Design and synthesis of parthenolide and 5-fluorouracil conjugates as potential anticancer agents against drug resistant hepatocellular carcinoma

Article abstract of DOI:10.1016/j.ejmech.2019.111706

A series of twenty-three parthenolide-5-fluorouracil (5-FU) conjugates ware synthesized and evaluated for their anti-cancer activities against human hepatocellular carcinoma cell line Bel-7402 and 5-fluorouracil resistant human hepatocellular carcinoma cell line Bel-7402/5-FU. The preliminary structure-activity relationships were discussed. The most active compound 15d showed high activity against Bel-7402/5-FU cell line with IC₅₀ value of 2.25 μM, which demonstrated 5.8-fold improvement compared to that of the parent compound parthenolide (IC₅₀ = 12.98 μM). The investigation of preliminary molecular mechanism of 15d revealed that 15d could reverse drug resistance by inhibiting MDR1, ABCC1 and ABCG2 to increase the intracellular drug accumulation and induce apoptosis of Bel-7402/5-FU cells through mitochondria mediated pathway. On the base of these results, compound 15d is deserved to be further investigated as a potential anti-HCC lead compound for ultimate discovery of pathenolide-based anti-cancer drug.

Full text of DOI:10.1016/j.ejmech.2019.111706

European Journal of Medicinal Chemistry 183 (2019) 111706
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design and synthesis of parthenolide and 5-fluorouracil conjugates as
potential anticancer agents against drug resistant hepatocellular
carcinoma
,
,
Yahui Ding a 1, Shengzu Li a 1, Weizhi Ge a, Zhongquan Liu a, Xuhai Zhang a,
Mengmeng Wang b, Tianyang Chen a, Yue Chen a **, Quan Zhang a
,
, *
a State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University,
Haihe Education Park, 38 Tongyan Road, Tianjin, 300353, People's Republic of China
b Accendatech Company, Ltd., Tianjin, 300384, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of twenty-three parthenolide-5-fluorouracil (5-FU) conjugates ware synthesized and evaluated
for their anti-cancer activities against human hepatocellular carcinoma cell line Bel-7402 and 5-
fluorouracil resistant human hepatocellular carcinoma cell line Bel-7402/5-FU. The preliminary
structure-activity relationships were discussed. The most active compound 15d showed high activity
Received 10 June 2019
Received in revised form
30 August 2019
Accepted 14 September 2019
Available online 16 September 2019
against Bel-7402/5-FU cell line with IC50 value of 2.25
compared to that of the parent compound parthenolide (IC50 ¼ 12.98
m
M, which demonstrated 5.8-fold improvement
M). The investigation of pre-
m
liminary molecular mechanism of 15d revealed that 15d could reverse drug resistance by inhibiting
MDR1, ABCC1 and ABCG2 to increase the intracellular drug accumulation and induce apoptosis of Bel-
7402/5-FU cells through mitochondria mediated pathway. On the base of these results, compound 15d is
deserved to be further investigated as a potential anti-HCC lead compound for ultimate discovery of
pathenolide-based anti-cancer drug.
Keywords:
Conjugation
Parthenolide
5-Fluorouracil
Hepatocellular carcinoma drug resistance
Structure-activity relationship
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction
poor overall prognosis for HCC [8]. The currently used chemo-
therapeutics for the treatment of HCC include doxorubicin,
Hepatocellular carcinoma (HCC) is the most common type of
primary liver cancer with a high global prevalence [1,2]. HCC is
highly fatal with only 18% 5-year relative survival rates [3]. In china,
HCC is the third leading causes of cancer death [4]. In the United
States, HCC is the fifth and eighth leading cause of cancer death in
men and women, respectively [3,5]. Most HCC cases are reported in
the developing countries, but there has been an alarming increase
in United States. Death rates of HCC are increasing at a faster pace
than any other cancer [1e5].
cisplatin, 5-fluorouracil (5-FU), gemcitabine, methotrexate agents,
sorafenib, regorafenib [8e11]. 5-FU was used in clinical practice as
first-line drug for the treatment of HCC. However, this chemo-
therapy comes with severe drug resistance, which led to limited or
no efficiency [8,9]. Thus, there is an urgent need to investigate
novel potential drugs which may be effective in the control of drug
resistant HCC.
Parthenolide (PTL, Fig. 1), a prominent naturally occurring ger-
macranolide originally isolated from Tanacetum parthenium L.,
showed cytotoxicity against various human cancer cells, while
sparing normal cells [12]. Even though PTL showed potent anti-
cancer activity, its poor pharmacologic properties limit its clinical
application [13]. To overcome this problem, many studies have
been devoted to investigation of its structure-activity relationships
(SARs) [14e30], which led to discovery of a PTL analogue dime-
thylaminoparthenolide (DMAPT) [14]. DMAPT showed an oral
bioavailability of 70% and was evaluated in a clinical trial for
treatment of AML [31,32]. Recently, we developed the other PTL
Surgical resection and liver transplant are curative treatments
for HCC, particularly for HCC at early stage [6,7]. However, most
cases of HCC are diagnosed in an advanced stage because of the
* Corresponding author.
** Corresponding author.
E-mail addresses: yuechen@nankai.edu.cn (Y. Chen), zhangquan@nankai.edu.cn
(Q. Zhang).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2019.111706
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

2
Y. Ding et al. / European Journal of Medicinal Chemistry 183 (2019) 111706
ester 6 (Scheme 2). Azide 7 was synthesized by reaction of com-
pound 5 and DPPA with the presence of DBU [23]. Then, azide 7 was
subjected to azide-alkyne cycloaddition with terminal alkyne 1
catalyzed by Cu2O nanoparticles (Cu2O-NPs), our previously
developed efficient catalyst for azide-alkyne cycloaddition [43], to
produce PTL-5-FU conjugate 8. Acid 9 was prepared by esterifaction
of alcohol 5 and Succinic anhydride catalyzed by DMAP in
dichloromethane. Acid 9 was treated with alcohol 2 in the presence
of DCC and DMAP to yield ester 10. Removal of Boc group of com-
pound 4a or 4b with trifluoroacetic acid, followed by conjugation
with acid 9 afforded the desired amide 11a or 11b, respectively.
Conjugates 12, 13a, 13b, 14a and 14b were synthesized through the
route outlined in Scheme 3. Alkylation of 6, 8, 11a and 11b with
benzyl bromide or 4-fluorobenzyl bromide provided conjugates 12,
13a, 13b, 14a and 14b, respectively. Compound 6 was treated with
appropriate different patterns of substituted benzyl halides or alkyl
halides with K2CO3 as base in DMF to afford conjugates 15ae15m
(Scheme 4).
Fig. 1. Structures of PTL, DMAPT, and ACT001.
derivative, ACT001 [26], to be in clinical trial in Australia and China
for treatment of glioblastoma [33].
Molecular combination strategy is a rational design of a new
chemical entity based structures of two or more known bioactive
molecules. The conjugates are commonly generated by connection
of the individual molecules via a chemically stable linker. Generally,
the conjugates could offer several advantages such as an
improvement of affinity and efficacy, an increase in the solubility,
multiple-targets in one molecule, or countering balance the known
side effects comparing to the one or both conjugated molecules.
Selection of two chemical entities for fusion is usually based on
their observed synergistic or additive pharmacological activities
[34e36].
Recently, it was reported that combination therapy with PTL and
5-FU had a synergistic anti-cancer effect both in vitro and in vivo
[37,38], which inspired us to evaluate the anti-cancer efficacy of PTL
and 5-FU conjugates. In our continuing efforts to develop PTL-based
potential anti-cancer drugs [23e30], we report herein the design
(Fig. 2) and synthesis of a series of conjugates of PTL and 5-FU, and
evaluation of their anti-HCC activities against human hepatocel-
lular carcinoma cell line Bel-7402 and 5-FU resistant human he-
patocellular carcinoma cell line Bel-7402/5-FU. The preliminary
structure-activity relationship was discussed, which led to the
discovery of the most potent compound 15d. The preliminary
molecular mechanism of compound 15d was also investigated.
2.2. Biological activities against HCC cell lines
All twenty-three synthesized conjugates were evaluated for
their anti-HCC activity against proliferation of human hepatocel-
lular carcinoma cell line Bel-7402 and 5-FU resistant human he-
patocellular carcinoma cell line Bel-7402/5-FU. PTL and 5-FU was
also included for comparison. Clinically used drug adriamycin, ADR,
was used as a positive control. These results were shown in Table 1
and Table 2.
5-FU showed potent activity against Bel-7402 cells with IC50
value of 7.36
5-FU cells (IC50 > 400
against Bel-7402 cells (IC50 ¼ 8.62
proliferative activity against Bel-7402/5-FU cells (IC50 ¼ 12.98
m
M. However, 5-FU had no potency against Bel-7402/
M). The parent compound PTL was potent
M), and maintained anti-
M).
m
m
m
Among twenty-three synthesized PTL-5-FU conjugates, sixteen
compounds showed more potent activity against Bel-7402/5-FU
cells than their parent compound PTL except for compounds 6, 8,
10, 11a, 11b, 14a and 14. Moreover, most of the conjugates were
more or comparable sensitive to Bel7402 and Bel-7402/5-FU cell
lines in contrast to 5-FU that was only active for Bel7402 while had
no efficacy to Bel-7402/5-FU cells.
2. Results and discussion
2.1. Chemistry
As shown in Table 1, for subseries of 6, 8, 10, 11a and 11b with no
substituent on 3-N-position of 5-FU, they lost activity against both
of Bel-7402 and Bel-7402/5-FU cell lines. Fortunately, when a
benzyl group was introduced to 3-N-5-FU moiety of conjugates, the
anti-HCC activity of conjugates 12, 13a, 14a and 14b was greatly
potentiated comparing to their counterparts 6, 8, 11a and 11b. The
result suggested that substituent on 3-N-5-FU moiety was signifi-
cant for their anti-HCC activity. Compound 13b, introducing of 4-
fluorobenzyl group to 3-N-5-FU moiety, was found to be approxi-
mately 1.86- and 1.82-fold more potent against Bel-7402 and Bel-
7402/5-FU cells, respectively, than compound 13a. For the subseries
12, 13a, 14a and 14b, compound 12 was most potent with IC50
The synthesis of the conjugates was illustrated in Schemes 1e4.
As shown in Scheme 1, the key intermediates 1, 2 and 3 were
prepared with 5-FU as starting material according to reported
procedures [39e41]. Conjugation of
3
with 1-Boc-1,6-
diaminohexane or 1-Boc-piperazine generated 4a or 4b, respec-
tively [42]. Compound 5 was readily obtained from PTL following
our previously reported route [25]. Reaction of compound 5 with
acid 3 in the presence of N,N-dicyclohexylcarbodiimide (DCC) and
4-dimethylaminopyridine (DMAP) in dichloromethane afforded
values of 3.98 mM and 4.17 mM for Bel-7402 and Bel-7402/5-FU cell
lines, respectively. Considering compound 12 showed the most
potent activity, more studies were devoted to explore its SAR.
To further investigate the effect of substituents at 3-N-5-FU
moiety on the anti-HCC activity and explore the SAR, a series of
compounds 15ae15m with different patterns of substituents on the
benzyl group or alkyl group at 3-N-5-FU moiety was synthesized
and evaluated for their anti-HCC activity. Generally, all the conju-
gates with 3-N-substitution of 5-FU moiety were more active than
parent compound PTL for Bel-7402/5-FU cells, and the conjugates
with substituted benzyl groups (15ae15k) with IC50 values ranging
from 2.25
mM to 4.5 mM for Bel-7402 cells and ranging from 2.25 mM
Fig. 2. Design of PTL-5-FU conjugates.
to 5.01 M for Bel-7402/5-FU cells were more active than the
m

