Synthesis and cytotoxicity of O,O′-dialkyl {[2-(substituted phenoxy)acetamido](substituted phenyl)methyl}phosphonates

Article abstract of DOI:10.1016/j.ejmech.2011.12.014

A series of O,O′-dialkyl {[2-(substituted phenoxy)acetamido] (substituted phenyl)methyl}phosphonates was synthesized and their cytotoxic activities were tested against various human tumor cell lines. Some compounds (5q, 5r, 5s, 5w, 5x and 5y) showed relatively high cytotoxicity. Especially, compounds 5x and 5q exhibited the best cytotoxicity against KB and CNE2 cells with IC₅₀ 7.1 and 11.4 μM, respectively. Their inhibitory activities against KB and CNE2 cells were even higher than that of fluorouracil.

Full text of DOI:10.1016/j.ejmech.2011.12.014

European Journal of Medicinal Chemistry 48 (2012) 379e384
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and cytotoxicity of O,O0-dialkyl {[2-(substituted phenoxy)
acetamido](substituted phenyl)methyl}phosphonates
,
,
,
a,
*
Lihong Ning a 1, Wei Wang a 1, Yongju Liang b, Hao Peng a, Liwu Fu b , Hongwu He
*
a Key Laboratory of Pesticide and Chemical Biology, Ministry of Education and College of Chemistry, Central China Normal University, Wuhan 430079, PR China
b State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-sen University, Guangzhou 510060, PR China
a r t i c l e i n f o
a b s t r a c t
A series of O,O0-dialkyl {[2-(substituted phenoxy)acetamido](substituted phenyl)methyl}phosphonates
was synthesized and their cytotoxic activities were tested against various human tumor cell lines. Some
compounds (5q, 5r, 5s, 5w, 5x and 5y) showed relatively high cytotoxicity. Especially, compounds 5x and
Article history:
Received 27 July 2011
Received in revised form
7 December 2011
Accepted 8 December 2011
Available online 16 December 2011
5q exhibited the best cytotoxicity against KB and CNE2 cells with IC50 7.1 and 11.4
inhibitory activities against KB and CNE2 cells were even higher than that of fluorouracil.
Ó 2011 Elsevier Masson SAS. All rights reserved.
mM, respectively. Their
Keywords:
Synthesis
Cytotoxicity
Phosphonate
1. Introduction
interesting that all of these compounds have poor herbicidal
activities, however, some of them showed potential cytotoxicity
against two human tumor cells. Here we would like to describe the
synthesis and cytotoxicity of O,O0-dialkyl {[2-(substituted phenoxy)
acetamido](substituted phenyl)methyl}phosphonates 5 from easily
Organophosphorus compounds have attracted intense interest
owing to their wide applications in the areas of industrial, agricul-
tural, and medicinal chemistry and their potential biological and
physical properties [1e4]. As mimics of
a-amino acids, the
a-ami-
accessible O,O0-dialkyl
a-aminophosphonates 4.
nophosphonic acids and their derivatives are an important class of
compounds that exhibited intriguing biological activities [5e14].
Indeed a number of potent antibiotics [5,6], enzyme inhibitors [7],
2. Results and discussion
antiviral, and antitumor agents [8,9] are a-aminophosphonic acids
2.1. Chemistry
or peptide analogues thereof. On the other hand, a variety of the
reports regarding synthetic studies of the amide derivatives have
been presented because they can serve not only as agrochemicals
such as fungicide, insecticide, and plant virucide, but also as medi-
cines such as antitumor agents [15e19]. Especially some amide
The synthesis of O,O0-dialkyl {[2-(substituted phenoxy)acet-
amido](substituted phenyl)methyl}phosphonates 5aez is outlined
in Scheme 1.
O,O0-dialkyl
N-(phenylmethylene)-
phenyl)methylphosphonates 2, obtained by reaction of
substituted benzaldehyde
with ammonia and O,O0-dialkyl
phosphite, were converted easily to O,O0-dialkyl
-amino-
a-amino-a-(substituted
derivatives containing
recently described as potent antifungal and antiviral agents [20].
In our previous work, some -(substituted phenoxyacetoxy)
a-aminophosphonate moiety have been
1
a
a
a-
alkylphosphonate derivatives were synthesized and shown to be
endowed with notable herbicidal activities [21]. To extend our
(substituted phenyl)methylphosphonate 4 via hydrolysis with p-
toluenesulfonic acid and neutralization of the sulfonate salt [22].
research work of developing novel herbicides, some
a-amino-
The
a-aminophosphonates 4 reacted with substituted phe-
noxyacetic chloride to give O,O0-dialkyl {[2-(substituted phe-
phosphonate derivatives were designed and synthesized. It is very
noxy)acetamido](substituted phenyl)methyl}phosphonates
satisfactory yields. The results are listed in Table 1.
