Novel camphor-based pyrimidine derivatives induced cancer cell death through a ROS-mediated mitochondrial apoptosis pathway

Article abstract of DOI:10.1039/c9ra05900h

A series of novel camphor-based pyrimidine derivatives (3a-3x) have been synthesized; their structures were determined by using conventional methods and compound 3f was further confirmed through single crystal XRD analysis. The cytotoxic activity of the target compounds against a panel of human normal (GES-1) and cancer cell lines (MDA-MB-231, RPMI-8226, A549) was evaluated by MTS assay. Here we found that compound 3f exhibited the strongest anti-tumor activity, comparable to that of etoposide, and had much lower cytotoxicity to normal GES-1 cells (IC₅₀ > 50 μM) than the reference drug (IC₅₀ = 8.89 μM). Subsequent mechanism studies in MDA-MB-231 cells revealed that compound 3f caused G0/G1 phase arrest and apoptosis in a dose dependent manner. Moreover, the loss of mitochondrial membrane potential and enhancement of cellular ROS levels were also observed upon 3f treatment, which indicated that 3f exerted cytotoxic activity by a ROS-mediated mitochondrial apoptosis pathway. This result was confirmed by a significant increase in the expression of pro-apoptotic proteins Bax, cytochrome C and caspase-3, and downregulation of anti-apoptosis protein Bcl-2. Overall, 3f can be adopted for further investigation in the development of antitumor agents based on natural products.

Full text of DOI:10.1039/c9ra05900h

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Novel camphor-based pyrimidine derivatives
induced cancer cell death through a ROS-mediated
Cite this: RSC Adv., 2019, 9, 29711
mitochondrial apoptosis pathway†
ac
b
Yan Zhang,a Yunyun Wang,a Yuxun Zhao,a Wen Gu,
Yongqiang Zhu
*
ac
*
and Shifa Wang
A series of novel camphor-based pyrimidine derivatives (3a–3x) have been synthesized; their structures
were determined by using conventional methods and compound 3f was further confirmed through
single crystal XRD analysis. The cytotoxic activity of the target compounds against a panel of human
normal (GES-1) and cancer cell lines (MDA-MB-231, RPMI-8226, A549) was evaluated by MTS assay.
Here we found that compound 3f exhibited the strongest anti-tumor activity, comparable to that of
etoposide, and had much lower cytotoxicity to normal GES-1 cells (IC50 > 50 mM) than the reference
drug (IC50 ¼ 8.89 mM). Subsequent mechanism studies in MDA-MB-231 cells revealed that compound 3f
caused G0/G1 phase arrest and apoptosis in a dose dependent manner. Moreover, the loss of
mitochondrial membrane potential and enhancement of cellular ROS levels were also observed upon 3f
treatment, which indicated that 3f exerted cytotoxic activity by a ROS-mediated mitochondrial apoptosis
pathway. This result was confirmed by a significant increase in the expression of pro-apoptotic proteins
Bax, cytochrome C and caspase-3, and downregulation of anti-apoptosis protein Bcl-2. Overall, 3f can
be adopted for further investigation in the development of antitumor agents based on natural products.
Received 30th July 2019
Accepted 15th September 2019
DOI: 10.1039/c9ra05900h
rsc.li/rsc-advances
death receptor pathway and the mitochondria apoptotic
pathway.5 In the latter case, an early trigger of the apoptotic
1. Introduction
pathway is an increase in the permeability of the mitochondria
membrane which is dened as sudden loss of the membrane
potential (DJm), causing release of apoptogenic factors
including cytochrome C.6 Under normal circumstances, cyto-
chrome C is bound to the cardiolipin from inner mitochondria
membrane, which is susceptible to reactive oxygen species
(ROS).7 Thus, the level of ROS could affect the dissociation of
cytochrome C from the mitochondrial membrane, and further
mediate the mitochondria apoptosis. Additionally, the release
of cytochrome C is controlled by the expression of Bcl-2 family
proteins that contains pro-apoptotic proteins (Bcl-2, Bcl-xL, etc.)
and anti-apoptotic proteins (Bax, Bak, etc.).8 Upon entering into
the cytosol from mitochondria, cytochrome C would activate
a cascade of caspases – a family of killer proteases, and even-
tually, result in cell death.9
The majority of the commercially available drugs are built on
heterocyclic scaffolds and these chemical fragments are
responsible for desired pharmacological activity.10 Pyrimidine,
a six membered heterocyclic ring with nitrogen atom, exhibits
various pharmacological activities such as anticancer,11–13 anti-
microbial,14,15 antiviral,16–18 anti-tubercular,19,20 anti-inamma-
tory,21,22 antimalarial,23,24 anti-diabetic25 and antipyretic.26
Among these diverse pyrimidine derivatives, amino-pyrimidine
plays a vital role in the therapeutic activities against cancers,
meanwhile, a large number of studies have been reported in
Cancer is one of leading causes of death globally. According to
the statistics released by the World Health Organization (WHO),
millions of new cancer patients are diagnosed each year
worldwide.1 Although there has been steady progress made in
reducing the mortality of cancer, the successful treatment of
cancer remains a challenge. Mostly, therapeutic effectiveness of
anti-cancer drugs is encumbered by some adverse side effects
including high toxicity and drug resistance. Therefore, it is
urgent to search for effective and safe agents for cancer
treatment.
Recently, it has become increasingly evident that cancer
therapy is mainly realized through the induction of apoptosis.2–4
Hence, developing reliable anticancer drugs, which can
successfully induce apoptosis, has become the new focus of
anti-tumor research. It's well-known that there are two major
semi-interdependent pathways contributing to apoptosis: the
aCollege of Chemical Engineering, Nanjing Forestry University, Nanjing, Jiangsu
210037, People's Republic of China. E-mail: wangshifa65@163.com; Fax: +86 25
85427812; Tel: +86 25 85427812
bJiangsu Chia Tai Fenghai Pharmaceutical Co. Ltd, Nanjing, 210046, P. R. China
cCo-Innovation Center of Efficient Processing and Utilization of Forest Resources,
Nanjing Forestry University, Nanjing, 210037, P. R. China
† Electronic supplementary information (ESI) available. CCDC 1838718. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c9ra05900h
This journal is © The Royal Society of Chemistry 2019
RSC Adv., 2019, 9, 29711–29720 | 29711

View Article Online
RSC Advances
Paper
recent years.27–30 Natural compounds are the most promising reacted respectively with guanidine hydrochloride under base-
source for the synthesis of biologically active substances in catalyzed cyclization conditions to obtain target compounds
medicinal chemistry. Camphor, as one of the important mon- 3a–3x in excellent yields. All desired pyrimidine analogs were
1
oterpenoids, is widespread in plants and has been used as puried by recrystallization and characterized by H NMR, 13C
chiral, enantiopure starting materials.31–33 Moreover, camphor- NMR and HRMS analysis. As exemplied with the 1H NMR
based derivatives exhibit a number of biological activities. For analysis
of
4-(4-chlorophenyl)-8,9,9-trimethyl-5,6,7,8-
instance, N-arylpiperazine camphor sulfonamides were discov- tetrahydro-5,8-methanoquinazolin-2-amine (3f), 1H NMR
ered as novel CXCR3 antagonists,34 camphor-based cyano- spectra revealed appearance of NH2 signal at d 5.27 ppm,
pyrrolidines were used as inhibitors for the treatment of type 2 meanwhile, two symmetric doublets at d 7.41–7.73 ppm were
diabetes mellitus,35 the camphane-based amides presented the corresponding to aromatic protons. The 13C NMR spectrum of
promising anti-mycobacterial activity.36 Aliphatic and alicyclic 3f displayed all characteristic peaks corresponding to molecular
camphor imines showed signicant activity against inuenza structure. For the pyrimidine ring, three aromatic carbons
virus H1N1,37 the platinum(II) complex possessing camphoryl adjacent to N atom at d 182.11, 161.75 and 154.87 ppm were
had antiproliferative activity.38 From the view of the chemical found, respectively. The presence of two high peaks at d 128.67
structure, camphor is a small molecule with chirality and and 129.63 ppm conrmed the para-substituted structure of p-
rigidity property which may bind to some active site, together chlorophenyl moiety. Besides, some representative spectra are
with its low cost and convenience to transform into syntheti- given in ESI.†
cally useful derivatives, it can serve as a good building block of
bioactive leading compounds.
2.2. Crystal structure of the compound 3f
In view of the above-mentioned facts, it was envisaged that
The single crystal of 3f was grown from chloroform solvent by
combining these two pharmacological scaffolds would
the slow evaporation at room temperature. The compound 3f
construct novel molecular templates which are likely to show
ꢁ
ꢀ
ꢀ
belongs to triclinic system with space group P1, a ¼ 6.9895(2) A,
interesting biological properties. Based on the previous studies
of anticancer drug in our research team, in this study, we
coupled amino-pyrimidine fragments to camphor scaffold by
a cyclization reaction to synthesize 24 novel camphor-based
pyrimidine derivatives. Besides, we evaluated the cytotoxic
activities of the target compounds by MTS in three different
human-derived cancer cell lines including breast cancer cells
(MDA-MB-231), multiple myeloma cells (RPMI-8226) and non-
small cell lung cancer cells (A549), and explored the possible
preliminary mechanism of the most potent compound 3f in
MDA-MB-231 cells.