Y. Ding et al. / European Journal of Medicinal Chemistry 183 (2019) 111706
3
Scheme 1. Synthesis of intermediates 1e4.
Reagents and conditions: (a) K2CO3, DMF, propargyl bromide, room temperature, 6 h, 31%; (b) formaldehyde, 60 ꢀC, 2 h, 87%; (c) KOH, bromoacetic acid, H2O, 40 ꢀC-60 ꢀC, 5 h, 72%; d)
EDCI, HOBt, N-Boc-1,6-diaminohexane or 1-Boc-piperazine, DMF, room temperature, 12 h, 65% for 4a, 56% for 4b.
Scheme 2. Synthesis of conjugates 6, 8, 10, 11a and 11b.
Reagents and conditions: (a) SeO2, t-BuOOH, CH2Cl2, room temperature, 4 d, 72%; (b) DCC, DMAP, compound 3, room temperature, 12 h, 50%; (c) DPPA, DBU, THF, 0 ꢀC, 12 h, 69%; (d)
Cu2O-NPs, THF-H2O, compound 1, room temperature, 12 h, 37%; (e) Succinic anhydride, DMAP, DCM, room temperature, 12 h, 88%; (f) compound 2, DCC, DMAP, DCM, room
temperature, 12 h, 44%; (g) step 1: compound 4a or 4b, TFA, DCM, room temperature, 3 h; step 2: EDCI, HOBt, DIPEA, DCM, room temperature, 12 h, 44% over two steps for 11a, 50%
over two steps for 11b.
aliphatic groups (15l and 15m). These results suggested that the
benzyl group at 3-N-5-FU moiety could accommodate diverse
patterns of substituents, and the substituted benzyl groups were
more favored than aliphatic groups. Among the subseries
15ae15m, compound 15d exhibited the highest activity against
(IC50 ¼ 8.56
m
M) and 16 (IC50 > 50
m
M) or combination of 5 and 16
(1:1) (IC50 ¼ 7.82
mM) were less potent than 15d (IC50 ¼ 2.25
mM).
These results suggest that the anti-HCC activity of 15d may be
attributed to the synergic effects of two moieties. The most potent
compound 15d was further evaluated for its toxicity against normal
3T3 cells (mouse embryonic fibroblast cell line). As shown in
Table 2, 15d had selective cytotoxicity against HCC cells
(IC50 ¼ 2.25
with therapeutic index (TI) value of 2.3.
Taking account that compound 15d showed the most potent
Bel-7402/5-FU cells with IC50 value of 2.25
fold improvement compared to that of the parent compound PTL
(IC50 ¼ 12.98 M), moreover, which indicated a comparable po-
tency with the clinically used anti-cancer drug ADR
(IC50 ¼ 1.14 M). Moreover, as shown in Fig. 3, compounds 5
mM, demonstrating 5.8-
m
mM) being compared with 3T3 cells (IC50 ¼ 5.2
mM)
m

4
Y. Ding et al. / European Journal of Medicinal Chemistry 183 (2019) 111706
Scheme 3. Synthesis of conjugates 12, 13a, 13b, 14a and 14b.
Reagents and conditions: (a) K2CO3, benzyl bromide, DMF, room temperature, 6 h, 78%; (b) K2CO3, CH3CN, 4-fluorobenzyl bromide or benzyl bromide, room temperature, 6 h, 90%
yield for 13a, 90% yield for 13b; (c) K2CO3, benzyl bromide, DMF, room temperature, 6 h, 78% for 14a, 55% for 14b.
anti-HCC activity and did not significantly affect the normal cells,
15d was further selected for investigation of its preliminary
mechanism.
assay. As shown in Fig. 6, after the treatment of 15d for 24 h in Bel-
7402/5-FU cells, the intracellular accumulation of ADR was signif-
icantly enhanced with a dose-dependent manner. This result sug-
gested that 15d could reverse drug resistance by increasing the
intracellular drug accumulation.
To further explore the mechanism of 15d in Bel-7402/5-FU cells,
cell apoptosis assay was performed. As shown in Fig. 7, the per-
centage of apoptosis was 3.37 0.47, 3.97 0.32, 4.90 0.98,
14.43 2.48 and 77.97 2.94 after the treatment of 15 d at a con-
2.3. Preliminary mechanism study of compound 15d
From the results of the MTT assay, 15d showed preferably ac-
tivity against 5-FU resistant Bel-7402/5-FU cells. To explore the
mechanism of 15d in Bel-7402/5-FU cells, we detected the
expression level of multi-drug resistant proteins including MDR1,
ABCC1 and ABCG2 in Bel-7402 and Bel-7402/5-FU cells, which were
recognized as efflux transporters and played important roles in
drug resistance. As shown in Fig. 4A, Bel-7402/5-FU cells showed a
marked up-regulation of MDR1, ABCC1 and ABCG2 than that of Bel-
7402 cells. As shown in Fig. 4BeD, after the treatment of increasing
concentrations of 15d for 24 h, the level of MDR1, ABCC1 and
ABCG2 were significantly decreased in a dose-dependent manner.
Moreover, from the result of flow cytometry in Fig. 5, the level of p-
gp was significantly decreased after the treatment of 15d for 24 h at
0.5 mM. These results indicated that 15d could reverse drug resis-
tance by downregulated expression of MDR1, ABCC1 and ABCG2 in
Bel-7402/5-FU cells.
It was reported that the over expression of MDR1, ABCC1 and
ABCG2 reduced the accumulation of drugs in cells [44]. To deter-
mine whether 15d could increase drug accumulation in Bel-7402/5-
FU cells to reverse drug resistance, we performed the accumulation
centration of 0, 0.5 mM, 1 mM, 2.5 mM and 5 mM, respectively.
Moreover, compound 15d exhibited stronger effect on induction of
cell apoptosis compared with that of PTL and 5-FU (Fig. 7). Mean-
while, the DAPI staining experiment showed that the percentage of
irregular nuclear morphology and apoptotic bodies were signifi-
cantly increased after the treatment of 15d with a dose-dependent
manner (Fig. 8). These results indicated that 15d could induce the
apoptosis of Bel-7402/5-FU cells in a dose dependent manner.
In order to further elucidate the mechanism of 15d for induction
of cell apoptosis, the mitochondria mediated apoptosis pathway
related proteins were analyzed by Western blot experiment. As
shown in Fig. 9, the expression levels of pro-apoptotic proteins Bax
and Bim were up-regulated, while the levels of anti-apoptosis
proteins Bcl-2 and Bcl-xl were greatly down-regulated after the
treatment of 15d. Then the level of cytochrome C which activated
the cleavage of caspase-3 and caspase-9 was clearly increased.
Moreover, the caspase-3 and caspase-9 were activated and

Y. Ding et al. / European Journal of Medicinal Chemistry 183 (2019) 111706
5
Scheme 4. Synthesis of conjugates 15ae15m.
Reagents and conditions: (a) K2CO3, DMF, different substituted halides, room temperature, 6 h, 45%e78%.
Table 1
Bel-7402/5-FU cells than their parent compound PTL. It is worth to
note that most of the conjugates were more or comparable sensi-
tive to Bel7402 and Bel-7402/5-FU cell lines in contrast to 5-FU that
was only active for Bel7402 while had no efficacy to Bel-7402/5-FU
cells. Based on the above structure and activity results, the
following preliminary SARs can be drawn: (1) substituent on 3-N-5-
FU moiety of PTL-5-FU conjugates was significant for their anti-HCC
activity; (2) the benzyl group at 3-N-5-FU moiety can accommodate
diverse patterns of substituents, and the substituted benzyl groups
were more favored than aliphatic groups.
The above investigation led to discovery of the most active
compound 15d, which demonstrated 5.8-fold improvement
compared to that of the parent compound PTL, and showed a
comparable potency with the clinically used anti-cancer drug ADR.
Compound 15d could induce apoptosis of Bel-7402/5-FU cells
through mitochondria mediated pathway and reverse drug resis-
tant by inhibiting MDR1, ABCC1 and ABCG2 to increase the intro-
cellular drug accumulation. On the base of these results, com-
pound 15d is deserved to be further investigated as a potential anti-
HCC lead compound for ultimate discovery of pathenolide-based
anti-cancer drug.
Anti-HCC activities of PTL-5-FU conjugates.
a
Compounds
IC50
(
mM)
Bel-7402b
Bel-7402/5-FUc
6
8
10
11a
11b
12
13a
13b
14a
14b
PTL
5-FU
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
3.98 2.39
9.16 1.02
4.91 1.41
18.29 0.83
>20
8.62 1.68
7.36 1.58
e
4.27 1.29
8.84 2.49
4.86 0.64
16.16 1.57
>20
12.98 3.96
>400
8.36 3.63
1.14 0.59
PTLþ5-FU (1:1)
ADRd
0.73 0.28
a
All values are the mean of three independent experiments.
Bel-7402: human hepatocellular carcinoma cell line.
Bel-7402/5-FU: 5-FU resistant human hepatocellular carcinoma cell line.
ADR, adriamycin, used as a positive control.
b
c
d
eventually induced apoptosis of Bel-7402/5-FU cells. These results
suggested that 15d could induce apoptosis of Bel-7402/5-FU cells
through mitochondria medicated pathway (Fig. 10).
4. Experimental
4.1. Chemistry
3. Conclusion
Unless otherwise mentioned, all reactions were carried out
under a nitrogen atmosphere with dry solvents under anhydrous
conditions. Reactions were monitored by thin-layer chromatog-
raphy carried out on 0.25 mm Tsingdao silica gel plates (60F-254).
Visualization was achieved using UV light, phosphomolybdic acid
in 1þ1ethanol or potassium permanganate in water, each followed
In summary, a series of twenty-three new parthenolide-5-
fluorouracil conjugates ware synthesized and evaluated for their
anti-HCC activities. Among twenty-three synthesized PTL-5-FU
conjugates, sixteen compounds showed higher activity against

