Synthesis and in vitro antitumour activity of 4(R)-methyl-3-O-phosphonomethyl-α-L-threose nucleosides

Article abstract of DOI:10.1016/j.ejmech.2021.113513

A series of novel α-L-threose nucleoside phosphonate analogs, 4(R)-methyl-3-O-phosphonomethyl-α-L-threose nucleosides, were synthesized in multistep sequences starting from D-xylose. The synthetic sequence consisted of the following key stages: (i) the multistep synthesis of 1,2-O-isopropylidenyl-4(R)-methyl-3-O-phosphonomethyl-L-threose, (ii) the transformation of 1,2-O-isopropylidenyl sugar into suitable 1,2-di-O-acyl L-threose precursor, and (iii) the construction of target α-L-threose nucleoside phosphonate analogs by Vorbrüggen glycosidation reaction, deprotection of acyl group, and hydrolysis of diethyl group on phosphonate. The target nucleoside phosphonates were evaluated for their antitumour activities in cell culture-based assays. Compound 8g, 2-fluroadenosine phosphonate, showed remarkable activity against human breast cancer cell lines (MCF-7 and MDA-MB-231) with IC₅₀ values of 0.476 and 0.391 μM, corresponding to 41- and 47-fold higher potency than the reference compound 5-FU, respectively. Subsequent investigations found that the compound 8g can inhibit the proliferation of breast cancer cells and cell cloning. The mechanistic studies indicated that compound 8g could cause DNA damage to breast cancer cells through the ATM-Chk1/Chk2-cdc25c pathway, leading to blockage of the G2/M phase cycle of breast cancer cells, which ultimately led to apoptosis. Moreover, 8g could inhibit the PI3K/AKT signaling pathway and induce apoptosis. These results indicate that compound 8g holds promising potential as an antitumour agent.

Full text of DOI:10.1016/j.ejmech.2021.113513

European Journal of Medicinal Chemistry 221 (2021) 113513
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Synthesis and in vitro antitumour activity of 4(R)-methyl-3-O-
phosphonomethyl-
a- -threose nucleosides
L
,
Feng-Wu Liu a *, Shujie Ji a, Yingying Gao a, Yao Meng a, Wenke Xu a, Haixia Wang a,
,
**
Jing Yang a, Hao Huang a, Piet Herdewijn b, Cong Wang a
a Key Laboratory of Advanced Drug Preparation Technologies, Ministry of Education of China, School of Pharmaceutical Sciences, Zhengzhou University,
Zhengzhou, 450001, PR China
b Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Herestraat 49, 3000, Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel
threose nucleosides, were synthesized in multistep sequences starting from
sequence consisted of the following key stages: (i) the multistep synthesis of 1,2-O-isopropylidenyl-4(R)-
methyl-3-O-phosphonomethyl- -threose, (ii) the transformation of 1,2-O-isopropylidenyl sugar into
suitable 1,2-di-O-acyl -threose precursor, and (iii) the construction of target -threose nucleoside
a
-
L
-threose nucleoside phosphonate analogs, 4(R)-methyl-3-O-phosphonomethyl-
a-L-
Received 3 January 2021
Received in revised form
16 April 2021
Accepted 27 April 2021
Available online 14 May 2021
D-xylose. The synthetic
L
L
a-L
phosphonate analogs by Vorbrüggen glycosidation reaction, deprotection of acyl group, and hydrolysis of
diethyl group on phosphonate. The target nucleoside phosphonates were evaluated for their antitumour
activities in cell culture-based assays. Compound 8g, 2-fluroadenosine phosphonate, showed remarkable
activity against human breast cancer cell lines (MCF-7 and MDA-MB-231) with IC50 values of 0.476 and
Keywords:
a
-
L
-threose nucleoside phosphonate
analogs
-xylose
0.391
mM, corresponding to 41- and 47-fold higher potency than the reference compound 5-FU,
D
respectively. Subsequent investigations found that the compound 8g can inhibit the proliferation of
breast cancer cells and cell cloning. The mechanistic studies indicated that compound 8g could cause
DNA damage to breast cancer cells through the ATM-Chk1/Chk2-cdc25c pathway, leading to blockage of
the G2/M phase cycle of breast cancer cells, which ultimately led to apoptosis. Moreover, 8g could inhibit
the PI3K/AKT signaling pathway and induce apoptosis. These results indicate that compound 8g holds
promising potential as an antitumour agent.
Antitumour activity
Mechanistic investigations
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
One main challenge encountered in the development of nucle-
oside drugs is their limited capacity to undergo in vivo stepwise
Modified nucleoside analogs have occupied an important posi-
tion within medicine for the treatment of viral disease and cancer
for decades. Development of novel nucleoside drugs mainly
focused on chemical modification of both sugar and nucleobase
moiety. Nucleosides modified at the base or sugar moiety generally
are prodrugs, which enter cells and are anabolized by nucleoside
kinases to their mononucleotides, then to the bioactive 5-
triphosphates via two subsequent phosphorylations. The triphos-
phate metabolites are incorporated into DNA during replication,
leading to termination of DNA chain elongation [1].
phosphorylation to their biologically active nucleoside triphos-
phate to express their therapeutic effect [2]. Within the nucleoside
analog phosphorylation process, the first phosphorylation to
nucleoside monophosphate is often the rate-limiting step [3].
Furthermore the nucleoside monophosphate is susceptible to hy-
drolysis by phosphatase leading to free nucleoside analog, which
results in the lower efficiency of first phosphorylation. In order to
address the problem, medicinal chemists make efforts to prepare
nucleoside phosphonate. As isosteric analog of
a nucleoside
monophosphate, phosphonate has the advantage over its phos-
phate counterpart due to its PeC bond being stable to phosphatase
hydrolysis and phosphonate being readily phosphorylated further
by cellular kinases [4]. Nucleoside phosphonates are commonly
used as the antiviral therapeutic agents such as (S)-HPMPC [5],
PMEA [6], (R)-PMPA [7], and d4AP [8]. Some acyclic nucleoside
* Corresponding author.
** Corresponding author.
E-mail addresses: fwliu@zzu.edu.cn (F.-W. Liu), wangcong@zzu.edu.cn
(C. Wang).
https://doi.org/10.1016/j.ejmech.2021.113513
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

F.-W. Liu, S. Ji, Y. Gao et al.
European Journal of Medicinal Chemistry 221 (2021) 113513
phosphonates have been reported to have anticancer activity also
such as PMEA [9], (S)-HPMPC [10], and PMEDAP [11]. But only a few
of anticancer cyclic nucleoside phosphonates have been reported
such as BCH-1868 [12].
2. Results and discussion
2.1. Chemistry
Threose nucleic acid (TNA) has received considerable interest as
a possible RNA progenitor because of the chemical simplicity of
threose relative to that of ribose and the ability of TNA to form
thermally stable duplexes with DNA and RNA that are comparable
to those of natural nucleic acid associations [13]. Moreover, Piet
Herdewijn and co-workers [14] have demonstrated that the L-20-
The target 4-methyl-3-O-phosphonomethyl-L-threose nucleo-
sides 8a-8h were synthesized via a multistep synthetic protocol
starting from commercially available -xylose. Firstly, selective
protection of both hydroxyl groups at C-1 and C-2 of -xylose with
an isopropylidene group allowed 1,2-O-isopropylidenyl- -xylose
D
D
D
(1). The free primary hydroxyl group at C-5 was then tosylated to
give 2 and following reductive elimination furnished intermediate
3. The phosphonate function is introduced using the tosylate of
diethylphosphonomethanol and NaH in THF to give 1,2-O-iso-
deoxythreose nucleoside phosphonates PMDTA (EC50 ¼ 2.5
mM)
and PMDTT (EC50 ¼ 6.59
m
M) selectively inhibit HIV without
affecting normal cell proliferation (Fig. 1). And also, they demon-
strated that the diphosphate of PMDTA is an efficient selective
substrate for HIV-1 reverse transcriptase. Being interested in the
propylidenyl-4-methyl-3-O-phosphonomethyl- -threose (4). Com-
L
pound 5 was obtained by acid hydrolysis of 4 in aqueous acetic acid.
Subsequent acetylation of 1,2-dihydroxyl groups of 5 with acetic
nucleoside phosphonate with modification of the
eton, and as a part of continuous research on the SAR of
nucleoside phosphonate, 4(R)-methyl-3-O-phosphonomethyl-
L
-threose skel-
L-threosyl
anhydride in pyridine provided
in Scheme 1.
L-threose precursor 6 as illustrated
a-L-
threose nucleosides were designed and synthesized. Bioactivity of
these target nucleoside phosphonates was evaluated as well. One
amongst them was found to be a potent inhibitor against prolifer-
ation of breast cancer cells.
We herein describe the synthesis of a series of 4-methylthreose
nucleoside phosphonates with various base moieties starting from
The precursor 6 was glycosidated via Vorbrüggen reaction with
silylated uracil in presence of SnCl4, then the acetyl group was
removed by saturated methanolic ammonia to yield 7a. Further
hydrolysis of the diethyl protecting groups with (TMS)Br at room
temperature gave 8a. However, when silylated thymine and sily-
lated N4-benzoylcytosine were used in the Vorbrüggen glyco-
sidation separately, corresponding protected nucleosides (7b and
7c) were not obtained (Scheme 2).
D
-xylose and the evaluation of their potential antitumour activities,
particularly against the human breast cancer. We also disclose the
mechanism of the potential compound 8g, 1-(6-amino-2-
fluoropurin-9-yl)-4-methyl-3-O-phosphonomethyl-L-threose,
Alternation of the glycosidation conditions were made to
attempt synthesis of other target nucleosides using intermediate 6.
When compound 6 was treated with thymine, 5-chlorouracil, and
2-fluoroadenine in the presence of N,O-bis(trimethylsilyl)
acetamide (BSA) and trimethylsilyl trifluoromethanesulfonate
(TMSOTf) at room temperature, the reaction did not work. While
the reaction mixture was heated to 65 ꢀC, newly generated spots
with higher polarity were observed by monitored on TLC. After
completion of the reaction, purification give corresponding prod-
ucts in low yields. Further structural elucidation showed that the
against MCF-7 and MDA-MB-231 tumour cell lines. In general, most
of the modified nucleoside analogs are incorporated into the DNA
or RNA molecules of tumor cells or viruses, which interfere with
cell replication, competitively inhibit DNA polymerase and other
functions, specifically interfere with nucleic acid synthesis and
cause DNA damage [15]. DNA damage activates the cell cycle
checkpoint, leading to cell cycle arrest and waiting for DNA repair.
When DNA damage cannot be repaired, apoptosis is triggered,
eventually leading to cell death. Therefore we investigated the ef-
fects of 8g at different concentrations on antiproliferation, colony
formation, cell cycle arrest, mitochondrial membrane potential and
cell apoptosis in human breast cancers. Furthermore, in order to
explore the anti-tumor mechanism of 8g, we investigated the ef-
fects of 8g on the expression of DNA damage proteins, cell cycle
arrest proteins, PI3K/AKT signaling pathway proteins, and
apoptosis pathway proteins. In summary, our study provides evi-
dence for the use of 8g as a promising anti-breast cancer
compound.
product were 2-O-acetyl-3-O-ethylphosphonomethyl-L-threose
nucleoside. One ethyl group was removed in the higher tempera-
ture. In consideration of the low yield of the reaction and the dif-
ficulty in purification, another alternative approach is needed in the
further synthesis.
It is known that 2-O-benzoyl sugar precursor possesses more
reactivity than 2-O-acetyl counterpart in the glycosylation because
the benzoyl group could give a more stable five-membered cyclic
transition state. Therefore 2-O-benzoyl-L-threose intermediate 11
was prepared using a three-step sequence from 4. Firstly, treatment
Fig. 1. Structure of PMDTT and PMDTA
2

