Discovery of N-phenyl-(2,4-dihydroxypyrimidine-5-sulfonamido) phenylurea-based thymidylate synthase (TS) inhibitor as a novel multi-effects antitumor drugs with minimal toxicity

Article abstract of DOI:10.1038/s41419-019-1773-0

Thymidylate synthase (TS) is a hot target for tumor chemotherapy, and its inhibitors are an essential direction for anti-tumor drug research. To our knowledge, currently, there are no reported thymidylate synthase inhibitors that could inhibit cancer cell migration. Therefore, for optimal therapeutic purposes, combines our previous reports and findings, we hope to obtain a multi-effects inhibitor. This study according to the principle of flattening we designed and synthesized 18 of N-phenyl-(2,4-dihydroxypyrimidine-5-sulfonamido)phenyl urea derivatives as multi-effects inhibitors. The biological evaluation results showed that target compounds could significantly inhibit the hTS enzyme, BRaf kinase and EGFR kinase activity in vitro, and most of the compounds had excellent anti-cell viability for six cancer cell lines. Notably, the candidate compound L14e (IC₅₀ = 0.67 μM) had the superior anti-cell viability and safety to A549 and H460 cells compared with pemetrexed. Further studies had shown that L14e could cause G1/S phase arrest then induce intrinsic apoptosis. Transwell, western blot, and tube formation results proved that L14e could inhibit the activation of the EGFR signaling pathway, then ultimately achieve the purpose of inhibiting cancer cell migration and angiogenesis in cancer tissues. Furthermore, in vivo pharmacology evaluations of L14e showed significant antitumor activity in A549 cells xenografts with minimal toxicity. All of these results demonstrated that the L14e has the potential for drug discovery as a multi-effects inhibitor and provides a new reference for clinical treatment of non-small cell lung cancer.

Full text of DOI:10.1038/s41419-019-1773-0

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https://doi.org/10.1038/s41419-019-1773-0
Cell Death & Disease
A R T I C L E
O p e n A c c e s s
Discovery of N-phenyl-(2,4-
dihydroxypyrimidine-5-sulfonamido)
phenylurea-based thymidylate synthase
(TS) inhibitor as a novel multi-effects
antitumor drugs with minimal toxicity
Xin-yang Li1, Ting-jian Zhang1, Mohamed Olounfeh Kamara1, Guo-qing Lu1, Hai-li Xu1, De-pu Wang1 and
Fan-hao Meng1
Abstract
Thymidylate synthase (TS) is a hot target for tumor chemotherapy, and its inhibitors are an essential direction for anti-
tumor drug research. To our knowledge, currently, there are no reported thymidylate synthase inhibitors that could
inhibit cancer cell migration. Therefore, for optimal therapeutic purposes, combines our previous reports and findings,
we hope to obtain a multi-effects inhibitor. This study according to the principle of flattening we designed and
synthesized 18 of N-phenyl-(2,4-dihydroxypyrimidine-5-sulfonamido)phenyl urea derivatives as multi-effects inhibitors.
The biological evaluation results showed that target compounds could significantly inhibit the hTS enzyme, BRaf
kinase and EGFR kinase activity in vitro, and most of the compounds had excellent anti-cell viability for six cancer cell
lines. Notably, the candidate compound L14e (IC50 = 0.67 μM) had the superior anti-cell viability and safety to A549
and H460 cells compared with pemetrexed. Further studies had shown that L14e could cause G1/S phase arrest then
induce intrinsic apoptosis. Transwell, western blot, and tube formation results proved that L14e could inhibit the
activation of the EGFR signaling pathway, then ultimately achieve the purpose of inhibiting cancer cell migration and
angiogenesis in cancer tissues. Furthermore, in vivo pharmacology evaluations of L14e showed significant antitumor
activity in A549 cells xenografts with minimal toxicity. All of these results demonstrated that the L14e has the potential
for drug discovery as a multi-effects inhibitor and provides a new reference for clinical treatment of non-small cell lung
cancer.
Introduction
Thymidylate synthase (TS) involves in the process of
is a critical well-
Numerous chemotherapeutic drugs harm healthy tis- DNA replication and repair1–4
,
sues while treating cancer, and a single targeted drug does recognized target for anticancer agents. The regulatory
not achieve satisfactory therapeutic effects. Therefore, we role of TS may be implicated in the synthesis of key
are committed to the development of a multi-effect anti- proteins that regulate the apoptotic process5. In our
tumor drug.
previous studies, we reported a series of TS inhibitors
based on N-phenyl-(2,4-dihydroxypyrimidine-5-sulfona-
mido)benzoyl hydrazide skeleton with IC50 values around
10–20 μM. And compound 10l, the most potent one in
Correspondence: Fan-hao Meng (fhmeng@cmu.edu.cn)
1School of Pharmacy, China Medical University, 77 Puhe Road, 110122
Shenyang, China
the series, had excellent anti-proliferation ability (IC50
=
Edited by M. Campanella
© The Author(s) 2019
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1.26 μM, against A549 cells), which were superior to compounds against human thymidylate synthase (hTS),
pemetrexed in the treatment of non-small cell lung cancer BRaf kinase and EGFR kinase was evaluated, and their
(NSCLC) cell6.
inhibition of the cell viability of the six cancer cells was
Many studies have shown that NSCLC is characterized further examined in vitro. In the subsequent studies, we
by high malignancy compared with other cancers, which investigated the proliferation, migration and apoptosis of
is closely associated with high expression of EGFR sig- A549 cells and H460 cells using MTT assay, transwell
naling pathway in NSCLC7,8. Evidently, dual targeting to migration assay, and flow cytometry, respectively. Besides,
TS and EGFR signaling pathway is an extremely attractive the expression levels of apoptotic proteins and EGFR-
treatment strategy for NSCLC9. This preliminary con- related proteins were measured. In vivo pharmacological
ception drives us to screen the potential effects of our evaluation of L14e was performed in A549 tumor xeno-
above-mentioned compounds on the EGFR pathway. grafts and toxicity studies.
