Synthesis and biological evaluation of novel 4-(2-fluorophenoxy)-2-(1h- tetrazol-1-yl)pyridines bearing semicarbazone moieties as potent antitumor agents

Article abstract of DOI:10.1002/ardp.201300188

A series of 4-(2-fluorophenoxy)-2-(1H-tetrazol-1-yl)pyridines bearing semicarbazone moieties were synthesized and evaluated for their in vitro antitumor potency. Some of the compounds (10b, 10c, 10e-10h, 10m-10p, 10r, and 11b) exhibited moderate to excellent antitumor activity as compared to sorafenib and PAC-1, as well as low levels of toxicity toward the human fetal lung fibroblast cell line WI-38. The most promising compound 10p (IC₅₀ = 0.08, 0.36, 0.97 μM) was 45.1-, 6.1-, and 2.4-fold more active than sorafenib (IC₅₀ = 3.61, 2.19, 2.32 μM), and 17, 3.2, and 2.9 times better than PAC-1 (IC₅₀ = 1.36, 1.17, 2.83 μM) against three cancer cell lines (HT-29, H460, and MKN-45), respectively. In addition, further studies examining enzymatic activity suggested that the marked pharmacological activity observed might be ascribed to an inhibitory action against CRAf kinase. A series of 4-(2-fluorophenoxy)-2-(1H-tetrazol-1-yl)pyridines bearing semicarbazone moieties were synthesized and evaluated for their cytotoxic activities in vitro. The most promising compound 10p was further examined for enzymatic activity, with the goal to investigate the molecular mechanisms of action.

Full text of DOI:10.1002/ardp.201300188

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40
Arch. Pharm. Chem. Life Sci. 2013, 346, 840–850
Full Paper
Synthesis and Biological Evaluation of Novel 4-(2-
Fluorophenoxy)-2-(1H-tetrazol-1-yl)pyridines Bearing
Semicarbazone Moieties as Potent Antitumor Agents
1
1
1
1
1
2
1
Mingze Qin , Weike Liao , Chen Xu , Baolin Fu , Jianguo Ren , Yucheng Gu , and Ping Gong
1
Key Laboratory of Structure-Based Drug Design and Discovery, Shenyang Pharmaceutical University,
Ministry of Education, Shenyang, P. R. China
Syngenta, Jealott’s Hill International Research Centre, Bracknell, Berkshire, UK
2
A series of 4-(2-fluorophenoxy)-2-(1H-tetrazol-1-yl)pyridines bearing semicarbazone moieties were
synthesized and evaluated for their in vitro antitumor potency. Some of the compounds (10b, 10c,
1
0e–10h, 10m–10p, 10r, and 11b) exhibited moderate to excellent antitumor activity as compared to
sorafenib and PAC-1, as well as low levels of toxicity toward the human fetal lung fibroblast cell line
WI-38. The most promising compound 10p (IC50 ¼ 0.08, 0.36, 0.97 mM) was 45.1-, 6.1-, and 2.4-fold more
active than sorafenib (IC50 ¼ 3.61, 2.19, 2.32 mM), and 17, 3.2, and 2.9 times better than PAC-1
(
IC50 ¼ 1.36, 1.17, 2.83 mM) against three cancer cell lines (HT-29, H460, and MKN-45), respectively. In
addition, further studies examining enzymatic activity suggested that the marked pharmacological
activity observed might be ascribed to an inhibitory action against CRAf kinase.
Keywords: Antitumor activity / SARs / Semicarbazone moiety / Sorafenib analogs
Received: May 19, 2013; Revised: August 9, 2013; Accepted: August 12, 2013
DOI 10.1002/ardp.201300188
Additional supporting information may be found in the online version of this article at the publisher’s web-site
:
Introduction
Sorafenib was approved by the U.S. FDA for the treatment
of advanced renal cancer in December 2005, and primary
hepatocellular carcinoma in November 2007 [5–7]. Thus,
considerable attention has been focused on sorafenib and its
derivatives [8–13]. The co-crystal structure of sorafenib in
complex with Raf kinase domains revealed that the pyridyl
ring and the diaryl urea framework played important roles in
the interaction with Raf kinase due to their binding modes
and the presence of hydrogen bonds [14]. Therefore, we chose
to modify these pharmacophores while maintaining their
actions with the kinase domains.
N-Acylarylhydrazone scaffold and its mimics were widely
used as building blocks in several antitumor agents,
exemplified by PAC-1 [15–17]. Its favorable biologic activity
might be attributed to the presence of hydrogen bond donors
and acceptors as well as a flexible skeleton. Therefore, the
Cancer is the major leading cause of death in the world.
Serious side effects and drug resistance render traditional
chemotherapeutics far from satisfactory. Therefore, it is
urgent to discover potent antitumor agents that perform
safely and more efficiently.
In recent years, multikinase inhibitors have emerged as
new approaches for cancer therapy because of their superior
antitumor activity as well as their diverse mechanisms of
action [1–4]. Among these, sorafenib, a novel multikinase
inhibitor against Raf, VEGFR, and PDGFR, demonstrated
outstanding inhibition across a panel of tumors, including
breast, hepatocellular, renal, and colorectal carcinomas.
Correspondence: Ping Gong, Key Laboratory of Structure-Based Drug
Design and Discovery, Shenyang Pharmaceutical University, Ministry of
Education, 103 Wenhua Road, Shenhe District 110016, P. R. China.
E-mail: gongpinggp@126.com
resultant semicarbazone moiety was introduced as
a
modification of the diaryl urea framework in an attempt to
provide more potent antitumor agents.
Fax: þ86 24 2398 6429
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Arch. Pharm. Chem. Life Sci. 2013, 346, 840–850
Semicarbazone-Containing Antitumor Agents
841
Optimization of substituents on pyridyl group has led to a
variety of interesting compounds [18–20]. Recently, Zhan
et al. [18] reported a series of sorafenib analogs that were
substituted by a trifluoromethyl imidazolyl group at the
pyridyl ring instead of the methylamide fragment. Of the
resulting compounds, the best exhibited an IC50 value of
thionyl chloride and subsequently treated with methanol to
give 2 as a light-yellow solid [26]. Aminolysis of 2 afforded 3,
which was then converted to 4 by means of classical Hofmann
degradation in the presence of NaOH in water [27]. A
nucleophilic substitution of 4 with 2-fluoro-4-nitrophenol
in refluxing chlorobenzene catalyzed by pyridine provided 5
using a reported modified condition [28], which was then
cyclized with sodium azide and triethyl orthoformatein in
acetic acid to furnish intermediate 6 [21]. Reduction of 6
by hydrazine hydrate led to the preparation of the key
intermediate 7. Amidation of 7 with phenyl chloroformate in
the presence of potassium carbonate in acetone afforded 8,
which then reacted with hydrazine hydrate to give compound
9. Finally, by a condensation reaction of hydrazide derivative 9
with aromatic aldehydes, the target compounds 10a–10r
and 11a–11c were prepared.
10.6 nM against CRAF, which was 2.3-fold more active than
sorafenib, and indicated that the pyridyl ring was well
tolerated for heteroaryl substituents. In view of the broad
bioactivities of the tetrazolyl moiety presented in several
compounds as well as its synthetic accessibility [21–23], this
moiety was introduced to the 2-position of the pyridyl ring,
creating a novel drug-like scaffold. In addition, encouraged by
the success of several kinase inhibitors such as regorafenib
and foretinib [4, 24, 25], we introduced a fluoro group to
the central phenyl ring in the preliminary investigation.
Accordingly, a novel series of (E)-N-(4-(2-(1H-tetrazol-1-yl)-
pyridin-4-yloxy)-3-fluorophenyl)-2-(substituted arylidene)hydra-
zinecarboxamide derivatives were designed (Fig. 1).