F.-W. Liu, S. Ji, Y. Gao et al.
European Journal of Medicinal Chemistry 221 (2021) 113513
Scheme 1. Synthesis of precursor 6. (a) acetone, conc. H2SO4; aqueous NaOH; (b) TsCl, pyridine, 67% from D-xylose (two steps); (c) LiAlH4, THF, 94%; (d) tosylate of dieth-
ylphosphonomethanol, NaH, THF, 83%; (e) 60% AcOH; (f) Ac2O, pyridine, 75% from 4 (two steps).
Scheme 2. Synthesis of 8a-8c. (a) 1) silylated nucleobase, SnCl4, MeCN; 2) saturated NH3 in MeOH, 52%; (b) (TMS)Br, 2,6-dimethylpyridine, MeCN, aqueous NaHCO3, 55%.
of 1,2-O-isopropylidenyl intermediate 4 with methanol under
catalysis of sulfuric acid allowed methyl glycoside 9. Then hydroxyl
group at C-2 of 9 was benzoylated with benzoyl chloride to give 10.
Finally transformation of 1-methoxy group of 9 into 1-acetoxyl
group with acetic acid and acetic anhydride provided 11 (Scheme
3).
Glycosidation of precursor 11 with various nucleobases as
starting materials in the presence of BSA/TMSOTf, and following a
sequence of deprotections, allowed the desired nucleosides 8b-8h
successfully as described in Scheme 4.
(5-FU) was used as a positive control. The cells were treated with
the test compounds for 72 h. The results are summarized in Table 2.
As shown in Table 2, compound 8g exhibited potent inhibitory
activity against all cancer cell lines with IC50 values in the
0.391e6.621
lines MCF-7 and MDA-MB-231 with IC50 values of 0.476 and
0.391 M, respectively, representing 41- and 47-fold higher po-
mM range, particularly the human breast cancer cell
m
tency than the reference compound 5-FU. Furthermore, compound
8g was less sensitive to GES-1, showing certain selectivity between
normal and human breast cancer cells.
2.2.2. The inhibitory effects of compound 8g on breast cancer cells
MTT assays were used to test the antiproliferative effects of
compound 8g on breast cancer cells. As shown in Fig. 2(A), cell
viability decreased with prolonging drug treatment time (24, 48
and 72 h) and increasing the concentration of compound 8g.
The inhibitory effect of 8g on MCF-7 and MDA-MB-231 cells was
detected by colony formation assay. As shown in Fig. 2(B), the
number of cell clones decreased significantly with increasing 8g
concentration. The results revealed that the colony formation of
breast cancer cells was inhibited in a concentration-dependent
manner.
2.2. Biology
2.2.1. Screening anticancer activity
The anticancer activity of the target nucleoside phosphonate
analogs (8a-8h) was preliminarily screened in a human cervical
carcinoma cell line (HeLa), a human breast cancer cell line (MCF-7),
and a human gastric cancer cell line (MGC-803) by employing the
MTT assay, respectively. The cells were treated with the test com-
pounds for 72 h. The results are presented in Table 1.
Based on the preliminary results of the evaluations, we noticed
that 8g was the most active compound in all three cell lines among
the eight compounds tested. Therefore further in vitro evaluation of
compound 8g was performed on eight cancer cell lines, including
human lung adenocarcinoma cell line (A549), HeLa cell line, human
colon cancer cell line (SW620), human esophageal carcinoma cell
line (EC109), human prostate cancer cell line (PC3), human gastric
epithelial cell lines (MGC-803) and human breast cancer cell lines
(MDA-MB-231, MCF-7), and normal cell line GES-1. 5-Fluorouracil
2.2.3. Cell cycle analysis by flow cytometry
The cell cycle profile of exponentially growing MCF-7 and MDA-
MB-231 cells treated with compound 8g for 48 h was analyzed by
flow cytometry in cells stained with propidium iodide. Untreated
cells served as a control. The results are presented in Fig. 3. After
exposure of the cells to compound 8g at different concentrations
3

F.-W. Liu, S. Ji, Y. Gao et al.
European Journal of Medicinal Chemistry 221 (2021) 113513
Scheme 3. Synthesis of sugar precursor 11. (a) MeOH, H2SO4; (b) benzoyl chloride, pyridine; (c) AcOH, Ac2O, H2SO4, 55% from compound 4 (three steps).
Scheme 4. Synthesis of target compound 8b-8h from precursor 11. (a) 1) BSA, TMSOTf, MeCN, nucleobase; 2) saturated methanolic ammonia; (b) (TMS)Br, 2,6-dimethylpyridine,
MeCN, aqueous NaHCO3.
apoptosis of both cell lines in a concentration-dependent manner.
The total apoptosis percentage of MCF-7 cells changed from 3.6% to
Table 1
Preliminary results of anticancer activity IC50 (mM) of compounds 8a-8h.
76.1% with increasing concentrations of 8g from 0 to 4
1.3% to 40.8% for MDA-MB-231 cells with increasing concentrations
of 8g from 0 to 9 M.
mM, and from
8a
8b
8c
8d
8e
8f
8g
8h
HeLa
MCF-7
MGC-803
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
>16
<8
<8
<8
>16
>16
>16
m
2.2.5. Effects of compound 8g on the mitochondrial membrane
JC-1 is an ideal fluorescent probe widely used to detect mito-
chondrial membrane potential. The decrease in mitochondrial
membrane potential is a landmark detection indicator in the early
stage of apoptosis [16]. MCF-7 and MDA-MB-231 breast cancer cells
were treated with different concentrations of compound 8g. After
48 h, the cells were collected, the samples were processed, and the
reagents of the JC-1 detection kit were incubated at 37 ꢀC for 20 min
after staining. The instrument detects JC-1 in MCF-7 and MDA-MB-
231 cells. As shown in Fig. 5, it can be seen from the flow histogram
that as the concentration of compound 8g increases, the mito-
chondrial membrane potential within the two cells gradually
decreases.
Table 2
The IC50 of 8g on the proliferation of cancer cell lines and normal cell line.
Cell lines
IC50 (mM)
Compound 8g
5-FU
A549
Hela
SW620
EC109
PC3
MGC-803
MDA-MB-231
MCF-7
GES-1
3.483 0.297
6.195 0.507
6.621 0.565
2.566 0.030
2.206 0.192
6.178 1.320
0.391 0.089
0.476 0.055
5.095 0.328
e
e
e
e
e
e
16.117 2.550
22.439 0.951
e
2.2.6. Effects of compound 8g on DNA damage/repair-related
proteins and cell cycle-related proteins
for 48 h, the percentage of cells in G2/M phase relative to the
control was increased as the concentration increased, showing that
8g induced G2/M cell cycle arrest in both the MCF-7 and MDA-MB-
231 cell lines.
ATM plays a vital role in maintaining the stability of the genome.
When ATM is activated by DNA damage, it can phosphorylate the
corresponding downstream proteins Chk1 and Chk2 to regulate cell
cycle checkpoints, induce cell cycle arrest, and finally repair DNA
damage [17]. But when DNA damage cannot be repaired, the
apoptotic process is triggered. The results are shown in Fig. 6. As the
concentration of compound 8g increased, the protein expression of
ATM, Thr68p-Chk2, Chk1, and Chk2 was upregulated. In addition, the
expression of protein cdc25c was up-regulated and the protein
CyclinB1 and p-cdc2 were down-regulated. These results indicate
that compound 8g can induce DNA damage to reach G2/M cycle
arrest in breast cancer cells.
2.2.4. Detection of apoptosis by flow cytometry
The effect of compound 8g on the apoptosis of two human
breast cancer cell lines was investigated using Annexin V-FITC/PI.
After treatment with different concentrations of compound 8g (0, 1,
2 and 4 mM for MCF-7 cells; 0, 3, 6 and 9 mM for MDA-MB-231 cells)
for 48 h, cells were stained with PI and FITC and then analyzed by
flow cytometry. As illustrated in Fig. 4, compound 8g induced
4