Surprisingly, in continued studies, we found that com-
pound 10l could inhibit A549 cells migration at high Results
doses, implying its potential role in the EGFR signaling Chemistry
pathway. The results inspired us to design TS multi-
effects inhibitors with 10l as a lead compound. Sorafenib ypyrimidine-5-sulfonamido)phenyl
The chemical synthesis of N-phenyl-(2,4-dihydrox-
urea derivatives
(Fig. 1) is an oral multi-kinase inhibitor that could act on (L13d-L13i, L14d-L14i, and L15d-L15i) was carried out
the EGFR pathway10. Interestingly, compound 10l pre- by the synthetic method illustrated in Scheme 1. Pre-
sented some structural similarity with sorafenib, which is paration of 2,4-dihydroxypyrimidine-5-sulfonylchloride
two aryl groups linking by a 3–4 atoms linker (diacylhy- (1) was done according to the reported method in our
drazine for the former and urea for the latter). Therefore, past work6.
sorafenib could be another excellent reference compound
for designing TS multi-effects inhibitors.
Compounds L3a-L3c underwent diazonium reaction in
the presence of NaNO2 and dilute hydrochloric acid to
According to the co-crystal structure of sorafenib/BRaf, obtain compound L4a-L4c at 0 °C. Then, the compound
diaryl urea played a vital role in binding with wild type L4a-L4c was subjected to Curtius rearrangement reaction
BRaf11, the lipophilic trifluoromethyl phenyl ring inserts at 80 °C to obtain compounds L5a-L5c which was also
into a hydrophobic pocket, the central phenyl ring inter- then reacted with the corresponding substituted aniline
acts with aliphatic side chains of Lys482, Leu513, and (L6d-L6i) to obtain a corresponding substituted nitro-
Thr528 and the urea group forms two hydrogen bonds phenylurea compound (L7d-L7i, L8d-L8i, and L9d-L9i).
with the protein. In this work, we retained a basic skeleton Also, the nitro group was reduced in the presence of Zn
of 10l, and fused the diaryl urea structure of sorafenib into and NH4Cl at 25 °C. Finally, the corresponding sub-
the molecule by replacing the diacylhydrazine fragment stituted aminophenyl urea compounds (L10d-L10i,
with a urea (Fig. 1). Then designed a series of N-phenyl- L11d-L11i, and L12d-L12i) were reacted with uracil
(2,4-dihydroxypyrimidine-5-sulfonamido)
derivatives as TS multi-effects inhibitors (Fig. 1).
phenylurea under pyridine as an acid binding agent at 25 °C to obtain
the target compounds (L13d-L13i, L14d-L14i, and L15d-
In summary, we firstly synthesized a total of 18 target L15i). Here, the post-treatment of the target compound
compounds. In vitro the inhibitory potency of the target involved the Hinsberg reaction and was purified according
to the method reported by Gris12. The overall synthetic
route is environmentally friendly, has no violent reaction,
and the yield is as high as 70% or more and provides
guarantee for mass production in the industry. Further-
more, when purifying the target compound, we found that
the solubility of the compound at a pH of 8.0 to 9.0 is
from 0.1 to 0.5 g/mL.
In vitro enzyme activity assay and molecular docking
In vitro human TS (hTS) enzyme activity, BRaf kinase
and EGFR kinase assays studies on 18 target compounds
synthesized, the inhibition of enzymes by target com-
pounds were summarized in Table 1. From the results, the
inhibition efficiency of compound L14e was significantly
improved after structural modification. Moreover, the
Fig. 1 Chemical structures of selected inhibitors and design of target
overall inhibition of TS by the compounds were superior
compounds
to the inhibitory effect on the BRaf kinase and were
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Scheme 1 Reagents and condition: (1) N2H4·H2O, CH3OH, 80 °C; (2) NaNO2, HCl, CH2Cl2, 0 °C; (3) CH2Cl2, 80 °C; (4) CH2Cl2, Corresponding substituted
aniline, 80 °C; (5) Zn, NH4Cl, 25 °C; (6) DMF, Pyridine, 25 °C
Table 1 IC50 values of compounds, PTX and Sorafenib
against human TS (hTS), BRaf and EGFR kinase
molecular modeling simulations of L14e in the binding
pocket of TS were performed with MOE (Molecular
Operating Environment, version 2016.08, Chemical
Computing Group Inc., Canada) software. The crystal
structure of TS in complex with PTX (PDB code: 1JUJ)
was adopted in the docking calculations. According to the
docking results (Fig. 2A), compound L14e located at the
similar position to that of the PTX crystal structure (Fig.
