, J ¼ 5.7 Hz, 2H), 7.91e
1
3
6
C NMR (600 MHz, DMSO-d ) d 165.33, 154.95, 154.62, 153.47,
7
.74 (m, 2H), 7.61e7.49 (m, 2H), 7.35 (t, J ¼ 9.0 Hz, 1H), 7.24 (s, 1H),
152.52, 151.73, 150.60, 149.98, 147.47, 139.87, 139.25, 134.50, 131.12
7.05 (m, 2H), 6.74 (d, J ¼ 7.9 Hz, 1H), 4.57 (t, J ¼ 6.7 Hz, 2H), 2.53 (t,
(
(
2C), 126.49, 123.97, 117.30, 113.30 (2C), 112.25, 109.18, 58.98, 45.31
2C), 43.59; ESI-MS m/z: 661.1 [MþH] ; Anal. calcd. for
13
J ¼ 6.9 Hz, 2H), 2.09 (s, 6H); C NMR (600 MHz, DMSO-d
55.01, 153.49, 152.58, 151.72, 150.61, 150.04, 147.92, 147.48, 146.03,
42.70, 139.39, 134.31, 126.22, 124.03, 120.23, 116.69, 116.02, 114.29,
6
) d 165.33,
þ
1
1
25 2 8 3
C H23Br FN O (%): C, 45.34; H, 3.50; N, 16.92. Found (%): C, 45.39;
H, 3.61; N, 16.86.
þ
1
12.22, 109.21, 59.23, 45.52 (2C), 43.83; ESI-MS m/z: 521.3 [MþH] ;
Anal. calcd. for C25 (%): C, 57.69; H, 4.84; N, 21.53. Found
%): C, 57.65; H, 4.78; N, 21.59.
H
8 4
25FN O
5
.1.12.15. (E)-2-((2-(4-(2-(4-(2-(dimethylamino)ethyl)-4H-1,2,4-
(
triazol-3-yl)pyridin-4-yloxy)-3-fluorophenylcarbamoyl)hydrazono)
methyl)phenyl nitrate (11l). A yellow solid; Yield: 42%; M.p. 125e
ꢀ
1
127 C; H NMR (300 MHz, DMSO-d
6
)
d
11.29 (s, 1H), 9.35 (s, 1H),
.65 (s, 1H), 8.61 (d, J ¼ 5.6 Hz, 1H), 8.49 (d, J ¼ 7.8 Hz, 1H), 8.42 (s,
H), 8.06 (d, J ¼ 8.3 Hz, 1H), 7.90 (d, J ¼ 12.9 Hz, 1H), 7.79 (m, 1H),
5.1.12.20. (E)-N-(4-(2-(4-(2-(Dimethylamino)ethyl)-4H-1,2,4-
triazol-3-yl)pyridin-4-yloxy)-3-fluorophenyl)-2-(3-hydroxy-4-
methoxybenzylidene)hydrazinecarboxamide (11q). A white solid;
8
1
ꢀ
1
7
3
.63 (m, 2H), 7.57 (s, 1H), 7.42 (t, J ¼ 8.8 Hz, 1H), 7.14 (dd, J ¼ 6.0,
Yield: 39%; M.p. 122e123 C; H NMR (300 MHz, DMSO-d ) d 10.69
6
.5 Hz, 1H), 4.64 (t, J ¼ 5.9 Hz, 2H), 2.63 (t, J ¼ 6.0 Hz, 2H), 2.17 (s,
(s,1H), 9.17 (s,1H), 9.06 (s,1H), 8.62 (m, 2H), 7.96e7.82 (m, 2H), 7.63
(d, J ¼ 8.1 Hz, 1H), 7.56 (s, 1H), 7.38 (m, 2H), 7.14 (m, 2H), 6.96 (d,
J ¼ 8.2 Hz,1H), 4.63 (t, J ¼ 6.8 Hz, 2H), 3.82 (s, 3H), 2.64 (t, J ¼ 6.7 Hz,
6
H); Anal. calcd. for C25 (%): C, 56.28; H, 4.53; N, 23.63.
