Synthesis, anticancer, and docking studies of salicyl-hydrazone analogues: A novel series of small potent tropomyosin receptor kinase A inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.11.005

A series of novel salicyl-hydrazone analogues were synthesized and evaluated for their in vitro cytotoxic activities in five human cancer cell lines, namely, lung cancer (A549), ovarian cancer (SK-OV-3), skin cancer (SK-MEL-2), colon cancer (HCT15) and pancreatic cancer (MIA-PaCa-2) cells, and for their in vitro tropomyosin receptor kinase A (TrkA) inhibitory activities. Each of the compounds showed significant cytotoxicity against all cancer cells. Compound 3i was found to be most potent against all cancer cell lines with IC₅₀values of 2.46 (A549), 0.87 (SK-OV-3), 1.43 (SK-MEL-2), 0.89 (HCT15), and 0.48 μM (MIA-PaCa-2), followed by compound 3l. Cytotoxicity of 3i was similar to that of doxorubicin (0.87 μM) against HCT15 cells. Compounds 3i and 3l also showed highest TrkA inhibitory activities with IC₅₀values of 0.231 and 0.380 μM, respectively. A SAR study of the series revealed that compounds with hydroxyl groups showed better cytotoxicity and TrkA inhibitory potency (in the following order 2,4-OH > 2,3,4-OH > 3,4-OH > 4-OH) than compounds possessing electron donating or withdrawing groups on the benzylidenephenyl ring. Docking studies of compounds 3i and 3l conducted on the crystal structure of TrkA receptor (a promising target for anticancer agents) showed both had a high docking score and similar order of experimental TrkA inhibitory activities. The formation of several hydrogen bonds involving N and O containing moieties contributed most significantly to ligand binding and stabilization at the active site of the receptor. In addition, ligand-receptor complexes were further stabilized by π-cation, π-anion, amide-π stacked, and van der Waal's interactions. Conformational analyses showed ligand molecules adopted similar conformations at the receptor active site during interactions, but that the low energy optimized conformations of compounds 3i and 3l differed.

Full text of DOI:10.1016/j.bmc.2016.11.005

Accepted Manuscript
Synthesis, anticancer, and docking studies of salicyl-hydrazone analogues: A
novel series of small potent tropomyosin receptor kinase A inhibitors
Mohammad Sayed Alam, Sang-Un Choi, Dong-Ung Lee
PII:
S0968-0896(16)31112-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.11.005
BMC 13371
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
11 September 2016
1 November 2016
2 November 2016
Please cite this article as: Sayed Alam, M., Choi, S-U., Lee, D-U., Synthesis, anticancer, and docking studies of
salicyl-hydrazone analogues: A novel series of small potent tropomyosin receptor kinase A inhibitors, Bioorganic
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Synthesis, anticancer, and docking studies of salicyl-hydrazone analogues: a
novel series of small potent tropomyosin receptor kinase A inhibitors
a,b
c
a,
Mohammad Sayed Alam , Sang-Un Choi , Dong-Ung Lee *
a
Division of Bioscience, Dongguk University, Gyeongju 780-714, Republic of Korea
b
Department of Chemistry, Jagannath University, Dhaka 1100, Bangladesh
c
Center for Drug Discovery Technology, Korea Research Institute of Chemical Technology, 141
Gajeongro, Daejeon 34114, Republic of Korea
*
Corresponding author. Tel. +82 54 742 2224; fax: +82 54 742 9833.
E-mail address: dulee@dongguk.ac.kr (D.U. Lee)

ABSTRACT
A series of novel salicyl-hydrazone analogues were synthesized and evaluated for their in vitro
cytotoxic activities in five human cancer cell lines, namely, lung cancer (A549), ovarian cancer
(
SK-OV-3), skin cancer (SK-MEL-2), colon cancer (HCT15) and pancreatic cancer (MIA-PaCa-
) cells, and for their in vitro tropomyosin receptor kinase A (TrkA) inhibitory activities. Each of
2
the compounds showed significant cytotoxicity against all cancer cells. Compound 3i was found
to be most potent against all cancer cell lines with IC50 values of 2.46 (A549), 0.87 (SK-OV-3),
1
.43 (SK-MEL-2), 0.89 (HCT15), and 0.48 M (MIA-PaCa-2), followed by compound 3l.
Cytotoxicity of 3i was similar to that of doxorubicin (0.87 M) against HCT15 cells.
Compounds 3i and 3l also showed highest TrkA inhibitory activities with IC50 values of 0.231
and 0.380 M, respectively. A SAR study of the series revealed that compounds with hydroxyl
groups showed better cytotoxicity and TrkA inhibitory potency (in the following order 2,4-OH>
2
,3,4-OH>3,4-OH> 4-OH) than compounds possessing electron donating or withdrawing groups
on the benzylidenephenyl ring. Docking studies of compounds 3i and 3l conducted on the crystal
structure of TrkA receptor (a promising target for anticancer agents) showed both had a high
docking score and similar order of experimental TrkA inhibitory activities. The formation of
several hydrogen bonds involving N and O containing moieties contributed most significantly to
ligand binding and stabilization at the active site of the receptor. In addition, ligand-receptor
complexes were further stabilized by -cation, -anion, amide- stacked, and van der Waal’s
interactions. Conformational analyses showed ligand molecules adopted similar conformations at
the receptor active site during interactions, but that the low energy optimized conformations of
compounds 3i and 3l differed.