ꢀ
ꢁ
ꢀ
ꢁ
b ¼ 8.6676(3) A, c ¼ 13.6291(5) A, a ¼ 76.606(2) , b ¼ 81.336(2) ,
ꢁ3
g ¼ 79.948(2) , Z ¼ 2, V ¼ 785.64(5) A . Molecular structure of
compounds 3f together with the atom labeling scheme is given
in Fig. 1, and the crystal data and structure renement details
are summarized in Table S1.† Crystallographic data of the
compound 3f was deposited with the Cambridge Crystallo-
graphic Data Centre (CCDC) No 1838718.
2.3. Biological evaluation of compounds assay
All target compounds were appraised for the anticancer activi-
ties against three human malignant cell lines (lung adenocar-
cinoma A549, breast cancer MDA-MB-231, multiple myeloma
RPMI-8226) by using MTS assay. The preliminary screening
results were presented graphically in Fig. 2.
2. Results and discussion
2.1. Synthesis and characterization
As depicted in Fig. 2, it is noticeable that most of compounds
displayed little cytotoxic effect against all tested cancer cell lines
at testing concentrations (50 mM and 10 mM), in contrast, only
several compounds showed strong growth inhibition (above
50% inhibition). Interestingly, for RPMI-8226 cell line, there is
no obvious difference in the growth inhibition with the decrease
of testing concentration from 50 mM to 10 mM, but this trend
With commercially available camphor 1 as the starting material,
a series of pyrimidine derivatives were synthesized via two
simple steps, following the route outlined in Scheme 1. Initially,
aldol condensation of the compound 1 with various substituted
aromatic aldehydes was carried out to prepare intermediates
2a–2x. Subsequently, without purication, intermediates 2a–2x
Scheme 1 Synthesis of compounds 3a–3x.
29712 | RSC Adv., 2019, 9, 29711–29720
Fig. 1 X-ray crystal structure of compound 3f.
This journal is © The Royal Society of Chemistry 2019

View Article Online
Paper
RSC Advances
Fig. 2 The cell viability of synthesized compounds against (a) MDA-MB-231 (b) RPMI-8226 (c) A549 cancer cell line at testing concentrations of
10 mM and 50 mM. The dashed line across the graph represents the threshold percentage for further IC50 determination. Cell viability was
measured by MTS assay.
was not observed in the other two cell lines. On the other hand, overall IC50 values (ranging from 5.00 to 8.64 mM) comparable to
considering the inhibitory selectivity of designed compounds etoposide. Meanwhile, introduction of nitro group on the
on selected cancer cell lines, these pyrimidine derivatives phenyl ring (3w) also resulted in good cytotoxicity with IC50
present a preferable MDA-MB-231 targeting structure because values similar to compound 3f. However, the substituents (3j–3r
the majority of tested analogs showed higher MDA-MB-231 and 3v) with the electron-donating group showed decreased or
inhibition compared with their respective activity on the other complete loss of activity. Besides, the replacement of phenyl
two cancer cell lines. In order to gain better insight into the role ring with pyridine ring (3s–3u) also largely weakened the activity
of different structural functionalities on cytotoxic effect, IC50 against three cancer cell lines. These results indicated that the
determination study was performed in triplicate and the results electron deciency of the phenyl ring is a favorable feature to
of the mean values were summarized in Table 1, in which eto- increase anticancer potential. In addition to substituting func-
poside was used as a positive control.
tional group, substitution position was also found to play a role
As illustrated with Table 1, the majority of compounds (3a– in altering the cytotoxicity of synthesized compounds. Close
3i) with halogen group showed improved inhibitory activity, observation of results from Table 1, para-substitution of phenyl
especially, of which compound 3f bearing chlorine atom was ring (3c, 3f, 3i, 3l) exerted the most potent activity compared to
identied as the most promising candidates due to their lowest their respective ortho- and meta-substituted analogs regardless
Table 1 Antiproliferative activities of compounds 3a–3x
IC50 (mM)
Compound
Ar
MBA-MD-231
RPMI-8226
A549
GES-1
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
3v
3w
3x
o-FC6H4
m-FC6H4
p-FC6H4
32.85 ꢁ 0.31
28.56 ꢁ 0.26
12.59 ꢁ 0.04
25.48 ꢁ 0.39
23.26 ꢁ 0.44
5.00 ꢁ 0.14
18.28 ꢁ 0.26
>50
8.66 ꢁ 0.17
26.20 ꢁ 0.40
20.00 ꢁ 0.09
15.68 ꢁ 0.14
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
8.89 ꢁ 0.70
24.35 ꢁ 0.01
12.63 ꢁ 0.02
16.93 ꢁ 0.50
10.63 ꢁ 0.09
7.65 ꢁ 0.21
23.32 ꢁ 0.03
>50
16.45 ꢁ 0.04
22.54 ꢁ 0.15
17.97 ꢁ 0.18
15.32 ꢁ 0.09
>50
9.30 ꢁ 0.34
11.42 ꢁ 0.18
10.36 ꢁ 0.35
8.64 ꢁ 0.24
>50
o-ClC6H4
m-ClC6H4
p-ClC6H4
o-BrC6H4
m-BrC6H4
p-BrC6H4
o-CH3C6H4
m-CH3C6H4
p-CH3C6H4
o-CH3OC6H4
m-CH3OC6H4
p-CH3OC6H4
o-HOC6H4
m-HOC6H4
p-HOC6H4
o-NC5H4
>50
18.94 ꢁ 0.08
>50
>50
13.86 ꢁ 0.09
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
16.28 ꢁ 0.26
13.98 ꢁ 0.16
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
m-NC5H4
p-NC5H4
p-N(CH3)2C6H4
p-NO2C6H4
p-C6H5
>50
>50
>50
6.21 ꢁ 0.14
29.88 ꢁ 0.16
3.97 ꢁ 0.45
9.58 ꢁ 0.11
10.84 ꢁ 0.09
>50
>50
Etoposide
—
1.92 ꢁ 0.17
10.89 ꢁ 0.79
This journal is © The Royal Society of Chemistry 2019
RSC Adv., 2019, 9, 29711–29720 | 29713

View Article Online
RSC Advances
Paper
to the substituting functional groups, except 3p which quantied by the Annexin V-APC/7-AAD double staining ow
dramatically increased the potency in two solid tumor cell lines cytometric assay, in which Annexin V may specically bind to
(A549 and MDA-MB-231). On top of this, meta-substituted the early apoptosis marker phosphatidylserine (PS) exposed on
derivatives (3b, 3e, 3k) performed better in reducing cancer the outside of the cell (green uorescence) and 7-AAD (7-amino-
cells viability than that of ortho-substitutions (3a, 3d, 3j), but actinomycin D) can stain DNA and enter only dead and late
the trend was reversed when only meta-brominated compound apoptotic cells (red uorescence). As shown in dot plot (Fig. 4),
3h was being considered. According to above ndings, we can a high percentage of the control (untreated) MDA-MB-231 cells
conclude that the tested pyrimidine derivatives with phenyl ring were negative for both Annexin V and 7-AAD (97.4%). Aer the
bearing electron withdrawing group at p-position have a posi- completion of 72 h incubation with 2.5 mM compound 3f, a little
tive inuence on antitumor activity. Furthermore, all amount of cell lines (3.78%) were in early stage of the apoptosis,
compounds were examined for possible toxicity against GES-1 and 5.69% of cancer cells were appeared in late apoptosis. A
(normal human gastric epithelial cell line). We found that similar pattern was observed in MDA-MB-231 cells treated with
none of compounds showed signicant toxicity toward GES-1, 5 mM of compound 3f as well. Exposure of MDA-MB-231 cells to
whereas the reference compound (etoposide) exhibited severe 10 mM of 3f resulted in signicant increase in percentage of late
cytotoxicity (IC50 ¼ 8.89 mM).
apoptotic cells by 38.4%, in contrast, caused no changes in the
levels of early apoptotic cells. In a word, the total (early and late)
ratios of apoptosis enhanced from 9.47% to 57.9%, indicating
that anti-proliferative effect of 3f depended on apoptosis.
2.4. Cell cycle assay
The cytotoxicity results, which were discussed earlier, suggest 3f
is the most potent compound, especially in MDA-MB-231 cells.
The perturbation of cell cycle is closely associated to the cyto-
toxicity, to determine whether 3f might trigger the arrest of cell
cycle, a ow-cytometric analysis of MDA-MB-231 cells stained
with propidium iodide (PI) was conducted. Aer the cells were
treated with various concentrations of 3f (2.5, 5 and 10 mM) for
72 h, signicant alternations in the cell phases were observed.
