Design, synthesis and biological evaluation of sphingosine-1-phosphate receptor 2 antagonists as potent 5-FU-resistance reversal agents for the treatment of colorectal cancer

Article abstract of DOI:10.1016/j.ejmech.2021.113775

5-Fluorouracil (5-FU) and its prodrugs are the essential clinical drugs for colorectal cancer (CRC) treatment. However, the drug resistance of 5-FU has caused high mortality of CRC patients. Thus, it is urgent to develop reversal agents of 5-FU resistance. Sphingosine-1-phosphate receptor 2 (S1PR2) was proved to be a potential target for reversing 5-FU resistance, but the activity of known S1PR2 antagonists JTE-013 were weak in 5-FU-resistant cell lines. To develop more potent S1PR2 antagonists to treat 5-FU-resistant cancer, a series of JTE-013 derivatives were designed and synthesized. The most promising compound 40 could markedly reverse the resistance in 5-FU-resistant HCT116 cells and 5-FU-resistant SW620 cells via inhibiting the expression of dihydropyrimidine dehydrogenase (DPD). The key was that compound 40 with improved pharmacokinetic properties significantly increased the inhibitory rate of 5-FU in the SW620/5-FU cells xenograft model with no observable toxicity by inhibiting the expression of DPD in tumor and liver tissues. Altogether, these results suggest that compound 40 may be a promising drug candidate to reverse 5-FU resistance in the treatment of CRC.

Full text of DOI:10.1016/j.ejmech.2021.113775

European Journal of Medicinal Chemistry 225 (2021) 113775
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, synthesis and biological evaluation of sphingosine-1-
phosphate receptor 2 antagonists as potent 5-FU-resistance reversal
agents for the treatment of colorectal cancer
,
,
d
,
,
Dongdong Luo a 1, Yuhang Zhang b 1, Shuang Yang a 1, Xiaochen Tian a, Yan Lv a,
Zhikun Guo c, Xiaochun Liu a, Gaitian Han a, Shuai Liu a, Wenyu Wang b, Shuxiang Cui c,
Xianjun Qu b **, Shengbiao Wan a
,
, *
a Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, Key Laboratory of Marine Drugs,
Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, Qingdao, 266003, China
b Department of Pharmacology, School of Basic Medical Sciences, Capital Medical University, Beijing, 100069, China
c Department of Toxicology and Sanitary Chemistry, School of Public Health, Capital Medical University, Beijing, 100069, China
d Institute of Clinical Pharmacology, Peking University First Hospital, Beijing, 100034, China
a r t i c l e i n f o
a b s t r a c t
Article history:
5-Fluorouracil (5-FU) and its prodrugs are the essential clinical drugs for colorectal cancer (CRC) treat-
ment. However, the drug resistance of 5-FU has caused high mortality of CRC patients. Thus, it is urgent
to develop reversal agents of 5-FU resistance. Sphingosine-1-phosphate receptor 2 (S1PR2) was proved to
be a potential target for reversing 5-FU resistance, but the activity of known S1PR2 antagonists JTE-013
were weak in 5-FU-resistant cell lines. To develop more potent S1PR2 antagonists to treat 5-FU-resistant
cancer, a series of JTE-013 derivatives were designed and synthesized. The most promising compound 40
could markedly reverse the resistance in 5-FU-resistant HCT116 cells and 5-FU-resistant SW620 cells via
inhibiting the expression of dihydropyrimidine dehydrogenase (DPD). The key was that compound 40
with improved pharmacokinetic properties significantly increased the inhibitory rate of 5-FU in the
SW620/5-FU cells xenograft model with no observable toxicity by inhibiting the expression of DPD in
tumor and liver tissues. Altogether, these results suggest that compound 40 may be a promising drug
candidate to reverse 5-FU resistance in the treatment of CRC.
Received 3 June 2021
Received in revised form
27 July 2021
Accepted 11 August 2021
Available online 12 August 2021
Keywords:
S1PR2 antagonists
Colorectal cancer therapy
5-FU-Resistance
Pharmacokinetic properties
Mouse xenograft model
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
deoxyuridine-50-monophosphate (FdUMP) via interacting with
phosphorylated sugars in presence of several enzymes to inhibit
Colorectal cancer (CRC), especially unresectable metastatic
colorectal cancer, is one of the most commonly diagnosed cancers
with a very high mortality rate [1,2]. Approximately,1.8 million new
cases of CRC and estimated 880,000 deaths worldwide were
recorded in 2018 [3]. 5-Fluorouracil (5-FU) and 5-FU pro-drugs are
the most essential agents in CRC treatment and the main constit-
uent of therapeutic combinations of multiple cytotoxic agents [4].
5-FU is converted into 5-fluorouridine-50-triphosphate (FUTP), 5-
fluoro-20-deoxyuridine-50-triphosphate (FdUTP), and 5-fluoro-20-
thymidylate synthase (TS) activity in cancer cells [5]. Unfortunately,
CRC cells could become resistant to 5-FU through multiple mech-
anisms, and several drug resistance targets have been identified
[6e15]. Among all drug resistance mechanisms, 85 % intracellular
5-FU is rapidly degraded into 5,6-dihydro-5-fluorouracil (DHFU) by
the over-expressed dihydropyrimidine dehydrogenase (DPD) in
cancer cells, ultimately converted to the a-fluoro-b-alanine (FBAL),
and excreted via the kidneys. This conversion of 5-FU is the main
approach by which cancer cells acquire drug resistance [8,15e20].
However, CDHP (gimeracil), the DPD inhibitor in the S-1 regimen,
could only inhibit the activity of DPD in the liver, but not in cancer
cells [21,22]. Our previous study [23] disclosed that the activation of
the downstream signaling (i.e. JMJD3) causes increased expression
of DPD after the endogenic sphingosine-1-phosphate (S1P) binding
with receptor 2 (S1PR2). S1PR2 was identified as a key receptor in
* Corresponding author.
** Corresponding author.
E-mail addresses: quxj@ccmu.edu.cn (X. Qu), biaowan@ouc.edu.cn (S. Wan).
These authors contribute equally to this work.
1
https://doi.org/10.1016/j.ejmech.2021.113775
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

D. Luo, Y. Zhang, S. Yang et al.
European Journal of Medicinal Chemistry 225 (2021) 113775
the mechanisms of resistance to 5-FU and targeted inhibition of
S1PR2 might be a new avenue to reverse drug resistance of 5-FU.
Two classes of S1PR2 antagonists have been reported [24e29]
(Fig. 1), and the well-known S1PR2 antagonist JTE-013 have been
proved that could effectively reverse 5-FU-resistance and decrease
the expression of DPD in 5-FU-resistant HCT116 (HCT116DPD) cells.
However, our further studies demonstrated that it has weak ac-
tivity in 5-FU-resistant SW620 (SW620/5-FU) cells. In this study, to
further develop efficient S1PR2 antagonists to reverse 5-FU-resis-
tance in vitro and in vivo, a series of JTE-013 derivatives were syn-
thesized. The surface plasmon resonance (SPR) kinetics of binding
to S1PR2 protein of these compounds were investigated, and the
changes of refractive index on the chips coated with the protein
were measured to estimate ligand binding. The efficacy of syn-
thesized compounds was evaluated in combination with 5-FU in
the proliferation of DPD over-expressed and 5-FU-resistant HCT116
(HCT116DPD) and 5-FU-resistant SW620 (SW620/5-FU) cells. The
inhibitory effects of DPD of these compounds were determined to
further investigate their ability to reverse 5-FU resistance. Mean-
while, the most potent compound 40 was also evaluated in nude
mouse xenograft models. Our results demonstrate a promising
approach to develop a safe and effective treatment of colorectal
cancer patients with 5-FU resistance.
pyrimidine scaffolds with hydrophobic substituents to afford
compounds 10, 14, and 18 (Scheme 1). Intermediates 5 and 12 were
synthesized from 2,4-dichloro-1H-pyrrolopyrimidine via a two-
step substitution reaction. Intermediate 16 was synthesized
through substitution reaction and Suzuki-Miyaura cross-coupling
reaction. Then, compounds 5, 12, and 16 reacted with 80 % hydra-
zine hydrate to produce key intermediates 6, 13, 17. 2,6-dichloro-4-
isocyanatopyridine (9) was gained from 2,6-dichloropyridine 4-
carboxylic acid (7) as we reported, and it reacted with com-
pounds 6, 13, and 17 to acquire compounds 10, 14, and 18. Subse-
quently, the linker semicarbazide of JTE-013 was simplified to urea,
and its pyrazolopyridine scaffold was transformed into pyrazolo
[1,5-a]pyridine with different substituents (i.e. phenyl and
bromine). The ionic type intermediates 21a-21b were gained from
nitriles 20a-20b, then the cyclization of compounds 21a-21b under
alkaline conditions produced amines 22a-22b. Compounds 24a and
24b were synthesized through Suzuki-Miyaura cross-coupling re-
action from compound 22b, then compounds 23a, 23b, 25a, and
25b were acquired as similar procedures (Scheme 2). Cyanuric
chloride and dichloropyrimidine produced hydrazine compounds
28 and 32 through multistep substitution reaction, then target
compounds 29 and 33 were gained (Scheme 3). 2,6-Dichloro-4-
isocyanatopyridine (9) reacted with different amines to produce
urea compounds 34e40 (Scheme 4). 2,6-Dichloropyridine 4-
carboxylic acid (7) reacted with various alcohols to acquire car-
boxylic acid 41a-41d. Compounds 41a-41d were transformed into
isocyanates 43a-43d, which reacted with 2,6-dichloropyridine 4-
amide to gain compounds 44a-44d (Scheme 4).
2. Results and discussion
2.1. The design strategy of compounds
As illustrated in Fig. 2, based on JTE-013, target compounds were
designed in four ways. In the first step, the pyrazolopyridine ring of
JTE-013 was changed into pyrrolopyrimidine ring possessed
various hydrophobic substituents which are expected to be more
potential in the light of our previous studies (for compounds 10, 14,
and 18). In the following step, the pyrazolopyridine scaffold was
transformed into pyrazolo[1,5-a]pyridine with different sub-
stituents and the linker was shortened (for compounds 23a-23b
and 25a-25b). In the next step, the fused ring was changed into a
single ring and various hydrophobic substituents were introduced
(for compounds 29 and 33). The final step to gain the target com-
pounds 34e40 and 44a-44d is the replacement of pyrazolopyridine
with sing rings bearing various functional groups and shortening of
semicarbazide linker.
2.3. Study of binding kinetics of compounds and structure-activity
relationships (SAR)
Surface plasmon resonance (SPR) is widely used in studying the
interactions of proteins with small molecules. Due to S1PR2 is a
class of GPCR proteins, it is difficult to directly study its activity.
Thus, an SPR based assay for S1PR2 was established to study the
interactions with small molecules. Firstly, the binding ability of
compounds was investigated on the S1PR2 protein at 10 mM dose.
The results were illustrated as a binding affinity in Table 1. Most
compounds showed strong binding abilities (KD) and the range was
from 10 nM to 2 mM. Among them, compounds 23a, 23b, 25a, 25b,
29, 34, 36, 39, 40, and 44a-44d showed nanomolar level binding
affinity. Especially, the binding affinities of compounds 23a, 23b,
2.2. Chemistry
25a, 25b, 34,39, and 40 were less than 0.1 mM, and the most potent
compound 40 showed a strong binding with S1PR2 (KD ¼ 13.2 nM).
Initially, the pyrazolopyridine ring was replaced by pyrrole-
The above results indicated that these compounds are potent
Fig. 1. The structures of reported S1PR2 antagonists.
2

