Synthesis and in vitro study of pseudo-peptide thioureas containing α-aminophosphonate moiety as potential antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2010.08.021

Twenty pseudo-peptide thioureas IIa-l containing α-aminophosphonate moiety were synthesized from the reaction of chiral α-amino carboxamide derivatives Ia-c with O,O′-dialkylisothiocyanato(phenyl)methylphosphonate 5. The synthesized compounds were completely characterized by elemental analysis, physical and spectral (IR, ¹H NMR, ¹³C NMR) data. According to the preliminary studies on antitumor activities, compounds IIa-l could inhibit tumor cells PC3, Bcap37 and BGC823. These compounds displayed low to high activity by MTT assays. Among them, L-IIk, D-IIa and D-IIe were identified as potent inhibitors, with IC₅₀ values ranging from 4.7 to 11.2 μM according to in vitro assay.

Full text of DOI:10.1016/j.ejmech.2010.08.021

European Journal of Medicinal Chemistry 45 (2010) 5108e5112
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro study of pseudo-peptide thioureas containing
a
-aminophosphonate moiety as potential antitumor agents
*
*
Jing-Zi Liu, Bao-An Song , Hui-Tao Fan, Pinaki S. Bhadury, Wen-Ting Wan, Song Yang , Weiming Xu,
Jian Wu, Lin-Hong Jin, Xue Wei, De-Yu Hu, Song Zeng
State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education,
Guizhou University, Guiyang 550025, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Twenty pseudo-peptide thioureas IIael containing a-aminophosphonate moiety were synthesized from
the reaction of chiral
methylphosphonate 5. The synthesized compounds were completely characterized by elemental anal-
ysis, physical and spectral (IR, 1H NMR, 13C NMR) data. According to the preliminary studies on antitumor
activities, compounds IIael could inhibit tumor cells PC3, Bcap37 and BGC823. These compounds dis-
played low to high activity by MTT assays. Among them, L-IIk, D-IIa and D-IIe were identified as potent
a
-amino carboxamide derivatives Iaec with O,O0-dialkylisothiocyanato(phenyl)
Received 19 March 2010
Received in revised form
9 August 2010
Accepted 9 August 2010
Available online 14 August 2010
inhibitors, with IC50 values ranging from 4.7 to 11.2
m
M according to in vitro assay.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Pseudo-peptide thiourea
Synthesis
Antitumor activity
1. Introduction
derivatives mimicking the pharmacophore and thus the activity of
the original peptide [4,5]. As isosteres of peptides, phosphono-
peptides containing a transition state analogue of the hydrolysis of
the amide bond represent another attractive approach for the
preparation of proteolytically stable peptides [6]. In addition to
increased stability, incorporation of a phosphonate moiety into the
peptide sequence also provides access to additional binding
interactions within the transition state conformation of the
enzyme/substrate complex [7]. A wide range of phosphonopep-
tides have been used to design very effective protease inhibitors
[8e10]. However, since there are only a limited number of
economically viable chemicals available for practical application in
biological science or agriculture [11], a great deal of scope still lies
ahead for further research in this field. In this context, in order to
find broad spectrum biologically active pseudo-peptide thiourea,
we have previously described preparation of certain chiral thio-
Peptides are among the most versatile bioactive molecules e.g.
many peptide hormones and analogous short peptides exert their
action by binding to membrane receptors [1]. Peptides and their
derivatives may also exhibit a broad spectrum of biological activ-
ities such as antimicrobial [2], antiviral, and anticancer activities
[3]. However, most natural peptides are composed of L-form
a-
amino acids and because of the ubiquitous prevalence of pepti-
dases, they have limited biostability, and consequently low
bioavailability. To overcome this problem, stable and at the same
time biologically active pseudo-peptides have been developed.