9 4
H24FN O
Found (%): C, 56.17; H, 4.60; N, 23.71.
13
6
2H), 2.18 (s, 6H); C NMR (600 MHz, DMSO-d ) d 165.31, 154.59,
5
3
.1.12.16. (E)-N-(4-(2-(4-(2-(dimethylamino)ethyl)-4H-1,2,4-triazol-
-yl)pyridin-4-yloxy)-3-fluorophenyl)-2-(3-hydroxybenzylidene)
153.43, 152.97, 151.73, 150.58, 149.99, 149.70, 147.51, 147.13, 142.30,
139.37, 134.27, 127.66, 124.06, 120.16, 116.71, 113.58, 112.22, 112.11,
þ
hydrazinecarboxamide (11m). A white solid; Yield: 51%; M.p. 129e
109.16, 59.14, 56.05, 45.45 (2C), 43.74; ESI-MS m/z: 535.3 [MþH] ;
ꢀ
1
131 C; H NMR (300 MHz, DMSO-d
6
)
d
10.84 (s, 1H), 9.68 (s, 1H),
8 4
Anal. calcd. for C26H27FN O (%): C, 58.42; H, 5.09; N, 20.96. Found
9
.24 (s,1H), 8.65 (s,1H), 8.62 (d, J ¼ 5.6 Hz,1H), 7.90 (m, 2H), 7.63 (d,
(%): C, 58.36; H, 5.15; N, 20.89.
J ¼ 8.6 Hz, 1H), 7.56 (s, 1H), 7.41 (t, J ¼ 8.9 Hz, 1H), 7.26 (m, 3H), 7.16
(
2
d
d, J ¼ 3.1 Hz, 1H), 6.83 (d, J ¼ 5.9 Hz, 1H), 4.64 (t, J ¼ 6.6 Hz, 2H),
5
3
.1.12.21. (E)-N-(4-(2-(4-(2-(dimethylamino)ethyl)-4H-1,2,4-triazol-
-yl)pyridin-4-yloxy)-3-fluorophenyl)-2-(4-hydroxy-3-
1
3
.66 (t, J ¼ 6.6 Hz, 2H), 2.19 (s, 6H); C NMR (600 MHz, DMSO-d
6
)
165.31,158.16,155.00,153.40,152.57,151.73,150.61,150.04,147.49,
methoxybenzylidene)hydrazinecarboxamide (11r). A white solid;
