Synthesis and evaluation of antitumor activities of 4-selenopyrimidine derivatives

Article abstract of DOI:10.1080/15257770.2020.1833342

Pyrimidine antimetabolic agents are the essential drugs in treatment of various tumors. Novel synthesis and biological evaluation of the pyrimidine derivatives incorporating selenium element and amino acid carrier as potential antitumor agents have not been tried and studied. Based on the biological significance of pyrimidine structure, these two additional elemental fragments maybe enhance the antitumor effect and reduce toxic side effects of pyrimidine agents. The aim of this paper is to synthesis a series of 4-selenopyrimidine derivatives in order to find more potent lead compounds against cancer. In this study, 12 new 4-selenopyrimidine derivatives that are unstable in acidic solutions but very stable in alkaline and neutral solutions avoiding light were synthesized, and the antitumor activities on HepG2 cell lines of these compounds were evaluated by MTT assay. The results have shown that these compounds could reduce the proliferation of HepG2 cells in a dose-dependent fashion, and the inhibitory activity of compounds a6 was greater than that of positive control 5-fluorouracil (5-FU), the IC₅₀ for a6 was 3.63 μM. In the comprehensive analysis of the structure–activity relationship, we could draw the antitumor effect of selenouracil derivatives is stronger than those of selenothymine derivatives. These results suggest that the substituent groups of selenium element and amino acid on the pyrimidine derivatives are vital for their antitumor activities on HepG2 cells.

Full text of DOI:10.1080/15257770.2020.1833342

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Synthesis and evaluation of antitumor activities of
4-selenopyrimidine derivatives
Mingxing Shi , Libo Wang , Long Zhang , Kexin Wang , Hualin Zhang , Yajing
Wang , Chang Li & Weina Han
To cite this article: Mingxing Shi , Libo Wang , Long Zhang , Kexin Wang , Hualin Zhang ,
Yajing Wang , Chang Li & Weina Han (2020): Synthesis and evaluation of antitumor
activities of 4-selenopyrimidine derivatives, Nucleosides, Nucleotides & Nucleic Acids, DOI:
10.1080/15257770.2020.1833342
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
https://doi.org/10.1080/15257770.2020.1833342
Synthesis and evaluation of antitumor activities of
4-selenopyrimidine derivatives
a
ꢀ
a
ꢀ
a
a
a
Mingxing Shi , Libo Wang , Long Zhang , Kexin Wang , Hualin Zhang ,
a,b
a
a
Yajing Wang , Chang Li , and Weina Han
a
Department of Medicinal Chemistry and Natural Medicine Chemistry, College of Pharmacy,
b
Harbin Medical University, Harbin, PR China; Department of Pharmacology, College of
Pharmacy, Shenyang Pharmaceutical University, Shenyang, PR China
ABSTRACT
ARTICLE HISTORY
Pyrimidine antimetabolic agents are the essential drugs in
treatment of various tumors. Novel synthesis and biological
evaluation of the pyrimidine derivatives incorporating selen-
ium element and amino acid carrier as potential antitumor
agents have not been tried and studied. Based on the bio-
logical significance of pyrimidine structure, these two add-
itional elemental fragments maybe enhance the antitumor
effect and reduce toxic side effects of pyrimidine agents. The
aim of this paper is to synthesis a series of 4-selenopyrimidine
derivatives in order to find more potent lead compounds
against cancer. In this study, 12 new 4-selenopyrimidine deriv-
atives that are unstable in acidic solutions but very stable in
alkaline and neutral solutions avoiding light were synthesized,
and the antitumor activities on HepG2 cell lines of these com-
pounds were evaluated by MTT assay. The results have shown
that these compounds could reduce the proliferation of
HepG2 cells in a dose-dependent fashion, and the inhibitory
activity of compounds a6 was greater than that of positive
control 5-fluorouracil (5-FU), the IC50 for a6 was 3.63 lM. In
the comprehensive analysis of the structure–activity relation-
ship, we could draw the antitumor effect of selenouracil deriv-
atives is stronger than those of selenothymine derivatives.
These results suggest that the substituent groups of selenium
element and amino acid on the pyrimidine derivatives are
vital for their antitumor activities on HepG2 cells.
Received 27 June 2020
Accepted 4 October 2020
KEYWORDS
Selenium; amino acid; 4-
selenopyrimidine; synthe-
sis; antitumor
1
. Introduction
There are many kinds of antitumor drugs, among which 5-fluorouracil (5-
FU) is an essential pyrimidine antimetabolic drug in treatment of various
malignant tumors. At present, increased modification of structure of pyr-
imidine has led to the development with strategies to increase its anticancer
activity. Despite these advances, chemotherapy damage remains a
CONTACT Weina Han
hanweina@163.com
Department of Medicinal Chemistry and Natural Medicine
Chemistry, College of Pharmacy, Harbin Medical University, Harbin,150081, PR China
ꢀ
These authors contributed equally.
ß 2020 Taylor & Francis Group, LLC

2
M. SHI ET AL.
significant limitation to the clinical application of pyrimidine antimetabolic
drug. A series of novel pyrimidine structure derivatives were designed and
[
1–3]
synthesized with pyrimidine structure as a lead compound.
Novel syn-
thesis and biological evaluation of the pyrimidine derivatives incorporating
selenium element and amino acid carrier as potential antitumor agents has
not been tried and studied.
Selenium was one of the essential trace elements in the human body, which
induced many physiological and biochemical processes and has a wide range
[4,5]
of biological activities.
Due to its extensive biological activities, organic sel-
enium compounds have become hot spots in the research and development of
many fields such as prevention and treatment of tumors, maintenance of car-
diovascular and cerebrovascular diseases, immunoregulation, antivirals, anti-
[6,7]
bacteria, conditioning endocrine and protection of nerves.
The relationship
between selenium and tumor has been thick as thieves, and the results of com-
prehensive and multi-faceted investigation sited that the intake of selenium
[8]
was significantly negatively correlated with the incidence of tumors.
Malignant cells are more sensitive to the cytotoxicity of selenium than benign
cells and appear to be present at lower doses. It is reported that various forms
of selenium can be applied to liver, lung, prostate, cervix, human acute mye-
loid, breast, colorectal, melanoma and lymphocytic leukemia cells. Selenium is
[9]
used in combination therapy, enhancing the death chance of tumor cells.
That is to say, selenium has great potential for development as an antitumor
[10]
agent.
With the continuous development of life science and information
technology, selenium element will play more biological functions in the struc-
tural modification of leading compounds.
As an endogenous substance of the body, amino acids have no adverse
[
11]
reactions to the human body and no immunogenicity.
Amino acids have
excellent biocompatibility and affinity, and could be administered in a variety
of ways, easy to penetrate the membrane barrier of tumor cells, easily
[
12]
absorbed by multiple channels and not easy to display drug resistance.
According to the growth characteristics of tumor cells, in the process of pro-
liferation, invasion, migration, and other processes, etc. the required
nutrients such as amino acids are much higher than normal cells, enhancing
the selectivity to tumor cells and alleviating the toxic effects of drugs on nor-
[
13,14]
mal cells.
Modified with amino acids, the efficacy of the drug is guaran-
teed, and the solubility, dissolution and efficacy of drug could be further
improved, these derivatives also could reduce dosage, decrease toxic and side
[
15]
effects, increase bioavailability.
Amino acids have been often utilized as a
pharmaceutical carrier for structural modification to improve the physical
and chemical properties, biological properties, and so on.
This experiment was inspired by the 5-FU structure modification of pyr-
imidine anti-neoplastic agents. The following synthetic strategies: using

