Isothiouronium modification empowers pyrimidine-substituted curcumin analogs potent cytotoxicity and Golgi localization

Article abstract of DOI:10.1016/j.ejmech.2016.07.071

Most of protein post-translational modifications occur in the Golgi and many human diseases are associated with abnormal Golgi function or improper post translational modifications of proteins in the Golgi. In this study, we designed and synthesized 4?×?6

Full text of DOI:10.1016/j.ejmech.2016.07.071

Accepted Manuscript
Isothiouronium modification empowers pyrimidine-substituted curcumin analogs
potent cytotoxicity and Golgi localization
Sheng Tong, Meng Zhang, Shixi Wang, Ruijuan Yin, Rilei Yu, Shengbiao Wan, Tao
Jiang, Lijuan Zhang
PII:
S0223-5234(16)30636-5
10.1016/j.ejmech.2016.07.071
EJMECH 8789
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 March 2016
Revised Date: 18 July 2016
Accepted Date: 28 July 2016
Please cite this article as: S. Tong, M. Zhang, S. Wang, R. Yin, R. Yu, S. Wan, T. Jiang, L. Zhang,
Isothiouronium modification empowers pyrimidine-substituted curcumin analogs potent cytotoxicity and
Golgi localization, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.07.071.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Isothiouronium modification empowers pyrimidine-substituted curcumin
analogs potent cytotoxicity and Golgi localization
Sheng Tong1, †, Meng Zhang1, †, Shixi Wang1, Ruijuan Yin1, Rilei Yu1, Shengbiao
Wan1, Tao Jiang1,*, and Lijuan Zhang1,2,*
† These authors contributed equally to this study
1
Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of
Medicine and Pharmacy, Ocean University of China, Qingdao, China. 5 Yushan
2
Road, Qingdao, China; Institute of Cerebrovascular Diseases, Affiliated Hospital
of Qingdao University, Qingdao China, Post code: 266003, Tel:
0086-532-82031615, Fax: 0086-532-82033054
* To whom correspondence should be addressed:
Tao Jiang, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan
Road, Qingdao, China; Post code: 266003, Tel: 0086-532-82032712, Fax:
0086-532-82033054, jiangtao@ouc.edu.cn.
Lijuan Zhang, School of Medicine and Pharmacy, Ocean University of China, 5
Yushan Road, Qingdao, China; Post code: 266003, Tel: 0086-532-82031615, Fax:
0086-532-82033054, lijuanzhang@ouc.edu.cn;
ABSTRACT
Most of protein post-translational modifications occur in the Golgi and many human
diseases are associated with abnormal Golgi function or improper post translational
modifications of proteins in the Golgi. In this study, we designed and synthesized 4×
6 series of novel isothiouronium-modified (E,E)-4,6-bis(styryl)-pyrimidine analogs
and found that they localized at the Golgi as visualized by the intrinsic fluorescence of
the analogs. The isothiouronium-modified analogs had potent cytotoxicity in both
normal (Chinese Hamster Ovary or CHO) and cancer cells. Furthermore,
permethylated isothiouronium-modified analogs showed cancer cell-selective
cytotoxicity. The molecular mechanisms underlying Golgi localization of
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isothiouronium-modified compounds were investigated using 7 CHO and 4 human
cancer cell lines and the results indicated that the compounds had binding partners in
the Golgi. Thus, isothiouronium-modified analogs might be promising anticancer
agents, novel Golgi staining reagents, and useful research tools for studying Golgi
functions in normal or cancer cells and in Golgi-related human diseases.
Key words: Isothiouronium; pyrimidine-substituted curcumin; Golgi; cytotoxicity;
(E,E)-4,6-bis(styryl)-pyrimidine; immuno-staining; cancer
1. Introduction
Curcumin is responsible for the therapeutic properties of turmeric in traditional
medicine. Its IC50s in cancer cells range from 3 to > 30 µM that are quite high
compared to most of anticancer drugs[1,2]. Dietary curcumin exhibits poor
bioavailability, rapid metabolism, low solubility, and extensive rapid excretion in
clinical trials [1-3]. We have previously reported a family of (E,E)-4,6-bis(styryl)-pyr-
imidine analogs that contain the pyrimidine ring instead of the β-dicarbonyl moiety of
curcumin. These analogs have significantly improved anticancer activity, chemical
stability, and solubility when compared to curcumin [4,5]. Furthermore, the V-shaped
D-π-A-π-D structure of the pyrimidine-substituted curcumin analogs generates
intensive fluorescence [6]. However, the fluorescent property has not been explored
for biological applications.
With only 20,000 to 30,000 human genes, the dynamic complexity of living
systems is achieved, in part, through over 400 known post-translational modifications
of proteins [7]. Most of protein post-translational modifications, especially N-linked
and O-linked glycosylations [8-10], occur in the Golgi and many human diseases are
associated with abnormal Golgi function or improper protein post translational
modifications in the Golgi [11]. At present, development of small molecule probes
that target the morphology and reveal the functions of Golgi is still in its infancy [12].
In fact, only limited numbers of small molecules with different backbone structures
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that perturb the function of Golgi have been reported [13-15]. We noticed that positive
charges in those small molecules seem to be associated with the Golgi localization
[13-15], which is consistent with the fact that Golgi is an acidic compartment with a
pH gradient ranging from 5 to 6. Remarkably, a series of isothiouronium-modified
compounds exhibit significant pharmacological activities, such as anesthetic [16],
antimicrobial [17], and antitumoral [18-21] with unknown mechanisms.
Based on these facts, we decided to introduce positively charged isothiouronium
group to pyrimidine-substituted curcumin analogs and to screen for Golgi-localized
compounds by taking advantage of the unique fluorescent properties of the
(E,E)-4,6-bis(styryl)-pyrimidine analogs. Indeed, our data showed that the isothiouro-
nium-modified analogs had greatly enhanced anticancer activities in addition to their
unique Golgi localization compared to the unmodified compounds.
2. Chemistry
We first designed and synthesized 4×6 series of isothiouronium-modified
(E,E)-4,6-bis-(styryl)-pyrimidine analogs in three steps as outlined in Scheme 1. The
compounds 1-4 were prepared from the appropriate aromatic aldehydes according to
the procedure we reported previously [4]. Subsequently, the compounds were allowed
to react individually with excessive 1,ω-dibromoalkane in the presence of K2CO3 to
yield the ω-bromoalkyl derivatives 1a~c-4a~c, which were separated by chromatogr-
aphy in 30%~50% yield. Then 1a~c-4a~c reacted with thiourea in boiling acetone to
generate the target compounds 1d~f-4d~f in 50%~70% yield.
We also synthesized three 1,1,3,3-tetramethylated isothiouronium derivatives by
mixing compounds 1a-3a with 1,1,3,3-tetramethylthiourea in boiling acetone in
50%~60% yield.
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Scheme 1. Synthesis of the isothiouronium derivatives of curcumin-pyrimidine analogs.
Reagents and conditions: (i) appropriate aromatic aldehydes, HCl, EtOH, rf, 50%~75%; (ii)
Br(CH2)nBr, K2CO3, DMF, 80°C, 30%~50%;(iii)Thiourea, acetone, rf, 50%~70%; (vi)
1,1,3,3-Tetramethylthiourea, acetone, rf, 50%~60%.
