Synthesis and anticancer activity evaluation of naphthalene-substituted triazole spirodienones

Article abstract of DOI:10.1016/j.ejmech.2020.113039

Building on our previous work that discovered 1,2,4-triazole–spirodienone as a promising pharmacophore for anticancer activity, we have further diversified 1,2,4–triazole- spirodienone derivatives and synthesized a series of novel naphthalene-substituted

Full text of DOI:10.1016/j.ejmech.2020.113039

Journal Pre-proof
Synthesis and Anticancer Activity Evaluation of Naphthalene-Substituted Triazole
Spirodienones
Lan Luo, Jing Jing Jia, Qiu Zhong, Xue Zhong, Shilong Zheng, Guangdi Wang, Ling
He
PII:
S0223-5234(20)31011-4
DOI:
https://doi.org/10.1016/j.ejmech.2020.113039
EJMECH 113039
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 September 2020
Revised Date: 7 November 2020
Accepted Date: 16 November 2020
Please cite this article as: L. Luo, J.J. Jia, Q. Zhong, X. Zhong, S. Zheng, G. Wang, L. He, Synthesis and
Anticancer Activity Evaluation of Naphthalene-Substituted Triazole Spirodienones, European Journal of
Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.113039.
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4T1 murine breast tumor
O
N
N
N
N
O
O
6a

Synthesis and Biological Evaluation of Naphthalene-Substituted Triazole Spirodienones
2
Lan Luo1, Jing Jing Jia1, Qiu Zhong 2, Xue Zhong1, Shilong Zheng , Guangdi Wang 2,* and
Ling He 1,*
1
Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry,
Sichuan Engineering Laboratory for Plant-Sourced Drug and Sichuan Research Center for
Drug Precision Industrial Technology, Department of Medicinal Chemistry, West China
School of Pharmacy, Sichuan University, Chengdu, Sichuan 610041, China;
2
Department of Chemistry and RCMI Cancer Research Center, Xavier University of Loui-
siana, New Orleans, LA 70125, USA; qzhong@xula.edu (Q.Z.); szheng@xula.edu (S.Z.)
* Correspondence: gwang@xula.edu (G.W.); heling2012@scu.edu.cn. (L.H.)
Abstract
Building on our previous work that discovered 1,2,4-triazole–spirodienone as a promising
pharmacophore for anticancer activity, we have further diversified 1,2,4–
triazole-spirodienone derivatives and synthesized a series of novel naphthalene-substituted
triazole spirodienones to explore their antineoplastic activity. Of these, compound 6a pos-
sesses remarkable in vitro cytotoxic activity by arresting cell cycle and inducing apoptosis in
MDA-MB-231 cells. Subsequently, acute toxicity assay showed that 6a at 20mg/kg has no
apparent toxicity to the major organ in mice. In addition, compound 6a in vivo suppressed
breast cancer 4T1 tumor growth. Taken together, these results indicate that compound 6a may
be a potential anticancer agent for further development.
Keywords
Naphthalene-substituted
triazole
spirodienones,
cytotoxicity,
anti-proliferation,
MDA-MB-231, 4T1 breast tumor.

Introduction
The naphthalene ring widely exists in biologically active natural products1, partaking in
anti-inflammatory2, antibacterial3, antioxidant4, and antifungal5 properties in molecules con-
taining the naphthalene moiety (Figure 1A). Moreover, naphthalene derivatives have been
reported as potent topoisomerase inhibitors6, cytoplasmic protein-tyrosine phosphatase inhib-
itors7, and microtubule inhibitors8. Naphthalene moiety has been found a good surrogate of
the benzene ring to improve chemical and metabolic stability of the active molecule while
retaining pharmacology9, 10 (Figure 1B).
Figure 1. (A) Naphthalene-containing drugs (B) The strategy of introducing naphthalene to
biologically active compounds.
There has been a growing interest in the synthesis of [4,5] heterocyclic hybrids gathered
via a spiro carbon11,12 owing to their potential antitumor activity13,14. Moreover,
1,2,4-triazole-containing analogues have been reported to possess considerable antineoplastic
activity15, 16, increase the solubility of the ligand, and significantly improve the pharmacolog-
ical profile of the drug17. A series of 1,2,4-triazole–spirodienone conjugates were synthesized
in our laboratory and evaluated for their antitumor activities, most of which were shown to
inhibit cell proliferation against MDA-MB-231, HeLa, A549 and MCF-7 cell lines, as repre-

sented by g-1p (Figure 2A)18. The proven antitumor activity of the triazole-spirodienone
skeleton warrants further exploration of structural modifications that could improve potency
as well as physicochemical properties.
We seek to further expand the study of structure–activity relationship of the lead tria-
zole–spirodienone compounds, by introducing a naphthalene or a benzoheterocyclic deriva-
tive to potentially modify physicochemical properties such as lipid-water partition coefficient
and to potentially increase metabolic sites of the compound structure such as the α position of
naphthalene is easy to oxidative metabolism. Such structural modifications could facilitate
the absorption, distribution, and metabolism to reduce the toxicity of the compounds. With
these in mind, we replace the A rings of the 1,2,4-triazole–spirodienones with naphthalene
and other benzoheterocycles (Figure 2B). Herein, we describe the synthesis and biological
evaluation of naphthalene/benzoheteroccyclics -substituted 1,2,4-triazole spirodienone con-
jugates.
Figure 2. (A) A representative 1,2,4-triazole-spirodienone g-1p from a previous study; (B)
introduction of a naphthalene or a benzoheterocycle moiety to triazole-spirodienones.
Result and discussion
1. Chemistry
The designed derivatives were prepared following the procedures described in Figure 3.
The substituted N-cyanoimidates (2) were prepared by an efficient one-pot procedure for the
cyanoimidation of aldehyde (1). Then, the cyclization reaction of the corresponding
N-cyanoinidates led to the formation of 1,2,4-triazole derivatives (3)19. The acid (4) was first
converted to acid chloride with oxalyl chloride in dichloromethane and the following acyla-
tion
between
amino
(3)
and
acid
chloride
gave
the

N-(1,3-disubstituent-1H-1,2,4-triazol-5-yl)-2- phenoxyacetamides (5). Subsequently, the re-
duction of 5 gave 1,3-disubstituent-N-(2-phenoxyethyl)-1H-1,2,4-triazol-5-amines (7)18. Next,
the target derivatives (6 and 8) were formed by an oxidative amination reaction of 5 and 7
using PhI(OAc)2 as oxidants and Cu(CF3SO3)2 as a catalyst. All reactions were carried out as
detailed in the experimental section, and the structures of all target conjugates were con-
firmed by analytical characterization using 1H NMR, 13C NMR, and HR-MS spectrometry.
Figure 3. Synthetic route of target derivatives
2. Antiproliferative activity and structure-activity relationships
All synthesized compounds were tested for their antiproliferative activity against the tri-
ple-negative human breast carcinoma MDA-MB-231, the cervical epithelioid carcinoma
HeLa, and a metastatic non-small cell lung carcinoma A549. The IC50 values are summarized
in Table 1. Bendamustine and Vorinostat were used as the reference compounds. As shown in

Table 1, all designed compounds displayed potent antiproliferative activity against three
cancer cell lines. Compounds exhibited the highest inhibitory activity against MDA-MB-231,
with IC50 values ranging from 0.03 to 0.26μM. Moreover, target compounds also show a
strong inhibitory effect on Hela (IC50 ranging from 0.07 to 0.72μM) and A549 (IC50 ranging
from 0.08 to 2.00μM) cell lines.
Table 1. Cytotoxic activity of synthesized compounds on selected tumor cell lines
IC50 (μM)
Compound
R1
R2
R3
MDA-MB
-231
Hela A549
6a
6aβ
6aa
6ab
6b
1-naphthalene
1-naphthalene
1-naphthalene
1-naphthalene
2-naphthalene
2-naphthalene
2-naphthalene
2-naphthalene
phenyl
phenyl
H
methyl
H
0.05
0.10
0.04
0.12
0.07
0.07
0.03
0.10
0.07 0.08
0.11 0.18
0.14 0.14
0.10 0.13
0.12 0.14
0.12 0.31
0.15 0.11
0.53 1.21
p-toluene
o-toluene
phenyl
H
H
6bβ
6ba
6baβ
phenyl
methyl
H
p-toluene
p-toluene
methyl
p-chlorobenze
6bc
2-naphthalene
H
0.07
0.72 0.40
ne
6bd
2-naphthalene
p-(trifluoromet
H
0.07
0.39 0.46
hyl)benzene
6bf
6bfβ
6bg
2-naphthalene
2-naphthalene
2-naphthalene
2-naphthalene
p-anisole
p-anisole
H
methyl
H
0.06
0.05
0.05
0.06
0.20 0.26
0.14 0.86
0.21 0.25
0.30 0.30
2-naphthalene
6bgβ
2-naphthalene methyl
1-bromo-2-napht
halene
6c
6d
phenyl
phenyl
H
H
0.05
0.10
0.29 0.10
0.07 0.17
6-[1,4-benzodiox

ane]
6e
6f
6-quinoline
phenyl
phenyl
phenyl
H
H
H
0.05
0.08
0.07
0.11 0.17
0.20 0.32
0.11 0.15
1-fluoro-4-naphth
alene
6g
2-9H-fluorene
6gd
2-9H-fluorene
p-(trifluoromet
H
0.08
0.13 0.18
hyl)benzene
8a
8aa
8b
1-naphthalene
1-naphthalene
2-naphthalene
2-naphthalene
2-naphthalene
phenyl
p-toluene
phenyl
H
H
0.11
0.26
0.23
0.13
0.16
13.28
3.52
0.15 0.43
0.57 1.52
0.37 0.36
0.26 0.32
0.52 2.00
H
8bβ
8ba
phenyl
methyl
H
p-toluene
bendamustine
Vorinostat
>20
-
-
4.52
Based on tumor cell inhibitory effects of reported conjugates, a preliminary struc-
ture-activity relationship was summarized. The introduction of different aromatic rings at R1
positions of 1,2,4-tirazole conferred significant anticancer activity. We then introduced me-
thyl, chloro, trifluoromethyl, and methoxy group into the phenyl ring of R2 position, and the
resulting compounds are less active. Compounds 6 were stronger cell proliferation inhibitors
than the corresponding compounds 8, indicating that the introduction of the carbonyl group
on 3-position of the spirodienone ring increased the antitumor activity. Methyl group at R3
position caused an obvious reduction of antiproliferative activity against A549 cell line.
Overall, compound 6a is the most active conjugate, so we conducted subsequent assays to
further assess compound 6a.
3. ADME/T profile in silico
In silico prediction of ADME/T properties of 25 target compounds was computed by Dis-
covery Studio. The results of these computations are summarized in Table 2. The aqua solu-
bility parameter (solubility-level) is related to absorption and distribution. The prediction
shows that most of our target compounds have low water solubility, prompting us to intro-
duce a hydrophilic group into the existing molecule in further research. Moreover, BBB-level
and absorption-level are all in the desirable range. Polar surface area (PSA) means Van der
Waals surface area of polar nitrogen and oxygen atoms, which should not be greater than 140
Å, and the values for the target molecules are in the range of 57.453 to 92.614 Å. It is evident
from Table 2 that the great majority of target compounds follow Lipinski's rule. In general,
these parameters of target molecules may help in the further development of new drug candi-

dates.
Table 2. In silico ADME parameters of target compounds
Solubili- BBB-
Absorp-
logP PSA-2D
MW
HBD HBA
ty-level
0~5
level
1~4
tion-level
Standard
0~3
-2~7
<140
<500
<5
<10
range
6a
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
2
1
2
1
2
1
1
1
1
1
2
2
1
4
1
3
1
1
1
4
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
0
0
4.132
4.616
3.41
74.754
74.754
86.015
74.754
74.754
74.754
74.754
74.754
74.754
74.754
83.684
83.684
74.754
74.754
74.754
92.614
74.754
74.754
74.754
74.754
57.453
57.453
57.453
434.44
448.47
435.43
448.47
434.44
448.47
448.47
462.49
468.89
502.44
464.47
478.49
484.50
498.53
513.34
442.42
448.47
452.43
472.49
540.49
420.46
434.48
420.46
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
5
5
6
5
5
5
5
5
5
5
6
6
5
5
5
7
5
5
5
5
5
5
5
6aβ
6aa
6ab
6b
4.618
4.132
4.616
4.618
5.102
4.797
5.074
4.116
4.6
6bβ
6ba
6baβ
6bc
6bd
6bf
6bfβ
6bg
6bgβ
6c
5.041
5.525
4.881
3.01
6d
6e
4.618
4.338
4.887
5.829
5.101
5.587
5.101
6f
6g
6gd
8a
8aa
8b