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
3
Scheme 1. Selenium amino amide derivatives.
uracil or thymine as the parent nucleus, and introducing the selenium
replace the oxygen atom of the pyrimidine carbonyl group according to
the principle of electron isosterism. It has been illustrated that the anti-
neoplastic activity with the four-position is better than two-position in the
previous experiments. At the same time, amino acids were introduced into
the carrier at N position, which participated in the design and synthesis of
precursor drugs due to its many advantages (Scheme 1). The above proper
and reasonable derivation of pyrimidine structural parent nucleus would
not only independently develop new antitumor agents, but also may bring
better antitumor effect.
Based on the biological significance of pyrimidine structure, the two add-
itional elemental fragments maybe enhance the antitumor effect and reduce
toxic side effects, etc. In this paper, 12 new 4-selenopyrimidine derivatives
were synthesized, and the antitumor activities on HepG2 cell lines of these
compounds were evaluated by MTT assay.
2
. Results and discussion
.1. Chemistry
The synthetic route and synthesis steps were determined by reverse synthesis
2
[16]
analysis.
Firstly, one-position of the pyrimidine was substituted with an
acetoxy group to gain pyrimidine acetic acid. Secondly, the methyl esterifica-
tion of amino acid followed with the acetyl as a bridge to introduce amino
acid carrier into amide. Finally, selenium replaces the carbonyl oxygen at the
four-position in the pyrimidine structure to give the target compounds
(Scheme 2). The intermediates and the final products were separated and
purified by recrystallization and silicone column chromatography. 12 new
compounds were synthesized and identified using chemical route and spec-
troscopy technology, including 4-selenouracil glycamide (a1), 4-selenouracil
leucamide (a2), 4-selenouracil alanamide (a3), 4-selenouracil methioamide
(a4), 4-selenouracil phenylalanamide (a5), 4-selenouracil isoleucamide (a6),

4
M. SHI ET AL.
Scheme 2. Synthesis route of 4-selenopyrimidine derivatives.
4-selenothymidine glycamide (b1), 4-Selenothymine leucamide (b2), 4-seleno-
thymidine alanamide (b3), 4-selenothymidine methioamide (b4), 4-selenothy-
midine phenylalanamide(b5), 4-selenothymidine isoleucamide (b6), the
content of each compound is over 95% determined by HPLC. Usually, the
compounds containing selenium were unstable, but we found selenium com-
pounds are relatively more stable through amino acid linkage.
Among the synthesized compounds, the white amide intermediates
changed into the final products of orange selenides, which was one of the
methods to prove the synthesis of the target compounds. In the synthesis
reaction, if original compounds were insoluble or slightly soluble in a solv-
ent, the reaction was difficult to carry out. In this experiment, amino acid
derivatives were difficult to change into amides. The amidation of amino
acid with selenopyrimidine used easily to remove solvent is not prospective.
At last, we choose DMF as the solvent of reaction. DMF has a strong solu-
bility in this experiment, but DMF needs to be removed by steaming at
ꢁ
8
0–90 C under reduced pressure with difficult post-treatment. So, we
firstly synthesized amidation of amino acid with pyrimidine, and then the
reaction of selenium replacing oxygen is carried out to avoid the degrad-
ation caused by the instability of selenium at high temperature. It appears
to be a long-term problem the reactions of amide bond formation have low
yield. The factors influencing are nothing more than the reactivity of the
carboxyl-activated state and the selectivity of the subsequent acylation reac-
tion. However, two reactions were not to exist simultaneously, and the
increase in reactivity often causes a decrease in selectivity, and an increase
in reaction selectivity may also result in a decrease in activity, which will
result in low yield or no reaction （Scheme 3）.
2
.2. Stability study in different pH
The stability of 12 new compounds were tested by UV spectrophotometer
at the same time every day under the conditions of pH ¼ 4, pH ¼ 7, and

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
5
.Scheme 3. 4-Selenopyrimidine derivative structure.
pH ¼ 11. It was found that the acid solution of the compounds(pH ¼ 4)
were unstable, but they were very stable in the alkaline(pH ¼ 11) and
neutral(pH ¼ 7) solutions which were protected from light (Figures 1–4).
In alkaline and neutral solutions which are dark from light, the images
almost overlap when the ultraviolet of compounds were measured the next
day. However, when the solutions were not dark, the degradation would
occur. With the increase of the pH value of the solution, even if the com-
pounds were not protected from light, the stability will increase. The acidic
solutions (pH ¼ 4) were unstable regardless of whether they were protected
from light or not. These results were consistent with the result of purifica-
tion of the target products. When we use acidic silica gel flash column
chromatography, the loss of selenium was very large. After purification, the
orange powder of the compounds were very stable, and the prepared solu-
ꢁ
tions were also very stable when stored at ꢂ20 C. However, the solution of
selenopyrimidine derivatives before amino acid substitutions were very

6
M. SHI ET AL.
Figure 1. The stability of compounds a1–a3 were tested by UV spectrophotometer.
Figure 2. The stability of compounds a4–a6 were tested by UV spectrophotometer.

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
7
Figure 3. The stability of compounds b1–b3 were tested by UV spectrophotometer.
Figure 4. The stability of compounds b4–b6 were tested by UV spectrophotometer.

8
M. SHI ET AL.
Table 1. Proliferatory activities of the target compounds against HepG2 cells.
a
a
Compounds
a1
R
1
IC50(mM)
Compounds
b1
R
2
IC50 (mM)
ꢂH
5.99 ± 0.61
ꢂH
45.40 ± 0.33
a2
5.5 0 ± 0.54
b2
30.10 ± 0.65
a3
a4
ꢂCH
3
8.19 ± 0.46
5.92 ± 0.38
b3
b4
ꢂCH
3
35.58 ± 0.23
19.88 ± 0.41
a5
a6
6.63 ± 0.24
b5
b6
18.20 ± 0.22
26.41 ± 0.31
3.63 ± 0.45
7.70 ± 0.36
5
-FU
a
IC50: concentration of the test compound that inhibits 50% of cell growth. Results are expressed as the
mean ± SD (n ¼ 6)
unstable and easy to oxidize. Therefore, it is believed that amino acid sub-
stitution plays a good role in the stability of s elenopyrimidine.
2
.3. The inhibition on HepG2 of 4-selenopyrimidine derivatives
The screening was performed in 96-well microtiter plates maintained at
ꢁ
3
7 C. The absorbance value (OD value) at a wavelength of 490 nm was
measured by an enzyme-linked immunosorbent assay. The corresponding
compound IC50 values (Table 1) and the inhibition rate were calculated
(Figures 5 and 6).
As shown from the data in the observation (Figure 1), the inhibitory
effect of the compounds a1–a6 on the hepatoma cell HepG2 at a concentra-
ꢂ3
ꢂ3
ꢂ1
ꢂ1
tion of 1.00 ꢃ 10 , 5.00 ꢃ 10 , 1.25 ꢃ 10 , 6.25 ꢃ 10 , 3.125 lg/mL was
observed, the inhibition effect was gradually enhanced. As shown from the
data in the observation (Figure 2), the effect of compounds b1–b6 on hepa-
ꢂ1
ꢂ1
toma cell HepG2 at a concentration of 3.125 ꢃ 10 , 6.25 ꢃ 10 , 1.25,
2
.50, 5.00, 10.00 lg/mL was changed from promotion to inhibition with
increasing concentration. the inhibition effect was gradually enhanced. On
the whole, selenothymine derivatives have stronger antitumor effect than
selenouracil derivatives. The inhibition rates of compounds a1, a4, a6 and
b5 on HepG2 cells in a certain concentration range could reach 70% ꢄ
90%. Compound a6 displayed the best antiproliferative activities against

