Study on anti-proliferative activity in cancer cells and preliminary structure–activity relationship of pseudo-peptide chiral thioureas

Article abstract of DOI:10.1002/bkcs.11383

In our previous studies, we have shown that thiourea compounds containing phosphate esters have potent antitumor activity and can be used as a novel strategy for the development of antitumor agents. Herein, a series of novel phosphonate thioureas 5–38 have been synthesized, which were fully characterized by ¹H NMR,¹³C NMR spectrum, elemental analysis. Three human cancer cell lines (Bcap-37, BGC-823, and PC-3) have been used to investigate these compounds’ antitumor activities. After the summarization of the structure–activity relationships, we found that the variation of R, R₁, and R₂ in these novel phosphonate thioureas contribute to the antitumor activities. All these SAR-guided efforts may lead to novel antitumor drugs in the market in the near future.

Full text of DOI:10.1002/bkcs.11383

Article
DOI: 10.1002/bkcs.11383
BULLETIN OF THE
P. Liao et al.
KOREAN CHEMICAL SOCIETY
Study on Anti-Proliferative Activity in Cancer Cells and Preliminary
Structure–Activity Relationship of Pseudo-Peptide Chiral Thioureas
†
†
Peng Liao,† Shi-Qin Hu,† Hong Zhang,‡ Liang-Bi Xu,‡ Jing-Zi Liu,†, Bin He, Shang-Gao Liao,
*
and Yong-Jun Li†
†School of Pharmacy, Guizhou Medical University; Engineering Research Center for the Development
and Application of Ethnic Medicine and TCM, Ministry of Education; Guizhou Provincial Key
Laboratory of Pharmaceutics, Guizhou Medical University, Guiyang 550004, Guizhou, China.
*E-mail: liujingz@126.com; 435096064@qq.com
‡The Affiliated Hospital of Guizhou Medical University, Guiyang 550004, Guizhou, China
Received October 24, 2017, Accepted December 28, 2017
In our previous studies, we have shown that thiourea compounds containing phosphate esters have potent
antitumor activity and can be used as a novel strategy for the development of antitumor agents. Herein, a
1
series of novel phosphonate thioureas 5–38 have been synthesized, which were fully characterized by H
NMR, 13C NMR spectrum, elemental analysis. Three human cancer cell lines (Bcap-37, BGC-823, and
PC-3) have been used to investigate these compounds’ antitumor activities. After the summarization of
the structure–activity relationships, we found that the variation of R, R1, and R2 in these novel phospho-
nate thioureas contribute to the antitumor activities. All these SAR-guided efforts may lead to novel anti-
tumor drugs in the market in the near future.
Keywords: Thiourea, Phosphonate, Pseudo-peptide, Antitumor agents, Structure–function relationship,
Human cancer cell lines
Introduction
(PD153035) in BGC823 cells, respectively.23 It is most
likely to discover more potent anticancer agents by further
varying the part of amino acid and keeping the parts of
α-aminophosphonate and thiourea unchanged. Thus, We
would like to update a series of newly synthesized phospho-
nate thioureas (Figure 1), their anticancer activities and
structure–activity relationships.
Herein, a series of novel phosphonate thioureas 5–38
have been synthesized, which were fully characterized by
1H NMR, 13C NMR spectrum, elemental analysis. Three
human cancer cell lines (Bcap-37, BGC-823, and PC-3)
have been used to investigate these compounds’ antitumor
activities. All these novel phosphonate thioureas have dem-
onstrated comparable antitumor activities compared to a
commercial anticancer drug, Adriamycin (ADM). After
the summarization of the structure–activity relationships,
we found that the variation of R, R1, and R2 in these novel
phosphonate thioureas contribute to the antitumor activities.