F.-W. Liu, S. Ji, Y. Gao et al.
European Journal of Medicinal Chemistry 221 (2021) 113513
Fig. 2. (A) The inhibitory effect of compound 8g in breast cancer cells. (B) Colony formation breast cancer cells after treatment with compound 8g for 7 days. (C) The results shown
are representative of three independent experiments. *p < 0.05, **p < 0.01.
Fig. 3. (A) Flow cytometry detection of G2/M phase cells after 48 h of treatment of breast cancer cells with compound 8g. (B) The results shown are representative of three in-
dependent experiments. *p < 0.05, **p < 0.01.
2.2.7. Effects of compound 8g on apoptosis-related proteins
231 cells using Western blotting. As shown in Fig. 7, as the con-
centration of compound 8g increased, the protein expression of the
antiapoptotic protein Mcl-1 decreased, and the protein expression
of the proapoptotic proteins Bad and Noxa increased, which caused
the proportion of Bax/Bcl-2 protein to increase. This result shows
that 8g regulates Bcl-2 family protein expression, thereby reducing
the mitochondrial membrane potential and ultimately inducing
apoptosis of breast cancer cells. The expression levels of Cleaved
DNA damage activates the cell cycle checkpoint, leading to cell
cycle arrest and waiting for DNA repair. When DNA damage cannot
be repaired, apoptosis is triggered, eventually leading to cell death
[18]. To explore the specific molecular mechanism of the effects of
8g on apoptosis, we measured the expression levels of PARP,
Cleaved PARP, Cleaved caspase3, Cleaved caspase7, Cleaved cas-
pase9, Bax, Bcl-2, Bad, Noxa and Mcl-1 in MCF-7 and MDA-MB-
5

F.-W. Liu, S. Ji, Y. Gao et al.
European Journal of Medicinal Chemistry 221 (2021) 113513
Fig. 4. The apoptosis rate of MCF-7 cells and MDA-MB-231 cells was determined by Annexin V-FITC/PI dual-staining assay. (A) MCF-7 cells were treated with compound 8g (0, 1, 2
and 4 M) for 48 h, MDA-MB-231 cells were treated with compound 8g (0, 3, 6 and 9 M) for 48 h, and flow cytometry analysis was performed after staining with Annexin V-FITC/
PI. (B) The results shown are representative of three independent experiments. *p < 0.05, **p < 0.01.
m
m
caspase9, Cleaved caspase3, Cleaved caspase7, and Cleaved PARP all
increased with increasing drug concentration. These results indi-
cate that compound 8g can induce apoptosis of breast cancer cells.
phosphatase cdc25c at the Ser216 site, allowing p-cdc25c to accu-
mulate in the nucleus, and finally activate the CyclinB-cdc2 com-
plex to allow mitosis to proceed normally [20]. According to the
above situation, we set up four groups of experiments (NC, 8g
group, 0.3
mM NSC663284 group, combination drug group) and
2.2.8. Effects of compound 8g on PI3K/Akt pathway-related proteins
in breast cancer cells
detected the cell cycle by flow cytometry after 48 h in two breast
cancer cell lines. It can be seen from Fig. 9(A), that when a high
concentration of compound 8g was added, the number of cells in
the G2/M phase of the cell cycle was significantly increased. When
the inhibitor NSC663284 was added, the number of cells in the G2/
M phase of the cell cycle was significantly reduced (similar to the
NC group). These results indicate that when the protein phospha-
tase cdc25c is inhibited, the G2/M phase cycle blockade caused by
compound 8g is converted to normal cell division. It was proven
that compound 8g induces G2/M phase cycle arrest of breast cancer
cells through the ATM-Chk1/Chk2-cdc25c pathway.
The PI3K/Akt signalling pathway is a classic antiapoptotic pro-
survival signalling pathway that is widely present in cells. After
being activated in the PI3K/Akt pathway, activated Akt can phos-
phorylate downstream related anti-apoptotic factors, such as Cas-
pase 3 of the Caspase family and Mcl-1 factor of the Bcl-2 family to
promote survival. However, when the PI3K/Akt signalling pathway
is inhibited, it will correspondingly inhibit downstream anti-
apoptotic factors and activate downstream apoptotic factors to play
a proapoptotic role [19]. MCF-7 and MDA-MB-231 breast cancer
cells were treated with different concentrations of compound 8g.
After 48 h, the cells were collected, and proteins were extracted for
Western blotting. The results are shown in Fig. 8. As the concen-
tration of compound 8g increased, protein 85ap-Akt and Ser473p-
PI3K expression was downregulated. These results indicate that
compound 8g can inhibit the expression of PI3K/Akt pathway-
related proteins in breast cancer cells.
According to the above situation, we set up four groups of ex-
periments (NC, 8g group, 0.3 mM NSC663284 group, combined drug
group). The apoptosis of MCF-7 and MDA-MB-231 cells was
detected by flow cytometry after 48 h. It can be seen from Fig. 9(B)
that when the high concentration of compound 8g was added, the
number of apoptotic cells increased significantly. When the inhib-
itor NSC663284 was added, the number of apoptotic cells
decreased significantly (similar to the NC group). It was proven that
the inhibitor NSC663284 can inhibit the proapoptotic effect of
compound 8g.
2.2.9. Effects of NSC663284 on compound 8g on the cell cycle and
apoptosis
NSC663284 can inhibit the phosphorylation of protein
6

F.-W. Liu, S. Ji, Y. Gao et al.
European Journal of Medicinal Chemistry 221 (2021) 113513
Fig. 5. Mitochondrial membrane potential dissipation induced by compound 8g. (A) Flow cytometry analysis of the cellular mitochondrial membrane potential level of breast
cancer cells treated with JC-1 and compound 8g for 48 h. (B) The results shown are representative of three independent experiments. *p < 0.05, **p < 0.01.
Fig. 6. Compound 8g induced activation of the DNA damage response and G2/M phase arrest. (A) Total and phosphorylated proteins associated with the DNA damage response in
MCF-7 and MDA-MB-231 cells treated with the indicated concentrations of compound 8g for 48 h were analyzed by western blotting. (B) The expression of G2/M phase-related
proteins was detected by western blotting. The results shown are representative of three independent experiments. *p < 0.05, **p < 0.01.
7

F.-W. Liu, S. Ji, Y. Gao et al.
European Journal of Medicinal Chemistry 221 (2021) 113513
Fig. 7. (A) The protein levels of Bax, Bcl-2, Bad, Noxa, and Mcl-1 in MCF-7 and MDA-MB-231 cells incubated with various concentrations of 8g. (B) The expression of Cleaved
caspase3, Cleaved caspase7, Cleaved caspase9, PARP and Cleaved PARP in MCF-7 and MDA-MB-231 cells after treatment with various concentrations of 8g for 48 h *p < 0.05,
**p < 0.01.
Fig. 8. (A) Effect of compound 8g on the levels of PI3K/Akt pathway-related proteins in the breast cancer cell. (compared with the NC group, *P < 0.05, **P < 0.01).
2.2.10. NSC663284 affects the protein expression changes of
compound 8g in the cell cycle pathway and apoptotic pathway
From the above results, we know that compound 8g caused G2/
M cycle arrest in breast cancer cells. According to the above situa-
tion, we set up four groups of experiments (NC, 8g group,
Fig. 10(A) shows that when a high concentration of compound 8g
was added, the expression of protein cdc25c was significantly
upregulated, and the expression of protein cdc25c and protein p-
cdc2 was significantly downregulated. However, when the inhibi-
tor NSC663284 was added to the cells, the expression of protein
cdc25c was significantly downregulated, and the expression of
NSC663284 group with 0.3 mM added alone, combined drug group).
8

F.-W. Liu, S. Ji, Y. Gao et al.
European Journal of Medicinal Chemistry 221 (2021) 113513
Fig. 9. (A) Detection of the cell cycle following treatment with compound 8g and inhibitors for 48 h in breast cancer cells (compared to the NC group, *P < 0.05, **P < 0.01;
compared with the combination treatment group, #P < 0.05, ##P < 0.01). (B) Flow cytometry was used to detect the apoptosis of breast cancer cells after treatment with compound
8g and inhibitors for 48 h (compared with the NC group, *P < 0.05, **P < 0.01; compared with the combination treatment group, #P < 0.05, ##P < 0.01).
protein cdc25c and protein p-cdc2 was significantly upregulated
(similar to the results of the NC group). This result shows that the
high concentration of compound 8g can promote the G2/M phase
cycle arrest of breast cancer cells, but when the inhibitor
NSC663284 is added, the cells gradually return to normal mitosis,
indicating that the inhibitor NSC663284 can attenuate compound
8g. Once again, it was proven that compound 8g caused DNA
damage to breast cancer cells through the ATM-Chk1/Chk2-cdc25c
pathway, leading to blockage of the G2/M phase of breast cancer
cells.
Further mechanistic studies indicated that compound 8g mainly
relied on causing DNA damage, inducing cell cycle arrest in G2/M
phase, inhibiting the PI3K/Akt pathway, and ultimately inducing
apoptosis. Compound 8g regulates downstream apoptosis by
downregulating p-cdc2 and CyclinB1, upregulating the G2/M phase
key protein Ser216p-cdc25c during cell cycle arrest, and down-
regulating 85ap-PI3K and Ser473p-Akt in the PI3K/AKT pathway.
Cdc25c plays an important role in regulating the G2/M phase of the
cell cycle. Increased phosphorylation activity of cdc25c can activate
the cdc2/CyclinB complex, promote the cell cycle and promote
mitosis. NSC663284 can inhibit the phosphorylation of protein
phosphatase cdc25c at the Ser216 site, allowing p-cdc25c to accu-
mulate in the nucleus, and finally activate the CyclinB-cdc2 com-
plex to allow mitosis to proceed normally. NSC663284 can reverse
the promotion effect of 8g on Ser216P- cdc25c protein expression.
Therefore, it is speculated that 8g may target Ser216P-cdc25c protein
and block the cell cycle in the G2/M phase. In addition, 8g can also
inhibit cell proliferation by inhibiting the PI3K/Akt pathway.
Moreover, 8g further upregulated Bad and Noxa and down-
regulated Bcl-2 and Mcl-1, which ultimately led to the release of
cytochrome C. Finally, caspase 9, caspase 7, caspase 3, and PARP
were activated to induce cell apoptosis. In addition, NSC663284, an
inhibitor of phosphorylation of the protein phosphatase cdc25c,
reversed cell cycle arrest in G2/M phase and apoptosis caused by
8g. These results indicate that compound 8g holds promising po-
tential as an antiproliferative agent with low toxicity.
We know that compound 8g can induce apoptosis of breast
cancer cells. According to the above situation, we set up four groups
of experiments (NC, 8g group, 0.3 mM NSC663284 group, combi-
nation drug group) and extracted proteins from two breast cancer
cell lines after 48 h for Western blot experiments. Fig. 10(B) shows
that when a high concentration of compound 8g was added, the
expression of caspase family-related proteins was significantly
increased. When the inhibitor NSC663284 was added, the expres-
sion of caspase family-related proteins was significantly reduced
(similar to the results of the NC group). The results show that a high
concentration of compound 8g can effectively promote the
apoptosis of breast cancer cells, but when the inhibitor NSC663284
is added, the number of apoptotic cells is significantly reduced,
indicating that the inhibitor NSC663284 can inhibit the proapo-
ptotic effect of compound 8g. It was proven again that compound
8g caused DNA damage to breast cancer cells through the ATM-
Chk1/Chk2-cdc25c pathway, leading to blockage of the G2/M phase
cycle of breast cancer cells, which ultimately led to apoptosis.
4. Experimental
3. Conclusion
4.1. General
Overall, novel 4-methyl-
analogs were synthesized starting from
sequence of reactions and evaluated for their activities against eight
selected human cancer cell lines and a normal human cell line.
Among all of the compounds, compound 8g was found to possess
the best antiproliferative activity in human breast cancer cells.
a
-
L
-threose nucleoside phosphonate
-xylose via a multistep
All reagents except anhydrous solvents were commercially
available and used without further purification. Dichloromethane
and acetonitrile was distilled over CaH2. THF was refluxed over
sodium and distilled. All reactions were conducted under an inert
atmosphere of nitrogen. 1H and 13C NMR spectra were recorded for
samples in DMSO‑d6 or CDCl3 or D2O on a Bruker Avance III 400
D
9