In Fig. 3, compared with 56.86% in the control, the percentage
of cells in the G0/G1 phase aer treatment with 2.5, 5, 10 mM of
compound 3f steadily increased to 64.89%, 78.02%, and
81.72%, accompanied by decreasing the proportion of cells at
G2/M and S phase of cell cycle. Therefore, these results illus-
trated that 3f induced an obvious G0/G1 arrest in a concentra-
tion-dependent manner.
2.6. Intracellular ROS level
Reactive oxygen species (ROS) are recognized as a pivotal factor
in intracellular signaling and regulation. Cell growth and
proliferation are encouraged with a condition of a modest rise
in intracellular ROS, on the other hand, the elevation of ROS is
consequent to disruption of intracellular redox homeostasis
and inhibition of biomacromolecules function, resulting in
promoting cell death. Since cancer cells are more sensitive to
enhanced intracellular ROS than the nontransformed cells, the
regulation of intracellular ROS levels could be exploited for
selective therapeutic approach of cancer. To determine whether
the anticancer effect of active compound 3f is involved in ROS-
mediated therapy, the level of ROS in MDA-MB-231 cells treated
with compound 3f at 10 mM concentration increased by almost
12 times (63.4%) in comparison to the control group (5.13%)
(Fig. 5). However, the promoted intracellular ROS accumulation
induced by 3f was distinctly suppressed by N-acetyl cysteine
(NAC), which is one of the ROS scavengers. On the basis of these
results, it clearly indicated that compound 3f can modulate the
2.5. Apoptosis analysis
To get further insight into the mechanism of cell death induced
by compound 3f, apoptosis in treated MDA-MB-231 cells was
Fig. 3 Influence of compound 3f on cell cycle distribution in MDA-
MB-231 cells. (a) Cells were treated with different concentrations (2.5,
5 and 10 mM) of compound 3f for 72 h, which then were stained with Fig. 4 Compound 3f induced apoptosis in MDA-MB-231 cells. (a)
propidium iodide (PI), and the DNA content were analyzed by flow Flow cytometry analysis of MDA-MB-231 cells were treated with
cytometry. (b) Percentages of cells in various phases of the cell cycle. compound 3f (2.5, 5 and 10 mM) for 72 h, respectively, and stained with
These data are from three independent experiments. *p < 0.05; **p < Annexin V-APC/7-AAD. (b) Quantitative analysis of apoptotic cells in
0.01, verse the negative control.
histogram. These data are from three independent experiments.
29714 | RSC Adv., 2019, 9, 29711–29720
This journal is © The Royal Society of Chemistry 2019

View Article Online
Paper
RSC Advances
percentage of cells with a collapsed DJm (from 3.9% to 10.4%,
33.6%, 53.2%). Based on such study, we concluded that the
compound 3f could mediate perturbation of the mitochondrial
membrane integrity with
a
consequent concentration-
dependent collapse of DJm, and its antiproliferative activities
might be associated with mitochondrial apoptosis pathway.
2.8. Western blot analysis
Fig. 5 Effect of compound 3f on ROS production. (a) Analysis of the
ROS levels by flow cytometry after the MDA-MB-231 cells were treated
with compound 3f at indicated concentrations or in presence of NAC
Aforementioned ndings indicated that compound 3f gave rise
to a preliminary disruption of the mitochondrial trans-
for 72 h and stained with DCFH-DA. (b) Quantification of the flow membrane potential (DJm), which would be the consequence
cytometric results. These data are from three independent experi-
ments. *p < 0.05 verse the negative control.
of mitochondrial membrane permeabilization. This per-
meabilization is chiey mediated and controlled by Bcl-2 family
members which mainly includes pro-survival group (Bcl-2, Bcl-
xL, etc.), pro-apoptotic group (Bax, Bak, etc.) and apoptosis
initiator group (Bid, Bim, etc.). The ratio of anti- to pro-
apoptotic molecules constitutes a rheostat that sets the
generation of intracellular ROS, and at least partly, explain the
anticancer activity of compound 3f.
threshold of susceptibility to apoptosis for the intrinsic
pathway. In the presence of apoptotic stimuli, the expression of
2.7. Mitochondrial membrane potential (MMP) analysis
pro-apoptotic proteins increased and the release of pro-survival
It's well-known that there are two major semi-interdependent
proteins is inhibited, which leads to cytochrome C release from
routes contributing to apoptosis. One is initiated by ligand
the intermembrane space of mitochondria to the cytoplasm,
binding to the death receptors at the cell surface (extrinsic
likely by forming a channel in the outer mitochondrial
apoptotic pathway), the other involves mitochondria (intrinsic
membrane. The released cytochrome C binds to apoptosis
apoptotic pathway). In the latter case, an early trigger of the
protease-activating factor 1 (Apaf-1) and procaspase-9, gener-
apoptotic pathway is an increase in the permeability of the outer
ating apoptosome. Consecutively, the executioner caspase-3 is
membrane, which leads to a preliminary reduction of the
mitochondrial membrane potential (DJm). Hence, in order to
understand the mechanism of apoptosis, we used JC-1 as
a uorescent probe, a liquid crystal-forming cationic dye which
recruited to the apoptosome, where it is activated by the resi-
dent caspase-9. Then, caspase-3 cleaves key substrates in the
cell to produce many of the cellular and biochemical events of
apoptosis. In the present study, several apoptosis associated
can reversibly change its color from red to green as membrane
molecules, such as Bcl-2, Bax, cytochrome C, and caspase-3,
potential decreases, to observe the effect of compound 3f on
were evaluated. Aer treatment with compound 3f (2.5 mM, 5
DJm. The results are presented in Fig. 6, in the control, the
mM and 10 mM) for 72 h, the levels of apoptosis inducing
percentage of green uorescent cells is 3.9%, whereas MDA-MB-
231 cells were exposed to 3f at different concentration (2.5 mM, 5
mM and 10 mM) for 72 h, there is a gradual increase of the
proteins in MDA-MB-231 cells were tested via western blot
assays. As depicted in Fig. 7, the expression of anti-apoptosis
protein Bcl-2 was downregulated dose-dependently, whereas
the expression of pro-apoptotic proteins Bax was up-regulated.
Besides, the upregulation of cytochrome C and caspase-3 were
Fig. 6 Determination of mitochondrial membrane potential in MDA- Fig. 7 Expression changes of apoptosis related proteins. (a) Western
MB-231 cells. (a) Cells were treated with compound 3f (2.5, 5 and 10 blot analysis of Bcl-2, Bax, cytochrome C, caspase-3 in MDA-MB-231
mM) for 72 h and observed the changes in mitochondrial membrane cells treated with compound 3f (2.5, 5 and 10 mM) for 72 h, b-actin was
potential by flow cytometry. (b) Bar graph represents the percentage used as the internal reference. (b) Statistical analysis of protein
distribution of cells with red fluorescence or green fluorescence. expression levels. These data are from three independent experiments.
These data are from three independent experiments.
*p < 0.05 verse the negative control.
This journal is © The Royal Society of Chemistry 2019
RSC Adv., 2019, 9, 29711–29720 | 29715

View Article Online
RSC Advances
Paper
also observed compared with the control. Thus, these results
conrmed that compound 3f can induce mitochondria-related
apoptosis.
3.1.2.2. 4-(3-Fluorophenyl)-8,9,9-trimethyl-5,6,7,8-tetrahydro-
5,8-methanoquinazolin-2-amine (3b). Yield 88%; mp 155.4–
155.9 ꢀC. 1H NMR (400 MHz, CDCl3) d: 7.49–7.55 (m, 2H), 7.39–
7.44 (m, 1H), 7.09–7.14 (m, 1H), 5.31 (s, 2H), 3.06 (d, J ¼ 4 Hz,
1H), 2.18–2.23 (m, 1H), 1.86–1.96 (m, 1H), 1.32–1.37 (m, 2H),
1.25 (s, 3H), 0.99 (s, 3H), 0.63 (s, 3H); 13C NMR (100 MHz, CDCl3)
d: 182.24, 164.11, 161.75, 154.70, 140.38, 129.98, 125.48, 123.96,
116.18, 115.33, 55.96, 54.25, 49.65, 31.76, 25.95, 19.96, 18.98,
10.03; HR-MS (ESI+): m/z calculated for C18H20FN3 [M + H]+
298.1720, found 298.1717.