142.24, 139.32, 136.00, 134.40, 130.00, 124.05, 118.64, 117.25, 116.82,
ꢀ
ꢃ1
Yield: 47%; M.p. 110e112 C; IR (KBr, cm ): 3439.0, 2919.5, 2850.4,
1
113.89, 112.22, 109.21, 59.23, 45.52 (2C), 43.82; ESI-MS m/z: 505.1
1
6
688.1, 1595.0, 1508.5; H NMR (300 MHz, DMSO-d ) d 10.80 (s, 1H),
þ
[
2
MþH] ; Anal. calcd. for C25
8 3
H25FN O (%): C, 59.52; H, 4.99; N,
9
.55 (s, 1H), 9.27 (s, 1H), 8.64 (s, 1H), 8.58 (d, J ¼ 4.9 Hz, 1H), 7.90 (d,
2.21. Found (%): C, 59.59; H, 4.93; N, 22.17.
J ¼ 13.3 Hz, 1H), 7.87 (s, 1H), 7.60 (d, J ¼ 7.0 Hz, 1H), 7.50 (d,
J ¼ 31.2 Hz, 2H), 7.39 (t, J ¼ 8.5 Hz, 1H), 7.12 (s, 2H), 6.83 (d,
J ¼ 7.7 Hz, 1H), 4.60 (t, J ¼ 6.1 Hz, 2H), 3.84 (s, 3H), 2.57 (t, J ¼ 6.3 Hz,
5
3
.1.12.17. (E)-N-(4-(2-(4-(2-(Dimethylamino)ethyl)-4H-1,2,4-triazol-
-yl)pyridin-4-yloxy)-3-fluorophenyl)-2-(4-hydroxybenzylidene)
13
6
2H), 2.12 (s, 6H); C NMR (600 MHz, DMSO-d ) d 165.32, 154.59,
hydrazinecarboxamide (11n). A white solid; Yield: 33%; M.p. 151e
153.49, 152.97, 151.72, 150.58, 150.00, 149.00, 148.40, 147.51, 142.45,
ꢀ
1
152 C; H NMR (300 MHz, DMSO-d
6
) d 10.68 (s, 1H), 9.83 (s, 1H),
139.29, 134.29, 126.14, 124.07, 122.03, 116.90, 115.78, 112.24, 110.32,
9
.13 (s, 1H), 8.68e8.54 (m, 2H), 7.91 (m, 2H), 7.73e7.61 (m, 3H), 7.56
þ
1
09.10, 59.14, 56.20, 45.45 (2C), 43.74; ESI-MS m/z: 535.3 [MþH] ;
Anal. calcd. for C26 (%): C, 58.42; H, 5.09; N, 20.96. Found
%): C, 58.36; H, 5.13; N, 20.92.
(
4
(
1
s, 1H), 7.39 (t, J ¼ 9.0 Hz, 1H), 7.14 (s, 1H), 6.82 (d, J ¼ 7.9 Hz, 2H),
8 4
H27FN O
13
.62 (t, J ¼ 6.0 Hz, 2H), 2.59 (t, J ¼ 5.9 Hz, 2H), 2.14 (s, 6H); C NMR
165.33,159.43,154.99,153.52,152.56,151.71,
50.60, 150.05, 147.49, 142.18, 139.29, 134.38, 129.31, 125.78, 124.02,
(
6
600 MHz, DMSO-d ) d
1
4
16.86, 115.93, 112.26, 109.17, 108.79, 108.57, 59.24, 45.53 (2C),
5.1.12.22. (E)-N-(4-(2-(4-(2-(Dimethylamino)ethyl)-4H-1,2,4-
triazol-3-yl)pyridin-4-yloxy)-3-fluorophenyl)-2-(4-hydroxy-3,5-
dimethylbenzylidene)hydrazinecarboxamide (11s). A white solid;
þ
3.83; ESI-MS m/z: 505.2 [MþH] ; Anal. calcd. for C25
8 3
H25FN O (%):
C, 59.52; H, 4.99; N, 22.21. Found (%): C, 59.59; H, 5.07; N, 22.24.
ꢀ
ꢃ1
Yield: 42%; M.p. 134e137 C; IR (KBr, cm ): 3380.3, 2919.0, 2850.6,
1
5
.1.12.18. (E)-2-(2,4-Dihydroxybenzylidene)-N-(4-(2-(4-(2-(dime-
6
1688.6, 1595.1; H NMR (300 MHz, DMSO-d ) d 10.66 (s, 1H), 9.12 (s,
thylamino)ethyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-
fluorophenyl)hydrazinecarboxamide (11o). A white solid; Yield:
1H), 8.63 (m, 2H), 8.60 (d, J ¼ 5.9 Hz, 1H), 7.92 (d, J ¼ 13.6 Hz, 1H),
7.83 (s, 1H), 7.65 (d, J ¼ 8.4 Hz, 1H), 7.56 (s, 1H), 7.44e7.36 (m, 3H),
7.14 (dd, J ¼ 6.0, 3.1 Hz,1H), 4.62 (t, J ¼ 6.3 Hz, 2H), 2.59 (t, J ¼ 6.2 Hz,
ꢀ
1
5
9
1
(
2
2%; M.p. 146e147 C; H NMR (300 MHz, DMSO-d ) d 10.54 (s, 1H),
6
13
.97 (s, 1H), 9.69 (s, 1H), 9.11 (s, 1H), 8.59 (s, 1H), 8.55 (d, J ¼ 5.6 Hz,
H), 8.13 (s, 1H), 7.84 (d, J ¼ 13.4 Hz, 1H), 7.71 (d, J ¼ 8.1 Hz, 1H), 7.52
6
2H), 2.21 (s, 6H), 2.14 (s, 6H); C NMR (600 MHz, DMSO-d )
d
165.33, 155.45, 154.58, 153.47, 152.96, 151.73, 150.58, 149.98,
m, 2H), 7.34 (t, J ¼ 9.0 Hz, 1H), 7.08 (dd, J ¼ 6.0, 3.0 Hz, 1H), 6.27 (m,
147.51, 142.60, 139.37, 134.36, 127.88 (2C), 125.62, 124.88 (2C),
13
H), 4.57 (t, J ¼ 6.0 Hz, 2H), 2.53 (t, J ¼ 6.2 Hz, 2H), 2.08 (s, 6H);
165.36, 160.62, 158.60, 156.14, 155.07,
53.29, 152.64, 151.73, 150.62, 150.03, 147.49, 141.58, 139.80, 134.01,
C
124.02, 116.89, 112.23, 109.13, 59.05, 45.37 (2C), 43.66, 17.08 (2C);
þ
NMR (600 MHz, DMSO-d
6
)
d
ESI-MS m/z: 533.3 [MþH] ; Anal. calcd. for C27
8 3
H29FN O (%): C,
1
60.89; H, 5.49; N, 21.04. Found (%): C, 60.82; H, 5.43; N, 21.09.