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
9
Figure 5. inhibition rate of HepG2cell proliferation of compounds a1–a6 (mean ± SD, n ¼ 6).
Figure 6. Inhibition rate of HepG2 cells proliferation of compounds b1–b6 (mean ± SD, n ¼ 6).
Figure 7. Inhibition rate of HepG2 cells proliferation of 10 mM compounds a1–b6
mean ± SD, n ¼ 4).
(

1
0
M. SHI ET AL.
Table 2. Calculated theoretical parameters of the Lipinski’s rule for compounds a1–b6.
Lipinski’s
rule
Entries
Compounds
M.W.(g/mol)
Log P
TPSA
HA
HD
violations
CMR
1
2
3
4
5
6
7
8
9
a1
a2
a3
a4
a5
a6
b1
b2
b3
b4
b5
b6
304.16
360.27
318.19
378.31
394.28
360.27
318.19
374.29
332.21
392.33
408.31
374.29
ꢂ1.1169
0.64908
ꢂ0.80792
ꢂ0.6599
0.61008
0.64908
ꢂ0.61792
1.14808
ꢂ0.30892
ꢂ0.16092
1.10908
1.14808
87.74
87.74
87.74
87.74
87.74
87.74
87.74
87.74
87.74
87.74
87.74
87.74
6
6
6
7
6
6
6
6
6
7
6
6
2
2
2
2
2
2
2
2
2
2
2
2
0
0
0
0
0
0
0
0
0
0
0
0
6.4976
8.3528
6.9614
8.6953
9.4726
8.3528
6.9614
8.8166
7.4252
9.1591
9.9364
8.8166
1
1
1
0
1
2
HepG2 cells with an IC50 value of 3.63 mM, respectively, which is less than
-FU to HepG2 cells IC .
5
5
0
In the MTT assay, it showed that some synthesized target compounds
were better than 5-FU in inhibiting HepG2 cell proliferation in a certain
concentration range (Figures 5 and 6). In addition, among the compounds
we synthesized, the antitumor effect of selenouracil derivatives were stron-
ger than those of selenothymine derivatives in MTT assay (Figure 7).
Selenium could provide nutrition for body at low concentration while sel-
enium becomes a typical oxidant with high cytotoxic activity at high dose
(Figures 1 and 2). We consider that unstable selenopyrimidine derivatives
could decompose and oxidize to obtain other selenium compounds. This
may be related to the effect of the target compounds on promoting the
growth of HepG2 cells at its corresponding low concentration and inhibit-
ing the growth of HepG2 cells at its high concentration in this experiment.
Meanwhile, the metabolic antagonism of selenopyrimidine derivatives was
another cause of antitumor.
Finally, an in silico drug-likeness evaluation was performed on the
studied compounds using ChemDraw Ultra software. The study consisted
in the calculation of Lipinski’s parameters for the 12 derivatives, which can
predict oral bioavailability and membrane permeability of synthetic com-
pounds. It is important because Lipinski’s rules help to describe the
molecular properties related to the pharmacokinetics in human such as
[
17]
absorption, distribution, metabolism, and excretion (ADME).
The results
are summarized in Table 2. In general, all tested derivatives displayed good
bioavailability properties, with a molecular weight <500 g/mol, The best
range of logP is 0–3, the water solubility of high logP compound is poor
and the permeability of low logP compound is poor. This high LogP may
compromise the absorption properties of these compounds in the biological
[
18]
system due its limited solubility in water.
Acceptor and donor hydrogen
bond interactions below 10 and 5. To complement our in silico evaluation,

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
11
we calculated other molecular descriptors such as the topological polar sur-
face area. This evaluation reflected the potential intestinal absorption, bio-
availability, and hematoencephalic barrier permeation of our compounds
[
19]
according to Veber’s rules (TPSA < 140).
In conclusion, an in silico
drug-likeness evaluation performed well on the synthetic compounds.
Compound a6, which has a half inhibition concentration of 3.63lM in the
experiment, shows a good inhibition activity, so it is a good lead compound.
We can determine the mode of its action target for the next step of the study
on the mechanism of action, so as to further optimize the structure.
3
. Conclusions
The contents of synthesized and identified 12 new compounds were over
5% by HPLC. Usually, the compounds containing selenium were unstable,
9
but we found selenium compounds are relatively more stable through amino
acid linkage, especially in neutral and alkaline solutions of avoiding light.
This means that the introduction of amino acids can increase the stability of
selenopyrimidine. All of the synthesized target compounds showed the inhib-
ition of HepG2 cells growth at a certain concentration range, and some of
the compounds showed a dose-dependent manner. Among them, the com-
pounds a1, a4, a6 and b5 have a cell inhibition rate of 70–90% against
HepG2. The IC50 of compound a6 was 3.63 lM, which was less than the
minimum value of 5-FU to HepG2 cells IC . Compound a6 with good
50
drug-likeness evaluation will optimize the experimental object next step.
4
. Experimental
4
.1. General methods
All chemicals and materials are from commercial suppliers without further
purification. The reaction was monitored by thin layer chromatography
(
TLC) and was carried out on plates using 2 mm layers of silica gel (Merck
GF254). All solvents were removed under reduced pressure. Column chro-
matography separation was carried out using a column packed with
1
13
2
00–300 mesh silica gel. H NMR spectra and C NMR spectra were car-
ried out on a Bruker AMX-600 NMR spectrometer in dimethyl sulfoxide
DMSO)-d as the solvent at room temperature.
(
6
4
.2. Synthesis of uracil-1-yl acetic acid
0
.02 mol of uracil was dissolved in 40 mL of a 2.50 M NaOH solution
ꢁ
(
water) with heating temperature for 50 C, after 10 min, 25 mL bromoace-
tic acid (1.02 mol/L) was slowly added dropwise in about 30 mins. TLC