3. Results and discussion
3.1 Cytotoxicity and IC50
Four human cancer cell lines A549 (lung), H1299 (lung), HCT116 (colon), and
HT29 (colon) were treated with 10 µM of compounds 1a, 1d, 1g, 2a, 2d, 2g, 3a, 3d,
and 3g or compound-free control for 48 h and the cell viability was measured as an
indicator for cytotoxicity of the compounds (Figure 1).
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160
120
80
*** *** *** ***
*** *** *** ***
*** *** *** ***
** ** ** **
*** *** *** ***
*
*
*
*
d
g
a
40
0
1
2
3
Concentration: 10 μM
Figure 1. Effects of the tested compounds on cell viability of four human cancer cell lines.
Percentage of viable cells (A549, H1299, HCT116, HT29) after 48 h exposure to the curcumin-
pyrimidine analogues (1a, 1d, 1g, 2a, 2d, 2g, 3a, 3d, and 3g) at a concentration of 10 µM
compared to the compound-free control received an equal volume of DMSO (100% viability)
(***P<0.001, **P<0.01, *P < 0.05; Student’s t-test). The analogues were formulated initially in
DMSO and then diluted in complete growth media. Each value was calculated from 2 independent
experiments performed in triplicate. Data are shown as mean ± SD.
To obtain IC50 values for each compound, all four cancer cells were treated with
series of concentrations of each compound and the data were summarized in Table 1.
All isothiouronium-modified compounds (1d, 1e, 1f, 2d, 2e, 2f, 3d, 3e, 3f, 4d, 4e, and
4f) had IC50 values at low µM ranges. The 1,1,3,3-tetramethylated isothiouronium-
modified compounds (1g, 2g, and 3g) had higher IC50 values compared to their
unmethylated counterparts (1d, 2d, and 3d). In contrast, all Br-modified compounds
(1a, 1b, 1c, 2a, 2b, 2c, 3a, 3b, 3c, 4a, 4b, and 4c) had IC50 values over 20 µM.
The IC50 values indicated that the compounds with one methoxyl group were
most toxic in all four cancer cell lines tested. In general, when the compounds were
modified by methoxyl groups, the fewer the number of methoxyl groups, the stronger
the cytotoxicity (1>2>3). In contrast, the toxicities of compounds with no methoxyl
modification were between the compounds with two and three methoxyl groups.
When the compounds had 4, 5, or 6 methylene spacer (-(CH2)-) separating
isothiouronium from the (E,E)-4,6-bis(styryl)-pyrimidine, the spacer effect on IC50
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values was minimal in all four cell lines tested. Furthermore, the cytotoxicity declined
when the isothiouronium was fully methylated (1g, 2g, and 3g).
Table 1. IC50 of the compounds in four human cancer cell lines
IC50 (µM)a
Comp
HT29
＞20
HCT116
＞20
H1299
＞20
A549
＞20
1a
1b
1c
1d
1e
1f
＞20
＞20
＞20
＞20
＞20
＞20
＞20
＞20
0.79±0.16
2.03±0.04
2.14±0.13
5.97±1.04
＞20
1.00±0.13
1.93±0.03
1.98±0.04
3.00±0.39
＞20
0.92±0.14
2.96±0.74
3.69±0.42
7.49±0.58
＞20
0.99±0.19
1.97±0.03
1.97±0.13
2.26±0.18
＞20
1g
2a
2b
2c
2d
2e
2f
＞20
＞20
＞20
＞20
＞20
＞20
＞20
＞20
0.61±0.17
1.91±0.13
1.72±0.26
4.10±1.35
＞20
1.86±0.05
1.81±0.05
2.02±0.19
3.89±1.4
＞20
1.53±0.62
4.04±0.44
3.75±0.25
5.81±0.91
＞20
2.36±0.70
1.99±0.05
2.09±0.14
9.71±0.94
＞20
2g
3a
3b
3c
3d
3e
3f
＞20
＞20
＞20
＞20
＞20
＞20
＞20
＞20
2.19±0.04
1.86±0.23
1.17±0.49
10.49±2.28
＞20
3.77±0.16
2.08±0.09
1.87±0.03
5.41±0.35
＞20
5.1±0.72
4.13±0.08
3.09±1.22
14.97±3.6
＞20
4.24±0.91
2.88±0.89
2.00±0.03
14.36±4.97
＞20
3g
4a
4b
4c
4d
4e
4f
＞20
＞20
＞20
＞20
＞20
＞20
＞20
＞20
2.19±0.04
1.86±0.23
1.17±0.49
3.77±0.16
2.08±0.09
1.87±0.03
5.1±0.72
4.13±0.08
3.09±1.22
4.24±0.91
2.88±0.89
2.00±0.03
a. Each IC50 value was calculated from 3 independent experiments performed in triplicate. Data
are shown as mean ± SD.
3. 2 Golgi localization of isothiouronium-modified pyrimidine-substituted
curcumin analogs
The potent cytotoxicity of the isothiouronium-modified (E,E)-4,6-bis(styryl)-
pyrimidine analogs (Figure 1 and Table 1) prompted us to consider that the negatively
charged DNAs in nucleus might be the molecular targets of the positively charged
analogs. To verify this hypothesis, we performed live cell imaging to evaluate the
uptake and subcellular localization of the pyrimidine-substituted curcumin analogs.
A549 or HCT116 cells were seeded on glass coverslips in complete cell culture media
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before being treated with or without the compounds 1a and 1d at 10 µM for 4 h at 37
ºC. The cells were imaged for the unique green fluorescence in living cells under laser
scanning confocal microscope. Surprisingly, in contrast to the even distribution of
compound 1a in cytoplasm, the isothiouronium-modified compound 1d had an
apparent Golgi localization.
To confirm the Golgi localization of isothiouronium-modified compound, we
used Golgi marker Golgin-97 and the nuclear marker DAPI to co-stain A549 and
HCT116 cells. The cells were seeded on glass coverslips in complete cell culture
media before being treated with 10 µM of 1a, 1d, 1e, 1f, and 1g for 4 h at 37 ºC.
Subsequently, the media were removed and the cells were fixed with 4%
formaldehyde followed by permeabilizing with 0.2% Triton-100 in PBS. After being
blocked with 5% bovine serum albumin (BSA), the cells were incubated with the
anti-Glogin-97 primary antibody followed by incubation with fluorescent secondary
antibody (red fluorescence). The nucleus was stained with DAPI (blue fluorescence).
The cells stained by the compounds (green fluorescence), Golgin-97 (red
fluorescence), and DAPI (blue fluorescence) were imagined by laser scanning
confocal microscope and the results were shown in Figure 2.
Merging the cell images in green (compounds), red (Golgin-97), and blue
(nucleus) channels showed the absence of green fluorescence and the presence of the
red fluorescence (Golgin-97) in the Golgi next to the nucleus (blue fluorescence) in
the blank control (Control, Figure 2). Br-modified compound 1a was evenly
distributed in the cytoplasm (1a, Figure 2). In contrast, isothiouronium-modified
compound 1d was mostly co-localization with Golgi marker Golgin-97 (Insert 1 and
2, Figure 2). Although compounds 1d, 1e, and 1f with four, five, and six methylene
spacer groups did not show significant difference in their IC50 values (Table 1), their
Golgi localization showed different patterns: the fluorescence generated by 1d was
mostly associated with the Golgi while the green fluorescence from 1e and 1f was
associated with vesicles radiated away from the Golgi in the order of 6 > 5 > 4 of
methylene spacer groups (Inserts 3, 4, 5, and 6, Figure 2), suggesting the methylene
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spacer groups in the compounds contributed to the subtle differences in their Golgi
localization.