D. Luo, Y. Zhang, S. Yang et al.
European Journal of Medicinal Chemistry 225 (2021) 113775
Fig. 2. The design strategy of novel S1PR2 antagonists.
Scheme 1. Synthetic route to compounds 10, 14, 18. Reagents and conditions: (a) (CH3)2NH$HCl, Et3N, DCM, r.t., overnight, 54 %; (b) CH3I, NaH, DMF, 0 ꢀC, 2 h, 79e85 %; (c)
hydrazine hydrate, EtOH, reflux, 2e3 h, 90e95 %; (d) DPPA, Et3N, 1,4-dioxane, r.t., 3 h, 67 %; (e) toluene, 80 ꢀC, 2 h; (f) 6, THF, 50 ꢀC, 12 h, 55 % over two steps; (g) cyclopropylboronic
acid, Cu(OAc)2, Na2CO3, 20-2-(C5H4N)2, DCM, reflux, 5 h, 45 %; (h) morpholine, Et3N, DCM, r.t., 5 h, 59 %; (i) toluene, 80 ꢀC, 2 h, then 9, THF, 50 ꢀC, 12 h, 39e57 % over two steps; (j)
phenylboronic acid, Pd(PPh3)4, Cs2CO3, 1,4-dioxane/H2O, 80 ꢀC, 10 h, 55 %.
antagonists of S1PR2.
5-FU cells were also determined and the results showed that it is
dramatically increased relative to parental HCT116 and SW620 cells
(Fig. S1B). Initially, compounds combining with 5-FU were screened
for their ability to impair the viability of colorectal cancer cells.
HCT116DPD and SW620/5-FU cells were concurrently treated with
2.4. Preliminary biological activities of the compounds against 5-
FU-resistant colorectal cancer cell lines via MTT assay and western
blotting
10 mM 5-FU and 10 mM each of the S1PR2 antagonists for 72 h.
Among them, most compounds effectively reduced the cell viability
and their inhibition rates were all over 50 % (Fig. S2). In general, not
all compounds were as effective in inhibiting SW620/5-FU cells
viability as HCT116DPD cells.
To further evaluate the activity of compounds in the 5-FU-
resistant colorectal cancer cells, two resistant cell lines (HCT116DPD
and SW620/5-FU) were established. The cytotoxic tests showed
they are all resistant to 5-FU (IC50 > 1000 mM, Fig. S1A), especially
Subsequently, all compounds were evaluated for dosage effects
SW620/5-FU cells, which were set up by continuously treating with
on cell proliferation via MTT assay. HCT116DPD, SW620/5-FU, and
5-FU. Besides, the levels of DPD protein in HCT116DPD and SW620/
3

D. Luo, Y. Zhang, S. Yang et al.
European Journal of Medicinal Chemistry 225 (2021) 113775
Scheme 2. Synthetic route to compounds 23a-23b, 25a-25b. Reagents and conditions: (a) DCM, r.t., 1e3 h, 30e59 %; (b) K2CO3, MeOH, r.t., 3 h, 40e67 %; (c) toluene, 80 ꢀC, 2 h, then
9, THF, 50 ꢀC, 12 h, 39e57 % over two steps; (d) arylboronic acid, Pd(PPh3)4, Cs2CO3, 1,4-dioxane/H2O, 80 ꢀC, 3 h, 51e59 %.
Scheme 3. Synthetic route to compounds 29 and 33. Reagents and conditions: (a) pyrrole, acetone, 0 ꢀC, 3 h, 60 %; (b) phenylamine, Et3N, acetonitrile, 0 ꢀC, 5 h, 52 %; (c) hydrazine
hydrate, EtOH, reflux, 3 h, 90e95 %; (d) toluene, 80 ꢀC, 2 h, then 9, THF, 50 ꢀC, 12 h, 55 % over two steps; (e) CH3NH2, THF, r.t., overnight, 33 %.
Scheme 4. Synthetic route to compounds 34e40 and 44a-44d. Reagents and conditions: (a) DPPA, Et3N, 1,4-dioxane, r.t., 3 h, 67 %; (b) toluene, 80 ꢀC, 2 h; (c) amine, THF, 50 ꢀC,
5e15 h, 44e67 % over two steps. (d) alcohol, 1 M NaOH, r.t.-70 ꢀC, 5e12 h, 60e85 %.
NCM460 cells were used as the model system, which were treated
with different concentrations of compounds combined with 20
5-FU for 72 h. The values of EC50 were calculated and shown in
Table 2 and most compounds showed enhanced activity. Com-
pounds 10, 14, and 18 possessed pyrrolopyrimidine skeleton all
showed no activity to reverse 5-FU-resistance in both resistant
cells. Compounds 23a, 23b, 25a, and 25d all have enhanced 5-FU-
resistance reversal activity in both cell lines compared to JTE-013.
Remarkably, compound 23a, being the most potent resistance
reversal agent with an EC50 value of 2.49 mM against SW620/5-FU
cell line. Monocyclic compounds (29 and 33) bearing semi-
mM
carbazide all showed low activity. Surprisingly, monocyclic
4

D. Luo, Y. Zhang, S. Yang et al.
European Journal of Medicinal Chemistry 225 (2021) 113775
Table 1
5-FU-resistance reversal activity (23a, 23b, 25a, 25b, 35, 39, 40,
44a-44d) were selected to test the ability to down-regulate DPD in
SW620/5-FU cells by western blotting and they all showed strong
activity to inhibit the expression of DPD (Fig. S2B). Among them,
compound 40 showed the strongest activity and deserved further
exploration.
Kinetics of the interactions between S1PR2 and compounds.
Compds
Ka (Mꢁ1sꢁ1
)
Kd (sꢁ1
)
KD (M)
JTE-013
10
14
8.21 ꢂ 104
7.55 ꢂ 104
6.12 ꢂ 104
5.21 ꢂ 104
7.93 ꢂ 106
8.31 ꢂ 107
4.59 ꢂ 106
9.12 ꢂ 106
7.15 ꢂ 105
5.56 ꢂ 104
5.69 ꢂ 106
3.26 ꢂ 104
1.98 ꢂ 105
5.36 ꢂ 104
3.65 ꢂ 104
6.24 ꢂ 106
5.28 ꢂ 106
3.21 ꢂ 105
1.96 ꢂ 105
9.14 ꢂ 105
4.49 ꢂ 105
1.61 ꢂ 10ꢁ1
1.02 ꢂ 10ꢁ1
1.33 ꢂ 10ꢁ1
1.36 ꢂ 10ꢁ1
1.16 ꢂ 10ꢁ1
1.68
1.96 ꢂ 10ꢁ6
1.85 ꢂ 10ꢁ6
2.17 ꢂ 10ꢁ6
2.61 ꢂ 10ꢁ6
1.46 ꢂ 10ꢁ8
2.02 ꢂ 10ꢁ8
4.38 ꢂ 10ꢁ8
1.22 ꢂ 10ꢁ8
1.86 ꢂ 10ꢁ7
4.24 ꢂ 10ꢁ6
2.57 ꢂ 10ꢁ8
3.56 ꢂ 10ꢁ6
5.35 ꢂ 10ꢁ7
3.30 ꢂ 10ꢁ6
5.50 ꢂ 10ꢁ6
5.00 ꢂ 10ꢁ8
1.32 ꢂ 10ꢁ8
6.82 ꢂ 10ꢁ7
5.20 ꢂ 10ꢁ7
1.30 ꢂ 10ꢁ7
3.99 ꢂ 10ꢁ7
18
23a
23b
25a
25b
29
33
34
35
36
37
38
39
40
44a
44b
44c
44d
2.5. Compound 40 inhibited the expressions of DPD and JMJD3 to
reverse 5-FU resistance
2.01 ꢂ 10ꢁ1
1.12 ꢂ 10ꢁ1
1.33 ꢂ 10ꢁ1
2.36 ꢂ 10ꢁ1
1.46 ꢂ 10ꢁ1
1.16 ꢂ 10ꢁ1
1.06 ꢂ 10ꢁ1
1.77 ꢂ 10ꢁ1
2.01 ꢂ 10ꢁ1
3.12 ꢂ 10ꢁ1
6.97 ꢂ 10ꢁ1
2.19 ꢂ 10ꢁ1
1.02 ꢂ 10ꢁ1
1.19 ꢂ 10ꢁ1
1.79 ꢂ 10ꢁ1
The efficacy of compound 40 to down-regulate the expression of
DPD was tested at low concentrations in HCT116DPD and SW620/5-
FU cells. Compound 40 could suppress the expression of DPD in a
dose-dependent manner (Fig. 3). Moreover, our previous study
ascertained that S-45 and JTE-013 could effectively restrain 5-FU-
induced S1PR2 internalization into the endoplasmic reticulum (ER)
and inhibit the expression of JMJD3. Enlightened from our previous
discoveries, the JMJD3 was also tested and it was found that com-
pound 40 effectively restrained the expression of JMJD3 (Fig. 3).
S1PR2 was reported with multiple biological effects in vivo and its
decrease in expression level may cause unexpected side effects.
Therefore, it was important to assess whether compound 40 could
affect the expression of S1PR2 to reduce the expression of DPD.
HCT116DPD and SW620/5-FU cells were used to explore the effect of
compound 40 on the expression of S1PR2 at different concentra-
tions. The data clearly showed that the expression of S1PR2 was
hardly affected by compound 40 (Fig. 3). Overall, the above data
demonstrated that compound 40 had more potential to reverse 5-
FU resistance in 5-FU-resistant colorectal cancer cell lines
compared with JTE-013.
Ka, association constant; Kd, dissociation constant; KD ¼ Kd/Ka.
Table 2
In vitro antiproliferative efficacy of synthetic compounds in three cell lines.
Compds
EC50 (mM)
SW620/5-FU
HCT116DPD
NCM460
JTE-013
10
14
58.58 1.21
48.25 1.57
436.27 34.74
528.12 13.52
2.49 0.26
11.72 1.56
9.25 0.52
9.62 1.82
31.95 1.42
122.24 2.72
234.14 1.72
12.24 0.72
548.25 1.57
67.21 1.81
31.15 1.37
8.25 1.57
0.21 0.03
14.21 1.01
8.15 0.37
5.17 0.12
6.15 1.43
>1000
17.13 1.25
41.50 0.73
449.27 41.74
660.12 29.52
5.13 0.41
5.26 0.87
5.13 0.33
5.35 0.19
23.16 0.93
23.40 1.54
86.21 0.34
11.40 1.54
141.50 7.73
94.21 3.25
16.13 1.78
41.50 0.73
0.18 0.04
11.18 0.65
6.13 0.78
5.23 0.56
5.12 0.97
>1000
61.34 1.78
58.53 0.57
549.21 11.33
683.23 45.21
10.23 2.15
8.94 0.92
18
2.6. Compound 40 increase intracellular concentration of 5-FU to
reduce the dosage of 5-FU
23a
23b
25a
25b
29
33
34
35
36
37
38
39
40
44a
44b
44c
44d
5-FUa
8.05 0.37
8.35 1.78
Intracellular 5-FU could be rapidly degraded by intracellular
DPD and to further verify whether compound 40 could prohibit the
reduction of intracellular concentration of 5-FU or not, we deter-
mined to test intracellular 5-FU levels in SW620/5-FU cells after
treatments with compound 40 and 5-FU. As expected, the HPLC
analysis demonstrated that the degradation of intracellular 5-FU
was hindered by compound 40 and JTE-013, which could increase
the concentration of 5-FU from 0.59 mg/L to 10.01 mg/L and
4.87 mg/L, respectively (Fig. 4A). The decrease levels of FBAL were
consistent with the degradation levels of 5-FU, which showed
0.17 mg/L and 1.37 mg/L FBAL in compound 40 and JTE-013 treated
SW620/5-FU cells compared with 2.88 mg/L FBAL in control
SW620/5-FU cells. These results suggested that compound 40 could
prevent the degradation of 5-FU into FBAL and significantly in-
crease the intracellular concentration of 5-FU in 5-FU-resistant
colorectal cancer cells. To further evaluate the activity of compound
40, SW620/5-FU cells were treated with compound 40 and the
inhibitory effects of different concentrations of 5-FU were tested. It
was found that the increased dosage of compound 40 could effec-
tively reduce the dosage of 5-FU to achieve the same inhibition rate
in SW620/5-FU cells (Fig. 4B).
65.23 4.32
65.26 0.89
576.34 2.13
55.42 0.89
521.13 1.25
134.21 1.23
55.34 1.61
27.32 0.55
9.44 0.13
33.21 0.53
25.34 0.51
21.12 3.15
21.43 2.12
43.32 1.98
Data are presented as mean SD at least three independent experiments and the
concentration of 5-FU is 20 M.
m
a
The value of EC50 (5-FU) is the median inhibitory concentration of single 5-FU
against three cell lines.
compounds containing chlorinated aromatic ring showed potent
activity in both resistant cells, which indicated that chlorine atom
may be the key to increase activity. Most notably, compound 40
possessing four chlorine atoms showed nanomolar level activity in
both resistance cells, and there are a 95-fold improvement in po-
2.7. Binding models for compound 40 in the S1PR2-binding pocket
tency against HCT116DPD cells (0.18
ment against SW620/5-FU cells (0.21
More importantly, it showed obvious selectivity towards normal
NCM460 cells (9.44 M). Although compounds 44a-44d also
showed potent activity (EC50 < 10 M) to reverse 5-FU-resistance
mM) and a 279-fold improve-
mM) compared with JTE-013.
It is reported that an allosteric site of S1PR2 was found close to
the orthosteric binding site [30]. Considering biological data of
compound 40, it was proposed that compound 40 may not bind to
the orthosteric binding site. The allosteric site was inferred (Fig. 5A)
and compound 40 was docked in silico into the allosteric binding
pocket to explore the differences of the binding modes between
compound 40 and JTE-013. Based on the known interactions, the
m
m
compared with JTE-013, but there are reductions of activity
compared with compound 40. These results further showed the
importance of the chlorine atom. In addition, compounds with well
5

