Synthesis and antitumor activity of α-aminophosphonates containing thiazole[5,4-b]pyridine moiety

Article abstract of DOI:10.1039/c2ob25875g

A new procedure is developed for the synthesis of α-aminophosphonates containing thiazole[5,4-b]pyridine moiety from conveniently available starting materials. The target compounds were characterized by infrared, ¹H NMR, ¹³C NMR, ³¹P NMR, mass spectrometry and elemental analysis. The newly synthesized compounds were evaluated for their anticancer activities against PC-3, Bcap-37, H460 cells in vitro by the MTT method. Compounds 3b and 3f are highly effective against PC-3, Bcap-37 cells and good to H460 cells. Further study is necessary to find out the potential antitumor activities.

Full text of DOI:10.1039/c2ob25875g

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PAPER
Synthesis and antitumor activity of α-aminophosphonates containing
thiazole[5,4-b]pyridine moiety†
Lijun Gu*a and Cheng Jinb
Received 7th May 2012, Accepted 3rd July 2012
DOI: 10.1039/c2ob25875g
A new procedure is developed for the synthesis of α-aminophosphonates containing thiazole[5,4-b]-
pyridine moiety from conveniently available starting materials. The target compounds were characterized
by infrared, 1H NMR, 13C NMR, 31P NMR, mass spectrometry and elemental analysis. The newly
synthesized compounds were evaluated for their anticancer activities against PC-3, Bcap-37, H460 cells
in vitro by the MTT method. Compounds 3b and 3f are highly effective against PC-3, Bcap-37 cells and
good to H460 cells. Further study is necessary to find out the potential antitumor activities.
Introduction
Results and discussion
α-Aminophosphonic acids and their corresponding α-amino-
phosphonates have received much interest in organic and
medicinal chemistry because they are analogous to both natural
and unnatural amino acids.1 Some of them have been used as
potent enzyme inhibitors, antimicrobial, antitumor and antiviral
agents.2–6 Among these compounds, studies are mainly concen-
trated on those containing heterocycle moieties.
Fused systems containing a pyridine ring, in particular thiazo-
lopyridines, are widely used as base structures in the design of
various biologically active compounds.7,8 There are six isomeric
thiazolopyridine systems reported in the literature.9 Thiazole[5,4-b]-
pyridine belong to an important class of compounds which show
a wide range of biological activities such as anticancer, antiviral,
antibacterial.10
Chemistry
The standard procedures for the syntheses of α-aminophospho-
nates containing heterocycle moieties mainly rely on the
Kabachnik–Fields reaction. The Kabachnik–Fields reaction is a
three-component reaction of a dialkyl phosphonate, a carbonyl
and an amino compound.11 The investigation was initiated by
using the reaction of diethyl phosphate, benzaldehyde and thia-
zolo[5,4-b]pyridin-2-yl amine. The desired product O,O′-diethyl-
α-(thiazolo[5,4-b]pyridin-2-yl)amino(phenylmethyl) phospho-
nate 3a was obtained in a moderate yield (57%) under solvent-
catalyst free condition. Although the reaction is easy to operate,
the starting materials thiazolo[5,4-b]pyridin-2-ylamine is not
readily available.
Recently, Ma and his group have developed a copper-cata-
lyzed three-component synthesis of 2-N-substituted benzothia-
zoles.12 According to the structure of O,O′-dialkyl-α-(thiazolo-
[5,4-b]pyridin-2-yl)amino(substituted phenylmethyl) phospho-
nate 3, we envisioned that 3-amino-2-bromopyridine 1 may be
used to react with primary 1-aminophosphonate 2 under the cata-
lysis of CuCl2·2H2O in presence of carbon disulfide, K2CO3 and
N,N-dimethylformamide (DMF) to synthesize such products.
Performing the reaction using the conditions similar to those
reported by Ma and coworkers, the desired product was obtained
in 86% yield Scheme 1.
In view of this research and our desire to develop new antican-
cer agents of high potency, we fused biological thiazole[5,4-b]-
pyridine and aminophosphonate structures to obtain compounds
possessing better biological activities. In this paper, we have
designed and synthesized a series of new α-aminophosphonates
containing thiazole[5,4-b]pyridine moiety.
aKey Laboratory of Chemistry in Ethnic Medicinal Resources, State
Ethnic Affairs Commission & Ministry of Education, Yunnan University
of Nationalities, Kunming, Yunnan, China, 650500.