F.-W. Liu, S. Ji, Y. Gao et al.
European Journal of Medicinal Chemistry 221 (2021) 113513
Fig. 10. (A) Effect of compound 8g and inhibitor on cell cycle-related protein expression in breast cancer cells (compared with the NC group, *P < 0.05, **P < 0.01; compared with
the combination group, #P < 0.05, ##P < 0.01). (B) Effect of compound 8g and inhibitors on the expression of caspase family-related proteins in breast cancer cells (compared to the
NC group, *P < 0.05, **P < 0.01; compared with the combination group, #P < 0.05, ##P < 0.01).
Digital NMR Spectrometer. High resolution mass spectra (HRMS)
were recorded under electrospray ion conditions using a Q-Tof
Micromass spectrometer. Thin-layer chromatography (TLC) was
performed on silica gel GF254 with detection by charring with 5%
phosphomolybdic acid in alcohol or by UV light. Melting points
were determined on a XT5B melting-point apparatus and are un-
corrected. Optical rotations were measured on a PerkineElmer 341
Polarimeter. Column chromatography was conducted on a column
of silica gel (200e300 mesh). All phosphonates were purified by C-
18 column chromatography eluting with H2O/MeOH or H2O/MeCN.
Fraction solutions were lyophilized.
cooling in ice-water bath. Then the cooling bath was removed, the
mixture was stirred at room temperature until disappearance of
D-
xylose by TLC detection. The pH value of the reaction mixture was
adjusted to 1e2 by dropwise addition of 30% aqueous sodium hy-
droxide. After stirring at ambient temperature for 2 h, solid sodium
hydroxide was added to the solution to adjust pH to 8.0. The
resultant mixture was concentrated under reduced pressure to
almost dryness. The residue was dispersed into acetone (80 mL)
and filtrated. The filtrate was dried over Na2SO4, and concentrated
in vacuo to give crude 1,2-O-isopropylidenyl-
colorless oil.
D
-xylose 1 as a
The crude 1 was dissolved in anhydrous pyridine (100 mL) and
p-toluenesulfonyl chloride (11 g, 58 mmol) was added in portions at
0 ꢀC. The reaction mixture was warmed to 10 ꢀC and stirred for 14 h.
The reaction was quenched with saturated NaHCO3 and concen-
trated in vacuo. The residue was partitioned between H2O (50 mL)
and EtOAc (100 mL). The organic layer was dried over Na2SO4, and
concentrated in vacuo. The residue was purified by
4.2. Chemical synthesis
4.2.1. 1,2-O-Diacetyl-4(R)-methyl-3-O-(diethylphosphonomethyl)-
L
-threose (6)
To a suspension of
(100 mL) was slowly added dropwise 98% sulfuric acid (8 mL) under
D-xylose (10 g, 66.7 mmol) in acetone
10