8bβ
1
1
1
1
0
0
5.478
5.587
57.453
57.453
434.48
434.48
0
0
5
5
8ba
4. Cell cycle analysis
As shown in Table 2, the MDA-MB-231 breast cancer cell line was more sensitive to our
target compound than the other cell lines. Many antineoplastic agents exert their inhibition of
cell proliferation by arresting the cell cycle. Therefore, we next observed cell cycle changes
of MDA-MB-231 cells exposed to 6a and 8c. As shown in Figure 4, treatment with 1 μM of
compound 6a and 8c caused the percentage of MDA-MB-231 cells in G1 phase to decrease
significantly (from 74.39% to 39.14% and 55.31%), and cells in S phase to increase (from
19.84% to 36.99% and 21.86%). The results indicated that compound 6a and 8c were able to
induce cell cycle arrest in the S phase.

Figure 4. Cell cycle analysis of MDA-MB-231 cells treated with (a) DMSO; (b) CA4; (c) 6a;
(d) 8c
5. Analysis of cell apoptosis
To evaluate the efficacy of compounds 6a and 8c in induction of cell death through apop-
tosis, we performed cell apoptosis assays in MDA-MB-231 cells treated with the compounds.
After 48 hours of treatment with 6a, 8c, or Comretastatin A4 (CA4) as a positive control, cell
cycle was analyzed by flow cytometry with FITC-Annexin V/PI staining. As shown in Fig-
ure 5, 6a and 8c could induce cell apoptosis in a dose-dependent manner. Compounds 6a and
8c induced both early and late apoptosis in MDA-MB-231 cells at 1 μM.
Figure 5. Effects of 6a, 8c and CA4 on the induction of cell apoptosis in MDA-MB-231 cells
6. Acute toxicity study
To evaluate the safety of compound 6a, four groups of Kunming mice (n = 8, half male and
half female) were administered either a single dose of compound 6a at escalating doses
(10mg/kg, 20mg/kg, 40mg/kg) or a single dose of the vehicle control by intraperitoneal injec-

tion. No mortality occurred during the test, indicating the maximum tolerated dose of 6a
should be greater than 40mg/kg. There were no remarkable clinical signs in the eyes, skin,
and fur among mice administered 40 mg/kg body weight of tested compounds. But the gain
of bodyweight of the highest dose group is lower than the other three groups (Figure 6). As-
cites were detected when we dissected female mice of the highest dose group, with decreased
liver volume (Figure S1, Supporting Information). These results indicate that 40mg/kg of
compound 6a may induce partial damage on the liver of female mice.
Figure 6. Body weights of all the groups measured daily post treatments.
7. Antitumor activity in vivo
The orthotopic 4T1 tumor-bearing mice were used to further evaluate the antitumor effi-
ciency in vivo. The mice were randomly divided into four groups (n = 6) and treated with 6a
at 0 mg/kg, 1 mg/kg, 2 mg/kg dose by intraperitoneal injection daily. The positive control
group was treated with doxorubicin at 1 mg/kg.
Mice treated with vehicle control showed significant tumor growth, reaching average

volumes of 644 mm3 (Figure 7A). In comparison, three different treatment groups showed
varying degree of tumor growth inhibition (Figure 7E). The antitumor effect of 2 mg/kg of
compound 6a (Figure 7C) was comparable with doxorubicin at 1 mg/kg (Figure 7D). Fur-
thermore, the gross appearance of tumors (Figure 7F) and the tumor weights (Figure 7G)
were consistent with the tumor volume. The inhibition ratio of tumor by 6a at 1 mg/kg, 6a at
2 mg/kg, doxorubicin at 1 mg/kg were 18.2%, 38.8%, and 45.1% when compared with the
saline control at the end of the study, respectively.
Figure 7. Tumor volume of each mouse in the groups treated with A) saline; B) 6a at 1mg/kg;
C) 6a at 2 mg/kg; D) dox at 1 mg/kg; E) average tumor volumes and H) Image of isolated
tumors; G) average weights of tumors; I) body weights change of all four groups. The signif-
icant differences have been indicated, and *, ** and *** represent p < .05, p < .01 and p
< .001, respectively.
The body weights of all four groups were barely changed during the experiment (Figure
7H), indicating all treatments had no apparent toxicity. In comparison with the control group,
the size of the spleen and liver of 6a at 2mg/kg group enlarged (Figure S2, Supporting In-
formation). The hematoxylin and eosin (H&E) stain slices of 6a at 2mg/kg groups showed

extramedullary hematopoiesis in the liver and spleen, indicating that 6a might have a slight
inhibitory action on bone marrow. In addition, the levels of apoptosis occurred in tumor tis-
sues were consistent with the results of antitumor effect (Figure. 8).
Figure 8. H&E images of major organ and tumor tissues after different treatment, scale bar =
50 μm
8. Potential target of promising compounds
To determine potential targets of these spirodienone compounds, all chemical structures
were submitted to HitPickV220, SuperPred21 and SwissTargetPrediction22, three different web
servers for target prediction of bioactive molecules, but only the SwissTargetPrediction is
feasible. Table 3 lists the common target of all 25 compounds, ADORA2A and PDE10A are
not the typical targets of antineoplastic agents. In addition, molecular docking was performed
by Discovery Studio to view the potential interactions between the promising compound 6a
and the remaining seven targets (Figure S3, Supporting Information). The docking results
showed that Libdock Scores of AURKA and MAPK10 are higher than 100. In more detail,
for these seven targets, the obtained docking poses of 6a were compared with the
co-crystallized compounds (Table S1, Supporting Information). In this sense, MAPK10
and JAK2 are the most promising target. 6a has the same six interactions with 2B1P that
co-crystallized compounds do: phenyl and naphthalene moiety of 6a interact with ILE70,
VAL196, ALA91, LEU206 and ILE124 residues of MAPK10 to build Pi-alkyl interaction,
and the naphthalene interacts with MET146 residue to build a Pi-sulfur interaction. Addition-

ally, 6a has the same five interactions with 4C61 that co-crystallized compound do: car-
bon-hydrogen bond with the side chain of ASN981、GLY993，Pi-alkyl interaction with the
side chain ALA880、LEU983，Pi-sigma interaction with the side chain LEU855.
Table 3. Predicting results of SwissTargetPrediction
PDB
Code
Libdock
Score
Target Name
AURKB
Typical Target of Anticancer Agent?
Y
4AF3
93.448
Target for immunosuppressive disorders,
inflammatory tissue damage, and neuro-
degenerative diseases.
ADORA2A
-
-
MAPK8
AURKA
MAPK10
IGF1R
Y
Y
Y
Y
4QTD
4BYI
2B1P
3I81
91.398
100.574
135.906
94.451
Possible target for new anti-psychotic or
-diabetic drugs.
PDE10A
-
-
KDR
Y
Y
5EW3
4C61
-
JAK2
96.227
Based on the in silico target prediction and docking study, we first tested possible inhibi-
tion of AURKA, MAPK10, and JAK2, using 6a and 6h as test cases (Table 4). These com-
pounds did not have any significant effect on AURKA and MAPK10 when tested at 100 μM,
but showed weak inhibition on the Janus kinases 2. The Janus kinases (JAK1, JAK2, JAK3,
and TYK2) belong to the family of intracellular protein tyrosine kinases, which play an es-
sential role in the signaling of a variety of cytokines23. Although the inhibitory potency of 6a
and 6h toward JAK2 is not sufficient evidence of direct target determination, additional func-
tion study could validate the mechanism of action for the test compounds.
Table 4. Inhibitory effect on AURKA, MAPK10 and JAK2

Target
6a
6h
Repeat 1
Repeat 2
Average
SD
>100µM
>100µM
>100µM
-
>100µM
>100µM
>100µM
-
AURKA
Repeat 1
Repeat 2
Average
SD
>100µM
>100µM
>100µM
-
>100µM
>100µM
>100µM
-
MAPK10
Repeat 1
Repeat 2
Average
SD
41.3µM
76.6µM
59.0µM
25
20.9µM
21.4µM
21.2µM
0.4
JAK2
Conclusion
Building on our previous 1,2,4-triazole spirodienones, we synthesized a series of novel
molecules with a naphthalene fragment as A ring and tested their antiproliferative activity.
The naphthalene substituted analogs all demonstrated moderate to strong inhibitory effect
against three cancer cell lines. Compound 6a exhibited the strongest anticancer activity by
inducing cell cycle arrest and cell apoptosis. Furthermore, compound 6a significantly inhib-
ited tumor growth in a metastatic 4T1murine breast cancer model. Preliminary target model-
ing and enzyme assay suggest that compound 6a is a potential JAK2 inhibitor for cancer
therapy.
Experimental section
1. Chemistry