E-mail: gulijun2005@126.com
bChangzhou Tianma Group Company Limited, Changzhou, Jiangsu,
China, 213127
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c2ob25875g
Scheme 1 Synthesis of α-aminophosphonate derivative 3a.
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Encouraged by the result, we replaced 3-amino-2-bromopyri-
dine 1 with 3-nitro-2-bromopyridine 4 and carried out the reac-
tion using the following reaction conditions: 3-nitro-2-
bromopyridine (1 equiv.), CS2 (1.5 equiv.), primary 1-aminophos-
phonate (1.4 equiv.), K2CO3 (3 equiv.), CuCl2·2H2O (1 equiv.)
and SnCl2·2H2O (3 equiv.) in DMF. To our delight, the desired
product was isolated with 72% yield after reacting for 8 h at
105 °C. Then, we briefly examined the effect of different temp-
eratures and ratio of 4/SnCl2·2H2O. The results showed that at
105 °C, the reaction preceded smoothly in high yield. To further
evaluate the influence of the ratio of 4/SnCl2·2H2O, the reaction
was carried out in DMF at 105 °C using a 1 : 1 to 1 : 5 ratio of
4/SnCl2·2H2O, leading to 3a in 31%, 46%, 72%, 82%, and
78% yields, respectively. We concluded the best ratio of
4/SnCl2·2H2O was 1 : 4.
Scheme 4 Synthesis of α-aminophosphonate derivative 9.
With the optimized conditions in hand, we then performed the
reaction of a variety of primary 1-aminophosphonates 5 and
3-nitro-2-bromopyridine 4 (Scheme 2). The results are summar-
ized in Table 1.
As shown in Table 1, we were pleased to find that the method
was applicable to a broad substrate scope on both substituted
primary 1-aminophosphonates 2 and 3-nitro-2-bromopyridine 4.
It can be seen that this protocol can be applied to the primary
1-aminophosphonates with electron-withdrawing groups or elec-
tron-donating groups. Meanwhile, it was particularly noteworthy
that the effects of different 3-nitro-2-bromopyridine 4 were also
investigated. 4-Substituted 3-nitro-2-bromopyridin 6 can also
delivered the desired product 7 (Scheme 3) in high yields (83%).
It was found that 3-nitro-2-bromobenzene 8 could undergo the
reaction. The synthetic sequence was depicted in Scheme 4.
Scheme 5 Proposed reaction mechanism.
1-aminophosphonates 2 gave O,O′-diethyl-α-(benzothiazole-2-
yl) amino(phenylmethyl) phosphonate 9 in 80% yield.
A plausible mechanism for the formation of the target
compounds was illustrated in Scheme 5. The reaction of primary
1-aminophosphonate 2 with carbon disulfide in the presence of
K2CO3 to give dithiocarbamate salts A, and 3-nitro-2-bromopyri-
dine 4 was reduced by tin(II) chloride to 3-amino-2-bromopyri-
dine 1. Under the catalysis of CuCl2·2H2O, dithiocarbamate salts
A can react with 3-amino-2-bromopyridine 1 to provide dithio-
carbamates B. The amine group in B could in turn attack the
C–S double bond to give cyclization products C which would
deliver the desired products 3 upon elimination of hydrogen
sulfide under the action of CuCl2·2H2O.
The reaction of 3-nitro-2-bromobenzene
8 with primary
The structure of α-aminophosphonate derivatives 3a–g and
7 was thoroughly characterized with spectral and elemental ana-
lyses. The infrared spectrum of compound 3a displayed bands at
3213 cm−1, 1241 cm−1 and 1011 cm−1 due to N–H, PvO, and
Scheme 2 Synthesis of α-aminophosphonate derivatives 3.
Table 1 Substrate scope of primary 1-aminophosphonates
1
P–O stretching frequencies, respectively. The H NMR spectrum
of 3a exhibited a multiplet appeared at δ 7.31–7.42, which
accounts for the aromatic protons of the phenyl ring; a doublet
appeared at δ 5.77, which accounts for CH proton; a multiplet
appeared at δ 4.05–4.27, which accounts for the CH2 proton.