D. Luo, Y. Zhang, S. Yang et al.
European Journal of Medicinal Chemistry 225 (2021) 113775
Fig. 3. Compound 40 reversed 5-FU resistance by inhibiting the expression of intracellular DPD. (A) The expressions of S1PR2, DPD, JMJD3 in HCT116DPD cells were determined after
treatments with compound 40. (B) The expressions of S1PR2, DPD, JMJD3 in SW620/5-FU cells were determined after treatments with compound 40.
Fig. 4. Compound 40 reversed 5-FU resistance by reducing DPD-catalyzed degradation of intracellular 5-FU. (A) HPLC analysis of intracellular 5-FU and FBAL in SW620/5-FU cells
after treatments with 5-FU combined with compound 40 or JTE-013. (B) The inhibition rates of dose-gradient compound 40 and JTE-013 were measured in SW620/5-FU cells after
treatments with 20, 10, and 5 mM 5-FU. Data are presented as mean SEM. *p < 0.05, **p < 0.01 and ***p < 0.001.
final ligand-receptor complexes were ranked according to the
docking score. With the docking results, we observed that JTE-013
interacts with Tyr18, Arg108, Glu109, and Val182 by hydrogen
bonds. The hydrocarbon alkyl and dichloropyridine tail are in the
Fig. 5. Molecular docking studies. (A) The homology model of human S1PR2 (light blue ribbons) from the crystal complex of S1PR1 (PDB ID: 3V2Y) and the putative orthosteric
binding sites (cyan sphere) and allosteric binding sites (carmine sphere). (B) Docked result of JTE-013 (yellow carbon sticks) at the orthosteric site and specific amino acid residues.
(C) Docked result of compound 40 (carmine carbon sticks) at the allosteric site and specific amino acid residues. (D) superposition of JTE-013 binding mode and compound 40
binding modes in S1PR2. Atom color code: oxygen ¼ red, nitrogen ¼ blue, hydrogen ¼ white, chlorine ¼ green. Hydrogen bonds are indicated by green dashed lines.
6

D. Luo, Y. Zhang, S. Yang et al.
European Journal of Medicinal Chemistry 225 (2021) 113775
Table 3
hydrophobic pocket (Fig. 5B). Compound 40 occupies another hy-
drophobic pocket and interacts with Asn89 by apparent hydrogen
bonds (Fig. 5C). Co-docking of JTE-013 and compound 40 suggested
that the receptor pocket could accommodate JTE-013 in the hy-
drophobic pocket close to the surface of the protein, while com-
pound 40 could reach the interior part of the protein (Fig. 5D). The
binding mode between 40 and S1PR2 reasonably explained the 5-
FU resistance reversal activities of compound 40 in HCT116DPD
and SW620/5-FU cells.
Pharmacokinetic parameters for 40 and JTE-013 in blood and plasma after a single
intravenous injection at 1 mg/kg in rats (n ¼ 3)a.
Parameter
40
JTE-013
Blood
Blood
Plasma
Plasma
C0 (ng/mL)
t1/2 (h)
AUC(0-t) (h ng/mL)
AUC(0-∞) (h ng/mL)
Vd (L/kg)
489.1
4.4
1894.6
1947.7
3.3
1110.1
4.7
3838.2
3956.8
1.7
1838.9
1.0
725.9
737.8
2.1
2020.1
1.1
481.7
484.0
3.1
CL (L/h/kg)
MRT(0-t) (h)
MRT(0-∞) (h)
0.51
6.0
6.7
0.25
6.2
7.0
1.4
0.9
1.0
2.1
0.9
0.9
2.8. Pharmacokinetic profile of compound 40
a
The pharmacokinetic profile of compound 40 and JTE-013 were
examined in in vivo and in vitro. The amount of compound 40 was
no changed significantly in the incubation of MLM and RLMs with
NADPH generation for the 97.9 % and 95.2 % remaining after 60 min
incubation. Mean plasma and blood concentration of compound 40
and JTE-013 versus time profiles are illustrated (Fig. 6), while mean
major pharmacokinetic parameters are presented in Table 3. The
concentration of compound 40 at all collection time points was
lower in whole blood than plasma. The concentration at time zero
(C0) and area under the concentration curve (AUC) of compound 40
in plasma were about 2-fold of those in whole blood. Meanwhile,
the elimination half-life (t1/2) and mean residence time (MRT) of
compound 40 were similar in whole blood (4.4 h) and plasma
(4.7 h). However, JTE-013 plasma and blood concentrations at 8 h
after dosing and beyond were below the limit of quantification
(LOQ, 1 ng/mL). The AUC of JTE-013 in whole blood was higher than
in plasma. In addition, the clearance of compound 40 was signifi-
cantly lower than JTE-013. Moreover, compound 40 and JTE-013
between whole blood and plasma appear strongly correlated with
Pearson's correlation coefficients of 0.9765 and 0.9313, respectively.
The median whole blood to plasma ratios measured at all collection
time points were 0.484 (0.285e0.671) for compound 40, which
differs significantly (p < 0.001) from JTE-013 with the median ratio
of 1.622 (0.859e3.442). It was suggested that there was no binding
between 40 and hemocytes, meanwhile, JTE-013 has an affinity to
the blood cells.
C0, concentration at time zero after intravenous dose; t1/2, half-life; AUC, area
under the concentration curve; Vd, apparent volume of distribution; CL, clearance;
MRT, mean residence time.
to lose weight after 12 days (Fig. 7A). The mice treated with 5-FU
plus compound 40 showed a significant effect on inhibiting tu-
mor growth and the inhibition rate increased from 18.54 % to
66.16 % compared with those treated with 5-FU alone (Fig. 7BeD).
In contrast, the effect of JTE-013 was not as effective as compound
40 and the inhibition rate of JTE-013 plus 5-FU was only 29.88 %
(Fig. 7D). Besides, the treatment with compound 40 alone hardly
inhibited the tumor growth with the inhibition rate of 7.50 %
(Fig. 7D).
In addition, to verify whether compound 40 could prevent DPD
expression in vivo, DPD expression levels in liver, colon, and tumor
of nude mice were also measured. The results of western blotting
suggested that compound 40 could largely inhibit DPD expression
in tumor and liver tissues (Fig. 8A). Immunohistochemistry assays
were also performed in the paraffin-embedded tumor and tissue
sections, and the staining of the tumor and liver showed that
compound 40 and JTE-013 strongly inhibited DPD expression in the
tumoral and liver specimens with no observable toxicities. The
scattered staining was demonstrated in colonic tissues, while DPD
staining was impaired by compound 40 but not JTE-013 (Fig. 8B).
These results suggested that compound 40 could effectively reverse
5-FU resistance through inhibiting DPD expression in vivo.
2.9. Compound 40 combined with 5-FU suppresses tumor growth in
SW620/5-FU xenograft mice
3. Conclusions
To evaluate the 5-FU resistance reversal activity of compounds
40 in vivo, the SW620/5-FU xenograft model was established. The
mice were treated intravenously with the combination of 5-FU
(20 mg/kg/day) and compound 40 (1.08 mg/kg/day) for consecu-
tive 24 days. The combination of 5-FU (20 mg/kg/day) and JTE-013
(12.50 mg/kg/day) was used as a positive control. The body weights
of mice in the 5-FU plus compound 40 group continuously leveled
off, whereas body weights in the positive control group continued
5-FU, the earliest antimetabolites for cancer therapy, has been
widely used in the therapy of various solid tumors, especially
gastrointestinal tumors. Yet, the emergence of drug resistance has
greatly limited its applications. Cancer cells could develop 5-FU
resistance through overexpression of DPD, which is the main
enzyme to degrade 5-FU. Our previous study revealed that S1PR2
up-regulated the expression of DPD to accelerate the degradation of
intracellular 5-FU, and S1PR2 inhibition could overcome resistance
Fig. 6. Mean concentration-time curves in plasma (black line) and whole blood (red line) after intravenous injection of 40 and JTE-013 at 1 mg/kg in mice (n ¼ 3).
7