1.1 General chemistry methods
All solvents and reagents were obtained from commercial sources unless otherwise men-
tioned, and the solvents used were dried by conventional methods. All reactions were per-
formed under a nitrogen atmosphere. The 1H and 13C NMR spectra were recorded on a Mer-
cury 400 MHz NMR, Varian, Palo Alto, CA, USA and 600 MHz NMR. (CDCl3 and
DMSO-d6 were the solvent and TMS was the internal standard). MS spectra were measured
on Bruker Daltonics Data Analysis 3.4 Mass Spectrometer, Bruker, Karlsruhe, Germany and
Thermo LTQ Orbitrap-XL Mass Spectrometer (Thermo Scientific, Waltham, MA, USA). A
YRT-3 melting point instrument (Tianda Tianfa Company, Tianjin, China) was used for
measuring melting points. HSGF 254 high-efficiency thin-layer chromatography silica gel
plates were purchased from Huiyou Development Co., Yantai, Shangdong, China. Ltd. HSGF
254 thin-layer silica gel (300 mesh ~ 400 mesh) was purchased from Ocean Chemical Plant
(Qingdao, Shangdong, China).
1.2 Synthetic procedures
1.2.1 General procedure of the synthesis of methyl N-cyanobenzimidate (2): To a 50 ml flask
were added benzaldehyde (1 mmol), cyanamide (3 mmol), t-BuONa (4 mmol), and MeOH (8
mL), the mixture was stirred for 0.5h at room temperature, then NBS (4 mmol) was added.
The resulting mixture was stirred for 12 h at 50°C. After reaction, the reaction mixture was
extracted with ethyl acetate; the organic layer was dried with anhydrous sodium sulfate and
concentrated under reduced pressure. The crude was purified by flash chromatography on
silica gel with petroleum ether/ethyl acetate as eluent to give 2.
1.2.1.1 Methyl N-cyano-1-naphthimidate (2a): white solid, 180 mg, yield 86%; 1H NMR (400
MHz, Chloroform-d) δ 8.05 (d, J = 8.2 Hz, 1H), 7.93 (d, J = 8.2 Hz, 1H), 7.84 (d, J = 8.2 Hz,
1H), 7.78 (d, J = 7.1 Hz, 1H), 7.64-7.55 (m, 3H), 4.20 (s, 3H).
1.2.1.2 Methyl N-cyano-2-naphthimidate (2b): white solid, 188 mg, yield 90%; 1H NMR (400
MHz, Chloroform-d) δ 8.73 (s, 1H), 8.06 (d, J = 8.1 Hz, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.95 (d,
J = 8.0 Hz, 1H), 7.90 (d, J = 8.1 Hz, 1H), 7.65 (t, J = 7.4 Hz, 1H), 7.59 (t, J = 7.4 Hz, 1H),
4.12 (s, 3H).
1
1.2.1.3 Methyl 1-bromo-N-cyano-2-naphthimidate (2c): yellow solid, 229 mg, yield 80%; H
NMR (400 MHz, Chloroform-d) δ 8.36 (d, J = 8.4 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.89 (d,
J = 8.4 Hz, 1H), 7.69 (t, J = 8.1 Hz, 1H), 7.64 (t, J = 8.1 Hz, 1H), 7.40 (d, J = 8.4 Hz, 1H),
4.17 (s, 3H).
1.2.1.4 Methyl N-cyano-2,3-dihydrobenzo[b][1,4]dioxine-6-carbimidate (2d): yellow solid,
152 mg, yield 70%; 1H NMR (400 MHz, Chloroform-d) δ 7.74 (dd, J = 8.6, 2.2 Hz, 1H), 7.64
(d, J = 2.2 Hz, 1H), 6.95 (d, J = 8.6 Hz, 1H), 4.34-4.27 (m, 4H), 4.01 (s, 3H).

1
1.2.1.5 Methyl N-cyanoquinoline-6-carbimidate (2e): yellow solid, 155 mg, yield 74%; H
NMR (400 MHz, Chloroform-d) δ 9.05 (s, 1H), 8.78 (s, 1H), 8.34 (dd, J = 8.2, 2.2 Hz, 1H),
8.27-8.22 (m, 2H), 7.56-7.52 (m, 1H), 4.14 (s, 3H).
1
1.2.1.6 Methyl N-cyano-4-fluoro-1-naphthimidate (2f): yellow solid, 180 mg, yield 86%; H
NMR (400 MHz, Chloroform-d) δ 8.20 (d, J = 8.0 Hz, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.78 (dd,
d, J = 8.0, 5.1 Hz 1H), 7.70 – 7.63 (m, 2H), 7.23 (d, J = 8.0 Hz, 1H), 4.20 (s, 3H).
1.2.1.7 Methyl -N-cyano-9H-fluorene-2-carbimidate (2g): yellow solid, 142 mg, yield 57%;
1H NMR (400 MHz, Chloroform-d) δ 8.30 (s, 1H), 8.14 (d, J = 8.0 Hz, 1H), 7.85 (t, J = 8.0
Hz, 2H), 7.58 (d, J = 6.6 Hz, 1H), 7.44-7.37 (m, 2H), 4.06 (s, 3H), 3.95 (s, 2H)
1.2.2 General procedure of the synthesis of 1,3-disubstituted-1,2,4-triazole-5-amine (3): A
mixture of triethylamine (1.5 mmol) and phenylhydrazine hydrochloride (1.5 mmol) in
methanol (3 mL) was stirred at room temperature for 30 min, then the N-cyanoimidates
(1mmol) was added and the mixture was refluxed for 4h. Then the solvent was removed in
reduces pressure and the residue was purified by flash chromatography on silica gel to afford
3.
1.2.2.1 3-(Naphthalen-1-yl)-1-phenyl-1H-1,2,4-triazol-5-amine (3a): brown solid, 175 mg,
yield 61%; 1H NMR (400 MHz, Chloroform-d) δ 9.09 (d, J = 8.4 Hz, 1H), 8.14 (d, J = 7.1 Hz,
1H), 7.90 (t, J = 7.6 Hz, 2H), 7.79 (d, J = 7.6 Hz, 2H), 7.59 – 7.48 (m, 5H), 7.43 (t, J = 7.4
Hz, 1H), 5.06 (s, 2H).
1.2.2.2 3-(Naphthalen-1-yl)-1-(p-tolyl)-1H-1,2,4-triazol-5-amine (3aa): yellow solid, 130 mg,
yield 43%; 1H NMR (400 MHz, Chloroform-d) δ 9.10 (d, J = 8.0 Hz, 1H), 8.14 (d, J = 8.0 Hz,
1H), 7.89 (t, J = 8.0 Hz, 2H), 7.58-7.48 (m, 5H), 7.35 (d, J = 8.0 Hz, 2H), 4.94 (s, 2H), 2.44
(s, 3H).
1.2.2.3 3-(Naphthalen-1-yl)-1-(o-tolyl)-1H-1,2,4-triazol-5-amine (3ab): yellow solid, 117 mg,
yield 39%; 1H NMR (400 MHz, Chloroform-d) δ 9.08 (d, J = 8.4 Hz, 1H), 8.12 (d, J = 8.4 Hz,
1H), 7.90 – 7.86 (m, 2H), 7.56 – 7.36 (m, 7H), 4.70 (s, 2H), 2.33 (s, 3H).
1.2.2.4 3-(Naphthalen-2-yl)-1-phenyl-1H-1,2,4-triazol-5-amine (3b): white solid, 232 mg,
yield 81%; 1H NMR (400 MHz, Chloroform-d) δ 8.53 (s, 1H), 8.16 (dd, J = 8.5, 1.3 Hz, 1H),
7.94 – 7.78 (m, 3H), 7.69 – 7.35 (m, 7H), 5.12 (s, 2H).
1.2.2.5 3-(Naphthalen-2-yl)-1-(p-tolyl)-1H-1,2,4-triazol-5-amine (3ba): yellow solid, 271 mg,
yield 90%; 1H NMR (400 MHz, Chloroform-d) δ 8.55 (s, 1H), 8.18 (d, J = 8.4 Hz, 1H), 7.95
– 7.79 (m, 3H), 7.52-7.47 (m, 4H), 7.33 (m, 2H), 4.85 (d, J = 7.8 Hz, 2H), 2.43 (s, 3H).
1.2.2.6 1-(4-Chlorophenyl)-3-(naphthalen-2-yl)-1H-1,2,4-triazol-5-amine (3bc): white solid,
192 mg, yield 60%; 1H NMR (400 MHz, Chloroform-d) δ 8.54 (s, 1H), 8.16 (dd, J = 8.5, 1.7

Hz, 1H), 7.94 – 7.82 (m, 3H), 7.65 – 7.58 (m, 2H), 7.55 – 7.46 (m, 4H), 4.95 (s, 2H).
1.2.2.7 3-(Naphthalen-2-yl)-1-(4-trifluoromethyl phenyl)-1H-1,2,4-triazol-5-amine (3bd):
yellow solid, 191 mg, yield 54%; 1H NMR (400 MHz, Chloroform-d) δ 8.54 (s, 1H), 8.16 (s,
1H), 7.90-7.82 (m, 7H), 7.50 (m, 2H), 5.03 (s, 2H).
1.2.2.8 1-(4-Methoxyphenyl)-3-(naphthalen-2-yl)-1H-1,2,4-triazol-5-amine (3bf): yellow
1
solid, 227 mg, yield 72%; H NMR (400 MHz, Chloroform-d) δ 8.54 (s, 1H), 8.17 (dd, J =
8.5, 1.7 Hz, 1H), 7.93 – 7.82 (m, 3H), 7.54-7.46 (m, 4H), 7.05 – 7.00 (m, 2H), 4.94 (s, 2H),
3.85 (s, 3H).
1.2.2.9 1,3-Di(naphthalen-2-yl)-1H-1,2,4-triazol-5-amine (3bg): yellow solid, 218 mg, yield
65%; 1H NMR (400 MHz, Chloroform-d) δ 8.59 (s, 1H), 8.21 (d, J = 8.5 Hz, 1H), 8.08 – 7.99
(m, 2H), 7.97 – 7.82 (m, 6H), 7.80 – 7.76 (m, 1H), 7.58 (m, 2H), 7.49 (dd, J = 6.1, 3.2 Hz,
2H), 5.05 (s, 2H).
1.2.2.10 3-(1-Bromonaphthalen-2-yl)-1-phenyl-1H-1,2,4-triazol-5-amine (3c): yellow solid,
255 mg, yield 72%; 1H NMR (400 MHz, Chloroform-d) δ 8.48 (d, J = 8.5 Hz, 1H), 7.88-7.83
(m, 3H), 7.72 – 7.60 (m, 3H), 7.57-7.53 (m, 3H), 7.43 (t, J = 7.4 Hz, 1H), 5.03 (s, 2H).
1.2.2.11 3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-1-phenyl-1H-1,2,4-triazol-5-amine (3d):
yellow solid, 232 mg, yield 79%; 1H NMR (400 MHz, Chloroform-d) δ 7.61 – 7.51 (m, 7H),
7.40 (t, J = 8.0 Hz, 1H), 6.90 (d, J = 8.0 Hz, 1H), 5.12 (s, 2H), 4.28 (m, 4H).
1.2.2.12 1-Phenyl-3-(quinolin-6-yl)-1H-1,2,4-triazol-5-amine (3e): yellow solid, 218 mg,
yield 76%; 1H NMR (400 MHz, Chloroform-d) δ 8.92 (dd, J = 4.3, 1.7 Hz, 1H), 8.54 (d, J =
1.9 Hz, 1H), 8.45 (dd, J = 8.8, 1.7 Hz, 1H), 8.25 (dd, J = 8.4, 1.9 Hz, 1H), 8.17 (d, J = 8.8 Hz,
1H), 7.65 (m, 2H), 7.56 (m, 2H), 7.44 (m, 2H), 4.90 (s, 2H).
1.2.2.13 3-(4-Fluoronaphthalen-1-yl)-1-phenyl-1H-1,2,4-triazol-5-amine (3f): yellow solid,
213 mg, yield 70%; 1H NMR (400 MHz, Chloroform-d) δ 9.12 (d, J = 8.0 Hz, 1H), 8.17 (d, J
= 8.0 Hz, 1H), 8.09 (t, J = 7.3 Hz, 1H), 7.68-7.53 (m, 6H), 7.43 (t, J = 8.0 Hz, 1H), 7.21 (t, J
= 8.0 Hz, 1H), 5.20 (s, 2H).
1.2.2.14 3-(9H-Fluoren-1-yl)-1-phenyl-1H-1,2,4-triazol-5-amine (3g): bright yellow solid,
1
156 mg, yield 48%; H NMR (400 MHz, Chloroform-d) δ 8.24 (s, 1H), 8.08 (d, J = 7.9 Hz,
1H), 7.84-7.79 (m, 2H), 7.63-7.61 (m, 2H), 7.56-7.54 (m, 3H), 7.45 – 7.28 (m, 3H), 5.04 (s,
2H), 3.94 (s, 2H).
1.2.2.15 3-(9H-Fluoren-1-yl)-1-(4-(trifluoromethyl)phenyl)-1H-1,2,4-triazol-5-amine (3gd):
1
bright yellow solid, 164 mg, yield 43%; H NMR (400 MHz, Chloroform-d) δ 8.25 (s, 1H),
8.10 (d, J = 8.0, 1H), 7.86 – 7.80 (m, 6H), 7.57 (d, J = 7.4 Hz, 1H), 7.40 (t, J = 7.4 Hz, 1H),
7.34 (t, J = 7.4, 1H), 4.91 (s, 2H), 3.97 (s, 2H).