Figure 2. Fluorescence images of A549 showing the location of Compounds. Laser scanning
confocal microscope images of A549 cells incubated with or without compounds (green) (1a, 1d,
1e, 1f, and 1g) followed by Golgi (red) and nucleus (blue) staining with Golgin-97 and DAPI.
Scale bars: 10 µm. Control: non-derivative treated cells recevied an equal volum of DMSO.
Sub-Figure 1-8: zoom in of the arrows in 1d-1g. The results have been repeated.
The IC50 values (Table 1) was obtained 48 h after the cells were treated with the
compounds. Within this time frame, the isothiouronium-modified compounds were
highly toxic. It raised the question if the compounds had binding partners in the Golgi
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or they were accumulated in the Golgi as a result of their Golgi-targeted cytotoxicity.
To address this issue, we conducted three sets of experiments.
First, we performed the live cell imaging using A549 cells treated with
compounds 1a and 1d for 1, 2, 4, and 12 h. We observed that compound 1a was
evenly distributed in cytoplasm whereas the isothiouronium-modified compound 1d
had the Golgi localization at all-time points tested, indicating that compound 1d may
have binding partners in the Golgi.
It was reported that the positively charged guanidinium-rich scaffolds bind to
negatively charged phosphate, sulfate, and carboxyl groups on cell surface through
hydrogen binding [22]. We reasoned that if the isothiouronium-modified compound
binds to its binding partners in the Golgi through hydrogen bond via isothiouronium,
such binding will be weakened if the isothiouronium is fully methylated. Therefore, in
the next experiment, we synthesized permethylated compounds 1g, 2g, and 3g, and
measured their cytotoxicity, followed by imaging the cellular localization of these
compounds. Indeed, not only the cytotoxicity of compounds 1g, 2g, and 3g in all four
cancer cell lines were decreased (Table 1), but also compound 1g almost lost the
Golgi localization (Inserts 1, 2, 3, 4, 5, 6, 7, and 8, Figure 2). These results suggested
that compounds 1d, 1e, and 1f bind to their binding partners in the Golgi through
hydrogen bonds.
The same Golgi localization and cytotoxicity (data not shown) were observed
when HCT116 cells were used in the same experiment, suggesting that the analogs
have the same molecular actions in different types of cancer cells.
Finally, we investigated if the binding partners of the isothiouronium-modified
(E,E)-4,6-bis(styryl)-pyrimidine analogs were glycosaminoglycans (GAGs), the most
negatively charged biopolymers assembled on the core protein of proteoglycans in the
Golgi since it was reported that the binding of positively charged guanidinoneomycin
to cells is exclusively dependent on the cell surface glycosaminoglycans [23]. The
normal fibroblast CHO cells used in current study include wild-type K1, GAG-defici-
ent 745, heparan sulfate-deficient 677, 3-O-sulfotransferase 1-overexpressing 3.1,
3-O-sulfotransferase 2-overexpressing 3.2, 3-O-sulfotransferase 3-overexpressing 3.3,
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3-O-sulfotransferase 4-overexpressing 3.4 that we have used and described in details
previously [24]. We evaluated the cytotoxicity of compounds 1a, 1d, 1g, 2a, 2d, and
2g in all the CHO cells and the lung cancer cells A549 (Figure 3). The isothiouronium
modified compounds 1d and 2d were highly toxic to all 8 cell lines including the
GAG-deficient 745 cells, indicating that the binding partners in the Golgi might not
be the GAGs (Figure 3). The Br-modified compound 1a was not toxic whereas 1d, 2a,
and 2d were toxic to all cell lines tested. This evidence further supported the
hypothesis that isothiouronium-modified compounds 1d and 2d targeted the common
partners in the Golgi both in normal and cancer cells. Even though permethylated
compounds 1g and 2g were toxic to all four cancer cells tested (Table 1, Figure 1, and
Figure 3), they stimulated CHO cell growth instead, suggesting that 1g and 2g had
methylation-related toxicity towards cancer cells only (Figure 3).
300
***
**
***
200
100
0
***
***
1a
1d
1g
2a
2d
2g
Concentration: 10 μM
K1
745
677
3.1
3.2
3.3
3.4
A549
Figure 3. Effects of the tested compounds on cytotoxicity to all the CHO cells and the lung
cancer cells A549. Percentage of viable cells (CHOK1, 745, 677, 3.1, 3.2, 3.3, 3.4, and human
cancer cells A549) after 48 h exposure to the curcumin-pyrimidine analogues (1a, 1d, 1g, 2a, 2d,
and 2g) at a concentration of 10 µM compared to the compound-free control received an equal
volume of DMSO (100% viability) (***P<0.001, **P<0.01, *P < 0.05; Student’s t-test). The
analogues were formulated initially in DMSO and then diluted in complete growth media. Each
value was calculated from 2 independent experiments performed in triplicate. Data are shown as
mean ± SD.
4. Conclusions
In current study, we designed and synthesized a series of novel isothiouronium-
modified pyrimidine-substituted curcumin analogs. Their unexpected Golgi
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localization, potent cytotoxicity, and the molecular mechanisms underlying Golgi
localization were revealed. These compounds provide several advantages over the
current tools to image and study the function of the Golgi. First, they could be used in
live cells and do not require fixation and permeation as common for the application of
conventional immunofluorescence methods. Second, the probes were localized in the
Golgi over a 12 h period that provided sufficient time frame to perform detailed
functional studies of the Golgi-associated post translational modification-defects
associated with various human diseases. Third, isothiouronium-modified
(E,E)-4,6-bis(styryl)-pyrimidine analogs were toxic to both CHO and cancer cells
whereas permethylated analogs were only toxic to cancer cells (Figure 3). Such
property might provide a novel way for selecting and developing permethylated
compounds as selective anti-cancer drugs.
In conclusion, identifying the precise binding partner(s) of the isothiouronium-
modified pyrimidine-substituted curcumin analogs in the Golgi will be our next goal.
This information is critical for understanding the isothiouronium-modified
(E,E)-4,6-bis(styryl)-pyrimidine analog-induced cell death. In addition, the chemical
and biological approach used in current study in developing Golgi targeting
fluorescent probes can be useful tools for the booming field of protein post
translational modification research in the near future.
5. EXPERIMENTAL SECTION
5.1. Chemical Synthesis
5.1.1. Materials and Methods
All chemicals were purchased from commercial vendors and used without further
purification unless otherwise noted. Thin-layer chromatography (TLC) was performed
on precoated E. Merck silica-gel 60 F254 plates. Column chromatography was
performed on silica gel (200-300 mesh). Melting points were determined on a
1
Mitamura-Riken micro-hot stage and were not corrected. H NMR and 13C NMR
spectra were obtained on a Jeol JNM-ECP 600 spectrometer with tetramethylsilane
(Me4Si) as the internal standard and chemical shifts were recorded as δ values in ppm.
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Following abbreviations are used: s = singlet, d = doublet, t = triplet, q= quartet, m =
multiplet, dd = double-doublet. Mass spectra were recorded on a Q-TOF Global mass
spectrometer.
4,6-Dimethyl-2-hydroxyl-pyrimidine hydrochloride was prepared according to
literature procedures [25]. The syntheses of (E,E)-4,6-bis(4-methoxystyryl)-2-hydrox-
yl-pyrimidine (1), (E,E)-4,6-bis(3,4-dimethoxystyryl)-2-hydroxyl-pyrimidine (2),
(E,E)-4,6-bis(3,4,5-trimethoxystyryl)-2-hydroxylpyrimidine (3) have been described
previously [4].