These novel compounds open up new perspectives in drug design
by providing an entire range of highly specific and non-toxic
pharmaceuticals. With growing application on their synthesis and
bioactivity, chemists and biologists in recent years have directed
considerable attention on the research of pseudo-peptide
urea derivatives containing
a-aminophosphonate moiety with
significant antiviral activity [12]. Herein, we further turned our
attention to prepare novel compounds with enhanced antitumor
activities by incorporating a-aminophosphonate moiety at the 1 or
Abbreviations: 1H NMR, 1H Nuclear Magnetic Resonance; 13C NMR, 13C Nuclear
Magnetic Resonance; 31P NMR, 31P Nuclear Magnetic Resonance; 19F NMR, 19F
Nuclear Magnetic Resonance; MTT, [3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl
tetrazoliumbromide] assay; PC3, Prostate cancer; BGC823, Human gastric cancer;
Bcap37, Breast cancer cell lines; ADM, Adriamycin.
* Corresponding authors. Tel.: þ86 851 362 0521; fax: þ86 851 362 2211.
E-mail addresses: songbaoan22@yahoo.com (B.-A. Song), yangsdqj@gmail.com
(S. Yang).
3-position of pseudo-peptide thiourea. The primary aim of this
study is to synthesize the title compounds and study their anti-
tumor activities for the development of a new inhibitor to PC3,
Bcap37 and BGC823 cells. To the best of our knowledge, this is the
first report on the synthesis and antitumor activity of pseudo-
peptide thioureas containing a-aminophosphonate moiety.
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.021

J.-Z. Liu et al. / European Journal of Medicinal Chemistry 45 (2010) 5108e5112
5109
Table 1
Comparison of the yields of 5aed in two different methods.
Compound
R2
Yield (%)
BTCb
a
POCl3
5a
5b
5c
5d
Et
60.3
61.5
54.2
57.8
78.6
70.5
67.4
75.1
n-Pr
i-Pr
n-Bu
a
The reported yields [12].
The yields of isolated products.
b
In order to establish whether the overall reaction from enan-
tiomer pure N-Boc-phenylalanine leads to any loss of optical purity
through partial racemization in presence of base, enantiomeric
excess (ee) values of L-Ia and diastereoisomeric excess (de) values
based on the stereoconfiguration of amino acid amide part of final
product L-IIa obtained from L-N-Boc-phenylalanine were deter-
mined. While Boc-deprotected product (free amine) showed an ee
of 99.4%, the target compound L-IIa arising through nucleophilic
Fig. 1. Synthetic pathway to target compounds IIael.
addition of L-Ia on
98.5% de. Thus, as expected, stereochemical configuration at
-carbon atom of the acid was practically unaffected and this
synthetic transformation from chiral -amino acid could be applied
a-phosphateisothiocyanate was obtained in
a
2. Results and discussion
a
to a wide range of compounds without undergoing any significant
loss of optical activity. Since both L- and D-isomers of N-Boc-
phenylalanine are easily accessible, it provides a useful route to
obtain both the enantiomeric forms of carboxamide Iaec which are
useful synthons to a variety of pharmaceuticals. The overall process
for novel (L)/(D)-pseudo-peptide thioureas IIael is convenient,
high yielding and atom-economic.
2.1. Chemistry
The synthetic route designed for the pseudo-peptide thiourea
analogues containing
summarized in Fig. 1. Boc-protected intermediate amide was first
generated from the reaction of phenylalanine and substituted
benzylamine in presence of O-benzotriazol-1-yl-N,N,N0,N0-tetra-
methyluronium hexafluorophosphate (HBTU) at room temperature.
Chiral a-amino carboxamide derivatives Iaec were then obtained
through deprotection of Boc with trifluoroacetic acid as shown in
the figure. The desired pseudo-peptide thiourea analogues IIael
a-aminophosphonate moiety IIael is
The yield of target compound IIael depended both on the
absolute configuration of initial N-Boc-phenylalanine and nature of
substituents present in the phenyl ring and phosphonate moiety
respectively. The structures of all the compounds were confirmed
by IR, 1H NMR, 13C NMR, 31P NMR, 19F NMR spectra, and elemental
analysis. The IR spectra of products IIael exhibited absorption
bands at 3281e3289 cmꢀ1, indicating the presence of amidic NeH.
