Synthesis and antitumor activities of chiral dipeptide thioureas containing an alpha-aminophosphonate moiety

Article abstract of DOI:10.3390/molecules22020238

Thiourea derivatives demonstrate potent cytotoxic activity against various leukemias and many tumor cell lines. In our previous study, the combination of thiourea and phosphonate has been proven as an effective strategy for developing antitumor agents. Herein, we synthesized and evaluated a series of novel chiral dipeptide thioureas containing an α-aminophosphonate moiety as antitumor agents. Finally, we developed novel dipeptide thioureas 11d and 11f that showed comparable inhibition with that of Cisplatin against BGC-823 and A-549 cells, respectively.

Full text of DOI:10.3390/molecules22020238

molecules
Article
Synthesis and Antitumor Activities of
Chiral Dipeptide Thioureas Containing
an Alpha-Aminophosphonate Moiety
Jingzi Liu 1,2,3,4,5,*, Peng Liao 1,2,3,4, Junfeng Hu 5, Hong Zhu 1,2,3,4, Yonglin Wang 1,2,3,4
,
Yongjun Li 1,2,3,4, Yan Li 6,* and Bin He 1,2,3,4,
*
1
Engineering Research Center for the Development and Application of Ethnic Medicine and TCM
(Ministry of Education), Guizhou Medical University, Guiyang 550004, Guizhou, China;
m17785598945@163.com (P.L.); Zhhgmu@21cn.com (H.Z.); yonglinwang@gmc.edu.cn (Y.W.);
m13765113985@163.com (Y.L.)
Guizhou Provincial Key Laboratory of Pharmaceutics, Guizhou Medical University, Guiyang 550004,
Guizhou, China
School of Pharmacy, Guizhou Medical University, Guiyang 550004, Guizhou, China
National Engineering Research Center of Miao’s Medicines, Guizhou Medical University, Guiyang 550004,
Guizhou, China
School of Pharmacy, Zunyi Medical College, Zunyi 563000, Guizhou, China; hjfgmu@21cn.com
School of Basic Medicine, Guizhou Medical University, Guiyang 550004, Guizhou, China
Correspondence: liujingz@126.com (J.L.); yanli@gmc.edu.cn (Y.L.); binhe@gmc.edu.cn (B.H.);
Tel.: +86-851-8691-0028 (B.H.)
2
3
4
5
6
*
Academic Editor: Jean Jacques Vanden Eynde
Received: 18 December 2016; Accepted: 30 January 2017; Published: 16 February 2017
Abstract: Thiourea derivatives demonstrate potent cytotoxic activity against various leukemias and
many tumor cell lines. In our previous study, the combination of thiourea and phosphonate has
been proven as an effective strategy for developing antitumor agents. Herein, we synthesized and
evaluated a series of novel chiral dipeptide thioureas containing an α-aminophosphonate moiety
as antitumor agents. Finally, we developed novel dipeptide thioureas 11d and 11f that showed
comparable inhibition with that of Cisplatin against BGC-823 and A-549 cells, respectively.
Keywords: dipeptide; thiourea; aminophosphonate; synthesis; antitumor agents
1. Introduction
It is well-known that thiourea derivatives demonstrate potent cytotoxic activity against various
leukemias and many tumor cell lines [
inhibitory activity against protein tyrosine kinases (PTKs) [
DNA topoisomerase [ 10], sirtuins [11], carbonic anhydrase [12], vanilloid receptor [13], a family
1,
2], and it is believed that this is due to their good
3–6
], receptor tyrosine kinases (RTKs) [ ],
7
8
–
of G protein-coupled receptors (sst1-5) [14], etc. In the past few decades, many thiourea derivatives
have been reported on regarding their synthesis, evaluation as anticancer agents, structure-activity
relationships, and mechanism of action [15
anticancer activities when thioureas are conjugated with amino acids [
amino acid and -aminophosphonate to develop novel thiourea derivatives, called pseudo-peptide
thioureas [19], which turned out to be another type of potential anticancer agents. Other functionalized
thioureas were structurally incorporated with other scaffolds [ ], such as benzensulfonamide [20],
–
18]. For example, the derivatives show enhanced
2]. We also combined
α
2
chlorocolchicine [21], podophyllotoxin [22], pyrazole [23], benzimidazole [24], benzothiazole [25], etc.
On the other hand, peptide conjugated derivatives have attracted much attention in recent years
because of their broad biological activity, biocompatibility, and the possibility of introducing structural
Molecules 2017, 22, 238; doi:10.3390/molecules22020238
www.mdpi.com/journal/molecules

Molecules 2017, 22, 238
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diversity in them, which became an effective strategy to obtain novel chemical entities when small
bioactive motifs are conjugated with peptides [26]. Over the years, the conjugated molecules, as novel
chemotherapeutics, have made significant progress due to the accessibility of combining the structural
features of two or more small bioactive motifs to achieve novel molecules with enhanced bioactivities.
Based on our previous study [19], we would like to further introduce one more amino acid
to the pseudo-peptide thiourea containing an
the synthesis of a series of novel chiral dipeptide thioureas containing
α
-aminophosphonate moiety. Herein, we describe
-aminophosphonate
α
moieties. The structures of the synthesized compounds were characterized by IR (Infrared spectrum),
1H-NMR (Nuclear Magnetic Resonance), 13C-NMR, 31P-NMR, 19F-NMR, and elemental analysis
studies. The anticancer potency of all of these novel thioureas was examined in the human gastric
cancer cell line BGC-823 and the human non-small cell lung cancer cell line A-549. To the best of
our knowledge, this is the first report on the synthesis and antitumor activity of these dipeptide
thioureas containing α-aminophosphonate moieties. These novel thioureas may provide promising
lead compounds for treating human cancer.