D. Luo, Y. Zhang, S. Yang et al.
European Journal of Medicinal Chemistry 225 (2021) 113775
Fig. 7. The inhibitory effects of compound 40 combined with 5-FU in the SW620/5-FU xenografted athymic mice, n ¼ 6. (A) Body weight changes of mice were measured every four
days during treatment. (B) Tumor volume changes of mice were measured every four days during treatment. (C) Representative tumor-bearing nude mice of every group. (D) Picture
of dissected SW620/5-FU tumor tissues of each group. Data are presented as mean SEM. *p < 0.05, **p < 0.01 and ***p < 0.001.
Fig. 8. Compound 40 prevented 5-FU resistance in vivo by downregulating DPD expression, n ¼ 6. (A) DPD expressions were analyzed in tumor and various tissues. Histogram
showed the ratios of their corresponding grayscale values to b-actin in the right panel. (B) DPD expressions were analyzed in tumor and various tissues of each group by
immunohistochemistry. Data are presented as mean SEM. *p < 0.05, **p < 0.01 and ***p < 0.001.
via reducing intracellular DPD. The previously identified S1PR2
antagonist S-45 has excellent in vitro activity compared with JTE-
013, but its in vivo activity was unsatisfactory.
that compound 40 could prevent the degradation of 5-FU into FBAL
in cells. More importantly, the 5-FU resistance reversal activity of
compound 40 was verified in the colorectal cancer xenograft mouse
model. The combination of compound 40 and 5-FU significantly
inhibited tumor growth and decreased tumor weight by 66.16 %,
which was greater than that of the control group (JTE-013 and 5-FU
group). Concurrently, the treatment with compound 40 did not
affect the body weight of the mice, suggesting that 40 was well
tolerated in vivo. Additionally, western blotting and IHC results
showed compound 40 decreased DPD expression in tumor and
liver, and colon tissues from 40-treated mice, consistent with our
in vitro findings. Overall, our results demonstrated that the most
potent compound 40 showed excellent reversion on 5-FU resis-
tance via reducing DPD expression levels in vitro and in vivo and
represents a promising lead candidate to treat 5-FU resistant
colorectal cancer patients.
In this study, using a structure-based approach, we designed
and synthesized novel small molecules that effectively inhibited
S1PR2, with verified effects in vitro and in vivo. The initial screening
included HCT116DPD cells, SW620/5-FU cells, and non-cancerous
Colonic NCM460 cells via MTT assay. Among all compounds, com-
pound 40 was selected for further investigations due to its superior
activity to reverse 5-FU resistance and inhibit DPD expression, and
reasonably good selectivity. Furthermore, compound 40 was
analyzed for its ability to decrease the dosage of 5-FU and inhibit
the JMJD3-H3K27me3-DPD pathway in 5-FU-resistant colorectal
cancer cells. And it was found that compound 40 could reduce the
use of 5-FU and suppress the expression of JMJD3 in
a
concentration-dependent manner. Further HPLC analysis showed
8

D. Luo, Y. Zhang, S. Yang et al.
European Journal of Medicinal Chemistry 225 (2021) 113775
4. Experimental section
cooled to 50 ꢀC, and compound 6 (152 mg, 0.74 mmol) in anhydrous
THF (3 mL) was added, and the mixture was allowed to stir for
another 8 h. Then the reaction mixture was concentrated in vacuo,
and the residue was subjected to column chromatography to afford
compound 10 (134 mg, 46 %) as a white solid.
4.1. Chemical reagents and synthesis procedures
The analytical grade chemicals and solvents were gained from
commercial companies. Unless otherwise stated, all commercial
reagents were used without additional purification. If needs be, the
solvents were purified and dried before use by standard methods.
Dichloromethane (DCM) was dehydrated by CaH2, and tetrahy-
drofuran (THF) was dried by sodium. Other solvents were dried by
using the dried 4 Å molecular sieves. Thin-layer chromatography
(TLC) was carried out by using silica gel plates (GF254) and visu-
alization of chromatographic spots was affected at 254 nm and
365 nm. The crude compounds were purified by crystallization and
column chromatography. 1H NMR and 13C NMR spectra were
recorded in CDCl3 or DMSO‑d6 at 400, 500 or 600 MHz on an Agi-
Compound 14 was prepared from compound 6 through the
similar procedure described for the synthesis of compound 10.
Compound 6 (1000 mg, 5.35 mmol) was mixed with CH3I
(1140 mg, 8.03 mmol), NaH (385 mg, 16.05 mmol) in DMF (15 mL)
and allowed to stir under an ice bath for 2 h the reaction mixture
was quenched with water and extracted with DCM. The extract was
combined and washed with saturated NaCl solutions, then dried
over anhydrous MgSO4, filtered, and concentrated in vacuo. The
crude product was purified by column chromatography to afford
the compound 15 (914 mg, 85 %) as a white solid. Compound 15
(900 mg, 4.48 mmol) was combined with Cs2CO3 (4378 mg,
13.43 mmol), PdCl2(PPh3)2 (154 mg, 0.22 mmol), and 4-
phenylboronic acid (547 mg, 4.48 mmol) in 1,4-dioxane/H2O
(20 mL, v/v 3/1). The mixture was degassed using two rounds of
vacuum evacuation followed by nitrogen fill, then the mixture was
allowed to stir at 80 ꢀC for 12 h. After the reaction was completed,
the mixture was washed with water and saturated NaCl solution,
and extracted with DCM. The extract was dried over anhydrous
MgSO4, filtered, and concentrated in vacuo, the residue was sub-
jected to column chromatography. Compound 16 was obtained as a
white solid (533 mg, 49 %).
lent spectrometer by using CDCl3 as
a reference standard
(
d
¼ 7.26 ppm) for 1H NMR and (
d
¼ 77.00 ppm) for 13C NMR or
DMSO‑d6 as a reference standard (
(
were recorded by using a Waters Xevo G2-XS QTOF spectrometer
d
¼ 2.50 ppm) for 1H NMR and
d
¼ 39.52 ppm) for 13C NMR. High-resolution mass spectra (HRMS)
with an ESI ionization source. In addition, the Agilent 1290 Infinity
HPLC and a reversed-phase C18 column (2.1 ꢂ 100 mm, 3.5
mm)
were used to estimate the purity (>95 %) of the compounds. The
compounds were dissolved in acetonitrile (1.5 mL), every sample
was injected at a volume of 4
mL and eluted with a mixture of
solvent acetonitrile and water (20/80, containing 0.1 % formic acid),
the flow rate was 1 mL/min and the detection wavelength was
280 nm under UV.
Compound 18 was prepared from 16 through the similar pro-
cedure described for the synthesis of compound 10.
2-Chloro-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (4).
White solid (54 %). 1H NMR (500 MHz, DMSO‑d6)
d 11.76 (s, 1H), 7.10
4.1.1. Procedure for preparation of compounds 10, 14, 18
(dd, J ¼ 3.5, 2.4 Hz, 1H), 6.60 (dd, J ¼ 3.6, 2.0 Hz, 1H). 13C NMR
Compound
3
(1000 mg, 5.35 mmol) was mixed with
(125 MHz, DMSO‑d6) d 157.90, 152.47, 121.44, 102.50, 101.27, 39.14.
(CH3)2NH$HCl (477 mg, 5.88 mmol), Et3N (1081 mg, 10.70 mmol) in
DCM (15 mL), and allowed to stir at room temperature for 8 h. Then
the mixture was washed with water and extracted with DCM, the
extract was combined and washed with saturated NaCl solution,
dried over anhydrous MgSO4, filtered, and concentrated in vacuo.
The crude product was purified by column chromatography to
afford the compound 4 (566 mg, 54 %) as a white solid. Compound 4
(500 mg, 2.55 mmol) was mixed with CH3I (543 mg, 3.83 mmol),
NaH (184 mg, 7.65 mmol) in dimethyl formamide (DMF, 15 mL) and
allowed to stir under ice bath conditions for 2 h. Then the reaction
was quenched with water and extracted with DCM, the extract was
combined and washed with saturated NaCl solutions, then dried
over anhydrous MgSO4, filtered, and concentrated. The crude
product was purified by column chromatography to afford the
compound 5 (423 mg, 79 %) as a white solid. 80 % Hydrazine hy-
drate (3012 mg, 60.24 mmol) was mixed with a solution of 5
(229 mg, 1.09 mmol) in ethanol (10 mL). The mixture was heated at
100 ꢀC for 3 h. Then the mixture was concentrated and washed with
saturated NaCl solutions and extracted with DCM. The extract was
dried over anhydrous MgSO4, filtered, and concentrated in vacuo to
yield compound 6 as a white solid (204 mg, 91 %).
2-Chloro-N,N,7-trimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine
(5). White solid (79 %). 1H NMR (500 MHz, DMSO‑d6)
d 7.08 (d,
J ¼ 3.6 Hz, 1H), 6.54 (d, J ¼ 3.6 Hz, 1H), 3.62 (s, 3H), 3.21 (s, 6H). 13
C
NMR (125 MHz, DMSO‑d6)
d 157.72, 152.50, 151.42, 125.40, 101.80,
101.32, 39.08, 31.48.
2-Hydrazinyl-N,N,7-trimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-
amine (6). White solid (92 %). 1H NMR (500 MHz, DMSO‑d6)
d
7.00
(s, 1H), 6.76 (d, J ¼ 3.6 Hz, 1H), 6.39 (d, J ¼ 3.6 Hz, 1H), 3.55 (s, 3H),
3.20 (s, 6H). 13C NMR (125 MHz, DMSO‑d6)
d 161.51, 157.81, 153.30,
121.71, 101.72, 96.96, 38.81, 31.01.
N-(2,6-dichloropyridin-4-yl)-2-(4-(dimethylamino)-7-methyl-7H-
pyrrolo[2,3-d]pyrimidin-2-yl)hydrazine-1-carboxamide (10). White
solid (46 %): mp 241.5e241.8 ꢀC; 1H NMR (500 MHz, DMSO‑d6)
d
9.58 (s, 1H), 8.44 (d, J ¼ 97.7 Hz, 1H), 8.08 (s, 1H), 7.85 (s, 1H), 6.85
(d, J ¼ 3.6 Hz, 1H), 6.45 (d, J ¼ 3.6 Hz, 1H), 3.53 (s, 3H), 3.19 (s, 6H).
13C NMR (125 MHz, DMSO‑d6)
159.16, 157.76, 152.87, 152.25,
d
149.71, 122.68, 111.66, 101.69, 98.27, 38.71, 31.05. HRESIMS m/z
417.0710 [MþNa]þ (calcd for C15H16Cl2N8ONa 417.0722).
2,4-Dichloro-7-cyclopropyl-7H-pyrrolo[2,3-d]pyrimidine
(11).
7.70 (d,
White solid (45 %). 1H NMR (500 MHz, DMSO‑d6)
d
J ¼ 3.7 Hz, 1H), 6.62 (d, J ¼ 3.7 Hz, 1H), 3.59 (td, J ¼ 6.9, 3.5 Hz, 1H),
A
mixture of 2,6-dichloroisonicotinic acid (1000 mg,
1.06 (dq, J ¼ 7.9, 2.7 Hz, 4H). 13C NMR (125 MHz, DMSO‑d6)
d 153.28,
5.20 mmol), Et3N (687 mg, 6.80 mmol), in 1,4-dioxane (10 mL) was
allowed to stir at 0 ꢀC and diphenyl phosphoryl azide (1900 mg,
6.80 mmol) was added dropwise. Then the mixture was warmed to
room temperature. After the reaction was completed, the mixture
was washed into 1 M NaHCO3 solution and extracted with DCM.
The extract was washed with saturated NaCl solution, dried over
anhydrous MgSO4, filtered, and concentrated in vacuo. The crude
product was purified by column chromatography to afford com-
pound 8 (753 mg, 67 %) as a white solid.
151.31, 150.69, 132.59, 116.92, 99.17, 27.61, 6.49.
4-(7-Cyclopropyl-2-hydrazinyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)
morpholine (13). White solid (95 %). 1H NMR (500 MHz, DMSO‑d6)
d
7.18 (d, J ¼ 3.7 Hz, 1H), 6.62 (d, J ¼ 3.8 Hz, 1H), 3.80 (dd, J ¼ 5.8,
4.0 Hz, 4H), 3.68 (dd, J ¼ 5.8, 3.9 Hz, 4H), 3.51e3.45 (m, 1H),
1.02e0.91 (m, 4H). 13C NMR (125 MHz, DMSO‑d6)
157.29, 153.36,
d
152.39, 124.71, 101.84, 101.21, 66.36, 45.85, 27.23, 6.43.
2-(7-Cyclopropyl-4-morpholino-7H-pyrrolo[2,3-d]pyrimidin-2-
yl)-N-(2,6-dichloropyridin-4-yl)hydrazine-1-carboxamide
(14).
A solution of 8 (159 mg, 0.74 mmol) in toluene (1 mL) was
refluxed for 2 h to produce the isocyanate 9. The mixture was
White solid (57 %): mp 241.4e241.9 ꢀC; 1H NMR (400 MHz,
DMSO‑d6) d 9.61 (s, 1H), 8.60 (s, 1H), 8.25 (s,1H), 7.85 (s, 2H), 6.88 (s,
9