5.1.2. Synthesis of Compound (E,E)-4,6-bisstyryl-2-hydroxyl-pyrimidine (4).
A mixture of 2-hydroxy-4,6-dimethylpyrimidine hydrochloride (0.16 g, 1 mmol),
benzaldehyde (306 µL, 3 mmol) in anhydrous ethyl alcohol (100 mL) was stirred
under an N2 atmosphere at room temperature for 1 h. Then hydrochloric acid (600 µL,
1.2 mmol) was added to the reaction mixture which was refluxed for 36 h. After
completion of the reaction, the solvent was evaporated under reduce pressure, and the
saturated NaHCO3 solution (250 mL) was added into the brown oil residue for
desalting. After stirring at room temperature for 5 h, the resulting precipitate was
filtered, washed with water followed with ether, and then purified by chromatography
1
on silica gel to give 4 as a yellow powder (0.15g , 50%). H NMR (500 MHz,
DMSO-d6) δ 11.73 (s, 1H), 7.85 (d, J = 16.3 Hz, 2H), 7.67 (d, J = 6.7 Hz, 4H),
7.52-7.35 (m, 6H), 7.05 (d, J = 16.2 Hz, 2H), 6.96 (s, 1H); MS (ESI) m/z ESI+ 301.1
[M+H]+.
5.1.3. General procedure for Compounds 1a~c-4a~c.
To a solution of 1-4 (1.0 mmol) in anhydrous N,N-dimethylformamide (DMF, 30
mL) at 80 °C was added anhydrous potassium carbonate (3.0 mmol), 1,ω-dibromoalk-
an (15.0 mmol) and the mixture was stirred for 12 h. After the reaction was judged
complete by TLC, the solvent was removed under reduced pressure, and the residue
was dissolved in CH2Cl2 and washed with water. The organic layer was separated,
dried over anhydrous Na2SO4, filtered, and concentrated. The resulting residue was
purified by chromatography on silica gel to give compounds 1a~c-4a~c.
5.1.4 (E,E)-2-(4-bromobutoxy)-4,6-bis(4-methoxystyryl)pyrimidine (1a)
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Yellow powder (193 mg, yield 39%); 1H NMR (500 MHz, CDCl3) δ 7.87 (s, 1H),
7.84 (s, 1H), 7.54 (d, J = 8.4 Hz, 4H), 6.92 (d, J = 8.4 Hz, 4H), 6.90 (s, 1H), 6.86 (s,
2H), 4.51 (t, J = 6.1 Hz, 2H), 3.84 (s, 6H), 3.52 (t, J = 6.5 Hz, 2H), 2.18-2.10 (m, 2H),
13
2.08-2.00 (m, 2H); C NMR (126 MHz, CDCl3) δ 165.22, 165.17, 160.60, 136.40,
129.12, 128.59, 123.58, 114.29, 110.71, 66.06, 55.35, 33.61, 29.41, 27.67; MS (ESI)
m/z ESI+ 495.1 [M+H]+.
5.1.5. (E,E)-2-(5-bromopentyloxy)-4,6-bis(4-methoxystyryl)pyrimidine (1b)
Yellow powder (178 mg, yield 35%); 1H NMR (500 MHz, CDCl3) δ 7.87 (s, 1H),
7.84 (s, 1H), 7.54 (d, J = 8.7 Hz, 4H), 6.92 (d, J = 8.7 Hz, 4H), 6.90 (s, 1H), 6.87 (s,
2H), 4.48 (t, J = 6.5 Hz, 2H), 3.85 (s, 6H), 3.45 (t, J = 6.8 Hz, 2H), 2.02-1.95 (m, 2H),
1.94-1.88 (m, 2H), 1.73-1.66 (m, 2H); 13C NMR (126 MHz, CDCl3) δ 165.29, 165.15,
160.57, 136.33, 129.10, 128.61, 123.63, 114.28, 110.62, 66.83, 55.35, 33.65, 32.60,
28.22, 24.88; MS (ESI) m/z ESI+ 509.1 [M+H]+.
5.1.6. (E,E)-2-(6-bromohexyloxy)-4,6-bis(4-methoxystyryl)pyrimidine (1c)
Yellow powder (161 mg, yield 31%); 1H NMR (500 MHz, CDCl3) δ 7.87 (s, 1H),
7.84 (s, 1H), 7.54 (d, J = 8.7 Hz, 4H), 6.92 (d, J = 8.7 Hz, 4H), 6.90 (s, 1H), 6.87 (s,
2H), 4.48 (t, J = 6.5 Hz, 2H), 3.85 (s, 6H), 3.45 (t, J = 6.8 Hz, 2H), 2.02-1.95 (m, 2H),
1.94-1.88 (m, 2H), 1.73-1.66 (m, 2H); 13C NMR (126 MHz, CDCl3) δ 165.11, 160.59,
136.40, 129.12, 128.61, 123.60, 114.28, 110.51, 67.08, 55.35, 33.89, 32.75, 28.87,
28.00, 25.36; MS (ESI) m/z ESI+ 523.1 [M+H]+.
5.1.7. (E,E)-2-(4-bromobutoxy)-4,6-bis(3,4-dimethoxystyryl)pyrimidine (2a)
Yellow powder (266 mg, yield 48%); 1H NMR (500 MHz, CDCl3) δ 7.84 (d, J =
15.8 Hz, 2H), 7.18-7.12 (m, 4H), 6.93-6.85 (m, 5H), 4.52 (t, J = 6.1 Hz, 2H), 3.94 (d,
J = 11.8 Hz, 12H), 3.53 (t, J = 6.5 Hz, 2H), 2.14 (dt, J = 13.4, 6.6 Hz, 2H), 2.04 (dt, J
13
= 12.7, 6.4 Hz, 2H); C NMR (126 MHz, CDCl3) δ 165.23, 165.09, 150.30, 149.15,
136.70, 128.80, 123.78, 121.72, 111.09, 110.53, 109.42, 66.09, 55.96, 33.67, 29.40,
27.64; MS (ESI) m/z ESI+ 555.1 [M+H]+.
5.1.8. (E,E)-2-(5-bromopentyloxy)-4,6-bis(3,4-dimethoxystyryl)pyrimidine (2b)
Yellow powder (210 mg, yield 37%); 1H NMR (500 MHz, CDCl3) δ 7.85 (s, 1H),
7.82 (s, 1H), 7.18-7.12 (m, 4H), 6.92-6.89 (m, 3H), 6.87 (d, J = 3.7 Hz, 2H), 4.49 (t, J
13

ACCEPTED MANUSCRIPT
= 6.5 Hz, 2H), 3.93 (d, J = 13.3 Hz, 12H), 3.45 (t, J = 6.8 Hz, 2H), 2.01-1.95 (m, 2H),
13
1.94-1.87 (m, 2H), 1.70 (dd, J = 15.3, 8.0 Hz, 2H); C NMR (126 MHz, CDCl3) δ
165.33, 165.07, 150.31, 149.18, 136.61, 128.86, 123.88, 121.70, 111.14, 110.40,
109.49, 77.26, 77.01, 76.76, 66.85, 55.95, 33.66, 32.58, 28.22, 24.87; MS (ESI) m/z
ESI+ 569.1 [M+H]+.