The thioureas belong to the urea derivatives, which demon-
strate a wide range of bioactivities including antimicrobial,1,2
antiviral,3,4 and antitumor activities.5–8 Among the urea
derivatives, the thiourea derivatives are most if not all attrac-
tive for the scientist according to their diverse anticancer
activity against various leukemias and solid tumors.9,10
Besides, the increasing evidence show that the thioureas der-
ivates are potent anticancer agents because of their potent
inhibition against DNA topoisomerase,11–13 protein tyrosine
kinase (PTKs),14–18 receptor tyrosine kinases (RTKs),19 car-
bonic anhydrase20 and sirtuins,21,22 etc. These important
findings will continuously encourage people to pursue the
novel design of thiourea compounds as anticancer drug can-
didates. Previously, people show that the thioureas conju-
gated with amino acids can generate series of newly
bioactive molecules.10 By using the combination principles
of medicinal chemistry, we also developed pseudo-peptide
thiourea derivatives containing α-aminophosphonate moiety
as potential anticancer agents,23 which generally consist of
three parts, like α-aminophosphonate, thiourea, and amino
acid. Based on our previous results, we are sure that thiourea
and α-aminophosphonate are very important pharmaco-
phores in these derivatives. Additionally, fluorinated deriva-
tives of D-IIe and L-IIk show the comparable cytotoxic
potency with Adriamycin (ADM) in PC-3 cells and 6,7-
dimethoxy-N-(3-bromophenyl)-4-aminoquiazoline
Experimental
Materials. The reagents used were analytically pure, from
Aldrich or Acros (Waltham, MA, USA), and were treated
in a conventional manner prior to use. The melting points
were determined by the X-4 type microcalorimetric analyzer
(the thermometer is not corrected). IR data were measured
by a Shimadzu IR Prestige-21 type Fourier transform infra-
red spectrometer (manufactured by Shimadzu Corporation,
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Article
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KOREAN CHEMICAL SOCIETY
Figure 1. The design of novel phosphonate thioureas.
1
Kyoto, Japan) (KBr tablet); H, 13C, 19F, 31P NMR data
Dissolved intermediate 3 (1 mmol, 1.0 equiv) into THF
(10 mL) and anhydrous DIPEA was added to the solution.
After stirring at room temperature for 5 min, O, O-dialkyl
isothiocyanate (phenyl) methyl phosphonate 4 (1 mmol, 1.0
equiv) was added slowly using a constant pressure drop-
ping funnel. After completion of the dropwise addition, the
reaction was stirred at room temperature for 1–2 h. The
crude product was purified by column chromatography
(developing solvent, dichloromethane/methanol) to give the
target compound as a yield of 82–98%. All the target com-
pounds were confirmed by IR, 1H NMR, 13C NMR and ele-
mental analysis.
were measured by JEOL-ECX400 type 400 (101, 162, 376)
MHz NMR (Chiyoda, Japan) (TMS as internal standard,
CDCl3 or DMSO-d6 as solvent); elemental analysis data
were determined by Elementar Vario type element analyzer;
optical rotation was determined by WZZ-2A automatic
polarimeter (Shanghai, China). TLC analysis was performed
using silica gel GF254.
PC-3, BGC-823, and Bcap-37 cells were adherent cells
and cultured in RPMI 1640 containing 10% (V/V) fetal
bovine serum (FBS) and high glucose DMEM medium at
37ꢀC, 5% CO2 in a saturated humidity incubator, 2 days
for a culture medium, 4–6 days pass a generation, take the
logarithmic growth phase cells as the experimental object.
Anti-proliferative activity in cancer cells. The inhibitory
rate of the compounds on cancer cells PC-3, BGC-823,
and Bcap-37 was determined by MTT colorimetric method
with DMSO as the reference. The logarithmic growth
phase cells were inoculated into 96-well culture plates at
2 × 104 cells/mL per well in 100 μL of RPMI 1640 or
DMEM medium containing 10% FBS, the rightmost col-
umn as a blank control group, pulsed serum RPMI 1640
medium without cells. The cells were incubated for 24 h
in a saturated humidity incubator at 37ꢀC and 5% CO2.
Removing the medium, added 200 μL complete medium
with different concentrations of the compounds per well,
blank control group per hole plus 200 μL complete
medium, noted that the final concentration of DMSO in
the medium cannot exceed 0.1%. According to the expo-
sure time of the experimental requirements, removed the
supernatant, then added 100 μL/well of 0.5 mg/mL MTT.