D. Luo, Y. Zhang, S. Yang et al.
European Journal of Medicinal Chemistry 225 (2021) 113775
1H), 6.45 (d, J ¼ 3.8 Hz, 1H), 3.76 (t, J ¼ 4.7 Hz, 4H), 3.69e3.61 (m,
4H), 3.38 (q, J ¼ 5.8 Hz, 1H), 0.90 (d, J ¼ 5.0 Hz, 4H). 13C NMR
13C NMR (125 MHz, DMSO‑d6)
d 151.48, 151.24, 150.09, 150.05,
139.56, 128.73, 127.68, 118.33, 111.49, 104.84, 87.00. HRESIMS m/z
(100 MHz, DMSO‑d6) d 159.03, 157.29, 154.74, 152.33, 149.77, 121.66,
399.9362 [MþH]þ (calcd for C13H9Cl2BrN5O 399.9368).
111.71, 100.98, 67.49, 66.51, 45.84, 26.99, 6.39. HRESIMS m/z
6-Phenylpyrazolo[1,5-a]pyridin-2-amine (24a). White solid
463.1121 [MþH]þ (calcd for C19H21Cl2N8O2 463.1165).
(51 %). 1H NMR (600 MHz, DMSO‑d6)
d
8.55 (s, 1H), 7.64 (dd, J ¼ 8.4,
2,4-Dichloro-7-methyl-7H-pyrrolo[2,3-d]pyrimidine (15). White
1.1 Hz, 2H), 7.42e7.39 (m, 2H), 7.35e7.26 (m, 3H), 5.63 (s, 1H), 5.31
solid (85 %). 1H NMR (500 MHz, DMSO‑d6)
d
7.71 (d, J ¼ 3.6 Hz, 1H),
(s, 2H). 13C NMR (150 MHz, DMSO‑d6)
d 129.51, 127.46, 126.46,
6.62 (d, J ¼ 3.6 Hz, 1H), 3.78 (s, 3H). 13C NMR (125 MHz, DMSO‑d6)
125.34, 123.43, 115.37, 81.85.
d
152.11, 151.26, 150.54, 133.75, 116.24, 99.15, 32.04.
1-(2,6-Dichloropyridin-4-yl)-3-(6-phenylpyrazolo[1,5-a]pyridin-
2-Chloro-7-methyl-4-phenyl-7H-pyrrolo[2,3-d]pyrimidine (16).
2-yl)urea (25a). White solid (42 %): mp 260.1e260.9 ꢀC; 1H NMR
White solid (55 %). 1H NMR (600 MHz, CDCl3)
d
8.07 (dd, J ¼ 7.7,
(600 MHz, DMSO‑d6) d 9.89 (s, 1H), 9.81 (s, 1H), 8.86 (s, 1H),
2.1 Hz, 2H), 7.52e7.48 (m, 3H), 7.16 (d, J ¼ 3.6 Hz, 1H), 6.74 (d,
7.72e7.70 (m, 2H), 7.64e7.62 (m, 1H), 7.59 (s, 2H), 7.54 (dd, J ¼ 9.2,
J ¼ 3.6 Hz, 1H), 3.83 (s, 3H). 13C NMR (125 MHz, CDCl3)
d 159.23,
1.6 Hz,1H), 7.44 (t, J ¼ 7.8 Hz, 2H), 7.34 (t, J ¼ 7.4 Hz,1H), 6.64 (s,1H).
153.61, 153.23, 136.97, 130.56, 129.01, 128.78, 114.40, 100.68, 31.46.
N-(2,6-dichloropyridin-4-yl)-2-(7-methyl-4-phenyl-7H-pyrrolo
[2,3-d]pyrimidin-2-yl)hydrazine-1-carboxamide (18). White solid
13C NMR (125 MHz, DMSO‑d6)
d 151.49, 151.27, 150.16, 150.06,
139.95, 136.89, 129.49, 128.02, 126.85, 125.73, 124.67, 124.47, 117.43,
111.51, 86.08. HRESIMS m/z 398.0575 [MþH]þ (calcd for
(39 %): mp 205.3e206.0 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d
9.72 (s,
C
19H14Cl2N5O 398.0575).
1H), 8.82 (d, J ¼ 43.4 Hz, 2H), 8.12 (d, J ¼ 6.3 Hz, 2H), 7.89 (s, 1H),
6-(3-Methoxyphenyl)pyrazolo[1,5-a]pyridin-2-amine
(24b).
7.55 (d, J ¼ 6.7 Hz, 3H), 7.34 (s,1H), 6.74 (s,1H), 3.70 (s, 3H). 13C NMR
White solid (59 %). 1H NMR (600 MHz, DMSO‑d6)
d
8.58 (s, 1H),
(100 MHz, DMSO‑d6)
d
159.83, 156.95, 154.03, 152.24, 149.85,
7.35e7.28 (m, 3H), 7.20 (dt, J ¼ 7.8, 1.2 Hz, 1H), 7.18e7.16 (m, 1H),
138.27, 130.53, 129.14, 128.89, 111.79, 100.09, 31.08. HRESIMS m/z
6.85 (dd, J ¼ 8.0, 2.7 Hz, 1H), 5.62 (s, 1H), 5.31 (s, 2H), 3.78 (s, 3H).
428.0711 [MþH]þ (calcd for C19H16Cl2N7O 428.0793).
13C NMR (150 MHz, DMSO‑d6)
d 160.36, 159.65, 140.82, 139.11,
130.53, 125.53, 123.51, 118.77, 115.25, 113.22, 111.86, 81.88, 55.67.
1-(2,6-Dichloropyridin-4-yl)-3-(6-(3-methoxyphenyl)pyrazolo
[1,5-a]pyridin-2-yl)urea (25b). White solid (57 %): mp
4.1.2. Procedure for preparation of compounds 23a-23b, 25a-25b
O-(mesitylsulfonyl)hydroxylamine (1000 mg, 4.65 mmol) was
mixed with 2-(pyridine-2-yl)acetonitrile (549 mg, 4.65 mmol) in
DCM (15 mL) and allowed to stir at room temperature for 1 h, then
the reaction mixture was filtered to afford compound 21a (465 mg,
30 %) as a white solid. Compound 21a (400 mg, 1.20 mmol) and
K2CO3 (332 mg, 2.40 mmol) were mixed in MeOH (10 mL) and
allowed to stir at room temperature for 3 h. The mixture was
washed with water and extracted with DCM, and the extract was
washed with saturated NaCl solution, dried over anhydrous MgSO4,
filtered, and concentrated in vacuo. The crude product was purified
by column chromatography to afford compound 22a (64 mg, 40 %)
as a white solid. Compound 23a was prepared from 22a through the
similar procedure described for the synthesis of compound 10, and
compound 23b was also prepared from compound 20b.
Compound 22b (300 mg, 1.42 mmol), Cs2CO3 (1391 mg,
4.27 mmol), Pd(PPh3)4 (819 mg, 0.71 mmol), and 4-phenylboronic
acid (173 mg, 1.42 mmol) were added into 1,4-dioxane/H2O
(20 mL, v/v 3/1). The reaction mixture was degassed using two
rounds of vacuum evacuation followed by nitrogen fill. The mixture
was stirred at 80 ꢀC for 3 h, and washed with water and saturated
NaCl solution, and extracted with DCM. The extract was dried over
anhydrous MgSO4, filtered, and concentrated in vacuo, and the
residue was subjected to column chromatography. Compound 24a
was obtained as a white solid (151 mg, 51 %). Compound 25a was
prepared from 24a through the similar procedure described for the
synthesis of compound 10, and compounds 25b was also prepared
from compound 22b.
246.2e246.9 ꢀC; 1H NMR (500 MHz, DMSO‑d6)
d 9.90 (s,1H), 9.83 (s,
1H), 8.92 (s,1H), 7.66e7.60 (m, 3H), 7.57 (dd, J ¼ 9.2,1.7 Hz,1H), 7.37
(t, J ¼ 7.9 Hz, 1H), 7.31e7.27 (m, 2H), 6.93 (dd, J ¼ 8.2, 1.7 Hz, 1H),
6.67 (s, 1H), 3.83 (s, 3H). 13C NMR (100 MHz, DMSO‑d6)
d 160.32,
151.52, 151.31, 150.10, 140.05, 138.39, 130.57, 125.93, 124.59, 119.15,
117.36, 113.80, 112.29, 111.54, 86.13, 55.68. HRESIMS m/z 428.0673
[MþH]þ (calcd for C20H16Cl2N5O2 428.0681).
4.1.3. Procedure for preparation of compounds 29, 33
Cyanuric chloride (1000 mg, 5.47 mmol), pyrrole (388 mg,
5.47 mmol) were dissolved in acetone (10 mL) and the mixture was
stirred at 0 ꢀC for 5 h. Then the mixture was diluted with water and
extracted with DCM. The extract was combined and washed with
saturated NaCl solution, dried over anhydrous MgSO4, filtered, and
concentrated in vacuo. The crude product was purified by column
chromatography to afford the compound 27 (620 mg, 52 %) as a
white solid; Compound 27 (500 mg, 2.29 mmol) was mixed with
phenylamine (213 mg, 2.29 mmol), Et3N (462 mg, 4.58 mmol) in
acetonitrile (10 mL) and stirred at 60 ꢀC for 3 h. Then the mixture
was poured into 1 M HCl solution, and extracted with DCM three
times. The extract was combined and washed with water and
saturated NaCl solutions, dried over anhydrous MgSO4, filtered, and
concentrated in vacuo to yield a crude product. Then the crude
product was purified by column chromatography to afford com-
pound 27 (378 mg, 60 %) as a white solid. 80 % Hydrazine hydrate
(3012 mg, 60.24 mmol) was mixed with a solution of 27 (300 mg,
1.09 mmol) in ethanol (10 mL). The mixture was heated at 100 ꢀC
for 3 h. Then the mixture was concentrated and washed with
saturated NaCl solutions and extracted with DCM. The extract was
dried over anhydrous MgSO4, filtered, and concentrated in vacuo to
yield compound 28 as a white solid (269 mg, 91 %).
Compound 21a-21b, 22a-22b were obtained as described in the
literature [31].
1-(2,6-Dichloropyridin-4-yl)-3-(pyrazolo[1,5-a]pyridin-2-yl)urea
(23a). White solid (39 %): mp 241.8e242.2 ꢀC; 1H NMR (600 MHz,
DMSO‑d6)
d
9.80 (s, 1H), 9.74 (s, 1H), 8.51 (d, J ¼ 7.7 Hz, 1H), 7.58 (s,
2H), 7.53 (dt, J ¼ 8.8, 1.1 Hz, 1H), 7.16 (ddd, J ¼ 8.7, 6.7, 1.0 Hz, 1H),
A solution of 8 (159 mg, 0.74 mmol) in toluene (1 mL) was
refluxed for 2 h to produce the isocyanate 9. The mixture was
cooled to 50 ꢀC, and compound 28 (200 mg, 0.74 mmol) in anhy-
drous THF (3 mL) was added, and the mixture was allowed to stir
for another 8 h. Then the reaction mixture was concentrated in
vacuo, and the residue was subjected to column chromatography to
afford compound 29 (187 mg, 55 %) as a white solid.
6.78e6.75 (m, 1H), 6.62 (s, 1H). 13C NMR (150 MHz, DMSO‑d6)
d
151.61, 151.41, 150.15, 149.75, 141.01, 128.69, 124.76, 117.43, 111.66,
111.58, 86.10. HRESIMS m/z 322.0260 [MþH]þ (calcd for
C
13H10Cl2N5O 322.0262).
1-(6-Bromopyrazolo[1,5-a]pyridin-2-yl)-3-(2,6-dichloropyridin-
4-yl)urea (23b). White solid (46 %): mp 277.1e277.7 ꢀC; 1H NMR
(600 MHz, DMSO‑d6)
2H), 7.54 (d, J ¼ 9.4 Hz, 1H), 7.29 (dd, J ¼ 9.3, 1.7 Hz, 1H), 6.68 (s, 1H).
d
9.93 (s, 1H), 9.82 (s, 1H), 8.90 (s, 1H), 7.57 (s,
Compounds 33 were prepared through a similar procedure from
compounds 30.
10