3.1.2.3. 4-(4-Fluorophenyl)-8,9,9-trimethyl-5,6,7,8-tetrahydro-
5,8-methanoquinazolin-2-amine (3c). Yield 91%; mp 197.0–
197.9 ꢀC. 1H NMR (400 MHz, CDCl3) d: 7.75–7.79 (m, 2H), 7.14 (t,
J ¼ 8 Hz, 2H), 5.27 (s, 2H), 3.03 (d, J ¼ 3.9 Hz, 1H), 2.16–2.22 (m,
1H), 1.86–1.95 (m, 1H), 1.32–1.36 (m, 2H), 1.25 (s, 3H), 0.99 (s,
3H), 0.63 (s, 3H); 13C NMR (100 MHz, CDCl3) d: 181.98, 164.66,
161.74, 155.07, 134.17, 130.24, 125.17, 115.32, 55.97, 54.21,
49.70, 31.76, 25.97, 19.95, 19.01, 10.03; HR-MS (ESI+): m/z
calculated for C18H20FN3 [M + H]+ 298.1720, found 298.1717.
3.1.2.4. 4-(2-Chlorophenyl)-8,9,9-trimethyl-5,6,7,8-tetrahydro-
5,8-methanoquinazolin-2-amine (3d). Yield 92%; mp 185.5–
185.8 ꢀC. 1H NMR (400 MHz, CDCl3) d: 7.45–7.48 (m, 3H), 7.31–
7.36 (m, 3H), 5.20 (s, 2H), 2.69 (d, J ¼ 4 Hz, 1H), 1.97–2.04 (m,
1H), 1.84–1.87 (m, 1H), 1.30–1.33 (m, 1H), 1.27 (s, 3H), 1.14–1.20
(m, 1H), 0.97 (s, 3H), 0.74 (s, 3H); 13C NMR (100 MHz, CDCl3) d:
181.27, 161.59, 155.60, 137.18, 132.15, 130.30, 129.82, 129.73,
127.53, 126.81, 56.14, 54.66, 48.82, 31.86, 25.87, 19.91, 18.93,
9.94; HR-MS (ESI+): m/z calculated for C18H20ClN3 [M + H]+
314.1424, found 314.1420.
3. Experimental
3.1. Chemistry
Starting materials and chemical reagents were purchased from
Aladdin or Energy, and used without further purication.
Solvents were purchased from common commercial suppliers.
Thin-layer chromatography (TLC) was utilized to monitor the
reaction progress, and components were visualized under
254 nm illumination. The purity of target compounds evaluated
biologically was greater than 98% by America Agilent 1260 high
1
performance liquid chromatography (HPLC) analysis. H (400
MHz) and 13C NMR (100 MHz) spectra were recorded on Bruker
Advance spectrometer in CDCl3, DMSO-d6 solutions. The proton
and carbon chemical shis (ppm) are given with respect to
tetramethylsilane (TMS). High resolution mass spectra (HRMS)
were obtained by using Agilent 6540 high-resolution mass
spectrometer. Melting points were measured on Optimelt-
MPA100 melting point apparatus (uncorrected). X-ray diffrac-
tion analysis was determined on a Bruker D8 Venture area
diffractometer.
3.1.1. Synthesis of compounds 2a–2x. In a 50 ml dried
three-necked ask equipped with a stirrer, thermometer and
condenser, camphor (8 mmol), aromatic aldehyde (10 mmol),
tert-butanol (35 ml), and potassium tert-butoxide (8 mmol) were
added in sequence. The mixture was reuxed for 6–12 h until
the conversion ratio of camphor exceeded 95% (monitored by
TLC). Aer completion of reaction, the reaction mixture was
evaporated under vacuum and extracted three times with 20 ml
ethyl acetate, then merged organic layers were washed, dried
and evaporated to afford the concentrated solution. Aer add-
ing a small amount of ethanol, the intermediates 2a–2x were
precipitated out, and obtained by ltration.
3.1.2. Synthesis of target compounds 3a–3x. Guanidine
hydrochloride (8 mmol), intermediates 2a–2x (2 mmol), potas-
sium tert-butoxide (12 mmol) were dissolved in tert-butanol (35
ml). The reaction mixture was reuxed for 24–48 h and moni-
tored by TLC until the spot of 2a–2x in silica gel plate almost
disappeared. Aer completion of reaction, the reaction mixture
was evaporated under vacuum and extracted three times with
20 ml ethyl acetate, then merged organic layers were washed,
dried and concentrated. Finally, the crude product was recrys-
tallized from methanol and ltered to give target compound 3a–
3x (the purity was above 98%).
3.1.2.5. 4-(3-Chlorophenyl)-8,9,9-trimethyl-5,6,7,8-tetrahydro-
5,8-methanoquinazolin-2-amine (3e). Yield 87%; mp 162.8–
1
ꢀ
163.2 C. H NMR (400 MHz, CDCl3) d: 7.78 (s, 1H), 7.61–7.64
(m, 1H), 7.38–7.39 (m, 1H), 5.25 (s, 2H), 3.05 (d, J ¼ 4 Hz, 1H),
2.17–2.23 (m, 1H), 1.86–1.96 (m, 1H), 1.34 (t, J ¼ 8 Hz, 2H), 1.25
(s, 3H), 0.99 (s, 3H), 0.63 (s, 3H); 13C NMR (100 MHz, CDCl3) d:
182.24, 161.73, 154.59, 139.87, 134.55, 129.66, 129.22, 128.39,
126.42, 125.56, 55.99, 54.25, 49.61, 31.73, 25.96, 19.96, 18.98,
10.02; HR-MS (ESI+): m/z calculated for C18H20ClN3 [M + H]+
314.1424, found 314.1423.
3.1.2.6. 4-(4-Chlorophenyl)-8,9,9-trimethyl-5,6,7,8-tetrahydro-
5,8-methanoquinazolin-2-amine (3f). Yield 94%; mp 192.3–
1
192.6 ꢀC. H NMR (400 MHz, CDCl3) d: 7.71 (d, J ¼ 12 Hz, 2H),
7.42 (d, J ¼ 12 Hz, 2H), 5.27 (s, 2H), 3.0 (d, J ¼ 4 Hz, 1H), 2.16–
2.22 (m, 1H), 1.86–1.96 (m, 1H), 1.29–1.35 (m, 2H), 1.25 (s, 3H),
0.99 (s, 3H), 0.62 (s, 3H); 13C NMR (100 MHz, CDCl3) d: 182.11,
161.75, 154.87, 136.48, 135.29, 129.63, 128.67, 125.34, 77.36,
77.04, 76.72, 55.98, 54.22, 49.70, 31.74, 25.96, 19.95, 19.00,
10.03; HR-MS (ESI+): m/z calculated for C18H20ClN3 [M + H]+
314.1424, found 314.1420.
3.1.2.1. 4-(2-Fluorophenyl)-8,9,9-trimethyl-5,6,7,8-tetrahydro-
5,8-methanoquinazolin-2-amine (3a). Yield 92%; mp 166.2–
166.4 ꢀC. 1H NMR (400 MHz, CDCl3) d: 7.57 (s, 1H), 7.40 (s, 1H),
7.13 (s, 2H), 5.39 (s, 2H), 2.79 (s, 1H), 2.28 (s, 1H), 2.18 (s, 1H),
1.90 (s, 1H), 1.32 (s, 1H), 1.25 (s, 3H), 0.97 (s, 3H), 0.66 (s, 3H);
13C NMR (100 MHz, CDCl3) d: 181.40, 161.89, 158.46, 151.97,
131.02, 130.70, 127.79, 125.87, 124.34, 116.03, 56.00, 54.64,
49.55, 31.78, 25.77, 19.83, 18.97, 10.04; HR-MS (ESI+): m/z
calculated for C18H20FN3 [M + H]+ 298.1720, found 298.1717.
3.1.2.7. 4-(2-Bromophenyl)-8,9,9-trimethyl-5,6,7,8-tetrahydro-
5,8-methanoquinazolin-2-amine (3g). Yield 88%; mp 172.5–
172.9 ꢀC. 1H NMR (400 MHz, CDCl3) d: 7.75–7.77 (m, 2H), 7.41–
7.48 (m, 3H), 5.28 (s, 2H), 3.07 (d, J ¼ 4 Hz, 1H), 2.14–2.22 (m,
1H), 1.87–1.94 (m, 1H), 1.32–1.39 (m, 2H), 1.25 (s, 3H), 0.98 (s,
3H), 0.63 (s, 3H); 13C NMR (100 MHz, CDCl3) d: 181.86, 161.79,
156.18, 138.05, 129.18, 128.44, 128.30, 125.48, 55.97, 54.23,
29716 | RSC Adv., 2019, 9, 29711–29720
This journal is © The Royal Society of Chemistry 2019

View Article Online
Paper
RSC Advances
49.67, 31.80, 26.05, 19.97, 19.04, 10.06; HR-MS (ESI+): m/z 7.03 (t, J ¼ 8 Hz, 1H), 6.95 (d, J ¼ 8 Hz, 1H), 5.32 (s, 2H), 3.78 (s,
calculated for C18H20BrN3 [M + H]+ 358.0919, found 358.0911.