The chemical structures of the target compounds were
1
13
confirmed by H NMR, C NMR, 2D NOESY NMR, and MS
1
spectra. In the H NMR spectra of compound 10p, exchange-
Here, we report the newly designed compounds and their
in vitro antitumor activities against three human cancer cell
lines. The enzymatic inhibitory activity was also evaluated
using the most promising compound 10p.
able singlet signals appeared at d 9.14 and 11.28 ppm that
were assigned to –NH–CO and –NH–N, respectively, and the
proton of –N–CH was observed at d 8.18 ppm, which indicated
the formation of the semicarbazone moiety. Moreover, the
characteristic signal for tetrazole proton was seen at d
10.19 ppm. All the other aromatic protons were observed with
the expected chemical shifts and integral values. The
chemical structures of the target compounds were further
Chemistry
The synthetic procedures are outlined in Scheme 1. The
commercially available picolinic acid was chlorinated in
1
3
13
verified by the C NMR experiments. The C NMR spectra of
Figure 1. Structures of multikinase inhibitors, PAC-1 and the target compounds.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 840–850
Scheme 1. Reagents and conditions: (a) SOCl
0°C; (e) 2-fluoro-4-nitrophenol, chlorobenzene, reflux; (f) triethyl orthoformate, NaN
h) phenyl chloroformate, K CO , acetone, r.t.; (i) NH NH · H
2
, NaBr, chlorobenzene, 85°C; (b) MeOH, toluene, 0–15°C; (c) NH
3
2
· H
NH
2
O, r.t.; (d) Br
2
, NaOH,
8
(
3
, HOAc, reflux; (g) NH
2
· H O, EtOH, reflux;
2
2
3
2
2
2
O, dioxane, 90°C; (j) substituted aldehyde, isopropanol, reflux.
compound 10p showed the presence of a low-field resonance
at d 167.05 ppm corresponding to C–O group. Simultaneous-
cancer), and MKN-45 (human gastric cancer) cell lines by the
MTT assay. In addition, several potent compounds were
further assessed against a non-malignant cell line WI-38
(human fetal lung fibroblasts). Using sorafenib and PAC-1 as
the positive controls, the bioactivity data summarized in
Table 1 are presented as IC50 values derived from the average
of at least three independent experiments.
As shown in Table 1, several target compounds exhibited
moderate to excellent antitumor potency against the tested
cancer cell lines as compared to sorafenib and PAC-1. Of
particular note, the most promising compound 10p showed
eminent activity against the HT-29, H460, and MKN-45 cell
lines with IC50 values of 0.08, 0.36, and 0.97 mM, respectively,
while 10c was the most potent against the MKN-45 cell line
(IC50 ¼ 0.38 mM). 10c as well as 11b were good candidates for
further investigation due to their marked inhibitory activity,
which was in the low micromolar range.
ly, the signal of C–Cl appeared at d 134.53 ppm, showing the
integration of two carbon atoms. In addition, the stereo-
chemistry of the imine double bond was determined. In
1
H NMR spectra, the –N–CH signal was seen as a single peak
for each compound, indicating that only one isomer was
generated. In order to determine the stereochemistry more
clearly, the NOESY experiment of compound 10p was carried
out, and an evident NOE signal was observed between the
proton of –NH–N– (d 11.28 ppm) and the proton of –N–CH–
(d 8.18 ppm), which existed only in the E isomer due to
the appropriate intramolecular H–H distance (Fig. 2 and see
Supporting Information). Thus, the target compounds were
unambiguously confirmed as the E isomers.
Biological evaluation and discussion
Preliminary structure–activity relationships (SARs) indicat-
ed that the substitution pattern of the benzylidene segment
was important for the potency. In general, compounds
with substituents on the phenyl ring showed an enhanced
activity as compared to 10a. In most cases, compounds with
In vitro cytotoxicity and structure–activity
relationships
The cytotoxic actions of the target compounds were evaluated
against HT-29 (human colorectal cancer), H460 (human lung
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Arch. Pharm. Chem. Life Sci. 2013, 346, 840–850
Semicarbazone-Containing Antitumor Agents
843
Figure 2. NOESY effect of the representative compound 10p.
one hydroxyl group on benzylidene (10b, 10c, 10e–10h, 10o)
exhibited marked antitumor potency, while the introduction
of more hydroxyl groups (10j–10l) resulted in a dramatic loss
in activity, suggesting that multi-hydroxyl groups were not
tolerated for this region. Comparing 10d and 10j with 10b
and 10e, we concluded that electron-withdrawing groups
In vitro enzymatic assays
In order to investigate the molecular mechanisms of action
preliminarily, the optimal compound 10p was screened
against four kinases, which are probably the biological targets
for the designed compounds [4, 24]. The results summarized
in Table 2 are expressed as percentage inhibition and are
derived from two independent experiments. As shown,
compound 10p inhibited CRAF kinase by 70.7% at a
concentration of 10 mM, whereas the impact on the other
tested kinases was negligible. These results indicated that
CRAF kinase might be a biological target for the synthesized
compounds.
(
EWGs) at the 3-position were benefit for the cytotoxicity.
Interestingly, the introduction of an additional electron-
donating group (EDG) at the 5-position (10c, 10o) led to an
improvement in antitumor activity, a result that might be
attributed to the electron density on the phenyl ring. In
addition, the position of a halogen substituent clearly
influenced the cytotoxic action, with the following rank
order of activity: 2,3-dichloro < 2,4-dichloro < 2,6-dichloro. A
case in point was that compound 10p with a 2,6-dichloro-
phenyl group possessed the best potency. With the exception
of 10h and 10o, all the tested compounds showed weaker
toxicity against the human fibroblast cell line WI-38 in
comparison to their effects on all three cancer cells. These
results indicated that the cancer cells were more sensitive to
the newly synthesized compounds than the non-malignant
cells.
Apoptosis analysis
To assess whether compound 10p acted by apoptosis, H460
cells were treated with 10p at different concentrations (0, 0.2,
1, 5, 25 mM) for 48 h, and the cleavage of PARP, a biomarker of
apoptosis, was analyzed by Western blot (Fig. 3). The enhanced
PARP cleavage demonstrated that compound 10p increased
apoptosis in a concentration-dependent manner. It has been
reported that CRAF kinase was involved in the protection of
cells from apoptosis [29–31]; thus, the inhibition of CRAF was
probably responsible for the cell apoptosis observed.
Compounds 11a–11c were designed to further evaluate
the impact of the phenyl segment on cytotoxicity. To our
delight, compound 11b, which contained a 2-hydroxylnaph- Conclusions
thalene moiety, exhibited superior potency as compared to
the reference compounds, demonstrating IC50 values of
less than single-digit micromole against all the tested cancer
cell lines. Further studies are currently underway in our
laboratory.
In summary, a series of novel 4-(2-fluorophenoxy)-2-(1H-
tetrazol-1-yl)pyridines bearing semicarbazone moieties were
synthesized and evaluated. Several target compounds showed
comparative or enhanced cytotoxicity as compared to the
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Arch. Pharm. Chem. Life Sci. 2013, 346, 840–850
Table 1. Cytotoxicity of target compounds against HT-29, H460, MKN-45 cancer cell lines, and WI-38 normal cell line in vitro
H
N
H
N
H
N
H
N
F
R
F
R
N
N
O
O
O
O
N
N
N
N
N
N
N
N
N
N
_
_
11a 11c
1
0a 10r
IC50 (mM)
Compd.
