Design, synthesis and biological evaluation of novel β-pinene-based thiazole derivatives as potential anticancer agents via mitochondrial-mediated apoptosis pathway

Article abstract of DOI:10.1016/j.bioorg.2018.12.010

A series of novel β-pinene-based thiazole derivatives were synthesized and characterized by HRMS, ¹H NMR, and ¹³C NMR analyses as potential antineoplastic agents. Derivatives were evaluated for their anticancer activities in vitro, and the data manifested that most target compounds showed potent anti-proliferative activities against three human cancer cell lines. Especially, compound 5g displayed excellent cytotoxic activity against Hela, CT-26, and SMMC-7721 cell lines with IC₅₀ values of 3.48 ± 0.14, 8.84 ± 0.16, and 6.69 ± 0.15 μM, respectively. To determine the underlying mechanism of compound 5g on cell viability, DAPI staining, Annexin-V/PI staining, JC-1 staining, DCFDA staining, and Western blot analysis were performed. Our data showed that compound 5g inhibited cell proliferation by inducing apoptosis and cell cycle arrest of Hela cells at the G0/G1 phase in a dose dependent manner. Further studies revealed that compound 5g enhanced levels of reactive oxygen species (ROS), caused a decrease in mitochondrial membrane potential, increased the release of mitochondrial cytochrome C, and affected the expression of Bax, Bcl-2, caspase-3 and caspase-9. Thus, our findings indicated that compound 5g induced apoptosis in Hela through ROS-mediated mitochondrial dysfunction signaling pathways.

Full text of DOI:10.1016/j.bioorg.2018.12.010

Accepted Manuscript
Design, synthesis and biological evaluation of novel β-pinene-based thiazole
derivatives as potential anticancer agents via mitochondrial-mediated apoptosis
pathway
Yunyun Wang, Chenliang Wu, Qiangjian Zhang, Yu Shan, Wen Gu, Shifa Wang
PII:
S0045-2068(18)30983-0
DOI:
https://doi.org/10.1016/j.bioorg.2018.12.010
YBIOO 2674
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
4 September 2018
27 November 2018
8 December 2018
Please cite this article as: Y. Wang, C. Wu, Q. Zhang, Y. Shan, W. Gu, S. Wang, Design, synthesis and biological
evaluation of novel β-pinene-based thiazole derivatives as potential anticancer agents via mitochondrial-mediated
apoptosis pathway, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.12.010
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Design, synthesis and biological evaluation of novel β-pinene-based thiazole
derivatives as potential anticancer agents via mitochondrial-mediated apoptosis
pathway
Yunyun Wang a, Chenliang Wu a, Qiangjian Zhang a, Yu Shan b, Wen Gu a, c*, Shifa Wang a, c*
a College of Chemical Engineering, Nanjing Forestry University, Nanjing, 210037, P. R. China
b
Institute of Botany, Jiangsu Province and Chinese Academy of Sciences (Nanjing Botanical Garden Mem. Sun
Yat-Sen), 210014, People’s Republic of China
c
Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, Nanjing Forestry University,
Nanjing, 210037, P. R. China
* Corresponding author at: College of Chemical Engineering, Nanjing Forestry University, Nanjing, 210037, P. R.
China.
E-mail address: wangshifa65@163.com (S. Wang).
1

Abstract
A series of novel β-pinene-based thiazole derivatives were synthesized and characterized by
1
HRMS, H-NMR, and 13C-NMR analyses as potential antineoplastic agents. Derivatives were
evaluated for their anticancer activities in vitro, and the data manifested that most target
compounds showed potent anti-proliferative activities against three human cancer cell lines.
Especially, compound 5g displayed excellent cytotoxic activity against Hela, CT-26, and SMMC-
7721 cell lines with IC50 values of 3.48 ± 0.14, 8.84 ± 0.16, and 6.69 ± 0.15 µM, respectively. To
determine the underlying mechanism of compound 5g on cell viability, DAPI staining, Annexin-
V/PI staining, JC-1 staining, DCFDA staining, and Western blot analysis were performed. Our
data showed that compound 5g inhibited cell proliferation by inducing apoptosis and cell cycle
arrest of Hela cells at the G0/G1 phase in a dose dependent manner. Further studies revealed that
compound 5g enhanced levels of reactive oxygen species (ROS), caused a decrease in
mitochondrial membrane potential, increased the release of mitochondrial cytochrome C, and
affected the expression of Bax, Bcl-2, caspase-3 and caspase-9. Thus, our findings indicated that
compound 5g induced apoptosis in Hela through ROS-mediated mitochondrial dysfunction
signaling pathways.
Keywords: β-pinene-based thiazole derivatives; anticancer; mitochondrial membrane potential;
reactive oxygen species
2

1. Introduction
Cancer continues to be a major threat to human health and is regarded as one of the most
common causes of death worldwide. Although significant achievements have been made in
anticancer agents, identifying effective, safe, and novel chemical entities is still a big challenge.
Inducing cell apoptosis has been an effective method for tumor treatment. It is well recognized
that the apoptotic pathways mainly involve two routes: the mitochondrial apoptosis pathway and
the death receptor pathway. As signaling molecules, reactive oxygen species (ROS) are effective
in mediating multiple biological events, including cellular survival and death [1,2]. Moreover,
mitochondria are, to a great extent, considered the major source of ROS production [3]. High
levels of ROS can mediate cell apoptosis, and even necrosis, through mitochondrial dysfunction,
loss of Mitochondrial membrane potential (Δψm), the release of cytochrome C (Cyt C), cleavage
of executioner caspases-3/9, and levels of the Bcl-2 family proteins [4-11]. Together, those events
ultimately result in cell apoptosis.
Essential oils and their derivatives, well known for medicinal purposes [12], play an
important role in drug discovery. For example, turpentine, obtained from resin, has been used as
permeation enhancer [13] and therapeutic agents [14-16] in drugs. β-Pinene, which is isolated
from the raw material of turpentine, is a consequential natural monoterpene resource that is widely
used for producing natural perfume [17]. Additionally, β-pinene is an important raw material for
industrial synthesis. β-Pinene derivatives have wide spread biological activities, including
anticancer, antibacterial, anticoagulation, and antimalarial activities [18-21]. Over the last few
decades, the application of β-pinene mainly concentrated on spices, and studies on β-pinene
pharmaceuticals were limited. Therefore, studying the pharmacological effects of β-pinene and its
3

derivatives is a field that is wide open and may have great potential.
The α, β-Unsaturated carbonyl structures have been recognized as one of the privileged
structures in drug discovery and were widely distributed in biologically active compounds that
have successfully been applied in cancer therapeutics [22,23] due to its low genotoxic effects and
limited reactivity toward cell hydroxyl or amino groups of functional ketone groups [24,25]. In
addition, the functional C=N group, as the necessary component of Schiff bases, is present in
several bioactive molecules, and is recognized as a key building block for the synthesis of small-
molecules with potential pharmaceutical activities [26-28]. Therefore, building functional C=N
groups based on the α, β-unsaturated carbonyl structure has high potential.
Moreover, heterocyclic groups, such as thiazole, are considered a widely-distributed
framework that is present in many pharmaceuticals and natural compounds. Thiazole and its
derivatives have been proven pharmacologically important heterocyclic nucleus in anti-HIV [29],
antibacterial [30], antimalarial [31], and anti-inflammatory [32] territories. In particular, the use of
thiazole hydrazone scaffolds as an active framework in the design of anticancer agents has been
the focus of numerous studies [33-35]. In most cases, the antitumor mechanism of action of
thiazole derivatives was limited to a mitochondria-dependent pathway [36,37]. Therefore, a series
of substituted thiazoles was synthesized and connected with the pinene moiety via the C=N group.
Our studies have long been fully committed to the synthesis of novel turpentine derivatives
and their application in chemistry and biology, such as anticancer [38,39], antimicrobial [40],
insecticidal activities [41], and fluorescent dyes [42,43]. Among them, many compounds exhibited
potent antitumor activities, especially β-pinene based thiosemicarbazone derivatives [44], which
showed strong inhibitory activity on the proliferation of MDA-MB-231, SMMC-7721, and Hela
4