5.1.9 (E,E)-2-(6-bromohexyloxy)-4,6-bis(3,4-dimethoxystyryl)pyrimidine (2c)
Yellow powder (257 mg, yield 44%); 1H NMR (500 MHz, CDCl3) δ 7.85 (s, 1H),
7.82 (s, 1H), 7.19-7.11 (m, 4H), 6.92-6.85 (m, 5H), 4.48 (t, J = 6.4 Hz, 2H), 3.93 (d, J
13
= 13.4 Hz, 12H), 3.43 (t, J = 6.7 Hz, 2H), 1.95-1.85 (m, 4H), 1.56 (s, 4H); C NMR
(126 MHz, CDCl3) δ 165.39, 165.05, 150.27, 149.15, 136.58, 128.85, 123.90, 121.72,
111.09, 110.38, 109.39, 67.07, 55.95, 33.94, 32.74, 28.90, 27.99, 25.38; MS (ESI)
m/z ESI+ 583.1 [M+H]+.
5.1.10. (E,E)-2-(4-bromobutoxy)-4,6-bis(3,4,5-trimethoxystyryl)pyrimidine (3a)
1
Red powder (221 mg, yield 36%); H NMR (500 MHz, CDCl3) δ 7.83 (s, 1H),
7.80 (s, 1H), 6.95 (s, 1H), 6.93 (s, 1H), 6.90 (s, 1H), 6.82 (s, 4H), 4.53 (t, J = 6.1 Hz,
2H), 3.90 (d, J = 13.8 Hz, 18H), 3.53 (t, J = 6.5 Hz, 2H), 2.14 (dd, J = 13.7, 6.2 Hz,
13
2H), 2.05 (dd, J = 14.0, 6.1 Hz, 2H); C NMR (126 MHz, CDCl3) δ 165.27, 164.94,
153.44, 139.40, 136.91, 131.32, 125.13, 110.79, 104.79, 66.17, 60.98, 56.15, 33.58,
29.39, 27.62; MS (ESI) m/z ESI+ 615.1 [M+H]+.
5.1.12. (E,E)-2-(5-bromopentyloxy)-4,6-bis(3,4,5-trimethoxystyryl)pyrimidine
(3b)
1
Red powder (270 mg, yield 43%); H NMR (500 MHz, CDCl3) δ 7.81 (d, J =
15.8 Hz, 2H), 6.94 (d, J = 6.1 Hz, 2H), 6.90 (s, 1H), 6.82 (s, 4H), 4.49 (t, J = 6.2 Hz,
2H), 3.90 (d, J = 13.9 Hz, 18H), 3.45 (t, J = 6.6 Hz, 2H), 2.02-1.94 (m, 2H), 1.94-1.87
13
(m, 2H), 1.74-1.66 (m, 2H); C NMR (126 MHz, CDCl3) δ 165.35, 164.90, 153.42,
139.31, 136.83, 131.35, 125.20, 110.74, 104.72, 66.96, 60.99, 56.14, 33.71, 32.55,
28.21, 24.87; MS (ESI) m/z ESI+ 629.1 [M+H]+.
5.1.12. (E,E)-2-(6-bromohexyloxy)-4,6-bis(3,4,5-trimethoxystyryl)pyrimidine (3c)
Red powder (264 mg, yield 41%); 1H NMR (500 MHz, CDCl3) δ 7.82 (d, J =
15.6 Hz, 2H), 6.94 (d, J = 16.6 Hz, 3H), 6.82 (s, 4H), 4.48 (t, J = 6.3 Hz, 2H), 3.89 (d,
14

ACCEPTED MANUSCRIPT
J = 13.4 Hz, 18H), 3.43 (t, J = 6.7 Hz, 2H), 1.90 (dt, J = 13.1, 6.7 Hz, 4H), 1.55 (s,
4H); 13C NMR (126 MHz, CDCl3) δ 164.81,153.41, 139.32, 138.7.11, 131.30, 125.02,
110.57, 104.72, 67.28, 61.00, 56.14, 33.97, 32.69, 28.85, 27.95, 25.35; MS (ESI) m/z
ESI+ 643.1 [M+H]+.
5.1.13. (E,E)-2-(4-bromobutoxy)-4,6-distyrylpyrimidine (4a)
1
White powder (148 mg, yield 34%); H NMR (500 MHz, CDCl3) δ 7.93 (s, 1H),
7.89 (s, 1H), 7.60 (d, J = 7.3 Hz, 4H), 7.40 (t, J = 7.4 Hz, 4H), 7.35 (t, J = 7.0 Hz, 2H),
7.03 (d, J = 15.9 Hz, 2H), 6.95 (s, 1H), 4.53 (t, J = 6.2 Hz, 2H), 3.53 (t, J = 6.6 Hz,
13
2H), 2.15 (dt, J = 14.5, 6.5 Hz, 2H), 2.05 (dt, J = 12.7, 6.3 Hz, 2H); C NMR (126
MHz, CDCl3) δ 165.28, 165.06, 136.95, 135.76, 129.30, 128.84, 127.64, 125.76,
111.20, 66.22, 33.56, 29.40, 27.65; MS (ESI) m/z ESI+ 435.1 [M+H]+.
5.1.14. (E,E)-2-(5-bromopentyloxy)-4,6-distyrylpyrimidine (4b)
1
White powder (207 mg, yield 46%); H NMR (500 MHz, CDCl3) δ 7.93 (s, 1H),
7.90 (s, 1H), 7.61 (d, J = 7.4 Hz, 4H), 7.40 (t, J = 7.3 Hz, 4H), 7.37-7.32 (m, 2H), 7.05
(s, 1H), 7.01 (s, 1H), 6.94 (s, 1H), 4.50 (t, J = 6.5 Hz, 2H), 3.46 (t, J = 6.8 Hz, 2H),
1.98 (dt, J = 15.3, 7.6 Hz, 2H), 1.92 (dd, J = 14.4, 7.0 Hz, 2H), 1.74-1.66 (m, 2H); 13C
NMR (126 MHz, CDCl3) δ 165.35, 165.02, 136.88, 135.78, 129.29, 128.84, 127.65,
125.80, 111.17, 67.00, 33.68, 32.60, 28.21, 24.88; MS (ESI) m/z ESI+ 449.1 [M+H]+.
5.1.15. (E,E)-2-(6-bromohexyloxy)-4,6-distyrylpyrimidine (4c)
1
White powder (143 mg, yield 31%); H NMR (500 MHz, CDCl3) δ 7.93 (s, 1H),
7.90 (s, 1H), 7.60 (d, J = 7.5 Hz, 4H), 7.40 (t, J = 7.3 Hz, 4H), 7.37-7.33 (m, 2H), 7.05
(s, 1H), 7.01 (s, 1H), 6.94 (s, 1H), 4.49 (t, J = 6.4 Hz, 2H), 3.48-3.40 (m, 2H), 1.91
(dd, J = 13.0, 6.5 Hz, 4H), 1.59 (d, J = 19.6 Hz, 4H); 13C NMR (126 MHz, CDCl3) δ
165.41, 165.00, 136.84, 135.79, 129.27, 128.84, 127.64, 125.83, 111.12, 67.21, 33.92,
32.75, 28.87, 28.01, 25.38; MS (ESI) m/z ESI+ 463.1 [M+H]+.
5.1.16 General procedure for 1d~f-4d~f
A mixture of compound 1a~c-4a~c (0.1 mmol) and thiourea (0.5 mmol) in
anhydrous acetone (1 mL) was heated to reflux under nitrogen for 12 h and monitored
with TLC. When the reaction was finished, the solvent was evaporated under reduce
pressure, and the resulting residue was purified by chromatography on silica gel to
15

ACCEPTED MANUSCRIPT
give compound 1d~f-4d~f.