R
HT-29
H460
MKN-45
WI-38
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0a
0b
0c
0d
0e
0f
0g
0h
0i
0j
0k
0l
0m
0n
0o
0p
0q
0r
H
25.82 ꢀ 0.27
1.12 ꢀ 0.13
0.96 ꢀ ꢀ 0.11
10.34 ꢀ 0.54
1.67 ꢀ 0.17
1.03 ꢀ 0.086
4.31 ꢀ 0.21
2.71 ꢀ 0.34
24.65 ꢀ 0.28
9.22 ꢀ 0.56
10.45 ꢀ 2.13
13.24 ꢀ 1.58
3.65 ꢀ 0.23
4.43 ꢀ 0.15
2.03 ꢀ 0.12
0.08 ꢀ 0.023
5.32 ꢀ 1.31
1.42 ꢀ 0.42
10.51 ꢀ 0.39
1.37 ꢀ 0.032
0.42 ꢀ 0.067
23.37 ꢀ 1.29
1.52 ꢀ 0.037
0.83 ꢀ 0.13
3.62 ꢀ 0.077
0.87 ꢀ 0.059
38.01 ꢀ 3.21
NA
6.17 ꢀ 1.02
9.61 ꢀ 0.29
2.92 ꢀ 0.13
3.17 ꢀ 0.039
5.37 ꢀ 1.17
0.36 ꢀ 0.036
6.10 ꢀ 0.34
1.13 ꢀ 0.071
NA
ND
3-Fluoro-4-hydroxy
2.62 ꢀ 0.12
0.38 ꢀ 0.063
13.76 ꢀ 1.13
2.54 ꢀ 0.27
2.33 ꢀ 0.22
2.92 ꢀ 0.089
6.52 ꢀ 1.02
25.37 ꢀ 2.28
6.65 ꢀ 0.37
8.35 ꢀ 0.91
16.34 ꢀ 2.62
1.29 ꢀ 0.12
4.13 ꢀ 0.097
2.37 ꢀ 0.35
0.97 ꢀ 0.17
8.91 ꢀ 1.75
2.04 ꢀ 0.63
3.27 ꢀ 0.67
2.28 ꢀ 0.22
ND
5.64 ꢀ 0.60
4.63 ꢀ 0.19
7.68 ꢀ 0.25
4.03 ꢀ 0.32
ND
4-Hydroxy-3,5-dimethyl
4-Hydroxy-3-methoxy
3-Bromo-4-hydroxy
3,5-Di-tert-butyl-2-hydroxy
2-Hydroxy
3-Allyl-2-hydroxy
4-Hydroxy
3,4-Dihydroxy
ND
ND
ND
2,4-Dihydroxy
3,4,5-Trihydroxy
3,4,5-Trimethoxy
2,4-Dimethoxy
6.37 ꢀ 0.63
8.39 ꢀ 0.31
3.38 ꢀ 0.15
3.96 ꢀ 0.28
ND
4-Hydroxy-3,5-dimethoxy
2,6-Dichloro
2,3-Dichloro
2,4-Dichloro
2.62 ꢀ 0.27
O
O
11a
13.31 ꢀ 0.87
4.12 ꢀ 0.25
14.27 ꢀ 0.88
ND
OH
1
1b
1c
0.70 ꢀ 0.087
10.56 ꢀ 1.21
0.63 ꢀ 0.13
0.96 ꢀ 0.031
17.06 ꢀ 0.53
2.67 ꢀ 0.56
1
17.50 ꢀ 1.72
ND
N
H
Sorafenib
PAC-1
3.61 ꢀ 0.36
1.36 ꢀ 0.18
2.19 ꢀ 0.11
1.17 ꢀ 0.79
2.32 ꢀ 0.25
2.83 ꢀ 0.17
7.54 ꢀ 0.78
5.60 ꢀ 0.67
NA: compound showing IC50 value >100 mM.
ND, not determined.
Bold values show the IC50 values of the most promising compound in the biological evalution.
positive controls. From the preliminary SARs, we may
conclude that multi-hydroxyl substituents on the benzylidene
moiety were not well tolerated. In contrast, halogen had a
positive impact on the cytotoxicity. Investigation into the
position of substituents demonstrated that the 2,6-dichloro
group was the optimal substitution pattern. In particular, the
result of Western blot assay showed that the 2,6-dichloro
derivative 10p caused a significant induction of apoptosis in a
concentration-dependent manner. Additionally, the discovery
of compound 11b highlighted the potential for further
optimization of the aryl moiety. The mechanism for the
antitumor potency of the designed compounds was not fully
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Arch. Pharm. Chem. Life Sci. 2013, 346, 840–850
Semicarbazone-Containing Antitumor Agents
845
Table 2. Percentage inhibition of compound 10p against four
kinases
the completion of reaction. The solvent was removed under
reduced pressure to give a residue, which was then dissolved in
toluene (300 mL) in an ice-bath; subsequently, anhydrous
methanol (80 mL) was added dropwise. Upon completion of
addition, the mixture was stirred at 15°C for 1.5 h. The precipitate
%
Inhibition at 10 mM
c-Met
KDR
13.5
C-Raf
PDGFRa
was filtered off and dissolved in CH
were washed with saturated aqueous K
Na SO , and evaporated to give 2 as a light-yellow solid (158.5 g,
yield 76.0%). m.p. 67–68°C; literature (hydrochloride form) m.p.
Cl
(500 mL). The organics
2
2
2
CO (300 mL), dried over
3
10p
1.7
–
70.7
97.2
–
7.3
–
2
4
Sorafenib
Foretinib
–
98.6
99.7
99.2
1
1
08–112°C. H NMR (300 MHz, DMSO-d
6
) d 8.65 (d, J ¼ 5.2 Hz, 1H,
ArH), 8.02 (d, J ¼ 1.7 Hz, 1H, ArH), 7.78 (dd, J ¼ 5.1, 1.6 Hz, 1H, ArH),
þ
3
.85 (s, 3H, –OCH
3
). ESI-MS m/z: 194.0 [MþNa] .
elucidated. However, the pattern of activity for 10p across the
tested kinases suggested that these compounds may be potent
inhibitors of CRAF kinase. Additional work will focus on
detailed SARs as well as clarifying molecular mechanism.
4
-Chloropicolinamide (3)
To a suspension of methyl 4-chloropicolinate 2 (15 g, 0.058 mol) in
water ammonia (100 mL) was added at room temperature, and
then the mixture was stirred for 3 h. The resulting solid was
separated by filtration and dried to give 3 as a white solid (11.5 g,
yield 84%). m.p. 160–162°C. H NMR (300 MHz, DMSO-d
1
6
) d 8.61 (d,
), 8.03 (d, J ¼ 2.0 Hz, 1H, ArH),
), 7.75 (dd, J ¼ 5.3, 2.1 Hz, 1H, ArH). ESI-MS m/z:
Experimental
J ¼ 5.3 Hz, 1H, ArH), 8.19 (s, 1H, –NH
2
7
1
.81 (s, 1H, –NH
79.0 [MþNa] .
2
þ
Chemistry
Unless otherwise noted, all materials were obtained from
commercially available sources and used without further
purification. All melting points were obtained on a Büchi Melting
Point B-540 apparatus (Büchi Labortechnik, Flawil, Switzerland)
4-Chloropyridin-2-amine (4)
Bromine (21.4 g, 0.135 mol) was added dropwise to 10% aqueous
NaOH (200 mL) at a rate that kept the internal temperature below
10°C with vigorous agitation. Compound 3 (17.6 g, 0.113 mol) was
added, and the mixture was allowed to stir at room temperature
for 1 h, then heated to 80°C for another 4 h. The mixture was
cooled to 0°C, and the precipitate was separated by filtration to
obtain a white solid, which was then dissolved in 2 N aqueous
HCl. Subsequently, the mixture was alkalized with 10% aqueous
NaOH to pH 12–13. The precipitate was filtered off and dried
1
13
and were uncorrected. H NMR (1D and 2D) and C NMR spectra
were recorded on Bruker ARX-300 or ARX-600 spectrometers
(
Bruker Bioscience, Billerica, MA, USA) with TMS as an internal
standard. Mass spectra (MS) were taken in ESI mode on Agilent
100 LC-MS (Agilent, Palo Alto, CA, USA). Elemental analysis was
determined on a Carlo-Erba 1106 Elemental analysis instrument
Carlo Erba, Milan, Italy).
1
(
under vacuum to give 4 as a white crystal (3.9 g, yield 26.8%). m.p.
1
1
1
1
30–131°C. H NMR (300 MHz, DMSO-d
6
) d 7.81 (d, J ¼ 5.2 Hz,
Methyl 4-chloropicolinate (2)
H, ArH), 6.52–6.39 (m, 2H, ArH), 6.18 (s, 2H, –NH
2
). ESI-MS m/z:
Compound 2 has been prepared according to [26] with minor
modifications. To a solution of picolinic acid (150 g, 1.22 mol) and
sodium bromide (20.1 g, 0.197 mol) in chlorobenzene (200 mL)
thionyl chloride (355 mL, 4.88 mol) was added slowly at room
temperature. The mixture was stirred at 50°C for 30 min, and
then heated to 85°C and stirred for another 20 h until TLC showed
þ
28.1 [MþH] .