D. Luo, Y. Zhang, S. Yang et al.
European Journal of Medicinal Chemistry 225 (2021) 113775
4-Chloro-N-phenyl-6-(pyrrolidin-1-yl)-1,3,5-triazin-2-amine
DMSO‑d6)
d
9.79 (s, 2H), 8.23 (d, J ¼ 5.6 Hz, 1H), 7.64 (d, J ¼ 1.8 Hz,
(27). White solid (60 %). 1H NMR (500 MHz, DMSO‑d6)
d
9.98 (s, 1H),
1H), 7.55 (s, 2H), 7.37 (dd, J ¼ 5.6, 1.9 Hz, 1H). 13C NMR (100 MHz,
7.72 (s, 2H), 7.28 (t, J ¼ 7.8 Hz, 2H), 7.03e6.96 (m, 1H), 3.48 (dt,
DMSO‑d6) d 151.71, 151.45, 150.94, 150.53, 150.08, 148.68, 112.77,
J ¼ 30.2, 6.2 Hz, 4H), 1.95e1.82 (m, 4H). 13C NMR (125 MHz,
112.39, 111.72. HRESIMS m/z 316.9763 [MþH]þ (calcd for
DMSO‑d6)
25.10, 24.96.
2,6-Dichloroisonicotinoyl azide (28). White solid (67 %). 1H NMR
(500 MHz, DMSO‑d6)
7.88 (s, 2H). 13C NMR (125 MHz, DMSO‑d6)
169.37, 150.92, 143.78, 122.71.
N-(2,6-dichloropyridin-4-yl)-2-(4-(phenylamino)-6-(pyrrolidin-
1-yl)-1,3,5-triazin-2-yl)hydrazine-1-carboxamide (29). White solid
(55 %): mp 243.6e243.9 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
9.72 (d,
d
162.89, 139.50, 128.99, 123.14, 120.24, 46.93, 46.71,
C
11H8Cl3N4O 316.9764).
1-(3,5-Dichloro-4-methoxyphenyl)-3-(2,6-dichloropyridin-4-yl)
urea (39): mp 237e237.5 ꢀC; White solid (49 %). 1H NMR (400 MHz,
d
DMSO‑d6)
d
9.74 (s, 1H), 9.37 (s, 1H), 7.57 (d, J ¼ 15.0 Hz, 4H), 3.79 (s,
d
3H). 13C NMR (100 MHz, DMSO‑d6)
d
152.04, 151.43, 150.04, 147.24,
136.46, 128.62, 119.66, 111.56, 61.12. HRESIMS m/z 379.9524
[MþH]þ (calcd for C13H10Cl4N3O2 379.9527).
d
1,3-Bis(2,6-dichloropyridin-4-yl)urea (40). White solid (55 %): mp
J ¼ 95.6 Hz, 1H), 9.16 (s, 1H), 8.72 (s, 2H), 7.99e7.47 (m, 4H), 7.06 (d,
245.3e246.1 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d 9.97 (s, 2H), 7.55 (s,
J ¼ 131.0 Hz, 3H), 3.49 (s, 4H), 1.90 (s, 4H). 13C NMR (150 MHz,
4H). 13C NMR (100 MHz, DMSO‑d6)
d 151.63, 150.84, 150.12, 111.93.
DMSO‑d6)
d
164.45, 163.76, 152.21, 140.95, 130.17, 128.78, 128.63,
HRESIMS m/z 350.9372 [MþH]þ (calcd for C11H7Cl4N4O 350.9374).
121.87, 120.05, 111.88, 46.52, 46.34, 31.82, 29.56, 29.24, 29.11, 25.32,
22.63, 14.50. HRESIMS m/z 460.1149 [MþH]þ (calcd for
4.1.5. Procedure for preparation of compounds 44a-44d
C
19H20Cl2N9O 460.1168).
A mixture of 2,6-dichloroisonicotinic acid (1000 mg, 5.24 mmol)
in methanol (15 mL) was stirred at room temperature and 1 M
NaOH (7 mL) was added dropwise, then the mixture was heated at
70 ꢀC for 8 h. The pH value of the reaction mixture was adjusted to
5. The mixture was washed with water, and extracted with DCM,
and the extract was washed with saturated NaCl solution, dried
over anhydrous MgSO4, filtered, and concentrated in vacuo. The
crude product was purified by column chromatography to afford
compound 41a (636 mg, 65 %) as a white solid. The acid azide 42a
was prepared from compound 41a through the similar procedure of
compound 7. Then compound 44a was prepared through a similar
procedure to compound 10, and compounds 44a-44d were also
prepared from compound 6.
2-Chloro-N,6-dimethylpyrimidin-4-amine (31). White solid
(33 %). 1H NMR (600 MHz, CDCl3)
d 6.35 (s, 1H), 6.07 (s, 1H), 2.92 (d,
J ¼ 4.6 Hz, 3H), 2.31 (s, 3H). 13C NMR (150 MHz, CDCl3)
d 165.06,
160.08, 159.99, 98.34, 29.76, 28.52, 23.92.
2-Hydrazinyl-N,6-dimethylpyrimidin-4-amine (32). White solid
(95 %). 1H NMR (600 MHz, DMSO‑d6)
d 7.26 (s, 1H), 6.68 (s, 1H), 5.56
(s, 1H), 3.96 (s, 2H), 2.69 (d, J ¼ 4.2 Hz, 3H), 2.02 (s, 3H). 13C NMR
(150 MHz, CDCl3)
d 169.63, 169.08, 32.24, 28.77.
N-(2,6-dichloropyridin-4-yl)-2-(4-methyl-6-(methylamino)pyr-
imidin-2-yl)hydrazine-1-carboxamide (33). White solid (51 %): mp
214.0e214.2 ꢀC; 1H NMR (500 MHz, DMSO‑d6)
d
9.53 (s, 1H), 8.58 (s,
1H), 8.19 (s, 1H), 7.68 (d, J ¼ 168.2 Hz, 2H), 6.92 (s, 1H), 5.76 (s, 1H),
2.68 (s, 3H), 2.05 (s, 3H). 13C NMR (125 MHz, DMSO‑d6)
164.39,
d
2-Chloro-6-methoxyisonicotinic acid (41a). White solid (65 %). 1H
163.37, 152.18, 149.75, 111.69, 23.90. HRESIMS m/z 342.0465
[MþH]þ (calcd for C12H14Cl2N7O 342.0637).
NMR (400 MHz, DMSO‑d6)
d
13.97 (s, 1H), 7.40 (d, J ¼ 1.0 Hz, 1H),
7.19 (d, J ¼ 1.0 Hz, 1H), 3.91 (s, 3H). 13C NMR (100 MHz, DMSO‑d6)
d
165.09, 164.51, 148.56, 144.77, 115.99, 109.78, 54.96.
4.1.4. Procedure for preparation of compounds 34e40
Compounds 34e40 were prepared from acid azide 7 and
different amines through the similar procedure described for the
synthesis of compound 10.
2-Chloro-6-ethoxyisonicotinic acid (41b). White solid (82 %). 1H
NMR (400 MHz, DMSO‑d6)
d
13.94 (s, 1H), 7.38 (d, J ¼ 1.0 Hz, 1H),
7.14 (d, J ¼ 1.0 Hz, 1H), 4.33 (q, J ¼ 7.0 Hz, 2H), 1.33 (t, J ¼ 7.0 Hz, 3H).
13C NMR (100 MHz, DMSO‑d6)
d 165.11, 164.10, 148.53, 144.74,
1-(2,6-Dichloropyridin-4-yl)-3-(2,6-dimethylpyridin-4-yl)urea
115.81, 109.83, 63.37, 14.65.
(34). White solid (59 %): mp 209.9e210.6 ꢀC; 1H NMR (400 MHz,
2-Chloro-6-isopropoxyisonicotinic acid (41c). White solid (85 %).
DMSO‑d6)
d
9.74 (s, 1H), 9.48e9.36 (m, 1H), 7.55 (s, 2H), 7.14 (s, 2H),
158.12, 151.87, 151.23,
1H NMR (400 MHz, DMSO‑d6)
d
13.89 (s, 1H), 7.34 (d, J ¼ 1.0 Hz, 1H),
2.37 (s, 6H). 13C NMR (100 MHz, DMSO‑d6)
d
7.08 (d, J ¼ 1.0 Hz,1H), 5.18 (h, J ¼ 6.2 Hz,1H),1.30 (d, J ¼ 6.2 Hz, 6H).
150.08, 146.86, 111.52, 109.58, 24.47. HRESIMS m/z 311.0468
13C NMR (100 MHz, DMSO‑d6)
d 165.10, 163.62, 148.49, 144.75,
[MþH]þ (calcd for C13H13Cl2N4O 311.0466).
115.53, 110.28, 70.15, 21.99.
1-(3,5-Dichlorophenyl)-3-(2,6-dichloropyridin-4-yl)urea
(35).
2-Chloro-6-(cyclopentyloxy)isonicotinic acid (41d). White solid
White solid (49 %): mp 243.8e244.9 ꢀC; 1H NMR (400 MHz,
(60 %). 1H NMR (400 MHz, DMSO‑d6)
d
13.86 (s, 1H), 7.33 (d,
DMSO‑d6)
d
9.58 (d, J ¼ 72.3 Hz, 2H), 7.55e7.50 (m, 4H), 7.18 (t,
J ¼ 1.0 Hz, 1H), 7.08 (d, J ¼ 1.0 Hz, 1H), 5.29 (td, J ¼ 5.9, 3.0 Hz, 1H),
J ¼ 1.9 Hz, 1H). 13C NMR (100 MHz, DMSO‑d6)
d 151.91, 151.26,
2.04e1.83 (m, 2H),1.78e1.51 (m, 6H). 13C NMR (100 MHz, DMSO‑d6)
150.04, 141.61, 134.59, 122.42, 117.47, 111.56. HRESIMS m/z 349.9423
d
165.09, 163.80, 148.50, 144.61, 115.54, 110.18, 79.56, 32.57, 23.82.
[MþH]þ (calcd for C12H8Cl4N3O 349.9421).
2-Chloro-6-methoxyisonicotinoyl azide (42a). White solid (58 %).
N-(2,6-Dichloropyridin-4-yl)-4-methylpiperazine-1-carboxamide
(36). White solid (44 %): mp 105.3e106.4 ꢀC; 1H NMR (400 MHz,
1H NMR (400 MHz, DMSO‑d6)
d
7.41 (d, J ¼ 1.1 Hz, 1H), 7.20 (d,
J ¼ 1.1 Hz, 1H), 3.92 (s, 3H). 13C NMR (100 MHz, DMSO‑d6)
d 170.24,
DMSO‑d6)
d
9.38 (d, J ¼ 4.5 Hz, 1H), 7.61 (t, J ¼ 4.0 Hz, 2H), 3.47 (q,
164.59, 149.02, 143.43, 115.11, 109.39, 55.17.
J ¼ 4.8 Hz, 4H), 2.33 (q, J ¼ 4.7 Hz, 4H), 2.21 (d, J ¼ 4.5 Hz, 3H). 13
C
2-Chloro-6-ethoxyisonicotinoyl azide (42b). White solid (50 %). 1H
NMR (100 MHz, DMSO‑d6)
d
153.73, 152.72, 149.70, 111.72, 54.73,
NMR (400 MHz, CDCl3)
d
7.39 (d, J ¼ 1.1 Hz, 1H), 7.18 (d, J ¼ 1.1 Hz,
46.08, 44.16. HRESIMS m/z 371.0673 [MþH]þ (calcd for
1H), 4.39 (q, J ¼ 7.1 Hz, 2H), 1.40 (t, J ¼ 7.1 Hz, 3H). 13C NMR
C
15H17Cl3N4O2 371.0678).
(100 MHz, CDCl3)
63.36, 14.32.
d 170.37, 164.24, 149.64, 142.38, 114.80, 109.39,
1-(2,6-Dichloropyridin-4-yl)-3-(1,3-dimethyl-1H-pyrazol-5-yl)
urea (37). White solid (67 %): mp 168.3e169.6 ꢀC; 1H NMR
2-Chloro-6-isopropoxyisonicotinoyl azide (42c). White solid
(400 MHz, DMSO‑d6)
d
9.73 (s, 1H), 8.96 (s, 1H), 7.55 (s, 2H), 5.98 (s,
151.76,
(81 %). 1H NMR (400 MHz, CDCl3)
d
7.36 (d, J ¼ 1.2 Hz, 1H), 7.13 (d,
1H), 3.59 (s, 3H), 2.10 (s, 3H). 13C NMR (100 MHz, DMSO‑d6)
d
J ¼ 1.2 Hz, 1H), 5.31 (p, J ¼ 6.2 Hz, 1H), 1.36 (d, J ¼ 6.1 Hz, 6H). 13
C
151.48, 150.04, 145.92, 136.63, 111.40, 98.62, 35.32, 14.10. HRESIMS
NMR (100 MHz, CDCl3)
109.84, 70.15, 21.74.
d 170.38, 163.80, 149.56, 142.33, 114.43,
m/z 300.0423 [MþH]þ (calcd for C11H12Cl2N5O 300.0419).
1-(2-Chloropyridin-4-yl)-3-(2,6-dichloropyridin-4-yl)urea (38).
White solid (56 %): mp 200.6e201.5 ꢀC; 1H NMR (400 MHz,
2-Chloro-6-(cyclopentyloxy)isonicotinoyl azide (42d). White solid
(79 %). 1H NMR (400 MHz, CDCl3)
d
7.35 (d, J ¼ 1.2 Hz, 1H), 7.13 (d,
11