6
Y. Ding et al. / European Journal of Medicinal Chemistry 183 (2019) 111706
Table 2
Anti-HCC activities of conjugates 15ae15m.
R3
H
IC50
(m
M)
a
Compounds
Bel-7402b
Bel-7402/5-FUc
6d
>20
3.98 2.39
>20
4.27 1.29
12d
15a
15b
15c
2.73 0.86
2.56 0.98
2.67 1.10
2.25 0.25
2.90 0.88
2.42 0.11
3.24 0.89
2.25 0.16
15de
15e
4.07 0.64
4.34 1.00
Fig. 3. Comparison of anti-HCC activity of 15d with that of 5, 16, and an equimolar
mixture of 5 and 16 against Bel-7402/5-FU cells.
15f
2.53 0.38
3.77 0.11
3.70 0.69
3.83 0.64
spectrometer. Data are reported as follows: chemical shift, multi-
plicity (s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, br. ¼ broad,
m ¼ multiplet), coupling constants and integration.
15g
15h
15i
3.67 0.62
4.50 1.20
5.01 1.17
4.98 0.79
4.1.1. ((1aR,7aS,10aS,10bS,E)-1a-Methyl-8-methylene-9-oxo-
1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[20,30:9,10]cyclodeca[1,2-
b]furan-5-yl)methyl 2-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)acetate (6)
A mixture of compound 3 (80.00 mg, 0.30 mmol), compound 5
(72.7 mg, 0.38 mmol), DCC (79.00 mg, 0.38 mmol) and 4-
dimethylaminopyridine (4.00 mg, 0.03 mmol) was stirred in
anhydrous dichloromethene (3.2 mL) for 12 h at room temperature.
The reaction mixture was filtered and filtrate was evaporated in
vacuo. The resulting crude product was further purified by column
chromatography on silica gel to give compound 6 (white ceraceous
15j
2.70 1.10
3.06 0.65
4.48 0.46
4.61 0.39
15k
solid 78 mg, yield 50%). 1H NMR (400 MHz, CDCl3)
d 10.14 (s, 1H),
7.38 (d, J ¼ 5.3 Hz, 1H), 6.17 (d, J ¼ 2.9 Hz, 1H), 5.71 (t, J ¼ 8.1 Hz, 1H),
5.58 (d, J ¼ 2.2 Hz, 1H), 4.75 (d, J ¼ 12.3 Hz, 1H), 4.56 (d, J ¼ 12.3 Hz,
1H), 4.52e4.38 (m, 2H), 3.83 (t, J ¼ 9.3 Hz, 1H), 2.93e2.77 (m, 2H),
2.47e2.09 (m, 6H), 1.70e1.63 (m, 1H), 1.51 (s, 3H), 1.07 (t,
15l
9.40 1.43
8.58 0.19
11.73 1.58
9.11 1.40
J ¼ 12.7 Hz, 1H). 13C NMR (100 MHz, CDCl3)
d 169.6, 167.2, 157.4 (d,
15m
J ¼ 26.2 Hz), 149.8, 140.4 (d, J ¼ 237.6 Hz), 138.6, 134.1, 131.9, 129.2
PTL
5-FU
e
e
e
e
8.62 1.68
7.36 1.58
e
12.98 3.96
>400
8.36 3.63
1.14 0.59
(d, J ¼ 33.2 Hz), 120.6, 81.1, 68.6, 63.2, 60.2, 49.2, 42.6, 36.4, 25.5,
24.4, 23.8, 17.9. 19F NMR (376 MHz, CDCl3)
d
ꢁ163.23. HRMS (ESI)
PTLþ5-FU (1:1)
ADRf
0.73 0.28
calcd for C21H23FN2NaOþ7 [MþNa]þ457.1382, found 457.1385.
a
All values are the mean of three independent experiments.
Bel-7402: human hepatocellular carcinoma cell line.
Bel-7402/5-FU: 5-FU resistant human hepatocellular carcinoma cell line.
Data from Table 1.
IC50 value for 3T3 cell line was 5.2 0.45
ADR, adriamycin, used as a positive control.
b
c
d
e
f
4.1.2. (1aR,7aS,10aS,10bS,E)-5-(Azidomethyl)-1a-methyl-8-
methylene-2,3,6,7,7a,8,10a,10b-octahydrooxireno[20,30:9,10]
cyclodeca[1,2-b]furan-9(1aH)-one (7)
To a solution of compound 5 (1.0 g, 3.80 mmol) in THF (37.6 mL)
was added diphenylphosphorous azidophosphate (1.6 g,
5.70 mmol) and 1,8-diazabicyclo[rho]el-7-ene (0.9 g, 5.70 mmol) at
0 ꢀC. The reaction mixture was stirred for 12 h at room temperature,
and then filtered. The filtrate was evaporated in vacuo, and the
mM.
by heating. Tsingdao silica gel (60, particle size 0.040e0.063 mm)
was used for flash column chromatography. NMR spectra were
recorded with
a
400 MHz (1H: 400 MHz, 13C: 100 MHz)

Y. Ding et al. / European Journal of Medicinal Chemistry 183 (2019) 111706
7
Fig. 4. Compound 15d reduced the expression level of MDR1, ABCC1 and ABCG2 in Bel-7402/5-FU cells. (A) The expression of MDR1, ABCC1 and ABCG2 in Bel7402 and Bel-7402/
5-Fu cells. (B) The expression of MDR1, ABCC1 and ABCG2 in Bel-7402/5-Fu cells after the treatment of 15 d at different concentrations. (C, D) The quantitative analysis of protein
expression and statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 5. Compound 15d inhibited the expression of p-gp. (A) The fluorescence intensity of p-gp with the treatment of 15 d at a concentration of 0.5
The statistics of the fluorescence intensity after the treatment of 15d in Bel7402/5-FU cells. *P < 0.05.
mM in Bel-7402/5-FU cells. (B)
resulting crude product was further purified by column chroma-
tography on silica gel to yield compound 7 was obtained as white
ceraceous solid 750.0 mg. Yield 69%. 1H NMR (400 MHz, CDCl3)
4.1.3. 5-Fluoro-1-((1-(((1aR,7aS,10aS,10bS,E)-1a-methyl-8-
methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno
[20,30:9,10]cyclodeca[1,2-b]furan-5-yl)methyl)-1H-1,2,3-triazol-4-
yl)methyl)pyrimidine-2,4(1H,3H)-dione (8)
To s stirred solution of compound 7 (117.7 mg, 0.70 mmol) in
THF (0.8 mL) was added compound 1 (100.0 mg, 0.35 mmol), H2O
(0.5 mL) and Cu2O-NPs (0.5 mL) under Ar at room temperature.
After being stirred for overnight, the reaction mixture was washed
with cold saturated NaCl and then water. The extract was dried over
Na2SO4, filtered and evaporated in vacuo. The crude product was
further purified by column chromatography on silica gel to give
d
6.15 (d, J ¼ 3.2 Hz, 1H), 5.61 (t, J ¼ 8.3 Hz, 1H), 5.50 (d, J ¼ 2.6 Hz,
1H), 3.83e3.73 (m, 2H), 3.64 (d, J ¼ 13.3 Hz, 1H), 2.80e2.63 (m, 2H),
2.43e2.34 (m, 1H), 2.34e2.31 (m, 1H), 2.27 (dd, J ¼ 14.7, 10.1 Hz,
2H), 2.22e2.14 (m, 1H), 2.10 (dd, J ¼ 12.8, 6.0 Hz, 1H), 1.70e1.59 (m,
1H), 1.48 (s, 3H), 1.03 (t, J ¼ 13.0 Hz, 1H). 13C NMR (100 MHz, CDCl3)
d
169.2, 138.6, 134.6, 131.0, 120.2, 80.9, 63.2, 59.9, 55.4, 42.5, 36.5,
25.2, 24.1, 23.7, 17.8. HRMS (ESI) calcd for C15H19N3NaO3 [MþNa]þ
312.1319, found 312.1322.

8
Y. Ding et al. / European Journal of Medicinal Chemistry 183 (2019) 111706
Fig. 6. Compond 15d could increase the accumulation of ADR in Bel-7402/5-FU cells. (A) The accumulation of ADR in Bel-7402/5-FU cells after the treatment of 15 d at different
concentrations of 0.5 M, 1 M, 2.5 M, 5 M, respectively. (B) The statistics of ADR accumulation in Bel-7402/5-FU cells after the treatment of 15 d at different concentrations of
0.5 M, 1 M, 2.5 M, 5 M, respectively. *P < 0.05, **P < 0.01, ***P < 0.001.
m
m
m
m
m
m
m
m
Fig. 7. Compound 15d induced the apoptosis of Bel-7402/5-FU cells. (A) The representative pictures of apoptosis after the treatment of 15 d at different concentrations by flow
cytometry. (B) The statistic of the apoptosis of Bel-7402/5-FU cells after the treatment of 15 d at different concentrations by flow cytometry. (C) The representative pictures of
apoptosis after the treatment of 15d, PTL and 5-FU at 5
respectively. *P < 0.05, **P < 0.01, ***P < 0.001.
mM, respectively. (D) The statistic of the apoptosis of Bel-7402/5-FU cells after the treatment of 15d, PTL and 5-FU at 5 mM,
compound 8 (colorless oil, 68 mg, yield 37%). 1H NMR (400 MHz,
CDCl3)
d
7.78 (d, J ¼ 3.0 Hz, 1H), 7.64 (dd, J ¼ 5.3, 1.7 Hz, 1H), 6.25 (d,
J ¼ 3.2 Hz, 1H), 5.74 (t, J ¼ 8.3 Hz, 1H), 5.63 (d, J ¼ 2.9 Hz, 1H), 5.12 (d,
J ¼ 14.5 Hz, 1H), 4.99 (d, J ¼ 15.0 Hz, 1H), 4.90 (d, J ¼ 15.0 Hz, 1H),
4.77 (d, J ¼ 14.5 Hz, 1H), 3.83 (t, J ¼ 9.3 Hz, 1H), 2.85 (d, J ¼ 9.4 Hz,
1H), 2.71 (t, J ¼ 8.7 Hz, 1H), 2.61e2.38 (m, 2H), 2.37e2.14 (m, 3H),
1.99 (d, J ¼ 15.1 Hz, 1H), 1.66 (t, J ¼ 12.5 Hz, 1H), 1.52 (s, 3H), 1.13 (t,
J ¼ 12.1 Hz, 1H). 13C NMR (100 MHz, CDCl3)
d 168.2, 159.8 (d,
J ¼ 25.5 Hz), 156.2, 148.6, 140.8, 138.4, 135.3 (d, J ¼ 378.5 Hz), 131.8,
127.6 (d, J ¼ 33.1 Hz), 122.7, 119.9, 79.8, 62.2, 58.9, 53.8, 42.4, 41.5,
35.4, 28.7, 23.0, 22.9, 16.9. 19F NMR (376 MHz, CDCl3)
d
ꢁ163.60, ꢁ163.62. HRMS (ESI) calcd for C22H24FN5NaOþ5 [MþNa]þ
480.1654, found 480.1658.
4.1.4. 4-(((1aR,7aS,10aS,10bS,E)-1a-Methyl-8-methylene-9-oxo-
1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[20,30:9,10]cyclodeca[1,2-
b]furan-5-yl)methoxy)-4-oxobutanoic acid (9)
A mixture of succinic anhydride (454.6 mg, 4.54 mmol), com-
pound 5 (1 g, 3.78 mmol), and DMAP (462.5 mg, 3.78 mmol) was
stirred in anhydrous DCM (50 mL) for 12 h at room temperature.
The mixutre was adjusted to pH 3 with hydrochloric acid. The
extract was dried over Na2SO4, filtered and the filtrate was evap-
orated in vacuo. The crude product was further purified by column
chromatography on silica gel to afford compound 9 as white
Fig. 8. The nuclear morphology of Bel-7402/5-FU cells after the treatment of 15 d at
different concentrations of 1 M, 2 M and 5 M.
m
m
m