4-(2-Fluoro-4-nitrophenoxy)pyridin-2-amine (5)
A mixture of 4 (3 g, 0.023 mol), 2-fluoro-4-nitrophenol (5.5 g,
0.035 mol), and a catalytic amount of pyridine in chlorobenzene
Figure 3. Compound 10p inhibits cell growth by inducing apoptosis in the H460 cell line. The apoptosis was assessed by Western blot.
Cleavage of PARP is considered as apoptosis.
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M. Qin et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 840–850
(
400 mL) was stirred under reflux for approximately 30 h. The
(s, 1H, tetrazole CH), 9.01 (s, 1H, –NH–NH
2
), 8.48 (d, J ¼ 5.8 Hz, 1H,
completion of reaction was detected by TLC, and then the mixture
was cooled to room temperature. The solvent was removed under
reduced pressure to afford a brown residue. The residue was
ArH), 7.81 (d, J ¼ 14.0 Hz, 1H, ArH), 7.59 (s, 1H, –NH–CO), 7.43
(m, 2H, ArH), 7.29 (t, J ¼ 9.0 Hz, 1H, ArH), 7.12 (dd, J ¼ 5.7, 2.1 Hz,
þ
1H, ArH), 4.39 (s, 2H, –NH–NH
2
). ESI-MS m/z: 353.1 [MþNa] .
dissolved in CH
2
Cl
2
, and washed with saturated aqueous K
2 3
CO
(
3 ꢁ 250 mL), then brined (200 mL), and dried over Na
2
SO . The
4
General procedure for the synthesis of compounds
10a–10r, 11a–11c
A mixture of 9, aromatic aldehydes, and a catalytic amount of
glacial acetic acid was refluxed in isopropanol for 4–8 h. The
completion of the reaction was detected by TLC. The mixture was
cooled to room temperature, and then the precipitate was filtered
off and dried under vacuum to afford the target compounds.
solvent was removed under reduced pressure to yield 5 as a pale
solid (3.7 g, yield 63.4%). m.p. 124–126°C. H NMR (300 MHz,
DMSO-d
2
1
6
) d 8.34 (dd, J ¼ 10.5, 2.7 Hz, 1H, ArH), 8.12 (ddd, J ¼ 9.0,
.6, 1.4 Hz, 1H, ArH), 7.85 (d, J ¼ 5.8 Hz, 1H, ArH), 7.53–7.42 (m, 1H,
ArH), 6.21 (dd, J ¼ 5.8, 2.3 Hz, 1H, ArH), 6.07 (s, 2H, –NH
2
), 5.96 (d,
þ
J ¼ 2.2 Hz, 1H, ArH). ESI-MS m/z: 250.1 [MþH] .
4
-(2-Fluoro-4-nitrophenoxy)-2-(1H-tetrazol-1-yl)pyridine
(
E)-N-(4-(2-(1H-Tetrazol-1-yl)pyridin-4-yloxy)-3-fluoro-
(
6)
phenyl)-2-benzylidenehydrazinecarboxamide (10a)
Yield: 66%. m.p. 207–209°C. H NMR (300 MHz, DMSO-d
A solution of 5 (3.7 g, 15 mmol) and triethyl orthoformate (22.2 g,
.15 mol) in glacial acetic acid (30 mL) was refluxed for 1 h. Upon
1
6
) d 10.94
0
(s, 1H, –NH–N), 10.19 (s, 1H, tetrazole CH), 9.26 (s, 1H, –NH–CO),
cooling to room temperature, sodium azide (2.9 g, 45 mmol) was
added cautiously, and the mixture was stirred under reflux for
another 5 h. The reaction was considered complete when <5%
starting material remained. The mixture was poured into water
8
2
1
.56 (d, J ¼ 5.8 Hz, 1H, ArH), 7.99 (s, 1H, –N–CH), 7.94 (dd, J ¼ 13.5,
.3 Hz, 1H, ArH), 7.87 (d, J ¼ 7.63 Hz, 2H, ArH), 7.67 (d, J ¼ 10.0 Hz,
H, ArH), 7.53 (d, J ¼ 2.2 Hz, 1H, ArH), 7.44 (m, 3H, ArH), 7.21 (dd,
ꢂ
J ¼ 5.8, 2.3 Hz, 1H, ArH). ESI-MS m/z: 417.0 [MꢂH] . Anal. calcd. for
(300 mL) and stirred for 1 h. The resulting precipitate was filtered
C
20
H15FN
8
O
2
(%): C, 57.41; H, 3.61; F, 4.54; N, 26.78; O, 7.65. Found
off, washed with diethyl ether, and dried under vacuum to afford
6
(%): C, 57.33; H, 3.66; N, 26.72.
as a light yellow solid (2.8 g, yield 62.2%). ESI-MS m/z: 303.1
þ
[
MþH] .
(E)-N-(4-(2-(1H-Tetrazol-1-yl)pyridin-4-yloxy)-3-fluoro-
phenyl)-2-(3-fluoro-4-hydroxybenzylidene)hydrazinecar-
4
-(2-(1H-Tetrazol-1-yl)pyridin-4-yloxy)-3-fluoroaniline (7)
At room temperature, to a suspension of 6 (3.02 g, 10 mmol),
active carbon (0.036 g, 3 mmol) and FeCl · 6H O in ethanol (0.41 g,
.5 mmol) 80% hydrazine hydrate (6.25 mL, 0.1 mol) was added
boxamide (10b)
1
3
2
Yield: 62%. m.p. 225–227°C. H NMR (300 MHz, DMSO-d
6
) d 10.78
1
(s, 1H, –NH–N), 10.23 (s, 1H, –OH), 10.16 (s, 1H, tetrazole CH), 9.19
(s, 1H, –NH–CO), 8.56 (d, J ¼ 5.8 Hz, 1H, ArH), 7.94 (dd, J ¼ 13.5,
2.4 Hz, 1H, ArH), 7.89 (s, 1H, –N–CH), 7.85 (d, J ¼ 1.8 Hz, 1H, ArH),
dropwise with vigorous agitation. The mixture was stirred for 3 h
under reflux until TLC showed the completion of the reaction.
The solvent was evaporated under reduced pressure, then poured
into water, and allowed to stir for 30 min. The precipitate was
collected by filtration and dried to yield 7 as a white solid (1.7 g,
yield 62.6%). m.p. 171–174°C. H NMR (300 MHz, DMSO-d
7.67 (d, J ¼ 8.9 Hz, 1H, ArH), 7.53 (d, J ¼ 2.2 Hz, 1H, ArH), 7.43 (t,
J ¼ 9.1 Hz, 1H, ArH), 7.37 (d, J ¼ 8.4 Hz, 1H, ArH), 7.20 (dd, J ¼ 5.7,
2.3 Hz, 1H, ArH), 6.99 (t, J ¼ 8.6 Hz, 1H, ArH). ESI-MS m/z: 450.7
1
ꢂ
6
) d 10.11
[MꢂH] . Anal. calcd. for C20
14 2 8 3
H F N O (%): C, 53.10; H, 3.12;
(
1
s, 1H, tetrazole CH), 8.46 (d, J ¼ 5.7 Hz, 1H, ArH), 7.38 (d, J ¼ 1.9 Hz,
F, 8.40; N, 24.77; O, 10.61. Found (%): C, 53.19; H, 3.17; N, 24.63.