The two ethoxy groups of the phosphonates are magnetically
nonequivalent, which is caused by the different shielding effects
of α-benzene on the two ethoxy groups. The mass spectrum dis-
played the molecular ion [M]+ peak at m/z: 377. All of the none-
quivalent carbon atoms were identified in 13C NMR and the total
number of protons calculated from the integration curve
accorded (in 1H NMR) with the assigned structures.
Entry
Ar
R
Product
Yield (%)
1
2
3
4
5
6
7
Ph
Et
Et
Et
Et
Et
Et
Me
3a
3b
3c
3d
3e
3f
82
80
83
85
81
80
83
o-F-C6H4
p-Me-C6H4
p-Cl-C6H4
p-MeO-C6H4
p-F-C6H4
Ph
3g
Biological activities
The antitumor activities were determined by the standard MTT
assay13 against three cancer cell lines PC-3, Bcap-37 and H460.
The screening results expressed as IC50 are summarized in
Scheme 3 Synthesis of α-aminophosphonate derivative 7.
Org. Biomol. Chem.
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Table 2 The antitumor activities of the target compounds
IC50 (μM)
determined on an XT-4 micro melting point apparatus and uncor-
rected. IR spectra were recorded on an EQUINOX-55 spec-
trometer on a KBr matrix. NMR spectra were recorded on an
INOVA-400 NMR instrument at room temperature using TMS as
internal standard. Coupling constants (J) were measured in Hz.
Chemical shift values (δ) are given in ppm. Elemental analyses
were performed on a Vario EL III CHNS analyzer. Electrospray
mass spectra were obtained with an MALDI-TOF Mass spec-
trometer. 200–300 Mesh silica gel was used for column
chromatography.
PC-3
Bcap-37
H460
Compd. 3a
Compd. 3b
Compd. 3c
Compd. 3d
Compd. 3e
Compd. 3f
Compd. 3g
Compd. 7
Compd. 9
6.13
0.84
12.02
2.97
25.18
1.04
8.91
8.49
1.72
17.75
6.62
33.71
0.81
0.96
12.55
3.19
9.26
5.13
23.10
2.23
14.64
11.69
27.21
5.65
24.77
8.41
22.83
Synthesis of target compound 3a (solvent-catalyst free con-
dition). A reaction mixture of benzaldehyde (3 mmol), thiazolo-
[5,4-b]pyridin-2-yl amine (3 mmol) and diethyl phosphate
(3 mmol) was stirred in silicone oil bath at 100 °C for 2 h. The
resultant viscous liquid was dissolved in ether and then washed,
first with saturated aqueous NaHCO3 and next with distilled
water. The organic layer was separated, followed by extraction of
the aqueous layer with ethyl acetate (3 × 10 mL). The combined
organic layers were dried over MgSO4 and concentrated under
reduced pressure to yield the crude product. The crude product
was purified by flash chromatography with ethyl acetate/pet-
roleum ether as eluent on silica gel to afford the desired product.
Table 2. The IC50 values were the average of three independent
experiments. According to the antitumor activity data, some of
the target compounds exhibited potential anticancer activities.
Compound 3f bearing a fluorine atom at 4-position of phenyl
ring showed good activity against the tested cancer cell lines,
removal of the fluorine atom of 3f to give 3a caused the inhi-
bition to decrease more than 5-fold, and replacement of the
fluorine atom with methyl or methoxyl substituent in the 4-posi-
tion of phenyl ring had a deactivating effect. Compound 7 with
methyl substituent in thiazole[5,4-b]pyridine moiety retained
micromolar activity against PC-3 and Bcap-37 cells. Interest-
ingly, the compound containing thiazole[5,4-b]pyridine
moiety was significantly more active than their benzothiazole-
containing analogue (3a vs. 9, Table 2), which suggested that the
thiazole[5,4-b]pyridine moiety was crucial for the potency
enhancements.