cells. In addition, it has been rarely reported that thiazole derivatives were synthesized from α,β-
unsaturated ketone. Encouraged by the above results and in continuation of our program, which is
aimed at the development of potential anticancer agents, in this study we designed and synthesized
β-pinene-based thiazole derivatives and studied their antitumor activities against human cancer
cell lines in vitro. In addition, we also preliminarily probed the underlying antitumor mechanisms
of the derivatives.
2. Results and discussion
2.1. Synthesis and characterization
The protocol for the synthesis of thiazole derivatives 5a-5x from β-pinene 1 involves four
steps, including oxidation, aldol condensation, addition condensation and Hantzsch cyclization
reactions. Compound 1 was treated with potassium permanganate in acetone at 0C to form
nopinone 2. Intermediates 3 were generated by aldol condensation of nopinone 2 with substituted
benzaldehydes in excellent yields using sodium ethoxide as the catalyst. Subsequently,
compounds 4 were prepared by condensation of intermediates 3 and thiosemicarbazide using
hydrochloric acid as the catalyst. The novel thiazole derivatives 5a-5x were prepared by
cyclization of compounds 4 with phenacyl bromide. The structures of target compounds were
characterized by 1H NMR, 13C NMR and HRMS. As exemplified with the 1H NMR analysis of 4-
(2-(2-(3-benzylidene-6,6-dimethylbicyclo [3.1.1] heptan-2-ylidene) hydrazinyl) thiazol-4-yl)
phenol (5f), singlet and doublets peaks, corresponding to aromatic protons, were observed at δ
7.16-8.26. For the thiazole ring, a singlet at δ 7.57 was found. NH proton appeared as singlet at δ
12.59. 13C NMR spectrum of 5f showed peaks of the aromatic carbons were found at δ 115.73-
146.74ꢀppm. The presence of peaks at δ 123.74 and 169.49ꢀppm, confirm the thiazole cyclisation.
5

1
Besides, representative H-NMR and 13C NMR spectrums of some compounds are presented in
Supplementary Material.
S
S
N
O
N
N
N
NH2
N
O
R2
(a)
(c)
(d)
(b)
H
H
3
2
1
4
5a-5x
R1
R1
R1
5i: R1= F,
5j: R1= F,
5k: R1= F,
5l: R1= F,
R2= H
R2= F
R2= OH
R2= OCH3
5q: R1= CH3, R2= H
5r: R1= CH3, R2= F
5s: R1= CH3, R2= OH
5t: R1= CH3, R2= OCH3
5a: R1= H,
5b: R1= H,
5c: R1= H,
5d: R1= H,
R2= H
R2= F
R2= OH
R2= OCH3
5m: R1= OCH3, R2= H
5n: R1= OCH3, R2= F
5o: R1= OCH3, R2= OH
5p: R1= OCH3, R2= OCH3
5u: R1= Cl,
5v: R1= Cl,
5w: R1= Cl,
5x: R1= Cl,
R2= H
R2= F
R2= OH
R2= OCH3
5e: R1= NO2, R2= H
5f: R1= NO2, R2= F
5g: R1= NO2, R2= OH
5h: R1= NO2, R2= OCH3
Scheme 1. Synthesis of β-pinene-based thiazole derivatives 5a-5x. Reagents and experimental
conditions were as follows: (a) KMnO4, H2SO4, acetone, ice bath to room temperature, 6 h. (b)
substituted benzaldehyde, C2H5ONa, ethanol, reflux, 8 h. (c) HCl, ethanol, reflux, 4-6 h. (d)
phenacyl bromide, reflux.
2.2. Anti-proliferative activity of thiazole derivatives (5a-5x) by MTT assay
The designed 24 compounds were evaluated for cytotoxicity against three cancer cell lines
(colon cancer CT-26 cells, human cervical carcinoma Hela cells and human hepatocarcinoma
SMMC-7721 cells) using the MTT assay with etoposide as a positive control. As illustrated in
Table 1, most designed compounds exhibited cytotoxic activities against the selected cells.
Among them, six compounds (5c, 5g, 5k, 5o, and 5w) showed strong inhibitory effects against the
three cancer cell lines selected. In addition, it was observed that the activities of compounds 5c
and 5w were slightly weaker towards SMMC-7721 cells when compared to other hydroxy-
substituted compounds (5g, 5k, and 5o). Moreover, we found that compound 5g was the most
effective in cervical cancer (Hela) cells. Additionally, several other compounds, including
compounds 5d, 5h, and 5x showed moderate anti-cancer activities, whereas eight compounds (5f,
5j, 5m, 5n, 5q, 5r, 5t, and 5v) did not show any promising anti-cancer activity in vitro.
Table 1
6

Antiproliferative activities of compounds 5a-5x
IC50(µM)
CT-26
Compounds
R1
R2
Hela
21.19±0.23
30.44±0.05
5.99±0.14
13.40±0.16
33.33±0.23
>80
SMMC-7721
66.94±0.0.69
52.52±0.05
12.98±0.52
31.15±0.12
50.47±0.13
>80
5a
5b
H
H
H
F
21.66±0.12
45.51±0.09
7.15±0.11
16.40±0.1.7
25.04±0.04
>80
5c
H
H
OH
OCH3
5d
NO2
5e
H
NO2
NO2
NO2
5f
F
5g
OH
3.48±0.14
19.91±0.19
37.79±0.05
>80
8.84±0.16
44.55±0.76
33.75±0.10
>80
6.69±0.15
41.62±0.09
>80
OCH3
5h
5i
F
F
F
H
F
5j
67.41±0.12
19.42±0.12
30.26±0.06
>80
5k
OH
OCH3
3.57±0.11
42.97±0.47
>80
9.01±0.21
39.95±0.06
>80
5l
F
OCH3
OCH3
5m
5n
H
F
>80
>80
>80
OCH3
5o
OH
OCH3
H
8.813±0.19
41.34±0.03
>80
10.71±0.11
38.45±0.07
>80
9.76±0.07
30.3±0.07
>80
5p
OCH3
CH3
CH3
CH3
CH3
Cl
5q
5r
F
>80
>80
>80
5s
OH
OCH3
H
8.224±0.05
>80
5.89±0.07
>80
7.46±0.11
>80
5t
5u
33.35±0.20
>80
48.82±0.10
>80
42.03±0.16
>80
5v
Cl
F
5w
5x
Cl
OH
OCH3
-
6.46±0.16
17.55±0.03
7.89±1.37
8.95±0.17
30.61±0.20
2.22±1.26
14.69±0.07
39.09±0.03
40.44±0.29
Cl
Etoposide
-
The activity results of compounds 5c, 5g, 5k, 5o and 5w showed that compounds bearing a
strong electron-donating group (R2 = OH) in the para-position (Table 1) were more active against
a wide range of tumor cell lines (CT-26, SMMC-7721, and Hela cells) when compared with other
substituent groups. Especially, compound 5g in which the electron-acceptor group (R1 = NO2) was
present, showed a lower IC50 value ranging from 3.48 µM to 8.84 µM. Moreover, the electron-
withdrawing group (R2 = F) greatly reduced the activity of the corresponding derivatives and even
lost their antitumor activities as reflected by compound 5b with an IC50 value ranging from 30.44
7