After incubation for 4 h, added 10 μL/well of 10% SDS.
After 10 h at 37ꢀC, the crystals were fully dissolved and
removed for micro oscillation 5 min, placed at room tem-
perature for 30 min, then measured the OD values at the
wavelength of A595. The results were analyzed by SPSS
software and the cell viability and inhibition rate were
calculated.
Synthesis. The Boc-protected amino acid 2 (5 mmol, 1.0
equiv) and the condensing agent benzotriazole-N, N, N0,
N0-tetramethyluronium
hexafluorophosphate
(HBTU,
5 mmol, 1.0 equiv) were added to a 100 mL dry three-
necked flask, then injection 30 mL freshly distilled dry
CH2Cl2. After stirring at room temperature for 5 min, N,
N-diisopropylethylamine (DIPEA, 10 mmol, 2.0 equiv)
and amine 1 (5.5 mmol, 1.1 equiv) were added thereto.
The reaction mixture was stirred at room temperature,
after 0.5 h, the reaction system gradually became clear
from the turbidity and the TLC was followed by the dis-
appearance of the amine. When the reaction was com-
plete, the reaction solution was dissolved in 40 mL of
CH2Cl2 then transferred to a 150 mL separatory funnel
and washed with 1 N HCl (3 × 20 mL), and the organic
phase dried over anhydrous sodium sulfate and concen-
trated to give a pale yellow oil. The yellowish oil was
dissolved with 30 mL of CH2Cl2 and transferred to a
100 mL three-necked flask, injected 75 mmol of trifluor-
oacetic acid (TFA) under ice bath and stirred for 1–2 h at
room temperature. The reaction solution was diluted with
30 mL of methylene chloride and then washed with a sat-
urated aqueous solution of sodium carbonate (3 ×
50 mL), the aqueous layer was extracted with dichloro-
methane (3 × 50 mL) and the combined organic phases
were dried over anhydrous sodium sulfate and the solvent
was removed under reduced pressure. The crude product
was purified by flash column chromatography on silica
gel (elution solvent: 4% MeOH/CH2Cl2, V/V) to afford
intermediate 3.
Results and Discussion
According to our previous report,23 the synthetic route of
novel phosphonate thioureas 5–38 was shown in
Figure 2. We started from different substituted benzyla-
mine analogues
1 and t-butyloxy carbonyl (Boc)
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Structure–Activity Relationship of Pseudo-Peptide Chiral Thioureas
KOREAN CHEMICAL SOCIETY
Table 1. Structure, yield and melting point of novel thiourea
pseudo-peptides.
protected α-amino acid 2 to synthesize the compound
3 under the conditions of adding the coupling reagent O-
benzotriazol-1-yl-N, N, N0,N0-tetramethyluronium hexa-
fluorophosphate (HBTU) and the racemization inhibitor
of 1-hydroxybenzotriazole (HOBt), followed by depro-
tection of trifluoroacetic acid (TFA). The desired phos-
phonate thioureas 5–38 were achieved by the addition of
the isothiocyanate 423 to the intermediate 3 in tetrahydro-
furan (THF) at room temperature.
Compd.