1
2
M. SHI ET AL.
tracking detection method was used for identification with silica gel GF254
plate and V (dichloromethane): V (methanol) ¼ 9 : 1 as a developing solv-
ent. After about 6–7 h at this temperature, when the origin uracil is con-
sumed, the mixture is allowed to stand at room temperature. Adjust the
pH to about 2 with hydrochloric acid, cool and crystallize to obtain a pre-
cipitate. It was washed with distilled water and dried to give a white solid.
1
Intermediate: uracil acetic acid, white solid 2.37 g, 76.85%. H-
NMR(600 MHz, DMSO-d ), d 13.13 (1H, s), 11.34 (1H, s), 7.61 (1H, d,
6
1
3
J ¼ 6.0 Hz), 5.59 (1H, d, J ¼ 6.0 Hz), 4.41 (2H, s). C-NMR (151 MHz,
DMSO-d ) d 169.94 (–COOH), 164.16 (C-4), 151.37 (C-2), 146.43 (C-5),
6
0
þ
1
01.23 (C-6), 48.95 (C-1 ). ESI-MS m/z: 170.03 [M þ H]
4
.3. Synthesis of thymine acetic acid
About 0.02 mol of thymidine was dissolved in 40 mL of 2.32 mol/L NaOH
ꢁ
solution (water), with a heating temperature of 50 C and stirring 10 min,
25 mL of 10 mL 1.08 mol/L aqueous solution of bromoacetic acid was
slowly added dropwise, and the mixture was dropped in about 30 min.
Control the reaction at this temperature for about 6–7 h, TLC tracking
detection, the developing agent is V (dichloromethane): V (methanol) ¼
6:1. After the thymine raw material is consumed, the mixture is allowed to
stand at room temperature. The pH was adjusted to about 2 with hydro-
chloric acid. The mixture was cooled, and the precipitate was precipitated,
washed with distilled water and dried to give a white solid.
1
Intermediate: thymine acetic acid, white solid 2.45 g, 66.56%. H-NMR
(
600 MHz, DMSO-d ) d 13.11 (1H, s), 11.34 (1H, s), 7.49 (1H, s), 4.37 (2H,
6
1
3
s), 1.75 (3H,s). C-NMR (151 MHz, DMSO-d ) d 170.03 (–COOH), 164.73
6
0
0
(
C-4), 151.36 (C-2), 142.18 (C-6), 108.69 (C-5), 48.77 (C-1 ), 12.27 (C-5 ).
þ
ESI-MS m/z: 184.04 [M þ H]
4
.4. Synthesis of amino acid methyl ester hydrochloride
About 0.01–0.03 mol of L-amino acid (glycine, leucine, alanine, methionine,
phenylalanine, isoleucine) were added to the reaction bottle and 25 mL
water-free methanol was added. Under the cooling of ice water bath, slowly
ꢁ
add (0.03–0.06) mol of thionyl chloride at 8–10 C, continue to react for
1
–2 h after the addition, and then slowly reflux for 4–5 h. TLC tracking
detection, the developing agent was V (dichloromethane): V (methanol) ¼
:1, and ninhydrin was developed. After the reaction was completed, the
6
solvent was distilled off with an excess of thionylchloride, and then recrys-
tallized from methylene chloride and petroleum ether to give a white solid.

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
13
1
Intermediate: glycine methyl ester, white solid 4.14 g, 96.54%. H-NMR
1
3
(
(
600 MHz, DMSO-d ) d 8.66 (3H, s), 3.77 (2H, s), 3.73 (3H, s). C-NMR
6
0
151 MHz, DMSO-d ) d 168.36 (C-1), 52.85 (C-1 ), 39.78 (C-2). ESI-MS m/
6
þ
z: 125.55 [M þ H]
1
Intermediate: leucine methyl ester, white solid 1.92 g, 88.34%. H-NMR
(
(
(
2
600 MHz, DMSO-d ) d 8.74 (3H, s), 3.91 (1H, t, J ¼ 7.1, J ¼ 14.1 Hz), 3.74
6
1
3
3H, s), 1.77 (1H, m), 1.67 (2H, m), 0.89 (6H, d, J ¼ 6.48 Hz). C-NMR
0
151 MHz, DMSO-d ) d 170.64(C-1), 53.05 (C-2), 50.84 (C-1 ), 39.48 (C-3),
6
0
þ
4.08 (C-4), 22.53 (C-4 ), 22.34 (C-5). ESI-MS m/z: 181.66 [M þ H]
1
Intermediate: alanine methyl ester, white solid 3.50 g, 96.68%. H-NMR
(
600 MHz,DMSO-d ) d 8.74 (3H, s), 4.05 (1H, m), 3.73 (3H, s), 1.43 (3H,
6
1
3
0
d, J ¼ 7.2 Hz) C-NMR (151 MHz, DMSO-d ) d 170.74 (C-1), 53.11 (C-1 ),
6
þ
4
8.13 (C-2), 16.02 (C-3). ESI-MS m/z: 139.58 [M þ H]
1
Intermediate: methionine methyl ester, white solid 1.78 g, 89.13%. H-
NMR (600 MHz, DMSO-d ) d 8.82 (1H, s), 4.08 (1H, t, J ¼ 6.1, 9.2 Hz),
6
1
3
3
1
.75 (3H, s), 2.63 (2H, m), 2.05 (3H, s). C-NMR (151 MHz, DMSO-d ) d
6
69.98 (C-1), 53.20 (C-2), 51.16 (C-1 ), 29.69 (C-3), 14.61 (C-3 ). ESI-MS
0
0
þ
m/z: 199.7 [M þ H]
1
Intermediate: phenylalanine methyl ester, white solid 3.02 g, 93.35%. H-
NMR (600 MHz, DMSO-d ) d 8.85 (3H, s), 7.31 (5H, m), 4.21 (1H, m),
6
1
3
3
1
5
.64 (3H, s), 3.18 (2H, m). C-NMR (151 MHz, DMSO-d ) d 169.71 (C-1),
6
35.18 (ph-1), 129.77 (ph-2, ph-6), 128.95 (ph-3, ph-4), 127.60 (ph-5),
0
þ
3.65 (C-2), 52.87 (C-1 ), 36.19 (C-3). ESI-MS m/z: 251.68 [M þ H]
1
Intermediate: isoleucine methyl ester, white solid 2.25 g, 82.57%. H-
NMR (600 MHz, DMSO-d ) d 8.62 (3H, s), 3.90 (1H, d, J ¼ 4.1 Hz), 3.74
6
1
3
(
3H, s), 1.94 (1H, m), 1.37 (2H, m), 0.89 (6H, m). C-NMR (151 MHz,
DMSO-d ) d 169.68 (C-1), 56.38 (C-2), 52.90 (C-1 ), 36.26 (C-3), 25.67 (C-
), 14.66 (C-3 ), 11.84 (C-5). ESI-MS m/z: 181.66 [M þ H]
0
6
0
þ
4
4
.5. Synthesis of uracil amino acid amide
About 3–5 mmol uracil acetic acid and 3–5 mmol 1-hydroxybenzotriazole
HOBt) were added to 100 mL two-diameter bottle at room temperature.
5–20 mL N,N-dimethylformamide (DMF) was dissolved, 3–5 mmol of vari-
ous amino acid methyl ester hydrochloride was added, and after dissolution,
1.2–1.8) mL of triethylamine (TEA) was added and stirring was continued.
(
1
(
Further, 4–7 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride (EDCI) and 10mL of DMF were added to dissolve, and the reaction
was carried out at room temperature. TLC tracking detection, the developing
agent was V (dichloromethane): V (methanol) ¼ 7:1. After the reaction was
completed, it was filtered, and the solvent DMF was evaporated under vacuo
to give a viscous yellow oil. The dry method is purified by silica gel column,
and the eluent is pure dichloromethane, V (dichloromethane): V (methanol)