The stretching frequency at 1653e1684 cmꢀ1 was assigned to C]O
vibrations and the characteristic absorptions at 1207e1224 cmꢀ1
and 1005e1024 cmꢀ1 were attributed to the presence of P]O
and the PeOeC group, respectively. In 1H NMR spectra, all phenyl
protons showed multiplet at 7.49e6.71 ppm. The main character-
istic of the 1H NMR spectra of IIael is the presence of high-
frequency downfield broad singlet dH 8.84e8.43 for amidic NeH
protons. The broad doublets at dH 8.12e7.97 presumably arise due
to the presence of deshielded NeH proton of thiourea linked to the
phosphonate moiety and benzene ring through an intervening
carbon atom. While the broad doublets at dH 6.45e6.32 are
assigned to the NeH proton of thiourea directly attached to the
containing
a-aminophosphonate moiety were prepared by the
addition of racemic O,O0-dialkylisothiocyanato(phenyl)methyl-
phosphonate 5 to Iaec in THF at room temperature.
The key intermediate O,O0-dialkylisothiocyanato(phenyl)meth-
ylphosphonate 5 was prepared according to the literature procedure
[12]. In order to improve the yield, the existing method for conver-
sion of 4 into 5 was further modified by using Bis(trichloromethyl)
carbonate (BTC) instead of phosphorus oxychloride as shown in
Fig. 2. The results obtained with BTC were compared with those
obtained with phosphorus oxychloride (Table 1). As could be
observed from the table, the yields phosphonate 5 in the former case
from different alkyl substituents ranged from 67.4e78.6%. The other
intermediates involved in the preparation of 5 were obtained as
depicted in Fig. 2 [12].
R2
R2
O
P
(E)
O
HP
R2
R2
O
S
(E)
O
O
N
N
O
O
(E)
O
OH
N
CHO
· H2O
NH3·H2O
70ºC-75ºC
1
2
R2
R2
R2
O
P
O
P
O
P
NH2
Et3N, CS2
O
O
O
NH3·H2O
O
O
O
O
R2
R2
R2
Cl3COCOCCl3, Et2O
NH3· OTs
N=C=S
4
5
3
Fig. 2. Synthetic route to the intermediates 5aed.

5110
J.-Z. Liu et al. / European Journal of Medicinal Chemistry 45 (2010) 5108e5112
Table 2
(adriamycin) and PD153035 [6,7-dimethoxy-N-(3-bromophenyl)-
Growth inhibition of selected cell lines.
4-aminoquiazoline] [13] were evaluated for their anti-proliferation
activities against three types of human cancer cell lines, breast
cancer, prostate cancer and stomach cancer cells as shown inTable 2.
Among the studied compounds, L-IIc (R1 ¼ H, R2 ¼ i-Pr), D-IIa
(R1 ¼ H, R2 ¼ Et), D-IIe (R1 ¼ o-F, R2 ¼ Et), L-IIj (R1 ¼ p-F, R2 ¼ n-Pr)
and L-IIk (R1 ¼ p-F, R2 ¼ i-Pr) exhibited moderate inhibitory activ-
ities of 50.1%, 56.9%, 73.1%, 56.7% and 58.6%, respectively against
R1
S
R2
H
N
O
P
O
O
N
H
N
H
R2
O
PC3-cell at 10 mM concentration. The rest of the compounds,
IIa-l
however, did not indicate any significant inhibition against PC3-
cell. The compounds D-IIe (R1 ¼ o-F, R2 ¼ Et), L-IIk (R1 ¼ p-F,
R2 ¼ i-Pr), D-IIa (R1 ¼ H, R2 ¼ Et) and D-IIb (R1 ¼ H, R2 ¼ n-Pr) also
displayed potential antitumor bioactivities of 70.2%, 53.1%, 52.3%
Compounda
R1
R2
Inhibition ratio(%)b
Bcap37c
PC3d
BGC823e
and 49.1% respectively against Bcap37 cells at 10 mM. From Table 2 it
is evident that compounds L-IIk and D-IIa bearing an electron
withdrawing fluorine atom at the 4-position of the aromatic ring
showed good antitumor activities with inhibition rate ranging from
55.7% to 89.1% against BGC823 cell lines. Remarkably, these values
were comparable to the inhibition rate exhibited by ADM (adria-
mycin) (98%). From the data, it may also be derived that the
cell growth inhibition bioactivity of L-IIk has appreciable effect
on BGC823 cells, being better than the commercial analogue
PD153035.