2A(a)), the uracil fragment formed two strong H-bonds
with Arg50 and Ala312 residues, and the phenyl group
attached to the sulfonamide group formed an H-Arene
conjugate with Gly222. Although the H-bond interactions
between Lys77 and glutamate residue of PTX were
missing, an alternate π-π stack interaction between
Phe225 and terminal benzyl ring were conducted to
remain the binding affinity (Fig. 2A(b)). This interaction
could explain the differences in TS inhibitory activities
between L14e and PTX.
a
Compounds IC50 (µM)
Compounds IC50 (µM)
hTS BRaf EGFR
hTS BRaf EGFR
L13d
L13e
L13f
2.36 2.39 4.22
4.55 3.64 4.41
6.30 3.59 4.65
9.25 >10 >10
4.57 3.53 3.99
7.39 4.91 5.48
1.91 1.62 2.39
L14h
L14i
3.52 3.61 4.28
6.98 5.45 >10
3.81 4.09 7.65
2.17 2.96 4.37
1.55 2.68 5.54
>10 >10 >10
7.16 6.38 7.10
8.3 7.23 8.67
L15d
L15e
L15f
L15g
L15h
L13g
L13h
L13i
L14d
L14e
L14f
1.06 1.09 1.92 L15i
2.12 1.71 2.23
6.47 7.41 >10
PTX
10l
2.71
–
–
L14g
Sorafenib
1.36 1.67 2.71
Furthermore, to investigate the possible interaction of
the compound with tyrosine kinase, the crystal structure
of human BRaf in complex with sorafenib (PDB code:
1UWH) was adopted in the docking calculations. L14e
overlaped with the bis- uracil ring of sorafenib and forms
–
0.57 8.12
aAll IC50 values (μM) are averages from triplicate assays
superior to the inhibition of EGFR kinase. Among them, a similar hydrogen bond. For example, the urea fragment
compound L14e had the most effective inhibitory effect formed three strong H bonds with the Glu500 and
on these enzymes. Since the active sites of tyrosine kinases Asp593 residues (Fig. 2A(c)). Since the sulfonamide
were universal (both ATP binding sites), we had mole- fragment and the urea fragment were in the ortho posi-
cular docking of hit compound L14e with TS and BRaf tion of the benzene ring, the molecular structure was
kinase to study their mode of action.
folded (not stretched), so that the urea fragment of L14e
To rationalize the structure-activity relationships cannot penetrate the active pocket like the pyridine
(SARs) observed in this study and to foresee the possible fragment of sorafenib (Fig. 2A(d)). In contrast, the pyr-
interactions of the synthesized compounds with TS, imidine fragment is on the outside of the active pocket
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Fig. 2 A Docking poses of compound L14e (a, c), PTX (b) and Sorafenib (d) within the protein binding pocket; B, C Comparison of
compound L14e with PTX and compound 10l for: inhibiting the proliferation of six cancer cells and inhibiting the proliferation of A549 and H460
cells at the similar concentrations
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and forms a hydrogen bond with His573. In addition, the the average of at least three independent experiments. As
terminal benzyl ring of L14e undergoes an alternating π-π shown in Table 2, 95% of the target compounds showed
stacking interaction with Phe594 to maintain binding in excellent ability to inhibit cell viability against A549,
the active pocket. These interactions supplied a unique OVCAR-3, SGC7901, HCT116, MDA-MB-231, and
ligand-receptor binding pattern that distinguishes sor- HepG2. Primarily, they proved to have high inhibitory
afenib and also reasonably explains the BRaf inhibitory potency against A549 cells when compared with the other
activity of L14e.
five cancer cells (Fig. 2B).
Further, based on the results of preliminary screening of
In vitro anti-proliferative activity studies and SAR study of MTT, the structure-activity relationship (SAR) of the
compounds compounds was analyzed. When X was replaced by
Taking PTX, Sorafenib and compound 10l as a refer- o-phenyl, R was replaced by 3-Cl; the anti-proliferative
ence compound, the target compounds (L13d-L13i, activity of the compound (L14e) was better than the
L14d-L14i, and L15d-L15i) were evaluated for the anti- replacement by 4-Cl (L14f), wherein 4-Cl replaced the R,
proliferative against six cancer cell lines: A549, OVCAR-3, however, the anti-proliferative activity of the compound
SGC7901, HCT-116, MDA-MB-231 and HepG2 by 3- was better than replaced by 2-Cl (L14d). This was con-
(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bro- sistent with when X is replaced by m-phenyl or p-phenyl.