µM to 52.52 µM. When the electron-donating group (CH3, OCH3) was introduced into the R1
position, most compounds (5m, 5n, 5q, 5r, and 5t) were inactive. Interestingly, when a non-
substituted group was present at the R1 position, compounds (5a-5d) obtained prominent
antitumor activities.
2.3. Apoptosis analysis by flow cytometry assay
Cell apoptosis analysis was carried out to evaluate whether the growth inhibitory effects of
these compounds on tumor cells were caused by cell apoptosis. Accordingly, compound 5g was
selected for Annexin V/PI apoptosis assay due to its high anticancer activity in the MTT assay. In
brief, Hela cells were treated with different doses of compound 5g for 72 h and evaluated for
apoptosis by flow cytometry using an Annexin V-APC/PI assay. Fig. 1 shows a significant
increase in apoptosis events at 2.5 µM of compound 5g when compared to the control. In addition,
the cell apoptosis rate increased when the radiation dose was increased. Combined, these results
suggested that compound 5g induced apoptosis in Hela cells in a dose-dependent manner.
Fig. 1. Flow cytometric analysis of Hela cells treated with compounds 5g. (a) exposure to 2.5, 5
8

and 10 µM of compound 5g for 72 h and detection and analysis of cell cycle by flow cytometry.
(b) the apoptosis rate in undergone treated by compound 5g.
2.4. Investigation of cell cycle distribution
To further investigate the antitumor mechanism of action, the effect of compound 5g on the
cell cycle was evaluated. Hela cells were harvested after treatment with compound 5g and
analyzed by flow cytometry using PI-stained cell populations. The consequent cell cycle
distributions are presented in Fig. 2. Hela cells treated with a low dose (2.5 µM) of compound 5g
showed an increase in the G0/G1 phase cell population at the expense of reducing the S phase
population when compared to the control. This accumulation in G0/G1 phase was maintained by
increasing the 5g concentration to 5 µM and 10 µM, respectively. Together, these results indicated
that compound 5g caused significant G0/G1 phase arrest.
Fig. 2. Cell cycle distribution of Hela cells (a) exposure to 2.5, 5 and 10 µM of compound 5g
for 24 h and detection and analysis of cell cycle by flow cytometry. (b) the cell cycle change in
undergone treated by compound 5g.
2.5. Intracellular ROS level in dose-dependent manner
9

ROS act as part of a secondary messenger in cellular signaling and are closely related to cell
survival and cell death. High ROS levels can cause mitochondrial dysfunction, resulting in
apoptosis of cancer cells [45]. To evaluate whether β-pinene-based thiazole derivatives induced
ROS accumulation in Hela cells, flow cytometric analysis and microscopic fluorescence imaging
were conducted. In brief, Hela cells were incubated with different concentrations of compound 5g
(2.5, 5, and 10 µM). As shown in Fig. 3, imaging analysis revealed that compound 5g strongly
generated ROS in a dose-dependent manner. It was found that treatment with 5g resulted in an
increase in DCF fluorescence intensity when compared to untreated control cells. Therefore, these
results suggested that compound 5g triggered the production and accumulation of intracellular
ROS in Hela cells in a dose-dependent manner.
10

Fig. 3. (a) HeLa cells were incubated with 0, 2.5, 5 and 10 µM of compound 5g for 72 h and
intracellular DCF fluorescence were observed by fluorescent microscope. (b) intracellular ROS
level were determined by flow cytometry. (c) ROS levels after exposing HeLa cells for 72 h and
distinctly higher ROS levels compared to untreated control cells.
2.6. Measurement of mitochondrial membrane potential
Mitochondria, as the energy center of the cell, are closely connected with cell apoptotic
events. Mitochondrial dysfunction is one of the most profound features of apoptosis and involves
the process, such as mitochondrial permeability transition or the reduction of mitochondrial
membrane potential (MMP) [46]. In this study, mitochondrial dysfunction was determined based
on the MMP index that was measured by flow cytometry using JC-1 dye as a fluorescent probe.
As shown in Fig. 4, after exposure to 2.5, 5, or 10 µM of compound 5g for 72 h, 5g-treated Hela
cells exhibited notably increased green fluorescence and the proportion of green fluorescence
increased from 28.62% to 65.70%. These results indicated that compound 5g could reduce the
mitochondria membrane potential of the cells, resulting in a dose-dependent loss in membrane
potential. As a consequence, 5g-induced apoptosis involved the mitochondrial pathway.
11

Fig. 4. HeLa cells after incubation with compound 5g in 2.5, 5 and 10 µM for 72 h. (a) the MMP
was measured by flow cytometry. (b) the percentages of loss of mitochondrial membrane
potential in Hela cells induced by different concentrations of compound 5g.
2.6. Western blot analysis
As shown previously, compound 5g induced a loss of ΔΨm in Hela cells. These results
suggested that the mitochondrial apoptosis pathway participated in 5g-induced Hela cell apoptosis.
In the course of mediating apoptosis of cancer cells through the mitochondrial pathway,
mitochondria release Cyt C into the cytosol, which is followed by induction of apoptosis factors
and activation of caspases [47]. The expression of apoptosis protein in Hela cells after treatment
with compound 5g for 72 h was detected by Western blot analysis. As shown in Fig. 5, the
immunoblot results confirmed that treatment with compound 5g decreased Bcl-2 expression, and
increased Bax in a concentration-dependent manner. As illustrated in Fig. 5 (b), the proportion of
Bax/Bcl-2 increased, indicating that compound 5g induced apoptosis by elevating the ratio of
Bax/Bcl-2. Moreover, caspase-3 and caspase-9 are the most important signaling proteins
12

associated with the mitochondria-mediated apoptosis pathway. The expression of cleaved caspase-
3 is a biochemical marker of apoptosis19. Fig. 5 (a) and (c) show that after treatment with
compound 5g, the expression of cleaved caspase-3, caspase-9, and Cyt C was increased when
compared to that in control cells. In summary, our results demonstrated that compound 5g induced
apoptosis by regulating Bcl-2, Bax, caspase-3, caspase-9, and Cyt C expression.
Fig. 5. The expression of Cyt C, caspases and Bcl-2 family proteins in Hela cells induced by 5
and 10 µM of compound 5g for 72 h, GAPDH was used to internal control. (a) the expression of
caspases and Bcl-2 family proteins detected by western blot; (b) the rate changes of Bax/Bcl-2
in treated Hela cells; (c) compound 5g induced the rise of Cyt C, caspases and Bcl-2 family
proteins levels in Hela cells.
3. Conclusion
In this study, 24 thiazole analogues were designed, synthesized, and evaluated for
antiproliferative activities. MTT analysis indicated that the antitumor properties of thiazole
containing a hydroxyl group on the phenyl group were increased, and that when thiazole contained
13

a fluorine-group, it was decreased. Among the compounds, compound 5g showed the highest
activity (3.48 µM) on Hela cells. These findings indicated that thiazole with a hydroxyl group on
the phenyl group was favorable for increasing of anti-cancer activity by structural optimization.
Mechanistic studies showed that compound 5g induced the formation of ROS, decreased the
mitochondrial membrane potential and released Cyt C into the cytoplasm. Moreover, compound
5g arrested the cell cycle in the G0/G1 phase and induced cancer cells to be in early apoptosis,
thereby altering the expression of Bax/Bcl-2 proteins. Together, these findings indicated that
compound 5g induced apoptosis via the mitochondria-mediated apoptosis pathway.
4. Experimental
4.1. Chemistry
The reagents (chemicals), all being of A.R. grade, were purchased from Energy Chemical
Reagent Company, Aladdin Reagent Company or Shanghai Chemical Reagent Company
(Shanghai, China). Reactions were monitored by thin layer chromatography (TLC), which was
carried out on silica gel IB-F flexible sheets from Mallinckrodt Baker Inc., Germany, and
visualized in UV light (254 nm). Silica gel (200-300 mesh) for column chromatography was
purchased from Qingdao Marine Chemical Factory (Qingdao, China). Nuclear magnetic resonance
(1H NMR and 13C NMR) spectra were accomplished in CDCl3 or C5D5N on Bruker AV-400 and
AV-500 NMR spectrometers using TMS as internal standard. The HR-MS spectra were recorded
on Agilent 6540 high-resolution mass spectrometer.
4.1.1. Synthesis of nopinone (2)
Nopinone (2) was synthesized according to a previous published method [48]. In brief, 30 mL
14