R1
R2
R
Yield (%)
Mp (ꢀC)
5
6
7
8
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
o-FBn
o-FBn
o-FBn
o-FBn
o-FBn
o-FBn
o-FBn
o-FBn
p-FBn
p-FBn
p-FBn
p-FBn
p-FBn
p-FBn
p-FBn
p-FBn
Bn
L-Bn
D-Bn
L-Bn
D-Bn
L-Bn
D-Bn
L-Bn
D-Bn
L-Bn
D-Bn
L-Bn
D-Bn
L-Bn
D-Bn
L-Bn
D-Bn
L-Bn
D-Bn
L-Bn
D-Bn
L-Bn
D-Bn
L-Bn
D-Bn
L-iBu
L-iBu
L-iBu
L-iBu
L-iBu
L-iBu
L-tertBu
L-tertBu
L-tertBu
L-tertBu
Et
Et
95
94
93
92
90
93
86
84
96
94
90
92
93
87
87
86
96
95
93
87
96
90
89
82
94
89
95
86
94
90
98
93
96
98
57–59
56–58
42–44
43–44
50–52
49–51
—
n-Pr
n-Pr
i-Pr
i-Pr
n-Bu
n-Bu
Et
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
By this general protocol,23 we have introduced different
amino acids with L- or D- configuration into the thiourea
pseudo-peptides, such as phenylalanine (R2 = benzyl, Bn),
leucine (R2 = iso-butyl, iBu), tert-leucine (R2 = tert-butyl,
tert-Bu). On the other hand, we conjugated benzyl amine (-
R1 = benzyl, Bn) or fluorinated benzyl amine (R1 = ortho-
fluorinated benzyl, o-FBn, or para-fluorinated benzyl,
p-FBn) to C-terminal of these amino acids. Besides, we
have also investigated the influence of different α-amino-
phosphonates, like ethyl (R = Et), n-propyl (R = n-Pr), iso-
propyl (R = i-Pr), and n-butyl (R = n-Bu) α-aminopho-
sphonates on antitumor activities. Eventually, we obtained
34 novel thiourea pseudo-peptides in total with excellent
yields. The structure, yield and melting point of all the syn-
thesized compounds are given in Table 1. All the synthe-
—
42–43
41–42
—
Et
n-Pr
n-Pr
i-Pr
i-Pr
n-Bu
n-Bu
Et
—
39–40
38–40
—
—
55–57
53–55
44–45
43–45
47–48
46–49
—
Et
n-Pr
n-Pr
i-Pr
i-Pr
n-Bu
n-Bu
Et
n-Pr
i-Pr
n-Bu
Et
i-Pr
Et
n-Pr
i-Pr
n-Bu
1
sized compounds were tested by H NMR, 13C NMR, IR,
and elemental analysis (see Appendix S1, Supporting
information).
—
Three human cancer cell lines of PC-3, BGC-823, and
Bcap-37, which are prostate cancer, stomach cancer, and
breast cancer cells, respectively, were used to test the anti-
tumor activity of phosphonate thiourea 5–38 with the final
concentration of 10 μM by comparing with the listed anti-
cancer drugs, adriamycin (ADM). As shown in Table 2,
was measured by the MTT method, phosphonates thiourea
5–38 showed good anti-proliferative activities for PC-3,
BGC-823, and Bcap-3 with a range of 12.6–73.4%,
11.1–89.1%, and 13.6–77.2%, respectively. Compounds 6,
14, and 25 demonstrate moderate to potent inhibitory
activities (from 51.2 to 89.1%) among tested three human
cancer cell lines. Compounds 6, 14, 23, 25, and 34
showed more than 50% inhibition against PC-3 cells,
while compounds 6, 14, and 25 showed more than 50%
inhibition against both of BGC-823 and Bcap-37 cells.
The rest compounds only showed lower inhibition against
these human cancer cell lines. Notably, compound 25
exhibited the comparable inhibitory potency to that of
43–45
—
Bn
Bn
Bn
p-FBn
p-FBn
Bn
Bn
Bn
51–53
—
31–32
43–44
157–159
171–172
176–177
122–124
Bn
ADM against stomach cancer cell line, BGC-823 (89.1
vs. 93.8% at 10 μM).
Encouraged by this, we picked up compounds 6, 14, 23,
and 25 further testing its effectiveness at various concentra-
tions. As shown in Table 3, the inhibition of these four
compounds against PC-3 cells was very potent. The IC50
value of compounds 6, 14, 23, and 25 were 11.3, 6.1, 15.6,
Figure 2. The synthetic route of novel phosphonate thioureas.