mide (MTT) assay. The results were expressed as IC50 Simultaneously, when R was replaced by 3-CH3 (L14h)
values and summarized in Table 2, and the values were the anti-proliferative activity of the compound was better
Table 2 IC50 values of compounds and PTX against A549, OVCAR-3, SGC7901, HCT-116, MDA-MB-231, and HepG2
cell lines
a
Compounds
IC50 (µM)
A549
OVCAR-3
SGC7901
HCT-116
MDA-MB-231
HepG2
L13d
L13e
L13f
2.01 0.14
0.95 0.05
0.99 0.07
4.68 0.11
1.85 0.13
3.48 0.13
3.29 0.30
0.67 0.22
1.38 0.27
2.09 0.72
1.45 0.06
1.37 0.45
1.87 0.12
0.87 0.28
1.01 0.11
8.75 0.61
1.46 0.50
4.18 0.46
3.29 0.15
1.26 0.16
>10
1.73 0.44
1.05 0.42
8.04 0.32
2.68 0.38
9.56 0.94
>10
5.30 0.28
3.38 0.18
3.98 0.05
6.39 0.19
6.35 0.52
4.92 0.23
5.22 0.18
1.95 0.15
3.98 0.33
5.40 0.09
3.30 0.15
5.13 0.44
5.25 0.81
2.58 0.43
4.28 0.67
7.04 0.87
4.78 0.62
6.96 1.53
6.96 0.43
1.82 0.08
>10
4.13 0.16
2.46 0.06
5.53 0.25
4.75 0.07
5.46 1.21
7.98 0.61
4.96 0.63
1.98 0.18
4.22 0.31
4.82 0.19
5.39 0.41
5.62 0.61
2.65 0.33
3.30 0.07
7.61 0.04
6.41 1.83
6.15 0.53
9.50 1.60
4.64 0.22
2.27 0.23
>10
>10
2.48 1.46
8.89 2.42
>10
6.56 0.15
2.81 0.16
4.35 0.45
9.16 0.12
5.83 0.18
8.25 0.25
2.20 0.06
1.36 0.13
1.62 0.10
5.59 0.64
>10
L13g
L13h
L13i
>10
>10
L14d
L14e
L14f
7.27 1.23
1.68 0.31
6.52 0.50
>10
6.71 0.18
2.60 0.08
4.54 0.41
>10
L14g
L14h
L14i
7.30 0.68
>10
7.16 0.90
7.30 0.10
2.04 0.29
3.13 0.09
3.20 0.62
>10
5.72 0.21
4.97 0.36
2.29 0.29
3.07 0.29
7.66 0.78
5.97 0.21
6.96 0.59
3.73 0. 31
7.66 0.77
8.21 0.34
L15d
L15e
L15f
6.30 0.25
1.19 0.49
1.49 0.28
>10
L15g
L15h
L15i
5.98 0.60
>10
6.41 0.41
9.06 1.13
4.18 0.14
2.04 0.37
>10
PTX
7.35 0.60
2.08 0.12
>10
10lb
Sorafenib
aInhibitory effect was reported as an IC50 value (IC50 = Mean SD). From MTT assay after 24h of treatment; the values were average from at least 3 independent
experiments
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than replaced by 4-CH3 (L14i), and R was replaced by 4- L14e could arrest the cell cycle in G1/S phase then affect
CH3 the anti-proliferative activity of the compound was
the proliferation of A549 and H460 cells
better than replaced by 2-CH3 (L14g), Whether X was
TS is a crucial enzyme in the DNA synthesis stage.
replaced by m-, p-, or o-phenyl. In summary, our results When it is inhibited, first to show cell cycle arrest at G1/S
indicated that the introduction of a urea group increases phase14. According to TS assays results, compounds could
the activity of the target compound, and the introduction significantly inhibit TS activity, and MTT assays results
of an electron withdrawing group was superior to the showed that L14e has nearly five times higher anti-
electron donating group and compound L14e had the proliferative potency than PTX to A549 and H460 cells.
best anti-proliferative activity (IC50 = 0.67 0.22 μM) To verify that the compound L14e’s inhibition of cell
than the other compounds.
viability is associated with cell cycle arrest, A549 cells and
H460 cells were treated with different concentrations of
compound L14e (0, 0.5, 1.0, 1.5 μM) for 24 h, then col-
lected and stained with PI staining15. Flow cytometry
show (Fig. 3), as the concentration of the compound
L14e had more anticancer activity and selectivity index
than PTX and compound 10l and had more potential of
medicinal research significance on A549 and H460
The most common types of NSCLC are adenocarci- increases, the cells in the G0/G1 phase increase, the cells
noma, large cell carcinoma and squamous cell carci- in the S phase and the G2/M phase decrease. Eventually,
noma13. The candidate compound L14e was subjected to the proliferation of cancer cells was inhibited by the G1/S
MTT assay on representative cell lines: A549, H460 cells, phase arrest of the compound L14e.
and SK-MES-1. The result, as shown in Fig. 2B(b), L14e
These results demonstrated that compound L14e could
was significantly more inhibitory to the proliferation of arrest the cell cycle in the G1/S phase, then affect A549
A549 and H460 cells than PTX and compound 10l. All of and H460 cell proliferation. And the result is significantly
them did not inhibit SK-MES-1 cells.
different (P < 0.001).
Further comparative analysis, as shown in Fig. 2C, we
found that the inhibition rate of L14e was better than L14e could further inhibit cell proliferation by inducing
PTX at each concentration point, and moreover L14e had apoptosis in A549 and H460 cells
a higher inhibitory capacity at low concentrations com-
Most chemotherapeutic drugs eventually induce apop-
pared to compound 10l, indicating that the compound tosis in cancer cells when they inhibit cell cycle progres-
L14e has an excellent anti-proliferative potency on A549 sion, thereby achieving anti-tumor effects16. Therefore, to
cells and H460 cells than compound 10l and PTX. Also, determine whether the cell cycle arrest caused by the
L14e had a higher selectivity index (SI) than PTX, its SI compound L14e could ultimately induce apoptosis, A549
even higher than compound 10l, which was shown in cells and H460 cells were treated with different con-
Table 3. This result also indicated that L14e had high centrations of compound L14e (0, 0.5, 1.0, 1.5 μM) for
inhibition of A549 and H460 at low concentrations and 24 h, then collected and stained with Annexin V/PI
low injury to healthy cells, which made it possible to staining15. Flow cytometry showed that the apoptotic rate
become a potential agent.
of A549 cells was 4.3%, 19.3%, 23.3%, and 43.5% for each
In summary, compound L14e had stronger ability to concentration respectively (Fig. 4a). With the increase in
inhibit cell viability and higher selectivity index than both concentration, the apoptotic rate increased, of which the
PTX and 10l and had more potential of medicinal late apoptosis rates were 2.8%, 17.7%, 27.5%, and 37.1%.
research significance on NSCLC, and this study mainly However, the apoptotic rate of H460 cells was 2.9%, 6.4%,
focused on the inhibitory effect of compound L14e on 21.8%, and 24.4% for each concentration respectively.