In addition, selected chiral compounds D-IIe, D-IIa, L-IIk, L-IIj
and L-IIc which displayed good antitumor activities were
bioassayed further to investigate their efficacies at different
concentrations with ADM and PD153035 serving as the commercial
controls. As shown in Table 3, the inhibition effects of these
compounds against PC3 were significant. The IC50 values of D-IIe,
L-IIa
L-IIb
L-IIc
L-IId
L-IIe
L-IIf
L-IIg
L-IIh
L-IIi
H
H
H
H
o-F
o-F
o-F
o-F
p-F
p-F
p-F
p-F
H
Et
16.9 ꢁ 10.9
35.4 ꢁ 4.7
24.4 ꢁ 4.1
34.5 ꢁ 6.5
25.9 ꢁ 4.7
44.7 ꢁ 9.2
44.4 ꢁ 18.1
32.1 ꢁ 7.8
26.1 ꢁ 11.7
24.0 ꢁ 16.4
53.1 ꢁ 9.2
48.4 ꢁ 6.7
52.3 ꢁ 4.3
49.1 ꢁ 7.2
22.9 ꢁ 8.4
46.6 ꢁ 8.9
70.2 ꢁ 3.9
17.2 ꢁ 4.8
27.8 ꢁ 8.1
34.0 ꢁ 7.4
34.0 ꢁ 8.6
12.6 ꢁ 8.4
27.0 ꢁ 5.8
35.8 ꢁ 7.3
20.1
33.5
50.1
22.8
19.3
18.1
26.8
22.5
27.9
56.7
58.6
19.7
56.9
30.8
30.5
21.7
73.1
30.6
14.3
18.0
32.4
30.6
34.8
33.6
18.3 ꢁ 8.1
35.7 ꢁ 4.7
27.9 ꢁ 4.0
21.1 ꢁ 11.6
30.1 ꢁ 3.8
11.1 ꢁ 26.3
47.9 ꢁ 5.0
30.1 ꢁ 21.7
22.9 ꢁ 7.4
21.0 ꢁ 6.3
89.1 ꢁ 1.2
26.1 ꢁ 9.5
55.7 ꢁ 10.0
24.4 ꢁ 11.5
29.0 ꢁ 10.9
22.7 ꢁ 12.9
30.1 ꢁ 3.8
11.1 ꢁ 26.3
51.6 ꢁ 5.0
30.1 ꢁ 21.7
44.5 ꢁ 12.6
53.9 ꢁ 0.0
20.8 ꢁ 14.7
33.2 ꢁ 4.2
93.8 ꢁ 2.1g
n-Pr
i-Pr
n-Bu
Et
n-Pr
i-Pr
n-Bu
Et
n-Pr
i-Pr
n-Bu
Et
n-Pr
i-Pr
n-Bu
Et
n-Pr
i-Pr
n-Bu
Et
n-Pr
i-Pr
n-Bu
L-IIj
L-IIk
L-IIl
D-IIa
D-IIb
D-IIc
D-IId
D-IIe
D-IIf
D-IIg
D-IIh
D-IIi
D-IIj
D-IIk
D-IIl
ADMf
H
H
H
o-F
o-F
o-F
o-F
p-F
p-F
p-F
p-F
D-IIa, L-IIk, L-IIc and L-IIj were 6.7, 11.2, 17.2, 19.5 and 22.1 mM,
respectively. Among these compounds, D-IIe showed highest anti-
tumor activity, comparable to that shown by PD153035
(IC50 ¼ 13.7
was that the activity displayed by L-IIk in terms of the average IC50
value (4.7 M) against BGC823 was also comparable to that of
M). This finding appears to be beneficial to
mM) against PC3. Also an interesting observation noted
m
a
These compounds were tested as the free base.
PD153035 (IC50 ¼ 6.9
m
b
The known numbers of cells (2.0 ꢂ 104) were incubated for 24 h in a 5% CO2
establish SAR of title pseudo-peptide thiourea analogues.
incubator at 37 ꢃC in the presence of different concentrations of test compounds.
After 24 h of drug incubation, the MTT solution was added, supernatant was dis-
carded, 100 ml DMSO was added in each well and absorbance was recorded at
595 nm by ELISA reader; determined by three separate experiments and each was
performed in triplicate.