Y. Ding et al. / European Journal of Medicinal Chemistry 183 (2019) 111706
9
Fig. 9. The Western blot analysis of apoptosis related proteins of mitochondrial pathway in Bel-7402/5-FU cells with the treatment of 15 d at different concentrations. (A)
The Western blot analysis of apoptosis related proteins. (B, C) The quantitative analysis of protein expression and statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig. 10. The preliminary anti-HCC mechanism of compound 15d.
amorphous solid (1.2 g, yield 88%). 1H NMR (400 MHz, CDCl3)
d
6.23
4.1.5. (5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl
(((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
(s, 1H), 5.69 (s, 1H), 5.55 (s, 1H), 4.69 (d, J ¼ 12.4 Hz, 1H), 4.48 (d,
J ¼ 12.3 Hz, 1H), 3.85 (t, J ¼ 9.1 Hz, 1H), 2.86 (t, J ¼ 10.6 Hz, 2H), 2.65
(d, J ¼ 24.1 Hz, 4H), 2.53e2.10 (m, 6H), 1.66 (t, J ¼ 12.1 Hz, 1H), 1.54
1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[20,30:9,10]cyclodeca[1,2-
b]furan-5-yl)methyl) succinate (10)
(s, 3H), 1.10 (t, J ¼ 12.6 Hz, 1H). 13C NMR (100 MHz, CDCl3)
d
177.7,
To a stirred solution of compound 2 (122.0 mg, 0.75 mmol) in
MeCN (2 mL), compound 9 (325.0 mg, 0.90 mmol), DCC (186.0 mg,
0.90 mmol) and DMAP (8.0 mg, 0.056 mmol) were added under Ar
at room temperature. After being stirred for overnight, the reaction
172.0, 169.6, 138.9, 134.8, 130.9, 120.5, 81.2, 67.3, 63.4, 60.2, 42.8,
36.7, 28.9, 28.8, 25.8, 24.6, 23.9, 18.1. HRMS (ESI) calcd for C19H23O7ꢁ
[M-H]- 363.1449, found 363.1445.