A549 and H460 cells.
With the increase in concentration, the apoptotic rate
increased, of which the early apoptosis rates were 1.8%,
3.9%, 15.1%, and 17.3%.
It was suggested that compound L14e could induce
apoptosis of A549 cells and H460 cells, eventually inhib-
ited cell proliferation. And the result is significantly dif-
ferent (P < 0.001).
Table 3 Selectivity index of compound L14e, compound
10l and PTX against HPAEpiC, IOSE80, GES-1, HCoEpiC,
HTB-125, LO2 cell lines
Compound SI (Selectivity index)a
HPAEpiC IOSE80 GES-1 HCoEpiC HTB-125 LO2
Effect of L14e on the apoptotic protein expression of P53,
caspase-3, bcl-2/bax, and cleaved caspase-3 in A549 and
H460 cells
According to previous studies, TS was a key enzyme in
DNA replication and repair17. Flow cytometry results
showed that the compound L14e was able to cause G1/S
L14e
10l
19.79
16.56
7.71
8.79
11.90
3.56
8.58 9.40
14.25 4.09
3.25 4.91
10.39
5.26
6.70
12.58
12.57
8.23
PTX
aSI = CC50/IC50
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Fig. 3 a, b Effects of compound L14e on cell cycle were evidenced by PI staining and FACS analysis. Three independent experiments were
performed, each column represented the mean SD of triplicate determinations (*P < 0.05; **P < 0.01 and ***P < 0.001. Compound L14e -treated
group VS control). L14e was able to further inhibit cell proliferation by inducing apoptosis in A549 and H460 cells
phase arrest in cancer cells. However, when the DNA eventually, induce apoptosis21. Therefore, when P53
structure was damaged, which will activate the p53 expression increases and the ratio of bcl-2/bax decreased
pathway cause mitochondria apoptosis18,19. The P53 gene and activated caspase-3 increases, the apoptosis
is one of tumor suppressor genes, which was activated increased22,23
.
that bax proteins will be motivated to over express20. Bax
To further investigate the expression of P53 and the
is a pro-apoptotic protein in the bcl-2 gene family that is expression of bcl-2, as well as the activation of caspase-3,
homologous to bcl-2 which is anti-apoptotic protein. A549 cells and H460 cells were treated with different
When bax is over express, it can antagonize the protective concentrations of (0, 0.5, 1.0, 1.5 μM) compound L14e for
effect of bcl-2, and the expression of bcl-2 is decreased, 24 h and western blot analysis was carried out. It was
then increase the expression of cleaved caspase-3, observed that over expression of P53, up-regulation of bax
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Fig. 4 (See legend on next page.)
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Fig. 4 a Compound L14e induces apoptosis in A549 and H460. Effects of compound L14e on cell death were evidenced by Annexin V-
FITC/PI double staining and FACS analysis. Apoptotic cells were Annexin V [+] and PI [−], late apoptotic cells were Annexin V [+] and PI [+],
nectotic cells were Annexin V [−] and PI [+] and living cells were Annexin V [−] and PI [−]. Three independent experiments were performed, each
column represented the mean SD of triplicate determinations (*P < 0.05; **P < 0.01 and ***P < 0.001. Compound L14e -treated group VS control).
b Effect of compound L14e on the apoptotic protein expression of P53, caspase-3, bcl-2/bax and cleaved caspase-3 in A549 and H460. The levels of
P53, caspase-3, bcl-2/bax and cleaved caspase-3 proteins were analyzed by western blot. Relative protein expression of bcl-2/bax, cleaved-caspase-3/
caspase-3, and P53 in A549 and H460 with or without compound L14e. Each column represented the mean SD of triplicate determinations (*P <
0.05; **P < 0.01 and ***P < 0.001. Compound L14e -treated group VS control)
and down-regulation of bcl-2, as well as the activity of cells26,27. Moreover, when VEGF secretion is reduced, it
caspase-3 which in turn caused an increased in cleaved directly affects the formation of blood vessels in tumor
caspase-3 protein levels as shown in Fig. 4b. And the tissues28. Consequently, to multiple verify the inhibitory
result is significantly different (P < 0.001).
effect of the L14e on the EGFR signaling pathway, we
detect the levels of VEGF secreted by tumor cells.
Since the inhibitory effect of the compound on A549
L14e could inhibit the migration of A549 and H460
In vitro kinase assay showed that the compound L14e cells was stronger than that of H460, the A549 cells were
has a stronger potential to inhibit EGFR activity than treated with different concentrations of L14e. Taking the
Sorafenib. To verify the multi-effects of the compound supernatant of the cells to detect the change of VEGF
L14e, we investigated the effects of L14e on A549 and content by ELISA assays, and the effect of the supernatant
H460 cells migration by wound healing assays and on the formation of endothelial cells was detected. The
transwell assays.
ELISA result showed that the L14e could significantly
The results showed that the L14e significantly inhibited reduce the secretion of VEGF in A549 cells (Fig. 6b).
the migration of A549 cells and H460 cells in a dose- Furthermore, the L14e inhibited tube formation by inhi-
dependent manner (Fig. 5). The inhibition of the migra- biting the expression of phosphorylated VEGFR-2 (p-
tion ability of A549 cells was particularly prominent. VEGFR-2, Tyr1175) in a dose-dependent manner.