2.3. Structureeactivity relationship (SAR)
Within the limits of experimental error, the title compounds
with D-configuration displayed better antitumor activities against
Bcap37 compared to their respective L-counterparts e.g. D-IIa
(52.3%) > L-IIa (16.9%), D-IIe (70.2%) > L-IIe (25.9%), D-IIi (34.0%) >
L-IIi (26.1%). Furthermore, the compound bearing a fluorine atom at
the 4-position of the benzyl ring linked to the nitrogen atom of the
amide (R1 ¼ p-F) showed good antitumor activities with inhibition
rate ranging from 55.7 to 89.1% against BGC823 cell lines. With
regard to the alkyl substituents (R2) in the phosphonate part of the
studied compounds, D-diastereoisomers with diethyl substituents
(R2 ¼ Et) displayed significant enhancement towards inhibition
against Bcap37, PC3 and BGC823 cells in comparison with D-O,O0-
dialkylphosphonates derived from n-propyl, isopropyl or n-butyl
groups. In particular, the compound D-IIe (R1 ¼ o-F, R2 ¼ Et) dis-
played most promising result amongst all. The L-diastereoisomers
of O,O0-diisopropylphosphonate e.g. L-IIk (R1 ¼ p-F, R2 ¼ i-Pr), L-IIc
(R1 ¼ H, R2 ¼ i-Pr), L-IIg (R1 ¼ o-F, R2 ¼ i-Pr), on the other hand,
revealed higher antitumor activities than the L-diastereoisomers of
phosphonates derived from ethyl, n-propyl or butyl groups (R2 ¼ Et,
n-Pr, Bu). Thus, the nature of stereochemical configuration (D or L)
of the amino acid amide part, type of substituents (R1) in the
aromatic ring as well as alkyl substituents (R2) in the phosphonate
moiety of the title compounds appear to be the key factors in
controlling the antitumor activity. While phosphonates derived
from branched alkyl chains were preferred over straight-chain
c
Breast cancer.
Prostate cancer.
Stomach cancer.
The standard compound used for comparison of activity.
d
e
f
g
The value was determined by using our assay protocol.
asymmetric carbon atom, another adjacent doublet at dH 6.33e6.12
appeared due to the CeH proton of NeCeP. The multiplet at dH
5.28e5.14 was assigned to the CeH of NeCeC group. The typical
low intense carbon resonance frequencies at dc 183.8e182.9 and dc
171.2e170.4 in the 13C NMR spectra of IIael also confirmed the
presence of C]S and C]O double bond respectively. As expected,
fluorine and phosphorus resonance appeared at dF ꢀ118.8 to 115.2
and dP 20.3e22.6 in their corresponding fluorine and phosphorus
NMR spectra, respectively. According to the 31P NMR spectra of all
target compounds, phosphorus atom of the phosphonate moiety
was coupled to adjacent CH.
2.2. Evaluation of antitumor activities
Some potential chiral thiourea derivatives incorporating phos-
phonate moiety were identified for cancer treatment by studying
their anticancer activities on
compounds in the series IIael and commercial drugs ADM
3 human tumor cells. All the

J.-Z. Liu et al. / European Journal of Medicinal Chemistry 45 (2010) 5108e5112
5111
Table 3
Cytotoxic activity of promising compounds in PC3 and BGC823 cell at different concentrations of drug exposure by MTT assay.
Compound
1
m
Ma
5
m
Ma
10
m
Ma
20
m
Ma
IC50
(
m
M)b
Cell line
L-IIc
L-IIj
L-IIk
D-IIa
2.7 ꢁ 0.2
12.8 ꢁ 1.4
19.8 ꢁ 4.2
17.3 ꢁ 1.9
21.2 ꢁ 0.9
45.6 ꢁ 3.1
89.4 ꢁ 2.1
21.8 ꢁ 3.2
28.0 ꢁ 1.9
31.4 ꢁ 3.1
56.9 ꢁ 3.1
73.1 ꢁ 2.1
98.0 ꢁ 3.1
50.14 ꢁ 4.9
51.2 ꢁ 2.3
66.7 ꢁ 3.0
73.8 ꢁ 4.0
97.2 ꢁ 0.2
100
19.5 ꢁ 2.1d
22.1 ꢁ 1.9d
17.2 ꢁ 2.1d
11.2 ꢁ 0.9d
6.7 ꢁ 0.1d
3.3 ꢁ 0.8d
13.7 ꢁ 1.4d
4.7 ꢁ 0.04e
6.9 ꢁ 1.1e
PC3
18.1 ꢁ 4.1
15.8 ꢁ 2.1
10.4 ꢁ 2.1
4.9 ꢁ 0.9
D-IIe
ADM (adriamycin)c
PD153035c
L-IIk
36.4 ꢁ 2.0
30.6 ꢁ 2.1
70.1 ꢁ 1.1
89.1 ꢁ 2.2
100
BGC823
PD153035c
a
The data represented the mean of three experiments in triplicate and were expressed as means ꢁSD; Only descriptive statistics were done in the text.