H, ArH), 7.12–6.99 (m, 2H, ArH), 6.51 (dd, J ¼ 13.3, 2.2 Hz, 1H,
ArH), 6.43 (d, J ¼ 2.4, 8.8 Hz, 1H, ArH). 5.54 (s, 2H, –NH
2
). ESI-MS
(E)-N-(4-(2-(1H-Tetrazol-1-yl)pyridin-4-yloxy)-3-fluoro-
þ
m/z: 274.3 [MþH] .
phenyl)-2-(4-hydroxy-3,5-dimethylbenzylidene)hydrazine-
carboxamide (10c)
Phenyl 4-(2-(1H-tetrazol-1-yl)pyridin-4-yloxy)-3-
1
Yield: 71%. m.p. 213–215°C. H NMR (300 MHz, DMSO-d
6
) d 10.65
fluorophenylcarbamate (8)
(s, 1H, –NH–N), 10.16 (s, 1H, tetrazole CH), 9.13 (s, 1H, –NH–CO),
8.62 (s, 1H, –OH), 8.56 (d, J ¼ 5.8 Hz, 1H, ArH), 7.95 (dd, J ¼ 13.4,
2.1 Hz, 1H, ArH), 7.84 (s, 1H, –N–CH), 7.67 (d, J ¼ 8.9 Hz, 1H, ArH),
2 3
To a solution of 7 (1.1 g, 4 mmol) and K CO (0.83 g, 6 mmol) in dry
acetone (20 mL) phenyl chloroformate (0.81 g, 5.2 mmol) was
added dropwise with stirring at 0°C. Upon completion of
addition, the reaction mixture was heated to room temperature
and stirred for 3 h. The reaction mixture was quenched by
addition of water; subsequently, the precipitate was filtered off
7.52 (d, J ¼ 2.0 Hz, 1H, ArH), 7.47–7.39 (m, 3H, ArH), 7.20 (dd,
1
3
J ¼ 5.7, 2.1 Hz, 1H, ArH), 2.22 (s, 6H, –CH
3 6
). C NMR (DMSO-d ) d
167.11, 155.44, 154.41, 153.51, 152.79, 151.67, 148.67, 142.58,
142.25, 139.50, 134.19, 127.92, 125.59, 124.82, 123.84, 116.93,
and dried under vacuum to generate 8 as a white solid (1.2 g, yield
112.72, 108.70, 102.08, 62.48, 25.93, 17.02. ESI-MS m/z: 460.9
þ
ꢂ
7
7.5%). ESI-MS m/z: 393.2 [MþH] .
[MꢂH] . Anal. calcd. for C22
8 3
H19FN O (%): C, 57.14; H, 4.14; F, 4.11;
N, 24.23; O, 10.38. Found (%): C, 57.21; H, 4.09; N, 24.29.
N-(4-(2-(1H-Tetrazol-1-yl)pyridin-4-yloxy)-3-fluorophenyl)-
hydrazinecarboxamide (9)
A solution of 8 (1 g, 2.6 mmol) and 80% hydrazine hydrate
(
E)-N-(4-(2-(1H-Tetrazol-1-yl)pyridin-4-yloxy)-3-fluoro-
phenyl)-2-(4-hydroxy-3-methoxybenzylidene)hydrazine-
(
0.33 mL, 5.3 mmol) in dioxane (20 mL) was stirred at 90°C for 3 h.
carboxamide (10d)
1
After cooling to room temperature, the precipitate was collected
Yield: 53%. m.p. 195–196°C. H NMR (300 MHz, DMSO-d
6
) d 10.70
by filtration, and dried to provide 9 as a white solid (0.71 g,
(s, 1H, –NH–N), 10.15 (s, 1H, tetrazole CH), 9.37 (s, 1H, –OH), 9.19
(s, 1H, –NH–CO), 8.55 (d, J ¼ 5.8 Hz, 1H, ArH), 7.94 (dd, J ¼ 13.5,
1
8
3.8%). m.p. 197–200°C. H NMR (300 MHz, DMSO-d
6
) d 10.12
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2013, 346, 840–850
Semicarbazone-Containing Antitumor Agents
847
2
7
.3 Hz, 1H, ArH), 7.88 (s, 1H, –N–CH), 7.64 (d, J ¼ 10.0 Hz, 1H, ArH),
J ¼ 7.5 Hz, 1H, ArH), 5.98 (dt, J ¼ 16.8, 8.4 Hz, 1H, –CH
–CH–CH
),
2 2
.53–7.48 (m, 2H, ArH), 7.42 (t, J ¼ 9.0 Hz, 1H, ArH), 7.19 (m, 2H,
5.11–5.00 (m, 2H, –CH
–CH
–CH–CH
2
–CH–CH
2
), 3.39 (d, J ¼ 6.2 Hz, 2H,
ꢂ
ArH), 6.82 (d, J ¼ 8.1 Hz, 1H, ArH), 3.87 (s, 3H, –OCH
3
). ESI-MS m/z:
(%): C, 54.31; H, 3.69;
2
2
). ESI-MS m/z: 472.8 [MꢂH] . Anal. calcd. for
ꢂ
4
62.9 [MꢂH] . Anal. calcd. for C21
H17FN
8
O
4
23 8 3
C H19FN O (%): C, 58.22; H, 4.04; F, 4.00; N, 23.62; O, 10.12.
F, 4.09; N, 24.13; O, 13.78. Found (%): C, 54.35; H, 3.77; N, 24.07.
Found (%): C, 58.31; H, 4.12; N, 23.77.
(E)-N-(4-(2-(1H-Tetrazol-1-yl)pyridin-4-yloxy)-3-fluoro-
(E)-N-(4-(2-(1H-Tetrazol-1-yl)pyridin-4-yloxy)-3-fluoro-
phenyl)-2-(3-bromo-4-hydroxybenzylidene)hydrazinecar-
phenyl)-2-(4-hydroxybenzylidene)hydrazinecarboxamide
boxamide (10e)
(10i)
1
1
Yield: 61%. m.p. 204–206°C. H NMR (300 MHz, DMSO-d
6
) d 10.79
Yield: 65%. m.p. 212–215°C. H NMR (300 MHz, DMSO-d
6
) d 10.69
(
(
1
7
s, 1H, –NH–N), 10.65 (s, 1H, –OH), 10.17 (s, 1H, tetrazole CH), 9.24
(s, 1H, –NH–N), 10.17 (s, 1H, –OH), 9.82 (s, 1H, tetrazole CH), 9.15
(s, 1H, –NH–CO), 8.55 (d, J ¼ 5.8 Hz, 1H, ArH), 7.94 (dd, J ¼ 13.7,
2.2 Hz, 1H, ArH), 7.88 (s, 1H, –N–CH), 7.67 (m, 3H, ArH),
s, 1H, –NH–CO), 8.56 (d, J ¼ 5.8 Hz, 1H, ArH), 8.11 (d, J ¼ 1.8 Hz,
H, ArH), 7.93 (dd, J ¼ 13.5, 2.3 Hz, 1H, ArH), 7.86 (s, 1H, –N–CH),
.69–7.58 (m, 2H, ArH), 7.52 (d, J ¼ 2.2 Hz, 1H, ArH), 7.43 (t,
7.52 (d, J ¼ 2.2 Hz, 1H, ArH), 7.42 (t, J ¼ 9.1 Hz, 1H, ArH), 7.20 (dd,
J ¼ 9.1 Hz, 1H, ArH), 7.20 (dd, J ¼ 5.7, 2.2 Hz, 1H, ArH), 6.99 (d,
J ¼ 5.7, 2.2 Hz, 1H, ArH), 6.82 (d, J ¼ 8.6 Hz, 2H, ArH). ESI-MS
1
3
ꢂ
J ¼ 8.4 Hz, 1H, ArH). C NMR (DMSO-d
6
) d 167.10, 155.78, 154.38,
m/z: 432.8 [MꢂH] . Anal. calcd. for C20
8 3
H15FN O (%): C, 55.30;
1
1
1
53.49, 151.69, 148.68, 142.27, 140.74, 139.43, 134.32, 131.51,
H, 3.48; F, 4.37; N, 25.80; O, 11.05. Found (%): C, 55.22; H, 3.55;
N, 25.66.