5 in
All the compounds in the series 5 were obtained as white solids
or yellowish oils after chromatography on silica gel with ethyl
acetate/petroleum ether. Their structures were fully characterized
by IR, 1H NMR, EI-MS and elemental analysis. For example, the IR
* Corresponding authors. Tel./fax: þ86 27 67867960.
E-mail addresses: fulw@mail.sysu.edu.cn (L. Fu), he1208@mail.ccnu.edu.cn
(H. He).
1
These authors contributed equally to this study.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.12.014

380
L. Ning et al. / European Journal of Medicinal Chemistry 48 (2012) 379e384
O
P
O
P
OR1
OR1
OR1
TsOH3N
C
H
N
1)NH4OH(aq)
OR1
CHO
P-TsOH H2O
R2
R2
O
P
2)
OR1
OR1
R2
R2
H
3
1
2
R3
O
P
OR1
O
P
R3
OR1
OR1
Cl
H
N
H2N
R2
O
OR1
O
NH4OH(aq)
O
O
R2
Et3N
5
4
Scheme 1. General synthetic route for compound 5aez.
spectrum of 5a revealed absorption bands at 1693 (C]O), 3067
(C6H5), and 3224 (NH) cmꢀ1. The corresponding 1H NMR spectrum
showed the two methoxy groups attached with phosphorus
appears as two doublet at 3.57 and 3.74, respectively. The chemical
shifts of the two methyl hydrogens differentiate due to the low rate
of environmental exchange caused by the slow rotation of the PeC
bond, and the magnetic nucleus phosphorus makes the signal of
both methyls split into a doublet. The signal corresponding to the
methylene group (eCH2e) flanked by the phenoxy group and
carbonyl group appears as a quartet, the outside lines smaller in
size, which belongs to the AB system with the difference in
chemical shift between the two mutually coupled protons A and B,
at 4.52 and 4.54 in the former series and at 4.59 and 4.62 in the
latter series, respectively. The other signals resonated at d(H) 5.57
(dd, 1H, PCH), 6.81e7.45 (m, 8H, Ar-H), 7.72 (d, 1H, NH). The
structures of 5a and the other analogs were further confirmed on
the basis of elemental analysis.
(5q, 5r, 5s, 5w, 5x and 5y) showed relatively high cytotoxicity.
Especially, compounds 5x and 5q exhibited the best cytotoxicity
against KB and CNE2 cells with IC50 7.1 and 11.4 mM, respectively.
Their inhibitory activity against KB and CNE2 cells were even higher
than that of fluorouracil.
4. Experimental
4.1. Chemistry
1H NMR spectra were recorded at 600 MHz, in CDCl3 on a Varian
Mercury-Plus 600 spectrometer and chemical shifts were recorded
in parts per million (ppm) with TMS as the internal reference. MS
spectra were determined using a TraceMS 2000 organic mass
spectrometry, and signals were given in m/z. Melting points were
determined using a X-4 apparatus and were uncorrected. Elemental
analysis (EA) was measured on a Vario ELIII CHNSO elemental
analyzer. Element analyses (C, H, N) indicated by the symbols of the
elements or functions were within ꢁ0.4% of the theoretical values.
All commercially available solvents and reagents were used as
supplied by Acros Organics unless otherwise stated. The silica gel
(200e300 meshes) for flash column chromatography was from
Qingdao Marine Chemical Factory in China.