F.-W. Liu, S. Ji, Y. Gao et al.
European Journal of Medicinal Chemistry 221 (2021) 113513
chromatography on a silica gel column (EtOAc:PE ¼ 1:2, Rf ¼ 0.2) to
21.05.
4.2.2. 1-(Uracil-1-yl)-4(R)-methyl-3-O-(diethylphosphonomethyl)-
afford 5-O-tosyl-1,2-O-isopropylidenyl- -xylose 2 (15.3 g, 67% from
D
D
-xylose) as a white solid. mp 127.4e127.9 ꢀC (literature [21]:
134e135 ꢀC); 1H NMR (400 MHz, CDCl3)
d
7.81 (d, J ¼ 8.3 Hz, 2H),
L
-threose (7a)
7.36 (d, J ¼ 8.0 Hz, 2H), 5.88 (d, J ¼ 3.6 Hz, 1H), 4.51 (d, J ¼ 3.6 Hz,
1H), 4.40e4.26 (m, 3H), 4.14 (q, J ¼ 9.0 Hz, 1H), 2.46 (s, 3H), 2.40 (d,
J ¼ 5.1 Hz, 1H), 1.46 (s, 3H), 1.30 (s, 3H). 13C NMR (101 MHz, CDCl3)
Uracil (0.403 g, 3.6 mmol) was dissolved in anhydrous toluene
(25 mL), ammonia sulfate (10 mg, 0.07 mmol) and HMDS (20 mL)
were added at room temperature under nitrogen. The mixture was
refluxed overnight and then concentrated in vacuo. To the residue
was added the solution of 6 (0.663 g, 1.8 mmol) in 15 mL of dry
d
145.3, 132.4, 130.0, 128.0, 112.1, 105.0, 85.0, 76.7, 74.3, 66.2, 26.8,
26.2, 21.7 (Identical with NMR data in the literature [21]).
To a solution of 2 obtained above (10.0 g, 29.0 mmol) in dry THF
(100 mL) was slowly added lithium aluminum hydride (1.65 g,
43.4 mmol) at 0 ꢀC in three portions. The mixture was continued to
stir vigorously under N2 until disappearance of 2. The reaction was
quenched cautiously with water (5 mL) at 0 ꢀC, and followed by
dropwise addition of 15% aqueous sodium hydroxide (5 mL). After
stirring for 30 min, the resultant slurry was filtered and the filtrate
was concentrated in vacuo. The residue was purified by chroma-
tography on a silica gel column (EtOAc:PE ¼ 1:2, Rf ¼ 0.25) to afford
MeCN followed by a dropwise addition of SnCl4 (840 mL, 7.2 mmol).
The reaction mixture was stirred for 4 h at ambient temperature.
The reaction was quenched with saturated aqueous NaHCO3 and
concentrated to dryness. The residue was dispersed in 30 mL of
DCM and filtered. The cake was washed with DCM (20 mL) twice.
The combined organic layer was dried over Na2SO4, and concen-
trated to yield a syrup. To the syrup was added saturated meth-
anolic ammonia (15 mL), the mixture was stirred at room
temperature overnight and evaporated. The residue was purified by
chromatography on a silica gel column (DCM:MeOH ¼ 30:1) to
afford 7a (0.351 g, 0.93 mmol, 52%) as a yellowish oil. Rf ¼ 0.40
5-deoxy-1,2-O-isopropylidenyl-
solid. mp 72.8e73.1 ꢀC (literature [21]: 81e83 ꢀC); 1H NMR
(400 MHz, CDCl3)
5.89 (d, J ¼ 3.8 Hz, 1H), 4.53 (d, J ¼ 3.8 Hz, 1H),
D
-xylose 3 (4.73 g, 94%) as a white
d
(DCM:MeOH ¼ 10:1). 1H NMR (400 MHz, CDCl3)
7.54 (d, J ¼ 8.2 Hz,
d
4.32 (qd, J ¼ 6.5, 2.5 Hz, 1H), 3.99 (s, 1H), 1.50 (s, 3H), 1.31 (s, 3H),
1H), 5.69 (s, 1H), 5.64 (d, J ¼ 8.2 Hz, 1H), 4.54e4.38 (m, 1H), 4.31 (s,
1H), 4.05 (dq, J ¼ 14.0, 7.1 Hz, 4H), 3.88e3.62 (m, 3H), 1.43e1.31 (m,
1.30 (d, J ¼ 6.5 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 111.4, 104.4,
85.5, 76.4, 75.9, 26.6, 26.1, 12.7 (Identical with the NMR data in the
literature [21]).
3H), 1.34e1.20 (m, 6H).13C NMR (101 MHz, CDCl3)
d 164.3, 151.0,
140.8, 101.4, 91.8, 86.2 (d, JC-P ¼ 10.5 Hz), 78.9, 78.4, 63.4 (d, JC-
To a solution of 3 (3.48 g, 20 mmol) in dried THF (50 mL) was
added sodium hydride (80% dispersion in mineral oil, 1.2 g,
40 mmol) at 0 ꢀC under N2. Then the solution of the tosylate of
diethylphosphonomethanol (12.9 g, 40 mmol) in dried THF (50 mL)
was dropwise added, and the reaction mixture was slowly warmed
to room temperature. After being stirred for 6 h, the reaction was
quenched with saturated NaHCO3 and concentrated. The residue
was partitioned between H2O (40 mL) and DCM (80 mL). The
organic layer was washed with water and brine, dried over Na2SO4,
and concentrated. The residue was purified by chromatography on
a silica gel column (EtOAc:PE ¼ 1:1, Rf ¼ 0.20) to offer 5-deoxy-3-O-
¼ 167 Hz), 62.5 (d, JC-P ¼ 6.9 Hz), 62.4 (d, JC-P ¼ 6.9 Hz), 16.2 (d, JC-
P
¼ 5.4 Hz), 13.1. 31P NMR (162 MHz, CDCl3)
d 20.7. HRMS: calcd. for
P
C
14H24N2O8P [MþH]þ: 379.1265, found: 379.1268.
4.2.3. 1-O-Acetyl-2-O-benzoyl-3-O-diethylphosphonomethyl-4(R)-
methyl- -threose (11)
L
To the solution of 4 (10 g, 31 mmol) in methanol (100 mL) was
dropwise added concentrated sulfuric acid (1.6 mL, 30 mmol) under
cooling in an ice-water bath, the mixture was stirred for 14 h, then
solid NaHCO3 was added slowly. When carbon dioxide was not
continued to be generated, addition was stopped. The mixture was
concentrated to dryness, and EtOAc (50 mL) was added. The organic
layer was dried over Na2SO4 and concentrated to offer 9 as a
colorless oil.
(diethylphosphonomethyl)-1,2-O-isopropylidenyl-
D
-xylose
4
5.76
(5.35 g, 83%) as a light yellow oil. 1H NMR (400 MHz, CDCl3)
d
(d, J ¼ 4.0 Hz, 1H), 4.51 (d, J ¼ 4.0 Hz, 1H), 4.33 (qd, J ¼ 9.1, 4.6 Hz,
1H), 4.11e4.04 (m, 4H), 3.82 (dd, J ¼ 13.6, 8.8 Hz, 1H), 3.72 (dd,
J ¼ 13.6, 7.6 Hz, 1H), 3.69 (d, J ¼ 2.4 Hz, 1H), 1.38 (s, 3H), 1.23 (m,
The compound 9 obtained above was dissolved in absolute
pyridine (80 mL), and benzoyl chloride (7 mL, 60 mmol) was
dropwise added under cooling in an ice-water bath. The mixture
was warmed to room temperature and stirred for 4 h, then the
reaction was quenched with saturated aqueous NaHCO3 and
concentrated. The residue was partitioned between EtOAc and
saturated brine. The organic layer was dried over Na2SO4 and
concentrated. The residue was purified by chromatography on a
silica gel column (PE:EtOAc ¼ 1:1, Rf ¼ 0.20) to allow methyl 2-O-
12H).13C NMR (101 MHz, CDCl3)
d
111.2, 104.6, 86.0 (d, JC-P ¼ 9.2 Hz),
82.3, 75.9, 64.2 (d, JC-P ¼ 168.1 Hz), 62.5 (d, JC-P ¼ 14.6 Hz), 26.5, 26.1,
16.4 (d, JC-P ¼ 5.2 Hz), 12.9.
The compound 4 (8.0 g, 24.7 mmol) was dissolved in 60%
aqueous acetic acid (40 mL), the mixture was heated to 70 ꢀC and
stirred for 18 h at this temperature. The resultant solution was
concentrated in vacuo. The residue was dissolved in EtOAc (60 mL),
dried over anhydrous Na2SO4, and concentrated to give 5 as a
yellowish oil. To the oily 5 was added pyridine (30 mL) and acetic
anhydride (10 mL). The mixture was stirred at ambient temperature
under N2 for 6 h. The reaction was quenched with saturated
NaHCO3 and concentrated in vacuo. The residue was partitioned
between H2O (50 mL) and EtOAc (100 mL). The organic layer was
washed with brine twice, dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel chro-
matography (EtOAc:PE ¼ 1:1, Rf ¼ 0.20) to offer 1,2-O-diacetyl-
benzoyl-4-methyl-3-O-diethylphosphonomethyl-
anoside 10 as a colorless oily mixture of two anomers. 1H NMR
(400 MHz, CDCl3) 8.13e8.00 (m, 4H), 7.63e7.55 (m, 2H), 7.00e7.42
L-threopentofur-
d
(m, 4H), 5.32 (s, 1H), 5.21 (d, J ¼ 4.5 Hz, 1H), 5.13 (t, J ¼ 4.5 Hz, 1H),
5.02 (s, 1H), 4.53e4.41 (m, 2H), 4.35 (dd, J ¼ 6.3, 4.6 Hz, 1H),
4.23e4.09 (m, 9H), 4.07 (d, J ¼ 5.0 Hz, 1H), 4.01e3.93 (m, 2H), 3.85
(dd, J ¼ 13.7, 8.8 Hz, 1H), 3.43 (s, 3H), 3.34 (s, 3H), 1.39 (d, J ¼ 6.6 Hz,
3H), 1.36e1.27 (m, 15H). 13C NMR (101 MHz, CDCl3)
d 165.9, 165.5,
133.5, 133.4, 129.8, 129.7, 129.4, 129.3, 128.51, 128.47, 107.2, 100.2,
84.6 (d, JC-P ¼ 8.9 Hz), 84.2 (d, JC-P ¼ 11.7 Hz), 80.9, 79.3, 78.0, 73.2,
64.3 (d, JC-P ¼ 167.4 Hz), 64.1 (d, JC-P ¼ 165.7 Hz), 62.64 (d, JC-
4(R)-methyl-3-O-(diethylphosphonomethyl)-
colorless oily mixture of - and
-anomers (6.8 g, 75%). 1H NMR
(400 MHz, CDCl3)
6.35 (d, J ¼ 4.7 Hz, 1H), 6.05 (s, 1H), 5.20 (t,
L-threose
6
as
a
a
b
d
¼ 5.3 Hz), 62.61 (d, JC-P ¼ 5.3 Hz), 62.5 (d, JC-P ¼ 5.3 Hz), 62.4 (d, JC-
P
J ¼ 4.3 Hz, 2H), 4.50e4.42 (m, 2H), 4.23e4.12 (m, 10H), 4.05 (dd,
J ¼ 9.2, 13.9 Hz, 1H), 3.95 (dd, J ¼ 9.2, 13.9 Hz, 1H), 3.89 (dd, J ¼ 8.0,
13.9 Hz, 1H), 3.83 (dd, J ¼ 8.3, 13.8 Hz, 1H), 2.11 (s, 3H), 2.10 (s, 3H),
2.09 (s, 3H), 2.07 (s, 3H), 1.36 (d, J ¼ 6.5 Hz, 3H), 1.35 (t, J ¼ 7.0 Hz,
¼ 5.3 Hz), 55.5, 55.4, 16.55e16.36 (m), 15.5, 14.9. 31P NMR
P
(162 MHz, CDCl3)
d 20.4, 20.0.
The compound 10 obtained above was dissolved in acetic acid
(100 mL), acetic anhydride (9.5 mL, 100 mmol) and H2SO4 (65 L,
1.25 mmol) was added in sequence at 0 ꢀC. The mixture was stirred
m
12H), 1.30 (t, J ¼ 6.5 Hz, 3H).31P NMR (162 MHz, CDCl3)
d 21.12,
11