10
Y. Ding et al. / European Journal of Medicinal Chemistry 183 (2019) 111706
mixture was washed with cold saturated NaCl and then water. The
extract was dried over Na2SO4, filtered and evaporated in vacuo.
The crude product was further purified by column chromatography
on silica gel to provide compound 10 as white amorphous solid
J ¼ 26.0 Hz), 150.1 (d, J ¼ 4.5 Hz), 140.3 (d, J ¼ 235.8 Hz), 139.1, 134.8,
130.1 (d, J ¼ 33.5 Hz), 129.8, 120.4, 81.4, 81.3, 66.9, 63.2, 60.3, 42.6,
42.1, 41.4, 36.6, 32.0, 27.8, 25.8, 24.7, 23.9, 18.1. 19F NMR (376 MHz,
CDCl3)
d
ꢁ163.8. HRMS (ESI) calcd for C29H35FN4NaOþ9 [MþNa]þ
(130.0 mg, yield 44%). 1H NMR (400 MHz, CDCl3)
d
7.60 (d, J ¼ 5.1 Hz,
625.2280, found 625.2283.
1H), 6.22 (d, J ¼ 2.3 Hz, 1H), 5.70 (t, J ¼ 7.8 Hz, 1H), 5.64 (s, 2H), 5.56
(s, 1H), 4.67 (d, J ¼ 12.5 Hz, 1H), 4.48 (d, J ¼ 12.5 Hz, 1H), 3.85 (t,
J ¼ 9.3 Hz, 1H), 2.93 (t, J ¼ 8.7 Hz, 1H), 2.86 (d, J ¼ 9.4 Hz, 1H), 2.69
(d, J ¼ 4.7 Hz, 2H), 2.63 (d, J ¼ 4.3 Hz, 2H), 2.50e2.12 (m, 7H), 1.67 (t,
J ¼ 11.6 Hz, 1H), 1.54 (s, 3H), 0.87 (t, J ¼ 6.6 Hz, 1H).13C NMR
4.1.8. General procedure for synthesis of compounds 12, 13a, 13b,
14a, 14b, and 15ae15o
To s stirred solution of compound 6, 8, 11a or 11b (0.05 mmol) in
DMF (1 mL) was added different substituted benzyl bromide
(100 MHz, CDCl3)
d
172.6, 171.9, 169.7, 157.1 (d, J ¼ 27.0 Hz), 149.3,
(13.7 mL, 0.12 mmol) and K2CO3 (0.18 mmol) at room temperature.
140.2 (d, J ¼ 239.5 Hz), 139.0, 134.7, 131.2, 128.6 (d, J ¼ 33.8 Hz),
After stirring for 6 h, the reaction mixture was washed with cold
saturated NH4Cl and then water. The extract was dried over Na2SO4,
filtered and the filtrate was evaporated in vacuo. The resulting
crude product was further purified by column chromatography on
silica gel to provide desired compounds.
120.3, 81.1, 70.1, 67.4, 63.3, 60.1, 42.7, 36.6, 29.7, 28.8, 25.8, 24.8,
23.9,18.1.19F NMR (376 MHz, CDCl3)
d
ꢁ164.60. HRMS (ESI) calcd for
C
24H27FN2NaO9 [MþNa]þ 529.1593, found 529.1596.
4.1.6. ((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[20,30:9,10]cyclodeca[1,2-
b]furan-5-yl)methyl 4-((6-(2-(5-fluoro-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)acetamido)hexyl)amino)-4-
oxobutanoate (11a)
4.1.8.1. 1((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[20,30:9,10]cyclodeca[1,2-
b]furan-5-yl)methyl 2-(3-benzyl-5-fluoro-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)acetate (12). Yield 78%. 1H NMR
The protective Boc group of compound 4a (98.3 mg, 0.27 mmol)
were removed using trifluoroacetic acid (0.4 mL) in DCM (4 mL) for
3 h at room temperature. The reaction mixture was filtered and the
filtrate was evaporated under reduced pressure. The product in dry
(400 MHz, CDCl3)
d
7.43 (dd, J ¼ 7.7, 1.4 Hz, 2H), 7.29 (d, J ¼ 7.4 Hz,
2H), 7.24 (d, J ¼ 4.9 Hz, 1H), 6.22 (d, J ¼ 3.5 Hz, 1H), 6.22 (d,
J ¼ 3.5 Hz, 1H), 5.71 (t, J ¼ 7.9 Hz, 1H), 5.55 (d, J ¼ 3.1 Hz, 1H), 5.11 (s,
2H), 4.79 (d, J ¼ 12.2 Hz, 1H), 4.57e4.47 (m, 1H), 4.40 (t, J ¼ 15.4 Hz,
2H), 3.83 (t, J ¼ 9.3 Hz, 1H), 2.81 (d, J ¼ 9.4 Hz, 2H), 2.42e2.13 (m,
6H), 1.68e1.59 (m, 1H), 1.53 (s, 3H), 1.09 (t, J ¼ 12.3 Hz, 1H). 13C NMR
dichloromethane (2 mL) was added compound
9 (137.7 mg,
0.18 mmol), DIPEA (94.1 L,0.54 mmol), EDCI (52.3 mg,0.27 mmol)
m
and HOBt (36.0 mg, 0.27 mmol). The reaction mixture was stirred
for 12 h at room temperature, filtered, and evaporated under
reduced pressure. The crude product was further purified by col-
umn chromatography on silica gel to yield compound 11a as white
ceraceous solid (50 mg, yield 44%). 1H NMR (400 MHz, DMSO‑d6)
(100 MHz, CDCl3)
d
169.4, 167.8, 167.1, 157.3 (d, J ¼ 25.6 Hz), 150.1,
140.4 (d, J ¼ 237.1 Hz), 138.7, 135.9, 134.2, 132.4, 131.1, 129.3, 129.0,
128.7, 126.9 (d, J ¼ 33.2 Hz), 120.6, 81.1, 71.9, 68.6, 63.4, 50.3, 45.4,
42.7, 36.6, 25.6, 24.4, 24.0, 19.3. 19F NMR (376 MHz, CDCl3)
d
ꢁ163.37. HRMS (ESI) calcd for C28H29FN2NaOþ7 [MþNa]þ 547.1851,
d
11.82 (s, 1H), 8.10 (s, 1H), 8.00 (d, J ¼ 4.2 Hz, 1H), 7.81 (s, 1H), 6.03
found 547.1855.
(s, 1H), 5.65 (s, 1H), 5.57 (s, 1H), 4.58 (d, J ¼ 11.7 Hz, 1H), 4.38 (d,
J ¼ 12.8 Hz, 1H), 4.23 (s, 2H), 4.08 (t, J ¼ 8.3 Hz, 1H), 3.16 (s, 1H),
3.11e2.91 (m, 5H), 2.83 (d, J ¼ 9.0 Hz, 1H), 2.41e2.00 (m, 8H), 1.61 (t,
J ¼ 10.3 Hz, 1H), 1.46 (s, 3H), 1.36 (s, 4H), 1.23 (s, 5H), 0.92 (t,
4.1.8.2. 3-Benzyl-5-fluoro-1-((1-(((1aR,7aS,10aS,10bS,E)-1a-methyl-
8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno
[20,30:9,10]cyclodeca[1,2-b]furan-5-yl)methyl)-1H-1,2,3-triazol-4-yl)
methyl)pyrimidine-2,4(1H,3H)-dione (13a). Yield 90%. 1H NMR
J ¼ 12.3 Hz, 1H). 13C NMR (100 MHz, DMSO‑d6)
d 172.3, 170.4, 169.5,
166.2, 157.6 (d, J ¼ 25.6 Hz), 149.7, 140.4, 139.3 (d, J ¼ 228.1 Hz),
135.0, 131.1 (d, J ¼ 33.9 Hz), 128.9, 119.4, 80.7, 66.4, 62.7, 60.0, 49.7,
48.6, 41.8, 38.6, 38.5, 36.3, 29.8, 29.1, 29.0, 26.1, 26.0, 24.7, 23.8, 23.2,
(400 MHz, CDCl3)
d
7.63 (s, 1H), 7.58 (d, J ¼ 5.0 Hz, 1H), 7.47e7.41
(m, 2H), 7.33e7.26 (m, 3H), 6.28 (d, J ¼ 3.3 Hz,1H), 5.76 (t, J ¼ 8.0 Hz,
1H), 5.64 (d, J ¼ 3.0 Hz, 1H), 5.15 (d, J ¼ 14.4 Hz, 1H), 5.10 (d,
J ¼ 3.1 Hz, 2H), 4.98 (d, J ¼ 15.0 Hz,1H), 4.92 (d, J ¼ 15.0 Hz,1H), 4.69
(d, J ¼ 14.4 Hz, 1H), 3.85 (t, J ¼ 9.3 Hz, 1H), 2.84 (d, J ¼ 9.4 Hz, 1H),
2.79e2.68 (m, 1H), 2.61e2.16 (m, 6H), 1.91 (d, J ¼ 15.1 Hz, 1H), 1.53
17.5. 19F NMR (376 MHz, CDCl3)
C
d
ꢁ163.85. HRMS (ESI) calcd for
31H41FN4NaO9 [MþNa]þ 655.2750, found 655.2755.
4.1.7. ((1aR,7aS,10aS,10bS,E)-1a-Methyl-8-methylene-9-oxo-
1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[20,30:9,10]cyclodeca[1,2-
b]furan-5-yl)methyl 4-(4-(2-(5-fluoro-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)acetyl)piperazin-1-yl)-4-oxobutanoate
(11b)
The Boc group of compound 4b (84 mg, 0.24 mmol) was
removed using trifluoroacetic acid (0.4 mL) in DCM (4 mL) for 3 h at
room temperature. The reaction mixture was filtered and evapo-
rated under reduced pressure.The product in dry dichloromethane
(2 mL) was added compound 9 (131.1 mg, 0.36 mmol), DIPEA
(s, 3H), 1.18e1.10 (m, 1H). 13C NMR (100 MHz, CDCl3)
d 169.2, 157.3
(d, J ¼ 25.3 Hz), 150.0, 142.2, 140.4 (d, J ¼ 236.6 Hz), 138.3, 136.1,
134.7, 133.1, 129.3, 128.6, 128.1, 126.7 (d, J ¼ 33.0 Hz), 123.3, 121.1,
80.9, 63.4, 59.9, 55.0, 45.2, 44.2, 42.7, 36.6, 25.2, 24.2, 23.7, 18.0. 19
F
NMR (376 MHz, CDCl3)
d
ꢁ163.17. HRMS (ESI) calcd for
C
29H30FN5NaOþ5 [MþNa]þ 570.2123, found 570.2128.
4.1.8.3. 5-Fluoro-3-(4-fluorobenzyl)-1-((1-(((1aR,7aS,10aS,10bS,E)-
1a-methyl-8-methylene-9-oxo-1a,2,3,6,7,7a,8,9,10a,10b-decahy-
drooxireno[20,30:9,10]cyclodeca[1,2-b]furan-5-yl)methyl)-1H-1,2,3-
triazol-4-yl)methyl)pyrimidine-2,4(1H,3H)-dione (13b). Yield 90%.
(125 mL,0.72 mmol), EDCI (69 mg,0.36 mmol) and HOBt (48 mg,
0.36 mmol). The reaction mixture was stirred for 12 h at room
temperature, filtered and evaporated in vacuo. The crude product
was further purified by column chromatography on silica gel to
yield compound 11b as white ceraceous solid (70 mg, yield 50%). 1H
1H NMR (400 MHz, CDCl3)
d
7.64 (s, 1H), 7.59 (d, J ¼ 5.0 Hz, 1H), 7.43
(dd, J ¼ 8.4, 5.5 Hz, 2H), 6.95 (t, J ¼ 8.6 Hz, 2H), 6.25 (d, J ¼ 3.3 Hz,
1H), 5.74 (t, J ¼ 8.1 Hz, 1H), 5.63 (d, J ¼ 3.0 Hz, 1H), 5.14 (d,
J ¼ 14.5 Hz, 1H), 5.07 (d, J ¼ 13.7 Hz, 1H), 5.02 (d, J ¼ 13.9 Hz, 1H),
4.98 (d, J ¼ 15.1 Hz, 1H), 4.92 (d, J ¼ 15.0 Hz, 1H), 4.70 (d, J ¼ 14.5 Hz,
1H), 3.84 (t, J ¼ 9.3 Hz, 1H), 2.84 (d, J ¼ 9.4 Hz, 1H), 2.80e2.67 (m,
1H), 2.59e2.14 (m, 6H), 1.63 (t, J ¼ 12.0 Hz, 1H), 1.52 (s, 3H), 1.12 (t,
NMR (400 MHz, CDCl3)
d
7.40 (d, J ¼ 4.1 Hz, 1H), 6.17 (d, J ¼ 2.8 Hz,
1H), 5.66 (d, J ¼ 7.5 Hz, 1H), 5.57 (d, J ¼ 2.4 Hz, 1H), 4.70e4.54 (m,
3H), 4.55e4.46 (m, 1H), 3.84 (t, J ¼ 9.3 Hz, 1H), 3.79e3.39 (m, 8H),
3.04 (s, 1H), 2.89e2.82 (m, 1H), 2.63 (s, 4H), 2.40 (d, J ¼ 6.6 Hz, 2H),
2.30e2.09 (m, 4H), 1.69e1.60 (m, 1H), 1.51 (s, 3H), 0.85 (t, J ¼ 6.6 Hz,
J ¼ 11.6 Hz, 1H). 13C NMR (100 MHz, CDCl3)
d 169.3, 163.7, 162.5 (d,
J ¼ 246.6 Hz), 157.2 (d, J ¼ 25.2 Hz), 149.9, 142.0, 141.4, 140.2 (d,
1H). 13C NMR (100 MHz, CDCl3)
d 173.1, 170.3, 169.9, 165.2, 157.7 (d,
J ¼ 236.4 Hz), 138.3, 131.9 (d, J ¼ 3.2 Hz), 131.3 (d, J ¼ 8.2 Hz), 126.9