1
4
M. SHI ET AL.
¼
40:1, 30:1, 20:1, respectively and the solvent is evaporated under reduced
pressure to obtain a solvent. White solid.
1
Intermediate: uracil glycine amide, white solid 0.50 g, 62.85%. H-NMR
(
7
600 MHz, DMSO-d ) d 11.28 (1H, s), 8.64 (1H, t, J ¼ 5.8 Hz, J ¼ 11.7 Hz),
6
.55 (1H, d, J ¼ 7.7 Hz), 5.56 (1H, d, J ¼ 7.7 Hz), 4.40 (2H, s), 3.90 (2H, d,
13
0
J ¼ 5.8 Hz), 3.64 (3H, s). C-NMR (151 MHz, DMSO-d ) d 170.43 (C-1 ),
6
1
67.86 (C-8), 164.26 (C-4), 151.36 (C-2), 146.88 (C-6), 100.98 (C-5), 52.15
0
þ
(
C-7), 49.58 (C-2 ), 40.94 (–OCH ). ESI-MS m/z:241.07 [M þ H]
3
1
Intermediate: uracil leucine amide, white solid 0.70 g, 70.74%. H-NMR
(
600 MHz, DMSO-d ) d 11.26 (1H, s), 8.60 (1H, d, J ¼ 7.8 Hz), 7.54 (1H, d,
6
J ¼ 7.9 Hz), 5.55 (1H, d, J ¼ 7.9 Hz), 4.38 (2H, s), 4.32 (1H, m), 3.63 (3H,
1
3
s), 1.63 (1H, m), 1.52 (2H, m), 0.87 (6H, dd, J ¼ 6.6, 12.0 Hz). C-NMR
0
(
151 MHz, DMSO-d ) d 173.07 (C-1 ), 167.41 (C-8), 164.25 (C-4), 151.32
6
0
(
C-2), 147.01 (C-6), 100.81 (C-5), 52.9 (C-7), 50.69 (C-2 ), 49.55 (–OCH ),
3
0
0
00
0
4
2
0.41 (C-3 ), 24.51 (C-4 ), 23.08 (C-4 ), 21.73 (C-5 ). ESI-MS m/z:
þ
97.13 [M þ H]
1
Intermediate: uracil alanine amide, white solid 0.36 g, 42.38%. H-NMR
(
600 MHz, DMSO-d ) d 11.27 (1H, s), 8.66 (1H, d, J ¼ 7.1 Hz), 7.54 (1H, d,
6
J ¼ 7.8 Hz), 5.55 (1H, d, J ¼ 7.8 Hz), 4.37 (2H, s), 4.30 (1H, m), 3.63 (3H,
1
3
m), 1.29 (3H, d, J ¼ 7.2 Hz). C-NMR (151 MHz, DMSO-d ) d 173.16 (C-
6
0
1
5
2
), 167.16 (C-8), 164.24 (C-4), 151.34 (C-2), 146.98 (C-6), 100.84 (C-5),
0
00
2.32 (C-7), 49.44 (C-2 ), 48.03 (–OCH ), 17.50 (C-2 ). ESI-MS m/z:
55.08 [M þ H]
3
þ
1
Intermediate: uracil methionine amide, white solid 0.55 g, 51.19%. H-
NMR (600 MHz, DMSO-d ) d 11.27 (1H, s), 8.64 (1H, d, J ¼ 7.8 Hz), 7.55
6
(
(
1H, d, J ¼ 7.1 Hz), 5.56 (1H, d, J ¼ 7.1 Hz), 4.43 (1H, m), 4.39 (2H, s), 3.64
13
3H, s), 2.48 (2H, m), 2.04 (3H, s), 1.94 (2H, m). C-NMR (151 MHz,
0
DMSO-d ) d 172.33 (C-1 ), 167.54 (C-8), 164.24 (C-4), 151.35 (C-2), 146.96
6
0
0
(
C-6), 100.88 (C-5), 52.43 (C-2 ), 51.31 (C-7), 49.65 (–OCH ), 31.14 (C-4 ),
3
0
þ
2
9.71 (–SCH ), 14.93 (C-3 ). ESI-MS m/z: 315.09 [M þ H]
3
1
Intermediate: uraci phenylalanine amide, white solid 0.60 g, 54.41%. H-
NMR (600 MHz, DMSO-d ) d 11.26 (1H, s), 8.72 (1H, d, J ¼ 7.6 Hz), 7.46
6
(
1H, d, J ¼ 7.8 Hz), 7.25 (5H, m), 5.53 (1H, d, J ¼ 7.8 Hz) , 4.48 (1H, m),
1
3
4
1
1
1
.35 (2H, s), 3.59 (3H, s), 2.95 (2H, m). C-NMR (151 MHz, DMSO-d ) d
6
0
71.99 (C-1 ), 167.41 (C-8), 164.22 (C-4), 151.30 (C-2), 146.91 (C-6),
37.24(ph-1), 129.51 (ph-2, ph-6), 128.68 (ph-3, ph-4), 127.02 (ph-5),
0
þ
0
00.82 (C-5), 54.19 (C-2 ), 52.27 (C-7), 49.47 (–OCH ), 37.17 (C-3 ). ESI-
3
MS m/z: 331.17 [M þ H]
1
Intermediate: uracil isoleucine amide, white solid 0.43 g, 43.48%. H-
NMR (600 MHz, DMSO-d ) d 13.72 (1H,s), 8.55 (1H, d, J ¼ 8.4 Hz), 7.54
6
(
(
1H, d, J ¼ 7.8 Hz), 5.54 (1H, d, J ¼ 7.8 Hz), 4.42 (2H, s), 4.26 (1H, m), 3.64
13
3H, s),1.61 (1H, m), 1.18 (2H, m), 0.84 (6H, m). C-NMR (151 MHz,

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
15
0
DMSO-d ) d 172.16 (C-1 ), 167.53 (C-8), 164.25 (C-4), 151.33 (C-2), 147.10
6
0
0
(
C-6), 100.72 (C-5), 56.75 (C-7), 52.10 (C-2 ), 49.56 (–OCH ), 37.01 (C-3 ),
3
5.06 (C-4 ), 15.74 (C-3 ), 11.50 (C-5 ). ESI-MS m/z: 297.13 [M þ H]
0
00
0
þ
2
4
.6. Synthesis of thymine amino acid amide
About 3–5 mmol thymidine acetic acid and 3–5 mmol 1-hydroxybenzotria-
zole (HOBt) were added to 100 mL two-diameter bottle at room tempera-
ture. (15–20) mL DMF was dissolved, (3 ꢄ 5) mmol of amino acid methyl
ester hydrochloride was added, and after dissolution, (1.2–1.8) mL of trie-
thylamine (TEA) was added and stirring was continued. Further, (4–7)
mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(
EDCI) and 10 mL of DMF were added and the mixture was reacted at
room temperature. TLC tracking detection, the developing agent was V
(
dichloromethane): V (methanol) ¼ 8:1. After the reaction was completed,
it was filtered, and the solvent DMF was evaporated under vacuo to give a
viscous yellow oil. Drying over silica gel column eluting with EtOAc.
1
Intermediate: thymidine glycine amide, white solid 0.50 g, 58.86%. H-
NMR (600 MHz, DMSO-d ) d 11.27 (1H, s), 8.61 (1H, dd, J ¼ 5.8, 11.7 Hz),
6
7
.43 (1H, s), 4.35 (2H, s), 3.89 (2H, d, J ¼ 6.42 Hz), 3.64 (3H, s), 1.75 (3H,
13
0
s). C-NMR (151 MHz, DMSO-d ) d 170.43 (C-1 ), 167.95 (C-8), 164.82
6
0
(
C-4), 151.34 (C-2), 142.61 (C-6), 108.42 (C-5), 52.14 (C-7), 49.43 (C-2 ),
þ
40.93 (–OCH ), 12.29(5-CH ). ESI-MS m/z: 255.08 [M þ H]
3
3
1
Intermediate: thymidine leucine amide, white solid 0.40 g, 38.61%. H-
NMR (600 MHz, DMSO-d ) d 11.26 (1H, s), 8.59 (1H, d, J ¼ 7.8 Hz), 7.43
6
(
1
1H, s), 4.34 (2H, s), 4.32 (1H, m), 3.64 (3H, s), 1.76 (3H, s), 1.64 (1H, m),
1
3
.53 (2H, m), 0.87 (6H, dd, J ¼ 6.6, 12.7 Hz). C-NMR (151 MHz, DMSO-
0
d ) d 173.09 (C-1 ), 167.51 (C-8), 164.81 (C-4), 151.31 (C-2), 142.77 (C-6),
6
0
1
2
3
08.23 (C-5), 52.28 (C-7), 50.69 (C-2 ), 49.39 (–OCH ), 24.52 (5-CH ),
3
3
0
0
00
0
3.07 (C-3 ), 21.74 (C-4 ), 12.27 (C-4 , C-5 ). ESI-MS m/z:
þ
11.14 [M þ H]
1
Intermediate: thymidine alanine amide, white solid 0.50 g, 55.80%. H-
NMR (600 MHz, DMSO-d ) d 11.26 (1H, s), 8.63 (1H, d, J ¼ 7.0 Hz), 7.42
6
(
1H, s), 4.33 (2H, s), 4.29 (1H, m), 3.63 (3H, s), 1.75 (3H, s), 1.29 (3H, d,
1
3
0
J ¼ 7.3 Hz). C-NMR (151 MHz, DMSO-d ) d 173.17 (C-1 ), 167.25 (C-8),
6
1
64.80 (C-4), 151.33 (C-2), 142.74 (C-6), 108.27 (C-5), 52.32 (C-7), 49.28
0
00
(
C-2 ), 48.02 (–OCH ), 17.49 (5-CH ), 12.27 (C-2 ). ESI-MS m/z:
3 3
þ
2
69.10 [M þ H]
Intermediate: thymidine methionine amide, white solid 0.55 g, 50.19%.
1
H-NMR (600 MHz, DMSO-d ) d 11.27 (1H, s), 8.62(1H, d, J ¼ 7.7 Hz),
6
7
.43 (1H, s), 4.42 (1H, s), 4.35 (2H, s), 3.64 (3H, s), 2.48 (2H, m), 2.04
1
3
(
3H, s), 1.92 (2H, m), 1.75 (3H, s). C-NMR (151 MHz, DMSO-d ) d
6