2. Results
According to our pseudo-peptide thioureas containing α-aminophosphonate moieties described
previously [19], we planned to introduce glycine or L-proline to obtain the novel thioureas 10 and 11
(Figure 1), respectively. The synthesis is outlined in Figure 1. Substituted benzylamine was
coupled with commercially available N-Boc protected glycine or N-Boc protected L-proline in
the presence of O-benzotriazol-1-yl-N,N,N’,N’-tetramethyluronium hexafluorophosphate (HBTU).
The Boc groups of the resulting compounds and were removed by trifluoroacetic acid (TFA)
in CH2Cl2. Similarly, the dipeptides and were achieved by coupling with amino acid after
the deprotection. The key intermediate, O,O’-dialkylisothiocyanato (phenyl) methylphosphonate
was prepared as described previously [19]. Finally, these novel thioureas 10 and 11 were obtained by
nucleophilic addition of -phosphonate isothiocyanate to the intermediate . The structures, yields,
1
2
3
4
5
7
8
6
9
,
α
9
and melting points of all these novel thioureas 10 and 11 are displayed in Table 1. All these novel
thioureas were obtained in modest to excellent yields (from 53.9% to 98.4%).
Table 1. Structure, yield, and melting point of novel thioureas 10 and 11.
◦
Compound
X
R1
R2
Yield (%)
m.p. ( C)
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
11a
11b
11c
11d
11e
11f
H
H
H
4-F
4-F
4-F
H
H
H
4-F
4-F
4-F
H
L-Ph
L-Ph
L-Ph
L-Ph
L-Ph
L-Ph
L-Bn
L-Bn
L-Bn
L-Bn
L-Bn
L-Bn
L-Bn
L-Bn
L-Bn
L-Bn
L-Bn
L-Bn
Et
n-Pr
i-Pr
Et
n-Pr
i-Pr
Et
n-Pr
i-Pr
Et
n-Pr
i-Pr
Et
91.6
81.5
78.6
93.3
75.5
77.8
66.8
71.4
70.4
98.7
94.3
95.0
92.6
90.2
81.8
93.5
53.9
82.4
179–180
155–157
203–204
74–75
133–134
176–177
167–168
148–149
72–73
75–76
95–96
62–63
103–104
117–118
89–90
97–98
107–108
83–84
H
H
4-F
4-F
4-F
n-Pr
i-Pr
Et
n-Pr
i-Pr

Molecules 2017, 22, 238
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Figure 1. Synthetic route for novel thioureas 10 and 11.
With these novel thioureas in hands, we then selected two human cancer cell lines, BGC-823 and
A-549, which are derived from stomach cancer and non-small cell lung cancer (NSCLC), respectively,
and they were used to evaluate the anti-proliferation activities of thioureas 10 and 11 with a series
concentration of 500 to 3.9 µM by comparison with a commercial anticancer drug, Cisplatin. As shown
in Table 2, by the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay, thioureas 10 and 11 both demonstrated the capability of inhibiting the proliferation of BGC-823
and A549 cells with IC50 values in the range of 20.9 to 103.6
µM and 19.2 to 112.5 µM, respectively.
Overall, thiourea 11 with the incorporation of L-proline demonstrated better anti-cancer activities in
these two cancer cell lines than that of thiourea 10 with the incorporation of glycine (10g
–
l
vs. 11a
–
f
,
0
Table 2). Among the O,O -Dialkylphosphonates derived from the ethyl, n-propyl, or iso-propyl groups
of these compounds, different substituted phosphonate esters demonstrated no significant effect on
the antitumor activities (Table 2). However, in some cases where R2 = n-propyl, the IC50 values were
higher with respect to the other cases (for example: 10b vs. 10a,c; 10g vs. 10h,i for BGC-823, Table 2),
which suggested that the n-propyl group may have a negative effect. Phenylalanine containing thiourea
10 or 11 (R1 = L-Ph) demonstrated better antitumor activity than that of phenylglycine containing
thiourea 10 or 11 (R1 = L-Bn) (10g
thioureas elicited superior antitumor activity to non-substituted benzyl thioureas (11d
10d vs. 10a 10j vs. 10g ; except for 11d vs. 11a for A549, Table 2). Notably, thiourea 11d showed
–
l
vs. 10a
–
f, Table 2). Additionally, the para-fluorinated benzyl
–
f
vs. 11a–c;
–
f
–
c;
–
l
–I
comparable inhibition with that of Cisplatin against BGC-823 cells (IC50 = 20.9 µM vs. 15.1 µM),
while thiourea 11f showed the highest inhibitory activity, close to that of Cisplatin, against A-549 cells
(IC50 = 19.2 µM vs. 17.6 µM).
Table 2. IC50 values of thioureas 10 and 11 for BGC-823 and A-549 cells.
−1
IC50/(µmol·L
BGC-823
)
Compound
R1
R2
X
A-549
10a
10b
10c
10d
10e
10f
10g
10h
10i
H
H
H
4-F
4-F
4-F
H
H
H
4-F
L-Ph
L-Ph
L-Ph
L-Ph
L-Ph
L-Ph
L-Bn
L-Bn
L-Bn
L-Bn
Et
n-Pr
i-Pr
Et
n-Pr
i-Pr
Et
n-Pr
i-Pr
Et
54.8 ± 3.2
87.3 ± 7.1
61.5 ± 2.2
51.9 ± 3.5
58.1 ± 2.0
47.2 ± 4.3
53.5 ± 1.8
103.6 ± 8.9
42.7 ± 2.1
38.3 ± 3.3
63.2 ± 2.1
112.5 ± 7.9
74.3 ± 6.6
51.5 ± 3.8
56.2 ± 4.2
43.4 ± 3.1
67.5 ± 3.4
58.3 ± 4.1
46.1 ± 3.3
35.2 ± 2.5
10j

Molecules 2017, 22, 238
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Table 2. Cont.