These all revealed that the L14e has a significant inhibi-
Summarize the results of inhibition of transwell and
tory effect on the migration of cancer cells. And the result tube formation, proved that L14e could inhibit the acti-
is significantly different (P < 0.001).
vation of the EGFR signaling pathway and ultimately
achieve the purpose of inhibiting cancer cell migration
and angiogenesis in cancer tissues. And the result is sig-
nificantly different (P < 0.001).
Effect of L14e on the activation of p-EGFR, p-AKT, and p-
ERK1/2 in A549 cells
Numerous studies proved that the migration capacity of
lung cancer is associated with high activation of EGFR
L14e displays minimal toxicity and significantly inhibited
signaling pathway24,25. To investigate the mechanism by tumor growth in vivo
which the compound L14e inhibits migration is associated
All in vitro studies had shown that the L14e has an
with signaling regulation of the EGFR signaling pathway, excellent anti-proliferative potency on A549 cells and has
cells were treated with different concentrations of L14e the potential to become an agent. Therefore, we studied
(0, 0.5, 1.0, 1.5 μM) for 24 h and western blot analysis was the toxicity and efficacy of the compound L14e in vivo.
carried out. As the results are shown in Fig. 6a, L14e To investigate the toxic effects of the compound L14e
significantly reduced the expression of phosphorylated in vivo, mice were injected with different concentrations
EGFR (p-EGFR, Tyr1068) and AKT (p-AKT, Ser473) in of compound L14e (0, 40, 80, 160 mg/kg) for 3 weeks29.
A549 and H460 cells. And down-regulate the expression The results showed that changes in the body weight of
of downstream p-ERK1/2. This effect was particularly L14e-treated mice were minimal compared with those of
pronounced in A549 cells.
control mice (Fig. 7A(a)). Simultaneously, the serum
Therefore, this result revealed that the inhibition of levels of aspartate aminotransferase (AST), alanine ami-
migration of the compound L14e was directly related to the notransferase (ALT), blood glucose (GLU) and creatinine
down-regulation of EGFR/AKT and EGFR/ERK signaling (CER) were evaluated (Fig. 7A(b)). The levels of these
pathway and the result is significantly different (P < 0.001). indicators in the serum collected from L14e-treated mice
were within the reference ranges (and no significant
L14e could reduce the secretion of VEGF in A549 cells and changes). The histopathological changes in liver, lung,
inhibit the tube formation of HUVECs
brain, and kidney from mice treated with PTX or L14e
Studies had shown that inhibition of EGFR signaling were evaluated by using HE-stained section of the tissues;
pathway directly affects the secretion of VEGF in cancer the results showed that there were no visible pathological
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Fig. 5 a, b L14e inhibited the migration of A549 and H460. The red line is the initial scratch and the black line is the cell migration edge. Cells
were seeded into the upper part of transwell chamber. The lower compartments were filled with 500 μL of RPMI 1640 supplemented with 10%
bovine serum albumin. After cells were incubated for 24 h, the migrated cells on the lower surface of the membrane were quantified by counting the
number of cells in ten random fields per membrane and expressed as cells/fields (mean SD). The scale bars indicate 100 μm. (*P < 0.05; **P < 0.01,
and ***P < 0.001. Compound L14e -treated group VS control)
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Fig. 6 a Effect of compound L14e on EGFR signaling pathway. Cells were treated in the presence of EGF (50 ng/ml) for 10 min at 37 °C prior to
lysis. (i) Relative protein expression of p-EGFR/EGFR, p-AKT/AKT, p-ERK/ERK in the A549 cells; (ii) Relative protein expression of p-EGFR/EGFR, p-AKT/
AKT, p-ERK/ERK in the H460 cells. b Effect of compound L14e on the secretion of VEGF in A549 cells. c L14e inhibited tube formation of HUVECs and
inhibited the activation of VEGFR-2. HUVECs were seeded in Matrigel-coated well with culture supernatant of A549 cells treated with different
concentrations of compound L14e (0, 0.5, 1.0, 1.5 μM) and incubated for 24 h. The tube formation of control HUVECs was normalized as 100%. Each
value represents the mean SD of three independent experiments. The scale bars indicate 100μm. (*P < 0.05; **P < 0.01 and ***P < 0.001. Compound
L14e -treated group VS control). The scale bars indicate 100 μm
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Fig. 7 Minimal toxicity of L14e in vivo and inhibitory effects of the L14e on the growth of xenograft tumors. Aa Changes in body weight
induced by treatment with L14e in mice (n = 5). Ab The level of ALT, AST, Glu and CRE in the serum. Ba Body weight changes and tumor volume
changes in PTX- or L14e-treated mice (n = 5); The Tumor Growth Inhibition value (TGI) of L14e. (*P < 0.05; **P < 0.01 and ***P < 0.001. Compound
L14e -treated group VS PTX- treated group). Bb IHC analyses for evaluating the expression of cleaved-caspase-3 and Ki67 in the tumors from PTX- or
L14e-treated nude mice
changes in each organ(the results were supplied in sup- treatment with L14e (Fig. 7Bb). Simultaneously, the
plemental materials). These results indicated that L14e expression of cleaved-caspase-3 in tumors was sig-
has minimal toxicity to mice.
nificantly increased.
Further research that evaluated the antitumor effect of
These results indicated that the L14e is the low injury
the L14e in A549 tumor xenograft model. Consistent with for healthy tissues and effective in inhibiting cancer pro-
the in vitro results, treatment with L14e significantly liferation in vivo. And the result is significantly different
suppressed the growth of xenograft tumors with negligible (P < 0.001).
changes in body weight (Fig. 7Ba). The therapeutic index
of the PTX is 63.42 at 80 mg/kg dose, and the therapeutic Discussion
index of the L14e is as high as 84.86 at the 80 mg/kg dose.