M. Qin et al. / European Journal of Medicinal Chemistry 81 (2014) 47e58
57
5
.1.12.23. (E)-2-(3,5-Di-tert-butyl-2-hydroxybenzylidene)-N-(4-(2-
Anal. calcd. for C28H31FN
8
O
5
(%): C, 58.12; H, 5.40; N, 19.37. Found
(
4-(2-(dimethylamino)ethyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-
(%): C, 58.16; H, 5.43; N, 19.40.
3
4
-fluorophenyl)hydrazinecarboxamide (11t). A white solid; Yield:
ꢀ
1
5%. M.p. 113e116 C; H NMR (300 MHz, DMSO-d
6
)
d
11.23 (s, 1H)
5.1.12.28. (E)-2-((1H-Indol-3-yl)methylene)-N-(4-(2-(4-(2-(dime-
thylamino)ethyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-
fluorophenyl)hydrazinecarboxamide (12a). A white solid; Yield:
1
1
0.63 (s, 1H), 9.43 (s,1H), 8.63 (s,1H), 8.60 (d, J ¼ 5.7 Hz,1H), 8.25 (s,
H), 7.79 (d, J ¼ 13.0 Hz, 1H), 7.56 (s, 1H), 7.40 (m, 2H), 7.28 (s, 1H),
ꢀ
1
7
.22 (s, 1H), 7.13 (dd, J ¼ 5.7, 2.4 Hz, 1H), 4.62 (t, J ¼ 6.0 Hz, 2H), 2.61
35%; M.p. 136e138 C; H NMR (300 MHz, DMSO-d ) d 11.58 (s, 1H),
6
(
6
t, J ¼ 6.2 Hz, 2H), 2.16 (s, 6H), 1.42 (s, 9H), 1.29 (s, 9H); ESI-MS m/z:
10.51 (s, 1H), 8.95 (s, 1H), 8.57 (m, 2H), 8.23 (m, 2H), 7.91e7.75 (m,
2H), 7.52 (s, 2H), 7.37 (m, 2H), 7.12 (m, 3H), 4.57 (t, J ¼ 7.5 Hz, 2H),
þ
17.3 [MþH] ; Anal. calcd. for C33
8 3
H41FN O (%): C, 64.27; H, 6.70; N,
þ
18.17. Found (%): C, 64.31; H, 6.77; N, 18.13.
2.53 (t, J ¼ 7.3 Hz, 2H), 2.08 (s, 6H); ESI-MS m/z: 528.2 [MþH] ;
9 2
Anal. calcd. for C27H26FN O (%): C, 61.47; H, 4.97; N, 23.90. Found
5
.1.12.24. (E)-2-(2,4-dimethoxybenzylidene)-N-(4-(2-(4-(2-(dime-
(%): C, 61.09; H, 5.35; N, 23.37.