5.1.17 (E,E)-4-(4,6-bis(4-methoxystyryl)pyrimidin-2-yloxy)butyl carbamimidoth-
ioate hydrobromide (1d)
1
Yellow powder (388 mg, yield 68%); H NMR (500 MHz, DMSO-d6 + D2O) δ
7.77 (s, 1H), 7.74 (s, 1H), 7.60 (t, J = 12.1 Hz, 4H), 7.17 (s, 1H), 6.98 (s, 1H),
6.96-6.92 (m, 5H), 4.36 (t, J = 5.7 Hz, 2H), 3.74 (s, 6H), 3.17 (t, J = 7.0 Hz, 2H),
1.87-1.81 (m, 2H), 1.77 (d, J = 6.9 Hz, 2H); 13C NMR (126 MHz, DMSO-d6 + D2O) δ
170.49, 165.29, 164.92, 160.73, 136.78, 129.77, 128.36, 123.68, 114.82, 110.92,
66.27, 55.65, 30.32, 27.57, 25.56; MS (ESI) m/z ESI+ 491.2 [M+H]+.
5.1.18. (E,E)-5-(4,6-bis(4-methoxystyryl)pyrimidin-2-yloxy) pentyl carbamimid-
othioate hydrobromide (1e)
1
Yellow powder (292 mg, yield 50%); H NMR (500 MHz, DMSO-d6 + D2O) δ
7.83 (s, 1H), 7.80 (s, 1H), 7.65 (d, J = 8.5 Hz, 4H), 7.24 (s, 1H), 7.05 (d, J = 16.0 Hz,
2H), 7.00 (d, J = 8.5 Hz, 4H), 4.39 (t, J = 5.8 Hz, 2H), 3.79 (s, 6H), 3.18 (t, J = 7.2 Hz,
13
2H), 1.83-1.75 (m, 2H), 1.73-1.65 (m, 2H), 1.56 (d, J = 6.5 Hz, 2H); C NMR (126
MHz, DMSO-d6 + D2O) δ 170.22, 165.33, 165.23, 160.78, 136.53, 129.76, 128.52,
124.06, 114.88, 110.95, 66.65, 55.74, 30.48, 28.61, 28.34, 25.04; MS (ESI) m/z ESI+
505.2 [M+H]+.
5.1.19. (E,E)-6-(4,6-bis(4-methoxystyryl)pyrimidin-2-yloxy)hexyl carbamimidot-
hioate hydrobromide (1f)
1
Yellow powder (316 mg, yield 53%); H NMR (500 MHz, DMSO-d6 + D2O) δ
7.80 (s, 1H), 7.77 (s, 1H), 7.62 (d, J = 8.1 Hz, 4H), 7.21 (s, 1H), 7.02 (s, 1H),
7.00-6.92 (m, 5H), 4.34 (s, 2H), 3.76 (s, 6H), 3.13-3.07 (m, 2H), 1.72 (s, 2H), 1.62 (s,
13
2H), 1.43 (s, 4H); C NMR (126 MHz, DMSO-d6 + D2O) δ 170.42, 165.30, 165.17,
160.74, 136.57, 129.76, 128.44, 123.92, 114.86, 110.90, 66.81, 55.70, 30.50, 28.65,
28.58, 27.89, 25.36; MS (ESI) m/z ESI+ 519.4 [M+H]+.
5.1.20. (E,E)-4-(4,6-bis(3,4-dimethoxystyryl)pyrimidin-2-yloxy)butyl carbamim-
idothioate hydrobromide (2d)
1
Yellow powder (321 mg, yield 51%); H NMR (500 MHz, DMSO-d6 + D2O) δ
7.79 (d, J = 15.8 Hz, 2H), 7.29 (s, 2H), 7.22 (d, J = 9.3 Hz, 3H), 7.07 (d, J = 15.8 Hz,
16

ACCEPTED MANUSCRIPT
2H), 6.98 (d, J = 7.9 Hz, 2H), 4.40 (s, 2H), 3.79 (d, J = 18.7 Hz, 12H), 3.21 (s, 2H),
13
1.83 (d, J = 30.3 Hz, 4H); C NMR (126 MHz, DMSO-d6 + D2O) δ 170.32, 165.35,
165.09, 150.55, 149.34, 137.03, 128.70, 124.07, 122.36, 112.11, 111.07,110.26, 66.20,
55.97, 30.31, 27.67, 25.71; MS (ESI) m/z ESI+ 551.1 [M+H]+.
5.1.21. (E,E)-5-(4,6-bis(3,4-dimethoxystyryl)pyrimidin-2-yloxy) pentyl carbami-
midothioate hydrobromide (2e)
1
Yellow powder (361 mg, yield 56%); H NMR (500 MHz, DMSO-d6 + D2O) δ
7.70 (s, 1H), 7.67 (s, 1H), 7.19 (s, 2H), 7.13 (d, J = 8.1 Hz, 2H), 7.09 (s, 1H), 6.97 (s,
1H), 6.95-6.90 (m, 3H), 4.29 (s, 2H), 3.72 (t, J = 18.8 Hz, 12H), 3.06 (t, J = 7.1 Hz,
13
2H), 1.69 (d, J = 6.6 Hz, 2H), 1.65-1.59 (m, 2H), 1.47 (d, J = 6.8 Hz, 2H); C NMR
(126 MHz, DMSO-d6 + D2O) δ 170.64, 165.23, 165.00, 150.35, 149.12, 136.97,
128.63, 123.89, 122.33, 111.94, 110.03, 66.75, 55.85, 30.50, 28.22, 28.12, 24.78; MS
(ESI) m/z ESI+ 565.3 [M+H]+.
5.1.22. (E,E)-6-(4,6-bis(3,4-dimethoxystyryl)pyrimidin-2-yloxy)hexyl carbamimi-
dothioate hydrobromide(2f)
1
Yellow powder (421 mg, yield 64%); H NMR (500 MHz, DMSO-d6 + D2O) δ
7.78 (d, J = 15.9 Hz, 2H), 7.29 (s, 2H), 7.20 (d, J = 8.3 Hz, 3H), 7.07 (d, J = 15.8 Hz,
2H), 6.97 (d, J = 7.2 Hz, 2H), 4.35 (s, 2H), 3.78 (d, J = 19.1 Hz, 12H), 3.11 (s, 2H),
1.72 (s, 2H), 1.61 (s, 2H), 1.43 (s, 4H); 13C NMR (126 MHz, DMSO-d6 + D2O) δ
170.03, 165.32, 165.24, 150.57, 149.39, 136.91, 128.75, 124.20, 122.30, 112.13,
110.91, 66.75, 55.98, 30.45, 28.71, 27.97, 26.89, 25.44; MS (ESI) m/z ESI+ 579.3
[M+H]+.
5.1.23. (E,E)-4-(4,6-bis(3,4,5-trimethoxystyryl)pyrimidin-2-yloxy)butyl carbami-
midothioate hydrobromide (3d)
1
Red powder (380 mg, yield 55%); H NMR (500 MHz, DMSO-d6 + D2O) δ 7.83
(s, 1H), 7.79 (s, 1H), 7.28 (s, 1H), 7.20 (s, 1H), 7.17 (s, 1H), 7.02 (s, 4H), 4.42 (t, J =
5.8 Hz, 2H), 3.82 (s, 12H), 3.68 (s, 6H), 3.23 (t, J = 7.1 Hz, 2H), 1.87 (dd, J = 14.0,
7.1 Hz, 2H), 1.81 (d, J = 6.9 Hz, 2H); 13C NMR (126 MHz, DMSO-d6 + D2O) δ
170.21, 165.24, 165.14, 153.52, 139.04, 137.13, 131.57, 125.73, 111.51, 105.50,
66.26, 60.61, 56.40, 30.26, 27.70, 25.78; MS (ESI) m/z ESI+ 611.1 [M+H]+.