F.-W. Liu, S. Ji, Y. Gao et al.
European Journal of Medicinal Chemistry 221 (2021) 113513
at room temperature for 8 h, quenched with saturated aqueous
NaHCO3 and then partitioned between EtOAc and water. The
organic layer was dried over Na2SO4 and concentrated. The residue
4.2.6. 1-(5-Chlorouracil-1-yl)-4(R)-methyl-3-O-
(diethylphosphonomethyl)- -threose (7d)
a-L
Compound 11 (108 mg, 0.25 mmol) was treated with 5-
chlorouracil (56 mg, 0.38 mmol) according to the procedure as
described in 4.2.4, to give compound 7d (63 mg, 0.16 mmol, 63%) as
a foamy solid. Rf ¼ 0.4 (DCM:MeOH ¼ 10:1). 1H NMR (400 MHz,
was purified by chromatography on
a silica gel column
(PE:EtOAc ¼ 1:1, Rf ¼ 0.20) to afford 11 (7.33 g, 55% from 4) as a
colorless oily mixture of two anomers (anomer-1: anomer-
2 ¼ 1:0.6). Anomer-1: 1H NMR (400 MHz, CDCl3)
d
8.05e8.00 (m,
CDCl3)
d
10.79 (s, 1H), 7.76 (s, 1H), 5.73 (s, 1H), 5.38 (d, J ¼ 3.2 Hz,
2H), 7.63e7.58 (m, 1H), 7.60e7.43 (m, 2H), 6.23 (s, 1H), 5.45 (t,
J ¼ 4.4 Hz, 1H), 4.60e4.51 (m, 1H), 4.24e4.10 (m, 5H), 4.07 (d,
J ¼ 5.0 Hz, 1H), 3.97 (dd, J ¼ 13.7, 7.9 Hz, 1H), 2.12 (s, 3H), 1.41 (d,
J ¼ 6.6 Hz, 3H),1.35 (t, J ¼ 7.1 Hz, 6H). Anomer-2: 1H NMR (400 MHz,
1H), 4.56 (qd, J ¼ 6.4, 3.2 Hz, 1H), 4.49 (d, J ¼ 2.0 Hz, 1H), 4.19e4.10
(m, 4H), 3.90 (d, J ¼ 8.8 Hz, 2H), 3.84 (d, J ¼ 3.2 Hz, 1H), 1.47 (d,
J ¼ 6.8 Hz, 3H), 1.34 (t, J ¼ 6.8 Hz, 3H), 1.33 (t, J ¼ 7.2 Hz, 3H). 13
C
NMR (101 MHz, CDCl3)
d 159.8, 150.2, 137.5, 108.4, 92.8, 86.0 (d, JC-
CDCl3)
d
8.05e8.00 (m, 2H), 7.63e7.58 (m, 1H), 7.60e7.43 (m, 2H),
¼ 10.0 Hz), 79.8, 78.5, 64.1 (d, JC-P ¼ 168.6 Hz), 62.9 (d, JC-
P
P
6.49 (d, J ¼ 4.6 Hz, 1H), 5.45 (t, J ¼ 4.4 Hz, 1H), 4.60e4.51 (m, 1H),
4.24e4.10 (m, 5H), 4.03 (dd, J ¼ 13.9, 8.9 Hz, 1H), 3.90 (dd, J ¼ 13.9,
8.1 Hz,1H),1.99 (s, 3H),1.35 (d, J ¼ 6.6 Hz, 3H),1.31 (t, J ¼ 7.1 Hz, 6H).
¼ 6.5 Hz), 62.6 (d, JC-P ¼ 6.7 Hz), 16.5 (d, JC-P ¼ 3.0 Hz), 16.5 (d, JC-
¼ 3.0 Hz), 13.3. 31P NMR (162 MHz, CDCl3)
d 20.3. HRMS: calcd. for
P
C
14H23ClN2O8P [MþH]þ: 413.0875, found: 413.0878.
13C NMR (101 MHz, CDCl
) d 169.8, 169.5, 165.35, 165.31, 133.8,
3
133.6, 129.8, 129.7, 129.1, 128.9, 128.6, 99.5, 94.0, 84.04 (d, JC-
4.2.7. 1-(5-Fluorouracil-1-yl)-4(R)-methyl-3-O-
(diethylphosphonomethyl)- -threose (7e)
Compound 11 (108 mg, 0.25 mmol) was treated with 5-
fluorouracil (50 mg, 0.38 mmol) according to the procedure as
described in 4.2.4, to give compound 7e (59 mg, 0.15 mmol, 61%) as
a foamy solid. Rf ¼ 0.4 (DCM:MeOH ¼ 10:1). 1H NMR (400 MHz,
¼ 10.0 Hz), 84.03 (d, JC-P ¼ 10.1 Hz), 80.3, 79.6, 77.2, 76.1, 64.3 (d, JC-
a-L
P
¼ 167.2 Hz), 62.7e62.5 (m), 21.2, 20.9,16.6e16.4 (m),15.0,14.5. 31
P
P
NMR (162 MHz, CDCl3)
d 20.1, 19.9.
4.2.4. 1-(Thymin-1-yl)-4(R)-methyl-3-O-
(diethylphosphonomethyl)- -threose (7b)
Thymine (48 mg, 0.38 mmol), N,O-bis(trimethylsilyl)acetamide
(196 L, 0.8 mmol), compound 11 (108 mg, 0.25 mmol), and
a-L
CDCl3)
d
10.74 (s,1H), 7.65 (d, J ¼ 6.4 Hz,1H), 5.74 (s,1H), 5.29 (s,1H),
4.52 (qd, J ¼ 6.4, 2.8 Hz, 1H), 4.45 (s, 1H), 4.20e4.06 (m, 4H), 3.90
(dd, J ¼ 13.9, 9.4 Hz, 1H), 3.86 (dd, J ¼ 13.9, 8.8 Hz, 1H), 3.81 (d,
J ¼ 3.2 Hz, 1H), 1.45 (d, J ¼ 6.4 Hz, 3H), 1.33 (t, J ¼ 7.0 Hz, 3H), 1.32 (t,
m
10 mL of MeCN were added to a dried flask. The mixture was heated
in an oil bath maintained at 65 ꢀC, until a clear solution was ob-
tained. After the mixture was cooled down to room temperature,
J ¼ 7.0 Hz, 3H). 13 C NMR (101 MHz, CDCl 3)
157.6 (d, JC-F ¼ 26.5 Hz),
d
149.5, 140.3 (d, JC-F ¼ 235.7 Hz), 125.0 (d, JC-F ¼ 35.1 Hz), 92.5, 86.0
(d, JC-P ¼ 10.1 Hz), 79.6, 78.6, 64.0 (d, JC-P ¼ 168.7 Hz), 62.8 (d, JC-
trimethylsilyl trifluoromethanesulfonate (200
mL, 1.1 mmol) was
added with stirring under N2. As soon as completion of addition,
the temperature was raised to 65 ꢀC again and maintained for 4 h.
The reaction was quenched with saturated aqueous NaHCO3 and
concentrated in vacuo. MeOH (30 mL) was added to the residue,
and stirred for 10 min. Resultant mixture was filtered and washed
with 10 mL of MeOH. The combined filtrate was dried over Na2SO4,
and concentrated in vacuo to give a syrup. The syrup was dissolved
in 15 mL of saturated methanolic ammonia, stirred at room tem-
perature overnight and evaporated. The residue was separated by
chromatography on a silica gel column (DCM:MeOH ¼ 20:1,
Rf ¼ 0.15) to afford 7b as a yellowish oil. 1H NMR (400 MHz,
¼ 6.7 Hz), 62.6 (d, JC-P ¼ 6.7 Hz), 16.4 (d, JC-P ¼ 5.8 Hz), 13.3. 19
F
P
NMR (376 MHz, CDCl3)
d d 20.3.
166.0. 31 P NMR (162 MHz, CDCl3)
HRMS: calcd. for C14H23FN2O8P [MþH]þ: 397.1171, found: 397.1175.
4.2.8. 1-(5-Fluorocytosin-1-yl)-4(R)-methyl-3-O-
(diethylphosphonomethyl)-a-L-threose (7f)
Compound 11 (129 mg, 0.3 mmol) was treated with 5-
fluorocytosine (58 mg, 0.45 mmol) according to the procedure as
described in 4.2.4, to give compound 7f (64 mg, 56%) as a white
solid. Rf ¼ 0.3 (DCM:MeOH ¼ 10:1). mp: 188.6e189.1 ꢀC. 1H NMR
(400 MHz, DMSO‑d6)
d
7.72 (s, 1H), 7.52 (d, J ¼ 6.8 Hz, 2H), 5.84 (d,
DMSO‑d6)
d
11.33 (s, H), 7.36 (d, J ¼ 1.2 Hz, 1H), 5.85 (d, J ¼ 4.4 Hz,
J ¼ 4.4 Hz, 1H), 5.65 (s, 1H), 4.30 (qd, J ¼ 6.4, 3.2 Hz, 1H), 4.12 (d,
J ¼ 4.0 Hz, 1H), 4.06e3.95 (m, 4H), 3.92 (dd, J ¼ 14.0, 9.6 Hz, 1H),
3.84 (dd, J ¼ 13.6, 8.8 Hz, 1H), 3.66 (d, J ¼ 2.8 Hz, 1H), 1.32 (d,
J ¼ 6.4 Hz, 3H),1.24 (t, J ¼ 7.0 Hz, 3H),1.21 (t, J ¼ 7.0 Hz, 3H). 13C NMR
1H), 5.71 (d, J ¼ 1.6 Hz, 1H), 4.28e4.23 (m, 1H), 4.16 (s, 1H),
4.10e3.98 (m, 5H), 3.88 (dd, J ¼ 13.6, 8.8 Hz, 1H), 3.70 (d, J ¼ 3.2 Hz,
1H), 1.80 (d, J ¼ 0.8 Hz, 3H),1.29 (d, J ¼ 6.4 Hz, 3H),1.25 (t, J ¼ 6.8 Hz,
3H), 1.24 (t, J ¼ 7.2 Hz, 3H). 13 C NMR (101 MHz, DMSO‑d6)
d
163.7,
(101 MHz, DMSO‑d6)
d
157.9 (d, JC-F ¼ 13.5 Hz), 153.8, 136.3 (d, JC-
150.5, 136.5, 109.3, 90.1, 86.5 (d, JC-P ¼ 12.7 Hz), 77.8, 77.2, 63.1 (d, JC-
¼ 241.6 Hz), 126.0 (d, JC-F ¼ 32.4 Hz), 92.1, 86.9 (d, JC-P ¼ 12. 3 Hz),
F
¼ 164.7 Hz), 61.8 (d, JC-P ¼ 6.3 Hz), 61.7 (d, JC-P ¼ 6.3 Hz), 16.3 (d, JC-
78.3 (d, JC-P ¼ 16.0 Hz), 63.7 (d, JC-P ¼ 164.1 Hz), 63.3, 62.2 (d, JC-
P
d
C
P
¼ 5.4 Hz), 13.3, 12.2. 31P NMR (162 MHz, DMSO‑d6)
d
20.8. HRMS:
¼ 6.2 Hz),16.7 (d, JC-P ¼ 5.7 Hz),13.8. 19F NMR (376 MHz, DMSO‑d6)
P
calcd. for C14H25N3O7P [MþH]þ: 393.1421, found: 393.1426.
ꢁ167.8. 31P NMR (162 MHz, DMSO‑d6)
d 20.7. HRMS: calcd. for
14H24FN3O7P [MþH]þ: 396.1330, found: 396.1333.
4.2.5. 1-(Cytosin-1-yl)-4(R)-methyl-3-O-
(diethylphosphonomethyl)-
a
-
L
-threose (7c)
4.2.9. 1-(2-Fluoroadenin-1-yl)-4(R)-methyl-3-O-
(diethylphosphonomethyl)- -threose (7g)
Compound 11 (108 mg, 0.25 mmol) was treated with N4-ben-
zoylcytosine (80 mg, 0.37 mmol) according to the procedure as
described in 4.2.4, to give compound 7c (60 mg, 63%) as a foamy
solid. Rf ¼ 0.20 (DCM:MeOH ¼ 10:1). 1H NMR (400 MHz, DMSO‑d6)
a-L
Compound 11 (112 mg, 0.26 mmol) was treated with 2-
fluoroadenine (60 mg, 0.39 mmol) according to the procedure as
described in 4.2.4, to give compound 7g (61 mg, 0.15 mmol, 58%) as
a white solid. Rf ¼ 0.25 (DCM:MeOH ¼ 10:1). mp: 205.5e206.5 ꢀC.
d
7.51 (d, J ¼ 7.6 Hz, 1H), 7.23 (s, 1H), 7.10 (s, 1H), 5.79 (d, J ¼ 3.6 Hz,
1H), 5.73e5.73 (m, 2H), 4.32e4.26 (m, 1H), 4.09 (s, 1H), 4.05e3.97
(m, 4H), 3.91 (dd, J ¼ 14.0, 9.2 Hz, 1H), 3.83 (dd, J ¼ 14.0, 8.4 Hz, 1H),
3.68 (d, J ¼ 3.2 Hz,1H),1.30 (d, J ¼ 6.4 Hz, 3H),1.24 (t, J ¼ 6.8 Hz, 3H),
1H NMR (400 MHz, DMSO‑d6)
d 8.09 (s, 1H), 7.84 (s, 2H), 6.04 (d,
J ¼ 4.4 Hz, 1H), 5.75 (s, 1H), 4.56 (s, 1H), 4.39 (m, 1H), 4.09e4.00 (m,
5H), 3.91 (dd, J ¼ 14.0, 8.8 Hz, 1H), 3.85 (d, J ¼ 3.2 Hz, 1H), 1.31 (d,
1.23 (t, J ¼ 6.8 Hz, 3H). 13 C NMR (101 MHz, DMSO‑d6)
d
165.5, 155.1,
J ¼ 6.4 Hz, 3H), 1.25 (t, J ¼ 6.8 Hz, 3H), 1.23 (t, J ¼ 6.8 Hz, 3H). 13
C
141.4, 93.6, 91.3, 86.5 (d, JC-P ¼ 11.7 Hz), 77.9, 77.5, 63.0 (d, JC-
NMR (101 MHz, DMSO‑d6)
d
158.7 (d, JC-F ¼ 205.0 Hz), 157.5 (d, JC-
¼ 164.1 Hz), 61.8 (d, JC-P ¼ 6.0 Hz), 16.3 (d, JC-P ¼ 5.5 Hz), 13.4. 31
P
¼ 21.4 Hz),150.4 (d, JC-F ¼ 20.2 Hz),139.2 (d, JC-F ¼ 2.5 Hz),116.9 (d,
P
F
NMR (162 MHz, CDCl3)
d
20.7. HRMS: calcd. for C14H25N3O7P
JC-F ¼ 3.9 Hz), 88.8, 86.4 (d, JC-P ¼ 12.0 Hz), 79.0, 77.8 (d, JC-
[MþH]þ: 378.1425, found: 378.1426.
¼ 13.7 Hz), 63.3 (d, JC-P ¼ 164.8 Hz), 61.8 (d, JC-P ¼ 6.4 Hz), 16.2 (d,
P
12