thylamino)ethyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-
fluorophenyl)hydrazinecarboxamide (11u). A white solid; Yield:
5.1.12.29. (E)-N-(4-(2-(4-(2-(dimethylamino)ethyl)-4H-1,2,4-triazol-
3-yl)pyridin-4-yloxy)-3-fluorophenyl)-2-((2-hydroxynaphthalen-1-
yl)methylene)hydrazinecarboxamide (12b). A yellow solid; Yield:
ꢀ
1
5
9
1%; M.p. 132e134 C; H NMR (300 MHz, DMSO-d
.13 (s, 1H), 8.63 (s, 1H), 8.60 (d, J ¼ 5.7 Hz, 1H), 8.25 (s, 1H), 8.12 (d,
J ¼ 8.3 Hz, 1H), 7.91 (dd, J ¼ 13.5, 2.3 Hz, 1H), 7.64 (d, J ¼ 9.0 Hz, 1H),
6
) d 10.70 (s, 1H),
ꢀ
1
6
56%; M.p. 169e171 C; H NMR (300 MHz, DMSO-d ) d 11.38 (s, 1H),
7
.57 (d, J ¼ 2.3 Hz,1H), 7.39 (t, J ¼ 9.1 Hz,1H), 7.13 (dd, J ¼ 5.7, 2.5 Hz,
10.74 (s, 1H), 9.41 (s, 1H), 8.95 (s, 1H), 8.59 (s, 1H), 8.55 (d, J ¼ 5.7 Hz,
1H), 8.39 (d, J ¼ 7.5 Hz, 1H), 7.80 (m, 3H), 7.53 (m, 2H), 7.45 (d,
J ¼ 10.4 Hz, 1H), 7.35 (m, 2H), 7.18 (d, J ¼ 8.8 Hz, 1H), 7.09 (s, 1H),
1
2
d
H), 6.61 (m, 2H), 4.62 (t, J ¼ 6.3 Hz, 2H), 3.86 (s, 3H), 3.83 (s, 3H),
13
.59 (t, J ¼ 6.3 Hz, 2H), 2.14 (s, 6H); C NMR (600 MHz, DMSO-d
165.33, 162.49, 159.23, 153.50, 152.56, 151.69, 150.61, 150.05,
47.47, 139.30, 137.50, 134.40, 127.84, 123.98, 116.89, 115.55, 112.24,
6
)
4.57 (t, J ¼ 6.0 Hz, 2H), 2.53 (t, J ¼ 6.1 Hz, 2H), 2.09 (s, 6H); ESI-MS
þ
1
m/z: 555.3 [MþH] ; Anal. calcd. for C29
8 3
H27FN O (%): C, 62.81; H,
1
09.19, 108.70, 106.72, 98.40, 59.22, 56.18, 55.86, 45.49 (2C), 43.81;
4.91; N, 20.21. Found (%): C, 62.96; H, 4.82; N, 20.33.
þ
ESI-MS m/z: 549.1 [MþH] ; Anal. calcd. for C27
8 4
H29FN O (%): C,
5
9.12; H, 5.33; N, 20.43. Found (%): C, 59.18; H, 5.37; N, 20.39.