Typical procedure for the synthesis of target compounds
(cascade reaction condition). A reaction mixture of 3-nitro-2-
bromopyridine 4 (1 mmol), CS2 (1.5 mmol), primary 1-amino-
phosphonate 5 (1.4 mmol), K2CO3 (3 mmol), CuCl2·2H2O
(1 mmol) and SnCl2·2H2O (4 mmol) in DMF (3.5 mL) was
stirred at 105 °C for 8 h. The reaction mixture was partitioned
between ethyl acetate and water. The organic layer was washed
with water and brine, and dried over anhydrous MgSO4 and con-
centrated under vacuum to yield the crude product. The crude
product was purified by flash chromatography with ethyl acetate/
petroleum ether as eluent on silica gel to afford the desired
product.
From the above results, some interesting structure–activity
relationships can be disclosed that: (i) the substituted position of
the phenyl group has no evident effect on the antitumor activity.
(ii) The introduction of fluoro groups was crucial for improving
their activity.
In conclusion, we have developed a new and efficient syn-
thetic route to α-aminophosphonates containing thiazole[5,4-b]-
pyridine moiety via a cascade three-component reaction from
conveniently available starting materials. The antitumor activities
of the target compounds were evaluated against three human
cancer cell lines (PC-3, Bcap-37 and H460). Most of the target
compounds displayed from moderate to excellent antitumor
activities against one or two cancer cell lines. Compounds 3b
and 3f were the most efficient, revealing a potential therapeutic
application of antitumor. Influence of subtle structural modifi-
cation and steric parameters on structure activity relationships for
identifying lead bioactive compound would be taken up in our
future course of investigation.
O,O′-Diethyl-α-(thiazolo[5,4-b]pyridin-2-yl)amino(phenylmethyl)-
phosphonate (3a). White powder, m.p. 114–116 °C; IR (KBr)
1
v: 3213, 2929, 2203, 1241 and 1011 cm−1. H NMR (CDCl3,
400 MHz): δ 8.42 (dd, J = 5.2 Hz, J = 1.2 Hz, 1 H), 8.11 (dd,
J = 7.2 Hz, J = 0.8 Hz, 1 H), 7.81 (dd, J = 6.8 Hz, J = 2.4 Hz,
1 H), 7.31–7.42 (m, 5 H), 5.77 (d, J = 20.8 Hz, 1 H), 4.67 (bs,
1H), 4.05–4.27 (m, 2 H), 3.83–3.92 (m, 2 H), 1.31 (t, J =
7.2 Hz, 3 H), 1.20 (t, J = 7.2 Hz, 3 H); 13C NMR (CDCl3,
75 MHz): δ 166.3, 160.1, 142.7, 138.4, 128.5, 128.3, 126.1,
126.0, 125.6, 123.2, 122.8, 121.2, 65.4 (d, J = 3.0 Hz), 59.0,
58.8, 16.6 16.1; 31P NMR (CDCl3, 200 MHz): δ 19.7. MALDI-
TOF MS: m/z = 377 (M)+. Anal. Calcd for C17H20N3O3PS:
C 54.10, H 5.34, N 11.13, S 8.50; found: C 54.13, H 5.38,
N 11.07, S 8.46.
Experimental
O,O′-Diethyl-α-(thiazolo[5,4-b]pyridin-2-yl)amino(2-fluorophenyl-
methyl)phosphonate (3b). White powder, m.p. 103–105 °C; IR
Synthesis
(KBr) v: 3234, 2981, 2208, 1233 and 1013 cm−1 1H NMR
.
Materials and equipment. Reagents were obtained commer-
cially and used as received. Solvents were purified and dried by
standard methods. Thiazolo[5,4-b]pyridin-2-yl amine was syn-
thesized according to a modified literature procedure.12 primary
1-aminophosphonates 2 was synthesized according to the
method described in the literature.13 The melting points were
(CDCl3, 400 MHz): δ 8.31 (dd, J = 4.4 Hz, J = 4.4 Hz, 1 H),
8.17 (dd, J = 6.0 Hz, J = 4.8 Hz, 1 H), 7.95 (dd, J = 2.0 Hz, J =
1.6 Hz, 1 H), 7.30–7.59 (m, 4 H), 5.82 (d, J = 22.8 Hz, 1 H),
4.59 (bs, 1H), 4.26–4.30 (m, 2 H), 3.98–4.17 (m, 2 H), 1.34 (t,
J = 6.4 Hz, 3 H), 1.26 (t, J = 6.4 Hz, 3 H); 13C NMR (CDCl3,
75 MHz): δ 163.8, 157.9, 140.7, 135.0, 129.5, 128.5, 128.1,
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O,O′-Dimethyl-α-(thiazolo[5,4-b]pyridin-2-yl)amino(phenylmethyl)-
phosphornate (3g). White powder, m.p. 102–104 °C; IR (KBr)
127.1, 126.6, 125.8, 125.1, 121.8, 68.6 (d, J = 3.6 Hz), 57.7,
56.1, 16.2, 16.1; 31P NMR (CDCl3, 200 MHz): δ 20.3.