H2SO4 (2 mol/L) was added to 250 ml acetone solution containing 0.41 mol β-pinene (1) in an ice
bath. Then 1.10 mol KMnO4 powder was slowly added to the above solution, and the reaction was
maintained at room temperature for 6 h. After completion of reaction, solid particles were filtered
from the solution, washed with acetone, and the acetone solution was collected. After removing
the solvent by reduced pressure distillation, the residue was diluted with 40 mL cyclohexane,
washed with distilled water, saturated to neutrality with brine, and dried with anhydrous sodium
sulfate. Nopinone was obtained as a yellowish limpid liquid through evaporating cyclohexane
(yield 80%).
4.1.2. Synthesis of compounds 3
A total of 13.7 mmol sodium ethoxide and corresponding substituted benzaldehyde (1.1 eq,
15.1 mol) was added to a solution of nopinone 2 (13.7 mmol) in ethanol, and the resulting mixture
was refluxed with stirring for 8 h. After cooling, the solvent was removed under reduced pressure.
The crude products were separated by silica gel (200-300 mesh) column chromatography and
eluted with ethyl acetate and petroleum ether (v: v = 3:1; yield 85%).
4.1.3. Synthesis of compounds 4
Compounds 3 (2 mmol) reacted with thiosemicarbazide (2 mmol) in the presence of 0.5 mL
HCl in absolute ethyl alcohol (20 mL) at reflux temperature. At completion of the reaction, the
obtained concentrated reaction solution was purified by a silica column using n-hexane/EtOAc
(8:1) to generate targeted compounds 4 (yield 80%).
4.1.4. synthesis of target compounds 5
An equimolar mixture of bromoacetophenone and compounds 4 (3.3 mmol) were dissolved in
15

20 ml ethanol and stirred for 2 h at room temperature. During the reaction process, the reaction
mixture turned into powder and remained suspended in solution. The powder was filtered and
washed with 5 mL petroleum ether to generate compounds 5a-5x.
4.1.4.1.
2-(2-(3-benzylidene-6,6-dimethylbicyclo[3.1.1]heptan-2-ylidene)hydrazinyl)-4-
phenylthiazole (5a)
1
Yield 78.4%; m.p. 176.5-179.6 C. H NMR (400 MHz, CDCl3) δ: 7.71 (m, 2H), 7.50 (s, 1H),
7.40 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.0 Hz, 2H), 7.15 (t, J = 8.6 Hz, 2H), 6.67 (s, 1H), 3.55 (t, J
= 5.4 Hz, 1H), 2.92 (s, 2H), 2.61 (m, 1H), 2.37 (s, 3H), 2.22 (m, 1H), 1.49 (s, 3H), 1.33 (d, J =
10.4 Hz, 1H), 0.93 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 169.38, 165.86, 140.59, 136.36,
130.99, 130.70, 130.40, 130.14, 129.64, 128.52, 128.17, 125.65, 100.82, 44.52, 42.34, 39.41,
31.75, 27.87, 26.10, 21.66. HR-MS (ESI+): m/z calculated for C25H25N3S [M+H]+ 400.1847, found
400.1853.
4.1.4.2. 2-(2-(3-benzylidene-6,6-dimethylbicyclo[3.1.1]heptan-2-ylidene)hydrazinyl) -4-(4-
fluorophenyl)thiazole (5b)
Yield 85.2%; m.p. 205.3-206.3 C. 1H NMR (400 MHz, CDCl3) δ: 12.56 (s, 1H), 7.74 (dd, J =
8.7, 5.0 Hz, 2H), 7.50 (m, 3H), 7.43 (t, J = 7.6 Hz, 2H), 7.31 (m, 1H), 7.19 (t, J = 8.5 Hz, 2H),
6.70 (s, 1H), 3.60 (t, J = 5.4 Hz, 1H), 2.97 (d, J = 2.8 Hz, 2H), 2.65 (m, 1H), 2.26 (m, 1H), 1.37
(d, J = 10.4 Hz, 1H), 0.97 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 169.31, 165.91, 139.57, 136.27,
131.00, 130.58, 130.07, 128.46, 128.13, 127.76, 127.67, 123.76, 116.92, 116.70, 100.44, 44.43,
42.30, 39.34, 31.66, 27.80, 26.04, 21.59. HR-MS (ESI+): m/z calculated for C25H24FN3S [M+H]+
418.1753, found 418.1761.
4.1.4.3. 4-(2-(2-(3-benzylidene-6,6-dimethylbicyclo[3.1.1]heptan-2-ylidene)hydrazinyl)thiazol-4-
16

yl)phenol (5c)
Yield 81.3%; m.p. 132.6-133.2 C.1H NMR (400 MHz, C5D5N) δ: 8.08 (d, J = 8.6 Hz, 2H),
7.61 (d, J = 7.4 Hz, 2H), 7.90 (s, 1H), 7.30 (t, J = 8.9 Hz, 3H), 7.45 (t, J = 7.7 Hz, 2H), 7.10 (s,
1H), 3.79 (t, J = 5.6 Hz, 1H), 2.92 (m, 1H), 2.83 (m, 1H), 2.46 (m, 1H), 2.04 (m, 1H), 1.24 (s, 3H),
1.22 (s, 1H), 0.84 (s, 3H); 13C NMR (100 MHz, C5D5N) δ: 170.73, 158.57, 154.83, 151.73, 137.57,
132.47, 130.01, 128.64, 127.69, 127.33, 127.25, 127.14, 116.22, 100.70, 42.66, 40.98, 39.57,
32.02, 28.18, 25.72, 21.29. HR-MS (ESI+): m/z calculated for C25H25N3OS [M+H]+ 416.1797,
found 416.1776.
4.1.4.4.
2-(2-(3-benzylidene-6,6-dimethylbicyclo[3.1.1]heptan-2-ylidene)hydrazinyl)-4-(4-
methoxyphenyl)thiazole (5d)
Yield 81.0%; m.p. 132.6-133.2 C.1H NMR (400 MHz, CDCl3) δ: 12.55 (s, 1H), 7.66 (m, 2H),
7.53 (m, 3H), 7.41 (dd, J = 8.4, 6.8 Hz, 2H), 7.34 (m, 1H), 6.99 (m, 2H), 6.59 (s, 1H), 3.84 (s, 3H),
3.61 (t, J = 5.4 Hz, 1H), 3.01 (m, 2H), 2.55 (m, 1H), 2.24 (m, 1H), 1.52 (s, 3H), 1.36 (d, J = 10.4
Hz, 1H), 0.96 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 169.28, 165.54, 161.18, 140.48, 136.41,
130.87, 130.74, 130.14, 128.52, 128.14, 127.20, 120.13, 115.01, 98.64, 55.53, 44.43, 42.31, 39.42,
31.76, 27.88, 26.10, 21.66. HR-MS (ESI+): m/z calculated for C26H27N3OS [M+H]+ 430.1953,
found 430.1957.
4.1.4.5. 2-(2-(6,6-dimethyl-3-(4-nitrobenzylidene)bicyclo[3.1.1]heptan-2-ylidene)hydrazinyl)-4-
phenylthiazole (5e)
1
Yield 87.2%; m.p. 206.4-207.1 C. H NMR (400 MHz, CDCl3) δ: 12.76 (s, 1H), 8.26 (d, J =
8.8 Hz, 2H), 7.72 (d, J = 6.8 Hz, 2H), 7.66 (d, J = 8.8 Hz, 2H), 7.47 (m, 3H), 7.58 (s, 1H), 6.80 (s,
1H), 3.67 (t, J = 5.3 Hz, 1H), 3.00 (m, 1H), 2.94 (m, 1H), 2.69 (m, 1H), 2.29 (m, 1H), 1.53 (s, 3H),
17