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Table 2. Anti-proliferation activities of phosphonate thioureas
5–38 against three human cancer cell lines.a
Table 3. IC50 values of selected compounds in PC-3 and
BGC-823.a
Inhibition ratio (%)
Compound
IC50 (μM)b
Compd.b
PC-3c
BGC-823d
Bcap-37e
6
14
23
25
11.3 ꢁ 0.9c
6.1 ꢁ 0.1c
15.6 ꢁ 1.9c
13.0 ꢁ 2.1c
3.2 ꢁ 0.8c
13.7 ꢁ 1.4c
4.8 ꢁ 0.0.4e
6.9 ꢁ 1.1e
5
6
7
8
20.1 ꢁ 5.1
55.9 ꢁ 7.3
33.5 ꢁ 3.3
30.8 ꢁ 5.2
50.1 ꢁ 8.3
30.5 ꢁ 4.9
22.8 ꢁ 2.4
21.7 ꢁ 2.3
19.3 ꢁ 3.4
73.4 ꢁ 1.7
18.1 ꢁ 5.1
30.6 ꢁ 2.3
26.8 ꢁ 2.5
14.3 ꢁ 1.1
22.5 ꢁ 2.3
18.0 ꢁ 4.4
27.9 ꢁ 2.9
32.4 ꢁ 5.3
56.7 ꢁ 6.7
30.6 ꢁ 7.3
58.6 ꢁ 8.1
34.8 ꢁ 2.3
19.7 ꢁ 3.7
33.6 ꢁ 0.3
31.8 ꢁ 2.2
16.2 ꢁ 0.6
34.4 ꢁ 1.3
18.3 ꢁ 0.7
49.4 ꢁ 4.2
51.2 ꢁ 3.1
26.9 ꢁ 1.3
12.6 ꢁ 3.4
29.1 ꢁ 2.0
15.7 ꢁ 1.4
90.5 ꢁ 10.2
18.3 ꢁ 8.1
67.8 ꢁ 10.0
35.7 ꢁ 4.5
24.4 ꢁ 11.5
27.8 ꢁ 4.0
29.0 ꢁ 10.9
21.1 ꢁ 11.6
22.7 ꢁ 12.9
30.4 ꢁ 3.8
54.6 ꢁ 3.7
11.1 ꢁ 26.3
11.8 ꢁ 24.3
47.9 ꢁ 5.0
51.6 ꢁ 5.0
31.2 ꢁ 21.7
30.1 ꢁ 21.7
22.9 ꢁ 7.4
44.5 ꢁ 12.6
21.0 ꢁ 6.3
53.9 ꢁ 0.1
89.1 ꢁ 1.2
20.8 ꢁ 14.7
26.1 ꢁ 9.5
33.2 ꢁ 4.2
—
16.9 ꢁ 10.9
52.5 ꢁ 4.3
35.4 ꢁ 4.7
49.1 ꢁ 7.2
34.5 ꢁ 6.5
46.6 ꢁ 8.9
24.4 ꢁ 4.1
22.9 ꢁ 8.4
25.9 ꢁ 4.7
77.2 ꢁ 3.9
44.7 ꢁ 9.2
17.2 ꢁ 4.8
50.4 ꢁ 18.1
27.8 ꢁ 8.1
32.1 ꢁ 7.8
34.0 ꢁ 7.4
26.1 ꢁ 11.7
35.0 ꢁ 8.6
24.0 ꢁ 16.4
13.6 ꢁ 8.4
53.1 ꢁ 9.2
27.0 ꢁ 5.8
48.4 ꢁ 6.7
35.8 ꢁ 7.3
—
ADM(adriamycin)d
PD153035d
9
25
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
ADM
PD153035d
a
The data represented the mean of three experiments in triplicate and
were expressed as means ꢁSD.
The IC50 value was defined as the concentration at which 50%
survival of cells was observed.
b
c
d
e
For PC-3 cells.
Control drug.
For BGC-823 cells.
value of compound 25 against BGC-823 cells was 4.8 μM
compared to the 6.9 μM of PD153035.