3H), 2.63 (d, J ¼ 4 Hz, 1H), 1.97–2.03 (m, 1H), 1.81–1.87 (m, 1H),
3.1.2.8. 4-(3-Bromophenyl)-8,9,9-trimethyl-5,6,7,8-tetrahydro- 1.27–1.33 (m, 1H), 1.23 (s, 3H), 0.94 (s, 3H), 0.65 (s, 3H); 13C
5,8-methanoquinazolin-2-amine (3h). Yield 83%; mp 187.2– NMR (100 MHz, CDCl3) d: 180.39, 161.87, 156.61, 154.90,
1
ꢀ
187.8 C. H NMR (400 MHz, CDCl3) d: 7.94 (t, J ¼ 4 Hz, 1H), 130.60, 130.08, 128.08, 127.44, 120.80, 111.00, 55.91, 55.17,
7.68, 7.65–7.68 (m, 1H), 7.53–7.56 (m, 1H), 7.32 (t, J ¼ 8 Hz, 1H), 54.47, 49.71, 32.01, 25.65, 19.85, 19.07, 10.07; HR-MS (ESI+): m/z
5.24 (s, 2H), 3.04 (d, J ¼ 4 Hz, 1H), 2.17–2.23 (m, 1H), 1.93, 1.91, calculated for C19H23N3O [M + H]+ 310.1919, found 310.1915.
1.86–1.96 (m, 1H), 1.29–1.36 (m, 2H), 1.25 (s, 3H), 0.99 (s, 3H),
3.1.2.14. 4-(3-Methoxyphenyl)-8,9,9-trimethyl-5,6,7,8-
0.63 (s, 3H); 13C NMR (100 MHz, CDCl3) d: 182.24, 161.72, tetrahydro-5,8-methanoquinazolin-2-amine (3n). Yield 90%; mp
154.48, 140.11, 132.14, 131.31, 129.91, 126.87, 125.57, 122.72, 183.2–183.4 ꢀC. 1H NMR (400 MHz, CDCl3) d: 7.31–7.38 (m, 3H),
56.00, 54.25, 49.60, 31.73, 25.96, 19.97, 18.99, 10.02; HR-MS 6.95–6.98 (m, 1H), 5.27 (s, 2H), 3.87 (s, 3H), 3.07 (d, J ¼ 4 Hz,
(ESI+): m/z calculated for C18H20BrN3 [M + H]+ 358.0919, found 1H), 2.15–2.21 (m, 1H), 1.87–1.92 (m, 1H), 1.32–1.36 (m, 2H),
358.0911.
1.25 (s, 3H), 0.98 (s, 3H), 0.63 (s, 3H); 13C NMR (100 MHz, CDCl3)
3.1.2.9. 4-(4-Bromophenyl)-8,9,9-trimethyl-5,6,7,8-tetrahydro- d: 181.91, 161.74, 159.72, 155.99, 139.43, 129.40, 125.52, 120.78,
5,8-methanoquinazolin-2-amine (3i). Yield 89%; mp 191.4– 115.07, 113.58, 55.94, 55.32, 54.24, 49.74, 31.80, 25.99, 19.97,
1
191.8 C. H NMR (400 MHz, CDCl3) d: 7.65 (d, J ¼ 8 Hz, 1H), 19.02, 10.05; HR-MS (ESI+): m/z calculated for C19H23N3O [M +
ꢀ
7.58 (d, J ¼ 8 Hz, 1H), 5.25 (s, 2H), 3.02 (d, J ¼ 2 Hz, 1H), 2.16– H]+ 310.1919, found 310.1919.
2.22 (m, 1H), 1.91 (t, J ¼ 12 Hz, 1H), 1.28–1.35 (m, 2H), 1.25 (s,
3.1.2.15. 4-(4-Methoxyphenyl)-8,9,9-trimethyl-5,6,7,8-
3H), 0.99 (s, 3H), 0.62 (s, 3H); 13C NMR (100 MHz, CDCl3) d: tetrahydro-5,8-methanoquinazolin-2-amine (3o). Yield 93%; mp
182.15, 161.73, 154.90, 136.91, 131.64, 129.90, 125.37, 123.64, 131.7–131.9 ꢀC. 1H NMR (400 MHz, CDCl3) d: 7.75 (d, J ¼ 8 Hz,
77.36, 77.04, 76.73, 55.99, 54.23, 49.68, 31.73, 25.95, 19.95, 2H), 6.98 (d, J ¼ 8 Hz, 2H), 5.27 (s, 2H), 3.85 (s, 3H), 3.06 (d, J ¼
19.01, 10.04; HR-MS (ESI+): m/z calculated for C18H20BrN3 [M + 4 Hz, 1H), 2.14–2.21 (m, 1H), 1.86–1.94 (m, 1H), 1.31–1.35 (m,
H]+ 358.0919, found 358.0913.
2H), 1.24 (s, 3H), 0.98 (s, 3H), 0.63 (s, 3H); 13C NMR (100 MHz,
3.1.2.10. 8,9,9-Trimethyl-4-(o-tolyl)-5,6,7,8-tetrahydro-5,8- ꢀ
CDCl3) d: 181.59, 161.72, 160.54, 155.82, 130.61, 129.76, 124.81,
methanoquinazolin-2-amine (3j). Yield 88%; mp 171.9–172.5 C. 113.85, 55.91, 55.33, 54.12, 49.83, 31.83, 25.97, 19.96, 19.06,
1H NMR (400 MHz, CDCl3) d: 7.20–7.30 (m, 3H), 7.15 (d, J ¼ 10.08; HR-MS (ESI+): m/z calculated for C19H23N3O [M + H]+
8 Hz, 1H), 5.39 (s, 2H), 2.61 (d, J ¼ 4 Hz, 1H), 2.27 (s, 3H), 1.98– 310.1919, found 310.1916.
2.05 (m, 1H), 1.82–1.88 (m, 1H), 1.27–1.33 (m, 1H), 1.24 (s, 3H),
3.1.2.16. 2-(2-Amino-8,9,9-trimethyl-5,6,7,8-tetrahydro-5,8-
1.12–1.18 (m, 1H), 0.93 (s, 3H), 0.70 (s, 3H); 13C NMR (100 MHz, methanoquinazolin-4-yl)phenol (3p). Yield 89%; mp 167.9–
1
ꢀ
CDCl3) d: 181.13, 161.53, 158.38, 137.51, 135.64, 130.53, 128.41, 168.3 C. H NMR (400 MHz, CDCl3) d: 7.65 (d, J ¼ 8 Hz, 1H),
126.91, 125.55, 55.99, 54.61, 48.71, 31.76, 26.26, 19.97, 19.87, 7.31 (t, J ¼ 8 Hz, 1H), 7.0 (d, J ¼ 8 Hz, 1H), 6.9 (d, J ¼ 16 Hz, 1H),
18.94, 9.97; HR-MS (ESI+): m/z calculated for C19H23N3 [M + H]+ 5.21 (s, 2H), 3.27 (d, J ¼ 4 Hz, 1H), 2.21–2.29 (m, 1H), 1.90–1.96
294.1970, found 294.1967.
3.1.2.11. 8,9,9-Trimethyl-4-(m-tolyl)-5,6,7,8-tetrahydro-5,8-
(m, 1H), 1.31–1.43 (m, 2H), 1.24 (s, 3H), 1.00 (s, 3H), 0.63 (s, 3H);
13C NMR (100 MHz, CDCl3): d 183.33, 159.85, 159.01, 155.66,
methanoquinazolin-2-amine (3k). Yield 86%; mp 156.8–157.3 ꢀC. 131.66, 129.11, 123.93, 119.21, 118.64, 118.22, 55.72, 54.22,
1H NMR (400 MHz, CDCl3) d: 7.60 (s, 1H), 7.50–7.52 (d, J ¼ 50.86, 31.88, 25.35, 19.94, 18.99, 10.03; HR-MS (ESI+): m/z
7.6 Hz, 1H), 7.34 (t, J ¼ 8 Hz, 1H), 7.23 (d, J ¼ 8 Hz, 1H), 5.24 (s, calculated for C18H21N3O [M + H]+ 296.1763, found 296.1758.
2H), 3.05 (d, J ¼ 4 Hz, 1H), 2.42 (s, 3H), 2.15–2.21 (m, 1H), 1.87–
3.1.2.17. 3-(2-Amino-8,9,9-trimethyl-5,6,7,8-tetrahydro-5,8-
1.94 (m, 1H), 1.32–1.36 (m, 2H), 1.25 (s, 3H), 0.98 (s, 3H), 0.63 (s, methanoquinazolin-4-yl)phenol (3q). Yield 87%; mp 262.0–
3H); 13C NMR (100 MHz, CDCl3) d: 181.76, 161.75, 156.39, 262.8 ꢀC. 1H NMR (400 MHz, DMSO-d6) d: 9.32 (s, 1H), 6.92–7.04
138.17, 137.98, 129.97, 128.87, 128.25, 125.52, 125.46, 55.96, (m, 3H), 6.59 (d, J ¼ 8 Hz, 1H), 6.10 (s, 2H), 2.78 (d, J ¼ 4 Hz, 1H),
54.23, 49.69, 31.80, 26.04, 21.57, 19.97, 19.04, 10.06; HR-MS 1.89–1.94 (m, 1H), 1.60–1.65 (m, 1H), 0.94–1.03 (m, 2H), 0.90 (s,
(ESI+): m/z calculated for C19H23N3 [M + H]+ 294.1970, found 3H), 0.70 (s, 3H), 0.31 (s, 3H); 13C NMR (100 MHz, DMSO-d6):
294.1967.