17

ACCEPTED MANUSCRIPT
5.1.24. (E,E)-5-(4,6-bis(3,4,5-trimethoxystyryl)pyrimidin-2-yloxy)pentyl carbami-
midothioate hydrobromide (3e)
1
Red powder (470 mg, yield 67%); H NMR (500 MHz, DMSO-d6 + D2O) δ 7.80
(s, 1H), 7.77 (s, 1H), 7.27 (s, 1H), 7.18 (s, 1H), 7.15 (s, 1H), 7.00 (s, 4H), 4.38 (s, 2H),
3.82 (s, 12H), 3.67 (s, 6H), 3.15 (t, J = 7.1 Hz, 2H), 1.77 (s, 2H), 1.68 (d, J = 7.0 Hz,
2H), 1.55 (d, J = 6.4 Hz, 2H); 13C NMR (126 MHz, DMSO-d6 + D2O) δ 170.39,
165.23, 153.52, 139.07, 137.09, 131.58, 125.78, 111.42, 105.52, 66.81, 60.64, 56.42,
30.51, 28.46, 28.29, 24.97; MS (ESI) m/z ESI+ 625.4 [M+H]+.
5.1.25. (E,E)-6-(4,6-bis(3,4,5-trimethoxystyryl)pyrimidin-2-yloxy)hexyl carbami-
midothioate hydrobromide (3f)
1
Red powder (452 mg, yield 63%); H NMR (500 MHz, DMSO-d6 + D2O) δ 7.68
(s, 1H), 7.65 (s, 1H), 7.13 (s, 1H), 7.04 (s, 1H), 7.01 (s, 1H), 6.88 (s, 4H), 4.27 (s, 2H),
3.75 (s, 12H), 3.63 (s, 6H), 3.02 (t, J = 7.1 Hz, 2H), 1.64 (s, 2H), 1.55 (s, 2H), 1.35 (s,
4H); 13C NMR (126 MHz, DMSO-d6 + D2O) δ 170.66, 165.07, 165.03, 153.33,
138.84, 136.95, 131.57, 125.55, 111.35, 105.29, 66.98, 60.68, 56.29, 30.49, 28.53,
28.37, 27.79, 25.25; MS (ESI) m/z ESI+ 639.3 [M+H]+.
5.1.26. (E,E)-4-(4,6-distyrylpyrimidin-2-yloxy)butyl carbamimidothioate hydro-
bromide (4d)
1
Yellow powder (261 mg, yield 51%); H NMR (500 MHz, DMSO-d6 ) δ 9.07 (s,
4H), 7.91 (d, J = 16.0 Hz, 2H), 7.73 (d, J = 6.7 Hz, 4H), 7.50-7.34 (m, 7H), 7.25 (d, J
13
= 16.0 Hz, 2H), 4.45 (s, 2H), 3.28 (s, 2H), 1.86 (d, J = 40.1 Hz, 4H); C NMR (126
MHz, DMSO-d6 ) δ 170.27, 165.23, 136.95, 135.91, 129.88, 129.40, 128.17, 126.47,
111.74, 66.29, 30.30, 27.80, 25.9; MS (ESI) m/z ESI+ 431.3 [M+H]+.
5.1.27. (E,E)-5-(4,6-distyrylpyrimidin-2-yloxy)pentyl carbamimidothioate hydro-
bromide (4e)
1
Yellow powder (346 mg, yield 66%); H NMR (500 MHz, DMSO-d6 + D2O) δ
7.85 (d, J = 16.0 Hz, 2H), 7.67 (d, J = 7.5 Hz, 4H), 7.42 (t, J = 7.3 Hz, 4H), 7.39-7.35
(m, 2H), 7.33 (s, 1H), 7.18 (d, J = 16.0 Hz, 2H), 4.37 (t, J = 6.0 Hz, 2H), 3.14 (t, J =
7.1 Hz, 2H), 1.82-1.72 (m, 2H), 1.67 (dd, J = 14.4, 7.2 Hz, 2H), 1.53 (d, J = 6.8 Hz,
2H); 13C NMR (126 MHz, DMSO-d6 + D2O) δ 170.38, 165.20, 136.99, 135.73,
18

ACCEPTED MANUSCRIPT
129.96, 129.44, 128.53, 128.10, 126.28, 111.59, 66.84, 30.47, 28.47, 28.23, 24.91;
MS (ESI) m/z ESI+ 445.3 [M+H]+.
5.1.28. (E,E)-6-(4,6-distyrylpyrimidin-2yloxy)hexyl carbamimidothioate hydro-
bromide (4f)
1
Yellow powder (350 mg, yield 65%); H NMR (500 MHz, DMSO-d6 + D2O) δ
7.85 (d, J = 16.0 Hz, 2H), 7.68 (d, J = 7.2 Hz, 4H), 7.42 (t, J = 7.2 Hz, 4H), 7.38 (d, J
= 7.1 Hz, 2H), 7.33 (s, 1H), 7.19 (d, J = 16.0 Hz, 2H), 4.37 (t, J = 5.9 Hz, 2H), 3.11 (t,
J = 7.1 Hz, 2H), 1.73 (s, 2H), 1.62 (s, 2H), 1.43 (s, 4H); 13C NMR (126 MHz,
DMSO-d6 + D2O) δ 170.33, 165.23, 165.19, 136.96, 135.75, 129.95, 129.44, 128.52,
128.10, 126.31, 111.35, 66.98, 30.48, 28.64,28.61, 27.91, 25.37; MS (ESI) m/z ESI+
459.3 [M+H]+.
5.1.29. General procedure for Compounds 1g-3g
A mixture of compound 1a-3a (0.1 mmol) and tetramethylthiourea (0.5 mmol) in
anhydrous acetone (1 mL) was heated to reflux under nitrogen for 12 h and monitored
with TLC. When the reaction was finished, the solvent was evaporated under reduce
pressure, and the resulting residue was purified by chromatography on silica gel to
give compound 1g-3g.
5.1.30. (E,E)-2-(4-(4,6-bis(4-methoxystyryl)pyrimidin-2-yloxy)butyl)-1,1,3,3-tetr-
amethylisothiouronium hydrobromide (1g)
1
Red powder (330 mg, yield 53%); H NMR (500 MHz, DMSO-d6 + D2O) δ 7.78
(d, J = 15.8 Hz, 2H), 7.61 (d, J = 7.9 Hz, 4H), 7.21 (s, 1H), 7.01 (s, 1H), 6.96 (d, J =
8.1 Hz, 5H), 4.39 (s, 2H), 3.73 (d, J = 22.5 Hz, 6H), 3.18 (s, 12H), 3.07 (s, 2H), 1.84
(s, 2H), 1.75 (s, 2H); 13C NMR (126 MHz, DMSO-d6 + D2O) δ 174.78, 165.35,
165.01, 160.76, 136.72, 129.79, 128.38, 123.78, 114.86, 110.99, 66.30, 55.68, 43.72,
33.98, 27.60, 26.41; MS (ESI) m/z ESI+ 547.2 [M+H]+.