Conclusion
According to the data from Table 2, we summarized the
structure–activity relationship (SAR) of this series of novel
thiourea pseudo-peptides shown in Figure 3. In general, the
configuration of amino acid in the desired compounds was
not obviously effective on the antitumor activity. However,
D-configurations in certain cases show better inhibition
(more than three folds) than that of L-configurations in
these three cancer cell lines (6 vs. 5 or 14 vs. 23 in
Table 2). The desired compounds when D- or L- phenylala-
nine conjugated demonstrated better cellular inhibitions
compared with those conjugated with L-leucine or 4-methyl
L-leucine. In general, in the D-configuration, when
R1 = Bn or o-FBn, the compounds containing ethyl group
of phosphate ester presented better anti-proliferation activi-
ties in tested cell lines than those containing bulky group
like propyl, iso-propyl or butyl (6 vs. 8, 10, and 12 or 14
vs. 16, 18, and 20 in Table 2). However, in the L-configura-
tion, when R1 = o-FBn or p-FBn, the compounds contain-
ing iso-propyl group showed better anti-proliferation
—
—
—
—
—
—
61.5 ꢁ 0.6
47.9 ꢁ 4.1
—
—
—
—
—
—
—
—
93.8 ꢁ 2.1
92.7 ꢁ 4.3
a
MTT assay.
b
10 μM for all tested compounds.
Prostate cancer cells.
Stomach cancer cells.
Breast cancer cells.
c
d
e
13.0 μM, respectively. They all presented the approximate
inhibition with that of PD153035 against PC-3 cells
(IC50 = 13.7 μM). The best one was compound 14, which
showed the highest inhibitory activity (IC50 = 6.1 μM)
close to that of ADM against PC-3 cells (IC50 = 3.2 μM).
Additionally, the inhibition of compound 25 was a slightly
better compared to that of commercial anticancer drug,
PD153035 against both PC-3 and BGC-823 cells. The IC50
value of compound 25 against PC-3 cells was 13.0 μM
compared to the 13.7 μM of PD153035, while the IC50
Figure 3. The structure–activity relationship (SAR) of these phos-
phonate novel thioureas.
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Structure–Activity Relationship of Pseudo-Peptide Chiral Thioureas
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activities in tested cell lines than other compounds contain-
ing ethyl, propyl, and butyl (17 vs. 13, 15, and 19 or 25
vs. 21, 23, and 27 in Table 2); When R1 = Bn, the ten-
dency is observed only in the case of PC-3 cells (9 vs. 5, 7,
and 11 in Table 2). In most cases, the compounds
conjugated with fluorinated benzyl amine (R1 = o-FBn and
p-FBn) demonstrated the comparable anti-proliferation
activities to that of those compounds with non-fluorinated
benzyl (R1 = Bn). But compound 14 (R1 = o-FBn) has the
best inhibitory activity on PC-3 (73.4% inhibition at
10 μM, Table 2) and Bcap-37 cells (77.2% inhibition at
10 μM, Table 2). Compound 25 (R1 = p-FBn) show the
best anti-BGC-823 cells activity (89.1% inhibition at
10 μM, Table 2).
In conclusion, the configuration of α-amino acids, the
types of amino acids and the alkyl structures of phospho-
nates in these compounds all have an influence on the anti-
proliferative activity in cancer cells. The presence of
fluorine-containing groups in the amino acid amide struc-
ture may enhance the anti-cancer activity of these
compounds.
Acknowledgments. We are grateful for financial supports
from National Natural Science Foundation of China
(No. 81160385, 21662010); Science and Technology
Department of Guizhou Province (QKHSYZ[2012]3103);
The Governor Special Foundation of Outstanding Talents
of Science and Technology Education in Guizhou Province
(QSZHZ[2012]89) and Guiyang Medical College Fund
(YJ2014–17); The Innovation Team of Natural Science
Foundation of Department of Education of Guizhou Prov-
ince (No. QJHRCTDZ[2015]57); The Biding Project of
Natural Science Foundation of Department of Education of
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Author Contributions. J.L. conceived and designed the
experiments; P.L. and S.H. performed the experiments; L.-
X. and H.Z. analyzed the data; J.L. and B.H. wrote the
paper; Y.L and S.L review.
Conflict of Interest. The authors declare no conflict of
interest.
Supporting Information. Additional supporting informa-
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Bull. Korean Chem. Soc. 2018
© 2018 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.bkcs.wiley-vch.de
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