Y. Ding et al. / European Journal of Medicinal Chemistry 183 (2019) 111706
11
(d, J ¼ 32.9 Hz), 121.0, 115.5, 115.3 (d, J ¼ 21.4 Hz), 80.9, 63.3, 59.9,
dihydropyrimidin-1(2H)-yl)acetate (15b). Yield 54%. 1H NMR
(400 MHz, CDCl3)
54.9, 44.5, 44.1, 42.5, 36.5, 29.7, 24.1, 23.7, 17.9. 19F NMR (376 MHz,
d
7.40 (d, J ¼ 8.4 Hz, 2H), 7.32e7.27 (m, 3H), 6.23
CDCl3)
d
ꢁ113.92, ꢁ163.32. HRMS (ESI) calcd for C29H29F2N5NaO5
(d, J ¼ 3.4 Hz, 1H), 5.73 (t, J ¼ 7.9 Hz,1H), 5.56 (d, J ¼ 3.1 Hz, 1H), 5.08
(s, 2H), 4.80 (d, J ¼ 12.2 Hz, 1H), 4.55 (d, J ¼ 12.2 Hz, 1H), 4.43 (q,
J ¼ 17.3 Hz, 2H), 3.85 (t, J ¼ 9.3 Hz, 1H), 2.92e2.78 (m, 2H),
2.49e2.11 (m, 6H), 1.65 (dd, J ¼ 14.4, 11.9 Hz, 1H), 1.55 (s, 3H), 1.09 (t,
[MþNa]þ 588.2029, found 588.2032.
4.1.8.4. ((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[20,30:9,10]cyclodeca[1,2-
b]furan-5-yl)methyl 4-((6-(2-(3-benzyl-5-fluoro-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)acetamido)hexyl)amino)-4-oxobutanoate
J ¼ 12.3 Hz, 1H).13C NMR (100 MHz, CDCl3)
d 169.5, 167.1, 157.2 (d,
J ¼ 25.4 Hz), 150.0, 140.2 (d, J ¼ 236.5 Hz), 138.8, 134.4, 134.1, 134.0,
132.4, 130.8, 128.8, 127.3 (d, J ¼ 33.2 Hz), 120.5, 81.1, 68.7, 63.3, 60.1,
50.3, 44.6, 42.7, 36.6, 25.7, 24.5, 24.0, 18.1.19F NMR (376 MHz, CDCl3)
(14a). Yield 78%. 1H NMR (400 MHz, CDCl3)
d
7.41 (d, J ¼ 7.2 Hz, 2H),
7.38 (d, J ¼ 4.9 Hz,1H), 7.28 (d, J ¼ 6.7 Hz,1H), 7.23 (d, J ¼ 6.9 Hz,1H),
6.72 (t, J ¼ 4.4 Hz, 1H), 6.20 (d, J ¼ 1.5 Hz, 1H), 5.86 (t, J ¼ 4.6 Hz, 1H),
5.63 (t, J ¼ 7.9 Hz, 1H), 5.58 (s, 1H), 5.10 (s, 2H), 4.58 (d, J ¼ 12.8 Hz,
1H), 4.51 (d, J ¼ 12.8 Hz, 1H), 4.38e4.27 (m, 2H), 3.85 (t, J ¼ 9.3 Hz,
1H), 3.26e3.16 (m, 4H), 3.02 (t, J ¼ 9.9 Hz, 1H), 2.85 (d, J ¼ 9.4 Hz,
1H), 2.62 (d, J ¼ 6.3 Hz, 2H), 2.47e2.40 (m, 3H), 2.38e2.10 (m, 5H),
1.65 (t, J ¼ 11.7 Hz, 1H), 1.52 (s, 3H), 1.50e1.41 (m, 4H), 1.29 (s, 3H),
d
ꢁ163.57. HRMS (ESI) calcd for C28H28ClFN2NaOþ7 [MþNa]þ
581.1461, found 581.14.
4.1.8.8. ((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[20,30:9,10]cyclodeca[1,2-
b]furan-5-yl)methyl 2-(3-(4-bromobenzyl)-5-fluoro-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)acetate (15c). Yield 45%. 1H NMR
1.09 (t, J ¼ 12.7 Hz, 1H).13C NMR (100 MHz, CDCl3)
d
173.0, 171.4,
(400 MHz, CDCl3)
d
7.41 (d, J ¼ 8.4 Hz, 2H), 7.32 (d, J ¼ 8.4 Hz, 2H),
170.0, 166.2, 157.5 (d, J ¼ 25.3 Hz), 150.3, 140.1 (d, J ¼ 233.4 Hz),
136.1, 134.6, 129.6, 129.1, 128.6, 128.1, 127.9 (d, J ¼ 33.0 Hz), 120.6,
81.4, 66.8, 63.2, 60.3, 51.4, 45.2, 42.6, 39.3, 38.9, 36.7, 30.7, 29.4, 29.4,
28.9, 25.9, 25.6, 25.6, 24.7, 23.8, 18.1. 19F NMR (376 MHz, CDCl3)
7.28 (d, J ¼ 5.0 Hz, 1H), 6.21 (d, J ¼ 3.4 Hz, 1H), 5.70 (t, J ¼ 7.9 Hz, 1H),
5.54 (d, J ¼ 3.1 Hz, 1H), 5.04 (s, 2H), 4.78 (d, J ¼ 12.2 Hz, 1H), 4.52 (d,
J ¼ 12.3 Hz, 1H), 4.41 (q, J ¼ 17.3 Hz, 2H), 3.83 (t, J ¼ 9.3 Hz, 1H),
2.88e2.75 (m, 2H), 2.47e2.06 (m, 6H), 1.68e1.57 (m, 1H), 1.53 (s,
d
ꢁ164.56. HRMS (ESI) calcd for C38H47FN4NaOþ9 [MþNa]þ
3H),1.07 (t, J ¼ 12.4 Hz,1H).13C NMR (100 MHz, CDCl3)
d 169.5,167.1,
745.3219, found 745.3223.
157.2 (d, J ¼ 25.4 Hz), 150.0, 140.2 (d, J ¼ 236.4 Hz), 138.8, 134.9,
134.1, 132.5, 131.7, 131.1, 127.3 (d, J ¼ 33.3 Hz), 122.2, 120.5, 81.1, 68.6,
63.3, 60.1, 50.3, 44.7, 42.7, 36.6, 25.6, 24.5, 24.0, 18.1.19F NMR
4.1.8.5. ((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[20,30:9,10]cyclodeca[1,2-
b]furan-5-yl)methyl 4-(4-(2-(3-benzyl-5-fluoro-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)acetyl)piperazin-1-yl)-4-oxobutanoate
(376 MHz, CDCl3)
C
d
ꢁ163.60. HRMS (ESI) calcd for
28H28BrFN2NaOþ7 [MþNa]þ 625.0956, found 625.0959.
(14b). Yield 55%. 1H NMR (400 MHz, CDCl3)
d
7.44 (d, J ¼ 7.0 Hz,
4.1.8.9. ((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[20,30:9,10]cyclodeca[1,2-
2H), 7.31 (d, J ¼ 6.1 Hz, 2H), 7.28 (d, J ¼ 2.8 Hz, 1H), 6.22 (d,
J ¼ 3.4 Hz, 1H), 5.75e5.61 (m, 1H), 5.57 (d, J ¼ 3.0 Hz, 1H), 5.18e5.06
(m, 2H), 4.68e4.43 (m, 4H), 3.84 (t, J ¼ 9.4 Hz, 1H), 3.78e3.39 (m,
8H), 3.12 (t, J ¼ 9.9 Hz, 1H), 2.86 (t, J ¼ 8.2 Hz, 1H), 2.73e2.54 (m,
4H), 2.49e2.11 (m, 6H), 1.68 (d, J ¼ 10.0 Hz, 2H), 1.53 (s, 3H),
b]furan-5-yl)methyl
benzyl)-3,4-dihydropyrimidin-1(2H)-yl)acetate (15d). Yield 63%. 1H
NMR (400 MHz, CDCl3)
2-(5-fluoro-2,4-dioxo-3-(4-(trifluoromethyl)
d
7.56 (s, 4H), 7.22 (d, J ¼ 4.8 Hz, 1H), 6.25 (d,
J ¼ 3.5 Hz, 1H), 5.74 (t, J ¼ 8.0 Hz, 1H), 5.55 (d, J ¼ 3.1 Hz, 1H), 5.17 (s,
2H), 4.80 (d, J ¼ 12.2 Hz, 1H), 4.55 (d, J ¼ 12.2 Hz, 1H), 4.41 (q,
J ¼ 17.3 Hz, 2H), 3.85 (t, J ¼ 9.3 Hz, 1H), 2.89e2.79 (m, 2H),
2.50e2.11 (m, 6H), 1.58 (s, 1H), 1.55 (s, 3H), 0.96 (t, J ¼ 7.4 Hz, 1H).
1.19e1.10 (m, 1H). 13C NMR (100 MHz, CDCl3)
d 172.9, 170.1, 169.7,
164.9, 157.4 (d, J ¼ 25.2 Hz), 150.3, 140.0 (d, J ¼ 234.3 Hz), 139.0,
136.0, 134.7, 129.8, 129.7, 128.7 (d, J ¼ 33.2 Hz), 128.2, 127.9, 120.3,
81.3, 66.7, 63.1, 60.2, 49.3, 45.2, 44.8, 44.6, 42.5, 41.9, 41.3, 36.6, 29.1,
13C NMR (100 MHz, CDCl3)
150.0, 140.2 (d, J ¼ 237.3 Hz), 139.7, 138.9, 134.1, 132.4, 131.1, 129.5,
129.0, 127.2 (d, J ¼ 33.2 Hz), 125.6 (d, J ¼ 3.8 Hz), 120.4, 81.0, 68.7,
63.3, 60.0, 50.3, 44.9, 42.7, 36.6, 25.7, 24.5, 24.0, 18.1. 19F NMR
d
169.4, 167.1, 157.2 (d, J ¼ 25.5 Hz),
27.7, 25.8, 24.6, 23.8, 18.0. 19F NMR (376 MHz, CDCl3)
d
ꢁ164.69.
HRMS (ESI) calcd for C36H41FN4NaOþ9 [MþNa]þ 715.2750, found
715.2753.
(376 MHz, CDCl3)
C
d
ꢁ62.60, ꢁ163.10. HRMS (ESI) calcd for
4.1.8.6. ((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[20,30:9,10]cyclodeca[1,2-
b]furan-5-yl)methyl 2-(5-fluoro-3-(4-fluorobenzyl)-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)acetate (15a). Yield 64%. 1H NMR
29H28F4N2NaO7 [MþNa]þ 615.1725, found 615.1728.
4.1.8.10. ((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[20,30:9,10]cyclodeca[1,2-
b]furan-5-yl)methyl 2-(5-fluoro-3-(4-nitrobenzyl)-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)acetate (15e). Yield 60%. 1H NMR
(400 MHz, CDCl3)
d
7.72 (dd, J ¼ 5.7, 3.3 Hz, 1H), 7.53 (dd, J ¼ 5.7,
3.3 Hz, 1H), 7.45 (dd, J ¼ 8.6, 5.4 Hz, 1H), 7.23 (d, J ¼ 4.9 Hz, 1H), 6.98
(t, J ¼ 8.7 Hz, 2H), 6.23 (d, J ¼ 3.5 Hz, 1H), 5.72 (t, J ¼ 7.9 Hz, 1H), 5.55
(d, J ¼ 3.1 Hz, 1H), 4.79 (d, J ¼ 12.2 Hz, 1H), 4.54 (d, J ¼ 12.3 Hz, 1H),
4.45 (d, J ¼ 17.3 Hz, 1H), 4.37 (d, J ¼ 17.3 Hz, 1H), 4.08 (d, J ¼ 6.7 Hz,
2H), 3.84 (t, J ¼ 9.3 Hz, 1H), 2.91e2.78 (m, 2H), 2.44e2.13 (m, 6H),
1.54 (s, 3H), 1.09 (t, J ¼ 12.2 Hz, 1H). 13C NMR (100 MHz, CDCl3)
(400 MHz, CDCl3)
d
8.16 (d, J ¼ 7.5 Hz, 2H), 7.59 (d, J ¼ 7.6 Hz, 2H),
7.28 (d, J ¼ 4.8 Hz, 1H), 6.22 (s, 1H), 5.74 (t, J ¼ 7.8 Hz, 1H), 5.54 (s,
1H), 5.25e5.13 (m, 2H), 4.80 (d, J ¼ 12.3 Hz, 1H), 4.55 (d, J ¼ 12.3 Hz,
1H), 4.46 (d, J ¼ 17.3 Hz, 1H), 4.37 (d, J ¼ 17.2 Hz, 1H), 2.88 (s, 1H),
2.81 (d, J ¼ 9.4 Hz, 1H), 2.49e2.13 (m, 7H), 1.72e1.65 (m, 1H), 1.54 (s,
d
169.40, 167.08, 162.65 (d, J ¼ 246.8 Hz), 157.22 (d, J ¼ 25.4 Hz),
3H), 1.08 (t, J ¼ 12.6 Hz, 1H). 13C NMR (100 MHz, CDCl3)
d 169.5,
150.10, 140.38 (d, J ¼ 237.1 Hz), 138.89, 134.14, 132.59, 131.75 (d,
J ¼ 3.3 Hz), 131.41 (d, J ¼ 8.2 Hz), 126.90 (d, J ¼ 33.3 Hz), 120.42,
115.52 (d, J ¼ 21.5 Hz), 81.07, 68.68, 63.39, 60.00, 50.32, 44.68, 42.77,
36.64, 25.73, 24.52, 24.04, 18.09. 19F NMR (376 MHz, CDCl3)
167.1, 157.1 (d, J ¼ 25.5 Hz), 148.9 (d, J ¼ 228.4 Hz), 142.9, 139.0,
134.0, 132.8, 131.1, 130.1, 127.4 (d, J ¼ 33.2 Hz), 124.0, 120.3, 81.1,
68.9, 65.8, 63.3, 60.1, 50.4, 44.6, 42.7, 36.6, 25.8, 24.8, 24.0, 18.1.19
F
NMR (376 MHz, CDCl3)
C
d
ꢁ163.20. HRMS (ESI) calcd for
d
C
ꢁ113.80, ꢁ113.84, ꢁ163.42, ꢁ163.46. HRMS (ESI) calcd for
28H28FN3NaOþ9 [MþNa]þ 592.1702, found 592.1706.
28H28F2N2NaO7 [MþNa]þ 565.1757, found 565.1760.
4.1.8.11. ((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[20,30:9,10]cyclodeca[1,2-
b]furan-5-yl)methyl 2-(5-fluoro-3-(3-fluorobenzyl)-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)acetate (15f). Yield 60%. 1H NMR
4.1.8.7. ((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[20,30:9,10]cyclodeca[1,2-
b]furan-5-yl)methyl 2-(3-(4-chlorobenzyl)-5-fluoro-2,4-dioxo-3,4-