b
c
The IC50 value was defined as the concentration at which 50% survival of cells was observed. The results are listed in the table.
Used as a positive control.
IC50 values on PC3.
IC50 values on BGC823.
d
e
alkyls for L-diastereoisomers, simple diethyl derived phosphonates
4.2. Preparation of intermediates Iaec
gave better result with D-diastereoisomers. Therefore, for ideal
activity, R1 should be a strong electron withdrawing group and for
L-diastereoisomers R2 should be i-Pr, whereas for D-diastereoiso-
mers R2 needs to be Et. Although our studies indicate the existence
of a definite relationship of antitumor activity with the nature of
substituents and type of configuration within the compound, the
role of steric, electronic and hydrophobic effects at physiological pH
on structureeactivity relationships could not be successfully
ascertained due to lack of structural diversity.
Phenylalanine (2.65 g, 0.01 mol,1.00 equiv.) and O-benzotriazol-
1-yl-N,N,N0,N0-tetramethyl- uronium hexafluorophosphate (HBTU,
3.80 g, 0.01 mol,1.00 equiv.) were loaded into an oven-dried round-
bottomed flask equipped with a magnetic stir bar, rubber septum,
and argon inlet. Anhydrous dichloromethane (50 mL) was then
added. After 3 min, anhydrous DIPEA (2.58 g, 0.02 mol, 2.0 equiv)
and benzylamine (0.011 mol, 1.10 equiv.) were sequentially added
and the reaction mixture was stirred at room temperature for
2e4 h. During the process, the state of the solution was seen to
change from slightly heterogeneous to homogeneous confirming
the consumption of HBTU. And the reaction was followed by TLC
(developing solvent: 4% MeOH/CH2Cl2, V/V). The reaction mixture
was poured into a separatory funnel containing 1 N HCl (50 mL),
and was then partitioned between dichloromethane and aqueous
hydrochloric acid solution. The organic layer was washed with 1 N
HCl (3 ꢂ 40 mL), dried over anhydrous sodium sulfate, filtered, and
concentrated in vacuo. The resulting light yellow oil was transferred
to a 100-mL flask and redissolved in dichloromethane (40 mL).
Trifluoroacetic acid (10 mL) was then added in one portion. After
2e4 h, the mixture was slowly partitioned with dichloromethane
and chilled, saturated aqueous sodium carbonate solution (50 mL)
was added. The aqueous layer was extracted with dichloromethane
(3 ꢂ 40 mL), and the combined organic extracts were dried over
anhydrous sodium sulfate, filtered, and concentrated in vacuo. The
crude product was purified by thin layer chromatography (TLC) on
a silica gel (developing solvent: 4% MeOH/CH2Cl2, V/V) to give the
intermediates Iaec. The structure was confirmed by 1H NMR, 13C
NMR, IR, and elemental analysis (see the Supporting Information).
3. Conclusion
A series of new pseudo-peptide thioureas IIael containing
a
-aminophosphonate moiety were synthesized in high yield
under mild conditions by the addition of racemic O,O0-dialkyl
isothiocyanato(phenyl)methylphosphonate 5 to chiral -amino
a
carboxamide derivatives in THF. The synthetic method from chiral
N-Boc-phenylalanine practically does not affect the stereochemical
configuration of a-carbon bearing the amino group. The synthe-
sized compounds IIael could inhibit tumor cells PC3, Bcap37 and
BGC823 and showed low to high activity by MTT assays. Among
them, L-IIk, D-IIa and D-IIe were found as potent inhibitors, with
IC50 values ranging from 4.7 to 11.2 mM by in vitro assay. The
present work demonstrates that the antitumor activity of pseudo-
peptide thioureas was significantly improved through introduction
of suitably substituted stereoisomer with Ds.-configuration. The
D-diastereoisomers, in general, displayed enhanced activity
compared to their corresponding L-diastereoisomers. Influence of
different substituents, minor structural modification and steric
parameters on structureeactivity relationships for identifying lead
bioactive compound would be taken up in our future investigation.