5.1.12.30. (E)-2-(Benzo[d][1,3]dioxol-4-ylmethylene)-N-(4-(2-(4-(2-
(
dimethylamino)ethyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-
5
.1.12.25. (E)-2-(2,5-dimethoxybenzylidene)-N-(4-(2-(4-(2-(dime-
fluorophenyl)hydrazinecarboxamide (12c). A white solid; Yield:
ꢀ
ꢃ1
thylamino)ethyl)-4H-1,2,4-triazol-3-yl)pyridin-4-yloxy)-3-
fluorophenyl)hydrazinecarboxamide (11v). A white solid; Yield:
40%; M.p. 143e145 C; IR (KBr, cm ): 3428.7, 2918.8, 2850.6,
1
6
1691.3, 1594.9; H NMR (300 MHz, DMSO-d ) d 10.75 (s, 1H), 9.13 (s,
ꢀ
1
4
9
7
(
2
1
1%; M.p. 114e117 C; H NMR (300 MHz, DMSO-d
.28 (s, 1H), 8.67e8.56 (m, 2H), 8.33 (s, 1H), 7.92 (d, J ¼ 13.0 Hz, 1H),
.75 (s, 1H), 7.68e7.55 (m, 2H), 7.46e7.35 (m, 1H), 7.13 (s, 1H), 7.01
m, 2H), 4.63 (t, J ¼ 6.0 Hz, 2H), 3.81 (m, 6H), 2.59 (t, J ¼ 6.1 Hz, 2H),
.14 (s, 6H); 13C NMR (600 MHz, DMSO-d
165.32, 154.98, 153.78,
53.43, 152.55, 152.33, 151.72, 150.60, 150.05, 147.50, 139.26, 137.27,
6
)
d
10.89 (s, 1H),
1H), 8.58 (s, 1H), 8.56 (d, J ¼ 5.6 Hz, 1H), 7.86 (m, 2H), 7.69 (s, 1H),
7.60 (d, J ¼ 8.8 Hz, 1H), 7.51 (s, 1H), 7.36 (t, J ¼ 8.5 Hz, 1H), 7.09 (d,
J ¼ 5.9 Hz, 2H), 6.91 (d, J ¼ 7.1 Hz,1H), 6.04 (s, 2H), 4.57 (t, J ¼ 6.0 Hz,
13
2H), 2.53 (t, J ¼ 5.9 Hz, 2H), 2.08 (s, 6H). C NMR (600 MHz, DMSO-
6
)
d
6
d ) d 164.96, 154.58, 153.10, 152.15, 151.36, 150.21, 149.57, 148.70,
148.06, 147.08, 141.29, 138.82, 134.13, 128.92, 123.60, 123.45, 116.75,
1
5
34.45, 124.04, 123.38, 117.04, 113.40, 112.24, 111.17, 109.16, 108.91,
111.92, 108.75, 108.24, 105.53, 101.48, 58.43, 44.75 (2C), 43.02; ESI-
þ
þ
9.23, 56.61, 56.07, 45.52 (2C), 43.82; ESI-MS m/z: 549.3 [MþH] ;
8 4
MS m/z: 533.3 [MþH] ; Anal. calcd. for C26H25FN O (%): C, 58.64;
Anal. calcd. for C27
H29FN
8
O
4
(%): C, 59.12; H, 5.33; N, 20.43. Found
H, 4.73; N, 21.04. Found (%): C, 58.70; H, 4.69; N, 21.23.
(
%): C, 59.17; H, 5.26; N, 20.49.
5.2. Pharmacology
5
.1.12.26. (E)-N-(4-(2-(4-(2-(Dimethylamino)ethyl)-4H-1,2,4-
triazol-3-yl)pyridin-4-yloxy)-3-fluorophenyl)-2-(2,3,4-
trimethoxybenzylidene)hydrazinecarboxamide (11w). A white solid;
Yield: 35%; M.p. 136e139 C; H NMR (300 MHz, DMSO-d ) d 10.77
6
5.2.1. MTT assay in vitro
The cytotoxic activities of compounds 10ae10c, 11ae11x and
12ae10c were evaluated against MKN-45, H460, HT-29, A549 and
U87MG cancer cell lines and WI-38 normal cell line by the standard
MTT assay in vitro. Sorafenib was used as the positive control. The
cancer cell lines were cultured in minimum essential medium
(MEM) supplement with 10% fetal bovine serum (FBS).