−1
IC50/(µmol·L
BGC-823
)
Compound
R1
R2
X
A-549
10k
10l
11a
11b
11c
11d
11e
11f
4-F
4-F
H
H
H
4-F
4-F
4-F
L-Bn
L-Bn
L-Bn
L-Bn
L-Bn
L-Bn
L-Bn
L-Bn
n-Pr
i-Pr
Et
n-Pr
i-Pr
Et
44.1 ± 3.1
24.8 ± 2.6
31.4 ± 2.0
45.2 ± 1.6
39.7 ± 3.3
20.9 ± 2.8
37.5 ± 3.7
25.6 ± 4.1
15.1 ± 2.3
49.5 ± 5.1
41.7 ± 3.0
23.7 ± 2.2
34.6 ± 2.1
40.8 ± 3.9
30.3 ± 1.8
29.5 ± 3.4
19.2 ± 2.3
17.6 ± 3.1
n-Pr
i-Pr
Cisplatin
3. Materials and Methods
3.1. Materials
Reagents were obtained from Aldrich or Acros (Waltham, MA, USA) in the highest purity available
and were used as supplied. The melting points of the products were determined on a XT-4 binocular
microscope (Beijing Tech Instrument Co., Beijing, China) and were not corrected. The IR spectra were
recorded on a Bruker VECTOR22 spectrometer in KBr disks (Billerica, MA, USA). 1H- and 13C-NMR
spectra were recorded on a JEOL-300 NMR spectrometer (Otemachi, Chiyoda, Tokyo, Japan) at room
temperature using tetramethylsilane (TMS) as an internal standard for 1H- and 13C-NMR while using
H3PO4 (85%) and CFCl3 as an external reference for 31P- and 19F-NMR, respectively. The reported
13C chemical shifts are those present in the spectrum, and the 31P-13C coupling (or 19F-13C coupling)
has not been considered. Elemental analysis was performed on an Elementar Vario-III CHN analyzer
(Elementar, Frankfurt, Germany). UV spectra were recorded on a VARIAN Cary-50 spectrometer
(Santa Clara, CA, USA) using a cell path length of 1 cm. BIO-RAD, Model 680 Microplate Reader
(Hercules, CA, USA) was used to record those absorptions after MTT assay. The reagents were all of
analytical grade or were chemically pure. Analytical thin layer chromatography (TLC) was performed
on silica gel GF254.
3.2. Synthesis
A solution of O,O0-dialkylisothiocyanato (phenyl) methylphosphonate
9 [19] (1 mmol) in
tetrahydrofuran (10 mL) was stirred, followed by drop wise addition of the intermediate
(1.1 mmol). The reaction mixture was stirred for 1 h at 23 C, the solvent was removed by evaporation,
and the crude product was purified by flash chromatography on silica using a mixture of petroleum
7 or 8
◦
ether and ethyl acetate as the eluent to yield the compounds 10a
–l and 11a–f in 53.9%–98.4% yields.
All final compounds were confirmed by 1H-NMR, 13C-NMR, IR, and elemental analysis.
◦
20
10a: C29H35N4O5PS, white solid, yield 91.6%, m.p. 179–180 C, [
α
]
= +57 (c = 0.12, CHCl3) IR
ν
:
;
D
3291, 3084, 3065, 3032, 2982, 2928, 2908, 1652, 1535, 1497, 1453, 1352, 1336, 1226, 1050, 1026 cm−1
1H-NMR (300 MHz, CDCl3)
δ
: 8.61 (s, 1H, NH), 8.02 (s, 1H, NH), 7.93 (s, 1H, NH), 7.45–7.09 (m, 15H,
ArH), 6.51 (dd, J = 22.3, 9.2 Hz, 1H, NH), 6.38 (dd, J = 22.2, 9.8 Hz, 1H, NCH-P), 4.93 (s, 1H, NCH),
4.18 (dd, J = 26.5, 4.8Hz, 4H, 2OCH2), 4.03–3.83 (m, 2H, NCH2-Ar), 3.70–3.52 (m, 2H, NCH2), 1.09 (t,
J = 7.0 Hz, 3H, CH3), 1.03–0.92 (m, 3H, CH3) ; 13C-NMR (75 MHz, CDCl3) δ: 183.7, 171.5, 168.5, 137.9,
137.4, 135.1, 128.9, 128.5, 128.4, 127.6, 127.3, 63.4, 62.6, 61.9, 54.0, 43.4, 43.1, 16.1; 31P-NMR
δ: 21.2 ppm;
Anal. Calcd. (Analysis Calculated) for C29H35N4O5PS: C 60.17, H 5.72, N 9.99; Found: C 59.78, H 6.05,
N 9.62.
◦
20
10b: C31H39N4O5PS, white solid, yield 81.5%, m.p. 155–157 C, [
ν
α
]
= +44 (c = 0.10, CHCl3) IR
D
: 3295, 3083, 3064, 3031, 2969, 2935, 1654, 1541, 1497, 1454, 1355, 1227, 1204, 1058, 1012 cm−1
;

Molecules 2017, 22, 238
5 of 10
1H-NMR (300 MHz, CDCl3)
δ: 8.10 (d, J = 96.3 Hz, 2H, 2NH), 7.48–6.94 (m, 15H, ArH), 6.33 (s, 1H,
NCH-P), 6.21 (s, 1H, NCH), 4.06 (dt, J = 82.5, 52.6 Hz, 2H, NCH2-Ar), 3.52 (s, 2H, NCH2), 3.31 (s, 2H,
OCH2), 2.93 (s, 2H, OCH2), 1.57 (s, 4H, 2CH2), 1.40–1.35 (m, 2H, 2NH), 0.87 (d, J = 7.4 Hz, 6H, 2CH3);
13C-NMR (75 MHz, CDCl3)
δ: 183.5, 171.5, 169.0, 138.0, 135.2, 129.0, 127.5, 127.1, 69.4, 69.0, 62.6, 53.8,
43.2, 42.1, 23.8, 23.6, 10.1, 9.9; 31P-NMR δ: 21.3 ppm; Anal. Calcd. for C31H39N4O5PS: C 60.60, H 6.61,
N 8.79; Found: C 60.97, H 6.44, N 9.17.