1
6
M. SHI ET AL.
0
1
1
(
72.34 (C-1 ), 167.64 (C-8), 164.81 (C-4), 151.33 (C-2), 142.72 (C-6),
0
0
08.30 (C-5), 52.42 (C-2 ), 51.31 (C-7), 49.49 (–OCH ), 31.11 (C-4 ), 29.72
3
0
þ
5-CH ), 14.93 (C-3 ), 12.28 (–SCH ). ESI-MS m/z: 329.11 [M þ H]
3
3
Intermediate: thymidine phenylalanine amide, white solid 0.60 g, 52.21%.
1
H-NMR (600 MHz, DMSO-d ) d 11.25 (1H,s), 8.68 (1H, d, J ¼ 7.3 Hz),
6
7
(
.43 (1H, s), 7.26 (5H, m), 4.45 (1H, m), 4.31 (2H, s), 2.96 (2H, m), 1.74
1
3
0
3H, s), 1.09 (3H, s). C-NMR (151 MHz, DMSO-d ) d 171.52 (C-1 ),
6
1
6
2
3
67.48 (C-8), 164.79 (C-4), 151.29 (C-2), 142.64 (ph-1), 137.24 (ph-2, ph-
), 129.56 (ph-3, ph-4), 128.65 (ph-5), 127.00 (C-6), 108.26 (C-5), 60.95 (C-
0
0
), 54.25 (C-7), 49.30 (C-3 ), 14.29 (–OCH ), 12.27 (5-CH ). ESI-MS m/z:
3
3
þ
45.13 [M þ H]
1
Intermediate: thymidine isoleucine amide, white solid 0.65 g, 62.74%. H-
NMR (600 MHz, DMSO-d ) d 11.25 (1H, s), 8.53,(1H, d, J ¼ 8.2 Hz), 7.42
6
(
1H, s), 4.35 (2H, s), 4.25 (1H, m), 3.64 (3H, s), 1.77 (1H, m), 1.74 (3H, s),
1
3
1
1
5
1
.30 (2H, m), 0.84 (6H, m). C-NMR (151 MHz, DMSO-d ) d 172.16 (C-
6
0
), 167.61 (C-8), 164.81 (C-4), 151.32 (C-2), 142.88 (C-6), 108.14 (C-5),
6.74 (C-7), 52.09 (C-2 ), 49.38 (–OCH ), 37.00 (C-3 ), 25.09 (5-CH ),
5.75 (C-4 ), 12.26 (C-3 ), 11.50 (C-5 ). ESI-MS m/z: 311.14 [M þ H]
0
0
3
3
0
00
0
þ
5
. Synthesis of NaHSe
Selenium powder 0.01 mol, sodium borohydride NaBH (6–8) mmol was
4
placed in a reaction flask, sealed, vacuumed, added with 5 mL of absolute
ethanol, and stirred in an ice water bath for 40–50 min.
5
.1. Synthesis of selenouracil amino acid amide
About 1 mmol of various uracil amino acid amides and 2.5 mmol of 4-
dimethylaminopyridine (DMAP) were placed in a reaction flask, sealed, an
vacuumed under an argon atmosphere to add 1.2 mL of triethylamine
(
TEA), then weighed 2,4,6-triisopropylbenzenesulfonyl chloride (TPS-Cl)
1
2
mmol small amount of anhydrous dichloromethane dissolved (about
mL) was added to the reaction flask, and then add appropriate amount of
solvent 2–4 mL Stir for 60 min. TLC tracking detection, the developing
agent is V (dichloromethane): V (methanol) ¼ 7:1, after the activation is
completed, NaHSe obtained by the reaction is added to the reaction flask,
and stirred at room temperature for 5–6 h. After the reaction was com-
pleted, it was filtered, and the filtrate was evaporated under reduced pres-
sure. The oil was purified by a wet-purified silica gel column eluting with
methylene chloride, V (dichloromethane): V (methanol) ¼ 50:1.
1
Compound a1, orange red solid 0.15 g, 49.34%. H-NMR (600 MHz,
DMSO-d ) d 13.29 (1H, s), 8.72 (1H, dd, J ¼ 5.8, 5.9 Hz), 7.56 (1H, d,
6