12
Y. Ding et al. / European Journal of Medicinal Chemistry 183 (2019) 111706
(400 MHz, CDCl3)
d
7.29 (d, J ¼ 7.8 Hz, 1H), 7.23 (dd, J ¼ 8.7, 6.3 Hz,
[MþNa]þ 583.1663, found 583.1667.
2H), 7.13 (d, J ¼ 9.6 Hz, 1H), 7.01e6.90 (m, 1H), 6.23 (d, J ¼ 3.5 Hz,
1H), 5.72 (t, J ¼ 7.9 Hz, 1H), 5.55 (d, J ¼ 3.1 Hz, 1H), 5.10 (s, 2H), 4.80
(d, J ¼ 12.2 Hz, 1H), 4.54 (d, J ¼ 12.2 Hz, 1H), 4.42 (q, J ¼ 17.3 Hz, 2H),
3.84 (t, J ¼ 9.3 Hz, 1H), 2.82 (t, J ¼ 7.1 Hz, 2H), 2.47e2.11 (m, 6H),
1.71e1.64 (m, 1H), 1.54 (s, 3H), 1.09 (t, J ¼ 12.1 Hz, 1H).13C NMR
4.1.8.15. ((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[20,30:9,10]cyclodeca[1,2-
b]furan-5-yl)methyl
3,4-dihydropyrimidin-1(2H)-yl)acetate (15j). Yield 76%. 1H NMR
(400 MHz, CDCl3)
2-(3-(3,4-difluorobenzyl)-5-fluoro-2,4-dioxo-
(100 MHz, CDCl3)
d
169.4, 167.0, 162.8 (d, J ¼ 246.1 Hz), 157.1 (d,
d
7.22 (d, J ¼ 8.6 Hz, 2H), 7.14 (s, 1H), 7.09e6.97
J ¼ 25.5 Hz), 150.0, 140.2 (d, J ¼ 236.8 Hz), 138.7, 138.1 (d, J ¼ 7.4 Hz),
132.3, 130.1 (d, J ¼ 8.2 Hz), 127.1 (d, J ¼ 33.2 Hz), 124.9, 120.6, 116.0
(d, J ¼ 22.0 Hz), 115.1 (d, J ¼ 20.9 Hz), 81.0, 68.6, 63.3, 60.1, 50.3, 44.7,
42.7, 36.5, 29.8, 25.6, 24.3, 23.9, 18.0.19F NMR (376 MHz, CDCl3)
(m, 1H), 6.16 (s, 1H), 5.66 (d, J ¼ 7.8 Hz, 1H), 5.50 (s, 1H), 4.99 (s, 2H),
4.75 (d, J ¼ 12.1 Hz, 1H), 4.49 (d, J ¼ 12.2 Hz, 1H), 4.45e4.29 (m, 2H),
3.79 (t, J ¼ 9.4 Hz, 1H), 2.77 (t, J ¼ 10.3 Hz, 2H), 2.43e2.05 (m, 6H),
1.61 (t, J ¼ 11.4 Hz, 1H), 1.48 (s, 3H), 1.03 (t, J ¼ 12.5 Hz, 1H). 13C NMR
d
ꢁ112.70, ꢁ163.41. HRMS (ESI) calcd for C28H28F2N2NaO7
(100 MHz, CDCl3)
d
169.5, 167.1, 157.1 (d, J ¼ 25.5 Hz), 150.2 (dd,
[MþNa]þ565.1757, found 565.176.
J ¼ 248.4, 6.7 Hz), 150.1 (dd, J ¼ 248.3, 6.8 Hz), 150.0, 140.2 (d,
J ¼ 236.7 Hz), 138.8, 134.1, 132.7 (dd, J ¼ 5.5, 4.1 Hz), 132.5, 127.3 (d,
J ¼ 33.3 Hz), 125.8 (dd, J ¼ 6.5, 3.7 Hz), 120.5, 118.5 (d, J ¼ 17.5 Hz),
117.3 (d, J ¼ 17.3 Hz), 81.1, 68.7, 63.3, 60.1, 50.3, 44.3, 42.7, 36.5, 25.6,
4.1.8.12. ((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[20,30:9,10]cyclodeca[1,2-
b]furan-5-yl)methyl 2-(5-fluoro-3-(2-fluorobenzyl)-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)acetate (15g). Yield 56%. 1H NMR
24.5,
24.0,
18.0.
19F
NMR
(376 MHz,
CDCl3)
d
ꢁ137.31, ꢁ138.44, ꢁ163.49.HRMS (ESI) calcd for C28H27F3N2NaO7þ
(400 MHz, DMSO‑d6)
d
8.28 (d, J ¼ 6.4 Hz, 1H), 7.37e7.27 (m, 1H),
[MþNa]þ 583.1663, found 583.1666.
7.23e7.15 (m, 1H), 7.15e7.05 (m, 2H), 6.02 (d, J ¼ 3.4 Hz, 1H),
5.67e5.60 (m, 1H), 5.58 (d, J ¼ 3.1 Hz, 1H), 5.13e4.98 (m, 2H), 4.73
(d, J ¼ 12.5 Hz, 1H), 4.66e4.54 (m, 2H), 4.51 (d, J ¼ 12.6 Hz, 1H), 4.09
(t, J ¼ 9.3 Hz, 1H), 3.04e2.91 (m, 1H), 2.83 (d, J ¼ 9.5 Hz, 1H),
2.34e2.20 (m, 3H), 2.16e2.02 (m, 3H), 1.60 (t, J ¼ 10.4 Hz, 1H), 1.47
4.1.8.16. ((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[20,30:9,10]cyclodeca[1,2-
b]furan-5-yl)methyl
3,4-dihydropyrimidin-1(2H)-yl)acetate (15k). Yield 64%. 1H NMR
(400 MHz, CDCl3)
2-(3-(2,5-difluorobenzyl)-5-fluoro-2,4-dioxo-
(s, 3H), 0.95e0.86 (m, 1H). 13C NMR (100 MHz, DMSO‑d6)
d
169.4,
d
7.32 (d, J ¼ 4.9 Hz, 1H), 7.08e6.96 (m, 1H),
167.5, 159.9 (d, J ¼ 245.0 Hz), 156.6 (d, J ¼ 28.7 Hz), 149.7, 140.0,
139.6, 137.7,134.4,130.0,129.6 (d, J ¼ 39.4 Hz), 128.3, 124.4, 123.0 (d,
J ¼ 13.9 Hz), 119.3, 115.3 (d, J ¼ 20.6 Hz), 80.6, 67.8, 62.6, 59.9, 49.9,
41.7, 38.3, 36.2, 24.4, 23.6, 23.2, 17.4. 19F NMR (376 MHz, DMSO‑d6)
6.96e6.84 (m, 2H), 6.21 (d, J ¼ 3.3 Hz, 1H), 5.71 (t, J ¼ 7.8 Hz, 1H),
5.55 (d, J ¼ 2.9 Hz, 1H), 5.18 (s, 2H), 4.80 (d, J ¼ 12.2 Hz, 1H), 4.49 (dt,
J ¼ 47.0, 14.8 Hz, 3H), 3.83 (t, J ¼ 9.3 Hz, 1H), 2.82 (t, J ¼ 10.4 Hz, 2H),
2.54e2.09 (m, 6H), 1.91 (d, J ¼ 9.4 Hz, 1H), 1.53 (s, 3H), 1.33 (d,
d
ꢁ118.15, ꢁ167.69. HRMS (ESI) calcd for C28H28F2N2NaOþ7 [MþNa]þ
J ¼ 12.7 Hz, 1H). 13C NMR (100 MHz, CDCl3)
d 169.5, 167.1, 158.7 (dd,
565.1757, found 565.1761.
J ¼ 242.5, 2.2 Hz), 157.1 (d, J ¼ 25.3 Hz), 156.7 (dd, J ¼ 242.4, 2.4 Hz),
150.0, 140.1 (d, J ¼ 237.0 Hz), 138.6, 134.1, 132.3, 127.5 (d,
J ¼ 33.3 Hz), 124.4 (dd, J ¼ 17.0, 7.8 Hz), 120.7, 116.7 (dd, J ¼ 24.6,
8.6 Hz), 116.0 (dd, J ¼ 18.3, 8.5 Hz), 115.7 (dd, J ¼ 14.7, 3.9 Hz), 81.1,
68.6, 63.3, 60.1, 50.4, 42.7, 39.0 (d, J ¼ 3.8 Hz), 36.6, 25.6, 24.3, 24.0,
4.1.8.13. ((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[20,30:9,10]cyclodeca[1,2-
b]furan-5-yl)methyl
3,4-dihydropyrimidin-1(2H)-yl)acetate (15h). Yield 73%. 1H NMR
(400 MHz, CDCl3) 7.33e7.27 (m, 1H), 7.25 (s, 1H), 6.89e6.73 (m,
2-(3-(2,4-difluorobenzyl)-5-fluoro-2,4-dioxo-
18.1. 19F NMR (376 MHz, CDCl3)
d
ꢁ118.37, ꢁ123.16, ꢁ163.41.HRMS
d
(ESI) calcd for
583.1668.
C
28H27F3N2NaO7þ [MþNa]þ 583.1663, found
2H), 6.23 (d, J ¼ 3.5 Hz,1H), 5.72 (t, J ¼ 7.9 Hz,1H), 5.56 (d, J ¼ 3.1 Hz,
1H), 5.16 (s, 2H), 4.78 (d, J ¼ 12.2 Hz, 1H), 4.55 (d, J ¼ 12.3 Hz, 1H),
4.43 (q, J ¼ 17.3 Hz, 2H), 3.84 (t, J ¼ 9.3 Hz, 1H), 2.87e2.75 (m, 2H),
2.48e2.14 (m, 6H),1.71e1.64 (m,1H),1.54 (s, 3H),1.09 (t, J ¼ 12.2 Hz,
4.1.8.17. ((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[20,30:9,10]cyclodeca[1,2-
b]furan-5-yl)methyl 2-(5-fluoro-3-methyl-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)acetate (15l). Yield 66%, 1H NMR
1H). 13C NMR (100 MHz, CDCl3)
d
169.4, 167.1, 162.6 (dd, J ¼ 249.1,
12.1 Hz), 161.0 (dd, J ¼ 251.2, 12.1 Hz), 157.2 (d, J ¼ 25.5 Hz), 149.9,
140.2 (d, J ¼ 237.2 Hz), 138.7, 134.1, 132.4, 131.2 (dd, J ¼ 14.0, 4.3 Hz),
127.2 (d, J ¼ 33.3 Hz), 120.6, 118.8 (dd, J ¼ 14.7, 3.8 Hz), 111.5 (dd,
J ¼ 21.2, 3.8 Hz), 104.1 (t, J ¼ 25.5 Hz), 81.1, 68.7, 63.3, 60.1, 50.3, 42.7,
38.7 (d, J ¼ 3.6 Hz), 36.6, 25.7, 24.4, 24.0, 18.1. 19F NMR (376 MHz,
(400 MHz, CDCl3)
d
7.25 (s, 1H), 6.24 (s, 1H), 5.74 (t, J ¼ 8.0 Hz, 1H),
5.58 (s, 1H), 4.78 (d, J ¼ 12.2 Hz, 1H), 4.58 (d, J ¼ 12.2 Hz, 1H),
4.52e4.37 (m, 2H), 3.85 (t, J ¼ 9.2 Hz, 1H), 3.37 (s, 3H), 2.90e2.78
(m, 2H), 2.52e2.14 (m, 6H), 1.69 (t, J ¼ 11.2 Hz, 1H), 1.54 (s, 3H), 1.10
CDCl3)
d
ꢁ110.18, ꢁ112.37. HRMS (ESI) calcd for C28H27F3N2NaO7
(t, J ¼ 12.8 Hz, 1H). 13C NMR (100 MHz, CDCl3)
d 169.5, 167.3, 157.5
[MþNa]þ 583.1663, found 583.1668.
(d, J ¼ 25.4 Hz), 150.3, 140.3(d, J ¼ 236.1 Hz), 139.1, 138.9, 134.2,
126.8 (d, J ¼ 33.3 Hz), 120.6, 81.2, 68.7, 63.4, 60.1, 50.3, 42.8, 36.7,
4.1.8.14. ((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[20,30:9,10]cyclodeca[1,2-
b]furan-5-yl)methyl
3,4-dihydropyrimidin-1(2H)-yl)acetate (15i). Yield 64%. 1H NMR
(400 MHz, CDCl3)
28.7, 25.8, 24.5, 24.1, 18.2. 19F NMR (376 MHz, CDCl3)
d
ꢁ163.85.
HRMS (ESI) calcd for C22H25FN2NaOþ7 [MþNa]þ 471.1538, found
2-(3-(2,6-difluorobenzyl)-5-fluoro-2,4-dioxo-
471.1543.
d
7.25e7.15 (m, 2H), 6.83 (t, J ¼ 7.7 Hz, 2H), 6.19 (s,
4.1.8.18. ((1aR,7aS,10aS,10bS,E)-1a-methyl-8-methylene-9-oxo-
1a,2,3,6,7,7a,8,9,10a,10b-decahydrooxireno[20,30:9,10]cyclodeca[1,2-
b]furan-5-yl)methyl 2-(5-fluoro-2,4-dioxo-3-propyl-3,4-
dihydropyrimidin-1(2H)-yl)acetate (15m). Yield 78%. 1H NMR
1H), 5.68 (t, J ¼ 7.8 Hz, 1H), 5.54 (s, 2H), 5.22 (s, 1H), 4.74 (d,
J ¼ 12.3 Hz, 1H), 4.52 (d, J ¼ 12.3 Hz, 1H), 4.41 (q, J ¼ 17.3 Hz, 2H),
3.81 (t, J ¼ 9.1 Hz, 1H), 2.79 (d, J ¼ 9.4 Hz, 2H), 2.48e2.06 (m, 6H),
1.64 (t, J ¼ 12.2 Hz, 1H), 1.51 (s, 3H), 1.07 (t, J ¼ 12.6 Hz, 1H). 13C NMR
(400 MHz, CDCl3)
d
7.21 (d, J ¼ 4.8 Hz, 1H), 6.25 (d, J ¼ 3.4 Hz, 1H),
(100 MHz, CDCl3)
d
169.5,167.1,161.6 (dd, J ¼ 250.2, 7.6 Hz),156.8 (d,
5.73 (t, J ¼ 8.0 Hz, 1H), 5.58 (d, J ¼ 3.0 Hz, 1H), 4.80 (d, J ¼ 12.2 Hz,
1H), 4.56 (d, J ¼ 12.3 Hz, 1H), 4.42 (q, J ¼ 17.2 Hz, 2H), 3.98e3.89 (m,
2H), 3.85 (t, J ¼ 9.3 Hz, 1H), 2.90e2.80 (m, 2H), 2.48e2.16 (m, 6H),
1.68e1.64 (m, 2H), 1.55 (s, 3H), 1.14e1.07 (m, 2H), 0.94 (t, J ¼ 7.4 Hz,
J ¼ 25.5 Hz),149.7,140.1 (d, J ¼ 236.3 Hz),138.6,134.1,132.0,129.7 (t,
J ¼ 10.4 Hz), 127.2 (d, J ¼ 33.3 Hz), 120.7, 111.5 (d, J ¼ 25.2 Hz), 111.5
(d, J ¼ 9.6 Hz), 81.1, 68.5, 63.3, 60.1, 50.2, 42.7, 36.6, 34.6 (t, J ¼ 4
.0 Hz), 34.8e34.4 (m), 25.6, 24.3, 24.0, 18.1. 19F NMR (376 MHz,
3H). 13C NMR (100 MHz, CDCl3)
d
169.4, 167.2, 157.2 (d, J ¼ 24.9 Hz),
CDCl3)
d
ꢁ114.09, ꢁ163.73. HRMS (ESI) calcd for C28H27F3N2NaO7þ
150.0, 140.3 (d, J ¼ 236.2 Hz), 138.7, 134.1, 132.3, 126.7 (d,