MALDI-TOF MS: m/z = 396 [M + H]+. Anal. Calcd for
C17H19FN3O3PS: C 51.64, H 4.84, N 10.68, S 8.11; found:
C 51.67, H 4.88, N 10.71, S 8.13.
1
v: 3219, 2941, 2217, 1252 and 1026 cm−1. H NMR (CDCl3,
400 MHz): δ 8.25 (dd, J = 6.4 Hz, J = 1.6 Hz, 1 H), 8.18 (dd,
J = 6.8 Hz, J = 1.2 Hz, 1 H), 8.09 (dd, J = 1.2 Hz, J = 2.4 Hz,
1 H), 7.27–7.42 (m, 5 H), 5.63 (d, J = 22.0 Hz, 1 H), 4.72 (bs,
1H), 3.83 (d, J = 10.8 Hz, 3 H), 3.63 (d, J = 10.8 Hz, 3 H);
13C NMR (CDCl3, 75 MHz): δ 163.5, 159.4, 142.7, 140.1,
129.1, 128.3, 127.7, 127.1, 126.3, 123.4, 122.1, 121.7, 63.8
(d, J = 8.7 Hz), 55.7, 53.2; 31P NMR (CDCl3, 200 MHz):
δ 20.4. MALDI-TOF MS: m/z = 349 (M)+. Anal. Calcd for
C15H16N3O3PS: C 51.57, H 4.62, N 12.03, S 9.18; found:
C 51.62, H 4.66, N 12.00, S 9.12.
O,O′-Diethyl-α-(thiazolo[5,4-b]pyridin-2-yl)amino(4-methylphenyl-
methyl)phosphonate (3c). White powder, m.p. 111–112 °C; IR
(KBr) v: 3285, 2988, 2202, 1231 and 1027 cm−1 1H NMR
.
(CDCl3, 400 MHz): δ 8.28 (dd, J = 2.4 Hz, J = 0.4 Hz, 1 H),
8.16 (dd, J = 1.2 Hz, J = 2.4 Hz, 1 H), 7.96 (dd, J = 1.2 Hz, J =
6.4 Hz, 1 H), 7.20 (d, J = 2.0 Hz, 2 H), 7.18 (d, J = 2.0 Hz,
2 H), 5.28 (d, J = 22.0 Hz, 1 H), 4.90 (bs, 1H), 3.77–4.20 (m,
4 H), 2.33 (s, 3 H), 1.29 (t, J = 7.2 Hz, 3 H), 1.16 (t, J = 7.2 Hz,
3 H); 13C NMR (CDCl3, 75 MHz): δ 164.8, 161.3, 139.7, 139.1,
129.5, 129.0, 128.4, 128.1, 127.7, 127.2, 126.3, 67.2 (d, J =
3.0 Hz), 59.7, 58.6, 20.8, 14.6, 14.4; 31P NMR (CDCl3,
200 MHz): δ 21.5. MALDI-TOF MS: m/z = 391 (M)+. Anal.
Calcd for C18H22N3O3PS: C 55.23, H 5.67, N 10.74, S 8.19;
found: C 55.22, H 5.70, N 10.71, S 8.21.
O,O′-Diethyl-α-(4-methylthiazolo[5,4-b]pyridin-2-yl)amino(phe-
nylmethyl)phosphonate (7). White powder, m.p. 107–109 °C; IR
(KBr) v: 3233, 2923, 2206, 1252 and 1019 cm−1 1H NMR
.