F.-W. Liu, S. Ji, Y. Gao et al.
European Journal of Medicinal Chemistry 221 (2021) 113513
JC-P ¼ 5.5 Hz), 13.7. 19F NMR (376 MHz, DMSO‑d6)
d
ꢁ52.1. 31P NMR
4.2.14. 1-(5-Chlorouracil-1-yl)-4(R)-methyl-3-O-
(phosphonomethyl)- -threose disodium salt (8d)
Compound 7d was hydrolyzed according to the procedure as
(162 MHz, DMSO‑d6)
d
20.6. HRMS: calcd. for C15H24FN5O6P
a-L
[MþH]þ: 420.1443, found: 420.1445.
20
described in 4.2.11 to afford 8d as a white solid in 62% yield. [a]
D ꢁ25.5 (c 0.27, H2O). 1H NMR (400 MHz, D2O)
d 7.80 (s, 1H), 5.77 (d,
J ¼ 2.8 Hz, 1H), 4.39e4.36 (m, 1H), 4.32 (s, 1H), 3.97 (s, 1H),
4.2.10. 1-(Adenin-1-yl)-4(R)-methyl-3-O-
(diethylphosphonomethyl)-a-L-threose (7 h)
3.47e3.44 (m, 2H), 1.33 (d, J ¼ 6.4 Hz, 3H). 13C NMR (101 MHz, D2O)
Compound 11 (112 mg, 0.26 mmol) was treated with adenine
(53 mg, 0.39 mmol) according to the procedure as described in
4.2.4, to give compound 7h (56 mg, 55%) as a white solid. Rf ¼ 0.25
(DCM:MeOH ¼ 10:1). mp: 183.5e184.0 ꢀC. 1H NMR (400 MHz,
d
163.1, 151.7, 138.8,108.8, 90.7, 85.1 (d, JC-P ¼ 9.1 Hz), 79.6, 77.9, 66.9
(d, JC-P ¼ 153.7 Hz), 13.1. 31P NMR (162 MHz, D2O)
d 14.6. HRMS:
calcd. for C10H13ClN2O8P[M-2Na þ H]-: 355.0104, found: 355.0106.
DMSO‑d6)
d
8.16 (s, 1H), 8.13 (s, 1H), 7.29 (s, 2H), 6.03 (d, J ¼ 4.00 Hz,
1H), 5.89 (s, 1H), 4.58 (s, 1H), 4.40e4.38 (m, 1H), 4.12e3.98 (m, 5H),
3.92 (dd, J ¼ 13.7, 8.9 Hz, 1H), 3.85 (d, J ¼ 2.4 Hz, 1H), 1.31 (d,
4.2.15. 1-(5-Fluorouracil-1-yl)-4(R)-methyl-3-O-
(phosphonomethyl)-
a-L-threose disodium salt (8e)
J ¼ 6.0 Hz, 3H), 1.25 (t, J ¼ 6.9 Hz, 3H), 1.23 (t, J ¼ 6.9 Hz, 3H). 13
C
Compound 7e was hydrolyzed according to the procedure as
20
NMR (101 MHz, DMSO‑d6)
d
155.9, 152.7, 149.2, 139.0, 118.4, 88.5,
described in 4.2.11 to afford 8e as a white solid in 60% yield. [a]
86.4 (d, JC-P ¼ 12.6 Hz), 77.8, 77.5, 63.3 (d, JC-P ¼ 164.3 Hz), 61.8 (d, JC-
D ꢁ10.4 (c 0.29, H2O). 1H NMR (400 MHz, D2O)
d
7.85 (d, J ¼ 6.1 Hz,
¼ 6.2 Hz), 16.3 (d, JC-P ¼ 5.4 Hz), 13.8. 31P NMR (162 MHz,
1H), 5.80 (s, 1H), 4.43e4.40 (m, 1H), 4.37 (s, 1H), 3.88 (s, 1H),
P
DMSO‑d6)
d
20.8. HRMS: calcd. for C15H25N5O6P [MþH]þ: 402.1537,
3.57e3.44 (m, 2H), 1.38 (d, J ¼ 6.3 Hz, 3H). 13C NMR (101 MHz, D2O)
found: 402.1540.
d
163.3 (d, JC-F ¼ 21.2 Hz), 153.3, 141.2 (d, JC-F ¼ 237 Hz), 125.8 (d, JC-
¼ 34.9 Hz), 90.5, 85.2 (d, JC-P ¼ 9.3 Hz), 79.1, 78.0, 67.4 (d, JC-
F
¼ 151 Hz), 13.1. 19F NMR (376 MHz, D2O)
d
ꢁ164.6. 31P NMR
P
4.2.11. 1-(Uracil-1-yl)-4(R)-methyl-3-O-(phosphonomethyl)-
threose disodium salt (8a)
a-L-
(162 MHz, D2O)
d 13.6. HRMS: calcd. for C10H13FN2O8P[M-
2Na þ H]-: 339.0399, found: 339.0400.
To a solution of 7a (100 mg, 0.26 mmol) in MeCN (5 mL) was
added 2,6-dimethylpyridine (290 L, 2.5 mmol) and bromo-
trimethylsilane (690
L, 5.2 mmol) in sequence at 0 ꢀC under N2.
m
m
4.2.16. 1-(5-Fluorocytosin-1-yl)-4(R)-methyl-3-O-
Then the reaction mixture was warmed to room temperature and
stirred overnight. The reaction was quenched with saturated
aqueous NaHCO3 and concentrated. The residue was purified by a
(phosphonomethyl)-
a-L-threose disodium salt (8f)
Compound 7f was hydrolyzed according to the procedure as
20
described in 4.2.11 to afford 8f as a white solid in 59% yield. [a]
C-18 column (MeOH:H2O ¼ 1:4) to offer 8a (52 mg, 55%) as a white
D þ3.13 (c 0.32, H2O). 1H NMR (400 MHz, D2O)
d
7.84 (d, J ¼ 6.3 Hz,
20
solid. [
a
]
D þ5.83 (c 0.34, H2O). 1H NMR (400 MHz, D2O)
d
7.77 (d,
1H), 5.74 (s, 1H), 4.41e4.39 (m, 1H), 4.31 (s, 1H), 3.82 (s, 1H),
J ¼ 7.8 Hz,1H), 5.81 (d, J ¼ 7.8 Hz,1H), 5.85 (s, 1H), 4.40 (m, 1H), 4.38
3.48e3.35 (m, 2H), 1.36 (d, J ¼ 6.4 Hz, 3H). 13C NMR (101 MHz, D2O)
(s, 1H), 3.87 (s, 1H), 3.46 (m, 2H), 1.36 (d, J ¼ 6.4 Hz, 3H).13C NMR
d
158.4 (d, JC-F ¼ 14.9 Hz), 155.8, 137.4 (d, JC-F ¼ 242.3 Hz), 126.7 (d,
(101 MHz, D2O)
d
171.3, 155.4, 142.4, 102.7, 90.1, 85.4 (d, JC-
JC-F ¼ 32.7 Hz), 91.3, 85.1 (d, JC-P ¼ 8.9 Hz), 79.6, 78.0, 67.3 (d, JC-
¼ 8.2 Hz), 78.8, 77.9, 67.8 (d, JC-P ¼ 150 Hz), 13.3. 31P NMR
¼ 151.8 Hz), 13.0. 19F NMR (376 MHz, D2O)
d
ꢁ165.2. 31P NMR
P
P
(162 MHz, D2O)
d
13.1. HRMS: calcd. for C10H14N2O8P[M-2Na þ H]-:
(162 MHz, D2O) d 13.4. HRMS: calcd. for C10H14N3FO7P[M-
321.0493, found: 321.0493.
2Na þ H]-: 338.0559, found: 338.0562.
4.2.12. 1-(Thymin-1-yl)-4(R)-methyl-3-O-(phosphonomethyl)-
threose disodium salt (8b)
a-L
-
4.2.17. 1-(2-Fluoroadenin-1-yl)-4(R)-methyl-3-O-
(phosphonomethyl)-
a-L-threose disodium salt (8g)
Compound 7b was hydrolyzed according to the procedure as
described in 4.2.11 to afford 8b as a white solid (33 mg, 35% from
Compound 7g was hydrolyzed according to the procedure as
described in 4.2.11 to afford 8g as a white solid in 53% yield. [a]
20
20
compound 11). [
a
]
D ꢁ15.5 (c 0.32, H2O). 1H NMR (400 MHz, D2O)
D ꢁ56.9 (c 0.28, H2O). 1H NMR (400 MHz, D2O)
d 8.12 (s,1H), 5.72 (d,
J ¼ 1.36 Hz, 1H), 4.55 (s, 1H), 4.46 (m, 1H), 3.85 (dd, J ¼ 3.6, 0.8 Hz,
d
7.51 (d, J ¼ 1.1 Hz, 1H), 5.77 (d, J ¼ 1.8 Hz, 1H), 4.39 (dq, J ¼ 6.4,
3.8 Hz, 1H), 4.33 (s, 1H), 3.77 (d, J ¼ 3.5 Hz, 1H), 3.64 (dd, J ¼ 13.1,
9.3 Hz, 1H), 3.54 (dd, J ¼ 13.1, 9.5 Hz, 1H), 1.81 (t, J ¼ 0.76 Hz, 3H),
1H), 3.63 (dd, J ¼ 13.1, 9.3 Hz, 1H), 3.56 (dd, J ¼ 13.1, 9.3 Hz, 1H), 1.33
(d, J ¼ 6.5 Hz, 3H). 13C NMR (101 MHz, D2O)
d 158.7 (d, JC-
1.36 (d, J ¼ 6.4 Hz, 3H). 13C NMR (101 MHz, D2O)
d
166.5,151.7,138.0,
¼ 211.6 Hz), 156.6 (d, JC-F ¼ 16.5 Hz), 149.6 (d, JC-F ¼ 18.9 Hz), 140.7,
F
111.2, 90.4, 85.7 (d, JC-P ¼ 11.8 Hz), 79.2, 77.9, 65.9 (d, JC-
116.1, 88.7, 85.7 (d, JC-P ¼ 11.6 Hz), 79.6, 78.1, 66.4 (d, JC-P ¼ 157.4 Hz),
¼ 157.4 Hz), 12.8, 11.6. 31P NMR (162 MHz, D2O)
d
15.1. HRMS:
13.1. 19F NMR (376 MHz, D2O)
d
ꢁ53.1. 31P NMR (162 MHz, D2O)
P
calcd. for C11H16N2O8P[M-2Na þ H]-: 335.0650, found: 335.0652.
d
12.9. HRMS: calcd. for C11H14FN5O6P[M-2Na þ H]-: 362.0671,
found: 362.0673.
4.2.13. 1-(Cytosin-1-yl)-4(R)-methyl-3-O-(phosphonomethyl)-
a-L-
threose disodium salt (8c)
4.2.18. 1-(Adenin-1-yl)-4(R)-methyl-3-O-(phosphonomethyl)-
a-L-
Compound 7c was hydrolyzed according to the procedure as
threose disodium salt (8 h)
20
described in 4.2.11 to afford 8c as a white solid in 57% yield. [
a
]
Compound 7h was hydrolyzed according to the procedure as
D þ12.0 (c 0.33, H2O). 1H NMR (400 MHz, D2O)
d
7.73 (d, J ¼ 7.6 Hz,
described in 4.2.11 to afford 8h as a white solid in 50% yield. [a]
20
1H), 5.92 (d, J ¼ 7.6 Hz, 1H), 5.78 (d, J ¼ 1.5 Hz, 1H), 4.38 (dq, J ¼ 6.4,
3.6 Hz, 1H), 4.29 (s, 1H), 3.75 (d, J ¼ 3.3 Hz, 1H), 3.48 (dd, J ¼ 12.7,
D ꢁ9.04 (c 0.33, H2O). 1H NMR (400 MHz, D2O)
d 8.35 (s, 1H), 8.05 (s,
1H), 5.88 (s, 1H), 4.68 (s, 1H), 4.50e4.47 (m,1H), 3.97 (s,1H), 3.50 (d,
8.8 Hz, 2H), 3.40 (dd, J ¼ 12.7, 9.2 Hz, 2H), 1.32 (d, J ¼ 6.4 Hz, 3H).13
C
J ¼ 8.4 Hz, 2H), 1.34 (d, J ¼ 6.8 Hz, 3H). 13C NMR (101 MHz, D2O)
NMR (101 MHz, D2O)
d
166.1, 157.6, 142.7, 96.1, 91.1, 85.4 (d, JC-
d
155.4, 152.6, 148.7, 141.0, 118.2, 87.8, 85.6 (d, JC-P ¼ 9.4 Hz), 79.2,
¼ 10.4 Hz), 79.4, 77.8, 66.7 (d, JC-P ¼ 154.4 Hz), 12.9. 31P NMR
78.3, 68.4 (d, JC-P ¼ 151.1 Hz), 13.6. 31P NMR (162 MHz, D2O)
d 13.0.
P
(162 MHz, D2O)
d
13.3. HRMS: calcd. for C10H15N35O7P[M-
HRMS: calcd. for C11H15N5O6P[M-2Na þ H]-: 344.0765, found:
2Na þ H]-: 320.0653, found: 320.0655.
344.0768.
13