28.78, 127.46, 123.85, 117.12, 116.60, 112.74, 110.47, 108.88,
ꢂ
02.12. ESI-MS m/z: 512.7 [MꢂH] . Anal. calcd. for C20
8 3
H14BrFN O
(
%): C, 46.80; H, 2.75; Br, 15.57; F, 3.70; N, 21.83; O, 9.35. Found (%):
(E)-N-(4-(2-(1H-Tetrazol-1-yl)pyridin-4-yloxy)-3-fluoro-
C, 45.89; H, 2.87; N, 21.52.
phenyl)-2-(3,4-dihydroxybenzylidene)hydrazinecarbox-
amide (10j)
(
E)-N-(4-(2-(1H-Tetrazol-1-yl)pyridin-4-yloxy)-3-fluoro-
1
Yield: 51%. m.p. 203–206°C. H NMR (300 MHz, DMSO-d
6
) d 10.60
phenyl)-2-(3,5-di-tert-butyl-2-hydroxybenzylidene)hydrazi-
(s, 1H, –NH–N), 10.16 (s, 1H, tetrazole CH), 9.38 (s, 1H, –OH), 9.14
necarboxamide (10f)
(s, 1H, –NH–CO), 8.96 (s, 1H, –OH), 8.55 (d, J ¼ 5.8 Hz, 1H, ArH),
1
Yield: 73%. m.p. 201–204°C. H NMR (300 MHz, DMSO-d
6
) d 11.16
7.92 (dd, J ¼ 13.5, 2.2 Hz, 1H, ArH), 7.82 (s, 1H, –N–CH), 7.64 (d,
(
(
7
s, 1H, –NH–N) 10.58 (s, 1H, –OH), 10.11 (s, 1H, tetrazole CH), 9.39
s, 1H, –NH–CO), 8.50 (d, J ¼ 5.8 Hz, 1H, ArH), 8.21 (s, 1H, –N–CH),
.81–7.74 (m, 1H, ArH), 7.48 (d, J ¼ 2.2 Hz, 1H, ArH), 7.38 (m, 2H,
J ¼ 8.8 Hz, 1H, ArH), 7.52 (d, J ¼ 2.2 Hz, 1H, ArH), 7.42 (t, J ¼ 9.0 Hz,
1H, ArH), 7.28 (d, J ¼ 1.7 Hz, 1H, ArH), 7.20 (dd, J ¼ 5.7, 2.3 Hz, 1H,
ArH), 7.08 (dd, J ¼ 8.2, 1.7 Hz, 1H, ArH), 6.79 (d, J ¼ 8.1 Hz, 1H, ArH).
ꢂ
ArH), 7.24 (d, J ¼ 2.1 Hz, 1H, ArH), 7.19–7.13 (m, 2H, ArH), 1.37 (s,
ESI-MS m/z: 448.8 [MꢂH] . Anal. calcd. for C20
8 4
H15FN O (%):
1
3
9
1
1
1
5
H, –CH
3
), 1.24 (s, 9H, –CH
3
). C NMR (DMSO-d
6
) d 167.08, 154.55,
C, 53.34; H, 3.36; F, 4.22; N, 24.88; O, 14.21. Found (%): C, 53.32;
H, 3.31; N, 24.92.
54.08, 152.55, 151.70, 148.68, 147.46, 142.28, 140.95, 139.57,
36.05, 134.26, 125.86, 125.42, 124.16, 118.10, 116.33, 112.74,
08.26,102.17, 35.11, 34.36, 31.78 (3C), 29.81 (3C). ESI-MS m/z:
(E)-N-(4-(2-(1H-Tetrazol-1-yl)pyridin-4-yloxy)-3-fluoro-
ꢂ
44.9 [MꢂH] . Anal. calcd. for C28
8 3
H31FN O (%): C, 61.53; H, 5.72;
phenyl)-2-(2,4-dihydroxybenzylidene)hydrazinecarbox-
F, 3.48; N, 20.50; O, 8.78. Found (%): C, 61.77; H, 5.61; N, 21.02.
amide (10k)
1
Yield: 59%. m.p. 209–211°C. H NMR (300 MHz, DMSO-d
6
) d 10.58
(
E)-N-(4-(2-(1H-Tetrazol-1-yl)pyridin-4-yloxy)-3-fluoro-
phenyl)-2-(2-hydroxybenzylidene)hydrazinecarboxamide
10g)
(
(
s, 1H, –NH–N), 10.18 (s, 1H, tetrazole CH), 10.00 (s, 1H, –OH), 9.73
s, 1H, –OH), 9.17 (s, 1H, –NH–CO), 8.56 (d, J ¼ 5.7 Hz, 1H, ArH),
(
8.19 (s, 1H, –N–CH), 7.91 (d, J ¼ 13.6 Hz, 1H, ArH), 7.76 (d,
1
Yield: 56%. m.p. 215–217°C. H NMR (300 MHz, DMSO-d
6
) d 10.74
J ¼ 8.6 Hz, 1H, ArH), 7.62 (d, J ¼ 9.4 Hz, 1H, ArH), 7.53 (s, 1H, ArH),
(
(
7
s, 1H, –NH–N), 10.11 (s, 1H, tetrazole CH), 9.99 (s, 1H, –OH), 9.18
7.42 (t, J ¼ 9.0 Hz, 1H, ArH), 7.25–7.17 (m, 1H, ArH), 6.33 (m, 2H,
ꢂ
s, 1H, –NH–CO), 8.50 (d, J ¼ 5.8 Hz, 1H, ArH), 8.27 (s, 1H, –N–CH),
ArH). ESI-MS m/z: 448.9 [MꢂH] . Anal. calcd. for C20
8 4
H15FN O (%):
.93 (d, J ¼ 6.9 Hz, 1H, ArH), 7.87 (dd, J ¼ 13.5, 2.4 Hz, 1H, ArH),
.58 (d, J ¼ 10.0 Hz, 1H, ArH), 7.48 (d, J ¼ 2.2 Hz, 1H, ArH), 7.38 (t,
C, 53.34; H, 3.36; F, 4.22; N, 24.88; O, 14.21. Found (%): C, 53.38; H,
3.32; N, 24.97.
7
J ¼ 9.0 Hz, 1H, ArH), 7.23–7.13 (m, 2H, ArH), 6.88–6.79 (m, 2H,
ꢂ
ArH). ESI-MS m/z: 432.9 [MꢂH] . Anal. calcd. for C20
8 3
H15FN O (%):
(E)-N-(4-(2-(1H-Tetrazol-1-yl)pyridin-4-yloxy)-3-fluoro-
C, 55.30; H, 3.48; F, 4.37; N, 25.80; O, 11.05. Found (%): C, 55.76; H,
.32; N, 25.87.
phenyl)-2-(3,4,5-trihydroxybenzylidene)hydrazinecarbox-
3
amide (10l)
1
Yield: 49%. m.p. 210–212°C. H NMR (300 MHz, DMSO-d
6
) d 10.57
(E)-N-(4-(2-(1H-Tetrazol-1-yl)pyridin-4-yloxy)-3-fluoro-
(
(
s, 1H, –NH–N), 10.17 (s, 1H, tetrazole CH), 9.44 (s, 2H, –OH), 9.20
s, 1H, –NH–CO), 8.55 (d, J ¼ 5.8 Hz, 1H, ArH), 8.44 (s, 1H, –OH),
phenyl)-2-(3-allyl-2-hydroxybenzylidene)hydrazinecarbox-
amide (10h)
8.17 (s, 1H, –N–CH), 7.89 (dd, J ¼ 13.5, 2.2 Hz, 1H, ArH), 7.58 (d,
1
Yield: 62%. m.p. 208–210°C. H NMR (300 MHz, DMSO-d
6
) d 10.75
J ¼ 8.9 Hz, 1H, ArH), 7.53 (d, J ¼ 2.2 Hz, 1H, ArH), 7.41 (t, J ¼ 9.1 Hz,
(
(
7
s, 1H, –NH–N), 10.44 (s, 1H, –OH), 10.17 (s, 1H, tetrazole CH), 9.42
s, 1H, –NH–CO), 8.55 (d, J ¼ 5.7 Hz, 1H, ArH), 8.27 (s, 1H, –N–CH),
1H, ArH), 7.23–7.13 (m, 2H, ArH), 6.38 (d, J ¼ 8.6 Hz, 1H, ArH). ESI-
ꢂ
MS m/z: 464.7 [MꢂH] . Anal. calcd. for C20
8 5
H15FN O (%): C, 51.51;
.84 (d, J ¼ 13.6 Hz, 1H, ArH), 7.54–7.39 (m, 4H, ArH), 7.20 (dd,
H, 3.24; F, 4.07; N, 24.03; O, 17.15. Found (%): C, 51.55; H, 3.17;
N, 23.95.