1.2.3 General procedure of the synthesis of N-(1,3-disubstituted-1,2,4-triazol-5-yl)-
2-phenoxyacetamides (5): Oxalyl chloride (6 mmol) was added dropwise into a solution of
4-methoxyphenoxyacetic acid (3 mmol) and DMF (1d) in dichloromethane (50 mL), the re-
action mixture was stirred for 2h at room temperature. The solution was concentrated to dry-
ness under reduced pressure to give the acid chloride. Then dichloromethane (5 ml) was add-
ed into the acid chloride, next, the acid chloride solution was slowly added to the solution of
3 (1 mmol) and triethylamine (3 mmol) in dichloromethane (20 mL), and the mixture was
stirred at room temperature overnight. The reaction mixture was quanched with water, and
the product was extracted with ethyl acetate, the organic layer was dried over anhydrous so-
dium sulfate and concentrated. The crude was purified by column chromatography on silica
gel to afford 5.
1.2.3.1 2-(4-Methoxyphenoxy)-N-(3-(naphthalen-1-yl)-1-phenyl-1H-1,2,4-triazol-5-yl) acet-
amide (5a): yellow solid, 160 mg, yield 36%; 1H NMR (400 MHz, Chloroform-d) δ 9.08 (d, J
= 8.3 Hz, 1H), 8.23 (d, J = 7.1 Hz, 1H), 8.00 – 7.89 (m, 2H), 7.68 – 7.39 (m, 8H), 6.94 – 6.77
(m, 4H), 4.59 (s, 2H), 3.79 (s, 3H).
1.2.3.2
2-(4-Methoxyphenoxy)-N-(3-(naphthalen-1-yl)-1-phenyl-1H-1,2,4-triazol-5-yl)
1
propenamide (5aβ): brown solid, 155 mg, yield 33%; H NMR (400 MHz, Chloroform-d) δ
9.03 (d, J = 8.4 Hz, 1H), 8.19 (dd, J = 7.2, 1.2 Hz, 1H), 7.97 – 7.86 (m, 2H), 7.59 – 7.50 (m,
5H), 7.48 – 7.45 (m, 2H), 6.84 (s, 4H), 4.69 (q, J = 6.8 Hz, 1H), 3.79 (s, 3H), 1.56 (d, J = 6.8
Hz, 3H).
1.2.3.3 2-(4-Methoxyphenoxy)-N-(3-(naphthalen-1-yl)-1-(p-tolyl)-1H-1,2,4-triazol-5-yl) ac-
1
etamide (5aa): yellow solid, 139 mg, yield 30%; H NMR (400 MHz, Chloroform-d) δ 9.11
(s, 1H), 8.83 (s, 1H), 8.25 (s, 1H), 7.94 (s, 2H), 7.54 (m, 5H), 7.33 (s, 2H), 6.87 (s, 4H), 4.62
(s, 2H), 3.81 (s, 3H), 2.46 (s, 3H).
1.2.3.4 2-(4-Methoxyphenoxy)-N-(3-(naphthalen-1-yl)-1-(o-tolyl)-1H-1,2,4-triazol-5-yl) ac-
1
etamide (5ab): brown solid, 162 mg, yield 35%; H NMR (400 MHz, Chloroform-d) δ 9.10
(d, J = 8.4 Hz, 1H), 8.61 (s, 1H), 8.28 (d, J = 7.1 Hz, 1H), 7.94-7.89 (m, 2H), 7.61 – 7.47 (m,
3H), 7.47 – 7.38 (m, 2H), 7.36-7.33 (m, 2H), 6.82 (d, J = 8.8 Hz, 2H), 6.73 (d, J = 8.8 Hz,
2H), 4.53 (s, 2H), 3.78 (s, 3H), 2.30 (s, 3H)。
1.2.3.5 2-(4-Methoxyphenoxy)-N-(3-(naphthalen-2-yl)-1-phenyl-1H-1,2,4-triazol-5-yl) acet-
1
amide (5b): yellow solid, 210 mg, yield 47%; H NMR (400 MHz, Chloroform-d) δ 8.87 (s,
1H), 8.68 (s, 1H), 8.25 (d, J = 8.6 Hz, 1H), 7.97 – 7.85 (m, 3H), 7.61 – 7.45 (m, 7H),
6.88-6.81(m, 4H), 4.60 (s, 2H), 3.79 (s, 3H).
1.2.3.6
2-(4-Methoxyphenoxy)-N-(3-(naphthalen-2-yl)-1-phenyl-1H-1,2,4-triazol-5-yl)
1
propenamide (5bβ): yellow solid, 186 mg, yield 40%, H NMR (400 MHz, Chloroform-d) δ

8.67 (s, 1H), 8.24 (d, J = 8.5 Hz, 1H), 7.94 – 7.84 (m, 3H), 7.52-7.44 (m, 7H), 6.86 – 6.79 (m,
4H), 4.70 (q, J = 6.8 Hz, 1H), 3.79 (s, 3H), 1.58 (d, J = 6.8 Hz, 3H).
1.2.3.7 2-(4-Methoxyphenoxy)-N-(3-(naphthalen-2-yl)-1-(p-tolyl)-1H-1,2,4-triazol-5-yl) ac-
1
etamide (5ba): yellow solid, 186 mg, yield 34%; H NMR (400 MHz, Chloroform-d) δ 8.62
(s, 1H), 8.20 (d, J = 8.5 Hz, 1H), 7.93 – 7.89 (m, 2H), 7.88 – 7.84 (m, 1H), 7.53 – 7.49 (m,
2H), 7.44 (d, J = 8.2 Hz, 2H), 7.30 – 7.25 (m, 2H), 6.84 (s, 4H), 4.56 (s, 2H), 3.78 (s, 3H),
2.43 (s, 3H).
1.2.3.8
2-(4-Methoxyphenoxy)-N-(3-(naphthalen-2-yl)-1-(p-tolyl)-1H-1,2,4-triazol-5-yl)
propenamide (5baβ): yellow solid, 144 mg, yield 30%; 1H NMR (400 MHz, Chloroform-d) δ
8.79 (s, 1H), 8.65 (s, 1H), 8.23 (d, J = 8.5 Hz, 1H), 7.93 – 7.83 (m, 3H), 7.51-7.48 (m, 2H),
7.34 (d, J = 8.0 Hz, 2H), 7.23 (d, J = 8.2 Hz, 2H), 6.85-6.79 (m, 4H), 4.69 (q, J = 6.8 Hz, 1H),
3.79 (s, 3H), 2.42 (s, 3H), 1.58 (d, J = 6.8 Hz, 3H).
1.2.3.9
N-(1-(4-Chlorophenyl)-3-(naphthalen-2-yl)-1H-1,2,4-triazol-5-yl)-2-(4-methoxy-
1
phenoxy)acetamide (5bc): white solid, 150 mg, yield 31%; H NMR (400 MHz, Chloro-
form-d) δ 8.63 (s, 1H), 8.27 – 8.17 (m, 1H), 7.95 – 7.90 (m, 2H), 7.88 – 7.84 (m, 1H), 7.54 –
7.48 (m, 4H), 7.44 (d, J = 8.4 Hz, 2H), 6.91 – 6.81 (m, 4H), 4.59 (s, 2H), 3.80 (s, 3H).
1.2.3.10
2-(4-Methoxyphenoxy)-N-(3-(naphthalen-2-yl)-1-(4-(trifluoromethyl)phenyl)-1H-
1
1,2,4-triazol-5-yl)acetamide (5bd): yellow solid, 171 mg, yield 33%; H NMR (400 MHz,
Chloroform-d) δ 8.63 (s, 1H), 8.23 – 8.18 (m, 1H), 7.96 – 7.91 (m, 2H), 7.88 (dd, J = 9.0, 4.3
Hz, 1H), 7.75-7.71 (m, 4H), 7.55 – 7.50 (m, 2H), 6.90-6.86 (m, 4H), 4.60 (s, 2H), 3.80 (s,
3H).
1.2.3.11 2-(4-Methoxyphenoxy)-N-(1-(4-methoxyphenyl)-3-(naphthalen-2-yl)-1H-1,2,4- tria-
zol-5-yl)acetamide (5bf): white solid, 178 mg, yield 37%; 1H NMR (400 MHz, Chloroform-d)
δ 8.66 (s, 1H), 8.23 (d, J = 9.6 Hz, 1H), 7.91 (t, J = 8.9 Hz, 2H), 7.87 – 7.83 (m, 1H),
7.51-7.49 (m, 2H), 7.45 (d, J = 8.5 Hz, 2H), 6.98 (d, J = 8.6 Hz, 2H), 6.86-6,80 (m, 4H), 4.59
(s, 2H), 3.87 (s, 3H), 3.78 (s, 3H).
1.2.3.12 2-(4-Methoxyphenoxy)-N-(1-(4-methoxyphenyl)-3-(naphthalen-2-yl)-1H-1,2,4- tria-
1
zol-5-yl)propenamide (5bfβ): yellow solid, 148 mg, yield 30%; H NMR (400 MHz, Chloro-
form-d) δ 8.81 (s, 1H), 8.67 – 8.63 (m, 1H), 8.22 (dd, J = 8.5, 1.7 Hz, 1H), 7.93 – 7.83 (m,
3H), 7.51 – 7.47 (m, 2H), 7.36 – 7.30 (m, 2H), 6.94 – 6.88 (m, 2H), 6.83 – 6.77 (m, 4H), 4.68
(d, J = 6.8 Hz, 1H), 3.83 (s, 3H), 3.77 (s, 3H), 1.58 (d, J = 6.8 Hz, 3H).
1.2.3.13 N-(1,3-Di(naphthalen-2-yl)-1H-1,2,4-triazol-5-yl)-2-(4-methoxyphenoxy) acetamide
1
(5bg): yellow solid, 160 mg, yield 32%; H NMR (400 MHz, Chloroform-d) δ 8.94 (s, 1H),
8.70 (s, 1H), 8.30 – 8.25 (m, 1H), 8.03 (s, 1H), 7.98 (d, J = 8.8 Hz, 1H), 7.93 (d, J = 7.9 Hz,

3H), 7.89 – 7.83 (m, 2H), 7.69 (d, J = 8.5 Hz, 1H), 7.62 – 7.56 (m, 2H), 7.53 – 7.50 (m, 2H),
6.80-6.74 (m, 4H), 4.58 (s, 2H), 3.76 (s, 3H).
1.2.3.14 N-(1,3-Di(naphthalen-2-yl)-1H-1,2,4-triazol-5-yl)-2-(4-methoxyphenoxy) propena-
mide (5bgβ): brown solid, 165 mg, yield 32%; 1H NMR (400 MHz, Chloroform-d) δ 8.79 (s,
1H), 8.34 (s, 1H), 7.99 – 7.92 (m, 5H), 7.88 (t, J = 4.7 Hz, 1H), 7.83 – 7.79 (m, 1H), 7.65 –
7.58 (m, 3H), 7.54 – 7.51 (m, 2H), 6.77 (s, 4H), 4.69 (s, 1H), 3.78 (s, 3H), 1.59 (d, J = 6.8 Hz,
3H).
1.2.3.15 N-(3-(1-Bromonaphthalen-2-yl)-1-phenyl-1H-1,2,4-triazol-5-yl)-2-(4-methoxy- phe-
noxy)acetamide (5c): brown solid, 200 mg, yield 39%; 1H NMR (400 MHz, Chloroform-d) δ
8.97 (s, 1H), 8.49 (d, J = 8.4 Hz, 1H), 7.89 (s, 3H), 7.71 – 7.40 (m, 7H), 6.83 (m, 4H), 4.59 (s,
2H), 3.78 (s, 3H).
1.2.3.16 N-(3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-1-phenyl-1H-1,2,4-triazol-5-yl)-2-(4-
1
methoxyphenoxy)acetamide (5d): brown solid, 192 mg, yield 42%; H NMR (400 MHz,
Chloroform-d) δ 7.67 – 7.64 (m, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.47 (m, 5H), 6.90 (d, J = 8.4
Hz, 1H), 6.85 – 6.78 (m, 4H), 4.55 (s, 2H), 4.28 (m, 4H), 3.77 (s, 3H).
1.2.3.17 2-(4-Methoxyphenoxy)-N-(1-phenyl-3-(quinolin-6-yl)-1H-1,2,4-triazol-5-yl) acet-
1
amide (5e): yellow solid, 147 mg, yield 36%; H NMR (400 MHz, Chloroform-d) δ 8.96 (s,
1H), 8.92 – 8.89 (m, 1H), 8.66 – 8.60 (m, 1H), 8.47 (dd, J = 8.8, 1.9 Hz, 1H), 8.22 (d, J = 8.3
Hz, 1H), 8.16 (d, J = 8.8 Hz, 1H), 7.55 – 7.40 (m, 6H), 6.81 (m, 4H), 4.58 (s, 2H), 3.76 (s,
3H).
1.2.3.18
N-(3-(4-Fluoronaphthalen-1-yl)-1-phenyl-1H-1,2,4-triazol-5-yl)-2-(4-methoxy-
1
phenoxy)acetamide (5f): yellow solid, 230 mg, yield 50%; H NMR (400 MHz, Chloro-
form-d) δ 9.10 (d, J = 8.3 Hz, 1H), 8.19 (t, J = 7.5 Hz, 2H), 7.66 – 7.48 (m, 7H), 7.25 – 7.19
(m, 1H), 6.84 (m, 4H), 4.60 (s, 2H), 3.79 (s, 3H).
1.2.3.19 N-(3-(9H-Fluoren-1-yl)-1-phenyl-1H-1,2,4-triazol-5-yl)-2-(4-methoxyphenoxy) ac-
1
etamide (5g): bright yellow solid, 106 mg, yield 22%; H NMR (400 MHz, Chloroform-d) δ
8.90 (s, 1H), 8.32 (s, 1H), 8.17 (dd, J = 7.9, 1.5 Hz, 1H), 7.83 (dd, J = 10.8, 7.7 Hz, 2H), 7.58
– 7.53 (m, 3H), 7.52 – 7.44 (m, 3H), 7.39 (td, J = 7.5, 1.2 Hz, 1H), 7.32 (td, J = 7.4, 1.2 Hz,
1H), 6.86 – 6.80 (m, 4H), 4.58 (s, 2H), 3.95 (s, 2H), 3.78 (s, 3H).
1.2.3.20
N-(3-(9H-Fluoren-1-yl)-1-(4-(trifluoromethyl)phenyl)-1H-1,2,4-triazol-5-yl)-2-(4-
1
methoxyphenoxy)acetamide (5gd): bright yellow solid, 111 mg, yield 20%; H NMR (400
MHz, Chloroform-d) δ 8.30 (s, 1H), 8.15 (d, J = 7.7 Hz, 1H), 7.85 (dd, J = 15.1, 7.7 Hz, 2H),

7.74 (s, 4H), 7.57 (d, J = 7.4 Hz, 1H), 7.41 (t, J = 7.4 Hz, 1H), 7.34 (t, J = 7.3 Hz, 1H), 6.88
(s, 4H), 4.60 (s, 2H), 3.97 (s, 2H), 3.80 (s, 3H).
1.2.4 General procedure for the synthesis of N-(1,3-disubstituted-1,2,4-triazol-5-yl)-
2-phenoxyethylamides (7). To a solution of 5 (0.1 mmol) in anhydrous THF (5 mL) was
added LiAlH4 (0.4 mmol) and AlCl3 (0.2 mmol) at -20 under nitrogen. The reaction mix-
ture was warmed to room temperature and continued to stir for 6 h. The reaction mixture was
washed with 10% H2SO4 solution and then diluted with ethyl acetate. The organic layer was
separated, dried over Na2SO4, and concentrated in reduced pressure. The residue was purified
by flash chromatography on silica gel to afford 7.
1.2.4.1
N-(2-(4-Methoxyphenoxy)ethyl)-3-(naphthalen-1-yl)-1-phenyl-1H-1,2,4-triazol-5-
amine (7a): white solid, 34 mg, yield 80%;. 1H NMR (400 MHz, Chloroform-d) δ 9.11 (d, J =
8.5 Hz, 1H), 8.20 (d, J = 7.0 Hz, 1H), 7.90 (t, J = 7.3 Hz, 2H), 7.65 (d, J = 7.8 Hz, 2H), 7.58
– 7.51 (m, 5H), 7.42 (t, J = 7.4 Hz, 1H), 6.84 (m, 4H), 4.21 (t, J = 5.2 Hz, 2H), 3.95 (q, J =
5.2 Hz, 2H), 3.77 (s, 3H).
1.2.4.2
N-(2-(4-Methoxyphenoxy)ethyl)-3-(naphthalen-1-yl)-1-(p-tolyl)-1H-1,2,4-triazol-5
1
-amine (7aa): white solid, 30 mg, yield 68%; H NMR (400 MHz, Chloroform-d) δ 9.13 –
9.07 (m, 1H), 8.19 (dd, J = 7.2, 1.4 Hz, 1H), 7.91 – 7.85 (m, 2H), 7.58 – 7.46 (m, 5H), 7.33 (d,
J = 8.0 Hz, 2H), 6.87 – 6.79 (m, 4H), 4.20 (t, J = 5.0 Hz, 2H), 3.93 (q, J = 5.0 Hz, 2H), 3.76 (s,
3H), 2.43 (s, 3H).
1.2.4.3
N-(2-(4-Methoxyphenoxy)ethyl)-3-(naphthalen-2-yl)-1-phenyl-1H-1,2,4-triazol-5-
amine (7b): white solid, 36 mg, yield 83%; 1H NMR (400 MHz, Chloroform-d) δ 8.63 (s, 1H),
8.24 (d, J = 8.5 Hz, 1H), 7.95 (d, J = 9.3 Hz, 1H), 7.90 (d, J = 8.6 Hz, 1H), 7.88 – 7.84 (m,
1H), 7.60 (d, J = 7.9 Hz, 2H), 7.50 (dd, J = 9.1, 4.9 Hz, 4H), 7.42 (t, J = 7.3 Hz, 1H), 6.88 –
6.81 (m, 4H), 4.21 (t, J = 5.0 Hz, 2H), 3.95 (q, J = 5.0 Hz, 2H), 3.76 (s, 3H).
1.2.4.4
N-(2-(4-Methoxyphenoxy)ethyl)-3-(naphthalen-2-yl)-1-(p-tolyl)-1H-1,2,4-triazol-
5-amine (7ba): white solid, 27 mg, yield 60%; 1H NMR (400 MHz, Chloroform-d) δ 8.62 (s,
1H), 8.22 (d, J = 8.5 Hz, 1H), 7.93 (d, J = 9.3 Hz, 1H), 7.91 – 7.81 (m, 2H), 7.51 – 7.42 (m,
4H), 7.32 (d, J = 7.7 Hz, 2H), 6.86-6.81 (m, 4H), 4.88 (s, 1H), 4.20 (s, 2H), 3.93 (s, 2H), 3.76
(s, 3H), 2.42 (s, 3H).
1.2.4.5
N-(2-(4-Methoxyphenoxy)propyl)-3-(naphthalen-2-yl)-1-phenyl-1H-1,2,4-triazol-
5-amine (7bβ): white solid, 32 mg, yield 72%; 1H NMR (400 MHz, Chloroform-d) δ 8.63 (s,
1H), 8.24 (d, J = 8.6 Hz, 1H), 7.96 – 7.93 (m, 1H), 7.88 (m, 2H), 7.51 (m, 6H), 7.39 (t, J =
7.1 Hz, 1H), 6.84 (q, J = 8.8 Hz, 4H), 4.95 – 4.88 (m, 1H), 4.64 (m, 1H), 3.95 (m, 1H), 3.76
(s, 3H), 3.60 (m, 1H), 1.37 (d, J = 6.1 Hz, 3H).
1.2.5 General procedure for synthesis of N-(1,3-disubstituted-1,2,4-triazol-5-yl) -1-oxa-4