Tumor proliferation and migration are important cau-
Immunohistochemistry analysis results showed the ses of cancer progression. TS inhibitors can effectively
expression of Ki67(a key indicator to measure the extent inhibit tumor cell proliferation, but the clinically applied
of malignancy) in tumors was significantly decreased by TS inhibitors have not inhibited tumor migration.
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Therefore, the development of TS inhibitors with pleio-
tropic inhibition is crucial. In this study, we first synthe- medium
sized a total of 18 target compounds. In vitro enzyme The HPLC system consisted of e2695 Separations
HPLC analysis of stability of compound in cell culture
activity assay results showed that target compounds could Module pump coupled to a 2489 UV/Visible detector
effectively inhibit TS enzyme, BRaf kinase, and EGFR (Waters Corp., Milford, MA, USA). Chromatograms was
kinase activity. And most of the compounds had excellent achieved on a Waters BDS C18 column (200 mm ×
ability to inhibit cell viability against six cancer cell lines. 4.6 mm, 5 um). Detection was performed at a wavelength
Notably, the compound L14e had the superior anti- of 260 nm at 37 °C. The mobile phase consisted of 0.1%
proliferative ability compared with PTX. This result phosphoric acid water /methanol (50/50, v/v) at a flow
demonstrated that the structural modification of the lead rate of 1.0 mL/min. A10 µL of sample was injected. The
compound greatly enhances the inhibitory effect of the results are loaded in the supplemental file.
target compound on the proliferation of cancer cells.
Further mechanism studies showed that the compound Cell culture
L14e inhibited the proliferation of A549 and H460 cells
The human non-small cell lung cancer cell line A549
by arresting cells in G1/S phase, then activating the P53- and H460, ovarian cancer cell line OVCAR-3, gastric
mitochondrial apoptotic pathway. Simultaneously, the carcinoma cell line SGC7901, the human colon cancer cell
results of the inhibition of transwell and tube formation line HCT-116, the human liver cancer cell line hepG2 and
proved that L14e could inhibit the activation of the EGFR the human breast cancer cell line MBA-MD-231 were
signaling pathway and ultimately achieve the purpose of purchased from the American Type Culture Collection
inhibiting cancer cell migration and angiogenesis. These (ATCC, Manassas, VA, USA). The cell lines were cultured
results suggested that the compound L14e has multiple according to the suppliers’ instructions. The cells were
effects of inhibiting proliferation and migration of cancer periodically authenticated by morphologic inspection and
cells. Besides, in vivo studies had shown L14e significantly tested for mycoplasma contamination.
inhibited tumor growth in xenograft tumors, the ther-
apeutic index of the L14e was as high as 84.86 at 80 mg/ Thymidylate synthase (TS) assays
kg dose, which was much higher than the therapeutic
Recombinant human thymidylate synthase purchased
index of PTX. And it had a low injury to healthy tissues from ProSpec-Tany Company. TS was assayed spectro-
with minimal toxicity in vivo. These results indicated that photometrically at 30 °C and pH 7.4 in a mixture con-
L14e is safer in treatment than PTX.
taining 0.1 M 2-mercaptoethanol, 0.0003 M (6R,S)-
All in vivo and in vitro results demonstrated that tetrahydrofolate, 0.012 M formaldehyde, 0.02 M MgCl2,
compound L14e has multiple effects of inhibiting tumor 0.001 M dUMP, 0.04 M Tris–HCl, 30 nM hTS, and
proliferation and migration, and has excellent value for 0.00075 M NaEDTA. This was the assay described by
further research and development. At the same time, the Wahba and Friedkin30, except that the dUMP con-
discovery of multiplexed TS inhibitors has brought good centration was increased 25-fold according to the method
news for the clinical treatment of cancer, especially non- of Davisson et al.31. The reaction was initiated by the
small cell lung cancer.
addition of an amount of enzyme yielding a change in
absorbance at 340 nm of 0.016/min in the absence of
inhibitor.
Experimental section
General procedures
1H-NMR and 13C-NMR spectra were recorded on a BRaf and EGFR kinase asays
Varian NMR spectrometers operating at 600 MHz for 1H,
The ADP-Glo™ kinase assay (Promega, Madison, WI)
and 150 MHz for 13C. All chemical shifts were measured was used to screen target compounds for their inhibition
in DMSO-d6 as solvents. All chemicals were purchased effects for BRaf, EGFR kinase. The kinase assay was car-
from Sinoreagent Chemical Reagent (Beijing, China) and ried out in a 96-well plate, in a volume of 25 μL HEPES
were used as received, unless stated otherwise. Analytical (pH 7.3) solvent containing 1.6 μg/mL EGF, and 20 ng of
TLC is performed on silica gel 60 F254 plates (Qingdao EGFR kinase, or 50 ng BRaf kinase, and 10 μM ATP
Haiyang Chemical Company) and visualized by UV and (Promega, Madison, WI). The compound (L13d-L13i,
potassium permanganate staining. Flash column chro- L14d-L14i, and L15d-L15i) were dissolved in 100%
matography is performed on gel 60 (40–63 mm) (Qingdao DMSO, added to the system to give a final DMSO con-
Haiyang Chemical Company). Melting points were centration of 1.0%. Reactions in each well were started
determined with an Electro thermal melting point appa- immediately by adding ATP and kept going for half an
ratus, are uncorrected. The results are loaded in the hour under 30 °C in a constant temperature incubator
supplemental file.