J ¼ 6.0, 1.6 Hz, 1H, ArH), 7.15 (d, J ¼ 7.1 Hz, 1H, ArH), 6.89 (t,
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
48
M. Qin et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 840–850
(
E)-N-(4-(2-(1H-Tetrazol-1-yl)pyridin-4-yloxy)-3-fluoro-
8.56 (d, J ¼ 5.8 Hz, 1H, ArH), 8.41 (s, 1H, –N–CH), 8.37 (d, J ¼ 7.9 Hz,
1
7
H, ArH), 7.91 (d, J ¼ 13.4 Hz, 1H, ArH), 7.72–7.62 (m, 2H, ArH),
phenyl)-2-(3,4,5-trimethoxybenzylidene)hydrazinecarbox-
.52 (s, 1H, ArH), 7.45 (t, J ¼ 8.0 Hz, 2H, ArH), 7.21 (d, J ¼ 3.7 Hz, 1H,
amide (10m)
ꢂ
1
ArH). ESI-MS m/z: 484.8 [MꢂH] . Anal. calcd. for C20
2 8 2
H13Cl FN O
Yield: 62%. m.p. 216–218°C. H NMR (300 MHz, DMSO-d
6
) d
(
%): C, 49.30; H, 2.69; Cl, 14.55; F, 3.90; N, 23.00; O, 6.57. Found (%):
1
–
0.96 (s, 1H, –NH–N), 10.18 (s, 1H, tetrazole CH), 9.30 (s, 1H,
NH–CO), 8.56 (d, J ¼ 5.8 Hz, 1H, ArH), 7.97 (m, 1H, ArH), 7.91
s, 1H, –N–CH), 7.64 (d, J ¼ 8.9 Hz, 1H, ArH), 7.51 (d, J ¼ 2.2 Hz, 1H,
C, 49.37; H, 2.57; N, 23.06.
(
ArH), 7.45 (t, J ¼ 9.0 Hz, 1H, ArH), 7.21 (dd, J ¼ 5.7, 2.2 Hz, 1H, ArH),
(E)-N-(4-(2-(1H-Tetrazol-1-yl)pyridin-4-yloxy)-3-fluoro-
7
.16 (s, 2H, ArH), 3.87 (s, 6H, –OCH
3
), 3.70 (s, 3H, –OCH
3
). ESI-MS
phenyl)-2-(2,4-dichlorobenzylidene)hydrazinecarbox-
ꢂ
m/z: 506.8 [MꢂH] . Anal. calcd. for C23
8 5
H21FN O
(%): C, 54.33; H,
amide (10r)
4
2
.16; F, 3.74; N, 22.04; O, 15.73. Found (%): C, 54.31; H, 4.20; N,
1.97.
1
Yield: 71%. m.p. 214–216°C. H NMR (300 MHz, DMSO-d
6
) d 11.19
(s, 1H, –NH–N), 10.17 (s, 1H, tetrazole CH), 9.35 (s, 1H, –NH–CO),
8
1
.56 (d, J ¼ 5.8 Hz, 1H, ArH), 8.42 (d, J ¼ 8.5 Hz, 1H, ArH), 8.34 (s,
(
E)-N-(4-(2-(1H-Tetrazol-1-yl)pyridin-4-yloxy)-3-fluoro-
H, –N–CH), 7.91 (dd, J ¼ 13.3, 2.1 Hz, 1H, ArH), 7.70 (d, J ¼ 1.9 Hz,
phenyl)-2-(2,4-dimethoxybenzylidene)hydrazinecarbox-
1H, ArH), 7.64 (d, J ¼ 9.2 Hz, 1H, ArH), 7.55–7.49 (m, 2H, ArH),
amide (10n)
7.45 (t, J ¼ 9.1 Hz, 1H, ArH), 7.21 (dd, J ¼ 5.8, 2.0 Hz, 1H, ArH).
1
ꢂ
ESI-MS m/z: 486.7 [MꢂH] . Anal. calcd. for C20
2 8 2
H13Cl FN O (%):
Yield: 57%. m.p. 212–214°C. H NMR (300 MHz, DMSO-d
6
) d 10.71
(s, 1H, –NH–N), 10.16 (s, 1H, tetrazole CH), 9.15 (s, 1H, –NH–CO),
C, 49.30; H, 2.69; Cl, 14.55; F, 3.90; N, 23.00; O, 6.57. Found (%):
C, 49.34; H, 2.61; N, 22.89.
8
1
.55 (d, J ¼ 5.8 Hz, 1H, ArH), 8.24 (s, 1H, –N–CH), 8.12 (d, J ¼ 8.4 Hz,
H, ArH), 7.93 (dd, J ¼ 13.5, 2.4 Hz, 1H, ArH), 7.65 (d, J ¼ 8.9 Hz, 1H,
ArH), 7.52 (d, J ¼ 2.2 Hz, 1H, ArH), 7.42 (t, J ¼ 9.1 Hz, 1H, ArH), 7.19
(E)-N-(4-(2-(1H-Tetrazol-1-yl)pyridin-4-yloxy)-3-fluoro-
(
3
dd, J ¼ 5.7, 2.3 Hz, 1H, ArH), 6.61 (d, J ¼ 8.4 Hz, 2H, ArH), 3.85 (s,
ꢂ
phenyl)-2-(benzo[d][1,3]dioxol-5-ylmethylene)hydrazine-
carboxamide (11a)
Yield: 53%. m.p. 231–233°C. H NMR (300 MHz, DMSO-d
H, –OCH
3
), 3.83 (s, 3H, –OCH
3
). ESI-MS m/z: 476.9 [MꢂH] . Anal.
calcd. for C22
1
H
19FN
8
O
4
(%): C, 55.23; H, 4.00; F, 3.97; N, 23.42; O,
1
3.38. Found (%): C, 55.27; H, 3.96; N, 23.50.
6
)
d 10.84 (s, 1H, –NH–N), 10.20 (s, 1H, tetrazole CH), 9.23 (s,
H, –NH–CO), 8.57 (d, J ¼ 5.8 Hz, 1H, ArH), 7.98–7.91 (m, 2H,
ArH), 7.77 (s, 1H, –N–CH), 7.69 (d, J ¼ 8.8 Hz, 1H, ArH),
1
(
E)-N-(4-(2-(1H-Tetrazol-1-yl)pyridin-4-yloxy)-3-fluoro-
phenyl)-2-(4-hydroxy-3,5-dimethoxybenzylidene)hydrazi-
necarboxamide (10o)
Yield: 61%. m.p. 217–219°C. H NMR (300 MHz, DMSO-d
7.54 (d, J ¼ 1.6 Hz, 1H, ArH), 7.46 (t, J ¼ 9.1 Hz, 1H, ArH), 7.22
(
(
d, J ¼ 3.7 Hz, 1H, ArH), 7.16 (d, J ¼ 8.2 Hz, 1H, ArH), 6.98
1
6
) d 10.76
d, J ¼ 8.0 Hz, 1H, ArH), 6.11 (s, 2H, –O–CH
–O–). ESI-MS
2
ꢂ
(s, 1H, –NH–N), 10.13 (s, 1H, tetrazole CH), 9.20 (s, 1H, –NH–CO),
m/z: 460.8 [MꢂH] . Anal. calcd. for C21
H
15FN
8
O
4
(%): C, 54.55;
8
1
.73 (s, 1H, –OH), 8.51 (d, J ¼ 5.7 Hz, 1H, ArH), 7.90 (d, J ¼ 13.5 Hz,
H, 3.27; F, 4.11; N, 24.23; O, 13.84. Found (%): C, 54.47; H, 3.23;
N, 24.29.