-azaspiro [4,5]deca-6,9-dien-3,8-diones (6) and N-(1,3-disubstituted- 1,2,4-triazol-5-yl)
-1-oxa-4-azaspiro[4,5]deca-6,9-dien-8-ones (8). A dried flask was charged, under a nitrogen
atmosphere, with 5 or 7 (0.1mmol), PhI(OAc)2 (0.25 mmol), and Cu(CF3SO3)2 (0.015 mmol).
The distilled dichloromethane (10 mL) was added. After stirred at room temperature for 2h.
The reaction mixture was concentrated in vacuum and the residue was purified by flash
chromatography on silica gel to give 6 or 8.
1.2.5.1 4-(3-(Naphthalen-1-yl)-1-phenyl-1H-1,2,4-triazol-5-yl)-1-oxa-4-azaspiro[4.5] de-
ca-6,9-diene-3,8-dione (6a): white solid, 19 mg, yield 90%, mp 182.2-185.1 ; 1H NMR (400
MHz, Chloroform-d) δ 8.86 (d, J = 8.2 Hz, 1H), 8.13 (d, J = 7.2 Hz, 1H), 7.94 (d, J = 8.2 Hz,
1H), 7.90 (d, J = 7.7 Hz, 1H), 7.62 – 7.46 (m, 8H), 6.65 (d, J = 10.0 Hz, 2H), 6.22 (d, J = 10.0
Hz, 2H), 4.59 (s, 2H); 13C NMR (151 MHz, Chloroform-d) δ 183.8, 170.5, 162.6, 142.3,
142.1, 136.7, 134.3, 131.5, 131.0, 130.9, 130.3, 130.0, 128.8, 128.5, 127.4, 127.0, 126.4,
126.3, 125.5, 125.1, 88.6, 66.4. HRMS (ESI): Calculated for C26H19N4O3, {[M+H]+}:
435.1452, found 435.1451
1.2.5.2 2-Methyl-4-(3-(naphthalen-1-yl)-1-phenyl-1H-1,2,4-triazol-5-yl)-1-oxa-4- azaspi-
ro-[4.5]deca-6,9-diene-3,8-dione (6aβ): yellow solid, 19 mg, yield 82%, mp 184.4-187.0 ;
1H NMR (400 MHz, Chloroform-d) δ 8.85 (d, J = 8.3 Hz, 1H), 8.13 (dd, J = 7.3, 1.3 Hz, 1H),
7.97 – 7.85 (m, 2H), 7.64 – 7.47 (m, 8H), 6.77 (dd, J = 10.0, 3.2 Hz, 1H), 6.45 (dd, J = 10.0,
3.2 Hz, 1H), 6.26 – 6.16 (m, 2H), 4.71 (q, J = 6.7 Hz, 1H), 1.57 (d, J = 6.7 Hz, 3H); 13C NMR
(151 MHz, Chloroform-d) δ 183.9, 172.8, 162.6, 143.3, 142.7, 142.5, 136.7, 134.3, 132.3,
131.0, 130.9, 130.5, 130.3, 129.9, 128.8, 128.5, 127.4, 127.1, 126.4, 126.3, 125.5, 125.0, 87.0,
73.4, 18.5. HRMS (ESI): Calculated for C27H20N4NaO3, {[M+Na]+}: 471.1427, found
471.1422.
1.2.5.3 4-(3-(Naphthalen-1-yl)-1-(p-tolyl)-1H-1,2,4-triazol-5-yl)-1-oxa-4-azaspiro[4.5]- de-
1
ca-6,9-diene-3,8-dione (6aa): yellow solid, 16 mg, yield 87%, mp 198.1-198.9 ; H NMR
(400 MHz, Chloroform-d) δ 8.85 (d, J = 8.2 Hz, 1H), 8.12 (d, J = 7.2 Hz, 1H), 7.93 (d, J = 8.2
Hz, 1H), 7.89 (d, J = 9.1 Hz, 1H), 7.57 – 7.50 (m, 3H), 7.47 (d, J = 8.2 Hz, 2H), 7.35 (d, J =
8.2 Hz, 2H), 6.66 (d, J = 10.1 Hz, 2H), 6.22 (d, J = 10.1 Hz, 2H), 4.57 (s, 2H), 2.48 (s, 3H);
13C NMR (101 MHz, Chloroform-d) δ 183.5, 170.1, 162.2, 141.9, 140.2, 134.0, 133.9, 131.1,
130.7, 130.5, 130.2, 128.5, 128.2, 127.0, 126.9, 126.1, 126.0, 125.1, 124.7, 88.3, 66.0, 21.3.
HRMS (ESI): Calculated for C27H20N4NaO3, {[M+H]+}: 449.1608, found 449.1614.
1.2.5.4 4-(3-(Naphthalen-1-yl)-1-(o-tolyl)-1H-1,2,4-triazol-5-yl)-1-oxa-4-azaspiro[4.5]- deca
-6,9-diene-3,8-dione (6ab): yellow solid, 19 mg, yield 80%, mp 200.6-203.0 ; 1H NMR (400
MHz, Chloroform-d) δ 8.88 (d, J = 8.1 Hz, 1H), 8.15 (d, J = 7.2 Hz, 1H), 7.94-7.88 (m, 2H),
7.57 – 7.33 (m, 7H), 6.66 (d, J = 10.0 Hz, 2H), 6.25 (d, J = 10.0 Hz, 2H), 4.50 (s, 2H), 2.25 (s,
13
3H); C NMR (101 MHz, Chloroform-d) δ 183.5, 169.9, 162.1, 143.0, 142.0, 136.0, 135.1,

134.0, 131.8, 131.2, 130.6, 130.5, 128.5, 128.2, 127.0, 126.8, 126.7, 126.4, 126.0, 125.1, 87.9,
65.9, 18.1. HRMS (ESI): Calculated for C27H20N4NaO3, {[M+Na]+}: 471.1427, found
471.1424..
1.2.5.5 4-(3-(Naphthalen-2-yl)-1-phenyl-1H-1,2,4-triazol-5-yl)-1-oxa-4-azaspiro[4.5]- de-
1
ca-6,9-diene-3,8-dione (6b): brown solid, 20 mg, yield 90%, mp 210.9-213.7 ; H NMR
(400 MHz, Chloroform-d) δ 8.58 (s, 1H), 8.16 (d, J = 9.8 Hz, 1H), 8.00 – 7.82 (m, 3H),
13
7.56-7.50 (m, 7H), 6.59 (d, J = 10.1 Hz, 2H), 6.15 (d, J = 10.0 Hz, 2H), 4.58 (s, 2H); C
NMR (151 MHz, Chloroform-d) δ 183.4, 170.3, 161.9, 142.5, 141.6, 136.2, 134.1, 133.2,
131.1, 130.0, 129.7, 128.6, 128.4, 127.8, 127.2, 126.8, 126.4, 126.1, 124.8, 123.6, 88.2, 66.0.
HRMS (ESI): Calculated for C26H18N4NaO3, {[M+Na]+}: 457.1271, found 457.1275.
1.2.5.6 2-Methyl-4-(3-(naphthalen-2-yl)-1-phenyl-1H-1,2,4-triazol-5-yl)-1-oxa-4- azaspi-
ro-[4.5]deca-6,9-diene-3,8-dione (6bβ): yellow solid, 17 mg, yield 71%, mp 186.0-189.7 ;
1H NMR (400 MHz, Chloroform-d) δ 8.57 (d, J = 1.7 Hz, 1H), 8.16 (dd, J = 8.4, 1.7 Hz, 1H),
7.93 – 7.84 (m, 3H), 7.55-7.49 (m, 5H), 7.53 – 7.48 (m, 2H), 6.71 (dd, J = 9.9, 3.2 Hz, 1H),
6.38 (dd, J = 9.9, 3.2 Hz, 1H), 6.18 – 6.11 (m, 2H), 4.71 (q, J = 6.7 Hz, 1H), 1.57 (d, J = 6.7
Hz, 3H); 13C NMR (101 MHz, Chloroform-d) δ 183.6, 172.6, 161.9, 142.9, 142.7, 142.1,
136.3, 134.1, 133.2, 131.9, 130.2, 130.0, 129.6, 128.7, 128.4, 127.8, 127.3, 126.8, 126.5,
126.1, 124.9, 123.6, 86.6, 73.1, 18.2. HRMS (ESI): Calculated for C27H20N4NaO3, [M+Na]+}:
471.1428, found 471.1421.
1.2.5.7 4-(3-(Naphthalen-2-yl)-1-(p-tolyl)-1H-1,2,4-triazol-5-yl)-1-oxa-4-azaspiro[4.5] de-
1
ca-6,9-diene-3,8-dione (6ba): yellow solid, 19 mg, yield 80%, mp 220.4-221.8 ; H NMR
(400 MHz, Chloroform-d) δ 8.59 (s, 1H), 8.17 (d, J = 8.6 Hz, 1H), 7.9– 7.84 (m, 3H), 7.53 (m,
2H), 7.44 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 6.62 (d, J = 9.8 Hz, 2H), 6.18 (d, J =
13
9.8 Hz, 2H), 4.60 (s, 2H), 2.50 (s, 3H); C NMR (101 MHz, Chloroform-d) δ 183.5, 170.3,
161.8, 142.4, 141.8, 140.4, 134.1, 133.8, 133.2, 131.1, 130.2, 128.7, 128.4, 127.8, 127.4,
126.8, 126.4, 126.1, 124.8, 123.6, 88.2, 66.0, 21.4. HRMS (ESI): Calculated for
C27H20N4NaO2, {[M+Na]+}: 471.1428, found 471.1426.
1.2.5.8 2-Methyl-4-(3-(naphthalen-2-yl)-1-(p-tolyl)-1H-1,2,4-triazol-5-yl)-1-oxa-4- azaspi-
ro[4.5]deca-6,9-diene-3,8-dione (6baβ): white solid, 17 mg, yield 70%, mp 193.1-195.0 ;
1H NMR (400 MHz, Chloroform-d) δ 8.59 – 8.54 (m, 1H), 8.15 (dd, J = 8.5, 1.7 Hz, 1H),
7.94 – 7.83 (m, 3H), 7.53 – 7.47 (m, 2H), 7.44 – 7.39 (m, 2H), 7.34 (d, J = 8.2 Hz, 2H), 6.71
(dd, J = 10.0, 3.1 Hz, 1H), 6.39 (dd, J = 10.0, 3.1 Hz, 1H), 6.20 – 6.11 (m, 2H), 4.71 (q, J =
13
6.5 Hz, 1H), 2.47 (s, 3H), 1.58 (d, J = 6.5 Hz, 3H); C NMR (101 MHz, Chloroform-d) δ
183.6, 172.6, 161.8, 143.0, 142.6, 142.2, 140.3, 134.1, 133.8, 133.2, 131.8, 130.2, 130.1,
128.7, 128.4, 127.8, 127.4, 126.8, 126.4, 126.1, 124.7, 123.6, 86.6, 73.1, 21.4, 18.2. HRMS
(ESI): Calculated for C28H22N4NaO3, {[M+Na]+}: 485.1584, found 485.1578.

1.2.5.9 4-(1-(4-Chlorophenyl)-3-(naphthalen-2-yl)-1H-1,2,4-triazol-5-yl)-1-oxa-4- azaspi-
ro-[4.5]deca-6,9-diene-3,8-dione (6bc): brown solid, 17 mg, yield 82%, mp 219.7-223.1 ;
1H NMR (400 MHz, Chloroform-d) δ 8.55 (s, 1H), 8.12 (d, J = 10.2 Hz, 1H), 7.95 – 7.82 (m,
3H), 7.59 – 7.46 (m, 6H), 6.65 (d, J = 10.1 Hz, 2H), 6.20 (d, J = 10.1 Hz, 2H), 4.59 (s, 2H);
13C NMR (101 MHz, Chloroform-d) δ 183.4, 170.3, 162.1, 142.6, 141.6, 136.0, 134.8, 134.2,
133.2, 131.3, 129.9, 128.7, 128.5, 127.8, 127.0, 126.9, 126.5, 126.2, 126.1, 123.5, 88.3, 66.0.
HRMS (ESI): Calculated for C26H17 Cl N4NaO3, {[M+Na]+}: 491.0881, found 491.0876.
1.2.5.10
4-(3-(Naphthalen-2-yl)-1-(4-(trifluoromethyl)phenyl)-1H-1,2,4-triazol-5-yl)-1-
oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione (6bd): yellow solid, 19 mg, yield 76%, mp
178.1-181.5 ; 1H NMR (400 MHz, Chloroform-d) δ 8.55 (s, 1H), 8.12 (d, J = 10.0 Hz, 1H),
7.94-7.84 (m, 5H), 7.73 (d, J = 8.3 Hz, 2H), 7.52 (dd, J = 7.0, 2.4 Hz, 2H), 6.68 (d, J = 10.1
Hz, 2H), 6.23 (d, J = 10.1 Hz, 2H), 4.61 (s, 2H); 13C NMR (101 MHz, CDCl3) δ 183.3, 170.2,
162.3, 142.8, 141.4, 139.4, 134.3, 133.2, 132.0, 131.7, 131.4,131.2, 128.7, 128.5, 127.9,
126.99, 126.96, 126.93, 126.89, 126.6, 126.3, 124.8, 123.5, 88.4, 66.0.HRMS (ESI): Calcu-
lated for C27H18F3N4O3, {[M+H]+}: 503.1326, found 503.1315.
1.2.5.11 4-(1-(4-Methoxyphenyl)-3-(naphthalen-2-yl)-1H-1,2,4-triazol-5-yl)-1-oxa-4- azaspi-
1
ro[4.5]deca-6,9-diene-3,8-dione (6bf): white solid, 19 mg, yield 80%, mp 188.8-191.0 ; H
NMR (400 MHz, Chloroform-d) δ 8.59 – 8.55 (m, 1H), 8.14 (dd, J = 8.5, 1.7 Hz, 1H), 7.93 –
7.88 (m, 2H), 7.87 – 7.83 (m, 1H), 7.52 – 7.48 (m, 2H), 7.47 – 7.43 (m, 2H), 7.06 – 7.01 (m,
2H), 6.64 – 6.57 (m, 2H), 6.20 – 6.14 (m, 2H), 4.57 (s, 2H), 3.90 (s, 3H); 13C NMR (101
MHz, CDCl3) δ 183.5, 170.4, 161.7, 160.7, 142.5, 141.8, 134.1, 133.2, 131.1, 129.0, 128.7,
128.4, 127.8, 127.4, 126.8, 126.5, 126.4, 126.1, 123.6, 114.7, 88.1, 66.0, 55.7: Calculated for
C27H20N4NaO4, {[M+Na]+}: 487.1377, found 487.1375.
1.2.5.12
4-(1-(4-Methoxyphenyl)-3-(naphthalen-2-yl)-1H-1,2,4-triazol-5-yl)-2-methyl-1-
oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione (6bfβ): white solid, 17 mg, yield 72%, mp
184.7-187.2 ; 1H NMR (400 MHz, Chloroform-d) δ 8.57 (dd, J = 1.7, 0.8 Hz, 1H), 8.15 (dd,
J = 8.5, 1.7 Hz, 1H), 7.94 – 7.82 (m, 3H), 7.55 – 7.49 (m, 2H), 7.49 – 7.41 (m, 2H), 7.08 –
6.99 (m, 2H), 6.75 – 6.67 (m, 1H), 6.44 – 6.35 (m, 1H), 6.23 – 6.11 (m, 2H), 4.70 (q, J = 6.7
Hz, 1H), 3.90 (s, 3H), 1.57 (d, J = 6.7 Hz, 3H); 13C NMR (151 MHz, Chloroform-d) δ 183.9,
172.9, 162.1, 161.0, 143.4, 143.1, 142.6, 134.4, 133.6, 132.1, 130.5, 129.4, 128.9, 128.7,
128.1, 127.7, 127.1, 126.8, 126.7, 126.4, 123.9, 114.9, 86.8, 73.4, 56.0, 18.6. HRMS(ESI):
Calculated for C28H22N4NaO4, {[M+Na]+}: 501.1533, found 501.1529.
1.2.5.13 4-(1,3-Di(naphthalen-2-yl)-1H-1,2,4-triazol-5-yl)-1-oxa-4-azaspiro[4.5]deca-6,9-
diene-3,8-dione (6bg): white solid, 22 mg, yield 89%, mp 184.3-186.8 ; 1H NMR (400 MHz,
Chloro-form-d) δ 8.60 (s, 1H), 8.18 (dd, J = 8.5, 1.7 Hz, 1H), 8.07 – 8.01 (m, 2H), 7.94 (m,
4H), 7.89 – 7.85 (m, 1H), 7.66 – 7.60 (m, 3H), 7.54-7.50 (m, 2H), 6.56 (d, J = 10.0 Hz, 2H),

13
6.13 (d, J = 10.0 Hz, 2H), 4.57 (s, 2H); C NMR (151 MHz, Chloroform-d) δ 183.8, 170.8,
162.4, 143.0, 142.0, 134.5, 133.9, 133.7, 133.6, 133.3, 131.4, 130.2, 129.0, 128.8, 128.4,
128.2, 128.1, 128.0, 127.6, 127.2, 126.8, 126.5, 124.1, 124.0, 122.7, 88.6, 66.4. HRMS (ESI):
Calculated for C30H20N4NaO3, {[M+Na]+}: 507.1428, found 507.1427.
1.2.5.14 4-(1,3-Di(naphthalen-2-yl)-1H-1,2,4-triazol-5-yl)-2-methyl-1-oxa-4-azaspiro[4.5]
1
deca-6,9-diene-3,8-dione (6bgβ): brown solid, 19 mg, yield 72%, mp 174.7-176.3 ; H
NMR (400 MHz, Chloroform-d) δ 8.61 (d, J = 1.7 Hz, 1H), 8.19 (dd, J = 8.5, 1.7 Hz, 1H),
8.07 – 8.00 (m, 2H), 8.00 – 7.83 (m, 5H), 7.69 – 7.59 (m, 3H), 7.56 – 7.47 (m, 2H), 6.70 (dd,
J = 10.1, 3.2 Hz, 1H), 6.31 (dd, J = 10.1, 3.1 Hz, 1H), 6.12 (m, 2H), 4.72 (q, J = 6.7 Hz, 1H),
13
1.57 (d, J = 6.7 Hz, 3H); C NMR (151 MHz, Chloroform-d) δ 183.9, 173.1, 162.4, 143.2,
142.5, 134.5, 133.9, 133.7, 133.6, 133.24, 132.17, 130.5, 130.2, 129.0, 128.8, 128.7, 128.4,
128.2, 128.1, 128.0, 127.7, 127.2, 126.8, 126.5, 124.0, 123.7, 122.6, 87.0, 73.4, 18.6. HRMS
(ESI): Calculated for C31H22N4NaO3, {[M+Na]+}: 521.1584, found 521.1584.
1.2.5.15 4-(3-(1-Bromonaphthalen-2-yl)-1-phenyl-1H-1,2,4-triazol-5-yl)-1-oxa-4- azaspi-
1
ro[4.5]deca-6,9-diene-3,8-dione (6c): brown solid, 18 mg, yield 70%, mp 210.9-214.9 ; H
NMR (400 MHz, Chloroform-d) δ 8.46 (d, J = 8.5 Hz, 1H), 7.87 (dd, J = 8.0, 3.9 Hz, 2H),
7.78 (d, J = 8.5 Hz, 1H), 7.679-7.60 (m, 7H), 6.63 (d, J = 10.0 Hz, 2H), 6.22 (d, J = 10.0 Hz,
13
2H), 4.59 (s, 2H); C NMR (101 MHz, Chloroform-d) δ 183.6, 170.2, 162.2, 141.7, 141.6,
136.2, 134.7, 132.6, 131.3, 130.0, 129.7, 129.2, 128.4, 128.2, 127.9, 127.8, 127.6, 127.5,
124.7, 123.5, 88.4, 66.1. HRMS (ESI): Calculated for C26H18BrN4O3, {[M+H]+}: 513.0557,
found 513.0555.
1.2.5.16
4-(3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-1-phenyl-1H-1,2,4-triazol-5-yl)-1
-oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione (6d): white solid, 19 mg, yield 82%, mp
1
200.7-202.4 ; H NMR (400 MHz, Chloroform-d) δ 7.58-7.48 (m, 7H), 6.90 (d, J = 8.4 Hz,
13
1H), 6.54 (d, J = 9.8 Hz, 2H), 6.12 (d, J = 9.8 Hz, 2H), 4.55 (s, 2H), 4.29 (s, 4H); C NMR
(101 MHz, Chloroform-d) δ 183.5, 170.3, 161.5, 145.2, 143.6, 142.1, 141.7, 136.3, 131.0,
129.8, 129.6, 124.8, 123.5, 119.8, 117.5, 115.6, 88.1, 66.0, 64.5, 64.3. HRMS (ESI): Calcu-
lated for 442.1277, C24H18N4NaO5, {[M+Na]+}: 465.1169, found 465.1170.
1.2.5.17
4-(1-Phenyl-3-(quinolin-6-yl)-1H-1,2,4-triazol-5-yl)-1-oxa-4-azaspiro[4.5]deca-
1
6,9-diene-3,8-dione (6e): brown solid, 17 mg, yield 78%, mp 196.6-198.0 ; H NMR (400
MHz, Chloroform-d) δ 8.93 (dd, J = 4.3, 1.7 Hz, 1H), 8.54 (d, J = 1.9 Hz, 1H), 8.40 (dd, J =
8.8, 1.9 Hz, 1H), 8.23 (dd, J = 8.4, 1.7 Hz, 1H), 8.17 (d, J = 8.8 Hz, 1H), 7.57-7.52 (m, 5H),
13
7.44 (dd, J = 8.4, 4.3 Hz, 1H), 6.61 – 6.51 (m, 2H), 6.19 – 6.08 (m, 2H), 4.57 (s, 2H); C
NMR (101 MHz, Chloroform-d) δ 183.4, 170.4, 161.2, 151.0, 148.7, 142.7, 141.6, 136.8,
136.2, 131.2, 130.1, 129.9, 129.7, 128.2, 128.1, 127.4, 125.9, 124.8, 121.7, 88.2, 66.0. HRMS
(ESI): Calculated for C25H18N5O3, {[M+Na]+}: 458.1224, found 458.1228.