2.2. Cytotoxicity
The cytotoxicity results of all of the compounds against KB and
CNE2 tumor cell lines are listed in Table 1. As shown in Table 1, some
compounds (5q, 5r, 5s, 5w, 5x and 5y) exhibited good to moderate
cytotoxicity. Especially, compounds 5x exhibited the best cytotox-
icity against KB cell with IC50 7.1 mM, which is even higher than that
4.2. General procedure for the preparation of O,O0-dialkyl {[2-
(substituted phenoxy)acetamido](substituted phenyl)methyl}
phosphonates 5
of fluorouracil. However, replacement of R1 n-butyl group in
phosphonate moiety of compound 5x with methyl, resulted in the
compound 5d with complete loss in activity (IC50 > 50 mM), and the
other compounds with methyl or ethyl as R1 substituent (5aej) also
showed no inhibitory activity. It could be concluded that n-butyl as
R1 is favorable to the cytotoxicity. As for CNE2, in the series of
compounds 5pe5z substituted at the R1 position with n-butyl
derivatives, compound 5q showed the best inhibitory activity with
A suspension of aromatic aldehyde (10 mmol) and ammonium
hydroxide (30%, 10 mL) was stirred for 5 h at reflux. The reaction
mixture was filtered to give the white precipitate 2, to which the
dialkyl phosphite (4 mmol) was added and the resulting solution
was stirred for 2e5 h at 70 ꢂC p-Toluenesulfonic acid (4 mmol) in
30 mL THF was added to the reaction mixture, which was stirred for
2 h at 0 ꢂC to give the precipitate 3. The precipitate was filtered and
washed with THF (15 mL), and then it was added to 10 mL aqueous
ammonium hydroxide (30%) and stirred for 30 min at room
temperature. Extraction with ethyl acetate (3 ꢃ 30 mL) and evap-
oration of the solvent gave the oils of 4, which was purified by
column chromatography on silica gel with ethyl acetate. To
a mixture of 4 and pyridine in CH2Cl2 (10 mL), the appropriate
substituted phenoxyacetic chloride was added at 2e5 ꢂC and the
mixture was stirred at ambient temperature for another 3 h, then
washed with saturated sodium hydrogen carbonate solution and
brine, dried with anhydrous sodium sulfate and evaporated. The
residue was purified by column chromatography on silica gel with
IC50 11.4 mM. The compounds 5q, 5r, 5w, and 5y with chlorine at 2-
position in benzene ring (R3) showed better inhibitory activity
against CNE2 compared to the compounds 5p, 5s, 5x with 2-methyl
or 2-unsubstituted 5t, 5v and 5z, which indicated that the chlorine
introduced was beneficial to cytotoxic activity.
3. Conclusion
We synthesized a new series of O,O0-dialkyl {[2-(substituted
phenoxy)acetamido](substituted phenyl)methyl}phosphonates via
reaction of functionalized
a-aminophosphonates with various
substituted phenoxyacetic chloride. Their cytotoxic activities were
tested against various human tumor cell lines. Some compounds

L. Ning et al. / European Journal of Medicinal Chemistry 48 (2012) 379e384
381
Table 1
6H, 2Ar-CH3), 3.51 (d, J ¼ 11.2 Hz, 3H, OCH3), 3.73 (d, J ¼ 10.8 Hz, 3H,
OCH3), 4.44e4.56 (q, AB system, 2H, OCH2CO), 5.54 (dd, J ¼ 10.0,
20.8 Hz, 1H, PCH), 6.81 (d, J ¼ 2.4 Hz, 1H, Ar-H), 6.71 (dd, J ¼ 2.4 Hz,
1H, Ar-H), 7.16e7.29 (m, 5H, Ar-H), 7.34 (d, 1H, NH). 13C NMR
Yields and in vitro cytotoxicity (IC50,
amM) of O,O0-dialkyl {[2-(substituted phenoxy)
acetamido](substituted phenyl)methyl}phosphonates 5.
Compd R1
R2
R3
Yieldb Cytotoxicity Cytotoxicity
(%)
against KBc against CNE2c
(150 MHz CDCl3)
d
(ppm): 20.3, 21.2, 49.0 (d, J ¼ 157.8 Hz, PCH), 53.7
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
Me
H
2,4-diCl
2,4-diCl
3eCH3ꢀ4ꢀCl 73
78
82
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
(d, J ¼ 83.3 Hz, POCH3), 67.5 (OCH2), 113.4, 117.4, 127.5127.9, 129.6,
129.9, 131.1, 137.5, 138.4, 155.5, 167.4 (CO). MS (70 ev) m/z (%): 411
(Mþ). Anal. C19H20ClNO5P (C, H, N).