D. Luo, Y. Zhang, S. Yang et al.
European Journal of Medicinal Chemistry 225 (2021) 113775
J ¼ 1.2 Hz, 1H), 5.41 (tt, J ¼ 6.1, 2.9 Hz, 1H), 2.06e1.91 (m, 2H),
1.85e1.73 (m, 4H), 1.71e1.58 (m, 2H). 13C NMR (100 MHz, CDCl3)
vertically transfer them to the PVDF membrane. After the block of
5 % BSA for 90 min, the membranes were incubated with the pri-
d
170.38, 164.01, 149.59, 142.23, 114.42, 109.69, 79.75, 32.62, 23.82.
1-(2-Chloro-6-methoxypyridin-4-yl)-3-(2,6-dichloropyridin-4-yl)
mary antibody of DPD (Abcam, ab180609) and b-actin (Sigma-
Aldrich, A5441) in 1 % BSA/PBS containing Tween-20 (TBST) 4 ꢀC
overnight. Afterward, all membranes were rinsed with TBST
(3 ꢂ 10 min) and cultured with the corresponding secondary
antibody at 37 ꢀC for 1 h, extensively washed with TBST for
3 ꢂ 10 min, enhanced signals on the membranes were detected by
ECL chemiluminescent detection system and quantified by the ratio
of density value.
urea (44a). White solid (59 %): mp 229.7e230.9 ꢀC; 1H NMR
(400 MHz, DMSO‑d6)
d
10.03 (s, 2H), 7.55 (s, 2H), 7.18 (d, J ¼ 1.5 Hz,
1H), 6.89 (d, J ¼ 1.5 Hz, 1H), 3.83 (s, 3H). 13C NMR (100 MHz,
DMSO‑d6)
d 164.59, 151.97, 151.32, 150.65, 150.08, 148.34, 111.74,
106.76, 96.87, 54.36. HRESIMS m/z 346.9868 [MþH]þ (calcd for
C
12H10Cl3N4O2 346.9869).
1-(2-Chloro-6-ethoxypyridin-4-yl)-3-(2,6-dichloropyridin-4-yl)
urea (44b). White solid (37 %): mp 223.5e224.1 ꢀC; 1H NMR
4.5. HPLC analysis of intracellular 5-FU and FBAL
(400 MHz, DMSO‑d6)
d
9.74 (d, J ¼ 50.9 Hz, 2H), 7.54 (s, 2H), 7.16 (d,
J ¼ 1.5 Hz, 1H), 6.82 (d, J ¼ 1.4 Hz, 1H), 4.24 (q, J ¼ 7.0 Hz, 2H), 1.30 (t,
Concentrations of intracellular 5-FU and FBAL were determined
by HPLC-UV analysis. Both 5-FU and FBAL standards (0.2, 1, 5, 10, 25,
50 mg/L) were used to calculate the corresponding standard curves.
J ¼ 7.0 Hz, 3H). 13C NMR (100 MHz, DMSO‑d6)
d 164.17, 151.70,
151.04, 150.49, 150.09, 148.32, 111.74, 106.63, 97.10, 62.57, 14.80.
HRESIMS m/z 361.0024 [MþH]þ (calcd for C13H12Cl3N4O2
361.0026).
Treated with 25 mg/L 5-FU plus 2 mM compound 40 or 2 mM JTE-013
to culture with 6 h, cell lysates were sonicated in the ice-water bath
for 30 min and vortex mixed for 30 s. Then centrifuge the cell ly-
sates at 2000 g for 10 min at 4 ꢀC, and load the supernatant into the
solid-phase styrene-divinylbenzene resin column. Evaporate the
elutes for dryness and dissolve them into the methanol. HPLC-UV
was then taken to compare and quantify the 5-FU and FBAL levels
of each group, as described previously [32].
1-(2-Chloro-6-methoxypyridin-4-yl)-3-(2,6-dichloropyridin-4-yl)
urea (44c). White solid (64 %): mp 108.4e109.4 ꢀC; 1H NMR
(400 MHz, DMSO‑d6)
d
9.81 (s, 2H), 7.55 (s, 2H), 7.16 (d, J ¼ 1.5 Hz,
1H), 6.79 (d, J ¼ 1.5 Hz, 1H), 5.12 (hept, J ¼ 6.1 Hz, 1H), 1.28 (d,
J ¼ 6.2 Hz, 6H). 13C NMR (100 MHz)
d 111.79, 40.62, 40.41, 40.20,
39.99, 39.78, 39.57, 39.37, 22.20. HRESIMS m/z 375.0180 [MþH]þ
(calcd for C14H14Cl3N4O2 375.0182).
1-(2-Chloro-6-(cyclopentyloxy)pyridin-4-yl)-3-(2,6-
dichloropyridin-4-yl)urea (44d). White solid (48 %): mp
4.6. Binding kinetics assay
117.8e118.3 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d
9.67 (d, J ¼ 63.3 Hz,
The affinity of compounds towards S1PR2 protein was deter-
mined using SPR assays on a GE BiacoreT200 (GE, USA). S1PR2
protein was immobilized on a CM5 chip (GE, USA) by amine
coupling procedures achieving a RU of 15000 and activated with
2H), 7.47 (s, 2H), 7.06 (s, 1H), 6.74 (s, 1H), 5.17 (tt, J ¼ 5.7, 2.6 Hz, 1H),
1.92e1.75 (m, 2H),1.73e1.45 (m, 6H). 13C NMR (100 MHz, DMSO‑d6)
d
163.91, 151.70, 151.05, 150.43, 150.08, 148.30, 111.71, 106.40, 97.54,
78.68, 32.69, 23.83. HRESIMS m/z 401.0338 [MþH]þ (calcd for
sterile buffer (EDC/NHS, 1/1) with a flow rate of 30 mL/min for
C
16H16Cl3N4O2 401.0339).
15 min, and the chip immobilized S1PR2 was sealed with etha-
nolamine for 8 min. The test compounds were dissolved in DMSO
and diluted with purified water, and the association of serial di-
lutions of the compounds was performed at 25 ꢀC with PBSP buffer
4.2. Cell culture
Colorectal cancer cell lines HCT116, SW620, and normal colonic
epithelial cell line NCM460 were gained from Shanghai Cell Bank,
Chinese Academy of Science (Shanghai, China) and were cultured in
Dulbecco's Modified Eagle's Medium (DMEM) or RPMI 1640 me-
dium which contained 10 % FBS, 50 IU penicillin and 50 mg/mL
streptomycin. The condition of cell culture is at 37 ꢀC in a humid
incubator with 5 % CO2. The resistant HCT116DPD cells were ob-
tained as previous study and resistant SW620/5-FU cells were
gained through continual treatment of 5-FU.
solution (1 % DMSO) with a flow rate of 30 mL/min for 60 s, whereas
dissociation of the compounds from the S1PR2 was determined for
60s. The kinetic parameters were calculated by the BiacoreT200
SPR evaluation software. All sinograms were fitted using a kinetic
fitting model provided by the BiacoreT200 SPR evaluation software,
and the association (Ka) and dissociation (Kd) rate constants were
used to calculate the equilibrium dissociation constants (KD).
4.7. 3D computational modeling and molecule docking
4.3. Antiproliferative activity in vitro
Molecular docking was initiated from the construction of the
model of S1PR2, which was from the crystal complex of S1PR1 (PDB
code: 3V2Y). Molecular docking simulations in the S1PR2 were run
using the LeDock due to its high accuracy in pose prediction and
very fast speed. Ligands were prepared with the ChemBio3D Ultra
14.0, followed by MM2 energy minimization. Protein structures
were also prepared with the LePro, which could automatically add
hydrogen atoms to proteins by explicitly considering the proton-
ation state of histidine. After this step, the orthosteric binding site
was gained from the known crystal complex of S1PR1 and its
ligand. The allosteric binding site was deduced as described in the
literature [30]. The ligands were docked to the S1PR2 using LeDock
through flexible docking mode. Top scoring function poses were
selected as representative of the simulations and were displayed
with PyMOL software.
Cell viability was routinely assessed by 3-[4,5-dimethylthiazol-
2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. Logarithmic
growth phase cells were cultured in the 96-well plates at a con-
centration of 3 ꢂ 103 cells/mL. 20
mL serum-containing media
(5 mg/mL) with MTT reagent was added in cell cultures and incu-
bate for 4 h at 37 ꢀC and analyzed by the microplate reader by the
490 nm wave. The cells were cultured with 20 mM 5-FU and com-
pounds for 48 h, and the experiment was repeated three times. The
growth inhibition rate was calculated as follows: Inhibition
rate ¼ 1- (ODDrug - ODBlank)/(ODControl - ODBlank). The values of EC50
(20 mM 5-FU plus compound) and IC50 (compound) were calculated
from the dose-response curves of the assays (Prism 7.0).
4.4. Western blotting analysis
4.8. Pharmacokinetic profile
Quantify the cell or tissue lysates by the BCA assay according to
the standard curve. Then separate the proteins by SDS-PAGE and
All animal care and experimental procedures were performed in
12

D. Luo, Y. Zhang, S. Yang et al.
European Journal of Medicinal Chemistry 225 (2021) 113775
accordance with the regulations for animal experimentation issued
by Institute Animal Care and Welfare Committee, and approved by
the Institutional Animal Care and Use Committee at Ocean Uni-
versity of China. Sprague-Dawley rats (adult male) weighing
180e220 g were obtained from Ji'nan Pengyue Laboratory Animal
Breeding Co., Ltd. (approval number: SCXK 20190003). The rats
were acclimated for seven days prior to the study on a 12 h light/
ion spray voltage, 4000 V; ion transfer tube temperature, 325 ꢀC;
vaporizer temperature, 275 ꢀC; sheath gas, nitrogen, 30 arb; aux
gas, nitrogen, 20 arb; sweep gas, nitrogen, 0.8 arb; collision gas,
argon, 2.0 mTorr.
4.11. Data analysis
12 h dark cycle at 22
2
ꢀC, 60 % relative humidity. They were
Pharmacokinetic parameters, based on plasma and whole blood
concentrations, were calculated by classical noncompartmental
analysis via WinNonlin Software (version 6.3, Pharsight Corpora-
tion, Mountain View, CA, USA).
allowed free access to water and a chow diet. Rats were given 40
(1.0 mg/kg) or JTE-013 (1.0 mg/kg) through the tail vein. The dosing
solutions were prepared by dissolving in solvent consists of 1%
DMSO, 4 % polyoxyethylene castor Oil, and 95 % distilled water.
Blood samples (350
mL) were collected into heparinized ice-bathed
4.12. Determination of the effect of compound 40 to reverse 5-FU
polythene tubes via the retrobulbar plexus bleeding under iso-
flurane anesthesia before dosing and at 2, 5, 15, and 30 min and 1, 2,
4, 6, 8, 12, and 24 h after intravenous administration. Immediately
resistance in vivo
2 ꢂ 106/mL SW620/5-FU cells were subcutaneously inoculated
into the left or right flanks of athymic nude mice. When the tumor
volume of each group reached approximately 150e200 mm3, all
mice were randomly divided into 5 groups (n ¼ 6), which were all
consecutively treated by tail vein injection for 24 days. Compound
40, JTE-013, and 5-FU were respectively administrated at the dose
of 1.08 mg/kg, 12.5 mg/kg, and 20 mg/kg. The solvent consists of
30 % PEG300, 5 % Tween 80, 2 % DMSO, and 63 % distilled water.
Tumor volumes and mice health conditions were daily recorded.
On day 24, each group of mice was sacrificed and liver, colon, and
tumoral tissues were all collected and fixed for further western
blotting and histological analysis. All animal experiments were
approved by the Animal Welfare Committee of Capital Medical
University for scientific purposes (permit no. AEEI-2016-043).
after collection, a 100 mL aliquot of blood was removed from each
sample, and the remaining heparinized whole blood was processed
to plasma by centrifugation at 1660g for 5 min. Blood and plasma
samples were stored at ꢁ40 ꢀC until analysis.
All samples (100 mL) were added to an internal standard (IS,
JTE-013 when analyzing 40, 40 when analyzing JTE-013, final
concentration 100 ng/mL, respectively) and 200 L ice-bathed
acetonitrile. All mixtures were vortexed and centrifuged at
17968 g for 10 min twice at 4 ꢀC to precipitate protein. A 5
m
m
L
aliquot of each supernatant was then injected into the LC-MS/MS
system for analysis.
4.9. In vitro incubation assays
Mice liver microsomes (MLMs) and rats liver microsomes
(RLMs) with a final protein concentration of 0.5 mg/mL were pre-
incubated with an NADPH-regenerating system (containing
4.13. Immunohistochemistry analysis
Multiple tissue samples were all fixed in 4 % formaldehyde so-
0.011 mol/L
b
-nicotinamide adenine dinucleotide phosphate,
lution and embedded in paraffin on 4 mm thick sections. After the
0.110 mol/L glucose 6-phosphate, and 10 U/mL glucose-6-
phosphate dehydrogenase) in 50 mmol/L Tris-HCl buffer (pH 7.4)
at 37 ꢀC for 5 min. Then, 40 was added at a final concentration of
blocking step, the slides were incubated with the primary antibody
of DPD (Abcam, ab180609) at 4 ꢀC overnight. Besides, all slides were
also incubated with the IgG isotype as the control. We used an
axioplan microscope to observe DAB staining and scanned the
slides by KF-PRO-OO5 slide viewer, which were then visually
inspected by two observers to avoid subjective bias. IHC scoring
was obtained based on the staining intensity and percentage of
stained cancer cells as previously described [33].
2 m
mol/L to initiate the reaction. All samples were placed in 37 ꢀC
for incubation and were quenched with two volumes of acetonitrile
with IS (final concentration 200 ng/mL) at 60 min and then vortex-
mixed and centrifuged at 18,880 g for 10 min. The supernatant was
subjected to LCꢁMS/MS analysis. The results were expressed as the
percentage of the concentration at 0 min.
Author's contribution
4.10. LC-MS/MS analysis
Shengbiao Wan and Xianjun Qu conceived and designed the
study. Dongdong Luo, Xiaochen Tian, Yan Lv designed and syn-
thesized all compounds, and the animals and cellular experiments
were performed by Yuhang Zhang, Shuang Yang, Zhikun Guo,
Xiaochun Liu, Gaitian Han, Shuai Liu and Wenyu Wang. Dongdong
Luo, Yuhang Zhang, and Shuang Yang wrote the manuscript, and all
authors edited it.
LC-MS/MS instrument (Thermo Fisher Scientific, Waltham, MA,
USA) consisted of a DIODEX UltiMate 3000 UHPLC system and TSQ
Quantiva triple quadrupole mass spectrometer with Xcalibur 2.2
software for data acquisition and processing. 40 and JTE-13 were
chromatographed using an Eclipse Plus C18 column (3.5
mm,
2.1 ꢂ 50 mm, Agilent, Santa Clara, CA, USA) at 30 ꢀC.
The mobile phase consisted of solvent A (0.1 % formic acid in
water) and solvent B (0.1 % formic acid in acetonitrile). Separation
was performed at a flow rate of 0.2 mL/min with the following
gradient elution: 0.0e1.5 min, 20 % solvent B; 1.5e1.6 min, a linear
gradient runs from 20 % to 80 % solvent B; 1.6e3.5 min, 80 % solvent
B; 3.5e3.6 min, a linear gradient runs from 80 % to 20 % solvent B;
3.6e5.0 min 20 % solvent B for re-equilibration. A H-ESI source was
used in the positive ion mode. The detection was operated in se-
lective reaction monitoring (SRM) with a dwell time of 100 ms for
each transition. The transition of 40 and JTE-013 were m/z
350.84 / 188.89 (collision energy: 18.6 V, RF lens: 189.7) and m/z
408.17 / 203.06 (collision energy: 22.3 V, RF lens: 94.5), respec-
tively. The mass spectrometric condition was optimized as follows:
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.
Acknowledgments
We are grateful for the support of the National Natural Science
Foundation of China (81973170, 91629303, 81673449, 81872884,
81973350); NSFC-Shandong Joint Fund for Marine Science Research
Centers (U1606403), and Beijing Natural Science Foundation and
13

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5-FU
CRC
S1PR2
FBAL
DPD
GPCRs
COX-2
PG
FUTP
FdUTP
FdUMP
TS
DHFU
CDHP
S1P
SPR
ER
5-Fluorouracil
colorectal cancer
sphingosine-1-phosphate receptor 2
a-fluoro-b-alanine
dihydropyrimidine dehydrogenase
G-protein-coupled receptors
cyclooxygenase-2
prostaglandin
5-fluorouridine-50-triphosphate
5-fluoro-20-deoxyuridine-50-triphosphate
5-fluoro-20-deoxyuridine-50-monophosphate
thymidylate synthase
5,6-dihydro-5-fluorouracil
gimeracil
sphingosine-1-phosphate
surface plasmon resonance
endoplasmic reticulum
TLC
HRMS
thin-layer chromatography
high-resolution mass spectra
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