1.36 (d, J = 10.6 Hz, 1H), 0.96 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 169.60, 164.74, 142.77,
140.85, 135.02, 130.59, 130.56, 129.70, 128.41, 125.68, 123.77, 100.98, 44.59, 42.41, 39.25,
31.93, 27.92, 26.00, 21.68. HR-MS (ESI+): m/z calculated for C25H24N4O2S [M+H]+ 445.1698,
found 445.1696.
4.1.4.6. 2-(2-(6,6-dimethyl-3-(4-nitrobenzylidene)bicyclo[3.1.1]heptan-2-ylidene)hydrazinyl)-4-
(4-fluorophenyl)thiazole (5f)
1
Yield 83.7%; m.p. 207.5-208.5 C. H NMR (400 MHz, CDCl3) δ: 12.59 (s, 1H), 8.25 (d, J =
8.7 Hz, 2H), 7.73 (dd, J = 8.8, 5.0 Hz, 2H), 7.65 (d, J = 8.8 Hz, 2H), 7.57 (s, 1H), 7.18 (t, J = 8.5
Hz, 2H), 6.77 (s, 1H), 3.63 (t, J = 5.4 Hz, 1H), 3.02 (m, 2H), 2.68 (m, 1H), 2.28 (m, 1H), 1.52 (s,
3H), 1.36 (d, J = 10.6 Hz, 1H), 0.96 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 169.49, 164.72,
146.74, 142.64, 139.71, 134.85, 130.51, 128.39, 127.79, 127.70, 123.67, 116.95, 116.73, 100.81,
44.45, 42.34, 39.16, 31.82, 27.82, 25.92, 21.59. HR-MS (ESI+): m/z calculated for C25H24N4O2FS
[M+H]+ 445.1698, found 445.1696.
4.1.4.7.
4-(2-(2-(6,6-dimethyl-3-(4-nitrobenzylidene)bicyclo[3.1.1]heptan-2-
ylidene)hydrazinyl)thiazol-4-yl)phenol (5g)
1
Yield 70.6%; m.p. 132.6-133.2 C. H NMR (400 MHz, C5D5N) δ: 8.29 (d, J = 8.9 Hz, 2H),
8.08 (d, J = 8.4 Hz, 2H), 7.84 (s, 1H), 7.67 (d, J = 8.6 Hz, 2H), 7.28 (d, J = 8.6 Hz, 2H), 7.13 (s,
1H), 3.81 (t, J = 5.6 Hz, 1H), 2.94 (m, 1H), 2.84 (m, 1H), 2.51 (m, 1H), 2.08 (tt, J = 3.0, 6.0 Hz,
1H), 1.26 (s, 3H), 1.23 (s, 1H), 0.85 (s, 3H); 13C NMR (100 MHz, C5D5N) δ: 171.92, 160.14,
155.25, 153.20, 147.59, 145.48, 138.57, 131.85, 129.21, 128.61, 126.26, 117.73, 102.39, 44.12,
42.43, 40.94, 33.72, 29.81, 27.11, 22.77. HR-MS (ESI+): m/z calculated for C25H24N4O3S [M+H]+
461.1647, found 461.1642.
18

4.1.4.8. 2-(2-(6,6-dimethyl-3-(4-nitrobenzylidene)bicyclo[3.1.1]heptan-2-ylidene)hydrazinyl)-4-
(4-methoxyphenyl)thiazole (5h)
Yield 77.1%; m.p. 143.8-146.1 C. 1H NMR (400 MHz, CDCl3) δ: 12.68 (s, 1H), 8.25 (dd, J =
11.0, 8.7 Hz, 4H), 7.62 (d, J = 8.7 Hz, 2H), 7.51 (s, 1H), 7.00 (d, J = 8.8 Hz, 2H), 3.86 (s, 3H),
3.67 (m, 1H), 2.99 (m, 2H), 2.70 (m, 1H), 2.28 (s, 1H), 1.54 (s, 3H), 1.37 (d, J = 9.2 Hz, 1H), 0.97
13
(s, 3H); C NMR (100 MHz, CDCl3) δ: 169.38, 164.34, 161.22, 156.01, 146.72, 142.92, 135.55,
130.32, 128.21, 127.16, 126.72, 123.67, 115.00, 98.59, 55.48, 42.31, 41.73, 39.23, 31.92, 27.87,
26.00, 21.51. HR-MS (ESI+): m/z calculated for C26H26N4O3S [M+H]+ 474.1726, found 474.1696.
4.1.4.9. 2-(2-(3-(4-fluorobenzylidene)-6,6-dimethylbicyclo[3.1.1]heptan-2-ylidene)hydrazinyl)-4-
phenylthiazole (5i)
1
Yield 80.4%; m.p. 194.7-196.2 C. H NMR (400 MHz, CDCl3) δ: 12.59 (s, 1H), 7.70 (d, J =
7.8 Hz, 2H), 7.45 (m, 6H), 7.36 (d, J = 8.5 Hz, 2H), 6.79 (s, 1H), 3.62 (t, J = 5.4 Hz, 1H), 2.90 (m,
2H), 2.63 (dt, J = 10.5, 5.5 Hz, 1H), 2.24 (m, 1H), 1.50 (s, 3H), 1.33 (d, J = 10.4 Hz, 1H), 0.94 (s,
3H); 13C NMR (100 MHz, CDCl3) δ: 169.31, 165.31, 140.52, 134.73, 133.86, 131.25, 130.31,
129.55, 129.50, 128.65, 127.33, 125.55, 100.94, 44.41, 42.25, 39.26, 31.67, 27.81, 25.99, 21.57.
HR-MS (ESI+): m/z calculated for C25H24FN3S [M+H]+ 418.1753, found 418.1753.
4.1.4.10. 2-(2-(3-(4-fluorobenzylidene)-6,6-dimethylbicyclo[3.1.1]heptan-2-ylidene)hydrazinyl)-4-
(4-fluorophenyl)thiazole (5j)
Yield 81.9%; m.p. 196.4-196.9 C. 1H NMR (400 MHz, CDCl3) δ: 12.12 (s, 1H), 7.71 (m, 2H),
7.48 (m, 3H), 7.16 (t, J = 8.5 Hz, 2H), 7.09 (t, J = 8.7 Hz, 2H), 6.74 (s, 1H), 3.58 (t, J = 5.4 Hz,
1H), 2.92 (m, 2H), 2.63 (m, 1H), 2.24 (m, 1H), 1.50 (s, 3H), 1.34 (d, J = 10.4 Hz, 1H), 0.94 (s,
3H); 13C NMR (100 MHz, CDCl3) δ: 169.30, 165.82, 164.96, 163.53, 162.46, 161.05, 139.61,
19

132.47, 132.44, 131.87, 131.79, 130.25, 130.23, 129.78, 127.76, 127.68, 123.74, 123.71, 116.95,
116.73, 115.61, 115.40, 100.33, 100.32, 44.44, 42.31, 39.31, 31.60, 27.80, 26.02, 21.57. HR-MS
(ESI+): m/z calculated for C25H23F2N3S [M+H]+ 436.1659, found 436.1668.
4.1.4.11.
4-(2-(2-(3-(4-fluorobenzylidene)-6,6-dimethylbicyclo[3.1.1]heptan-2-
ylidene)hydrazinyl)thiazol-4-yl)phenol (5k)
Yield 69.8%; m.p. 180-183.2 C. 1H NMR (400 MHz, C5D5N) δ: 8.10 (d, J = 8.6 Hz, 2H), 7.83
(s, 1H), 7.58 (dd, J = 5.6, 8.6 Hz, 2H), 7.24 (t, J = 8.6 Hz, 1H), 7.12 (s, 1H), 3.81 (t, J = 5.6 Hz,
1H), 2.89 (m, 1H), 2.79 (m, 1H), 2.48 (m, 1H), 2.06 (m, 1H), 1.26 (s, 3H), 1.24 (d, J = 2.9, 1H),
0.86 (s, 3H); 13C NMR (100 MHz, C5D5N) δ: 170.76, 163.08, 160.63, 158.64, 151.78, 154.79,
134.00, 133.97, 132.23, 132.21, 131.88, 131.81, 127.75, 127.28, 125.94, 116.29, 115.61, 115.40,
100.74, 42.71, 41.05, 31.92, 28.24, 25.77, 21.34. HR-MS (ESI+): m/z calculated for C25H24FN3OS
[M+H]+ 434.1702, found 434.1540.
4.1.4.12. 2-(2-(3-(4-fluorobenzylidene)-6,6-dimethylbicyclo[3.1.1]heptan-2-ylidene)hydrazinyl)-4-
(4-methoxyphenyl)thiazole (5l)
1
Yield 81.5%; m.p. 213.6-215.7 C. H NMR (400 MHz, CDCl3) δ: 12.57 (s, 1H), 7.66 (d, J =
8.8 Hz, 2H), 7.50 (m, 3H), 7.11 (t, J = 8.6 Hz, 2H), 7.00 (d, J = 8.9 Hz, 2H), 6.59 (s, 1H), 3.85 (s,
3H), 3.63 (t, J = 5.4 Hz, 1H), 2.87 (m, 2H), 2.66 (m, 1H), 2.26 (m, 1H), 1.53 (s, 3H), 1.36 (d, J =
10.4 Hz, 1H), 0.96 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 169.31, 165.31, 140.52, 134.73,
133.86, 131.25, 130.31, 129.55, 129.50, 128.65, 127.33, 125.55, 100.94, 44.41, 42.25, 39.26,
31.67, 27.81, 25.99, 21.57. HR-MS (ESI+): m/z calculated for C26H26FN3OS [M+H]+ 448.1859,
found 448.1867.
4.1.4.13. 2-(2-(3-(4-methoxybenzylidene)-6,6-dimethylbicyclo[3.1.1]heptan-2-ylidene)hydrazinyl)-
20

4-phenylthiazole (5m)
1
Yield 70.3%; m.p. 180.8-184.9 C. H NMR (400 MHz, CDCl3) δ: 12.56 (s, 1H), 7.71 (d, J =
7.0 Hz, 2H), 7.46 (m, 6H), 6.94 (d, J = 8.7 Hz, 2H), 6.76 (s, 1H), 3.85 (s, 3H), 3.60 (t, J = 5.3 Hz,
1H), 2.93 (s, 2H), 2.63 (m, 1H), 2.24 (m, 1H), 1.51 (s, 3H), 1.35 (d, J = 10.3 Hz, 1H), 0.94 (s, 3H);
13C NMR (100 MHz, CDCl3) δ: 169.21, 166.14, 159.55, 140.52, 131.77, 130.60, 130.35, 129.62,
129.17, 128.37, 127.46, 125.63, 114.02, 100.69, 55.42, 44.46, 42.25, 39.47, 31.89, 27.97, 26.13,
21.65. HR-MS (ESI+): m/z calculated for C26H27N3OS [M+H]+ 430.1953, found 430.1974.
4.1.4.14. 4-(4-fluorophenyl)-2-(2-(3-(4-methoxybenzylidene)-6,6-dimethylbicyclo[3.1.1]heptan-2-
ylidene)hydrazinyl)thiazole (5n)
Yield 81.5%; m.p. 180.8-184.9 C.1H NMR (400 MHz, CDCl3) δ: 12.50 (s, 1H), 7.73 (dd, J =
8.8, 5.0 Hz, 2H), 7.51 (s, 1H), 7.49 (d, J = 8.8 Hz, 2H), 7.18 (t, J = 8.5 Hz, 2H), 6.95 (d, J = 8.7
Hz, 2H), 6.69 (s, 1H), 3.86 (s, 3H), 3.58 (t, J = 5.3 Hz, 1H), 2.94 (s, 2H), 2.64 (m, 1H), 2.26 (m,
1H), 1.51 (s, 3H), 1.36 (d, J = 10.4 Hz, 1H), 0.95 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 169.12,
166.24, 162.40, 159.51, 139.45, 131.71, 130.62, 129.06, 128.23, 127.75, 127.66, 123.80, 123.76,
116.90, 116.68, 113.96, 100.40, 100.39, 55.35, 44.36, 42.20, 39.39, 31.80, 27.89, 26.06, 21.57.
HR-MS (ESI+): m/z calculated for C26H27N3OS [M+H]+ 448.1859, found 448.1860.
4.1.4.15.
4-(2-(2-(3-(4-methoxybenzylidene)-6,6-dimethylbicyclo[3.1.1]heptan-2-
ylidene)hydrazinyl)thiazol-4-yl)phenol (5o)
1
Yield 81.5%; m.p. 205.6-207.6 C. H NMR (400 MHz, C5D5N) δ: 8.11 (d, J = 8.6 Hz, 2H),
7.94 (s, 1H), 7.63 (d, J = 8.7 Hz, 2H), 7.28 (d, J = 8.6 Hz, 2H), 7.10 (m, 3H), 3.80 (t, J = 5.6 Hz,
1H), 3.72 (s, 3H), 2.92 (m, 2H), 2.48 (m, 1H), 2.08 (m, 1H), 1.29 (s, 1H), 1.26 (s, 3H), 0.87 (s,
3H); 13C NMR (100 MHz, C5D5N) δ: 172.28, 160.62, 160.02, 156.70, 153.28, 133.01, 131.85,
21

131.59, 129.20, 128.87, 128.39, 117.67, 115.71, 102.07, 56.54, 44.13, 42.45, 41.16, 33.61, 29.73,
27.25, 22.78. HR-MS (ESI+): m/z calculated for C26H27N3O2S [M+H]+ 446.1902, found 446.1871.
4.1.4.16. 2-(2-(3-(4-methoxybenzylidene)-6,6-dimethylbicyclo[3.1.1]heptan-2-ylidene)hydrazinyl)-
4-(4-methoxyphenyl)thiazole (5p)
Yield 74.4%; m.p. 193.6-196.3 C.1H NMR (400 MHz, CDCl3) δ: 12.51 (s, 1H), 7.65 (d, J =
8.9 Hz, 2H), 7.50 (s, 1H), 7.49 (d, J = 8.9 Hz, 2H), 6.97 (dd, J = 15.5, 8.8 Hz, 4H), 6.57 (s, 1H),
3.85 (d, J = 7.0 Hz, 6H), 3.60 (t, J = 5.4 Hz, 1H), 2.93 (s, 2H), 2.64 (m, 1H), 2.25 (m, 1H), 1.51 (s,
3H), 1.36 (d, J = 10.4 Hz, 1H), 0.95 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 169.11, 165.89,
161.16, 159.54, 140.39, 131.77, 130.49, 129.22, 128.42, 127.19, 120.14, 115.01, 114.02, 98.50,
55.53, 55.43, 44.39, 42.24, 39.48, 31.91, 27.99, 26.14, 21.66. HR-MS (ESI+): m/z calculated for
C27H29N3O2S [M+H]+ 460.2059, found 460.2053.
4.1.4.17. 2-(2-(6,6-dimethyl-3-(4-methylbenzylidene)bicyclo[3.1.1]heptan-2-ylidene)hydrazinyl)-
4-phenylthiazole (5q)
1
Yield 76.3%; m.p. 163.6-168.5 C. H NMR (400 MHz, CDCl3) δ: 12.58 (s, 1H), 7.71 (d, J =
7.0 Hz, 2H), 7.52 (s, 1H), 7.41 (m, 5H), 7.22 (d, J = 7.9 Hz, 2H), 6.76 (s, 1H), 3.61 (t, J = 5.4 Hz,
1H), 2.94 (d, J = 2.8 Hz, 2H), 2.63 (m, 1H), 2.38 (s, 3H), 2.24 (m, 1H), 1.51 (s, 3H), 1.35 (d, J =
10.4 Hz, 1H), 0.95 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 169.35, 165.83, 140.89, 138.31,
133.64, 130.92, 130.36, 130.19, 129.79, 129.65, 129.29, 127.67, 125.69, 100.71, 44.47, 42.31,
39.48, 31.89, 27.97, 26.16, 21.69, 21.44. HR-MS (ESI+): m/z calculated for C26H27N3S [M+H]+
414.2004, found 414.2004.
4.1.4.18. 2-(2-(-6,6-dimethyl-3-(-4-methylbenzylidene)bicyclo[3.1.1]heptan-2-ylidene)hydrazinyl)-
4-(4-fluorophenyl)thiazole (5r)
22

1
Yield 79.0%; m.p. 177.0-180.8 C. H NMR (400 MHz, CDCl3) δ: 7.73 (dd, J = 8.8, 5.0 Hz,
2H), 7.53 (s, 1H), 7.42 (d, J = 8.0 Hz, 2H), 7.23 (d, J = 7.9 Hz, 2H), 7.18 (t, J = 8.6 Hz, 2H), 6.70
(s, 1H), 4.94 (s, 2H), 3.57 (t, J = 5.4 Hz, 1H), 2.95 (s, 2H), 2.64 (m, 1H), 2.39 (s, 3H), 2.25 (m,
1H), 1.51 (s, 3H), 1.36 (d, J = 10.4 Hz, 1H), 0.95 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 169.24,
165.76, 164.87, 162.37, 139.87, 138.24, 133.50, 130.88, 130.10, 129.62, 129.19, 127.76, 127.68,
124.06, 124.02, 116.85, 116.63, 100.58, 100.56, 44.29, 42.21, 39.36, 31.77, 27.85, 26.04, 21.58,
21.34. HR-MS (ESI+): m/z calculated for C26H26FN3S [M+H]+ 432.1910, found 432.1917.
4.1.4.19.
4-(2-(2-(6,6-dimethyl-3-(4-methylbenzylidene)bicyclo[3.1.1]heptan-2-
ylidene)hydrazinyl)thiazol-4-yl)phenol (5s)
1
Yield 61.0%; m.p. 159.3-161.6 C. H NMR (400 MHz, C5D5N) δ: 8.10 (d, J = 8.6 Hz, 2H),
7.94 (s, 1H), 7.57 (m, 3H), 7.28 (t, J = 7.9 Hz, 4H), 7.11 (s, 1H), 3.80 (t, J = 5.6 Hz, 1H), 2.90 (m,
13
2H), 2.47 (m, 1H), 2.27 (s, 3H), 2.06 (m, 1H), 1.27 (s, 4H), 1.25 (s, 4H), 0.86 (s, 3H); C NMR
(100 MHz, C5D5N) δ: 172.01, 159.81, 156.21, 153.01, 138.29, 132.71, 131.30, 130.59, 128.94,
128.40, 117.45, 101.86, 58.36, 43.88, 42.19, 40.85, 33.36, 29.46, 26.98, 22.53, 22.19. HR-MS
(ESI+): m/z calculated for C26H27N3OS [M+H]+ 430.1953, found 430.1935.
4.1.4.20. 2-(2-(6,6-dimethyl-3-(4-methylbenzylidene)bicyclo[3.1.1]heptan-2-ylidene)hydrazinyl)-
4-(4-fluorophenyl)thiazole (5t)
1
Yield 69.2%; m.p. 207.6-209.4 C. H NMR (400 MHz, CDCl3) δ: 12.53 (s, 1H), 7.66 (d, J =
8.8 Hz, 2H), 7.53 (s, 1H), 7.43 (d, J = 8.0 Hz, 2H), 7.23 (d, J = 7.9 Hz, 2H), 7.00 (d, J = 8.8 Hz,
2H), 6.57 (s, 1H), 3.85 (s, 3H), 3.62 (t, J = 5.4 Hz, 1H), 2.96 (s, 2H), 2.65 (dt, J = 10.9, 5.7 Hz,
1H), 2.40 (s, 3H), 2.25 (tt, J = 6.0, 3.2 Hz, 1H), 1.52 (s, 3H), 1.36 (d, J = 10.3 Hz, 1H), 0.96 (s,
3H); 13C NMR (100 MHz, CDCl3) δ: 169.21, 165.79, 161.19, 140.48, 138.29, 133.64, 130.88,
23

130.19, 129.79, 129.28, 127.21, 120.15, 115.04, 98.51, 55.55, 44.45, 42.30, 39.47, 31.89, 27.96,
26.15, 21.68, 21.44. HR-MS (ESI+): m/z calculated for C27H29N3OS [M+H]+ 444.2110, found
444.2116.
4.1.4.21. 2-(2-(3-(4-chlorobenzylidene)-6,6-dimethylbicyclo[3.1.1]heptan-2-ylidene)hydrazinyl)-
4-phenylthiazole (5u)
1
Yield 85.2%; m.p. 185.6-189.9 C. H NMR (400 MHz, CDCl3) δ: 12.68 (s, 1H), 7.74 (d, J =
6.9 Hz, 2H), 7.58 (m, 6H), 7.12 (t, J = 8.7 Hz, 2H), 6.75 (s, 1H), 3.64 (t, J = 5.4 Hz, 1H), 2.93 (m,
2H), 2.67 (m, 1H), 2.26 (m, 1H), 1.53 (s, 3H), 1.37 (d, J = 10.4 Hz, 1H), 0.97 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ: 169.42, 165.47, 140.75, 134.84, 133.97, 131.37, 131.34, 130.45, 129.67,
129.61, 128.75, 127.45, 125.67, 100.78, 44.53, 42.36, 39.37, 31.77, 27.91, 26.09, 21.67. HR-MS
(ESI+): m/z calculated for C25H24ClN3S [M+H]+ 434.1497, found 434.1538.
4.1.4.22. 2-(2-(3-(4-chlorobenzylidene)-6,6-dimethylbicyclo[3.1.1]heptan-2-ylidene)hydrazinyl)-
4-(4-fluorophenyl)thiazole (5v)
Yield 87.6%; m.p. 199.2-201.3℃.1H NMR (400 MHz, CDCl3) δ: 12.59 (s, 1H), 7.74 (dd, J =
8.8, 5.0 Hz, 2H), 7.50 (m, 3H), 7.20 (t, J = 8.5 Hz, 2H), 7.11 (t, J = 8.6 Hz, 2H), 6.69 (s, 1H),
3.61 (t, J = 5.4 Hz, 1H), 2.93 (m, 2H), 2.66 (m, 1H), 2.27 (m, 1H), 1.53 (s, 3H), 1.37 (d, J = 10.4
Hz, 1H), 0.96 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 169.30, 165.82, 164.96, 163.53, 162.46,
161.05, 139.61, 132.47, 132.44, 131.87, 131.79, 130.25, 130.23, 129.78, 127.76, 127.68, 123.74,
123.71, 116.95, 116.73, 115.61, 115.40, 100.33, 100.32, 44.44, 42.31, 39.31, 31.60, 27.80, 26.02,
21.57. HR-MS (ESI+): m/z calculated for C25H23ClFN3S [M+H]+ 452.1363, found 418.1753.
4.1.4.23. 4-(2-(2-(3-(4-chlorobenzylidene)-6,6-dimethylbicyclo[3.1.1]heptan-2-
ylidene)hydrazinyl)thiazol-4-yl)phenol (5w)
24

Yield 62.3%; m.p. 205.6-207.3℃.1H NMR (400 MHz, C5D5N) δ: 8.11 (d, J = 8.7 Hz, 2H), 7.82
(s, 1H), 0.85 (s, 3H), 7.54 (d, J = 8.7 Hz, 2H), 7.48 (d, J = 8.6 Hz, 2H), 7.28 (d, J = 8.7 Hz, 2H),
7.12 (s, 1H), 3.80 (t, J = 5.6 Hz, 1H), 2.89 (dd, J = 2.4, 17.1 Hz, 1H), 2.79 (m, 1H), 2.48 (m, 1H),
2.06 (m, 1H), 1.25 (s, 3H), 1.24 (s, 1H), 0.87 (s, 3H); 13C NMR (100 MHz, C5D5N) δ: 172.14,
160.11, 155.88, 153.26, 137.73, 134.75, 134.03, 132.87, 130.16, 129.21, 128.77, 127.17, 117.71,
102.19, 44.12, 42.45, 41.02, 33.44, 29.70, 27.18, 22.76. HR-MS (ESI+): m/z calculated for
C25H24ClN3OS [M+H]+ 450.1407, found 450.1370.
4.1.4.24. 2-(2-(3-(4-chlorobenzylidene)-6,6-dimethylbicyclo[3.1.1]heptan-2-ylidene)hydrazinyl)-
4-(4-methoxyphenyl)thiazole (5x)
Yield 76.1%; m.p. 205.6-207.3℃.1H NMR (400 MHz, C5D5N) δ: 8.08 (d, J = 8.8 Hz, 2H), 7.81
(s, 1H), 7.54 (d, J = 8.6 Hz, 2H), 7.48 (d, J = 8.6 Hz, 2H), 7.17 (s, 1H), 7.04 (d, J = 8.8 Hz, 2H),
3.81 (t, J = 5.6 Hz, 1H), 3.68 (s, 3H), 2.88 (m, 1H), 2.80 (m, 1H), 2.48 (m, 1H), 2.06 (m, 1H),
1.26 (s, 3H), 1.23 (s, 1H), 0.85 (s, 3H); 13C NMR (100 MHz, C5D5N) δ: 172.20, 160.96, 156.02,
152.80, 137.68, 134.72, 134.04, 132.87, 130.17, 130.14, 128.92, 127.21, 115.74, 103.10, 56.53,
44.15, 42.46, 41.01, 33.44, 29.70, 27.19, 22.78. HR-MS (ESI+): m/z calculated for C26H26ClN3OS
[M+H]+ 464.1563, found 464.1581.
4.2. Biology
4.2.1. MTT activity
Three cancer cell lines (Hela, SM7721, and CT-26 cells) were incubated in DMEM and RPMI-
1640 medium (Gibco, Milano, Italy) containing 10% FBS at 37℃ in a 5% CO2 humidified
incubator. Cells were grown in 96-well plates at a density of 5×103 cells/well and incubated
overnight at 37°C, 5% CO2. compounds 5a-5g were dissolved to 10 mM DMSO and diluted in
25

serum free medium to the final concentrations. After treatment with different concentrations of
compounds for 72 h, a volume of 10 μL MTT solution was added to each well and cells were
incubated for 4 h. Subsequently, the solution was removed and formazan crystals were dissolved
in 100 μL of DMSO. Absorbance was measured at 540 nm wavelength using a microplate.
4.2.2. Cell apoptosis analysis
Hela cells were seeded at 2 × 105/ wells and incubated overnight at 37 °C, 5% CO2. The
compounds were dissolved DMSO to 10 mM, and diluted in DMEM medium to the needed
concentrations (2.5, 5, or 10 μM). After treatment with different concentrations of compound 5g
for 72 h, cells were harvested and washed twice with cold phosphate-buffered saline (PBS). Then,
cells were stained with Annexin Ⅴ-APC/ PI solution at room temperature for 15 min in the dark.
Finally, stained cells were analyzed for apoptosis using flow cytometry (BD Accuri C6).
4.2.3. Cell cycle analysis
For flow cytometric analysis of DNA content, 5 × 105 Hela cells were seeded in six well plates.
After overnight incubation at 37 °C, cells were treated with different concentrations of compounds
5g (2.5, 5, or 10 μM) for 72 h. Then, cells were collected, centrifuged, and fixed overnight with
ice cold ethanol (70%) at -20 °C. Then, cells were treated with 100 μg/mL RNase A buffer for 30
min and stained with PI in the dark at 4 °C for 30 min. Finally, samples were analyzed for DNA
content by flow cytometry (BD FACS Canto II).
4.2.4. Mitochondrial membrane potential assay
Hela cells were treated with different concentration of compound 5g for 72 h in 6-well plates.
Then, cells were washed with cold PBS and incubated with 1 µg/mL of JC-1 (5,5',6,6'-tetrachloro-
26

1,1',3,3'-tetrethylbenzimidalylcarbocyanineiodide) working solution in culture medium without
FBS for 20 min at 37 °C in the dark. The red/green fluorescence intensity was determined by flow
cytometry (Becton-Dickinson FACS Calibur).
4.2.5. Analysis of reactive oxygen species
After treatment of 1×106 Hela cells with compound 5g (2.5, 5, or 10 μM) in 6-well plates for 72
h, the medium was discarded and cells were washed twice with PBS. Then, fresh FBS-free
medium, containing ROS indicator 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA, 10 μM)
was added to each well and cells were incubated for 20-30 min at 37 C in the dark. Next, cells
were visualized and imaged under an inverted fluorescence microscopy. For intracellular ROS
analysis, treated cells were collected by trypsinization and centrifugation, washed with PBS, and
stained with Carboxy-DCFDA (10 µM). The DCF fluorescent intensity was determined by flow
cytometry (Becton-Dickinson FACS Calibur).
4.2.5. Western blot analysis
For Western blot analysis, Hela cells (1×106 cells/ml) were treated with 0, 5, and 10 µM of
compound 5g for 72 h. After treatment, cells were washed twice with PBS, 200 L of lysis buffer
was added, and cells were resuspended. The lysate was centrifuged at 13000 rpm for 10 min at 4
C. Then, supernatants were collected and the protein concentrations were determined using a
BCA Protein Assay Kit (KeyGEN Biotech., China). Equal amounts of protein (50 g) were
separated by electrophoresis using a 12% SDS–PAGE gel and transferred to a nitrocellulose
membrane (Millipore, Billerica, MA, USA). Membranes were blocked with 5% nonfat milk in
Tris-buffered saline, containing 0.1% Tween-20 (TBST) at room temperature for 1 h, washed
27

three times with TBST and incubated overnight with monocloonal primary antibodies from Cell
Signaling Technology (Beverly, MA, USA) at 4 C. After being washed three times with TBST,
membranes were incubated with anti-mouse or anti-rabbit horseradish peroxidase (HRP)-
conjugated secondary antibodies (1:5000) for 2 h at room temperature and blocked with 5% nonfat
milk in TBST. To visualize proteins, membranes were washed three times with TBST and
exposed to an enhanced chemiluminescence (ECL) detection reagent. BioMax film was used for
Western blot analysis. Blots were quantified by laser scanning densitometry.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
This study was supported by the National Natural Science Foundation of China (No. 31470592),
Jiangsu Provincial Key Lab for the Chemistry and Utilization of Agro-Forest Biomass, and the
Priority Academic Program Development of Jiangsu Higher Education Institutions (Jiangsu,
China).
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