3.1.2.12. 8,9,9-Trimethyl-4-(p-tolyl)-5,6,7,8-tetrahydro-5,8-
d 180.89, 161.97, 157.31, 154.84, 139.11, 129.28, 123.15, 118.75,
116.10, 114.81, 55.00, 53.62, 49.26, 31.62, 25.84, 19.65, 18.68,
methanoquinazolin-2-amine (3l). Yield 90%; mp 197.5–197.9 C. 10.19; HR-MS (ESI+): m/z calculated for C18H21N3O [M + H]+
ꢀ
1H NMR (400 MHz, CDCl3) d: 7.67 (d, J ¼ 8 Hz, 2H), 7.26 (d, J ¼ 296.1763, found 296.1761.
8 Hz, 2H), 5.24 (s, 2H), 3.06 (d, J ¼ 4 Hz, 1H), 2.40 (s, 3H), 2.14–
3.1.2.18. 4-(2-Amino-8,9,9-trimethyl-5,6,7,8-tetrahydro-5,8-
2.21 (m, 1H), 1.86–1.92 (m, 1H), 1.32–1.35 (m, 2H), 1.24 (s, 3H), methanoquinazolin-4-yl)phenol (3r). Yield 89%; mp 264.1–
0.98 (s, 3H), 0.62 (s, 3H); 13C NMR (100 MHz, CDCl3) d: 181.70, 264.8 ꢀC. 1H NMR (400 MHz, DMSO-d6) d: 9.79 (s, 1H), 7.65 (d, J
161.73, 156.20, 139.27, 135.22, 129.15, 128.24, 125.26, 77.36, ¼ 8 Hz, 2H), 6.87 (d, J ¼ 8 Hz, 2H), 6.26 (s, 2H), 3.04 (d, J ¼ 4 Hz,
77.04, 76.72, 55.94, 54.18, 49.74, 31.81, 26.01, 21.38, 19.96, 1H), 2.13–2.18 (m, 1H), 1.83–1.89 (m, 1H), 1.17–1.25 (m, 2H),
19.05, 10.07; HR-MS (ESI+): m/z calculated for C19H23N3 [M + H]+ 1.14 (s, 3H), 0.94 (s, 3H), 0.56 (s, 3H); 13C NMR (100 MHz,
294.1970, found 294.1967.
DMSO-d6): d 180.56, 161.87, 158.52, 154.75, 129.49, 128.62,
3.1.2.13. 4-(2-Methoxyphenyl)-8,9,9-trimethyl-5,6,7,8-
122.24, 115.12, 54.95, 53.50, 49.33, 31.59, 25.77, 19.66, 18.72,
tetrahydro-5,8-methanoquinazolin-2-amine (3m). Yield 91%; mp 10.22; HR-MS (ESI+): m/z calculated for C18H21N3O [M + H]+
175.2–175.8 ꢀC. 1H NMR (400 MHz, CDCl3) d: 7.34–7.41 (m, 2H), 296.1763, found 296.1759.
This journal is © The Royal Society of Chemistry 2019
RSC Adv., 2019, 9, 29711–29720 | 29717

View Article Online
RSC Advances
Paper
31.80, 26.05, 19.97, 19.04, 10.07. HR-MS (ESI+): m/z calculated
3.1.2.19. 8,9,9-Trimethyl-4-(pyridin-2-yl)-5,6,7,8-tetrahydro-
for C18H21N3 [M + H]+ 280.1814, found 280.1810.
5,8-methanoquinazolin-2-amine (3s). Yield 89%; mp 174.7–
1
ꢀ
175.2 C. H NMR (400 MHz, CDCl3) d: 8.72 (d, J ¼ 8 Hz, 1H),
8.10 (d, J ¼ 8 Hz, 1H), 7.76–7.80 (m, 1H), 7.29–7.31 (m, 1H), 5.18
(s, 2H), 3.62 (d, J ¼ 4 Hz, 1H), 2.15–2.21 (m, 1H), 1.85–1.93 (m,
3.2. Cell imaging
1H), 1.28–1.36 (m, 2H), 1.26 (s, 3H), 1.00 (s, 3H), 0.65 (s, 3H); 13
C
3.2.1. Cells culture. The cells (MDA-MB-231, RPMI-8226,
A549, and GES-1) were supplied by the Cell Bank of the
Chinese Academy of Sciences. The cells were cultured in RPMI-
1640 medium (Gibco) supplemented with 5% fetal bovine
serum (FBS, Gibco), 1% penicillin and streptomycin (Gibco) in
NMR (100 MHz, DMSO-d6): d 177.83, 156.26, 151.19, 148.60,
144.14, 131.07, 121.59, 118.33, 117.43, 72.10, 71.78, 71.46,
50.06, 48.96, 44.84, 26.70, 20.51, 14.84, 13.70, 4.80; HR-MS
(ESI+): m/z calculated for C17H20N4 [M + H]+ 281.1766, found
281.1760.
ꢀ
a humidied atmosphere with 5% CO2 at 37 C. The medium
3.1.2.20. 8,9,9-Trimethyl-4-(pyridin-3-yl)-5,6,7,8-tetrahydro-
5,8-methanoquinazolin-2-amine (3t). Yield 85%; mp 197.2–
197.7 ꢀC. 1H NMR (400 MHz, CDCl3) d: 8.99 (s, 1H), 8.66, 8.65 (d,
J ¼ 4 Hz, 1H), 8.13 (d, J ¼ 8 Hz, 1H), 7.39–7.42 (m, 1H), 5.25 (s,
2H), 3.07 (d, J ¼ 4 Hz, 1H), 2.19–2.29 (m, 1H), 1.90–1.97 (m, 1H),
was updated every two days. Aer reaching about 70–80%
conuence, cells were trypsinized and harvested for further
process. Stock solutions of the candidate compounds (50 mM)
were prepared in DMSO.
3.2.2. MTS assay. Cell cytotoxicity was evaluated by the MTS
assay. Briey, the harvested cells were seeded in 96-well plates at
density of 1 ꢂ 105 cells per well and incubated overnight for
adherence. Fresh medium with different concentrations of the
candidate compounds were added into cells in triplicate, and
etoposide and assay medium were treated as positive and blank
controls respectively. The nal concentration of DMSO in the
well was kept at 0.1% in overall experiment. Upon complication
of 72 h incubation, 10 mL MTS solution was added to each well
and incubated for additional 0.5–1 h under same condition.
Thereaer, the plates were gently mixed and the optical density
(OD) was measured on a microplate reader at a wavelength of
490 nm. The percentage of inhibition was calculated by using
the below equation, and the values of IC50 were analyzed
through GraphPad Prism 5.
1.34 (d, J ¼ 8 Hz, 1H), 1.26 (s, 3H), 1.00 (s, 3H), 0.64 (s, 3H); 13
C
NMR (100 MHz, CDCl3): d 182.34, 161.82, 153.16, 150.10,
149.46, 135.60, 133.74, 125.83, 123.45, 56.07, 54.28, 49.59,
31.65, 26.04, 19.96, 18.97, 9.99; HR-MS (ESI+): m/z calculated for
C
17H20N4 [M + H]+ 281.1766, found 281.1762.
3.1.2.21. 8,9,9-Trimethyl-4-(pyridin-4-yl)-5,6,7,8-tetrahydro-
5,8-methanoquinazolin-2-amine (3u). Yield 90%; mp 207.9–
1
ꢀ
208.1 C. H NMR (400 MHz, CDCl3) d: 8.72 (d, J ¼ 4 Hz, 2H),
7.66 (d, J ¼ 4 Hz, 2H), 5.35 (s, 2H), 3.08 (d, J ¼ 4 Hz, 1H), 2.19–
2.25 (m, 1H), 1.89–1.98 (m, 1H), 1.30–1.37 (m, 2H), 1.26 (s, 3H),
1.01 (s, 3H), 0.63 (s, 3H); 13C NMR (100 MHz, CDCl3): d 182.70,
161.85, 153.26, 150.16, 145.40, 126.10, 122.52, 56.00, 54.27,
49.55, 31.67, 25.93, 19.96, 18.94, 9.96; HR-MS (ESI+): m/z
calculated for C17H20N4 [M + H]+ 281.1766, found 281.1763.