(Bluepard, Shanghai, China). After the plate cooled for
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5 min at room temperature, 25 μl of ADP-Glo reagent was using a 200 μL pipette tip. Add serum-free RPMI-1640
added into each well to stop the reaction and consume the medium containing different concentrations of drugs. At
remaining ADP within 40 min. At the end, 50 μl of kinase 24 h after culturing in serum-free medium, wound closure
detection reagent was added into the well and incubated images were captured in the same field under magnifi-
for 1 h to produce a luminescence signal.
cation. Cell healing rates were calculated by the fraction of
cell coverage across the line. These experiments were
performed in triplicate and repeated three times.
MTT assays
Details are given in Supplementary Materials and
Methods. Using probability unit and weighted regression Tube formation in vitro
method to calculate the IC50 value of compounds.
Matrigel was thawed at 4 °C and 50 μL of the solution
were added to each well in a 96-well plate and formed a
gel at 37 °C for 30 min. HUVECs were suspended at 1 ×
Annexin V/propidium iodide (PI) staining
The density of the A549 cells and H460 in the loga- 104 cells in 100 μL of A549 cell supernatant with or
rithmic growth phase was adjusted to 1 × 106 cells/mL, without L14e (0, 0.5, 1.0, and 1.5 μM), and then added to
and a total of 1 mL of cell suspension and 1 mL of L14e at each well. After 24h-incubation, the degree of tube for-
different concentrations were added into each well with mation was determined by counting the number of areas
the final compound L14e concentration in each group as surrounded by tubes contained in 10 random fields, and
0, 0.5, 1.0, and 1.5 μM. Details are given in Supplementary expressed as mean SD. Using Image J for data analysis.
Materials and Methods.
Western blot analysis
Toxicity test
A549 cells, H460 cells and HUVECs were treated with
FVB mice were treated with vehicle, L14e (40, 80, and
different concentrations of compound L14e for 24 h. 160 mg/kg), or PTX (80 mg/kg) every day for 3 weeks.
Primary antibodies (100 μL/cm2): P53(10442-1-AP, Pro- Blood was collected from euthanized mice under
teintech Group),caspase-3 (19677-1-AP, Proteintech isoflurane-induced deep anesthesia by cardiac puncture.
Group), bax (50599-2-lg, Proteintech Group), bcl-2 After allowing blood coagulation at 4 °C, serum was col-
(12789-1-AP, Proteintech Group), cleaved caspase-3 lected by centrifugation at 3000 rpm for 10 min at 4 °C.
(25546-1-AP, Proteintech Group), β-actin (60008-1-lg, Analysis of the level of ALT, AST, Glu and CRE in the
Proteintech Group), EGFR (ab21074, Abcam), AKT serum was performed using a veterinary hematology
(#2920, Cell Signaling Technology), ERK1/2 (#4370, Cell analyzer (Fuji DRI-Chem 3500 s, Fujifilm, Tokyo, Japan)
Signaling Technology), VEGFR-2 (#9698, Cell Signaling according to the manufacturer’s provided protocols. The
Technology), p-EGFR (ab40815, Abcam), p-AKT (#4060, histopathological changes in liver, lung, brain, and kidney
Cell Signaling Technology), p-ERK1/2 (#4695, Cell Sig- were evaluated by using HE-stained section of the tissues
naling Technology), p-VEGFR-2 (#3770, Cell Signaling (This part of the results is in the supplementary
Technology), and GAPDH (ab181603, Abcam). The cel- document).
lular levels of proteins were determined by standard
Western blotting. Details were given in Supplementary
Materials and Methods.
Tumor xenograft model
Five-week-old immunodeficient BABL/c female nude
mice, weighing 18 to 20 g, were purchased and maintained
under specific pathogen-free conditions. They were
Cell migration and wound healing assays
Cell migration ability was measured using transwell implanted subcutaneously with 1 × 107 A549 cells. Tumor
chambers (8-μm pore size; Corning Costar, Cambridge, sizes were assessed using the two largest perpendicular
MA, USA). For the transwell assay, A549 and H460 cells axes. Tumor volume was calculated using the formula
suspended in serum-free RPMI-1640 medium containing V = (a × b2)/2, where a is length and b is width. The
different concentrations of L14e (0, 0.5, 1.0, and 1.5 μM) tumor volume was measured every 3 days. When tumor
were seeded into the upper chamber. The lower chamber volumes reached 50 mm3, the mice were randomized to
contained RPMI-1640 medium supplemented with 20% drug-treated or vehicle groups (five mice per group).
serum. After 24 h incubation, the filters were fixed in Tumor growth was monitored every 3 days using a Ver-
methanol and stained with 0.1% crystal violet. The upper nier caliper. For a total of 3 weeks, the L14e (80 mg/kg)
faces of the filters were gently abraded, and the lower and PTX (80 mg/kg) was intraperitoneal injection, after
faces with cells migrated across the filters were imaged which the mice were euthanized and the xenograft tumors
and counted under the microscope. For wound healing were dissected. All animal studies were conducted with
assays, cells were placed into 6-well plates and cultured the approval of the Laboratory Animal Welfare and
until 100% confluence. An artificial scratch was created Ethical Committee of China Medical University.
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HE staining
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This work was financially supported by the National Natural Science
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Conflict of interest
The authors declare that they have no conflict of interest.
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Received: 2 January 2019 Revised: 11 June 2019 Accepted: 26 June 2019
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