Keywords: Salicyl-hydrazones; Cytotoxicity; TrkA inhibitor; Docking study; Conformational
analysis
1
.
Introduction
Tropomyosin receptor kinases (Trks) are a small subfamily of the largest family of protein
kinases, and are central regulators of signal transduction and controllers of various complex
1
,2
cellular processes. Trk receptor plays a crucial role in the development and maintenance of the
central and peripheral nervous system by activating neurotrophins (a family of high affinity
2
growth factors). The Trk subfamily includes three highly homologues isoforms, namely, TrkA,
TrkB and TrkC (also known as NTRK1, NTRK2 and NTRK3, respectively), which are activated
3
by nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), by and neurotrophin-
4
5
4
/5 (NT-4/5) or neurotrophin-3 (NT-3), respectively. TrkA protein is highly expressed in
sympathetic, trigeminal, and dorsal root ganglia and in cholinergic neurons of the basal forebrain
6
,7
and striatum. TrkB is expressed in the central and peripheral nervous systems, whereas TrkC is
8
,9
widely found in mammalian neural tissues. Non-neuronal tissues, such as, salivary glands,
stomach, intestines, pancreas, bone marrow, adrenal glands, prostate, ovary, uterus, skeletal
muscle and skin also produce Trk receptors in man. All three Trk receptors contain three
segments, specifically, an extracellular ligand binding domain, a transmembrane domain, and an
intracellular kinase domain. The binding of specific ligands activates receptor oligomerization,
induces phosphorylation of specific tyrosine residues in the receptor kinase domain, and
activates downstream signal transduction pathways, including survival, proliferation, and
1
0,11
differentiation pathways in normal and neoplastic neuronal cells.
In addition, experimental
evidence accumulated over the last two decades shows Trks are also involved in malignant
transformation, chemotaxis, metastasis, and survival signaling in human cancers, including,

1
2
13
14
15
16
17
prostate, pancreatic, colon, papillary thyroid, and lung cancer, breast carcinoma, acute
1
8
19
myelogenous leukemia, and neuroblastoma. Therefore, Trks are viewed as a promising target
for development of new drugs for the treatment of different human cancers.
A literature survey revealed much effort has been devoted over the past few decades to the
development of TrkA inhibitors as novel anticancer and antinociceptive drugs. These endeavors
have identified various TrkA inhibitors possessing pyrimidine, 4-amino-pyrazolopyrimidine
amide, pyridoquinoxaline, pyrrolidinyl urea, pyrrolopyrimidine, and alkyline-pyrimidine amide,
2
0,21
and isothiazole scaffold as a major structural feature,
The majority of TrkA inhibitors were
identified by the high-throughput screening of small molecule libraries, by using structural
tailoring of known inhibitors, or by in silico screening of chemical libraries of small molecules
using docking simulations. Figure 1 shows some TrkA inhibitors reported in the literature with a
common amide (-NHCO-) functional group and N and O donor moieties.

Figure 1. Chemical structures of several reported TrkA inhibitors and their reported IC50 values.
Small bioactive molecules are attracting growing interests in the therapeutic chemistry field.
Salicylic acid (2-hydroxybenzoic acid) derivatives are a class of small molecules with
2
2
surprisingly complex biological properties. Chemically, salicylic acid possesses an aromatic
ring with a hydroxyl group or a functionalized hydroxyl group. This scaffold is found in a
variety of natural and synthetic bioactive compounds used as pharmaceuticals and
agrochemicals. Salicylic acid and its functional analogues have attracted continued interest in
medicinal chemistry field during the last several decades due to their wide ranging biological

activities. For example, acetylsalicylic acid (aspirin), salicylamide, methyl salicylate, diflunisal,
-amino salicylic acid, salicylic acid azides (mesalazine, olsalizine, and balsazide), furo-salicylic
4
acid, salicin, saligenin, salicyl-hydrazones, -pyrazoles, and -oxadiazoles are used as analgesic,
antipyretic, antiinflammatory, antibacterial, antifungal, antiulcerogenic, antiplatelet,
2
3-31
32
antitubercular, antioxidant, and sedative-hypnotic agents.
Furthermore, Gasco et al.
suggested o-acyl salicylic acid derivatives could be use as anticancer agents, particular for the
treatment of colon, bladder, and prostate cancer.
During our continuing studies on the identification of novel biologically active molecules,
we synthesized a series of salicyl-hydrazone analogues possessing an amide (-NHCO-)
functional group and N and O donor moieties and evaluated their in vitro anticancer activities
against five human cancer cell lines, namely, lung cancer (A549), ovarian cancer (SK-OV-3),
skin cancer (SK-MEL-2), colon cancer (HCT15) and pancreatic cancer (MIA-PaCa-2) cells. The
TrkA inhibitory activities of the synthesized compounds were evaluated using in vitro enzyme
assay to investigate the mechanism responsible for their anticancer effects. Finally, docking
simulation was performed using the X-ray crystallographic structure of TrkA receptor to
investigate binding at its active site. To the best of our knowledge, this report is the first example
to describe the TrkA inhibitory effects of salicylic acid derivatives.
2
2
. Results and discussion
.1. Synthesis and characterization
A series of new salicyl-hydrazone analogues (3a-l) were synthesized using a convenient and
straightforward two-step reaction process under mild condition in ethanolic solution (Scheme 1).

Scheme 1. The synthesis of salicyl-hydrazone analogues.
Overall isolated yields of the synthesized compounds were excellent (68.37- 87.5 %).
1
Structural characterizations were performed by FT-IR, H NMR, mass spectrometry, and
elemental analysis. The FT-IR spectra of compounds 3a-l showed characteristic amide >C=O
-1
and –NH- group stretch absorption bands around 1628–1665 and 3112-3268 cm , respectively.
-1
1
Phenolic –OH stretch absorption bands were observed at around 3200-3561 cm . In the H NMR
spectra of compounds 3a-l, azomethine (–CH=N-) and amide (-NHCO-) protons showed
characteristic singlets at 8.30-8.87 and 11.60-12.17 ppm, respectively, equivalent to one proton

each. The N,N-dimethyl (-N(CH
3
)
2
) and aromatic methyl (Ar-CH ) protons of 3e and 3f
3
appeared as singlets at 2.97 and 2.35 ppm equivalent to six and three protons, respectively, while
the methoxy protons of compounds 3h and 3j were observed at around 3.83-3.89 ppm as a
singlet equivalent to three proton each. Other aromatic protons expected peaks that depended on
substitution pattern. In their EI-mass spectra, compounds 3a-l produced molecular ion peaks with
intensities of 7-93%.
2
.2. Cytotoxic activities
The in vitro cytotoxic activities of salicyl-hydrazone analogues (3a-l) were screened against
five human carcinoma cell lines, that is, lung cancer (A549), ovarian cancer (SK-OV-3), skin
cancer (SK-MEL-2), colon cancer (HCT15) and pancreatic cancer (MIA-PaCa-2) cells using the
3
3
sulforhodamine-B (SRB) method. To evaluate cytotoxic activities, inhibitions of net cell
proliferation were measured after incubating cells with test samples for 48 h. Inhibitions are
expressed as percentages of non-treated controls. The cytotoxicities of compounds (3a-l) are
presented in Table 1, and their activities were compared with doxorubicin (a standard anticancer
agent), which was used as a positive control. All 12 compounds studies showed cytotoxic
activities against the four cancer cell lines. Among them, compound 3i exhibited greatest
cytotoxic effect against all cancer cell lines, followed in decreasing order by compounds 3l, 3g
3
a, and 3k. Compound 3i had a cytotoxic potency similar to doxorubicin against HCT15 cancer
cells (IC50 0.89 M vs 0.872 M), but lower potency against A549 (2.46 M), SK-OV-3 (IC50
.87 M) SK-MEL-2 (1.43 M) and MIA-PaCa-2 (0.48 M) cancer cells (corresponding IC50
values for doxorubicin were 0.021, 0.74, 0.001 and 0.049 M, respectively).
0

Table 1
In vitro cytotoxicity data of the salicyl-hydrazone analogues (3a-l)
a
IC50 values/M
Comp.
b
c
d
e
f
A549
.78
2.19
SK-OV-3
SK-MEL-2
HCT15
MIA-PaCa-2
2.46
3
3
a
2
5.94
9.83
9.77
14.28
9.34
21.31
20.85
22.67
34.59
38.39
26.91
8.60
b
7.19
2
3
c
6.73
8
.72
8.80
3
d
14.04
20.67
4.45
2
2
3.15
1.48
19.61
18.52
9.81
19.40
19.33
13.05
5.46
3
e
3
f
6
.74
.86
3
3
g
1.18
2
9.10
h
8.64
1
3
2.92
14.36
0.87
15.17
1.43
28.06
0.89
3
i
0.48
2
.46
3
j
11.32
4.74
1.27
14.52
9.14
19.61
8.86
25.74
10.03
3
k
3
.15
.16
3
l
2.21
7
1.96
1.84
3.71
Doxorubicin 0.021
0.074
0.001
0.872
0.049
a
IC50 values were obtained using dose response curves by nonlinear regression using a curve
b
c
d
fitting program (OriginPro 7.5). human lung cancer, human ovarian cancer, human skin cancer,
e
f
human colon cancer and human pancreatic cancer.
The structure activity relationship study revealed that the natures and positions of
substituents on the benzylidenephenyl ring have significant effects on cytotoxic activities.
Compound 3i with hydroxyl groups (OH) at ortho- and para- positions in benzylidenephenyl
ring showed greater cytotoxicity against all five cell lines than 3a, which has a non-substituted
benzylidenephenyl ring. Whereas, compound 3k with hydroxyl groups at meta- and para-

position in its benzylidenephenyl ring had less cytotoxic effect on all cell lines than compound 3i.
However, compound 3k showed similar activity against A549 (IC50 3.15 M) and SK-MEL-2
(
IC50 8.86 M) cells, but less and more activity against SK-OV-3 (IC50 9.14 M), HCT15 (IC50
0.03 M) and MIA-PaCa-2 (IC50 4.74 M) cells than 3a (Table 1). Interestingly, the
introduction of an additional hydroxyl group at the ortho-position in benzylidenephenyl ring of
k to produce 3l resulted in a 2-5 fold increase in activity against SK-OV-3 (IC50 1.96 M),
1
3
SK-MEL-2 (IC50 1.84 M), HCT15 (IC50 3.71 M) and MIA-PaCa-2 (IC50 2.21 M) cells but
reduced activity against A549 cells (IC50 7.16 M) by 2.5 fold. Compound 3g with one
hydroxyl group at the para-position of the benzylidenephenyl ring exhibited 2-3 times greater
activity against SK-MEL-2 (IC50 5.46 M), HCT15 (IC50 8.60 M) and MIA-PaCa-2 (IC50 1.18

M) cells but similar activity against A549 (IC50 2.86 M), and less activity against SK-OV-3
(
IC50 9.10 M), respectively, than 3a (IC50 values 9.77, 21.31, 2.78 , 5.94 and 2.46 M for SK-
MEL-2, HCT15, A549, SK-OV-3 and MIA-PaCa-2, respectively). Methylation of 3g and 3i to
produce 3h and 3j, respectively, resulted in significant activity reductions against all cell lines.
Introduction of an electron donating group to 3a, namely, N,N-dimethylamino (3e), methyl (3f),
or methoxy (3h), or an electron withdrawing group, namely, chloro (3b) or nitro (3c), at the
para-position of benzylidenephenyl ring reduced activity against all five cell lines. The above
results show the presence of a hydroxy (OH) group at the ortho- and para- positions in the
benzylidenephenyl ring increased cytotoxic activity against the cell lines used in the present
study. Additional cytotoxicity assays on different salicyl-hydrazone analogues are required to
confirm and expand structure activity relationships.
2
.3. TrkA inhibitory activity

The TrkA inhibitory activities of the salicyl-hydrazones (3a-l) were also examined. Results
are presented in Table 2. Compound 3i, which had greatest cytotoxic activity, also showed
greatest TrkA inhibitory activity (IC50: 0.231 M) followed by compound 3l (IC50: 0.380 M).
Table 2
TrkA kinase inhibitory activities of the salicyl-hydrazone analogues (3a-l)
TrkA
TrkA
Compound
Compound
a
a
inhibition (%)
inhibition (%)
-
3
a
3g
20.62
-
3
b
6.92
3h
b
3
c
-
3i
92.38 (0.2315)
7.61
3
d
-
-
3j
b
b
b
3
e
3k
3l
92.88 (2.545)
90.90 (0.380)
>99.0 (0.003)
3
f
3.70
GW441756
a
b
Inhibition rate at 1 M. Values in parentheses represent IC50 (M). “-” : Not active at a
concentration of 1 M
Compound 3k also showed significant TrkA inhibitory activity with an IC50 of 2.545 M. The
other nine compounds exhibited low or no activity at 1 M. GW441756 (1,3-Dihydro-3-[(1-
3
4,35
methyl-1H-indol-3-yl)methylene]-2H-pyrrolo[3,2-b]pyridin-2-one),
an oxindole analogue
has been used as a positive control to compare the TrkA inhibitory activity of the synthesized
compounds. GW441756 is well-known potent selective TrkA inhibitor which displays 100-fold
greater selectivity over a range of other kinases. However, GW441756 exerted lower IC50 value
of 3 nM compare to that of salicyl-hydrazone analogues. Interestingly, compounds with a

hydroxyl group in the benzylidenephenyl ring exhibited excellent TrkA inhibitory activities in
the following order 2,4-OH> 2,3,4-OH>3,4-OH> 4-OH.
2
.4. Docking studies
To predict interaction modes and affinities and provide a guide for further SAR studies,
simulations were performed on the docking of compounds 3i and 3l into the active site of TrkA
receptor. The crystal structure of TrkA receptor was obtained from the Protein Data Bank (PDB
ID: 4f0i), and a summary of the docking studies is presented in Table 3.
Table 3
Docking energies and molecular interactions of salicyl-hydrazone ligands with TrkA receptor.
Binding
affinity
Energy contribution
Average
conpair
H-bonding residues
(distance, Å)
Entry
Other interacting residues
vdW
H-bond
-49.075
(
Kcal/mol)
Glu560, Leu564, Phe589,
Phe646, Asp650, Gly667,
Asp668, Met671, Ser672,
Tyr680
His648 (2.65) Asn655
(2.63) Gly670 (2.38)
Arg673(2.54)
3
3
i
l
-107.506
-103.194
-58.431
23.350
22.905
His648 (2.35)
Asn655 (2.74)
Asp668 (2.76)
Ser672 (2.38)
Glu560, Leu564, Phe589,
Phe646, Ile666, Asp650,
Gly667, Gly670, Arg673,
Met671, Tyr680
-60.873
-42.321
The results obtained showed compounds 3i and 3l bind to the same active site as previously
3
6,37
reported for TrkA inhibitors
by hydrogen bonding and hydrophobic, electrostatic, covalent,
and Van der Wall’s interactions. Calculated binding modes and interactions of 3i and 3l are
shown in Figs. 2 and 3, respectively.

(a)
(b)
MET671
GLY670
ASP668
2
.38 4.24
4
.81
2
.54
2
.65
3
.80
2
.63
3
.23
ARG673
ASN655
HIS648
(d)
(c)
MET671
GLY670
ASP668
ARG673
ASN655
HIS648
Figure 2. (a) A general view of a ligand/TrkA receptor complex. The black square shows the zone of
ligand-TrkA interactions. (b) Binding of compound 3i with TrkA receptor (Protein Data Bank ID: 4f0i).
The green dotted lines represent hydrogen bonds, the gold dotted lines -cation or anion interactions, the
pink dotted line shows an amide- stacked interaction, and the light green dotted line a -donor
interaction. (c) 2D ligand interaction diagram with TrkA receptor obtained using the Discovery Studio
program; essential amino acid residues at the binding site are circled. The purple circles show amino
acids involved in electrostatic and covalent interactions and the green circles show amino acids exhibiting
van der Waals interactions. (d) Hydrogen bond interaction showing donor (pink) and acceptor surfaces
(green).

ASP668
(a)
(b)
SER672
ASN655
.74
2
.76
2
2
.38
2.35
4
.09
HIS648
ARG673
ASP668
(c)
(d)
SER672
ASN655
HIS648
ARG673
Figure 3. (a) A general view of a ligand-TrkA receptor complex. The black square shows the zone of
ligand-kinase interactions. (b) Binding of compound 3l with TrkA receptor (Protein Data Bank ID: 4f0i).
The green dotted lines represents hydrogen bonds, and the gold dotted lines -cation or anion interactions.
(c) 2D ligand interaction diagram with TrkA receptor obtained using the Discovery Studio program;
essential amino acid residues at the binding site are circled. The purple circles show amino acids involved
in electrostatic and covalent interactions and the green circles amino acids involved in van der Waals
interactions. (d) Hydrogen bond interaction showing donor (pink) and acceptor surfaces (green).
Both compounds interacted with the same amino acid residues with few exceptions, and
both formed four hydrogen bonds, which appeared to contribute most to ligand stabilization in
the binding site. Binding scores for compounds 3i, and 3l were calculated using iGEMDOCK
3
8
software and their binding affinities with TrkA receptor were found to be -107.506 and -
-1
1
03.194 kcalmol , respectively, with hydrogen bond contributions of -49.075 and -42.321

-
1
-1
kcalmol , respectively, and van der Waal contributions of 58.431 and 60.873 kcalmol ,
respectively. Specifically, 3i forms hydrogen bonds using its carbonyl oxygen atom with His648
(
HO=C<: 2.65Å), and the ortho- and para-hydroxyl groups of its benzylidene phenyl ring
bond with Asn655 (OHO: 2.63Å), Gly670 (HOH: 2.38Å), and Arg673 (OHO: 2.54Å)
amino acid residues. On the other hand, compound 3l hydrogen bonds using the ortho-hydroxyl
group of its benzoyl phenyl ring with His648 (HOH: 2.35Å) and Asp668 (OHO: 2.76Å),
and using the meta- and para-hydroxyl groups of its benzylidene phenyl ring with the amino acid
residues of Asn655 (OHO: 2.74Å) and Ser672 (HOH: 2.38Å). Compounds 3i and 3l
were further stabilized in the binding site by a -cation interaction between the
benzylidenephenyl ring and Arg673 and by -anion interaction between the benzoylphenyl ring
of 3i and benzylidenephenyl ring of 3l and Asp668. In addition to these interactions, compounds
3
i and 3l also exhibited van der Waal’s interactions with Glu560, Leu564, Phe589, Phe646 (only
for 3i), Asp650, Gly667, Met671, Ser672 (only for 3i), and Tyr680 and Ile666 (only for 3l). In
addition, an amide- stacked interaction was also observed between Gly670 and the
benzylidenephenyl ring of 3i. The above results suggest the additional electrostatic and van der
Waal’s interactions of 3i explain its lower total binding energy and resulted in highest potency
(
TrkA IC50 231 nm) compare to that of 3l (TrkA IC50 380 nm).
2
.5. Conformational analysis
Conformational analysis was performed to better understand the bioactive conformations of
the salicyl-hydrazone analogues by superimposing the lowest energy optimized and docked
bioactive conformations of compounds 3i and 3l. A comparison of the optimized and bioactive
conformations of compounds 3i and 3l are shown in Fig. 4. The lowest energy conformer was

3
9
obtained by calculation using the MM2 semi-empirical molecular orbital method and bioactive
conformer was obtained by docking simulation allowing free rotation about single bonds of
ligands.
(a)
(b)
(c)
C6
C6
C5
C7
C7
C8
O4
C5
C4
C12
C13
O2
N1
C13
O4
C4
C8
C11
C14
C9
C14
C3
C1
Overlay
C2
N2
C3
N2
C12
C11
C10
C1
O2
N1
C2
C9
O3
C10
O1
O1
O3
(d)
(e)
(f)
C6
C5
O2
C5
C7
O4
O5
C11
C6
O4
O3
C4
C3
Overlay
C12
C11
O2
C13
C8
C7
C4
C1
C10
C9
C14
N2
O5
C12
N2
C3
C1
C2
C2
C10
O3
N1
C8
C9
N1
C13
O1
C14
O1
Figure 4. 3D geometries (a and d) of optimized and (b and e) of bioactive conformations of compounds
i and 3l, respectively. (c and f) Show overlays of the optimized (yellow) and bioactive (green)
3
conformations of compounds 3i and 3l, respectively.
Biologically active molecules can adopt several conformations of nearly equal torsion
energy, which is related to torsion angles produced by rotating about single bonds to achieve
interactions in receptor-active sites. As shown in Fig. 4, the bioactive conformers of compounds
3
i and 3l obviously differed from their corresponding optimized conformers. In particular, for 3i,
torsion angles around the N2-C2-C9-C14 bond of the optimized and bioactive conformers were
.18, and 74.12, respectively, and torsion angles around the O1-C1-C3-C4 bond were -163.86
0
and 54.72, respectively. While for 3l, optimized and bioactive torsion angles around the N2-C2-
C9-C14 bond were 0.26, and -125.08, respectively, and torsion angles around the O1-C1-C3-C4

bond were -163.89 and 63.25, respectively. Analysis of these conformations indicated both the
benzylidene and benzoyl phenyl ring of 3l deviated (by 125.34 and 227.14, respectively) during
docking to adopt the bioactive conformation, whereas deviations of 3i were 73.94 and 218.58,
-
1
respectively. MM2 calculated torsion energies were -6.4865 and -10.4168 kcalmol for 3i and
l, respectively. The above results indicate the optimized conformer of 3l is more stable, and
3
therefore, requires more energy to adopt the bioactive conformation, and that this results in lower
receptor binding affinity than that of 3i. This observation is consistent with our docking and
experimental results. It is also worth noting compounds 3i and 3l adopted similar bioactive
conformations (Fig. 5) at the receptor binding site, though their optimized conformations were
quite different.
Figure 5. Superimposition of the bioactive conformations of compound 3i (green) and 3l (violet).
3
. Conclusion
A new series of salicyl-hydrazone analogues were synthesized and evaluated for in vitro
cytotoxic activity against five human cancer cell lines, that is, lung cancer (A549), ovarian
cancer (SK-OV-3), skin cancer (SK-MEL-2), colon cancer (HCT15) and pancreatic cancer

(
MIA-PaCa-2) cell lines. The in vitro TrkA inhibitory activities of these analogues were screened
to examine their modes of action. All 12 tested compounds showed significant cytotoxicities
against the four cancer cell lines. Compound 3i showed greatest cytotoxic potency against all
cancer cell lines, followed in decreasing order by compounds 3l, 3g, 3a and 3k. Compound 3i,
which had hydroxyl groups at the ortho- and para-positions of its benzylidenephenyl ring
exhibited a potency similar to doxorubicin against HCT15 cancer cells with an IC50 value of 0.89

M, but showed lower potency against A549, SK-OV-3, SK-MEL-2 and MIA-PaCa-2 cells (IC50
values for 3i were 2.46, 0.87, 1.43, 0.48 M, and those of doxorubicin were 0.872, 0.021, 0.74,
.001 and 0.049 M, respectively). TrkA inhibitory assays also showed compound 3i was most
0
potent with an IC50 value of 231 nM was followed in decreasing order by 3l, 3k and 3g. The
structure-activity relationship study revealed that salicyl-hydrazone analogues with hydroxyl
groups on the benzylidenephenyl ring showed better cytotoxic and TrkA inhibitory activities in
the order 2,4-OH> 2,3,4-OH>3,4-OH> 4-OH than analogues bearing electron donating (e.g. –
N(CH
3
)
2
, -CH
3
, and –OCH
3
) or withdrawing (e.g. -NO , and -Cl) groups on the
2
benzylidenephenyl ring. Docking studies were performed to investigate interactions between the
highest potent analogues (3i and 3l) and the active site of TrkA receptor. These studies revealed
that 3i and 3l bound in the same manner as has been reported for TrkA inhibitors with a high
docking score. Conformational analysis led us to speculate that compounds 3i and 3l adopt
similar conformations during interaction with TrkA receptor, despite quite different optimized
conformations. These results provide a valuable starting point for the design and synthesis of
salicyl-hydrazone analogues that inhibit TrkA as potential anticancer agents.
4
4
. Experimental
.1. General

Melting points of the twelve synthesized compounds were determined using a Stuart SMP3
apparatus and results were uncorrected. FT-IR spectra were obtained using a Bruker Tensor 37
spectrometer and KBr discs. NMR spectra were recorded using an AM-400 MHz (Bruker, USA)
spectrometer in DMSO using TMS as the internal standard. Mass spectra were acquired using a
Jeol JMS700 high-resolution mass spectrometer at the Korean Basic Science Center (Daegu,
South Korea). Elemental analyses (C, H, N) were performed on a Flash EA 2000 series (Thermo
Fisher) elemental analyzer.
4
.2. General procedure for the preparations of salicyl-hydrazide (1) and salicyl-hydrazone
analogues (3a-l).
4
0
Salicyl-hydrazide (2-hydroxybenzohydrazide) was prepared as we previously described.
Briefly, methyl salicylate (15.2 g, 0.1 M) was refluxed with hydrazine hydrate (12.5 g, 0.25 M)
in approximately 50 mL of ethanol for 3 h. Reaction progress was monitored by TLC.
Precipitated salicyl-hydrazide was filtered and recrystallized from an ethanol-water mixture.
Physical data were compared with previously reported values to confirm structures.
3
7
Next, salicyl-benzohydrazone analogues (3a-l) were prepared as previously described.
Briefly, salicyl-hydrazide (1, 2 mM) in 30 mL ethanol was added drop-wise to 20 mL of suitably
substituted benzaldehydes (2, 2 mM) dissolved in ethanol. Mixtures were stirred and refluxed for
1
.5-3.0 h and reaction progress was monitored by TLC. After cooling reaction mixtures to
ambient temperature, they were filtered to give solid crude products, which were crystallized
from ethanol to provide the pure compounds.
2
7
4
.2.1. (E)-N'-(benzylidene)-2-hydroxybenzohydrazide (3a)
White crystals. Yield: 74.6%, mp 244-245 C. IR (KBr) max: 3242 (OH), 3185 (NH), 2934
-1
1
(
CH), 1630 cm (C=O). H NMR: (400 MHz; DMSO-d ) δ 6.89-7.05 (2H, m, Ar-H), 7.37-7.59
6

(
5H, m, Ar-H), 7.69-7.98 (2H, m, Ar-H), 8.43 (1H, s, CH), 11.81 (1H, s, NH). EI-MS m/z (%):
+
2
1
40 (M , 20), 137 (26), 121 (100), 120 (37). Anal. Calcd for C14
H
12
N
2
2
O C 69.99; H 5.03; N
1.66%. Found C 69.42; H 5.14; N 11.31%.
4
.2.2. (E)-N'-(4-chlorobenzylidene)-2-hydroxybenzohydrazide (3b)
White crystals. Yield 85.0%, mp 265-266C. IR (KBr) max: 3257 (OH), 3167 (NH), 2925
-
1
1
(
(
CH), 1629 cm (C=O). H NMR (400 MHz; DMSO-d ) δ 6.91-7.00 (2H, m, Ar-H), 7.40-7.56
6
3H, m, Ar-H), 7.76-7.90 (3H, m, Ar-H), 8.45 (1H, s, CH), 11.92 (1H, s, NH). EI-MS m/z (%):
+
2
4
74 (M , 12), 154 (16), 137 (22), 121 (100), 120 (25). Anal. Calcd for C14
H11ClN
2
2
O C 61.21; H
.04; N 10.20%. Found C 61.28; H 4.23; N 10.11%.
4
.2.3. (E)-N'-(4-nitrobenzylidene)-2-hydroxybenzohydrazide (3c)
Yellow crystals. Yield 68.4%, mp 105-106C. IR (KBr) max: 3379 (OH), 3249 (NH),
-
1
1
2
7
8
1
3
937 (CH), 1632 cm (C=O). H NMR (400 MHz; DMSO-d ) δ 6.99-7.02 (2H, m, Ar-H), 7.48-
6
.55 (1H, m, Ar-H), 7.92-8.09 (2H, m, Ar-H), 8.20-8.24 (1H, m, Ar-H), 8.36-8.50 (2H, m, Ar-H),
+
.62 (1H, s, CH), 12.12 (1H, s, NH). EI-MS m/z (%): 285 (M , 12), 179 (5), 137 (13), 122 (12),
21 (100), 120 (19). Anal. Calcd for C14
.99; N 14.18%.
H
11
N
3
4
O C 58.95; H 3.89; N 14.73%. Found C 58.65; H
4
.2.4. (E)-N'-(2-nitrobenzylidene)-2-hydroxybenzohydrazide (3d)
Yellow crystals. Yield 67.4%, mp 228-229C IR (KBr) max: 3294 (OH), 3212 (NH),
-
1
1
3
7
064 (CH), 1634 cm (C=O). H NMR (400 MHz; DMSO-d
6
) δ 6.94-7.02 (2H, m, Ar-H), 7.42-
.51 (1H, m, Ar-H), 7.64-7.89 (3H, m, Ar-H), 8.04-8.16 (2H, m, Ar-H), 8.87 (1H, s, CH), 12.17

+
(
(
1H, s, NH). EI-MS m/z (%): 285 (M , 7), 179 (5), 137 (14), 122 (9), 121 (100), 120 (14), 118
7). Anal. Calcd for C14 C 58.95; H 3.89; N 14.73%. Found C 59.00; H 4.03; N 16.05%.
H
11
N
3
O
4
4
.2.5. (E)-N'-(4-dimethylaminobenzylidene)-2-hydroxybenzohydrazide (3e)
Yellow crystals. Yield 83%, mp 259-260 C. IR (KBr) max: 3282 (OH), 3210 (NH), 2917
-1
1
(
CH), 1632 cm (C=O). H NMR (400 MHz; DMSO-d
6
) δ 2.97 (6H, s, N(CH
3
2
) ), 6.70-6.81 (2H,
m, Ar-H), 6.90-7.01 (2H, m, Ar-H), 7.38-7.46 (1H, m, Ar-H), 7.51- 7.61 (2H, m, Ar-H), 7.85-
+
8
1
.94 (1H, m, Ar-H), 8.30 (1H, s, CH), 11.60 (1H, s, NH). EI-MS m/z (%): 283 (M , 93), 164 (11),
63 (100), 161 (41), 146 (75), 145 (15), 121 (56), 118 (10). Anal. Clcd for C16
H
17
N
3
2
O C 67.83;
H 6.05; N 14.83% Found C 67.60; H 6.21; N 15.97%
4
1
4
.2.6. (E)-N'-(4-methylbenzylidene)-2-hydroxybenzohydrazide (3f)
White crystals. Yield 86.0%, mp 246-247 C. IR (KBr) max: 3271 (OH), 3235 (NH), 2922
-1
1
(
CH), 1655 cm (C=O). H NMR (400 MHz; DMSO-d
6
) δ 2.35 (3H, s, Ar-CH ), 6.90- 7.02 (2H,
3
m, Ar-H), 7.20- 7.46 (3H, m, Ar-H), 7.60-7.73 (2H, m, Ar-H), 7.84-7.92 (1H, m, Ar-H), 8.42
+
(
(
1H, s, CH), 11.78 (1H, s, NH). EI-MS m/z (%): 254 (M , 23), 137 (39), 134 (67), 121 (100), 119
18), 120 (39). Anal. Calcd for C15
H
14
N
2
O C 70.85; H 5.55; N 11.02%. Found C 70.78; H 5.67;
2
N 11.60%.
4
.2.7. (E)-N'-(4-hydroxybenzylidene)-2-hydroxybenzohydrazide (3g)
Yellow crystals. Yield 72.6%, mp 177-178 C. IR (KBr) max: 3346 (OH), 3268 (NH),
-
1
1
2
7
947 (CH), 1669 cm (C=O). H NMR (400 MHz; DMSO-d
6
) δ 6.88-7.06 (3H, m, Ar-H), 7.45-
.66 (3H, m, Ar-H),7.74-7.92 (2H, m, Ar-H), 8.37 (1H, s, CH), 11.72 (1H, s, NH). EI-MS m/z

+
(
%): 256 (M , 25), 137 (42), 136 (68), 121 (100), 120 (35). Anal. Calcd for C14
H
12
N
2
3
O C 65.62;
H 4.72; N 10.93%. Found C 62.62; H 5.24; N 9.12%.
4
1
4
.2.8. (E)-N'-(4-methoxybenzylidene)-2-hydroxybenzohydrazide (3h)
Yellow crystals. Yield 73.3%, mp 211-212 C. IR (KBr) max: 3263 (OH), 3158 (NH),
-1
1
2
954 (CH), 1654 cm (C=O). H NMR (400 MHz; DMSO-d
6
) δ 3.85 (3H, s, OCH ), 6.94-7.16
3
(
4H, m, Ar-H), 7.42-7.51 (1H, m, Ar-H), 7.69-7.95 (3H, m, Ar-H), 8.42 (1H, s, CH), 11.77 (1H,
+
s, NH). EI-MS m/z (%): 270 (M , 33), 268 (11), 150 (100), 149 (12), 137 (47), 134 (10), 121 (77),
1
1
20 (40). Anal. Calcd for C15
0.80%.
H
14
N
2
3
O C 66.66; H 5.22; N 10.36%. Found C 66.74; H 5.36; N
4
.2.9. (E)-N'-(2,4-dihydroxybenzylidene)-2-hydroxybenzohydrazide (3i)
Yellow crystals. Yield 87.5%, mp 155-156 C. IR (KBr) max: 3561 (OH), 3468(OH), 3200
-1
1
(
NH), 2913 (CH), 1628 cm (C=O). H NMR (400 MHz; DMSO-d
6
) δ 6.29-6.41 (2H, m, Ar-H),
6
1
.92-7.04 (2H, m, Ar-H), 7.29-7.49 (2H, m, Ar-H), 7.82-7.94 (1H, m, Ar-H), 8.55 (1H, s, CH),
+
1.37 (1H, s, NH). EI-MS m/z (%): 272 (M , 43), 152 (51), 138 (51), 137 (81), 121 (100), 120
(
30). Anal. Calcd for C14
H
12
N
2
O
4
C 61.76; H 4.44; N 10.29%. Found C 58.57 H, 4.82 N, 9.50%.
4
2
.2.10. (E)-N'-(2,4-dimethoxybenzylidene)-2-hydroxybenzohydrazide (3j)
Yellow crystals. Yield 74.3%, mp 155-156 C. IR (KBr) max: 3324 (OH), 3242 (NH),
-1
1
963 (CH), 1650 cm (C=O). H NMR (400 MHz; DMSO-d
6
) δ 3.83 (3H, s, OCH
3
), 3.89 (3H, s,
OCH
3
), 6.58-6.71 (2H, m, Ar-H), 6.89-7.01 (2H, m, Ar-H), 7.36-7.47 (1H, m, Ar-H), 7.67-7.87
+
(
2H, m, Ar-H), 8.70 (1H, s, CH), 11.77 (1H, s, NH). EI-MS m/z (%): 300 (M , 55), 180 (26), 179

(
12), 163 (81), 150 (10), 149 (90), 137 (45), 121 (100), 120 (45). Anal. Calcd for C16
H
16
N
2
4
O C
6
3.99; H 5.37; N 9.33%. Found C 64.21; H 5.66; N 7.68%.
4
.2.11. (E)-N'-(3,4-dihydroxybenzylidene)-2-hydroxybenzohydrazide (3k)
Brown powder. Yield 68.7%, mp 216-217 C. IR (KBr) max: 3330 (OH), 3112 (NH), 3061
-
1
1
(
(
CH), 1645 cm (C=O). H NMR (400 MHz; DMSO-d ) δ 6.81-7.02 (3H, m, Ar-H), 7.26-7.34
6
2H, m, Ar-H), 7.43-7.50 (1H, m, Ar-H), 7.89-7.97 (1H, m, Ar-H), 8.30 (1H, s, CH), 11.60 (1H,
+
s, NH). EI-MS m/z (%): 272 (M , 21), 152 (26), 138 (11), 137 (22), 121 (100), 120 (17). Anal.
Calcd for C14
H
12
N
2
4
O C 61.76; H 4.44; N 10.29%. Found C 60.99; H 4.55; N 7.72%.
4
.2.12. (E)-N'-(2,3,4-trihydroxybenzylidene)-2-hydroxybenzohydrazide (3l)
White crystals. Yield 68.5%, mp 250-251C. IR (KBr) max: 3379 (OH), 3243 (NH), 2925
-
1
1
(
(
CH), 1630 cm (C=O). H NMR (400 MHz; DMSO-d ) δ 6.40-6.52 (1H, m, Ar-H), 6.82-7.03
6
3H, m, Ar-H), 7.43-7.52 (1H, m, Ar-H), 7.88- 7.93 (1H, m, Ar-H), 8.53 (1H, s, CH), 11.92 (1H,
+
s, NH). EI-MS m/z (%): 288 (M , 39), 168 (35), 153 (11), 137 (12), 121 (100), 120 (22). Anal.
Calcd for C14
H
12
N
2
5
O C 58.33; H 4.20; N 9.72%. Found C 55.27; H 4.77; N 8.04%.
4
4
.3. Biological assay
.3.1. Cytotoxicity assays
The cytotoxicities of the 12 test compounds on the four tumor cell lines were determined using
the SRB (sulforhodamine-B) method, which is currently adopted by the NCI (National Cancer
3
3
Institute, UK) for in vitro anti-cancer drug screening. Inhibition of cell proliferation was
estimated after continuous exposure to test compounds for 48 h. All samples were tested in

triplicate, and mean IC50 values (mg/mL) (concentration resulting in 50% inhibition of cell
proliferation) were calculated.
4
.3.2. Tropomyosin receptor kinase A (TrkA) inhibitory activity
Purified TrkA protein was purchased from Carna Biosciences (Kobe, Japan). Protein kinase,
o
biotinylated substrate peptide, and TrkA inhibitors were pre-incubated for 15 min. at 25 C in
3
84-well plates. The phosphorylation reaction of TrkA enzyme was started by adding ATP (100
M), and 30 min later an anti-phosphotyrosine antibody (1 M) labeled with europium (Eu)

cryptate and 125 nM streptavidin labeled with fluorophore XL665 were added to reaction
mixtures. After incubation for 1h, fluorescences were measured using an Envision multi-label
reader (Perkin Elmer, Waltham, MA, USA) using an excitation wavelength of 320nm and
emission wavelengths of 620 (Eu-labeled antibody) and 665nm (XL665-labeled streptavidin).
Homogeneous time-resolved fluorescence resonance energy transfer (FRET) between Eu
cryptate and XL665 was measured to quantify substrate peptide phosphorylation. The ratios of
the fluorescence intensities (λ620nm/λ665nm) were calculated at 12 different serial dilutions
(
0.03~10000 nM) of the compounds tested. The % inhibition data was plotted against tested
compound concentration to determine IC50 values, which were calculated by using the Prizm
program (La Jolla, CA, USA). GW441756 (R&D System, Minneapolis, MN, USA), a well-
known TrkA inhibitors, was used as a positive control.
4
.4. Docking studies
The three-dimensional conformations of compounds 3i and 3l were constructed using standard
bond lengths and angles using the ChemBio3D ultra Ver. 14 molecular modeling program
Cambridge Soft Corporation, Cambridge, MA 02140 USA), and energies were optimized using
(

3
9
the semi-empirical molecular orbital MM2 method using 1810 (for 3i) and 2264 (for 3l)
iterations and a minimum RMS gradient of 0.10. The crystal structure of tropomyosin receptor
3
7
kinase A (TrkA) was obtained from Protein Data Bank (PDB code: 4f0i) for docking studies.
All bound water in the crystal structure of TrkA receptor was eliminated and polar hydrogen
atoms and Kollman-united charges were added to molecules. The pdb and pdbqt files of ligands
4
2
and the receptor were prepared using AutoDock 4.2 software. Docking simulations were
4
3
performed using the standard method implemented by AutoDock Vina in PyRx 0.8 software
and free rotations were allowed about single bonds during docking. Geometries of resulting
complexes and their conformations were investigated using Discovery Studio 4.0 (Accelrys, San
Diego, CA, USA) and GaussView 5.0 (Gaussian Inc., Wallingford, CT, USA).
Acknowledgements
This work was supported by the Research Fund of Dongguk University (2015).
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Graphical Abstract
TrkA inhibitor (IC ) 230 nM
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