◦
10c: C31H39N4O5PS, white solid, yield 78.6%, m.p. 203–204 C, [
3269, 3089, 3031, 2980, 2932, 1678, 1639, 1537, 1512, 1453, 1224, 1002 cm−1; 1H-NMR (300 MHz, CDCl3)
: 8.26 (s, 2H, 2NH), 8.03 (s, 2H, 2NH), 7.58–7.11 (m, 15H), 6.32 (d, J = 9.0 Hz, 1H, NCH-P), 4.73 (s, 1H,
NCH), 4.49 (s, 2H, 2OCH), 4.31 (t, J = 30.3Hz, 2H, NCH2-Ar), 4.11 (d, J = 5.7 Hz, 2H, NCH2), 1.21–1.00
α] ν: 3323,
20 = +69 (c = 0.14, CHCl3) IR
D
δ
(m, 12H, 4CH3); 13C-NMR (75 MHz, CDCl3)
δ: 171.4, 168.5, 138.0, 135.5, 129.0, 127.8, 127.3, 127.0, 72.7,
72.4, 61.8, 53.7, 43.4, 43.1, 24.4, 23.9, 23.6, 23.2; 31P-NMR
C 61.31, H 6.18, N 9.43; Found: C 60.97, H 6.44, N 9.17.
δ: 19.4 ppm; Anal. Calcd. for C31H39N4O5PS:
◦
20
10d: C29H34FN4O5PS, white solid, yield 93.3%, m.p. 74–75 C, [
α
]
= +98 (c = 0.11, CHCl3) IR ν: 3294,
D
3065, 3033, 2987, 2931, 2910, 1673, 1539, 1510, 1221, 1204, 1049, 1024, 977 cm−1; 1H-NMR (300 MHz,
CDCl3) δ: 8.68 (s, 1H, NH), 8.45 (s, 1H, NH), 7.48–7.06 (m, 12H, ArH), 7.00 (s, 1H, ArH), 6.88 (s, 1H,
ArH), 6.79 (s, 1H, NCH-P), 5.26 (s, 1H, NCH), 4.33 (m, 2H, NCH2-Ar), 3.78 (m, 2H, NCH2), 3.59 (d,
J = 6.8 Hz, 2H, OCH2), 3.39 (s, 2H, OCH2), 2.35 (s, 1H, NH), 2.01 (s, 1H, NH), 1.22 (t, J = 6.1 Hz, 3H,
CH3), 0.98–0.85 (m, 3H, CH3); 13C-NMR (75 MHz, CDCl3)
δ
: 183.7, 171.5, 169.0, 160.8, 137.1, 135.0,
133.8, 129.6, 129.1, 128.45, 128.0, 127.5, 127.3, 115.4, 115.1, 63.5, 62.8, 62.2, 53.8, 42.7, 42.2, 14.3, 12.0;
31P-NMR : 21.1; 19F-NMR
115.7 ppm; Anal. Calcd. for C29H34FN4O5PS: C 58.24, H 5.45, N 9.51;
Found: C 57.99, H 5.71, N 9.33.
δ
δ
:
−
◦
20
10e: C31H38FN4O5PS, white solid, yield 75.5%, m.p. 133–134 C, [
IR
1H-NMR (300 MHz, CDCl3)
α
]
= +113 (c = 0.13, CHCl3)
D
ν
: 3290, 3066, 3036, 2972, 2938, 1673, 1540, 1511, 1454, 1352, 1337, 1223, 1062, 1010 cm−1
;
δ
: 8.00 (s, 1H, NH), 7.82 (s, 1H, NH), 7.45–7.10 (m, 13H, ArH), 7.05 (s, 1H,
ArH), 6.87 (d, J = 29.2 Hz, 2H, 2NH), 6.35 (s, 2H, NCH-P+ NCH), 4.42–3.95 (m, 2H, NCH2-Ar), 3.80 (s,
2H, NCH2), 3.64 (s, 2H, OCH2), 3.43 (d, J = 73.9 Hz, 2H, OCH2), 1.62 (s, 2H, CH2), 1.43 (d, J = 12.9 Hz,
2H, CH2), 0.79 (dt, J = 16.7, 7.4 Hz, 6H, 2CH3); 13C-NMR (75 MHz, CDCl3)
δ: 183.8, 171.5, 168.6, 163.2,
137.1, 135.1, 129.5, 128.9, 128.7, 128.2, 128.1, 127.3, 115.4, 115.1, 69.5, 69.2, 62.7, 53.8, 42.1, 23.9, 23.6, 10.2,
10.0; 31P-NMR
N 9.12; Found: C 59.22, H 6.09, N 8.91.
δ
: 21.4; 19F-NMR
δ
:
−
115.8 ppm; Anal. Calcd. for C31H38FN4O5PS: C 59.36, H 5.87,
20
10f: C31H38FN4O5PS, white solid, yield 77.8%, m.p. 176–177 ◦C, [
α
]
= +85 (c = 0.14, CHCl3) IR
ν
:
:
D
1
3321, 3289, 3089, 3067, 2981, 2932, 1641, 1533, 1511, 1222, 999 cm−1; H-NMR (300 MHz, CDCl3)
δ
8.98 (s, 1H, NH), 8.45 (s, 1H, NH), 8.32 (d, J = 33.4 Hz, 2H, NH), 7.48–7.06 (m, 14H, ArH), 6.79 (s, 1H,
NCH-P), 5.26 (s, 1H, NCH), 4.33 (m, 2H, NCH2Ar), 3.78 (m, 2H, NCH2), 3.59 (d, J = 6.8 Hz, 2H, OCH),
3.39 (s, 2H, OCH), 1.50–0.98 (m, 12H, 4CH3); 13C-NMR (75 MHz, CDCl3)
δ: 183.6, 171.6, 168.6, 160.7,
138.2, 135.3, 133.9, 130.3, 129.5, 128.7, 127.9, 126.2, 115.9, 114.5, 73.4, 62.3, 55.3, 55.1, 43.1, 42.7, 24.7,
24.2, 23.6, 23.2; 31P-NMR δ: 19.5; 19F-NMR δ: −115.6 ppm; Anal. Calcd. for C31H38FN4O5PS: C 59.36,
H 6.30, N 9.12; Found: C 59.22, H 6.09, N 8.91.
◦
10g: C30H37N4O5PS, white solid, yield 66.8%, m.p. 167–168 C, [
3084, 3064, 3029, 2983, 2927, 1653, 1539, 1454, 1225, 1049, 1024, 976 cm−1; 1H-NMR (300 MHz, CDCl3)
8.35 (s, 2H, 2NH), 8.04 (s, 2H, 2NH), 7.52–7.09 (m, 15H, ArH), 6.51 (s, 1H, NCH-P), 5.39 (d, J = 35.5 Hz
α] ν: 3292,
20 = +47 (c = 0.10, CHCl3) IR
D
δ
:
,
1H, NCH), 4.39 (s, 2H, NCH-Ar), 4.10 (dd, J = 38.8, 23.9 Hz, 4H, 2OCH2), 3.77 (s, 1H, NCH), 3.57 (s,
1H, NCH), 2.99 (s, 1H, CHAr), 2.92 (s, 1H, CHAr), 1.06 (t, J = 7.0 Hz, 6H, 2CH3); 13C-NMR (75 MHz,
CDCl3) δ: 183.2, 172.1, 168.2, 138.1, 136.5, 135.5, 129.4, 128.6, 128.1, 127.8, 127.3, 127.0, 63.7, 63.4, 59.8,
53.6, 43.4, 36.6, 16.2; 31P-NMR
Found: C 60.39, H 6.25, N 9.39.
δ: 21.3 ppm; Anal. Calcd. for C30H37N4O5PS: C 60.61, H 6.07, N 9.86;

Molecules 2017, 22, 238
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20
10h: C32H41N4O5PS, white solid, yield 71.4%, m.p. 148–149 ◦C, [
α
]
= +54 (c = 0.12, CHCl3) IR ν:
D
3294, 3084, 3064, 3031, 2969, 2935, 1654, 1542, 1497, 1454, 1355, 1336, 1227, 1056, 1007 cm−1; 1H-NMR
(300 MHz, CDCl3) δ: 8.32 (d, J = 44.4 Hz, 2H, 2NH), 7.99 (s, 1H, NH), 7.51–7.03 (m, 15H, ArH), 6.92 (s,
1H, NH), 6.49 (s, 1H, NCH-P), 5.29 (s, 1H, NCH), 4.47 (m, 2H, NCH2-Ar), 4.38 (m, 2H, NCH2), 3.82 (m,
2H, OCH2), 3.62 (d, J = 30.8 Hz, 2H, OCH2), 3.19–2.85 (m, 2H, CH2Ar), 1.61 (d, J = 11.1 Hz, 2H, CH2),
1.49–1.29 (m, 2H, CH2), 0.98–0.79 (m, 3H, CH3), 0.71 (t, J = 7.3 Hz, 3H, CH3); 13C-NMR (75 MHz,
CDCl3)
43.4, 39.5, 23.7, 10.3, 10.1; 31P-NMR
N 9.04; Found: C 61.52, H 6.61, N 8.97.
δ
: 183.2, 172.0, 168.1, 138.1, 136.5, 135.6, 129.4, 128.7, 128.2, 127.8, 127.5, 127.1, 69.2, 68.7, 53.8,
δ: 21.4 ppm; Anal. Calcd. for C32H41N4O5PS: C 61.15, H 6.87,
10i: C32H41N4O5PS, white solid, yield 70.4%, m.p. 72–73 ◦C, [
α
]
20 = +76 (c = 0.11, CHCl3) IR
ν
: 3296,
D
3085, 3064, 3031, 2981, 2932, 1651, 1536, 1225, 998 cm−1; 1H-NMR (300 MHz, CDCl3)
δ: 8.35 (s, 2H,
2NH), 8.04 (s, 2H, 2NH), 7.38–6.90 (m, 15H, ArH), 6.51 (s, 1H, NCH-P), 5.39 (d, J = 35.5 Hz, 1H, NCH),
4.39 (s, 2H, NCH-Ar), 3.99 (d, J = 4.4 Hz, 2H, 2OCH), 3.77 (s, 1H, NCH), 3.57 (s, 1H, NCH), 2.99 (s, 1H,
CHAr), 2.92 (s, 1H, CHAr), 1.06 (t, J = 7.0Hz, 6H, 2CH3) ; 13C-NMR (75 MHz, CDCl3)
δ: 183.1, 171.8,
171.2, 137.9, 136.3, 135.7, 129.2, 128.6, 128.3, 128.1, 127.5, 127.1, 126.9, 72.7, 72.3, 60.4, 53.6, 43.3, 42.6,
39.2, 24.1, 23.7, 23.0; 31P-NMR
Found: C 61.52, H 6.61, N 8.97.
δ: 19.6 ppm; Anal. Calcd. for C32H41N4O5PS: C 61.91, H 6.28, N 9.35;
◦
10j: C30H36FN4O5PS, white solid, yield 98.7%, m.p. 75–76 C, [
3077, 3065, 3032, 2987, 2931, 1673, 1615, 1542, 1511, 1221, 1049, 1025, 976 cm−1; 1H-NMR (300 MHz,
CDCl3) : 8.24 (s, 1H, NH), 7.97 (s, 1H, NH), 7.47–6.77 (m, 14H), 6.44 (s, 1H, NCH-P), 5.28 (s, 1H,
α] ν: 3291,
20 = +107 (c = 0.12, CHCl3) IR
D
δ
NCH), 4.36–3.84 (m, 4H, NCH2Ar+ NCH2), 3.60 (d, J = 56.9 Hz, 4H, 2OCH2), 3.03 (d, J = 37.2 Hz, 2H,
CH2-Ar), 2.19–1.86 (m, 2H, 2NH), 1.24 (dd, J = 13.2, 6.2 Hz, 3H, CH3), 1.01 (d, J = 1. 8 Hz, 3H, CH3) ;
13C-NMR (75 MHz, CDCl3)
δ
: 183.4, 171.9, 171.4, 163.3, 136.4, 135.5, 134.0, 129.8, 128.9, 128.5, 128.1,
127.1, 115.4, 115.2, 60.6, 60.1, 53.7, 42.8, 42.1, 39.1, 17.6, 14.4; 31P-NMR : 21.1; 19F-NMR
115.5 ppm;
Anal. Calcd. for C30H36FN4O5 PS: C 58.35, H 6.13, N 8.87; Found: C 58.62, H 5.90, N 9.11.
δ
δ:
−
◦
10k: C32H40FN4O5PS, white solid, yield 94.3%, m.p. 95–96 C, [
3066, 3032, 2970, 2935, 1653, 1540, 1511, 1454, 1353, 1338, 1223, 1062, 1009 cm−1; 1H-NMR (300 MHz,
CDCl3) : 8.27 (s, 2H, 2NH), 7.82 (s, 2H, 2NH), 7.56–6.77 (m, 14H, ArH), 6.49 (s, 1H, NCH-P), 5.30 (s,
α] ν: 3294,
20 = +131 (c = 0.13, CHCl3) IR
D
δ
1H, NCH), 4.39 (s, 2H, NCH2-Ar), 3.89–3.78 (m, 4H, 2OCH2), 3.51 (d, J = 7.2 Hz, 2H, NCH2), 3.02 (d,
J = 54.6 Hz, 2H, CH2-Ar), 1.62 (d, J = 10.4 Hz, 2H, CH2), 1.50–1.41 (m, 2H, CH2), 1.00–0.83 (m, 3H,
CH3), 0.73 (t, J = 6.2 Hz, 2H, CH3); 13C-NMR (75 MHz, CDCl3)
δ: 183.3, 171.9, 168.2, 160.9, 136.4,
135.6, 133.9, 129.7, 129.3, 128.8, 128.2, 127.2, 115.5, 115.2, 69.3, 69.2, 60.1, 53.8, 42.7, 39.4, 24.0, 23.8, 10.1;
31P-NMR
Found: C 59.80, H 6.27, N 8.72.
δ
: 21.4; 19F-NMR
δ
:
−
115.6; ppm; Anal. Calcd. for C32H40FN4O5PS: C 59.59, H 6.01, N 8.94;
20
10l: C32H40FN4O5PS, white solid, yield 95.0%, m.p. 62–63 ◦C, [
α
]
= +86 (c = 0.12, CHCl3) IR
ν
: 3300,
D
3066, 3032, 2981, 2931, 2878, 1651, 1540, 1511, 1224, 1102, 999 cm−1; H-NMR (300 MHz, CDCl3)
δ:
1
8.37 (s, 1H, NH), 8.12 (s, 1H, NH), 7.48–6.97 (m, 12H), 6.86 (d, J = 20.7 Hz, 2H, 2ArH), 4.53 (s, 1H,
NCH-P), 4.28 (s, 1H, NCH), 4.07 (dd, J = 14.3, 7.2 Hz, 2H, NCH2-Ar + NCH2), 3.97 (s, 2H, 2OCH),
2.97 (d, J = 56.4 Hz, 2H, CH2Ar), 2.29 (s, 2H, 2NH), 1.27–1.11 (m, 12H, 4CH3); 13C-NMR (75 MHz,
CDCl3)
53.6, 42.7, 39.3, 21.2; 31P-NMR
C 59.62, H 6.46, N 8.47; Found: C 59.80, H 6.27, N 8.72.
δ
: 183.3, 171.9, 171.3, 163.2, 136.4, 135.9, 134.0, 129.4, 128.5, 128.1, 127.0, 115.4, 115.2, 72.7, 60.5,
δ
: 19.3; 19F-NMR
δ:
−
115.4 ppm; Anal. Calcd. for C32H40FN4O5PS:
◦
11a: C33H41N4O5PS, white solid, yield 92.6%, m.p. 103–104 C, [
3087, 3064, 3031, 2985, 2928, 2888, 1677, 1542, 1453, 1394, 1357, 1203, 1025 cm−1; 1H-NMR (300 MHz,
CDCl3) : 8.81 (s, 1H), 8.58 (s, 1H), 8.43 (s, 1H), 7.47–6.98 (m, 15H), 6.27 (s, 1H), 5.40 (s, 1H),
α] ν: 3300,
20 = +115 (c = 0.12, CHCl3) IR
D
δ
4.54 (d, J = 57.8 Hz, 2H), 4.24 (s, 1H), 3.78 (d, J = 6.5 Hz, 2H), 3.60 (dd, J = 10.1, 6.6 Hz, 4H), 3.02 (d,
J = 3.3 Hz, 2H), 1.92 (dd, J = 68.6, 32.5 Hz, 4H), 0.99 (dt, J = 28.1, 6.9 Hz, 6H); 13C-NMR (75 MHz,
CDCl3) δ: 183.3, 172.2, 171.5, 138.6, 136.1, 135.9, 129.4, 128.83, 128.2, 128.0, 127.7, 127.5, 126.8, 64.0, 63.5,

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60.2, 57.6, 53.6, 43.0, 42.0, 38.6, 24.9, 18.4, 11.9; 31P-NMR
C 62.41, H 6.85, N 8.46; Found: C 62.25, H 6.49, N 8.80.
δ: 21.5 ppm; Anal. Calcd. for C33H41N4O5PS:
◦
11b: C35H45N4O5PS, white solid, yield 90.2%, m.p. 117–118 C, [
3087, 3060, 3033, 2993, 2955, 2887, 1676, 1542, 1456, 1405, 1364, 1204, 1030 cm−1; 1H-NMR (300 MHz,
CDCl3) : 8.47 (d, J = 5.5 Hz, 2H), 8.23 (s, 1H), 7.53 (s, 1H), 7.33 (s, 1H), 7.27–6.99 (m, 13H), 6.35 (s, 1H),
α] ν: 3423,
20 = +133 (c = 0.13, CHCl3) IR
D
δ
5.23 (s, 1H), 4.33 (d, J = 19.6 Hz, 2H), 4.20 (s, 1H), 3.62–3.50 (m, 4H), 3.35 (d, J = 26.1 Hz, 2H), 3.10 (s,
2H), 2.75–2.56 (m, 4H), 1.80 (dd, J = 71.0, 33.4 Hz, 3H), 1.49–1.33 (m, 3H); 13C-NMR (75 MHz, CDCl3)
δ
: 170.8, 161.2, 160.8, 138.5, 134.3, 129.6, 129.2, 128.9, 128.4, 128.2, 127.6, 127.2, 126.7, 118.2, 115.3, 60.5,
58.8, 56.0, 53.4, 52.9, 42.8, 41.9, 38.3, 28.1, 24.6, 22.1, 17.5, 11.7; 31P-NMR
δ: 21.2 ppm; Anal. Calcd. for
C35H45N4O5PS: C 63.10, H 6.66, N 8.62; Found: C 63.23, H 6.82, N 8.43.
11c: C35H45N4O5PS, white solid, yield 81.8%, m.p. 89–90 ◦C, [
3085, 3064, 3031, 2983, 2935, 2884, 1675, 1542, 1454, 1386, 1359, 1203, 999 cm−1; 1H-NMR (300 MHz,
CDCl3) : 8.83 (d, J = 12.3 Hz, 2H), 8.30 (s, 1H), 7.98–6.59 (m, 15H), 6.19 (s, 1H), 5.53 (s, 1H), 4.68 (d,
J = 13.0 Hz, 2H), 4.46 (s, 1H), 4.33 (d, J = 15.4 Hz, 2H), 4.16 (s, 1H), 3.36 (s, 1H), 3.23 (s, 1H), 3.05 (s, 1H),
1.98 (dd, J = 81.3, 17.0 Hz, 4H), 1.40–0.88 (m, 12H); 13C-NMR (75 MHz, CDCl3)
: 183.6, 172.7, 172.6,
α] ν: 3301,
20 = +89 (c = 0.12, CHCl3) IR
D
δ
δ
139.1, 139.1, 136.6, 129.8, 129.6, 128.8, 128.5, 128.4, 128.4, 127.3, 127.2, 126.7, 72.8, 72.4, 60.3, 57.8, 55.8,
48.2, 43.0, 39.1, 29.6, 25.1, 24.3, 23.5; 31P-NMR
H 6.66, N 8.71; Found: C 63.23, H 6.82, N 8.43.
δ: 19.9 ppm; Anal. Calcd. for C35H45N4O5PS: C 63.48,
◦
11d: C33H40FN4O5PS, white solid, yield 93.5%, m.p. 97–98 C, [
3085, 3063, 3031, 2990, 2953, 2887, 1680, 1542, 1453, 1404, 1357, 1203, 1026, 720; 1H-NMR (300 MHz,
CDCl3) : 9.03–8.50 (m, 2H), 8.33 (s, 1H), 7.19–6.92 (m, 12H), 6.72 (d, J = 8.7 Hz, 2H), 6.14 (s, 1H), 5.22 (s,
α] ν: 3302,
20 = +121 (c = 0.11, CHCl3) IR
D
δ
1H), 4.56–4.02 (m, 3H), 3.87 (d, J = 7.1 Hz, 2H), 3.64 (dd, J = 19.5, 10.5 Hz, 4H), 3.15 (s, 1H), 2.96 (d,
J = 13.5 Hz, 1H), 1.81 (d, J = 9.7 Hz, 2H), 1.69 (d, J = 30.6 Hz, 1H), 0.91–0.81 (td, J = 16.0, 7.9 Hz, 6H);
13C-NMR (75 MHz, CDCl3)
δ
: 183.0, 171.6, 170.7, 160.5, 135.8, 135.4, 134.2, 128.5, 128.1, 127.6, 127.3,
126.5, 114.7, 114.5, 63.6, 63.1, 63.0, 59.9, 57.4, 41.8, 38.1, 29.1, 24.6, 20.6, 15.7, 13.8; 31P-NMR
: 21.2;
19F-NMR
115.8 ppm; Anal. Calcd. for C33H40FN4O5PS: C 60.40, H 6.37, N 8.95; Found: C 60.54,
H 6.16, N 8.56.
δ
δ:
−
11e: C35H44FN4O5PS, white solid, yield 53.9%, m.p. 107–108 ◦C, [
3296, 3087, 3065, 3031, 2970, 2932, 2882, 1662, 1539, 1454, 1383, 1357, 1222, 1012; 1H-NMR (300 MHz,
CDCl3) : 8.66–8.49 (m, 2H), 8.35 (s, 1H), 7.45–7.05 (m, 12H), 6.92 (d, J = 8.4 Hz, 2H), 6.10 (s, 1H), 5.25 (s,
1H), 4.51 (s, 1H), 4.30 (d, J = 46.8 Hz, 2H), 3.98 (d, J = 58.8 Hz, 2H), 3.53 (dd, J = 51.6, 32.8 Hz, 4H), 3.05
(s, 1H), 2.92 (s, 1H), 2.20 (s, 1H), 1.96–1.75 (m, 3H), 1.38 (dd, J = 17.7, 7.2 Hz, 2H), 1.21 (t, J = 7.1 Hz
3H), 0.98–0.60 (m, 3H); 13C-NMR (75 MHz, CDCl3)
: 173.1, 171.0, 169.5, 160.8, 136.4, 136.1, 134.3,
129.2, 128.6, 127.2, 115.5, 115.3, 69.2, 68.8, 60.3, 59.1, 54.3, 47.3, 45.4, 36.8, 28.4, 25.0, 22.5, 21.1, 10.0, 9.9;
31P-NMR : 21.6; 19F-NMR
115.9 ppm; Anal. Calcd. for C35H44FN4O5PS: C 61.34, H 6.77, N 8.02;
Found: C 61.57, H 6.50, N 8.21.