5.1.31. (E,E)-2-(4-(4,6-bis(3,4-dimethoxystyryl)pyrimidin-2-yloxy)-butyl)-1,1,3,3-
tetramethylisothiouronium hydrobromide (2g)
Red powder (376 mg, yield 55%); 1H NMR (500 MHz, CD3OD+ D2O) δ 7.80 (s,
1H), 7.77 (s, 1H), 7.24 (d, J = 1.9 Hz, 2H), 7.18 (d, J = 1.8 Hz, 1H), 7.16 (d, J = 1.8
Hz, 1H), 7.14 (s, 1H), 6.97 (d, J = 4.5 Hz, 2H), 6.94 (d, J = 3.0 Hz, 2H), 4.50 (t, J =
19

ACCEPTED MANUSCRIPT
5.9 Hz, 2H), 3.88 (s, 6H), 3.85 (s, 6H), 3.30 (s, 12H), 3.16 (t, J = 7.2 Hz, 2H), 1.96
(dd, J = 13.1, 6.2 Hz, 2H), 1.91 (dd, J = 14.6, 7.5 Hz, 2H); 13C NMR (126 MHz,
CD3OD+ D2O) δ 175.77, 165.52, 164.78, 150.69, 149.36, 137.16, 128.86, 123.12,
121.85, 111.36, 110.00, 109.87, 66.07, 55.04, 42.89, 33.95, 27.54, 26.37; MS (ESI)
m/z ESI+ 607.1 [M+H]+.
5.1.32. (E,E)-2-(4-(4,6-bis(3,4,5-trimethoxystyryl)pyrimidin-2-yloxy)-butyl)-
1,1,3,3-tetramethylisothiouronium hydrobromide (3g)
1
Red powder (388 mg, yield 52%); H NMR (500 MHz, DMSO-d6 + D2O) δ 7.77
(d, J = 15.9 Hz, 2H), 7.25 (s, 1H), 7.14 (d, J = 15.9 Hz, 2H), 6.97 (s, 4H), 4.40 (s, 2H),
3.80 (s, 12H), 3.64 (d, J = 18.9 Hz, 6H), 3.18 (s, 12H), 3.06 (dd, J = 16.0, 9.2 Hz, 2H),
13
1.84 (d, J = 6.3 Hz, 2H), 1.76 (d, J = 6.8 Hz, 2H); C NMR (126 MHz, DMSO-d6 +
D2O) δ 174.80, 165.22, 165.04, 153.47, 139.02, 137.14, 131.56, 125.64, 105.46, 66.41,
60.67, 56.40, 43.72, 33.99, 27.64, 26.41; MS (ESI) m/z ESI+ 667.2 [M+H]+.
5.2. Bioactivity Study
5.2.1. Cell culture
Both human colon cancer cells (HCT116 and HT29) and lung cancer cells
(H1299 and A549) were obtained from Shanghai Cell Bank of Chinese Academy of
Sciences (Shanghai, China). McCoy’s 5A medium was used for colon cancer cell
culture and RPMI 1640 medium was used for lung cancer cell culture, respectively.
Both media were supplemented with 5% fetal bovine serum (HyClone), penicillin
(100 units/mL), and streptomycin sulfate (100 µg/mL) (HyClone). Chinese hamster
ovary (CHO) cell lines (K1, 745, 677, 3.1, 3.2, 3.3 and 3.4) were described in detail
during the past by both Esko [26] and by us [24,27]. The CHO cells were cultured
with Ham's F-12 medium (Hyclone) supplemented with 10% FBS and antibiotics. All
the cells were cultured under an atmosphere of 5% CO2 and 100% relative humidity at
37 ºC, and passaged with trypsin every 2-4 days and revived periodically from frozen
stocks.
5.2.2 Antibodies
Anti-Golgin-97 (human) antibody was purchased from Life technologies, USA.
Golgin-97 is a trans-Golgi network peripheral membrane protein and localizes
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exclusively on the cytoplasmic face of the Golgi. Golgin-97 acts as an essential player
to the cell during the trafficking from the trans-Golgi network to the recycling
endosome and/or early endosome.
5.2.3 Cellular viability assay and IC50 calculation
All synthesized curcumin-pyrimidine analogues were dissolved in DMSO
(Solarbio, CN) in a final concentration of 0.1% initially and further diluted with cell
culture medium to desired mol/L concentrations for cellular viability assay.
For cellular viability assay, a 96-wells plate was seeded with 2000 cells/well in
100 µL complete cell culture medium. After 24 h, 100 µL complete medium
containing serial concentrations of each compound was added to each well. Either
cancer or CHO cells were cultured 48 h, followed by adding 20 µL of resazurin (2
mg/mL dissolved in water, catalog no. R7017-5G, Sigma) to the media for 16 h. The
fluorescent signal was monitored using 544 nm excitation wavelength and 595 nm
emission wavelength by Spectramax M5 plate reader (Molecular Devices). The
relative fluorescence unit (RFU) generated from the assay was proportional to the
number of living cells in each well [28]. The IC50 value of each drug was calculated
by the Logit approach.
5.2.4. Confocal analysis
The confocal analysis was performed according to Hao et al [29]. For confocal
imaging, A549 or HCT116 cells were seeded on glass coverslips in complete cell
culture media overnight before treated with or without the compounds at the
concentration of 10 µM for 4 h at 37 ºC. After that, the media were removed and the
cells were fixed with 4% formaldehyde in complete media for 15 min followed by
permeabilized with 0.2% Triton-100 in PBS for 30 min. After blocked with 5%
bovine serum albumin (BSA, BovoMax) for 60 min, the cells were incubated with
Golgin-97 primary antibody (Invitrogen) overnight followed by incubated with
fluorescent secondary antibody (LI-COR IRDye® 680LT) for 45 min. The nucleus
was stained with DAPI (Beyotime, CN) for 15 min. Among each steps, the cells were
washed with PBS for 3 times. The fluorescence of the compounds and secondary
antibody were measured at 488 nm and 680 nm excitation wavelength by Laser
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Scanning Confocal Microscope (Zeiss LSM 710, GER), respectively.
5.2.5. Statistical analysis
The statistical significance of differences between the groups was assessed by
Student’s t-test. P values < 0.05 were considered as statistically significant.
Acknowledgements
This research was supported by the Natural Science Foundation of China (Grant No.
81373322, 91129706 and 81502977), the Key International Cooperation Project of
CMST (Grant No. 2013DFG32160) and NSFC-Shandong Joint Fund (U1406402).
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Title Caption
Table 1. IC50 of the tested compounds in four human cancer cell lines.
Figure 1. Effects of the tested compounds on cell viability of four human cancer cell
lines.
Figure 2. Fluorescence images of A549 showing the location of Compounds.
Figure 3. Effects of the tested compounds on cytotoxicity to all the CHO cells and the lung cancer
cells A549.
Scheme 1. Synthesis of the isothiouronium derivatives of curcumin-pyrimidine analogs.
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Highlights:
1. Novel isothiouronium-modified pyrimidine-substituted curcumin analogs were
synthesized.
2. Isothiouronium modifications greatly enhanced anticancer activities of the analogs.
3. Only the fluorescence compound with isothiouronium modification showed unique Golgi
localization.
4. The isothiouronium analogs were novel Golgi staining compounds.
5. The isothiouronium analogs were useful in studying the biological functions of Golgi.