3.1.2.22. 4-(4-(Dimethylamino)phenyl)-8,9,9-trimethyl-5,6,7,8-
tetrahydro-5,8-methanoquinazolin-2-amine (3v). Yield 93%; mp
198.1–198.6 ꢀC. 1H NMR (400 MHz, CDCl3) d: 7.76 (d, J ¼ 8 Hz,
2H), 6.79 (d, J ¼ 8 Hz, 2H), 5.24 (s, 2H), 3.12 (d, J ¼ 4 Hz, 1H),
3.03 (s, 2H), 2.17–2.22 (m, 1H), 1.87–1.92 (m, 1H), 1.30–1.41 (m,
2H), 1.25 (s, 3H), 0.99 (s, 3H), 0.64 (s, 3H); 13C NMR (100 MHz,
CDCl3): d 181.14, 161.65, 156.35, 151.15, 129.47, 125.73, 124.28,
111.77, 55.84, 54.00, 50.01, 40.29, 31.92, 25.94, 19.97, 19.11,
10.12; HR-MS (ESI+): m/z calculated for C20H26N4 [M + H]+
323.2236, found 323.2231.
ODdrug ꢃ ODblank
Inhibition rate ð%Þ ¼
ꢂ 100%
ODcontrol ꢃ ODblank
3.2.3. Annexin V-APC/7-ADD apoptosis assay. Cellular
apoptosis was measured by ow cytometry using Annexin V-
APC/7-AAD double staining cell apoptosis detection kit. MDA-
MB-231 cell suspensions in the logarithmic growth phase
were seeded into a 6-well plates at 2 ꢂ 105 cells per well. Aer
incubation overnight, the adherent cells were exposed to
different concentrations (2.5, 5, and 10 mM) of compound 3f for
72 h. Then cells were harvested and centrifuged (2000 rpm, 5
min) to discard supernatant, followed by washing twice with
cold PBS. Subsequently, binding buffer (500 mL) and Annexin V-
APC (5 mL) were added and the cell mixture was incubated at
room temperature in the dark for 15 minutes. Prior to analysis
using ow cytometer, 5 mL of 7-AAD was applied to stain cells for
5 minutes.
3.2.4. Cell cycle assay. Cell cycle phase distribution was
evaluated by measuring the cellular DNA content with ow
cytometer. MDA-MB-231 cells (2 ꢂ 105 per well) were plated in
a 6-well culture plate and incubated at 37 ꢀC for 24 h. Then the
medium was removed and replaced with freshly prepared
solution of the tested compound 3f at different concentrations
(2.5, 5, and 10 mM). Aer exposure for 72 h, the cells were
washed twice with PBS, collected by centrifugation, and xed in
ice-cold 70% ethanol at 4 ꢀC for a whole night. Aerwards, the
3.1.2.23. 8,9,9-Trimethyl-4-(4-nitrophenyl)-5,6,7,8-tetrahydro-
5,8-methanoquinazolin-2-amine (3w). Yield 82%; mp 138.7–
1
ꢀ
139.1 C. H NMR (400 MHz, CDCl3) d: 8.33 (d, J ¼ 8 Hz, 2H),
7.97 (d, J ¼ 8 Hz, 2H), 5.07 (s, 2H), 3.07 (d, J ¼ 4 Hz, 1H), 2.22–
2.25 (m, 1H), 1.94 (m, 1H), 1.33–1.40 (m, 1H), 1.28 (s, 3H), 1.03
(s, 3H), 0.65 (s, 3H); 13C NMR (100 MHz, CDCl3): d 182.76,
161.66, 153.40, 145.72, 142.31, 130.14, 126.27, 123.90, 57.19,
49.33, 46.65, 30.46, 25.92, 20.64, 18.19, 9.21; HR-MS (ESI+): m/z
calculated for C18H20N4O2 [M + H]+ 325.1665, found 325.1659.
3.1.2.24. 8,9,9-Trimethyl-4-phenyl-5,6,7,8-tetrahydro-5,8-
methanoquinazolin-2-amine (3x). Yield 94%; mp 172.9–173.2 ꢀC.
1H NMR (400 MHz, CDCl3) d: 7.75–7.77 (m, 2H), 7.41–7.48 (m,
3H), 5.33 (s, 2H), 3.06 (d, J ¼ 4 Hz, 1H), 2.16–2.21 (m, 1H), 1.87–
1.92 (m, 1H), 1.32–1.35 (m, 2H), 1.25 (s, 3H), 0.98 (s, 3H), 0.63 (s,
3H); 13C NMR (100 MHz, CDCl3): d 181.85, 161.81, 156.19,
138.05, 129.18, 128.43, 128.30, 125.45, 55.96, 54.24, 49.67,
29718 | RSC Adv., 2019, 9, 29711–29720
This journal is © The Royal Society of Chemistry 2019

View Article Online
Paper
RSC Advances
cells were washed with pure PBS and resuspended in PBS comparable to etoposide, and had much lower cytotoxicity to
mixture containing RNase A (1 mg mlꢃ1) and propidium iodide normal GES-1 cells (IC50 > 50 mM) than the reference drug (IC50
(PI) (10 mg mlꢃ1). Half an hour later, the DNA content in ¼ 8.89 mM). Meanwhile, the overall result suggested that
samples was analysed immediately by ow cytometry.
electron-withdrawing group at para-position on aromatic rings
3.2.5. Mitochondrial membrane potential assay. The loss is crucial for cytotoxicity. Subsequently, compound 3f was
of the mitochondrial potential during apoptosis was assayed by selected for further anticancer mechanisms in a battery of
ow cytometry using dye JC-1 (5,50,6,60-tetrachloro-1,10,3,30- assays. Flow cytometry experiments demonstrated that
tetrethylbenzimidalylcarbocyanineiodide). MDA-MB-231 cells compound 3f induced G0/G1 phase arrest and apoptosis in
were cultured in 6-well plate for 72 h prior exposure to 2.5, 5, MDA-MB-231 cells, and the DCFH-DA assays revealed that
and 10 mM compound 3f for 24 h, cells without 3f treatment apoptosis was mediated through intracellular ROS stimulation.
were used as a control. Following treatment, the collected cells Moreover, compound 3f could also cause the depolarization of
were washed with cold PBS (phosphate buffer, pH ¼ 7.4) twice, mitochondrial membrane potentials, which indicated that
then JC-1 working solution was added to each tube and the cells compound 3f could activate the mitochondrial apoptosis
were allowed for incubation at 37 ꢀC in darkness. Finally, pathway. This mechanistic pathway was conrmed by upregu-
uorescent intensity of JC-1 was determined by ow cytometry lation of pro-apoptotic proteins Bax, cytochrome C and caspase-
aer another two times wash with PBS.
3, whereas the expression of anti-apoptosis protein Bcl-2 was
3.2.6. Analysis of reactive oxygen species. Effects of downregulated. On the basis of above studies, it can be
compound 3f on ROS generation were assessed by ow cytom- concluded that compound 3f exhibited potent anticancer effi-
etry using DCFH-DA as uorescent probe. MDA-MB-231 cells ciency via ROS-mediated mitochondrial apoptosis pathway. So
were seeded in a 6-well plate with 1 ꢂ 106 cells each well, and far, plenty of designed compounds against breast cancer cell,
incubated overnight. Subsequently, cells were treated with including ones based on pyrimidine rings, have been reported,
compound 3f at different concentration (2.5, 5, or 10 mM) or and their anti-tumor mechanism is mostly related to mito-
vehicle alone as a control. Aer 24 h treatment, the medium was chondrial apoptosis pathway.39–41 However, there is no effective
changed to serum-free medium containing 10 mM of DCFH-DA drug for triple-negative breast cancer (MDA-MB-231) because of
ꢀ
and incubated at 37 C in the dark for 20–30 min. The intra- its high metastasis and drug resistance. We hope these ndings
cellular ROS accumulation was quantied by measuring DCF in this paper will provide some valuable hints for exploring
uorescent intensity on ow cytometry. To investigate the role more effective drugs for breast cancer therapy.
of ROS in compound 3f induced cell apoptosis, N-acetylcysteine
(NAC, 5 mM) which as a ROS inhibitor was added to culture
medium prior to the treatment with compound 3f (5 mM).
Conflicts of interest
3.2.7. Western blot analysis. MDA-MB-231 cells were reac-
ted with compound 3f at indicated concentrations (0, 2.5, 5, and
The authors declare no competing interests.
10 mM) for 72 h, then collected cells were washed twice with PBS
and lysed in ice-cold lysis buffer over 30 min. The supernatant Acknowledgements
was obtained from lysate which was centrifuged for 15 min at
4 C. The BCA Protein Assay Kit was used for determining the
This study was supported by the National Natural Science
Foundation of China (No. 31470592), Major Project of Jiangsu
Province University Natural Science (No.14KJ220001) and Open
Funding of Jiangsu Key Laboratory of Biomass Energy and
Materials (No. JSBEM2014010). Besides, sincere gratitude to
Jiangsu Chia Tai Fenghai Pharmaceutical Co. Ltd for support in
cytotoxicity evaluation assay.