Y. Ding et al. / European Journal of Medicinal Chemistry 183 (2019) 111706
13
J ¼ 33.2 Hz), 120.5, 81.0, 68.6, 63.3, 60.0, 50.3, 43.7, 42.7, 36.6, 34.0,
4.7. The drug accumulation assay
29.8, 25.0, 20.8, 18.1, 11.3.19F NMR (376 MHz, CDCl3)
d
ꢁ163.61.
HRMS (ESI) calcd for C24H29FN2NaOþ7 [MþNa]þ 499.1851, found
Bel-7402/5-FU cells were seeded in 24 well plate with
499.1855.
1 ꢂ 105 cells/well/1 mL. After 12 h, 15d was added with the con-
centration of 0.5 mM, 1 mM, 2.5 mM and 5 mM and incubated for 48 h.
4.2. Cell culture
Then the cells were harvested, washed with PBS for 2 times and
incubated with ADR (1
m
M) for 1 h at 37 ꢀC. After that the cells were
The human liver cancer cell line Bel-7402 and its corresponding
drug resistance cell line Bel-7402/5-FU were purchased from Key-
Gen Biotech (Nanjing, China) and maintained in RPMI-1640 sup-
plemented with 10% fetal bovine serum (FBS) and 1% penicillin/
streptomycin in a humidified environment containing 5% CO2 at a
constant temperature of 37 ꢀC.
collected, resuspended with PBS and analyzed with flow cytometry.
4.8. P-gp expression assay
Bel-7402/5-FU cells with or without 15d treatment were
collected and washed with PBS buffer for 3 times. Then the cells
were resuspended and incubated with 5 mL p-gp-PE for 30 min at
room temperature. After that the cells were analyzed with flow
4.3. MTT assay
cytometry.
Bel-7402 and Bel-7402/5-FU cells were seeded into 96-well
plate respectively with 5000 cells each well. After 12 h, com-
pounds with different concentrations were added and incubated
Acknowledgments
for 72 h. Then 20
well and incubated for 4 h. The supernatant was discarded and the
crystal violet was dissolved with 200 L DMSO. The observance of
OD 570 nm was measured using a micro-plate reader. The IC50
values were calculated by GraphPad Prism 5.
mL solution of MTT (5 mg/mL) was added into each
We acknowledge the support of the National Natural Science
Foundation of China (NO. 81573308 and NO. 81872764 to Q.Z.; NO.
81573282 to Y.C.), The National Science Fund for Distinguished
Young Scholars (NO. 81625021) to Y.C., Natural Science Foundation
of Tianjin (NO. 17JCQNJC13400) to Q.Z., the Fundamental Research
Funds for the Central Universities to Y. D. (Nankai University, NO.
63191407), and the Fundamental Research Funds for the Central
Universities to Q.Z. (Nankai University, NO. 63191519).
m
4.4. Cell apoptosis assay
Bel-7402/5-FU cells were seeded in 24 well plate with
1 ꢂ 105 cells/well/1 mL. After 12 h, 15d was added at the concen-
Appendix A. Supplementary data
tration of 0.5 mM, 1 mM, 2.5 mM, 5 mM, respectively, and incubated
for 48 h. Cell apoptosis was evaluated using a FITC-Annexin V
apoptosis detection kit. Briefly, the Bel-7402/5-FU cells were har-
vested and washed with cold PBS, then the cells were resuspended
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2019.111706.
in binding buffer and incubated with 5
mL of a FITC-conjugated
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m
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