ꢀ
1
(
8
s, 1H), 9.16 (s, 1H), 8.64 (s, 1H), 8.61 (d, J ¼ 5.8 Hz, 1H), 8.21 (s, 1H),
.01e7.83 (m, 2H), 7.64 (d, J ¼ 9.3 Hz, 1H), 7.58 (s, 1H), 7.41 (t,
J ¼ 9.0 Hz, 1H), 7.14 (dd, J ¼ 5.4, 2.4 Hz, 1H), 6.91 (d, J ¼ 8.9 Hz, 1H),
4
.63 (t, J ¼ 6.0 Hz, 2H), 3.87 (s, 3H), 3.85 (s, 3H), 3.80 (s, 3H), 2.60 (t,
13
3
J ¼ 6.3 Hz, 2H), 2.15 (s, 6H); C NMR (600 MHz, DMSO-d6)
65.32, 155.20, 153.41, 152.74, 151.71, 150.61, 150.06, 147.47, 141.93,
39.27, 137.62, 134.51, 134.39, 124.02, 121.66, 120.90, 116.90, 112.25,
d
170.79,
Approximate 4 ꢁ 10 cells, suspended in MEM medium, were
1
1
plated onto each well of a 96-well plate and incubated in 5% CO
2
at
ꢀ
37 C for 24 h. The tested compounds were added to the culture
medium and the cell cultures were continued for 72 h. Fresh MTT
1
09.19, 108.97, 62.21, 60.94, 60.21, 59.24, 45.51 (2C), 43.83; ESI-MS
þ
m/z: 579.2 [MþH] ; Anal. calcd. for C28
H31FN
8
O
5
(%): C, 58.12; H,
was added to each well at a terminal concentration of 5
incubated with cells at 37 C for 4 h. The formazan crystals were
m
g/mL, and
ꢀ
5
.40; N, 19.37. Found (%): C, 58.19; H, 5.37; N, 19.32.
dissolved in 100 mL DMSO each well, and the absorbency at 492 nm
5
3
.1.12.27. (E)-N-(4-(2-(4-(2-(dimethylamino)ethyl)-4H-1,2,4-triazol-
- y l ) p y r i d i n - 4 - y l o x y ) - 3 - fl u o r o p h e n y l ) - 2 - ( 3 , 4 , 5 -
(for absorbance of MTT formazan) and 630 nm (for the reference
wavelength) was measured with an ELISA reader. All of the com-
pounds were tested three times in each of the cell lines. The results
expressed as IC50 (inhibitory concentration 50%) were the averages
of at least three determinations and calculated by using the Bacus
Laboratories Incorporated Slide Scanner (Bliss) sofeware.
trimethoxybenzylidene)hydrazinecarboxamide (11x). A white solid;
Yield: 55%; M.p. 117e120 C; H NMR (300 MHz, DMSO-d
ꢀ
1
6
) d 10.98
(
s, 1H), 9.30 (s, 1H), 8.65 (m, 2H), 7.92 (m, 2H), 7.70e7.38 (m, 4H),
7.18 (s, 2H), 4.63 (s, 2H), 3.88 (s, 6H), 3.71 (s, 3H), 2.59 (s, 2H), 2.14 (s,
13
6
H); C NMR (600 MHz, DMSO-d6)
d 165.32, 155.01, 153.58 (2C),
153.44, 152.58, 151.72, 150.59, 150.04, 147.50, 141.87, 139.24, 139.17,
5.2.2. Enzymatic assay
1
5
34.47, 130.28, 124.11, 117.10, 112.27, 109.09, 104.99 (2C), 60.57,
9.19, 56.53 (2C), 45.48 (2C), 43.79; ESI-MS m/z: 579.2 [MþH] ;
The kinase inhibitory measurements versus c-Met, VEGFR-2, B-
Raf, Raf-1 and PDGFRa were evaluated using homogeneous time-
þ

58
M. Qin et al. / European Journal of Medicinal Chemistry 81 (2014) 47e58
resolved fluorescence (HTRF) assay. All experiments were per-
formed in NUNC 264706 white 384-well low-volume plates (Cat.
[2] N. Jiang, X. Zhai, Y. Zhao, Y. Liu, B. Qi, H. Tao, P. Gong, European Journal of
Medicinal Chemistry 54 (2012) 534e541.
[3] S. Zhang, Y. Zhao, Y. Liu, D. Chen, W. Lan, Q. Zhao, C. Dong, L. Xin, P. Gong,
6
007290, PerkinElmer).
European Journal of Medicinal Chemistry 45 (2010) 3504e3510.
In B-Raf and Raf-1 kinase assay, a mixture of peptide substrates,
[4] T. O’Hare, M.W.N. Deininger, C.A. Eide, T. Clackson, B.J. Druker, Clinical Cancer
Research 17 (2010) 212e221.