ꢀ
protein contents, while SDS-PAGE gel electrophoresis was
employed to separate proteins according to their molecular
weight. Aer being transferred to a nitrocellulose membrane,
the protein was blocked in TBST buffer with 5% skim milk at
room temperature for 60 min. Subsequently, the membranes
were incubated with appropriate primary antibodies overnight
ꢀ
at 4 C. Thenceforth, the membranes were rinsed three times
with TBST and allowed for incubated with secondary antibodies
at 25 ꢀC for 1 h. Clean the membrane again and the blots were
visualized via an improved chemiluminescence detection
reagent, while the amount of proteins of each lane was indi-
cated by b-actin.
References
¨
1 B. Vainshelboim, J. Muller, R. M. Lima, K. T. Nead,
C. Chester, K. Chan, P. Kokkinos and J. Myers, Prev. Med.,
2017, 100, 89–94.
2 K. Yang, Y. Li, Q. Tang, L. Zheng and D. He, Eur. J. Med.
Chem., 2019, 170, 45–54.
3 D. Wan, B. Tang, Y. J. Wang, B. H. Guo, H. Yin, Q. Y. Yi and
Y. J. Liu, Eur. J. Med. Chem., 2017, 139, 180–190.
4 S. Gupta, G. E. Kass, E. Szegezdi and B. Joseph, J. Cell. Mol.
Med., 2009, 13, 1004–1033.
5 Z. Jin and W. S. El-Deiry, Cancer Biol. Ther., 2005, 4, 139–163.
6 J. C. Martinou, S. Desagher and B. Antonsson, Nat. Cell Biol.,
2000, 2, 41–43.
4. Conclusion
In summary, we synthesized a series of novel pyrimidine
derivatives based on camphor and evaluated their anticancer
activities against three human cancer cell lines (MDA-MB-231,
RPMI-8226, and A549). The majority of synthesized
compounds possessed moderate to good growth inhibition,
particularly, compound 3f appeared to be the most active
This journal is © The Royal Society of Chemistry 2019
RSC Adv., 2019, 9, 29711–29720 | 29719

View Article Online
RSC Advances
Paper
7 P. Giuseppe, F. M. Ruggiero and P. Giuseppe, FASEB J., 2003, 24 S. Manohar, U. C. Rajesh, S. I. Khan, B. L. Tekwani and
17, 2202. D. S. Rawat, ACS Med. Chem. Lett., 2012, 3, 555–559.
8 S. Xiong, T. Mu, G. Wang and X. Jiang, Protein Cell, 2014, 5, 25 Z. Yang, Y. Fang and H. Park, Bioorg. Med. Chem. Lett., 2017,
737–749.
27, 2515–2519.
9 E. Finkel, Science, 2001, 292, 624–626.
10 S. Cherukupalli, G. A. Hampannavar, S. Chinnam,
26 R. S. Keri, K. M. Hosamani, R. V. Shingalapur and
M. H. Hugar, ChemInform, 2010, 45, 2597–2605.
B. Chandrasekaran, N. Sayyad, F. Kayamba, A. R. Reddy 27 Y. Addepalli, X. Yang, M. Zhou, D. P. Reddy, S. L. Zhang,
and R. Karpoormath, Bioorg. Med. Chem., 2018, 26, 309–339. Z. Wang and Y. He, Eur. J. Med. Chem., 2018, 151, 214–225.
11 A. M. Fargualy, N. S. Habib, K. A. Ismail, A. M. M. Hassan 28 G. Joshi, H. Nayyar, S. Kalra, P. Sharma, A. Munshi, S. Singh
and M. T. M. Sarg, ChemInform, 2013, 66, 276–295. and R. Kumar, Chem. Biol. Drug Des., 2017, 90, 995–1006.
12 Y. Ouyang, W. Zou, L. Peng, Z. Yang, Q. Tang, M. Chen, S. Jia, 29 M. Murahari, K. V. Prakash, G. J. Peters and Y. C. Mayur, Eur.
H. Zhang, Z. Lan and P. Zheng, Eur. J. Med. Chem., 2018, 154,
29–43.
13 M. Allam, A. K. D. Bhavani, A. Mudiraj, N. Ranjan,
J. Med. Chem., 2017, 139, 961–981.
30 G. Luo, Z. Tang, K. Lao, X. Li, Q. You and H. Xiang, Eur. J.
Med. Chem., 2018, 150, 783–795.
M. Thippana and P. P. Babu, Eur. J. Med. Chem., 2018, 156, 31 M. Coffinet, F. Massicot, J. Joseph, J. B. Behr, F. Jaroschik
43–52. and J. L. Vasse, Chem. Commun., 2016, 53, 111–114.
14 Y. Yi, G. Yang, Z. Chao, J. Chen, J. Liang and R. Shang, Eur. J. 32 G. Blay, V. Hernandez-Olmos and J. R. Pedro, ChemInform,
Med. Chem., 2015, 101, 179–184. 2008, 40, 4840–4842.
15 E. Scocchera, S. M. Reeve, S. Keshipeddy, M. N. Lombardo, 33 F. W. Lewis, T. C. Mccabe and D. H. Grayson, Tetrahedron,
B. Hajian, A. E. Sochia, J. B. Alverson, N. D. Priestley, 2011, 67, 7517–7528.
A. C. Anderson and D. L. Wright, ACS Med. Chem. Lett., 34 Y. Wang, J. Busch-Petersen, F. Wang, T. J. Kiesow,
´
2016, 7, 692–696.
16 P. Gao, L. Zhang, L. Sun, T. Huang, J. Tan, J. Zhang, Z. Zhou,
T. L. Graybill, J. Jin, Z. Yang, J. J. Foley, G. E. Hunsberger
and D. B. Schmidt, Bioorg. Med. Chem. Lett., 2009, 19, 114–
118.
´
T. Zhao, L. Menendez-Arias and C. Pannecouque, Bioorg.
Med. Chem., 2017, 25, 5779–5789.
35 S. O. Kuranov, I. P. Tsypysheva, M. V. Khvostov,
L. F. Zainullina, S. S. Borisevich, Y. V. Vakhitova,
O. A. Luzina and N. F. Salakhutdinov, Bioorg. Med. Chem.,
2018, 26, 4402–4409.
ˇ
´
ˇ´
17 M. Krecmerova, M. Dracınsk´y, R. Snoeck, J. Balzarini,
K. Pomeisl and G. Andrei, Bioorg. Med. Chem., 2017, 25,
4637–4648.
18 J. Wang, L. Zhang, J. Zhao, Y. Zhang, Q. Liu, C. Tian, 36 G. Stavrakov, V. Valcheva, I. Philipova and I. Doytchinova,
Z. Zhang, J. Liu and X. Wang, Bioorg. Med. Chem., 2018,
100, 89–94.
Eur. J. Med. Chem., 2013, 70, 372–379.
37 A. M. Montana, F. J. Bernal, L. Julia, F. Carlos, B. Consuelo,
´
M. J. Prieto, M. Virtudes, F. X. Aviles, J. M. Mesas and
19 E. S. Zuniga, A. Korkegian, S. Mullen, E. J. Hembre,
P. L. Ornstein, G. Cortez, K. Biswas, N. Kumar, J. Cramer
´
A. Marıa-Teresa, Bioorg. Med. Chem., 2008, 16, 1721–1737.
and T. Masquelin, Bioorg. Med. Chem., 2017, 25, 3922–3946. 38 M. L. Circu and T. Y. Aw, Free Radicals Biol. Med., 2010, 48,
20 P. S. Shirude, B. Paul, C. N. Roy, C. Kedari, B. Bandodkar and
B. G. Ugarkar, ACS Med. Chem. Lett., 2012, 3, 736–740.
21 K. Shah, X. Lin, S. F. Queener, V. Cody, J. Pace and
A. Gangjee, Bioorg. Med. Chem., 2018, 26, 2640–2650.
749–762.
39 M. Zhao, H. Ren, J. Chang, D. Zhang, Y. Yang, Y. He, C. Qi
and H. Zhang, Eur. J. Med. Chem., 2016, 119, 183–196.
40 E. F. Abdelhaleem, M. K. Abdelhameid, A. E. Kassab and
M. M. Kandeel, Eur. J. Med. Chem., 2017, 143, 1807–1825.
22 D.
Giles,
K.
Roopa,
F.
R.
Sheeba,
P. M. Gurubasavarajaswamy, G. Divakar and T. Vidhya, 41 D. Yao, Y. Zhou, L. Zhu, L. Ouyang, J. Zhang, Y. Jiang,
Eur. J. Med. Chem., 2012, 58, 478–484.
23 H. Kaur, J. Balzarini, C. D. Kock, P. J. Smith, K. Chibale and
K. Singh, Eur. J. Med. Chem., 2015, 101, 52–62.
Y. Zhao, D. Sun, S. Yang and Y. Yu, Eur. J. Med. Chem.,
2017, 140, 155–171.
29720 | RSC Adv., 2019, 9, 29711–29720
This journal is © The Royal Society of Chemistry 2019