H, ArH), 7.83 (s, 1H, –N–CH), 7.60 (d, J ¼ 9.3 Hz, 1H), 7.47 (s, 1H,
ArH), 7.39 (t, J ¼ 9.0 Hz, 1H, ArH), 7.16 (d, J ¼ 5.7 Hz, 1H, ArH), 7.08
ꢂ
(s, 2H, ArH), 3.80 (s, 6H, –OCH
3
). ESI-MS m/z: 492.9 [MꢂH] . Anal.
calcd. for C22
H
19FN
8
O
5
(%): C, 53.44; H, 3.87; F, 3.84; N, 22.66; O,
(E)-N-(4-(2-(1H-Tetrazol-1-yl)pyridin-4-yloxy)-3-fluoro-
1
6.18. Found (%): C, 53.39; H, 3.91; N, 22.72.
phenyl)-2-((2-hydroxynaphthalen-1-yl)methylene)hydrazi-
necarboxamide (11b)
1
(
E)-N-(4-(2-(1H-Tetrazol-1-yl)pyridin-4-yloxy)-3-fluoro-
Yield: 46%. m.p. 221–225°C. H NMR (300 MHz, DMSO-d
6
) d 10.78
(
9
s, 1H, –NH–N), 10.19 (s, 1H, tetrazole CH), 9.46 (s, 1H, –NH–CO),
.01 (s, 1H, –N–CH), 8.56 (d, J ¼ 5.7 Hz, 1H, ArH), 8.44 (d, J ¼ 8.5 Hz,
phenyl)-2-(2,6-dichlorobenzylidene)hydrazinecarbox-
amide (10p)
1
1H, ArH), 7.89 (dd, J ¼ 9.1, 4.0 Hz, 3H, ArH), 7.63–7.56 (m, 1H, ArH),
Yield: 56%. m.p. 223–226°C. H NMR (600 MHz, DMSO-d
6
) d 11.28
7
.54 (s, 1H, –OH), 7.52–7.34 (m, 4H, ArH), 7.23 (m, 2H, ArH). ESI-MS
(s, 1H, –NH–N), 10.19 (s, 1H, tetrazole CH), 9.14 (s, 1H, –NH–CO),
ꢂ
m/z: 482.9 [MꢂH] . Anal. calcd. for C24
8 3
H17FN O (%): C, 59.50; H,
8
2
.55 (d, J ¼ 5.8 Hz, 1H, ArH), 8.18 (s, 1H, –N–CH), 7.89 (dd, J ¼ 13.3,
3
2
.54; F, 3.92; N, 23.13; O, 9.91. Found (%): C, 59.53; H, 3.51; N,
3.08.
.4 Hz, 1H, ArH), 7.60–7.55 (m, 3H, ArH), 7.53 (d, J ¼ 2.2 Hz, 1H,
ArH), 7.43 (dt, J ¼ 15.5, 8.5 Hz, 2H, ArH), 7.20 (dd, J ¼ 5.7, 2.3 Hz,
1
3
1
1
1
6
H, ArH). C NMR (DMSO-d ) d 167.05, 153.14, 152.82, 151.69,
48.67, 142.30, 139.12, 136.76, 134.53 (2C), 131.46, 130.81,
(
E)-2-((1H-Indol-3-yl)methylene)-N-(4-(2-(1H-tetrazol-1-yl)-
pyridin-4-yloxy)-3-fluorophenyl)hydrazinecarboxamide
11c)
29.55 (2C), 124.03, 116.72, 112.74, 108.63, 108.48, 102.18. ESI-MS
ꢂ
m/z: 484.7 [MꢂH] . Anal. calcd. for C20
2 8 2
H13Cl FN O (%): C, 49.30;
(
H, 2.69; Cl, 14.55; F, 3.90; N, 23.00; O, 6.57. Found (%): C, 49.35;
H, 2.67; N, 23.07.
1
Yield: 52%. m.p. 219–221°C. H NMR (300 MHz, DMSO-d
6
) d 11.57
(s, 1H, –NH–N), 10.55 (s, 1H, indole NH), 10.19 (s, 1H, tetrazole
CH), 8.97 (s, 1H, –NH–CO), 8.57 (s, 1H, –N–CH), 8.23 (m, 2H,
ArH), 7.96 (d, J ¼ 12.7 Hz, 1H, ArH), 7.84 (m, 1H, ArH), 7.60–7.55
(
E)-N-(4-(2-(1H-Tetrazol-1-yl)pyridin-4-yloxy)-3-fluoro-
phenyl)-2-(2,3-dichlorobenzylidene)hydrazinecarbox-
(m, 2H, ArH), 7.45 (m, 2H, ArH), 7.21 (m, 3H, ArH). ESI-MS
ꢂ
amide (10q)
m/z: 455.7 [MꢂH] . Anal. calcd. for C22
9 2
H16FN O (%): C, 57.77;
1
H, 3.53; F, 4.15; N, 27.56; O, 7.00. Found (%): C, 57.73; H, 3.58;
N, 27.53.
Yield: 63%. m.p. 220–222°C. H NMR (300 MHz, DMSO-d
6
) d 11.23
(s, 1H, –NH–N), 10.17 (s, 1H, tetrazole CH), 9.35 (s, 1H, –NH–CO),
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 840–850
Semicarbazone-Containing Antitumor Agents
849
Pharmacology
calculated using the following equation: % inhibition ¼ 100
ꢂ [(activity of enzyme with tested compounds ꢂ min)/(max
ꢂ min)] ꢁ 100. The results were expressed as percentage
inhibition from two independent experiments.
MTT assay in vitro
The antitumor activities of compounds 10a–10r, 11a–11c
were evaluated against HT-29, H460, MKN-45 cancer cell lines,
and WI-38 normal cell line by the standard MTT assay in vitro,
with sorafenib and PAC-1 as references. The cancer cell lines
were cultured in minimum essential medium (MEM)
supplemented with 10% fetal bovine serum (FBS).
Western blot analysis
7
About 1 ꢁ 10 H460 cells were gathered after pre-treatment
with indicated concentrations of compound 10p for 48 h.
Protein lysates were prepared using lysis buffer (10ꢁ) (Cell
Signalling Technology, Danvers, MA, USA) and protease
inhibitor cocktail (Sigma–Aldrich). All samples were sonicated
at 10 Hz for 10 s. An equal amount of total protein extracts
from cultured cells or tissues were fractionated by 10% SDS–
PAGE and electrically transferred onto polyvinylidene difluor-
ide (PVDF) membranes. Mouse or rabbit primary antibodies
and horseradish peroxidase (HRP)-conjugated appropriate
secondary antibodies were used to detect the designated
proteins. The bound secondary antibodies on the PVDF
membrane were reacted with ECL detection reagents (Thermo
Scientific) and exposed to X-ray films. The proportion of cleaved
caspase-3 was normalized to the internal control b-actin.
3
Approximately 4 ꢁ 10 cells, suspended in MEM medium,
were plated onto each well of a 96-well plate and incubated in
5
% CO at 37°C for 24 h. The tested compounds were added to
2
the culture medium and the cell cultures were continued for
2 h. Fresh MTT was added to each well at a terminal
concentration of 5 mg/mL and incubated with cells at 37°C for
h. The formazan crystals were dissolved in 100 mL of DMSO
7
4
each well, and the absorbency at 492 nm (for absorbance of
MTT formazan) and 630 nm (for the reference wavelength) was
measured with an ELISA reader. All the compounds 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
This work was supported by Program for Innovative Research Team of
the Ministry of Education and Program for Liaoning Innovative
Research Team in University. Also, we were supported by a grant from
National Natural Science Foundation of China (No. 21002065 and
81102470) and a grant from National High Technology Research and
Development Program of China (No. 2012AA020305). The PhD
studentship was provided by Syngenta Ltd.
(
Bliss) software.
Enzymatic assay
The kinase inhibitory measurements versus c-Met (Cat: 14-526,
Millipore), K < PREFIX > DR </PREFIX > (Cat: K2643, Sigma),
CRAF (Cat: PV3805, Invitrogen), PDGFRa (Cat: PV3811,
Invitrogen) were evaluated using homogeneous time-resolved
fluorescence (HTRF) assay. All experiments were performed in
NUNC 264706 white 384-well low-volume plates (Cat.
The authors have declared no conflict of interest.
6
007290, PerkinElmer).
In CRAF kinase assay, a mixture of peptide substrates, ATP
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