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
17
J ¼ 7.1 Hz), 6.57 (1H, d, J ¼ 7.1 Hz), 4.41 (2H, s), 3.91 (2H, d, J ¼ 5.8 Hz),
1
3
0
3
1
(
.63 (3H, s). C-NMR (151 MHz, DMSO-d ) d191.34 (C-4), 170.35 (C-1 ),
6
66.75 (C-8), 148.69 (C-2), 141.88 (C-6), 117.01 (C-5), 52.17 (C-7), 50.71
0
þ
C-2 ), 40.98 (–OCH ). ESI-MS m/z: 304.98 [M þ H]
3
1
Final product: Compound a2, orange red solid 0.40 g, 38.61%. H-NMR
(
600 MHz, DMSO-d ) d 13.27 (1H, s), 8.68 (1H, d, J ¼ 7.8 Hz), 7.56 (1H, d,
6
J ¼ 7.2 Hz), 6.57 (1H, d, J ¼ 7.2 Hz), 4.40 (2H, s), 4.33 (1H, m), 3.63 (3H,
1
3
s), 1.63 (1H, m), 1.53 (2H, m), 0.87 (6H, dd, J ¼ 6.6, 12.1 Hz). C-NMR
0
(
151 MHz, DMSO-d ) d 191.28 (C-4), 172.99 (C-1 ), 166.30 (C-8), 148.66
6
0
(
C-2), 142.00 (C-6), 116.89 (C-5), 52.33 (C-7), 50.75 (C-2 ), 50.71 (–OCH ),
3
0
0
00
0
4
3
0.40 (C-3 ), 24.52 (C-4 ), 23.09 (C-4 ), 21.72 (C-5 ). ESI-MS m/z:
þ
61.05 [M þ H]
1
Compound a3, orange red solid 0.20 g, 62.69%. H-NMR (600 MHz,
DMSO-d ) d 13.28 (1H, s), 8.74 (1H, d, J ¼ 7.1 Hz), 7.56 (1H, d,
6
J ¼ 7.2 Hz), 6.57 (1H, d, J ¼ 7.1 Hz), 4.39 (2H, s), 4.31(1H, m), 3.63 (3H, s),
1
3
1
1
(
.29 (3H, d, J ¼ 7.3 Hz). C-NMR (151 MHz, DMSO-d ) d 191.31 (C-4),
6
0
73.07 (C-1 ), 166.07 (C-8), 148.73 (C-2), 141.99 (C-6), 116.91 (C-5), 52.36
C-7), 50.64 (C-2 ), 48.09 (–OCH ), 17.50 (C-2 ). ESI-MS m/z:
3
0
00
3
19 [M þ H]þ
1
Compound a4, orange red solid 0.20 g, 52.77%. H-NMR (600 MHz,
DMSO-d ) d 13.29 (1H, s), 8.72 (1H, d, J ¼ 7.8 Hz), 7.56 (1H, d,
6
J ¼ 7.1 Hz), 6.57 (1H, d, J ¼ 7.1 Hz), 4.45 (1H, m), 4.40 (2H, s), 3.65 (3H,
1
3
s), 2.49 (2H, m), 2.04 (3H, s), 1.92 (2H, m). C-NMR (151 MHz, DMSO-
0
d ) d 191.30 (C-4), 172.24 (C-1 ), 166.44 (C-8), 148.69 (C-2), 141.95 (C-6),
6
0
0
1
(
16.95 (C-5), 52.46 (C-2 ), 51.35 (C-7), 50.80 (–OCH ), 31.12 (C-4 ), 29.68
3
0
þ
–SCH ), 14.92 (C-3 ). ESI-MS m/z: 379 [M þ H]
3
1
Compound a5, orange-red solid 0.16 g, 40.50%. H-NMR (600 MHz,
DMSO-d ) d 13.28 (1H, s), 8.80 (1H, d, J ¼ 7.6 Hz), 7.49 (1H, d,
6
J ¼ 7.2 Hz), 7.25 (5H, m), 6.55 (1H, d, J ¼ 7.2 Hz), 4.49 (1H, m), 4.37 (2H,
1
3
s), 3.60 (1H, s), 2.98 (2H, m). C-NMR (151 MHz, DMSO-d ) d 191.29
(
6
0
C-4), 173.07 (C-1 ), 166.05 (C-8), 150.53 (C-2), 148.67 (C-6), 141.96 (ph-
1
5
3
), 116.91 ((ph-2, ph-6), 94.28 (ph-3, ph-4), 62.51 (ph-5), 52.36 (C-5),
0
0
0.60 (C-2 ), 48.09 (C-7), 17.49 ((–OCH ), 14.45 (C-3 ). ESI-MS m/z:
3
þ
95.03 [M þ H]
1
Compound a6, orange red solid 0.20 g, 55.40%. H-NMR (600 MHz,
DMSO-d ) d 13.27 (1H, s), 8.63 (1H, d, J ¼ 8.3 Hz), 7.56 (1H, d,
6
J ¼ 7.0 Hz), 6.56 (1H, d, J ¼ 7.0 Hz), 4.43 (2H, s), 4.26 (1H, m), 3.64 (3H,
1
3
s), 1.61 (1H, m), 1.18 (2H, m), 0.84 (6H, m). C-NMR (151 MHz, DMSO-
0
d ) d 191.23 (C-4), 172.08 (C-1 ), 166.42 (C-8), 148.66 (C-2), 142.10 (C-6),
6
0
0
1
16.83 (C-5), 56.80 (C-7), 52.13 (C-2 ), 50.73 (–OCH ), 37.02 (C-3 ), 25.04
3
0
00
0
þ
(
C-4 ), 15.74 (C-3 ), 11.49 (C-5 ). ESI-MS m/z: 361.05 [M þ H]

1
8
M. SHI ET AL.
5
.2. Synthesis of selenothymidine amino acid amide
Weigh 1 mmol of thymine amino acid amide and 2.5 mmol of DMAP in a
reaction flask, seal, vacuum and add 1.2 mL of triethylamine (TEA) under
argon protection, then added 2,4,6-triisopropylbenzen-esulfonyl chloride
(
TPS-Cl) with anhydrous dichloromethane dissolved (about 2 mL) was to
the reaction flask, and then add appropriate amount of solvent (2–4) mL to
stir 60 Min. TLC tracking detection, the developing agent is V (dichlorome-
thane): V (methanol) ¼ 8:1 After the activation is completed, the NaHSe
obtained by the reaction is added to the reaction flask, and stirred at room
temperature for 5–6 h. After the reaction is completed, the mixture is fil-
tered, and the filtrate is evaporated under reduced pressure. The residue is
purified by silica gel column eluting with methylene chloride, V (dichloro-
methane): V (methanol) ¼ 50:1. The solvent was removed to give an
orange-red crystal.
Compound b1, orange red solid 0.15 g, 47.02%.
1
H-NMR (600 MHz, DMSO-d ) d 13.30 (1H, s), 8.72(1H, dd, J ¼ 5.9,
6
1
2
1
1.8 Hz), 7.78 (1H, s), 4.39 (2H, s), 3.91(2H, d, J ¼ 5.8 Hz), 3.64 (3H, s),
1
3
0
.07 (3H, s). C-NMR (151 MHz, DMSO-d ) d 193.18 (C-4), 170.36 (C-1 ),
6
66.81 (C-8), 148.63 (C-2), 139.63 (C-6), 120.86 (C-5), 52.17 (C-7), 50.58
0
þ
(
C-2 ), 40.98 (–OCH ), 19.18 (5-CH ). ESI-MS m/z: 319 [M þ H]
3
3
Final product: Compound b2, orange red solid 0.17 g, 55.87%.
1
H-NMR (600 MHz, DMSO-d ) d 13.28 (1H, s), 8.68 (1H, d, J ¼ 7.8 Hz),
6
7
.77 (1H, s), 4.37 (2H.d, J ¼ 1.7 Hz), 4.32 (1H, m), 3.63 (3H, s), 2.06 (3H,
1
3
s), 1.63 (2H, m), 1.53 (1H, m), 0.87 (6H, dd, J ¼ 6.5, 30.1 Hz). C-NMR
0
(
151 MHz, DMSO-d ) d 193.10 (C-1 ), 173.00 (C-8), 166.36 (C-4), 148.61
6
0
(
C-2), 139.78 (C-6), 120.74 (C-5), 52.33 (C-7), 50.76 (C-2 ), 50.57 (–OCH ),
3
0
0
00
0
24.53 (5-CH ), 23.07 (C-3 ), 21.73 (C-4 ), 19.12 (C-4 , C-5 ). ESI-MS m/z:
3
þ
375.06 [M þ H]
1
Compound b3, orange red solid 0.22 g, 63.06%. H-NMR (600 MHz,
DMSO-d ) d 13.29 (1H, s), 8.73 (1H, d, J ¼ 7.1 Hz), 7.76 (1H, s), 4.37 (2H,
6
1
3
s), 4.31 (1H, m), 3.63 (3H, s), 2.06 (3H, s), 1.29 (3H, d, J ¼ 7.3 Hz). C-
0
NMR (151 MHz, DMSO-d ) d 193.11 (C-4), 173.08 (C-1 ), 166.10 (C-4),
6
0
þ
1
(
48.62 (C-2), 139.74 (C-6), 120.78 (C-5), 52.36 (C-7), 50.47 (C-2 ), 48.10
0
0
–OCH ), 19.12 (5-CH ), 17.49 (C-2 ). ESI-MS m/z:333.02[M þ H]
3
3
1
Compound b4, orange red solid 0.14 g, 35.62%. H-NMR (600 MHz,
DMSO-d ) d 13.30 (1H, s), 8.71 (1H, d, J ¼ 7.74 Hz), 7.78 (1H, s), 4.43 (1H,
6
m), 4.38 (2H, s), 3.65 (3H, s), 2.50 (2H, m), 2.07 (3H, s), 2.04 (2H, m),
1
3
0
1
1
(
(
.92 (3H, s). C-NMR (151 MHz, DMSO-d ) d 193.10 (C-4), 173.00 (C-1 ),
6
0
66.36 (C-8), 148.61 (C-2), 139.78 (C-6), 120.74(C-5), 52.33 (C-2 ), 50.76
C-7), 50.57 (–OCH ), 24.53 (C-4 ), 23.07 (5-CH ), 21.73 (C-3 ), 19.12
–SCH ). ESI-MS m/z: 393.02 [M þ H]
0
0
3
3
þ
3