Me
Me
Me
Me
Et
Et
Et
Et
4eCH3
4eCH3
4eOCH3 2,3-diCH3
4eOCH3 2,4-diCl
4eOCH3 2,4-diCl
4eOCH3 4-Cl
63
80
75
77
88
80
4.2.4. O,O0-Dimethyl {[2-(2,3-dimethylphenoxy)acetamido](4-
methoxyphenyl)methyl}phosphonate(5d)
4eOCH3 3eCF3
4eOCH3 4eNO2
White solid. m.p. 141e142 ꢂC. IR(KBr)
n
: 3225, 3055, 1696(C]O),
>50
>50
1554, 1471, 1249 cmꢀ1 1H NMR (600 MHz CDCl3)
d(ppm): 2.27 (s,
Et
4eOCH3 2eCH3ꢀ4ꢀCl 95
>50
>50
n-Pr
n-Pr
n-Pr
H
H
H
2,4-diCl
4-Cl
58
75
61
74
60
93
78
64
16.0 ꢁ 0.9
23.0 ꢁ 1.1
21.6 ꢁ 1.1
17.4 ꢁ 0.8
19.8 ꢁ 1.0
18.2 ꢁ 0.9
15.8 ꢁ 0.8
17.8 ꢁ 0.9
12.3 ꢁ 0.6
18.1 ꢁ 0.9
15.9 ꢁ 0.8
25.5 ꢁ 1.3
12.3 ꢁ 0.6
7.1 ꢁ 0.4
14.5 ꢁ 0.7
13.3 ꢁ 0.6
9.0 ꢁ 0.6
26.0 ꢁ 1.4
42.9 ꢁ 2.1
41.8 ꢁ 2.0
27.2 ꢁ 1.4
38.5 ꢁ 2.0
25.1 ꢁ 1.3
11.4 ꢁ 0.6
13.8 ꢁ 0.7
19.9 ꢁ 1.0
43.0 ꢁ 2.2
>50
41.2 ꢁ 2.0
13.1 ꢁ 0.6
20.7 ꢁ 1.0
13.4 ꢁ 0.7
31.1 ꢁ 1.5
13.5 ꢁ 1.0
3H, Ar-CH3), 2.31 (s, 3H, Ar-CH3) 3.55 (d, J ¼ 10.8 Hz, 3H, OCH3), 3.73
(d, J ¼ 10.2 Hz, 3H, OCH3), 4.48e4.57 (q, AB system, 2H, OCH2CO),
5.52 (dd, 1H, PCH, J ¼ 9.6, 20.4 Hz), 6.64(d, 1H, J ¼ 7.8 Hz, Ar-H),
6.86e7.30 (m, 6H, Ar-H), 7.48 (d, 1H, NH). 13C NMR (150 MHz
3eCF3
n-Pr 4eOCH3 2,4-diCl
n-Pr 4eOCH3 3eCF3
CDCl3)
d
(ppm): 11.9, 20.1, 48.6 (d, J ¼ 157.8 Hz, PCH), 53.7 (d,
n-Bu
n-Bu
n-Bu
n-Bu
n-Bu
n-Bu
n-Bu
n-Bu
H
H
H
H
H
H
H
H
2,3-diCH3
2-Cl
2,4-diCl
J ¼ 69.8 Hz, POCH3), 55.3, 67.7 (OCH2), 109.5, 114.3, 123.8, 125.1,
126.1, 126.3, 129.0, 138.4, 155.1, 159.6, 167.8 (CO). MS (70 ev) m/z (%):
407 (Mþ). Anal. C20H26NO6P (C, H, N).
5s
5t
2eCH3ꢀ4ꢀCl 80
3eCH3ꢀ4ꢀCl 75
5u
5v
5w
5x
5y
5z
2,4,5-triCl
4eOCH3
2-Cl-4-F
65
78
66
68
60
92
4.2.5. O,O0-Dimethyl {[2-(2,4-dichlorophenoxy)acetamido](4-
methoxyphenyl)methyl}phosphonate(5e)
n-Bu 4eOCH3 2,3-diCH3
n-Bu 4eOCH3 2-Cl
n-Bu 4eOCH3 4eC(CH3)3
White solid. m.p. 122e123 ꢂC. IR(KBr)
n
: 3222, 3053, 1698(C]O),
1552, 1482, 1240 cmꢀ1 1H NMR (600 MHz CDCl3)
d(ppm): 3.58 (d,
J ¼ 10.8 Hz, 3H, OCH3), 3.75 (d, J ¼ 10.8 Hz, 3H, OCH3), 3.80 (s, 3H,
Ar-OCH3), 4.50e4.61 (q, AB system, 2H, OCH2CO), 5.51 (dd, J ¼ 9.0,
20.4 Hz, 1H, PCH), 6.82 (d, J ¼ 8.4 Hz, 1H, Ar-H), 6.81e7.44 (m, 6H,
Fluorouracil
a
IC50 is the concentration of compound required to inhibit the cell growth by 50%
compared to an untreated control.