α] ν:
20 = +137 (c = 0.13, CHCl3) IR
D
δ
,
δ
δ
δ
:
−
◦
11f: C35H44FN4O5PS, white solid, yield 82.4%, m.p. 83–84 C, [
3087, 3066, 3031, 2980, 2934, 2879, 1676, 1542, 1453, 1386, 1353, 1223, 999; 1H-NMR (300 MHz, CDCl3)
α] ν: 3299,
20 = +107 (c = 0.12, CHCl3) IR
D
δ
:
8.73–8.55 (s, 2H), 8.34 (s, 1H), 7.58–7.01 (m, 12H), 6.88 (d, J = 12.5 Hz, 2H), 6.17 (s, 1H), 5.45 (s, 1H),
4.54 (d, J = 37.1 Hz, 2H), 4.30 (s, 1H), 4.06 (d, J = 7.2 Hz, 2H), 3.62 (s, 1H), 3.46 (s, 1H), 3.03 (d, J = 7.5 Hz,
2H), 2.03–1.83 (m, 4H), 1.41–1.30 (m, 3H), 1.20 (t, J = 7.1 Hz, 3H), 1.17–1.08 (m, 3H), 0.86–0.81 (m, 3H);
13C-NMR (75 MHz, CDCl3)
δ
: 183.3, 172.3, 171.2, 160.6, 136.4, 136.0, 135.9, 128.9, 128.5, 127.7, 126.9,
115.2, 115.1, 72.6, 72.5, 60.4, 57.6, 53.6, 42.3, 42.0, 38.9, 28.3, 24.9, 24.2, 24.1, 23.2, 21.1; 31P-NMR
: 19.8;
19F-NMR
115.7 ppm; Anal. Calcd. for C35H44FN4O5PS: C 61.36, H 6.82, N 8.00; Found: C 61.57,
H 6.50, N 8.21.
δ
δ:
−

Molecules 2017, 22, 238
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3.3. Antitumor Activity
All tested compounds were dissolved in DMSO and subsequently diluted in the culture medium
in indicated final concentrations before treatment of the cultured cells. Tested cells were plated in
96-well plates at a density 2
with the compounds at 3.9–500
×
104 cells/well/100
µ
L of the proper culture medium and were treated
µM for 72 h. In parallel, the cells treated with 0.1% DMSO served
as a control. An MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was
performed 4 h later, according to the instructions. This assay was based on the cellular cleavage
of MTT into formazane which is soluble in the cell culture medium. Any absorbance caused by
formazan was measured at 595 nm with a microplate reader (BIO-RAD, model 680), which was
directly proportional to the number of living cells in the culture. Two types of cells were used in these
assays, BGC-823 (human gastric cancer) and A-549 (non-small cell lung cancer) cell lines, provided
by ATCC (American Type Culture Collection) and cultivated in RPMI 1640 (for BGC-823 and A-549)
supplemented with 10% fetal bovine serum. Tissue culture reagents were obtained from Gibco BRL.
The experiment was performed in triplicate.
4. Conclusions
In summary, this study is based on our previous work [19]. Our attempt was to incorporate
glycine or rigid amino acids such as L-proline into the previous pseudo-peptide thiourea containing
an
moieties 10a
tumor cell lines (BGC-823 and A-549) below 100
α
-aminophosphonate moiety. A series of novel chiral dipeptide thioureas containing
and 11a were synthesized in high yield (Table 1). All these novel thioureas could inhibit
M (Table 2) by the MTT assay. Although MTT is
α-aminophosphonate
–l
–f
µ
not a direct reflection on cell proliferation specifically and other orthogonal assays may need to be
performed [27], the thioureas 11d and 11f showed comparable inhibition with that of Cisplatin against
BGC-823 and A-549 cells, respectively (Table 2). Finally, we can conclude their structure-activity
relationship; overall, the rigid amino acid of incorporated L-proline is helpful for antitumor
activity. L-phenylalanine containing thioureas also showed better antitumor activities than that
of L-phenylglycine containing thioureas. Our results also indicate that the antitumor activity could be
improved by introducing an electron-withdrawing group in the para position of the terminal phenyl
group of the dipeptide thioureas. This will guide us to design and obtain more and more potent
antitumor agents. The mechanism of antitumor activity for these novel dipeptide thioureas is under
further investigation.
Acknowledgments: We are grateful for financial support from the National Natural Science Foundation of China
(Nos. 81160385, 21302027 and 21662010), One Thousand Talents Program of Guizhou Province (2013), the Innovation
Team of Natural Science Foundation of the Department of Education of Guizhou Province (No. QJHRCTDZ[2015]57),
the Projects of Natural Science Foundation of the Department of Education of Guizhou Province (No. QJHKYZ[2015]371,
QKHSYZ[2012]3103 and QSZHZ[2012]89), and the Innovation Team of Science and Technology of the Natural Drug
Resources Research of Guizhou Province (No. QKHRCTD[2015]4025).
Author Contributions: J.L. and Y.W. conceived and designed the experiments; P.L. and J.H. performed
the experiments; Y.L. (Yongjun Li) and H.Z. analyzed the data; B.H. and Y.L. (Yan Li) wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 11d and 11f are available from the authors.
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