1.2.5.18 4-(3-(4-Fluoronaphthalen-1-yl)-1-phenyl-1H-1,2,4-triazol-5-yl)-1-oxa-4- azaspi-
1
ro[4.5]deca-6,9-diene-3,8-dione (6f): brown solid, 19 mg, yield 82%, mp 192.1-195.5 ; H
NMR (400 MHz, Chloroform-d) δ 8.98 – 8.84 (m, 1H), 8.19-8.10 (m, 2H), 7.62-7.53 (m, 7H),
13
7.22 (s, 1H), 6.65 (d, J = 8.6 Hz, 2H), 6.22 (d, J = 8.6 Hz, 2H), 4.59 (s, 2H); C NMR (151
MHz, Chloroform-d) δ 183.5, 170.1, 161.7, 160.8, 159.1, 142.0, 141.8, 136.3, 132.1, 131.1,
130.0, 129.7, 128.4, 128.3, 128.0, 126.4, 126.1, 124.7, 124.0, 123.9, 123.0, 122.9, 120.9,
120.8, 109.1, 109.0, 88.2, 66.0. HRMS (ESI): calculated for C26H17FN4NaO3, {[M+Na]+}:
475.1177, found 475.1180.
1.2.5.19 4-(3-(9H-Fluoren-2-yl)-1-phenyl-1H-1,2,4-triazol-5-yl)-1-oxa-4-azaspiro[4.5]- de-
1
ca-6,9-diene-3,8-dione (6g): yellow solid, 20 mg, yield 82%, mp 236.1-239.6 ; H NMR
(400 MHz, Chloroform-d) δ 8.24 (s, 1H), 8.10 (dd, J = 8.0, 1.6 Hz, 1H), 7.83 (dd, J = 8.0, 6.2
Hz, 2H), 7.59 – 7.51 (m, 6H), 7.40 (t, J = 7.4 Hz, 1H), 7.36 – 7.31 (m, 1H), 6.60 – 6.54 (m,
2H), 6.17 – 6.12 (m, 2H), 4.58-4.57 (m, 2H), 3.96 (s, 2H); 13C NMR (101 MHz, Chloro-
form-d) δ 183.5, 170.3, 162.2, 143.8, 143.6, 143.4, 142.3, 141.7, 141.2, 136.3, 131.1, 129.9,
129.7, 128.3, 127.2, 126.9, 125.3, 125.1, 124.8, 123.0, 120.3, 120.0, 88.2, 66.0, 36.9. HRMS
(ESI): Calculated for C29H20N4NaO3, {[M+Na]+}: 495.1428, found 495.1433.
1.2.5.20
4-(3-(9H-Fluoren-2-yl)-1-(4-(trifluoromethyl)phenyl)-1H-1,2,4-triazol-5-yl)-1-
oxa-4-azaspiro[4.5]deca-6,9-diene-3,8-dione (6gd): yellow solid, 22 mg, yield 78%, mp
1
248.6-251.0 ; H NMR (400 MHz, Chloroform-d) δ 8.22 (s, 1H), 8.11 – 8.04 (m, 1H), 7.85
(dd, J = 8.2, 4.4 Hz, 4H), 7.72 (d, J = 8.2 Hz, 2H), 7.58 (d, J = 7.4 Hz, 1H), 7.43 – 7.32 (m,
13
2H), 6.73 – 6.61 (m, 2H), 6.28 – 6.17 (m, 2H), 4.60 (s, 2H), 3.97 (s, 2H); C NMR (101
MHz, Chloroform-d) δ 183.4, 170.2, 162.5, 143.8, 143.7, 143.6, 142.5, 141.4, 141.1, 139.3,
131.9, 131.6, 131.4, 127.9, 127.5, 127.3, 126.93, 126.92(126.98, 126.95, 126.91, 126.87, J =
150 Hz), 125.3, 125.2, 124.7, 123.0 , 120.3, 120.1, 88.3, 65.9, 36.9. HRMS (ESI): Calculated
for C30H19F3N4NaO3, {[M+Na]+}: 563.1301, found 563.1296.
1.2.5.21 4-(3-(Naphthalen-1-yl)-1-phenyl-1H-1,2,4-triazol-5-yl)-1-oxa-4-azaspiro[4.5]- de-
1
ca-6,9-dien-8-one (8a): yellow solid, 10 mg, yield 48%, mp 179.4-180.6 ; H NMR (400
MHz, Chloro-form-d) δ 9.08 (d, J = 8.4 Hz, 1H), 8.19 (d, J = 7.2 Hz, 1H), 7.96 – 7.81 (m,
2H), 7.69 – 7.37 (m, 8H), 6.57 (d, J = 10.1 Hz, 2H), 5.97 (d, J = 10.1 Hz, 2H), 4.29 (t, J = 6.2
Hz, 2H), 3.91 (t, J = 6.2 Hz, 2H); 13C NMR (101 MHz, Chloroform-d) δ 184.8, 160.5, 152.9,
143.8, 137.6, 134.0, 130.8, 130.1, 129.5, 129.1, 128.8, 128.4, 127.8, 127.4, 126.8, 126.4,
125.9, 125.5, 125.2, 87.9, 66.1, 50.0. HRMS (ESI): Calculated for C26H21N4O2, {[M+H]+}:
421.1659, found 421.1665.
1.2.5.22 4-(3-(Naphthalen-1-yl)-1-(p-tolyl)-1H-1,2,4-triazol-5-yl)-1-oxa-4-azaspiro[4.5] de-
1
ca-6,9-dien-8-one (8aa): brown solid, 9 mg, yield 40%, mp 170.6-172.1 ; H NMR (400
MHz, Chloroform-d) δ 9.07 (d, J = 8.2 Hz, 1H), 8.20 – 8.15 (m, 1H), 7.88 (t, J = 9.0 Hz, 2H),

7.60 – 7.46 (m, 3H), 7.43-7.41 (m, 2H), 7.25 (s, 2H), 6.58 (d, J = 10.1 Hz, 2H), 6.00 (d, J =
13
10.1 Hz, 2H), 4.28 (t, J = 6.2 Hz, 2H), 3.87 (t, J = 6.2 Hz, 2H), 2.43 (s, 3H); C NMR (151
MHz, Chloroform-d) δ 184.87, 160.29, 152.73, 143.84, 139.03, 135.13, 134.02, 130.81,
130.00, 129.18, 128.37, 127.70, 127.44, 126.73, 126.45, 125.83, 125.38, 125.16, 87.76, 66.13,
49.86, 21.24. HRMS (ESI): Calculated for C27H23N4O2, {[M+H]+}: 435.1815, found
435.1815.
1.2.5.23 4-(3-(Naphthalen-2-yl)-1-phenyl-1H-1,2,4-triazol-5-yl)-1-oxa-4-azaspiro[4.5] de-
1
ca-6,9-dien-8-one (8b): yellow solid, 13 mg, yield 60%, mp 186-9-191.0 ; H NMR (400
MHz, Chloro-form-d) δ 8.57 – 8.52 (m, 1H), 8.15 (dd, J = 8.6, 1.7 Hz, 1H), 7.94 – 7.83 (m,
3H), 7.52 – 7.38 (m, 7H), 6.55 – 6.47 (m, 2H), 5.96 – 5.87 (m, 2H), 4.29 (t, J = 6.2 Hz, 2H),
13
3.93 (t, J = 6.2 Hz, 2H); C NMR (101 MHz, Chloroform-d) δ 184.8, 159.9, 153.8, 143.5,
137.6, 133.9, 133.3, 129.6, 129.0, 128.9, 128.6, 128.3, 128.1, 127.8, 126.5, 126.3, 125.7,
125.6, 123.8, 87.9, 66.1, 50.1. HRMS (ESI): Calculated for C26H20N4NaO2, {[M+Na]+}:
443.1478, found 443.1482.
1.2.5.24 4-(3-(Naphthalen-2-yl)-1-(p-tolyl)-1H-1,2,4-triazol-5-yl)-1-oxa-4-azaspiro[4.5] de-
1
ca-6,9-dien-8-one (8ba): brown solid, 10 mg, yield 47%, mp 163.6-165.2 ; H NMR (400
MHz, Chloroform-d) δ 8.53 (s, 1H), 8.14 (d, J = 10.1 Hz, 1H), 7.94 – 7.83 (m, 3H), 7.49 (dt,
J = 6.2, 3.4 Hz, 2H), 7.36 (d, J = 8.2 Hz, 2H), 7.24 (d, J = 8.2 Hz, 2H), 6.52 (d, J = 10.1 Hz,
2H), 5.95 (d, J = 10.1 Hz, 2H), 4.28 (t, J = 6.2 Hz, 2H), 3.89 (t, J = 6.2 Hz, 2H), 2.41 (s, 3H);
13C NMR (151 MHz, Chloroform-d) δ 185.0, 159.8, 153.6, 143.6, 139.1, 135.1, 133.9, 133.3,
130.0, 129.1, 128.6, 128.2, 128.1, 127.8, 126.4, 126.2, 125.7, 125.5, 123.8, 87.8, 66.1, 50.0,
21.2. HRMS (ESI): Calculated for C27H22N4NaO2, {[M+Na]+}: 457.164035, found 457.1629.
1.2.5.25 2-Methyl-4-(3-(naphthalen-2-yl)-1-phenyl-1H-1,2,4-triazol-5-yl)-1-oxa-4- azaspi-
1
ro[4.5]deca-6,9-dien-8-one (8bβ): brown solid, 12 mg, yield 53%, mp 186.6-189.2 ; H
NMR (400 MHz, Chloroform-d) δ 8.54 (s, 1H), 8.15 (dd, J = 8.5, 1.7 Hz, 1H), 7.96 – 7.81 (m,
3H), 7.54 – 7.38 (m, 7H), 6.54 (dd, J = 10.1, 3.1 Hz, 1H), 6.49 (dd, J = 10.0, 3.1 Hz, 1H),
5.96 (dd, J = 10.0, 2.1 Hz, 1H), 5.82 (dd, J = 10.1, 2.1 Hz, 1H), 4.62 – 4.55 (m, 1H), 3.95 (m,
1H), 3.53 (t, J = 9.3 Hz, 1H), 1.45 (d, J = 6.0 Hz, 3H); 13C NMR (101 MHz, Chloroform-d) δ
184.9, 160.0, 153.8, 144.6, 143.6, 137.7, 133.9, 133.3, 129.6, 129.5, 128.8, 128.6, 128.3,
128.2, 128.1, 127.8, 126.5, 126.3, 125.7, 125.6, 123.8, 88.2, 73.9, 56.4, 18.5. HRMS (ESI):
Calculated for C27H22N4NaO2, {[M+Na]+}: 457.1635, found 457.1641.
2. Cell proliferation assay
Breast cancer cell lines, MDA-MB-231, the cervical cancer cell line, HeLa, and the
non-small cell lung carcinoma cell line, cell lines were purchased from ATCC. A549 were
routinely cultured in DMEM medium supplemented with 10% FBS, 4 mM glutamine, 1 mM
sodium pyruvate, 100 IU/mL penicillin, 100 μg/mL streptomycin and 0.25 μg/mL amphoteri-

cin. Cultures were maintained in 5% CO2 at a temperature of 37oC. The cells were plated in
24-well plates at a density of 20,000 per well in 10% FBS DMED medium. The cells were
then treated with synthesized triazole-spirodienone conjugates separately at 5 different doses
ranging from 0.01 mM to 1 nM for 5 days, while equal treatment volumes of DMSO were
used as vehicle control. Cell numbers were counted with a cell viability analyzer (Beckman-
eCoulter). The ratio of drug-treated viable cell numbers to vehicle- treated viable cell num-
bers were defined as percentage viability. IC50 values were obtained from dose-response
curves for each tested compound.
3. Cell cycle assay.
Cells (5 × 105 cells/mL) were seeded in six-well plates and treated with compounds at
different concentrations for 24 h. The cells were then harvested by trypsinization and washed
twice with cold PBS. After centrifugation and removal of the supernatants, cells were resus-
pended in 400μL of 1×PBS buffer. After adding 10μL of PI the cells were incubated at room
temperature for 15 min in the dark. The stained cells were analyzed by a flow cytometer (BD
Accuri C6).
4. Apoptosis detection assay.
Cells (5 × 105 cells/mL) were seeded in six-well plates and treated with compounds at
different concentrations for 24 h. The cells were then harvested by trypsinization and washed
twice with cold PBS. After centrifugation and removal of the supernatants, cells were resus-
pended in 400μL of 1×binding buffer which was then added to 5μL of annexin V-FITC and
incubated at room temperature for 15 min. After adding 10μL of PI the cells were incubated
at room temperature for another 15 min in the dark. The stained cells were analyzed by a flow
cytometer (BD Accuri C6).
5. In vivo antitumor effect
The orthotopic 4T1 tumor-bearing mice were divided into four groups (n = 6) randomly.
When the tumor volume reached around 100 mm2, mice were respectively treated with vehi-
cle control, 6a at 1 mg/kg, 6a at 2 mg/kg, and doxorubicin at 1 mg/kg by intraperitoneal in-
jection every day. While body weight and tumor volumes were measured and recorded every
day. At the end of treatment, the major organs and tumors were harvested and photographed.
Sections prepared from tumor and organs were histopathologically examined and H&E
stained, and then imaged by microscope.
6. Statistical analysis
All data were presented as mean ± standard deviation (SD). Statistical significance was
performed by two-tailed Student's t-tests for two groups and one-way ANOVA for multiple