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
19
1
Compound b5, orange red solid 0.25 g, 61.12%. H-NMR (600 MHz,
DMSO-d ) d 11.27 (1H, s), 8.69 (1H, d, J ¼ 7.6 Hz), 7.35 (1H, s), 7.26 (5H,
6
m), 4.46 (1H, m), 4.32 (2H, s), 2.97 (2H, m), 1.75 (3H, s), 1.10 (3H, s).
13
0
C-NMR (151 MHz, DMSO-d ) d 193.11 (C-4), 171.43 (C-1 ), 166.33 (C-
6
8
1
), 148.59 (C-2), 139.67 (ph-1), 137.16 (ph-2, ph-6), 129.56 (ph-3, ph-4),
0
28.67 (ph-5), 127.02 (C-6), 109.00 (C-5), 61.00 (C-2 ), 54.32 (C-7), 50.47
0
þ
(
C-3 ), 19.13 (–OCH ), 14.29 (5-CH ). ESI-MS m/z: 409.05 [M þ H]
3
3
1
Compound b6, orange red solid 0.13 g, 34.66%. H-NMR (600 MHz,
DMSO-d ) d 13.29 (1H, s), 8.64 (1H, d, J ¼ 8.2 Hz), 7.78 (1H, s), 4.43 (2H,
6
s), 4.27 (1H, m), 3.65 (3H, s), 2.07 (3H, s), 1.58 (2H, m), 1.19 (1H, m),
1
3
0
1
5
1
.86 (6H, m). C-NMR (151 MHz, DMSO-d ) d 193.12 (C-4), 172.26 (C-
6
0
), 166.48 (C-8), 148.63 (C-2), 139.72 (C-6), 120.80 (C-5), 52.46 (C-7),
1.37 (C-2 ), 50.67 (–OCH ), 40.44 (5-CH ), 31.11 (C-3 ), 29.69 (C-4 ),
9.13 (C-3 ), 14.92 (C-5 ). ESI-MS m/z: 375.06 [M þ H]
0
0
0
0
3
3
0
0
þ
6
. Antiproliferative activity
6
.1. Cell culture
ꢁ
The HepG2 cells were taken out from the liquid nitrogen, placed in a 37 C
water bath, and slightly shaken to melt. Transfer to a 15 mL centrifuge tube
and add 10 mL of RPMI-1640 medium to mix. Centrifuge at a speed of
1
000 rpm ꢃ 5 min and discard the supernatant. After the addition of the
RPMI-1640 medium containing the double antibody, the cells were sus-
pended and transferred to a culture flask. Gently shake the cells from side
to side to evenly distribute the cells and incubate in an incubator at 37 C,
ꢁ
5
% CO , and saturated humidity.
2
6
.2. MTT assay for cell viability
Briefly, the grown HepG2 cells were isolated by enzymatic digestion and
plated at a density of about 100 cells/mL in a 96-well microliter plates, and
the marginal wells were filled with PBS. After the plates were incubated at
ꢁ
3
7 C under a water-saturated atmosphere of 5% CO 2 for 2 days, HepG2
cells were exposed to increasing concentrations between 3.125 and
ꢂ3
ꢁ
1
ꢃ 10 lg/mL of each of the target compounds for 24 h at 37 C. The tar-
get compounds of a1–a6 dissolved in DMSO to the culture system (3.125,
ꢂ1
ꢂ1
ꢂ2
ꢂ3
ꢂ3
6
.25 ꢃ 10 ,
1.25 ꢃ 10 ,
2.5 ꢃ 10 ,
5 ꢃ 10 ,
1 ꢃ 10 lg/mL).
HepG2 cells were exposed to increasing concentrations between 10 and
ꢁ
0
.3125 lg/mL of each of the target compounds for 24 h at 37 C. The target
ꢂ1
ꢂ1
compounds b1–b6 (10, 5, 2.5, 1.25, 6.25 ꢃ 10 , 3.125 ꢃ 10 lg/mL).
Controls contained 0.4% of DMSO and medium. Set six parallel well for
each concentration. After incubation, the medium was removed and the

2
0
M. SHI ET AL.
ꢁ
cells were treated with 100 mL 0.4 mg/mL MTT for 4 h at 37 C.
Subsequently, 100 mL DMSO was added to the mixture. The solubilized for-
mazan product was quantified with the help of a microtiter plate reader
TECAN-sunrise at 490 nm. The inhibition rate of the target compounds on
HepG2 cell proliferation was calculated. In all cases, the tested compounds
were dissolved in DMSO to a final concentration in the culture medium
lower than 0.4%.
6
.3. Statistical analysis
All experiments were performed at least three times. The results are
expressed as mean ± SD. ANOVA test was performed. Dunnett’s test
p < 0.01 was considered to be statistically significant. For the cells prolifer-
ation of compounds, the dose–response curves were plotted with GraphPad
Prism v.5.02 software program, and 50% growth inhibitory concentrations
of synthetic products or chemotherapeutic drugs were determined by a
non-linear regression of individual experiments calculated also through
computation with GraphPad Prism v.5.02 software program.
Funding
This project supported by Hei Long Jiang Postdoctoral Foundation (LBH-Q15111) and Hei
Long Jiang University Students Innovation Entrepreneurship Training Program Platform
(
201810226035).
ORCID
Weina Han
http://orcid.org/0000-0001-7241-1608
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