(CDCl3, 400 MHz): δ 8.45 (d, J = 2.8 Hz, 1 H), 8.33 (d, J =
2.8 Hz, 1 H), 7.37–7.08 (m, 5 H), 5.65 (d, J = 22.4 Hz, 1 H),
4.54 (bs, 1H), 3.76–4.24 (m, 4 H), 2.22 (s, 3 H), 1.32 (t, J =
7.2 Hz, 3 H), 1.23 (t, J = 7.2 Hz, 3 H); 13C NMR (CDCl3,
75 MHz): δ 167.1, 162.3, 152.2, 141.2, 138.4, 130.0, 129.1,
128.1, 125.6, 123.4, 122.6, 121.2, 66.7 (d, J = 3.8 Hz), 59.1,
58.0, 21.3, 16.2 16.0; 31P NMR (CDCl3, 200 MHz): δ 21.4.
MALDI-TOF MS: m/z = 414 [M + Na]+. Anal. Calcd for
C18H22N3O3PS: C 55.23, H 5.67, N 10.74, S 8.19; found:
C 55.34, H 5.74, N 10.81, S 8.10.
O,O′-Diethyl-α-(thiazolo[5,4-b]pyridin-2-yl)amino(4-chloro phe-
nylmethyl)phosphonate (3d). White powder, m.p. 121–123 °C; IR
(KBr) v: 3233, 2931, 2211, 1239 and 1038 cm−1 1H NMR
.
(CDCl3, 400 MHz): δ 8.29 (dd, J = 4.8 Hz, J = 2.4 Hz, 1 H),
8.17 (dd, J = 4.0 Hz, J = 1.2 Hz, 1 H), 7.92 (dd, J = 5.6 Hz, J =
0.8 Hz, 1 H), 7.47 (d, J = 2.4 Hz, 2 H), 7.32 (d, J = 2.4 Hz,
2 H), 5.66 (d, J = 21.6 Hz, 1 H), 4.63 (bs, 1H), 3.87–4.23 (m,
4 H), 1.44 (t, J = 5.6 Hz, 3 H), 1.39 (t, J = 5.6 Hz, 3 H);
13C NMR (CDCl3, 75 MHz): δ 162.6, 158.7, 143.2, 140.1,
130.5, 129.1, 128.6, 128.0, 127.3, 126.9, 126.1, 63.2 (d, J =
3.0 Hz), 60.4, 59.2, 14.8, 14.0; 31P NMR (CDCl3, 200 MHz):
δ 19.9. MALDI-TOF MS: m/z = 434 [M + Na]+. Anal. Calcd for
C17H19ClN3O3PS: C 49.58, H 4.65, N 10.20, S 7.79; found:
C 49.61, H 4.58, N 10.27, S 7.82.
O,O′-Diethyl-α-(thiazolo[5,4-b]pyridin-2-yl)amino(4-methoxyl phe-
nylmethyl)phosphonate (3e). White powder, m.p. 107–109 °C; IR
(KBr) v: 3224, 2979, 2201, 1232 and 1026 cm−1. δ 8.06–8.30
(m, 3 H), 7.30 (d, J = 8.4 Hz, 2 H), 6.90 (d, J = 8.4 Hz, 2 H),
5.49 (d, J = 22.0 Hz, 1 H), 4.81 (bs, 1H), 3.79–4.23 (m, 7 H),
1.32 (t, J = 7.2 Hz, 3 H), 1.19 (t, J = 7.2 Hz, 3 H); 13C NMR
(CDCl3, 75 MHz): δ 163.4, 159.1, 142.5, 140.2, 131.6, 130.3,
129.8, 128.2, 127.7, 127.1, 126.7, 65.9 (d, J = 3.75 Hz), 58.7,
57.6, 55.3, 16.4, 16.2; 31P NMR (CDCl3, 200 MHz): δ 20.9.
MALDI-TOF MS: m/z = 430 [M + Na]+. Anal. Calcd for
C18H22N3O4PS: C 55.23, H 5.67, N 10.74, S 8.19; found:
C 53.06, H 5.44, N 10.31, S 7.87.