Ar-H), 7.63 (m, 1H, NH). 13C NMR (150 MHz CDCl3)
d(ppm): 48.7 (d,
b
Isolated yields based on
KB cells were the drug sensitive human oral carcinoma cells and CNE2 cells were
a
-aminophosphonates 4.
c
J ¼ 156.7 Hz, PCH), 53.7 (d, J ¼ 66.0 Hz, POCH3), 55.3, 68.2 (OCH2),
114.3, 114.8, 123.9, 126.1, 127.5, 127.9, 129.1, 130.2, 151.5, 159.6, 166.4
(CO). MS (70 ev) m/z (%): 448 (Mþ). Anal. C18H20Cl2NO6P (C, H, N).
the nasopharyngeal carcinoma cells.
petroleum ether/ethyl acetate (3:1) to give the pure title
4.2.6. O,O0-Diethyl {[2-(2,4-dichlorophenoxy)acetamido](4-
compounds 5 as white solids or yellowishish oils.
methoxyphenyl)methyl}phosphonate(5f)
White solid. m.p. 101e102 ꢂC IR(KBr)
n
: 3443, 3224, 1695(C]O),
4.2.1. O,O0-dimethyl {[2-(2,4-dichlorophenoxy)acetamido](phenyl)
1610, 1565, 1473, 1318 cmꢀ1 1H NMR (600 MH
Z
CDCl3) (ppm):
d
methyl}phosphonate(5a)
1.16e1.30 (m, 6H, 2CH3), 3.80 (s, 3H, OCH3), 3.83e4.12 (m, 4H,
OCH2), 4.50e4.60 (q, AB system, 2H, OCH2CO), 5.47 (dd, J ¼ 9.6,
19.8 Hz, 1H, PCH), 6.81e7.44 (m, 7H, Ar-H), 7.65 (m, 1H, NH). 13C
White solid. m.p. 150e152 ꢂC. IR(KBr)
n
: 3224, 3067, 1693(C]O),
1557, 1458, 1228 cmꢀ1 1H NMR (600 MHz CDCl3)
d(ppm): 3.57 (d,
J ¼ 10.8 Hz, 3H, OCH3), 3.74 (d, J ¼ 10.8 Hz, 3H, OCH3), 4.52e4.62 (q,
AB system, 2H, OCH2CO), 5.57 (dd, J ¼ 9.6, 20.4 Hz,1H, PCH), 6.82 (d,
J ¼ 9.0 Hz 1H, Ar-H), 7.20e7.45 (m, 7H, Ar-H), 7.72 (d, 1H, NH). 13C
NMR (150 MHz CDCl3)
d
(ppm): 16.4, 49.1 (d, J ¼ 153.0 Hz, PCH),
55.2, 63.1 (d, J ¼ 52.7 Hz, POCH2), 68.1 (OCH2), 114.1, 114.7, 123.8,
126.5, 127.3, 127.9, 129.0, 130.1, 151.5, 159.5, 166.3 (CO). MS (70 ev)
m/z (%): 477 (Mþ). Anal. C20H24Cl2NO6P (C, H, N).
NMR (150 MHz CDCl3)
d
(ppm): 49.1 (d, J ¼ 159.2 Hz, PCH), 53.5 (d,
J ¼ 60.0 Hz, POCH3), 67.9 (OCH2), 114.5, 123.7, 127.3, 127.6, 127.7,
128.3, 128.7, 130.0, 133.9, 151.2, 166.3 (CO). MS (70 ev) m/z (%): 417
(Mþ). Anal. C17H18Cl2NO5P (C, H, N).
4.2.7. O,O0-Diethyl {[2-(4-chlorophenoxy)acetamido](4-
methoxyphenyl)methyl}phosphonate(5g)
White solid. m.p. 119e120 ꢂC. IR(KBr)
n
: 3227, 3060, 1694(C]O),
CDCl3) (ppm): 1.11e1.29
4.2.2. O,O0-dimethyl {[2-(2,4-dichlorophenoxy)acetamido](4-
1553, 1490, 1218 cmꢀ1 1H NMR (600 MH
Z
d
methylphenyl)methyl}phosphonate(5b)
(m, 6H, 2CH3), 3.80 (s, 3H, OCH3), 3.72e4.10 (m, 4H, OCH2),
4.46e4.55 (q, AB system, 2H, OCH2CO), 5.46 (dd, J ¼ 9.6, 20.4 Hz,1H,
PCH), 6.86e7.33 (m, 8H, Ar-H, 1H, NH). 13C NMR (150 MHz CDCl3)
White solid. m.p. 165e166 ꢂC. IR(KBr)
n
: 3224, 3054, 1697(C]O),
1552, 1481, 1225 cmꢀ1 1H NMR (600 MHz CDCl3)
d(ppm): 2.34 (s,
3H, Ar-CH3), 3.57 (d, J ¼ 7.8 Hz, 3H, OCH3), 3.74 (d, J ¼ 7.8 Hz, 3H,
OCH3), 4.51e4.61 (q, AB system, 2H, OCH2CO), 5.53 (dd, 1H, PCH,
J ¼ 8.4, 21.6 Hz), 6.81e7.44 (m, 7H, