Synthesis and biological evaluation of N-(4-hydroxy-3-mercaptonaphthalen-1- yl)amides as inhibitors of angiogenesis and tumor growth

Article abstract of DOI:10.1016/j.ejmech.2013.03.043

A series of N-(4-hydroxy-3-mercaptonaphthalen-1-yl)amides were synthesized and investigated for their in vitro antiangiogenic activity. Among these compounds, 6d, which possesses an ortho-nitro group at the benzene ring, exhibited potent inhibitory effect

Full text of DOI:10.1016/j.ejmech.2013.03.043

Accepted Manuscript
Synthesis and biological evaluation of N-(4-hydroxy-3-mercaptonaphthalen-1-
yl)amides as inhibitors of angiogenesis and tumor growth
Fuming Xu, Yuping Jia, Qingli Wen, Xuejian Wang, Lei Zhang, Yingjie Zhang,
Kanghui Yang, Wenfang Xu
PII:
S0223-5234(13)00203-1
10.1016/j.ejmech.2013.03.043
EJMECH 6081
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 December 2012
Revised Date: 18 March 2013
Accepted Date: 24 March 2013
Please cite this article as: F. Xu, Y. Jia, Q. Wen, X. Wang, L. Zhang, Y. Zhang, K. Yang, W. Xu,
Synthesis and biological evaluation of N-(4-hydroxy-3-mercaptonaphthalen-1-yl)amides as inhibitors
of angiogenesis and tumor growth, European Journal of Medicinal Chemistry (2013), doi: 10.1016/
j.ejmech.2013.03.043.
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Graphical Abstract:
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Synthesis
and
biological
evaluation
of
N-(4-hydroxy-3-mercaptonaphthalen-1-yl)amides as inhibitors of
angiogenesis and tumor growth
Fuming Xua, Yuping Jiab, Qingli Wenc, Xuejian Wanga, Lei Zhanga, Yingjie Zhanga,
Kanghui Yanga, Wenfang Xua,*
aDepartment of Medicinal Chemistry, School of Pharmacy, Shandong University,
Ji’nan, Shandong 250012, PR China
bShandong Institute of Pharmaceutical Industry, No. 989 High-tech Industrial
Development Zone Xinluo road, Ji’nan, Shandong 250101, PR China
cAffiliated Hospital of Weifang Medical University, Weifang, Shandong 261031, PR
China
Abstract
A series of N-(4-hydroxy-3-mercaptonaphthalen-1-yl)amides were synthesized and
investigated for their in vitro antiangiogenic activity. Among these compounds, 6d,
which possesses an ortho-nitro group at the benzene ring, exhibited potent inhibitory
effect on the proliferation of HUVECs, A549, K562, PC-3, HCT116, MDA-MB-231
and MCF-7 cells (IC50 = 5.34, 40.53, 10.81, 52.52, 10.19, 21.37 and 2.81 ꢀM,
respectively). Meanwhile, compound 6d inhibited in vitro angiogenesis markedly in
both HUVECs tube formation assay and the rat thoracic aorta rings test. Further
kinase assay study showed that compound 6d had good VEGFR2, ALK, AKT1 and
ABL inhibitory activities and moderate EGFR and PDGFR-β inhibitory activities. The
data supports the further investigation of this class of compounds as potential
antiangiogenic and anticancer agents.
Keywords: angiogenesis; HUVECs; tube formation; rat thoracic aorta rings; inhibitor;
kinase
*
Corresponding author. Tel./fax: +86 531 88382264. E-mail address:
wfxu@yahoo.cn (W. Xu).
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1. Introduction
Aberrant angiogenesis, the process that leads to the formation of newly abnormal
vessels, is a prerequisite for tumor cell proliferation, invasion and metastasis [1-3].
Tumor-induced blood capillaries can help to create nutrient supply, to remove
metabolic waste and to facilitate metastasis formation for several solid tumors and
haematological malignancies [2, 4]. Protein kinases, especially protein tyrosine
kinases (PTKs) such as vascular endothelial growth factor receptors (VEGFRs) [5, 6],
epidermal growth factor receptor (EGFR) [7] and platelet-derived growth factor
receptors (PDGFRs) [8] are closely associated with aberrant signaling leading to
increased angiogenesis, tumor cell proliferation and metastasis [5, 9, 10].
It has been demonstrated that blockade of angiogenesis is one of the more attractive
approaches for the treatment of cancer [11, 12], so design of small molecule inhibitors
that target the adenosine triphosphate (ATP) binding site of protein kinases is
currently of great interest [13-15]. In the past decade, there are several small-molecule
angiogenesis inhibitors approved or in late stage clinical trials such as Sunitinib
(SU11248) [16, 17], Sorafenib (BAY 43-9006) [18], Pazopanib [19], PTK787
(ZK222584, vatalanib) [20, 21], ZD6474 (vandetanib) [22, 23], Semaxanib (SU5416)
[24], Imatinib (Gleevec®, STI571) [25], and so on. Recent preclinical studies show
that inhibition of multiple PTKs by single-agents has the potential to increase
antitumor activities [26-28].
Herein we report the antitumor and antiangiogenic activity for the newly synthesized
19 compounds. In the scaffold of our target compounds, naphthalene was selected as a
hydrophobic domain because its distinctly plane geometrical conformation and
structure could form special interaction with ATP binding site of protein kinases. In
addition, naphthalene has been employed increasingly in anticancer drug
design[29-32], which encourages us to search novel naphthalene-containing structures
as inhibitors of angiogenesis and tumor growth. The possible mechanism for the
antiangiogenic and antitumor activity was examined under in vitro condition. Among
these compounds, 6d exhibits potent inhibitory effects on both the proliferation of
several tumor cell lines and in vitro angiogenesis by application of tube formation
assay using HUVECs [33, 34], the rat thoracic aorta rings model [33, 35] and kinase
inhibition assay.
2. Chemistry
The synthetic pathway for target compounds was illustrated in Scheme 1. We first
introduced hydroxy into nitronaphthalene 1 in the presence of t-BuOOH and KOH [36,
37]. The nitro-group on compound 2 was then reduced to the amino group by Pd-C/H2
in methanol solution [38] to give compound 3, which was acylated using various
substituted acyl chlorides [39] to form compounds 4a-4m. Then, compounds 4a-4m
were oxidated using NaIO4/SiO2 [40] to obtain quinone type structures 5a-5m. At last
compounds 5a-5m took Michael addition reaction [41] to gain the target compounds
6a-6s with different arylthiol groups. The spectral characterization of all target
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compounds is the chemical shift of three active hydrogens (1 × phenolic OH and 2 ×
aromatic NH) with (DMSO-d6) δ 9.50-14.25. The chemical shift assignment
mentioned above was illustrated in the appropriate part.
(Scheme 1 should be listed here)
3. Results and discussion
3.1. In vitro antiproliferative activity
In
vitro
antiproliferative
activity
of
the
19
novel
N-(4-hydroxy-3-mercaptonaphthalen-1-yl)amides was initially evaluated against
MDA-MB-231, PC-3 and HCT116 cell lines by MTT assay using Sorafenib as a
positive control. From the data shown in Table 1, most compounds exhibited weak
antiproliferative activity with high IC50 (>100 ꢀM) values, while compounds 6c, 6d,
6q and 6r showed moderate activity. We can see that compounds that possess a
para-chloro group or an ortho-nitro group on the benzene ring of the amide part
displayed better antiproliferative activity than that of the other compounds.
Comparing compounds 6n-6q and 6c, when the 1H-1,2,4-triazole ring was replaced
by 5-methyl-1,3,4-thiadiazole ring or 1,3,4-thiadiazole ring or 1-methyl-1H-tetrazole
ring, efficacy in inhibiting proliferation almost disappeared. Meanwhile, when the
7H-purine took place of 1H-1,2,4-triazole ring in the compounds, a slight decrease of
the antiproliferative activity was perceived (for instance, the IC50 of the three cancer
cell lines mentioned in this section of the compound 6c/6q are 25.71/22.97,
5.52/66.43, and 43.27/>100 ꢀM, respectively).
Taking the good in vitro activity of compounds 6c, 6d, 6q and 6r against
MDA-MB-231, PC-3 and HCT116 cell lines into consideration, we further
investigated the potential antiproliferative effect of these compounds against another
four cell lines, HUVECs, MCF-7, A549 and K562 cells, which are all related to
tumor-induced angiogenesis. The results were illustrated in Table 2, which showed
that compounds 6d and 6q also exhibit low ꢀM activities against the growth of these
four cell lines.
(Table 1 should be listed here)
(Table 2 should be listed here)
3.2. In vitro antiangiogenesis assay
3.2.1. In vitro HUVECs tuber formation assay
In an effort to study the antiangiogenic effect of all the compounds reported above, we
were interested in in vitro tube formation assay using HUVECs which were plated on
Matrigel in order to mimic in vivo HUVEC associated angiogenesis. While the
HUVECs were plated, elongated and robust tube-like structures were well established
after incubation for 6 h in the negative control group; but treatment of HUVECs with
each compound at the concentration of 50 ꢀM inhibited the formation of tubular
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structures in different ranks. As shown in Fig. 1A, significant inhibition (>80%) was
observed with compounds 6d, 6r, 6b, 6n, 6o, 6k and 6l. However, compound 6c
possessing good cytotoxic activity showed moderate inhibitory efficacy at 50 ꢀM
(65%). In order to determine whether compounds 6d, 6r, 6b, 6n, 6o, 6k, 6l and 6c
could inhibit the HUVECs tuber formation in a concentration-dependent fashion, we
employed another two concentrations (10 and 20 ꢀM) for further tuber formation
inhibition test. Surprisingly, almost all the tested compounds exhibited
dose-dependent inhibition of tube formation (see Fig. 1B). Compound 6d and 6n were
found to be the most potent and efficacious, inhibiting tube formation at 10 ꢀM by
83% and 81% respectively, while compound 6c showed faint tuber formation
inhibitory activity at this concentration (data was not shown on Fig. 1B).
Representative tube images are shown in Fig. 2.
(Fig 1 should be listed here)
(Fig 2 should be listed here)
3.2.2. Rat thoracic aorta rings (TARs) assay
TARs model is more close to in vivo condition compared with HUVECs tuber
formation model in view of multi-steps involved in angiogenesis: sprouting,
proliferation, migration and differentiation. Herein, TARs assay was employed to
evaluate the antiangiogenic activity of all the synthesized compounds. The area of
angiogenic sprouting, reported in number of pixels, was quantified using Adobe
PhotoShop. The results are presented in Fig. 3. Compounds 6d, 6r, 6n and 6q showed
outstanding angiogenesis inhibition ratio at the concentration of 50 ꢀM (95%, 96%,
91% and 92%, respectively), while the rest compounds turned up to be moderate or
weak ones (see Fig. 3A). Especially, compound 6c having weak HUVECs tuber
formation inhibitory efficacy still could not reduce microvessel outgrowth at 50 ꢀM.
Fig. 3B showed that Compounds 6d, 6r, 6n and 6q appeared to reduce microvessel
outgrowth in a concentration-dependent manner. At 1 ꢀM, compound 6d, 6r and 6q
demonstrated >70% inhibition of angiogenesis, comparable with Pazopanib. Among
them, compound 6d was more potent than compound 6r and 6q, with 91% reduction
of angiogenesis at 1 ꢀM. Interestingly, compound 6n failed to inhibit microvessel
outgrowth when the concentration was below 20 ꢀM (23% and 18% at 10 ꢀM and 1
ꢀM, respectively). Representative images of aortic rings are illustrated in Fig. 4.
(Fig 3 should be listed here)
(Fig 4 should be listed here)
3.3. In vitro protein kinase assay
As many tyrosine and serine/threonine kinases are involved in oncogenic signaling,
the in vitro kinase assay was carried out to test the kinase inhibition activity of all the
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synthesized compounds at the concentration of 10 ꢀM against EGFR, ABL, AKT1,
and ALK kinases by measuring the levels of phosphorylation of the kinase-specific
ligand peptides. The pan-kinase inhibitor Staurosporine was used as a positive
reference compound. As shown in Table 3, most of the compounds showed moderate
EGFR, ABL and AKT1 inhibitory activity but no obvious ALK inhibition effect. The
only exception was compound 6d, which still retained potency against ALK (56.83%
at 10ꢀM). Compounds 6d, 6r and 6q were found to be more effective than the rest
compounds against EGFR, ABL and AKT1. This result was consistent with cell
cytotoxicity activity and antiangiogenic effect. Specifically, The most potent
compound 6d inhibited 36.79% , 87.18% and 85.77% of EGFR, ABL and AKT1
activity at 10 ꢀM, respectively. It is interesting that compound 6n which showed
moderate antiangiogenic activity disclosed slight kinase inhibitory effect. A SAR
study for all the synthesized compounds was undertaken. The phenyl group of R1
contributed more to the activity rather than bigger substitutes (such as 2-naphthyl
group) or longer substitutes (such as cinnamenyl group). For example, compounds 6a
and 6b showed less kinase inhibition activity than compounds 6c and 6d. Compounds
containing electron-withdrawing groups (for instance, -NO2/-Cl group of 6d/6c)
attached to the phenyl ring of R1 displayed much more inhibitory performances than
compounds with electron-donating groups (for instance, -OMe/-Me group of 6e/6i).
Comparing compounds 6n-6p and 6c, when the 1H-1,2,4-triazole ring was replaced
by 5-methyl-1,3,4-thiadiazole ring or 1,3,4-thiadiazole ring or 1-methyl-1H-tetrazole
ring, efficacy in inhibiting EGFR, ABL and AKT1 kinases almost disappeared.
Meanwhile, when the 7H-purine took place of 1H-1,2,4-triazole ring in the
compounds (e.g. 6q and 6c, 6r and 6d), apparent decrease of kinase inhibitory activity
was perceived (for instance, the inhibition rates(%) of the compound 6d/6r against
EGFR, ABL, AKT, and ALK are 36.79/24.73, 87.18/43.69, and 85.77/50.73,
respectively). The inhibition values of compound 6d against VEGFR2 and PDGFR-β
which are both closely related to tumor-induced angiogenesis were further determined.
Encouragingly, compound 6d exhibited 65.23% and 37.93% of VEGFR2 and
PDGFR-β inhibitory activity at 10 ꢀM respectively, which indicated that 6d was a
promising lead compound against tumor angiogenesis for further study.
(Table 3 should be listed here)
3.4. Molecular docking
To further investigate the antiangiogenesis mechanism of compound 6d, a molecular
docking study of 6d in the ATP-binding pocket of the ABL (PDB ID: 2HYY), EGFR
(PDB ID: 2ITY), ALK (PDB ID: 3LCS) and VEGFR2 (PDB ID: 2QU5) kinase
domains was performed using SYBYL 8.0 (Fig. 5). Compound 6d interacted with the
ATP binding pocket of the catalytic site of the four kinases mainly through
hydrophobic interaction and hydrogen bonds. In this binding mode, the naphthalene
ring structure of 6d sitted in a lipophilic pocket and the two -NH groups interacted
with Ile360 and Asp381 of ABL, which was proved to be an important binding site of
5

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ABL inhibitor (see Fig. 5A). As shown in Fig. 5B, 6d was nicely bound to the EGFR
binding domain, of which the ortho-nitro group formd a hydrogen bond with the
crucial amino acid MET97. It was consistent with the result that only compound 6d
showed more potent than any other ortho-nitro free compound as an EGFR kinase
inhibitor. Comparing the molecular docking of compounds 6d with ALK and
VEGFR2 kinases displayed in Fig. 5C and 5D, the –OH and -S- groups of 6d could
also form hydrogen bonds with different amino acid side chains of the two kinases.
The data supports the further investigation of this class of compounds as potential
kinase inhibitors.
(Fig 5 should be listed here)
4. Conclusions
In general, we presented for the first time the synthesis of 19
N-(4-hydroxy-3-mercaptonaphthalen-1-yl)amide derivatives. In light of the biological
activity assay results, it is concluded that some of these compounds are selective
against the proliferation of HUVECs and several cancer cell lines, microvessel tube
formation using HUVECs and endothelial sprouting of TARs. The SAR indicated that
electron-withdrawing substituent at the benzene ring of R1 and 1H-1,2,4-triazole ring
(R2) contributed much to the cytotoxicity and kinase inhibition activity. The results of
kinase
assay
and
molecular
docking
studying
exhibited
that
N-(4-hydroxy-3-mercaptonaphthalen-1-yl)amide derivatives were novel ABL, AKT1,
and VEGFR2 kinases inhibitors. Among all the compounds, 6d comprehensively
showed strong inhibitory effect, which deserved further in vitro and in vivo studies on
its mechanism of anti-angiogenesis activity.
5. Experimental part
5.1. Chemistry
Unless otherwise noted, all solvents and reagents were commercially available and
used without further purification. All reactions were monitored by thin-layer
chromatography on 0.25 mm silica gel plates (60 GF-254) and visualized with UV
light, or iodine vapor. Proton NMR spectrums were determined on a Brucker DRX
spectrometer (600/300 MHz), δ in parts per million and J in Hertz, using TMS as an
internal standard. Measurements were made in DMSO-d6 solutions. ESI-MS were
determined on an API 4000 spectrometer. Melting points were determined on an
electrothermal melting point apparatus and were uncorrected.
5.1.1. 4-nitronaphthalen-1-ol (2)
To a solution of compound 1 (0.86 g, 5 mmol) in 13 ml of DMSO, was added a
solution of KOH (1.2 g, 20 mmol) in water (5 mL) at 0-5 . The mixture was stirred
for 5 min before t-BuOOH (1.8 g, 10 mmol) in DMSO (2 mL) was added [36, 37].
After stirring at room temperature for 2 h, the mixture was poured into 1N HCl (50mL)
slowly, with yellow solid separation. Crude product compound 2 was obtained after
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filtering to remove aqueous phase and then being allowed to dry in air. The crude
product was at last recrystallized with ethyl acetate/petroleum ether to give 0.76 g of
title compound 2 as a faint yellow powder. Yield: 90 %, mp: 166-168 . 1H NMR
(DMSO-d6) δ 7.00 (d, J = 9.00 Hz, 1H, aromatic H), 7.65 (t, J = 7.20 Hz, 1H,
aromatic H), 7.82 (t, J = 7.20 Hz, 1H, aromatic H), 8.34 (d, J = 9.00 Hz, 1H, aromatic
H), 8.42 (d, J = 9.00 Hz, 1H, aromatic H), 8.67 (d, J = 9.00 Hz, 1H, aromatic H),
11.95 (s, 1H, phenolic OH). ESI-MS m/z 190.4 [M+H] +
.
5.1.2. 4-aminonaphthalen-1-ol (3)
To a solution of compound 2 (1.89 g, 10 mmol) in 50 ml of CH3OH, was added Pd/C
catalyst (0.2 g) [38]. The mixture was stirred for 12h with hydrogen under 10 times
standard atmospheric pressure, then the catalyst was filtered and washed with 20 mL
of CH3OH, and the filtrate was evaporated under vacuum. The black residues were
purified by ethyl ether to obtain white solid 3. Yield: 85 %, mp: 198 . 1H NMR
(DMSO-d6) δ 6.93 (d, J = 8.04 Hz, 1H, aromatic H), 7.52 (d, J = 8.08 Hz, 1H,
aromatic H), 7.58 (t, J = 7.80 Hz, 1H, aromatic H), 7.67 (t, J = 7.80 Hz, 1H, aromatic
H), 7.95 (d, J = 8.40 Hz, 1H, aromatic H), 8.22 (d, J = 8.40 Hz, 1H, aromatic H),
10.56 (s, 2H, aromatic NH2), 10.69 (s, 1H, phenolic OH). ESI-MS m/z 160.4 [M+H] +
.
5.1.3. N-(4-hydroxynaphthalen-1-yl)-2-naphthamide (4a)
Under the condition of ice bath, compound 3 (1.59 g, 10 mmol) was added to a
solution of NaHCO3 (2.52g, 30mmol) in THF/H2O (50 mL/1mL). After 2-naphthoyl
chloride (2.28 g, 12 mmol) was added drop by drop, the mixture was stirred at room
temperature for 5 h (detected by TLC) [39]. Solvents were evaporated with the
residues being taken up in EtOAc (50 mL). Then the organic layer was washed with
saturated 1 N HCl (20 mL × 2), distilled water (20 mL × 2), brine (20 mL × 2), dried
over MgSO4 and evaporated under vacuum. The residues were purified by EtOH/H2O
to give 2.66 g of desired compound 4a as white solid. Yield: 82 %, mp: 226-227 . 1H
NMR (DMSO-d6) δ 6.92 (d, J = 7.80 Hz, 1H, aromatic H), 7.38(d, J = 8.40 Hz, 1H,
aromatic H), 7.48-7.54 (m, 2H), 7.62-7.67 (m, 2H), 7.90 (d, J = 8.40 Hz, 1H, aromatic
H), 8.03 (d, J = 7.80 Hz, 1H, aromatic H), 8.06-8.13 (m, 3H), 8.20 (d, J = 7.80 Hz,
1H, aromatic H), 8.71 (s, 1H, aromatic H), 10.24 (s, 1H, phenolic OH),10.36 (s, 1H,
13
aromatic NH). C NMR (150 MHz, DMSO-d6) δ: 122.75, 123.68, 124.97, 125.11,
125.26, 125.36, 125.57, 126.65, 127.23, 128.11, 128.16, 128.42, 128.47, 129.41,
131.19, 132.39, 132.65, 134.72, 152.39, 166.43, 166.73. ESI-MS m/z 314.1 [M+H] +.
Compounds 4b-4m were synthesized following the procedure described above.
1
5.1.3.1. N-(4-hydroxynaphthalen-1-yl)cinnamamide (4b). Yield: 68 %. H NMR
(DMSO-d6) δ 6.87 (d, J = 8.40 Hz, 1H, aromatic H), 7.07 (d, J = 15.60 Hz, 1H,
Ar-CH=CH-CO-), 7.40-7.43 (m, 1H), 7.45-7.48 (m, 2H), 7.49-7.55 (m, 2H), 7.59 (d,
J = 15.60 Hz, 1H, Ar-CH=CH-CO-), 7.66 (d, J = 7.20 Hz, 2H, 2 × aromatic H), 7.95
(d, J = 9.00 Hz, 1H, aromatic H), 8.17 (d, J = 8.40 Hz, 1H, aromatic H), 9.90 (s, 1H,
phenolic OH), 10.15 (s, 1H, aromatic NH). ESI-MS m/z 290.3 [M+H] +.
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5.1.3.2. 4-chloro-N-(4-hydroxynaphthalen-1-yl)benzamide (4c). Yield: 75 %. 1H NMR
(DMSO-d6) δ 6.89 (d, J = 8.40 Hz, 1H, aromatic H), 7.32 (d, J = 7.80 Hz, 1H,
aromatic H), 7.46-7.52 (m, 2H, 2 × aromatic H), 7.63 (d, J = 8.40 Hz, 2H, 2 ×
aromatic H), 7.82 (d, J = 8.40 Hz, 1H, aromatic H), 8.09 (d, J = 8.40 Hz, 1H, aromatic
H), 8.18 (d, J = 7.80 Hz, 1H, aromatic H), 10.23 (s, 1H, phenolic OH), 10.26 (s, 1H,
aromatic NH). ESI-MS m/z 298.4 [M+H] +.
1
5.1.3.3. N-(4-hydroxynaphthalen-1-yl)-2-nitrobenzamide (4d). Yield: 81 %. H NMR
(DMSO-d6) δ 6.77 (d, J = 7.80 Hz, 1H, aromatic H), 7.05 (d, J = 8.40 Hz, 1H,
aromatic H), 7.34-7.40 (m, 2H, 2 × aromatic H), 7.62-7.65 (m, 2H, 2 × aromatic H),
7.78-7.80 (m, 1H, aromatic H), 7.88 (d, J = 7.80 Hz, 1H, aromatic H), 7.94 (d, J =
7.80 Hz, 1H, aromatic H), 8.08 (d, J = 7.80 Hz, 1H, aromatic H), 10.19 (s, 1H,
phenolic OH), 10.37 (s, 1H, aromatic NH). ESI-MS m/z 309.4 [M+H] +.
1
5.1.3.4. N-(4-hydroxynaphthalen-1-yl)-4-methoxybenzamide (4e). Yield: 77 %. H
NMR (DMSO-d6) δ 3.85 (s, 3H, OCH3), 6.88 (d, J = 7.80 Hz, 1H, aromatic H), 7.07
(d, J = 7.80 Hz, 2H, 2 × aromatic H), 7.29 (d, J = 8.40 Hz, 1H, aromatic H), 7.45-7.51
(m, 2H, 2 × aromatic H), 7.81 (d, J = 7.80 Hz, 1H, aromatic H), 8.06 (d, J = 8.40 Hz,
1H, aromatic H), 8.17 (d, J = 7.80 Hz, 1H, aromatic H), 10.02 (s, 1H, phenolic OH),
10.18 (s, 1H, aromatic NH). ESI-MS m/z 294.3 [M+H] +.
5.1.3.5. 3-(3,4-dimethoxyphenyl)-N-(4-hydroxynaphthalen-1-yl)acrylamide (4f). Yield:
1
82 %. H NMR (DMSO-d6) δ 3.81 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 6.87 (d, J =
8.40 Hz, 1H, aromatic H), 6.94 (d, J = 15.60 Hz, 1H, Ar-CH=CH-CO-), 7.03 (d, J =
8.40 Hz, 1H, aromatic H), 7.20 (d, J = 8.40 Hz, 1H, aromatic H), 7.25 (s, 1H,
aromatic H), 7.46-7.54 (m, 4H), 7.95 (d, J = 8.40 Hz, 1H, aromatic H), 8.17 (d, J =
7.80 Hz, 1H, aromatic H), 9.79 (s, 1H, phenolic OH), 10.12 (s, 1H, aromatic NH).
ESI-MS m/z 350.4 [M+H] +.
1
5.1.3.6. N-(4-hydroxynaphthalen-1-yl)-3-nitrobenzamide (4g). Yield: 68 %. H NMR
(DMSO-d6) δ 6.91 (d, J = 7.80 Hz, 1H, aromatic H), 7.36 (d, J = 7.80 Hz, 1H,
aromatic H), 7.48-7.53 (m, 2H, 2 × aromatic H), 7.85-7.88 (m, 2H, 2 × aromatic H),
8.19 (d, J = 7.80 Hz, 1H, aromatic H), 8.47 (d, J = 8.40 Hz, 1H, aromatic H), 8.51 (d,
J = 7.80 Hz, 1H, aromatic H), 8.90 (s, 1H, aromatic H), 10.27 (s, 1H, phenolic OH),
10.56 (s, 1H, aromatic NH). ESI-MS m/z 309.4 [M+H] +.
1
5.1.3.7. N-(4-hydroxynaphthalen-1-yl)-3-methoxybenzamide (4h). Yield: 65 %. H
NMR (DMSO-d6) δ 3.86 (s, 3H, OCH3), 8.90 (d, J = 7.80 Hz, 1H, aromatic H), 7.18
(d, J = 7.80 Hz, 1H, aromatic H), 7.32 (d, J = 7.80 Hz, 1H, aromatic H), 7.45-7.53 (m,
3H, 3 × aromatic H), 7.62 (s, 1H, aromatic H), 7.67 (d, J = 7.20 Hz, 1H, aromatic H),
7.82 (d, J = 8.40 Hz, 1H, aromatic H), 8.19 (d, J = 8.40 Hz, 1H, aromatic H), 10.17 (s,
1H, phenolic OH), 10.22 (s, 1H, aromatic NH). ESI-MS m/z 294.4 [M+H] +.
1
5.1.3.8. N-(4-hydroxynaphthalen-1-yl)-3-methylbenzamide (4i). Yield: 60 %. H
NMR (DMSO-d6) δ 2.42 (s, 3H, CH3), 6.89 (d, J = 8.00 Hz, 1H, aromatic H), 7.30 (d,
J = 8.00 Hz, 1H, aromatic H), 7.41-7.52(m, 4H, 4 × aromatic H), 7.81 (d, J = 7.76 Hz,
1H, aromatic H), 7.85-7.88 (m, 2H, 2 × aromatic H), 8.17 (d, J = 7.60 Hz, 1H,
aromatic H), 10.12 (s, 1H, phenolic OH), 10.21 (s, 1H, aromatic NH). ESI-MS m/z
278.4 [M+H] +.
8

ACCEPTED MANUSCRIPT
5.1.3.9. N-(4-hydroxynaphthalen-1-yl)-3-(4-nitrophenyl)acrylamide (4j). Yield: 65 %.
1H NMR (DMSO-d6) δ 6.89 (d, J = 7.80 Hz, 1H, aromatic H), 7.26 (d, J = 15.60 Hz,
1H, Ar-CH=CH-CO-), 7.50 (d, J = 7.80 Hz, 1H, aromatic H), 7.55 (d, J = 7.20 Hz, 2H,
2 × aromatic H), 7.71 (d, J = 15.60 Hz, 1H, Ar-CH=CH-CO-), 7.92 (d, J = 8.40 Hz,
1H, aromatic H), 7.96 (d, J = 9.00 Hz, 1H, aromatic H), 8.18 (d, J = 8.40 Hz, 1H,
aromatic H), 8.32 (d, J = 7.80 Hz, 1H, aromatic H), 10.05 (s 1H, phenolic OH), 10.20
(s, 1H, aromatic NH). ESI-MS m/z 335.5 [M+H] +.
1
5.1.3.10. 3-chloro-N-(4-hydroxynaphthalen-1-yl)benzamide (4k). Yield: 63 %. H
NMR (DMSO-d6) δ 6.89 (d, J = 7.80 Hz, 1H, aromatic H), 7.32 (d, J = 7.80 Hz, 1H,
aromatic H), 7.47-7.52 (m, 2H, 2 × aromatic H), 7.59 (t, J = 7.80 Hz, 1H, aromatic H),
7.69 (d, J = 8.40 Hz, 1H, aromatic H), 7.83 (d, J = 8.40 Hz, 1H, aromatic H), 8.03 (d,
J = 7.20 Hz, 1H, aromatic H), 8.11 (s, 1H, aromatic H), 8.18 (d, J = 7.80 Hz, 1H,
aromatic H), 10.24 (s, 1H, phenolic OH), 10.30 (s, 1H, aromatic NH). ESI-MS m/z
298.4 [M+H] +.
1
5.1.3.11. N-(4-hydroxynaphthalen-1-yl)-4-nitrobenzamide (4l). Yield: 78 %. H NMR
(DMSO-d6) δ 6.92 (d, J = 8.00 Hz, 1H, aromatic H), 7.37 (d, J = 8.00 Hz, 1H,
aromatic H), 7.47-7.54 (m, 2H, 2 × aromatic H), 7.86 (d, J = 8.00 Hz, 1H, aromatic
H), 8.20 (d, J = 7.68 Hz, 1H, aromatic H), 8.30 (d, J = 8.68 Hz, 2H, 2 × aromatic H),
8.40 (d, J = 8.72 Hz, 2H, 2 × aromatic H), 10.30 (s, 1H, phenolic OH), 10.52 (s, 1H,
aromatic NH). ESI-MS m/z 309.4 [M+H] +.
5.1.3.12. 3-(4-chlorophenyl)-N-(4-hydroxynaphthalen-1-yl)acrylamide (4m). Yield:
1
83 %. H NMR (DMSO-d6) δ 6.87 (d, J = 8.40 Hz, 1H, aromatic H), 7.07 (d, J =
15.60 Hz, 1H, Ar-CH=CH-CO-), 7.47-7.51 (m, 2H, aromatic H), 7.53 (d, J = 8.40 Hz,
3H, aromatic H), 7.58 (d, J = 15.60 Hz, 1H, Ar-CH=CH-CO-), 7.68 (d, J = 7.80 Hz,
2H, aromatic H), 7.94 (d, J = 8.40 Hz, 1H, aromatic H), 8.17 (d, J = 7.20 Hz, 1H,
aromatic H). 9.92 (s, 1H, phenolic OH), 10.17 (s, 1H, aromatic NH). ESI-MS m/z
324.4 [M+H] +.
5.1.4. (E)-N-(4-oxonaphthalen-1(4H)-ylidene)-2-naphthamide (5a)
To a solution of compound 4a (3.13 g, 10.0 mmol) in 200 mL of CH2Cl2, was added
22 g (15 mmol) of NaIO4/SiO2(0.68mmol of NaIO4/1g of SiO2) [40]. After stirring at
room temperature for 2 h, the solid particles were filtered from the solution and
washed with 20 mL of CH2Cl2, and the filtrates were evaporated in vacuum to obtain
reddish-brown crude compound 5a which was then purified by recrystallization using
1
EtOAc. Yield: 88 %, mp: 148-149 . H NMR (DMSO-d6) δ 6.85 (d, J = 10.20 Hz,
1H, aromatic H), 7.22 (d, J = 10.20 Hz, 1H, aromatic H), 7.62 (t, J = 7.80 Hz, 1H,
aromatic H), 7.70 (t, J = 7.80 Hz, 1H, aromatic H), 7.89 (t, J = 7.80 Hz, 1H, aromatic
H), 7.94 (t, J = 7.80 Hz, 1H, aromatic H), 8.01-8.05 (m, 2H), 8.10 (d, J = 7.80 Hz, 2H,
2 × aromatic H), 8.15 (d, J = 7.80 Hz, 2H, aromatic H), 8.42 (d, J = 7.80 Hz, 1H,
13
aromatic H), 8.57 (s, 1H, aromatic H). C NMR (150 MHz, DMSO-d6) δ: 124.86,
126.31, 127.08, 128.00, 128.04, 128.21, 128.26, 128.47, 129.31, 132.00, 132.12,
132.62, 134.62, 134.66, 135.05, 139.16, 168.42, 185.28. ESI-MS m/z 312.1 [M+H] +.
Compounds 5b-5m were synthesized following the procedure described above.
9

ACCEPTED MANUSCRIPT
1
5.1.4.1. (E)-N-(4-oxonaphthalen-1(4H)-ylidene)cinnamamide (5b). Yield: 90 %. H
NMR (CDCl3) δ 6.70-6.74 (m, 2H, 2 × aromatic H), 7.11 (d, J = 10.28 Hz, 1H,
Ar-CH=CH-CO-), 7.38-7.39 (m, 3H), 7.52-7.56 (m, 3H), 7.71-7.77 (m, 2H), 8.11 (dd,
J = 7.40 Hz, 1.72 Hz, 1H, aromatic H), 8.32 (dd, J = 7.56 Hz, 1.44 Hz, 1H, aromatic
H). ESI-MS m/z 288.1 [M+H] +.
5.1.4.2. (E)-4-chloro-N-(4-oxonaphthalen-1(4H)-ylidene)benzamide (5c). Yield: 89 %.
1H NMR (DMSO-d6) δ 6.74 (d, J = 10.28 Hz, 1H, aromatic H), 7.09 (d, J = 10.32 Hz,
1H, aromatic H), 7.46 (d, J = 8.64 Hz, 1H, aromatic H), 7.73-7.80 (m, 2H , 2 ×
aromatic H), 7.91 (d, J = 8.56 Hz, 2H, 2 × aromatic H), 8.17 (d, J = 7.16 Hz, 1H,
aromatic H), 8.37 (d, J = 7.24 Hz, 1H, aromatic H). ESI-MS m/z 296.3 [M+H] +.
5.1.4.3. (E)-2-nitro-N-(4-oxonaphthalen-1(4H)-ylidene)benzamide (5d). Yield: 72 %.
1H NMR (CDCl3) δ 6.80 (d, J = 10.44 Hz, 1H, aromatic H), 7.53 (d, J = 10.32 Hz, 1H,
aromatic H), 7.66-7.73 (m, 4H), 7.84 (d, J = 7.20 Hz, 1H, aromatic H), 7.95 (d, J =
9.60 Hz, 1H, aromatic H), 8.10 (d, J = 8.40 Hz, 1H, aromatic H). ESI-MS m/z 393.3
[M+H] +.
5.1.4.4. (E)-4-methoxy-N-(4-oxonaphthalen-1(4H)-ylidene)benzamide (5e). Yield: 85
%. 1H NMR (DMSO-d6) δ 3.84 (s, 3H, OCH3), 6.83 (d, J = 10.20 Hz, 1H, aromatic H),
7.08 (d, J = 9.60 Hz, 2H, 2 × aromatic H), 7.13 (d, J = 9.60 Hz, 1H, aromatic H),
7.85-7.91 (m, 4H), 8.07 (d, J = 7.20 Hz, 1H, aromatic H), 8.33 (d, J = 7.80 Hz, 1H,
aromatic H). ESI-MS m/z 292.3 [M+H] +.
5.1.4.5.
(2E,NE)-3-(3,4-dimethoxyphenyl)-N-(4-oxonaphthalen-1(4H)-ylidene)acrylamide (5f).
Yield: 80 %. 1H NMR (CDCl3) δ 3.86 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 6.60 (d, J =
15.96 Hz, 1H, Ar-CH=CH-CO-), 6.73 (d, J = 10.44 Hz, 1H, aromatic H), 6.87 (d, J =
8.28 Hz, 1H, aromatic H), 7.07-7.6 (m, 3H), 7.49-7.52 (m, 1H), 7.72-7.77 (m, 3H),
8.14 (d, J = 7.40 Hz, 1H, aromatic H), 8.35 (d, J = 7.56 Hz, 1H, aromatic H). ESI-MS
m/z 348.4 [M+H] +.
5.1.4.6. (E)-3-nitro-N-(4-oxonaphthalen-1(4H)-ylidene)benzamide (5g). Yield: 88 %.
1H NMR (DMSO-d6) δ 6.78 (d, J = 10.28 Hz, 1H, aromatic H), 7.15 (d, J = 10.32 Hz,
1H, aromatic H), 7.72 (t, J = 8.00 Hz, 1H, aromatic H), 7.78-7.82 (m, 2H, 2 ×
aromatic H), 7.20 (dd, J = 7.08 Hz, 2.00 Hz, 1H, aromatic H), 8.35 (d, J = 6.48 Hz,
1H, aromatic H), 8.40 (dd, J = 7.12 Hz, 4.76 Hz, 1H, aromatic H), 8.48 (d, J = 8.24
Hz, 1H, aromatic H), 8.80 (s, 1 H, aromatic H). ESI-MS m/z 393.3 [M+H] +.
5.1.4.7. (E)-3-methoxy-N-(4-oxonaphthalen-1(4H)-ylidene)benzamide (5h). Yield: 91
%. 1H NMR (DMSO-d6) δ 3.89 (s, 3H, OCH3), 6.75 (d, J = 10.32 Hz, 1H, aromatic H),
7.12 (d, J = 10.28 Hz, 1H, aromatic H), 7.16-7.19 (m, 1H, aromatic H), 7.39 (t, J =
10.28 Hz, 1H, aromatic H), 7.50 (dd, J = 6.52 Hz, 1.16 Hz, 1H, aromatic H), 7.57
(s,1H, aromatic H), 7.74-7.82 (m, 2H, 2 × aromatic H), 8.20 (d, J = 7.28 Hz, 1H,
aromatic H), 8.41 (d, J = 7.36 Hz, 1H, aromatic H). ESI-MS m/z 292.3 [M+H] +.
5.1.4.8. (E)-3-methyl-N-(4-oxonaphthalen-1(4H)-ylidene)benzamide (5i). Yield: 88 %.
1H NMR (DMSO-d6) δ 2.41 (s, 3H, CH3), 6.72 (d, J = 10.32 Hz, 1H, aromatic H),
7.09 (d, J = 10.60 Hz, 1H, aromatic H), 7.26-7.38 (m, 1H, aromatic H), 7.57 (t, J =
12.80 Hz, 1H, aromatic H), 7.64 (s, 1H, aromatic H), 7.73-7.77 (m, 2H, 2 × aromatic
10

ACCEPTED MANUSCRIPT
H), 8.09 (dd, J = 6.82 Hz, 2.82 Hz, 1H, aromatic H), 8.7 (d, J = 7.32 Hz, 1H, aromatic
H), 8.39 (d, J = 7.24 Hz, 1H, aromatic H). ESI-MS m/z 276.4 [M+H] +.
5.1.4.9.
(2E,NE)-3-(4-nitrophenyl)-N-(4-oxonaphthalen-1(4H)-ylidene)acrylamide
(5j). Yield: 83 %. 1H NMR (CDCl3) δ 6.75 (d, J = 10.28 Hz, 1H, aromatic H), 6.84 (d,
J = 16.12 Hz, 1H, Ar-CH=CH-CO-), 7.13 (d, J = 10.28 Hz, 1H, aromatic H), 7.62 (d,
J = 16.12 Hz, 1H, Ar-CH=CH-CO-), 7.75-7.77 (m, 2H, 2 × aromatic H), 8.14 (d, J =
7.24 Hz, 1H, aromatic H), 8.23 (d, J = 8.80 Hz, 1H, 2 × aromatic H), 8.33 (d, J = 7.24
Hz, 1H, aromatic H). ESI-MS m/z 333.4 [M+H] +.
5.1.4.10. (E)-3-chloro-N-(4-oxonaphthalen-1(4H)-ylidene)benzamide (5k). Yield: 90
%. 1H NMR (DMSO-d6) δ 6.74 (d, J = 10.28 Hz, 1H, aromatic H), 7.09 (d, J = 10.28
Hz, 1H, aromatic H), 7.43 (t, J = 10.28 Hz, 1H, aromatic H), 7.58 (dd, J = 8.00 Hz,
0.96 Hz, 1H, aromatic H), 7.77-7.81 (m, 2H, 2 × aromatic H), 7.85 (d, J = 7.72 Hz,
1H, aromatic H), 7.95 (s, 1H, aromatic H), 8.17 (d, J = 7.12 Hz, 1H, aromatic H), 8.39
(d, J = 8.00 Hz, 1H, aromatic H). ESI-MS m/z 296.4 [M+H] +.
5.1.4.11. (E)-4-nitro-N-(4-oxonaphthalen-1(4H)-ylidene)benzamide (5l). Yield: 81 %.
1H NMR (CDCl3) δ 6.79 (d, J = 10.28 Hz, 1H, aromatic H), 7.12 (d, J = 10.40 Hz, 1H,
aromatic H), 7.77-7.83 (m, 2H, 2 × aromatic H), 8.16-8.20 (m, 3H, 3 × aromatic H),
8.35 (d, J = 8.88 Hz, 2H, 2 × aromatic H), 8.40 (d, J = 9.00 Hz, 1H, aromatic H).
ESI-MS m/z 307.4 [M+H] +.
5.1.4.12. (2E,NE)-3-(4-chlorophenyl)-N-(4-oxonaphthalen-1(4H)-ylidene)acrylamide
1
(5m). Yield: 92 %. H NMR (CDCl3) δ 6.70 (d, J = 16.12 Hz, 1H, Ar-CH=CH-CO-),
6.74 (d, J = 10.36 Hz, 1H, aromatic H), 7.12 (d, J = 10.28 Hz, 1H, aromatic H), 7.37
(d, J = 8.56 Hz, 2H, 2 × aromatic H), 7.48-7.54 (m, 3H), 7.72-7.76 (m, 2H), 8.13 (d, J
= 7.28 Hz, 1H, aromatic H), 8.32 (d, J = 7.40 Hz, 1H, aromatic H). ESI-MS m/z 322.1
[M+H] +.
5.1.5. N-(3-((1H-1,2,4-triazol-5-yl)thio)-4-hydroxynaphthalen-1-yl)-2-naphthamide
(6a)
To a solution of 1H-1,2,4-triazole-5-thiol (2.53 g, 25 mmol) in 10 mL of DMF, was
added 3.11 g (10 mmol) of compound 5a, slowly [41]. After stirring at room
temperature for 4 h, the solution was poured into 100 mL of distilled water with slight
yellow solid precipitation. After standing for 20 minutes the slight yellow solid was
filtered with suction, pressed to remove excess water, and allowed to dry in air. The
dry residues were recrystallized with MeOH to give 2.84 g of title compound 6a as
white powder. Yield: 69 %, mp: 229-230 . 1H NMR (DMSO-d6) δ 7.43 (s, 1H,
aromatic H), 7.50-7.68 (m, 5H), 7.89-8.12 (m, 6H), 8.29 (q, J = 3.20 Hz, 1H, aromatic
H), 8.54 (s, 1H, aromatic H), 8.70 (s, 1H, aromatic H), 10.43 (s, 1H, aromatic NH).
13C NMR (150 MHz, DMSO-d6) δ: 110.4, 122.7, 123.5, 124.4, 125.1, 125.6, 125.7,
126.4, 126.7, 126.9, 127.6, 127.7, 127.9, 128.1, 128.9, 130.6, 131.6, 132.1, 134.3,
146.3, 151.4, 155.2, 166.3. ESI-MS m/z 413.6 [M+H] +.
Target compounds 6b-6s were synthesized following the procedure described above.
5.1.5.1.
N-(3-((1H-1,2,4-triazol-5-yl)thio)-4-hydroxynaphthalen-1-yl)cinnamamide
(6b). Yield: 85 %, mp: 307-309 . 1H NMR (DMSO-d6) δ 7.13 (d, J = 15.76 Hz, 1H,
11

ACCEPTED MANUSCRIPT
Ar-CH=CH-CO-), 7.39-7.48 (m, 4H), 7.57-7.66 (m, 6H), 7.77 (s, 1H, aromatic H),
8.07 (d, J = 7.32 Hz, 1H, aromatic H), 8.37 (d, J = 7.28 Hz, 1H, aromatic H), 9.42 (s,
1H, phenolic OH), 10.04 (s, 1H, aromatic NH). 13C NMR (150 MHz, DMSO-d6) δ:
119.6, 122.4, 122.6, 122.9, 125.1, 125.3, 126.1, 127.6, 128.4, 128.9, 129.6, 134.9,
139.8, 147.1, 164.3. ESI-MS m/z 389.4 [M+H] +.
5.1.5.2.
N-(3-((1H-1,2,4-triazol-5-yl)thio)-4-hydroxynaphthalen-1-yl)-4-chlorobenzamide (6c).
1
Yield: 70 %., mp: 146-148 . H NMR (DMSO-d6) δ 7.38 (s, 1H, aromatic H),
7.51-7.67 (m, 4H), 7.81-7.85 (m, 1H, aromatic H), 8.06 (d, J = 8.40 Hz, 2H, aromatic
H), 8.26-8.31 (m, 1H, aromatic H), 8.53 (s, 1H, aromatic H), 8.89 (s, 1H, aromatic
13
NH), 10.33 (s, 1H, phenolic OH), 10.60 (s, 1H, aromatic NH). C NMR (150 MHz,
DMSO-d6) δ: 110.3, 122.7, 123.4, 125.6, 125.7, 126.0, 126.9, 128.0, 128.4, 129.6,
130.5, 133.0, 136.4, 146.3, 151.5, 155.2, 165.1. ESI-MS m/z 397.4 [M+H] +.
5.1.5.3.
N-(3-((1H-1,2,4-triazol-5-yl)thio)-4-hydroxynaphthalen-1-yl)-2-nitrobenzamide (6d).
1
Yield: 91 %, mp: 265-266 . H NMR (DMSO-d6) δ 7.55 (s,1H, aromatic H),
7.57-7.64 (m, 2H, 2 × aromatic H), 7.74-7.78 (m, 1H, aromatic H), 7.87-7.94 (m,1H,
aromatic H), 8.05 (d, J = 7.84 Hz,1H, aromatic H), 8.16 (d, J = 8.04 Hz,1H, aromatic
H), 8.29 (d, J = 7.64 Hz,1H, aromatic H), 8.64 (s, 1H, aromatic H), 10.21 (s, 1H,
aromatic NH), 10.51 (s, 1H, phenolic OH), 14.25 (s, 1H, aromatic NH). 13C NMR
(150 MHz, DMSO-d6) δ: 123.2, 123.6, 124.7, 125.7, 125.09, 126.3, 127.5, 128.1,
129.9, 130.0, 130.6, 131.3, 133.2, 134.5, 145.6, 147.1, 165.7. ESI-MS m/z 408.4
[M+H] +.
5.1.5.4.
N-(3-((1H-1,2,4-triazol-5-yl)thio)-4-hydroxynaphthalen-1-yl)-4-methoxybenzamide
(6e). Yield: 92 %, mp: 142-143 . 1H NMR (DMSO-d6) δ 3.84 (s, 3H, CH3), 7.06 (d,
J=8.70 Hz, 2H, 2 × aromatic H), 7.34 (s, 1H, aromatic H), 7.53~7.57 (m, 2H,
aromatic H), 8.02 (d, J=8.70 Hz, 2H, 2 × aromatic H), 8.24~8.27 (m, 1H, aromatic H),
8.61 (s, 1H, aromatic H), 10.08 (s, 1H, aromatic NH), 10.13 (s, 1H, phenolic OH),
13
14.23 (s, 1H, aromatic NH). C NMR (150 MHz, DMSO-d6) δ: 55.3, 110.6, 113.6,
122.6, 123.5, 125.6, 125.7, 126.3, 126.5, 126.7, 128.0, 129.5, 130.6, 146.5, 151.2,
161.8, 165.6. ESI-MS m/z 393.3 [M+H] +.
5.1.5.5.
(E)-N-(3-((1H-1,2,4-triazol-5-yl)thio)-4-hydroxynaphthalen-1-yl)-3-(3,4-dimethoxyph
enyl)acrylamide (6f). Yield: 93 %, mp: 252-253 . 1H NMR (DMSO-d6) δ 3.80 (s, 3H,
OCH3), 3.83 (s, 3H, OCH3), 6.92 (d, J = 15.60 Hz, 1H, Ar-CH=CH-CO-), 7.03 (d, J =
8.40 Hz, 1H, aromatic H), 7.20 (d, J = 8.40 Hz, 1H, aromatic H), 7.24 (s, 1H,
aromatic H), 7.49-7.63 (m, 4H), 8.00 (d, J = 7.80 Hz, 1H, aromatic H), 8.26 (d, J =
6.90 Hz, 1H, aromatic H), 8.63 (s, 1H, aromatic H), 9.85 (s, 1H, aromatic NH), 10.03
(s, 1H, phenolic OH), 14.23 (s, 1H, aromatic NH). 13C NMR (150 MHz, DMSO-d6) δ:
55.4, 55.5, 110.1, 111.0, 111.7, 119.7, 121.6, 122.7, 122.8, 125.5, 125.7, 126.3, 126.7,
127.6, 128.9, 140.1, 145.1, 148.9, 150.3, 157.4, 164.5. ESI-MS m/z 449.5 [M+H] +
12

ACCEPTED MANUSCRIPT
5.1.5.6.
N-(3-((1H-1,2,4-triazol-5-yl)thio)-4-hydroxynaphthalen-1-yl)-3-nitrobenzamide (6g).
1
Yield: 57 %, mp: 155-157 . H NMR (DMSO-d6) δ 7.42 (s, 1H, aromatic H),
7.55-7.61 (m, 2H), 7.83-7.89 (m, 2H), 8.28-8.33 (m, 1H, aromatic H), 8.44-8.51 (m,
1H, aromatic H), 8.57 (s, 1H, aromatic H), 8.88 (s, 1H, aromatic H), 10.66 (s, 1H,
13
phenolic OH). C NMR (150 MHz, DMSO-d6) δ: 110.2, 122.5, 122.7, 123.4, 125.6,
125.8, 126.1, 127.0, 128.1, 130.1, 130.4, 134.1, 135.7, 146.2, 147.7, 151.7, 155.1,
164.1. ESI-MS m/z 408.5 [M+H] +.
5.1.5.7.
N-(3-((1H-1,2,4-triazol-5-yl)thio)-4-hydroxynaphthalen-1-yl)-3-methoxybenzamide
(6h). Yield: 81 %, mp: 202-204 . 1H NMR (DMSO-d6) δ 3.84 (s, 3H, OCH3), 7.16
(dd, J = 8.10 Hz, 2.10Hz, 1H, aromatic H), 7.36 (s, 1H, aromatic H), 7.45 (t, J = 8.10
Hz, 1H, aromatic H), 7.55-7.64 (m, 4H), 7.81-7.84 (m, 1H, aromatic H), 8.25-8.28 (m,
1H, aromatic H), 8.62 (s, 1H, aromatic H), 10.14 (s, 1H, aromatic NH), 10.22 (s, 1H,
13
phenolic OH), 14.23 (s, 1H, aromatic NH). C NMR (150 MHz, DMSO-d6) δ: 55.3,
110.7, 112.7, 117.5, 119.9, 122.6, 123.5, 125.6, 125.7, 126.2, 126.8, 128.0, 129.5,
130.5, 135.6, 145.1, 151.3, 159.2, 165.8. ESI-MS m/z 393.3 [M+H] +.
5.1.5.8.
N-(3-((1H-1,2,4-triazol-5-yl)thio)-4-hydroxynaphthalen-1-yl)-3-methylbenzamide (6i).
Yield: 97 %, mp: 195-196 . 1H NMR (DMSO-d6) δ 2.40 (s, 3H, CH3), 7.36 (s, 1H,
aromatic H), 7.42 (d, J=4.50 Hz, 1H, aromatic H), 7.54~7.58 (m, 2H), 7.82~7.86 (m,
3H), 8.25~8.28 (m, 1H, aromatic H), 8.62 (s, 1H, aromatic H), 10.11 (s, 1H, aromatic
13
NH), 10.19 (s, 1H, phenolic OH), 14.25 (s, 1H, aromatic NH). C NMR (150 MHz,
DMSO-d6) δ: 20.9, 110.9, 122.6, 123.5, 124.8, 125.6, 125.7, 126.3, 126.8, 128.2,
130.5, 132.1, 134.2, 137.6, 145.0, 151.2, 166.3. ESI-MS m/z 377.5 [M+H] +.
5.1.5.9.
(E)-N-(3-((1H-1,2,4-triazol-5-yl)thio)-4-hydroxynaphthalen-1-yl)-3-(4-nitrophenyl)ac
rylamide (6j). Yield: 79 %, mp: 202-204 . 1H NMR (DMSO-d6) δ 7.25 (d, J = 15.60
Hz, 1H, Ar-CH=CH-CO-), 7.57-7.70 (m, 4H), 7.91 (d, J = 8.40 Hz, 2H, 2 × aromatic
H), 8.01 (d, J = 7.80 Hz, 1H, aromatic H), 8.27 (d, J = 7.80 Hz, 1H, aromatic H), 8.31
(d, J = 8.40 Hz, 2H, aromatic H), 8.54 (s, 1H, aromatic H), 10.09 (s, 1H, aromatic
13
NH), 10.20 (s, 1H, phenolic OH), 14.31 (s, 1H, aromatic NH). C NMR (150 MHz,
DMSO-d6) δ: 111.3, 123.2, 124.6, 125.9, 126.0, 126.3, 126.4, 126.9, 127.3, 129.1,
129.3, 138.0, 141.9, 148.0, 151.0, 164.0. ESI-MS m/z 434.4 [M+H] +.
5.1.5.10.
N-(3-((1H-1,2,4-triazol-5-yl)thio)-4-hydroxynaphthalen-1-yl)-2-naphthamide
(6k).
1
Yield: 88 %, mp: 209-212 . H NMR (DMSO-d6) δ 7.38 (s, 1H, aromatic H),
7.51-7.67 (m, 3H), 7.68 (d, J = 8.40 Hz, 1H, aromatic H), 7.92-7.98 (m, 1H, aromatic
H), 7.99 (d, J = 7.50 Hz, 1H, aromatic H), 8.16 (s, 1H, aromatic H), 8.26-8.28 (m, 1H,
aromatic H), 8.62 (s, 1H, aromatic H), 10.15 (s, 1H, aromatic NH), 10.35 (s, 1H,
phenolic OH), 14.23 (s, 1H, aromatic NH). 13C NMR (150 MHz, DMSO-d6) δ: 110.7,
122.6, 123.4, 125.5, 125.8, 125.9, 126.4, 126.9, 127.5, 128.1, 130.3, 130.4, 131.3,
133.2, 136.2, 145.3, 151.5, 157.2, 164.8. ESI-MS m/z 397.4 [M+H] +.
13

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5.1.5.11.
N-(3-((1H-1,2,4-triazol-5-yl)thio)-4-hydroxynaphthalen-1-yl)-4-nitrobenzamide (6l).
1
Yield: 87 %, mp: 243-244 . H NMR (DMSO-d6) δ 7.43 (s, 1H, aromatic H),
7.57-7.59 (m, 2H), 7.87 (s, 1H, aromatic H), 8.25 (s, 1H, aromatic H), 8.28 (d, J =
9.00 Hz, 2H, 2 × aromatic H), 8.39 (d, J=9.00 Hz, 2H, 2 × aromatic H), 8.63 (s, 1H,
aromatic H), 10.21 (s, 1H, aromatic NH), 10.58 (s, 1H, phenolic OH), 14.24 (s, 1H,
13
aromatic NH). C NMR (150 MHz, DMSO-d6) δ: 123.2, 123.8, 124.0, 126.0, 126.1,
126.3, 127.5, 128.6, 129.7, 130.8, 140.5, 149.6, 152.1, 162.7, 165.1. ESI-MS m/z
408.4 [M+H] +.
5.1.5.12.
(E)-N-(3-((1H-1,2,4-triazol-5-yl)thio)-4-hydroxynaphthalen-1-yl)-3-(4-chlorophenyl)a
crylamide (6m). Yield: 83 %. 1H NMR (DMSO-d6) δ 7.18 (d, J = 15.60 Hz, 1H,
Ar-CH=CH-CO-), 7.58-7.65 (m, 4H), 7.84 (d, J = 7.80 Hz, 2H, 2 × aromatic H), 7.93
(d, J = 7.80 Hz, 2H, 2 × aromatic H), 7.99 (d, J = 7.80 Hz, 1H, aromatic H), 8.26 (d, J
= 7.80 Hz, 1H, aromatic H), 8.64 (s, 1H, aromatic H), 10.09 (s, 1H, aromatic NH),
10.19 (s, 1H, phenolic OH),14.25 (s, 1H, aromatic NH). 13C NMR (150 MHz,
DMSO-d6) δ: 111.7, 112.0, 119.1, 123.2, 126.0, 126.1, 126.3, 126.6, 127.2, 128.8,
129.3, 133.3, 138.5, 139.9, 145.6, 150.9, 157.8, 164.1. ESI-MS m/z 423.4 [M+H] +.
5.1.5.13.
4-chloro-N-(4-hydroxy-3-((5-methyl-1,3,4-thiadiazol-2-yl)thio)naphthalen-1-yl)benza
mide (6n). Yield: 86 %, mp: 229-230 . 1H NMR (DMSO-d6) δ 2.58 (s, 3H, CH3),
7.61-7.68 (m, 5H), 7.93 (d, J = 8.40 Hz, 1H, aromatic H), 8.09 (d, J = 8.40 Hz, 2H, 2
× aromatic H), 8.35 (d, J = 8.40 Hz, 1H, aromatic H), 10.41 (s, 1H, phenolic OH),
13
10.69 (s, 1H, aromatic NH). C NMR (150 MHz, DMSO-d6) δ: 15.6, 109.1, 123.8,
124.0, 126.1, 126.6, 127.1, 128.6, 128.9, 129.8, 130.1, 132.3, 133.5, 136.9, 154.4,
165.7, 166.3, 168.4. ESI-MS m/z 428.5 [M+H] +.
5.1.5.14.
N-(3-((1,3,4-thiadiazol-2-yl)thio)-4-hydroxynaphthalen-1-yl)-4-chlorobenzamide (6o).
Yield: 70 %, mp: 254 . 1H NMR (DMSO-d6) δ 7.61-7.70 (m,5H), 7.95 (d, J = 8.00 Hz,
1H, aromatic H), 8.09 (d, J = 8.28 Hz, 2H, 2 × aromatic H), 8.36 (d, J = 7.80 Hz, 1H,
aromatic H), 9.42 (s, 1H, aromatic H), 10.40 (s, 1H, phenolic OH), 10.69 (s, 1H,
13
aromatic NH). C NMR (150 MHz, DMSO-d6) δ: 108.8, 123.8, 124.1, 126.1, 126.7,
127.1, 128.7, 128.9, 129.9, 130.1, 132.4, 133.5, 136.9, 154.5, 154.7, 165.7, 169.2.
ESI-MS m/z 414.4 [M+H] +.
5.1.5.15.
4-chloro-N-(4-hydroxy-3-((1-methyl-1H-tetrazol-5-yl)thio)naphthalen-1-yl)benzamide
(6p). Yield: 91 %, mp: 255-257 . 1H NMR (DMSO-d6) δ 4.05 (s, 3H, CH3), 7.50 (s,
1H), 7.60-7.65 (m, 4H), 7.91 (d, J = 8.40 Hz, 1H, aromatic H), 8.08 (d, J = 8.40 Hz,
1H, aromatic H), 8.31 (d, J = 9.60 Hz, 1H, aromatic H), 10.40 (s, 1H, phenolic OH),
13
10.47 (s, 1H, aromatic NH). C NMR (150 MHz, DMSO-d6) δ: 34.5, 107.1, 123.4,
124.1, 126.0, 126.6, 126.6, 126.9, 128.1, 128.9, 129.0, 130.1, 131.7, 133.4, 136.9,
153.0, 153.3, 165.7. ESI-MS m/z 412.5 [M+H] +.
14

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5.1.5.16. N-(3-((7H-purin-6-yl)thio)-4-hydroxynaphthalen-1-yl)-4-chlorobenzamide
(6q). Yield: 69 %, mp: 275-276 . 1H NMR (DMSO-d6) δ 7.59-7.68 (m, 3H), 7.94 (d, J
= 8.12 Hz, 1H, aromatic H), 8.11 (d, J = 8.08 Hz, 1H, aromatic H), 8.21 (s, 1H,
aromatic H), 8.35 (d, J = 8.08 Hz, 1H, aromatic H), 8.41 (s, 1H, aromatic H), 8.54 (d,
J = 9.20 Hz, 1H, aromatic H), 10.17 (s, 1H, aromatic H), 10.33 (s, 1H, aromatic H),
10.40 (s, 1H, aromatic NH), 10.54 (s, 1H, phenolic OH), 13.76 (s, 1H, aromatic NH).
13C NMR (150 MHz, DMSO-d6) δ: 105.6, 105.7, 123.6, 123.9, 126.0, 126.1, 128.0,
128.9, 129.6, 130.1, 130.2, 131.6, 132.1, 133.6, 136.8, 144.1, 144.9, 145.1, 151.0,
152.0, 155.3, 165.7, 171.3. ESI-MS m/z 448.4 [M+H] +.
5.1.5.17.
N-(3-((7H-purin-6-yl)thio)-4-hydroxynaphthalen-1-yl)-2-nitrobenzamide
(6r). Yield: 78 %, mp: 209-212 . 1H NMR (DMSO-d6) δ 7.59-7.67 (m, 3H), 7.95 (d, J
= 7.80 Hz, 1H, aromatic H), 8.29 (d, J = 9.00 Hz, 2H, 2 × aromatic H), 8.33 (d, J =
7.80 Hz, 1H, aromatic H), 8.39 (d, J = 9.00 Hz, 2H, 2 × aromatic H), 8.49 (s, 1H,
aromatic H), 8.51 (s, 1H, aromatic H), 10.18 (s, 1H, aromatic NH), 10.63 (s, 1H,
phenolic OH), 13.58 (s, 1H, aromatic NH). 13C NMR (150 MHz, DMSO-d6) δ: 105.6,
123.7, 123.8, 124.0, 125.8, 126.0, 126.2, 128.1, 129.7, 131.6, 131.9, 140.6, 144.1,
149.6, 152.0, 155.5, 165.2. ESI-MS m/z 459.3 [M+H] +.
5.1.5.18.
N-(3-((7H-purin-6-yl)thio)-4-hydroxynaphthalen-1-yl)-4-nitrobenzamide
(6s). Yield: 74 %, mp: 262-263 . 1H NMR (DMSO-d6) δ 7.59 (s, 1H, aromatic H),
7.62 (t, J = 7.80 Hz, 1H, aromatic H), 7.67 (t, J = 7.80 Hz, 1H, aromatic H), 7.95 (d, J
= 7.80 Hz, 1H, aromatic H), 8.30 (d, J = 8.40 Hz, 2H, 2 × aromatic H), 8.34 (d, J =
7.80 Hz, 1H, aromatic H), 8.40 (d, J = 7.80 Hz, 1H, aromatic H), 8.49 (s, 1H,
aromatic H), 8.52 (s, 1H, aromatic H), 10.19 (s, 1H, aromatic NH), 10.63 (s, 1H,
phenolic OH), 13.58 (s, 1H, aromatic NH). 13C NMR (150 MHz, DMSO-d6) δ: 105.7,
123.7, 123.8, 123.9, 124.0, 125.8, 126.0, 126.2, 128.1, 129.7, 129.9, 131.5, 131.9,
140.6, 144.1, 149.7, 152.0, 155.5, 165.2. ESI-MS m/z 459.3 [M+H] +.
5.2. Biological assay
5.2.1. MTT assay
HUVECs, A549, K562, PC-3,HCT116, MDA-MB-231 and MCF-7 cells were
respectively grown in RPMI 1640 medium containing 10 % FBS at 37 °C in 5 % CO2
humidified incubator. Cell proliferation was determined by the MTT
(3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) assay. Briefly,
cells were plated in a 96-well plate at 5,000 cells per well, cultured for 4 h in
complete growth medium, then treated with serial dilutions of the compounds for 48 h.
During the last 4 h, 10 ꢀL of 0.5 % MTT solution was added to each well. After
further incubation for 4 h, formazan formed from MTT was extracted by adding 200
ꢀL of DMSO and mixed for 15 min. Optical density was read with an ELISA reader at
570 nm.
5.2.2. In vitro HUVECs tuber formation assay
Forty eight-well slide chambers were coated with 100 ꢀL of Matrigel (BD
Biosciences, NJ) and allowed to gel at 37 °C and 5 % CO2 for 30 min. HUVECs were
15

ACCEPTED MANUSCRIPT
then seeded at 40,000 cells/well in M199 (5 % FBS) containing either the vehicle (0.5
% DMSO), Pazopanib or synthesized compounds and incubated at 37 °C and 5 %
CO2 for 6 h. The morphological changes of the cells and HUVEC tubes formation
were observed under a phase-contrast microscope and photographed at ×200
magnification. The corresponding area was measured as the number of pixels using
Adobe PhotoShop [33, 34]. Experiments were repeated three times.
5.2.3. Rat thoracic aorta rings (TARs) assay
Forty eight-well tissue culture plates were coated with 100 ꢀL of Matrigel (BD
Biosciences, NJ) and allowed to gel for 30 min at 37 °C and 5 % CO2. Thoracic aortas
were excised from 8- to 10-week-old male Sprague Dawley rats. After careful
removal of fibroadipose tissues, the aortas were cut into 1-mm-long cross-sections,
placed on Matrigel-coated wells, and covered with an additional 100 ꢀL of Matrigel.
After the second layer of Matrigel set, the rings were incubated for 30 min at 37 °C
and 5 % CO2. Aorta rings were treated every other day with either the vehicle (0.5 %
DMSO), Pazopanib, or synthesized compounds for 6 days and photographed on the
7th day at ×200 magnification. The area of angiogenic sprouting, reported in the
number of pixels, was quantified using Adobe PhotoShop [33, 35]. Experiments were
repeated three times.
5.2.4. In vitro kinase activity
The kinase selectivity of compounds (at 10 ꢀM) was profiled by screening as follows:
The test compounds, reference compound or water (control) were mixed with the
appropriate enzyme in a buffer containing Hepes/Tris (pH 7.4), EGTA/Tris, MgCl2,
DTT and 0.008 % Tween 20. Thereafter, the reaction was initiated by adding the
substrate and ATP, and the mixture was incubated for 30 min at room temperature. For
control basal measurements, the enzyme was omitted from the reaction mixture.
Following incubation, the reaction was stopped by adding EDTA. After 5 min, the
anti-phopho-PT66 antibody labeled with europium chelate was added. After 60 more
min, the fluorescence transfer was measured using a microplate reader (Envision,
Perkin Elmer). The results were expressed as a percent inhibition of the control
enzyme activity. The standard inhibitory reference compound was Staurosporine. The
experiment was performed in duplicate.
5.2.5. Docking study
Compound 6d was docked to the active site of the modeled ABL, EGFR, ALK and
VEGFR2, respectively. Surflex dock program[42, 43] in Sybyl 8.0 software was used,
ten poses was generated for each ligand. The protomol was generated based on the
ligand in the crystal structure. Other parameter was set referring the default values.
Acknowledgments
This work was supported by National Nature Science Foundation of China (grant no.
21172134), National Scientific and Technological Major Project of Ministry of
16

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Science and Technology of China (grant no. 2011ZX09401-015), and Academic
Award for Doctoral Postgraduate of Ministry of Education of China (grant no.
20110131110037).
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Figure captions
Fig. 1. The tuber formation inhibitory effects of all the synthesized compounds using
bar graphs of the total tube area in the microscopic field measured as number of pixels
compared to the vehicle control (0.5% DMSO): (A) Tube formation assay of all the
synthesized compounds at 50 ꢀM; (B) The tuber formation inhibitory effects of
compounds 6d, 6r, 6b, 6n, 6o, 6k , 6l and 6c at concentrations of 50, 20 and 10 ꢀM,
respectively. Bar graph represents the mean ± SD of three independent experiments.
Fig. 2. Representative images of the tube formation assay depicting the formation of a
HUVEC capillary-like tubular network by treatment with synthesized compounds,
pazopanib (positive control) and 0.5% DMSO (vehicle control).
Fig. 3. The thoracic aorta rings (TARs) inhibitory effects of all the synthesized
compounds using bar graphs of the total microvessel area in the microscopic field
measured as number of pixels compared to the vehicle control (0.5% DMSO): (A)
TARs assay of all the synthesized compounds at 50 ꢀM; (B) The TARs inhibitory
effects of compounds 6r, 6d, 6b, 6q and 6n at concentrations of 50, 20, 10 and 1 ꢀM,
respectively. Bar graph represents the mean ± SD of three independent experiments.
Fig. 4. Representative images of rat aortic rings treated with synthesized compounds,
pazopanib (positive control) and 0.5% DMSO (vehicle control).
Fig. 5. Calculated binding modes of compound 6d in the ATP-binding pocket of the
ABL (A), EGFR (B), ALK (C) and VEGFR2 (D) kinase domains. The structural
framework of compound 6d was colored according to the atomic coloring scheme (S
in yellow, C in green, O in red and N in blue). Hydrogen bonds were indicated by
dash lines.
Scheme 1
Scheme 1. Reagents and conditions䟛(a) t-BuOOH, KOH, DMSO/H2O(3/1); (b) Pd-C,
H2, MeOH; (c) THF/H2O (50/1), NaHSO3; (d) NaIO4/SiO2, DCM; (e) DMF, R2SH.
22

ACCEPTED MANUSCRIPT
> A series of N-(4-hydroxy-3-mercaptonaphthalen-1-yl)amides were synthesized.
> The compounds were evaluated as inhibitors of angiogenesis and tumor growth.
> Some of them exhibited remarkably in vitro antiangiogenic activity.
> Inhibitory activity of 6d was similar to clinically used drug pazopanib.

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Table 1
The structures and in vitro antiproliferative activity of the target compounds and
Sorafenib
IC50 (µM)
MDA-MB-231 PC-3
Cmpd R1
R2
HCT116
>100
45.08±0.97
>100
>100
6a
6b
6c
>100
>100
25.71±0.84
5.52±0.36
43.27±1.35
21.37±0.72
52.52±1.15 10.19±0.65
6d
>100
>100
>100
>100
>100
6e
6f
52.45±1.17
48.00±1.02
>100
26.91±0.46 70.22±2.96
42.13±1.87 >100
6g
6h
6i
>100
59.54±2.77 >100
>100
>100
>100
>100
>100
6j
6k
6l
85.61±1.26 >100
53.51±0.95 >100

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>100
>100
>100
6m
6o
6p
6q
6r
>100
49.53±1.01 >100
>100
>100
>100
>100
>100
>100
22.97±0.74
54.18±1.46
66.43±3.14 >100
>100
>100
31.73±0.87
6s
>100
>100
6t
14.62±0.96
24.20±1.03 8.08±0.86
Sorafenib
Table 2.
Antiproliferative activity of compounds against HUVECs, MCF-7, A549 and K562
cells
IC50 (µM)
HUVEC
>100
Cmpd
MCF-7
A549
K562
>100
41.85±1.82
40.53±1.92
38.39±1.88
>100
18.76±2.43
10.81±0.19
16.94±1.53
37.18±1.75
13.85±0.89
6c
5.34±0.76
7.53±0.69
ND a
2.81±0.55
14.43±1.26
>100
6d
6q
6r
6.42±1.13
7.33±0.65
12.54±0.95
Sorafenib
a Not determined.
Table 3
Enzyme inhibitory activity assay results
Kinase inhibition (%) at 10 µM
Cmpd
EGFR
14.96
10.66
21.94
36.79
14.40
ABL
28.40
18.51
36.97
87.18
27.83
AKT1
41.64
26.15
50.13
85.77
30.84
ALK
NAa
NAa
NAa
56.83
NAa
6a
6b
6c
6d
6e

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26.41
20.64
11.76
11.15
20.86
17.94
14.60
21.32
1.20
7.93
4.34
37.77
24.73
97.60
1.77
31.65
29.26
19.38
20.20
40.59
45.96
37.34
31.84
8.48
14.87
NAa
NAa
NAa
NAa
NAa
3.37
0.70
9.25
4.20
NAa
NAa
NAa
97.89
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
31.30
12.82
24.08
41.21
52.37
30.08
30.92
13.93
8.07
16.72
32.80
43.69
94.87
7.50
13.10
21.72
50.73
96.52
6r
Staurosporine
a Not active.

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Figure 1
Figure 2

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Figure 3
Figure 4