ATP, kinase, and diluted compound was added to the kinase reac-
tion buffer (50 mM Hepes/NaOH pH 7.5, 10 mM MgCl , 1 mM EGTA,
.01% BRIJ-35) in each well. As a negative control, the tested com-
[
[
5] L. Liu, Y. Cao, X. Zhang, Cancer Research 66 (2006) 11851e11858.
6] G.M. Schroeder, Y. An, Z.W. Cai, X.T. Chen, C. Clark, L.A.M. Cornelius, J. Dai,
J. Gullo-Brown, A. Gupta, B. Henley, J.T. Hunt, R. Jeyaseelan, A. Kamath, K. Kim,
J. Lippy, L.J. Lombardo, V. Manne, S. Oppenheimer, J.S. Sack, R.J. Schmidt,
G. Shen, K. Stefanski, J.S. Tokarski, G.L. Trainor, B.S. Wautlet, D. Wei,
D.K. Williams, Y. Zhang, Y. Zhang, J. Fargnoli, R.M. Borzilleri, Journal of Me-
dicinal Chemistry 52 (2009) 1251e1254.
2
0
pound was dissolved in 4% DMSO (v/v). The incubation reactions
ꢀ
were performed at 25 C for 1 h. All the results were read using
Spectra M5(MD) instrument at 340 nm and 395 nm following the
[7] C. Durante, D. Russo, A. Verrienti, S. Filetti, Expert Opinion on Investigational
Drugs 20 (2011) 407e413.
addition of 10
mL working solution diluted with EDTA and Tb anti
pMAP2K1. In enzymatic assay against c-Met, VEGFR-2, PDGFR
a
, the
[8] E. Bello, G. Colella, V. Scarlato, P. Oliva, A. Berndt, G. Valbusa, S.C. Serra,
M. D’lncalci, E. Cavalletti, R. Giavazzi, G. Damia, G. Camboni, Cancer Research
solution of peptide substrates, ATP, appropriate kinase, and diluted
compound was mixed with the kinase reaction buffer (250 mM
71 (2011) 1396e1405.
[
9] C.A. Eide, L.T. Adrian, J.W. Tyner, M.M. Partlin, D.J. Anderson, S.C. Wise,
B.D. Smith, P.A. Petillo, D.L. Flynn, M.W.N. Deininger, T. O’Hare, B.J. Druker,
Cancer Research 71 (2011) 3189e3195.
HEPES pH 7.0, 0.1% NaN
mM DTT), subsequently reacted at 25 C for 20 min. As a negative
3
, 0.05% BSA, 0.5 mM orthovanadate, and
ꢀ
1
control, the tested compound was dissolved in 2.5% DMSO (v/v).
The HTRF readings were performed on ENVISION (Perkinelmer)
[10] T.H. Marsilje, W. Pei, B. Chen, W. Lu, T. Uno, Y. Jin, T. Jiang, S. Kim, N. Li,
M. Warmuth, Y. Sarkisova, F. Sun, A. Steffy, A.M.C. Pferdekamper, A.G. Li,
S.B. Joseph, Y. Kim, B. Liu, T. Tuntland, X. Cui, N.S. Gray, R. Steensma, Y. Wan,
J. Jiang, G. Chopiuk, J. Li, W.P. Gordon, W. Richmond, K. Johnson, J. Chang,
T. Groessl, Y.Q. He, A. Phimister, A. Aycinena, C.C. Lee, B. Bursulaya,
D.S. Karanewsky, H.M. Seidel, J.L. Harris, P.Y. Michellys, Journal of Medicinal
Chemistry 56 (2013) 5675e5690.
instrument by the addition of 5
Tk Antibody europium Cryptate solution. The inhibition rate (%)
was calculated using the following equation:
inhibition 100 [(Activity of enzyme with tested
mL of Streptavidin-XL665 and 5 mL
%
¼
ꢃ
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Conflict of interest
The authors have declared no conflict of interest.
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This work was supported by grants from National Natural Sci-
ence Foundation of China (No. 21002065 and 81102470) and Na-
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(
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.059.
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