O,O′-Diethyl-α-(benzothiazole-2-yl)amino(phenylmethyl)phospho-
nate (9). White powder, m.p. 112–114 °C (lit.14 m.p. 112 °C);
IR (KBr) v: 3210, 2907, 1256 and 1031 cm−1 1H NMR
.
(CDCl3, 400 MHz): δ 7.54 (dd, J = 7.2 Hz, J = 7.2 Hz, 4 H),
7.06–7.39 (m, 5 H), 5.54 (d, J = 22.4 Hz, 1 H), 4.84 (bs, 1H),
3.98–4.22 (m, 4 H), 1.30 (t, J = 7.2 Hz, 3 H), 1.15 (t, J =
7.2 Hz, 3 H); 13C NMR (CDCl3, 75 MHz): δ 151.9, 135.0,
131.0, 128.5, 128.2, 128.1, 125.8, 121.8, 120.7, 119.3, 63.6
(d, J = 10.7 Hz), 56.7, 55.1, 16.4, 16.2; 31P NMR (CDCl3,
200 MHz): δ 22.7. MALDI-TOF MS: m/z = 399 [M + Na]+.
Anal. Calcd for C18H21N2O3PS: C 57.44, H 5.62, N 7.44,
S 8.52; found: C 57.56, H 5.69, N 7.41, S 8.49.
Antitumor activities assay in vitro
The antitumor activities of the target compounds (3a–3g, 7)
were evaluated with human prostate cancer cell line (PC-3),
human breast cancer cell line (Bcap-37), and human large cell
lung cancer cell line (H460). PC-3, Bcap-37 and H460 were
obtained from the American Type Culture Collection (Manassas,
VA). Cells were cultured in RPMI 1640 and maintained in a
Thermo incubator (Waltham, MA) with a humidified air contain-
ing of 5% CO2, 95% air. All culture media contained 10% fetal
bovine serum (FBS) and 1% penicillin–streptomycin solution
(10 000 units of penicillin and 10 mg of streptomycin in 0.9%
NaCl). The cancer cell lines were cultured in minimum essential
medium (MEM). Four-thousand cells (per well) suspended in
MEM, were plated onto each well of a 96-well plate and incu-
bated for 24 h. The tested compounds at the indicated final con-
centrations were added to the culture medium and the cell
cultures were continued for 72 h. Fresh MTT was added to each
well at the terminal concentration of 5 μg mL−1 and incubated
O,O′-Diethyl-α-(thiazolo[5,4-b]pyridin-2-yl)amino(4-fluoro phe-
nylmethyl)phosphonate (3f). White powder, m.p. 119–121 °C; IR
(KBr) v: 3221, 2911, 2209, 1251 and 1026 cm−1 1H NMR
.
(CDCl3, 400 MHz): δ 8.24 (dd, J = 2.4 Hz, J = 1.2 Hz, 1 H),
8.11 (dd, J = 2.0 Hz, J = 0.8 Hz, 1 H), 7.92 (dd, J = 2.4 Hz, J =
1.2 Hz, 1 H), 7.59 (d, J = 2.0 Hz, 2 H), 7.48 (d, J = 2.0 Hz,
2 H), 5.57 (d, J = 22.4 Hz, 1 H), 4.81 (bs, 1H), 3.83–4.24 (m,
4 H), 1.32 (t, J = 7.2 Hz, 3 H), 1.18 (t, J = 7.2 Hz, 3 H);
13C NMR (CDCl3, 75 MHz): δ 163.7, 159.2, 140.8, 139.9,
130.2, 129.6, 129.0, 128.3, 127.5, 126.2, 125.7, 65.1 (d, J =
7.4 Hz), 59.7, 58.9, 14.4, 14.1; 31P NMR (CDCl3, 200 MHz):
δ 21.9. MALDI-TOF MS: m/z = 418 [M + Na]+. Anal. Calcd for
C17H19FN3O3PS: C 51.64, H 4.84, N 10.63, S 8.11; found:
C 51.71, H 4.90, N 10.66, S 8.12.
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Acknowledgements
This work was supported by The State Ethnic Affairs Commis-
sion, opening fund of Key Laboratory of Ethnic Medicine
Resource Chemistry (MZY 1117), the Youth Fund of Yunnan
University of Nationalities (No. 09QN04).
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