4.3. Preparation of title chiral compounds IIael
A
solution of O,O0-dialkylisothiocyanato(phenyl)methyl-
4. Experimental section
phosphonate 5 (1 mmol) in tetrahydrofuran (10 mL) was stirred,
followed by drop wise addition of chiral amine Iaec (1.1 mmol).
The reaction mixture was stirred for 1 h at 23 ꢃC, the solvent was
removed by evaporation, and the crude product was purified by
chromatography on silica using a mixture of petroleum ether and
tetrahydrofuran as the eluent to give the title compounds IIael in
84e98% yields (see the Supporting Information).
4.1. Analysis and instruments
The melting points of the products were determined on a XT-4
binocular microscope (Beijing Tech Instrument Co., China) and
were not corrected. The IR spectra were recorded on a Bruker
VECTOR22 spectrometer in KBr disks. 1H and 13C NMR (solvent
DMSO-d6) spectra were recorded on a JEOL-ECX 500 NMR spec-
trometer at room temperature using TMS as an internal standard.
Elemental analysis was performed on an Elementar Vario-III CHN
analyzer. UV spectra were recorded on a VARIAN Cary-50 spec-
trometer using a cell path length of 1 cm. BIO-RAD, Model 680
Microplate Reader. The reagents were all of analytical grade or
chemically pure. Analytical TLC was performed on silica gel GF254.
4.3.1. O,O0-diethyl(3-(L-1-(benzylamino)-1-oxo-3-phenylpropan-
2-yl)thioureido)(phenyl)methyl-phophonate (L-IIa)
White solid, mp 57e59 ꢃC, yield, 95%; ½a 2D0
ꢄ
[ 11.4ꢃ (c ¼ 0.1,
acetone); IR (KBr, cmꢀ1
) n: 3287, 3082, 2978, 2926,1684,1522,1454,
1352, 1211, 1024, 978, 698; 1H NMR (500 MHz, CDCl3, ppm)
d: 8.76
(br s, 1H, NH), 8.02 (br d, 1H, J ¼ 18.5 Hz, NH), 7.29e7.13 (m, 15H,
ArH), 6.33 (br d, 1H, J ¼ 9.4 Hz, NH), 6.21 (d, 1H, J ¼ 8.1 Hz, NCHP),

5112
J.-Z. Liu et al. / European Journal of Medicinal Chemistry 45 (2010) 5108e5112
5.21 (d, 1H, J ¼ 8.1 Hz, CH), 4.18e4.14 (m, 2H, NCH2), 3.96e3.70 (m,
4H, 2 OCH2), 3.04e3.02(m, 2H, CH2Ar),1.28e1.09 (m, 6H, 2CH3); 13
NMR (125 MHz, CDCl3, ppm) : 183.3, 170.8, 137.6, 137.1, 136.9,
129.4, 128.6, 128.5, 125.5, 64.0, 59.8, 54.3, 43.4, 38.3, 16.2; 31P NMR
(200 MHz, CDCl3, ppm) : 22.4; Anal. Calcd for C28H34N3O4PS (540):
Acknowledgments
C
d
The authors wish to thank the National Key Project for Basic
Research (2010CB126105) and the National Natural Science Foun-
dation of China (20872021) for the financial support.
d
C, 62.32; H, 6.35; N, 7.79%. Found: C, 62.53; H, 6.86; N 7.38%.
Supporting information
4.4. MTT assay against cancer cell proliferation
Supporting information associated with this article can be
found, in the online version, at doi:10.1016/j.ejmech.2010.08.021.
All tested compounds were dissolved in DMSO (1e100 mM
solution) and subsequently diluted in the culture medium before
treatment of the cultured cells. Tested cells were plated in 96-well
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