F.-W. Liu, S. Ji, Y. Gao et al.
European Journal of Medicinal Chemistry 221 (2021) 113513
4.3. Cell culture
4.8. Western blot analysis
Human breast cancer cells (MCF-7) and (MDA-MB-231),
esophageal cancer (EC109), lung cancer (A549), breast cancer
(MCF-7), prostate cancer (PC3), cervical cancer (HeLa), colon cancer
(SW620), stomach cancer (MGC-803) cell lines, human gastric
epithelial cell line (GES-1), the above cells were purchased from
China Center for Type Collection (CCTCC, China) and grown in
RPMI-1640 medium (Corning, USA) with 10% fetal bovine serum
(FBS). Cells were cultured in a humid atmosphere of 5% CO2 and 95%
air at 37 ꢀC.
The effect of compound 8g on the cell cycle and apoptosis of
breast cancer was detected by Western blot. For example, the
expression levels of DNA damage-related protein, cell cycle regu-
lators, and apoptosis related proteins. The protein samples were
separated by sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis (SDS-PAGE; 5%e15% gels), with protein standards used as
molecular weight markers. After electrophoresis, proteins were
transferred to nitrocellulose filter membranes. The membranes
were blocked with 5% nonfat-dried milk in phosphate-buffered
saline with 0.1% Tween 20 for 2 h and incubated with primary
antibodies overnight at 4 ꢀC. After the primary antibodies had been
incubated overnight, the membranes were washed three times in
phosphate-buffered saline with 0.1% Tween 20 for 15 min, incu-
bated with biotin-conjugated secondary antibodies for 1 h with
gentle shaking, and washed again in phosphate-buffered saline
with 0.1% Tween 20. Blots were visualized with X-ray film. Protein
expression was measured with ImageJ software.
4.4. Determination of in vitro anticancer activity
For evaluating the cytotoxic effect of tested compounds. The
cells were plated at a density of 3000 exponentially growing cells
per well seeded into 96-well plate and incubated at 37 ꢀC. After 12 h
incubation, remove medium and add 200 mL fresh medium con-
taining different concentration of candidate compounds to each
well for another 72 h incubation. Then, 20 mL MTT solution (5 mg/
mL) was added to each well and allowed to react for 4 h. Removal of
the medium containing MTT, followed by addition of 150 mL DMSO
to each well and agitation until the dark blue crystal was dissolved
completely. Absorbance (A) was determined at 570 nm with a
reference wavelength of 630 nm. Finally, calculate the relative
percentage inhibition rate of viable cells.
4.9. Statistical analysis
SPSS 18.0 statistical software was used for variance testing. All
results were presented as mean standard deviation. The signifi-
cance of difference between the control group and the three
experimental groups was analyzed with one-way analysis of vari-
ance. Differences were considered to be statistically significant
when P < 0.05. All of the analyses were performed with Graph Pad
8.0.
4.5. Colony formation assay
Cancer cells were cultured at a density of 1 ꢂ 103 in six-well
plates. After an overnight incubation, the medium was removed
and the cells were treated with 3 mL fresh medium containing
various concentrations of compound 8g dissolved in DMSO for 24 h
incubation. After 24 h, the medium was removed, the plates were
incubated for another 7 days. After treatment, the cell colonies
were washed twice by PBS, fixed with 4% paraformaldehyde for
30 min, and stained with 0.1% crystal violet for 30 min. After
washing away the crystal violet with PBS, the plates were photo-
graphed and counted. Finally, the number of cell clones was
analyzed using ImageJ.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by Natural Science Foundation of
Henan Province, China (No. 162300410281) and National Natural
Science Foundation of China (No. U1904156).
4.6. Flow cytomety measure for cells apoptosis and cell cycle
Appendix A. Supplementary data
For apoptotic assay, using different concentrations of compound
8g, after 48 h treatment, cells were collected and used the
AnnexinV-FITC/PI apoptosis kit (KeyGEN BioTECH, USA) according
to the manufacturer's instructions to detect apoptosis cells. About
ten thousand events were collected for each sample and were
analyzed by flow cytometry (BD Bioscience, USA).
For cell cycle distribution assay, using different concentrations
of compound 8g, after 48 h treatment, the cells were collected and
washed with ice-cold PBS, fixed with 70% ice-cold ethanol at 4 ꢀC
overnight. Before analyzed by Instrument LSRForteassa (BD
Bioscience, USA), the cells were washed by PBS and stained with
RNase and PI (KeyGEN BioTECH, USA) for 30 min in dark condition
according to the protocol.
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113513.
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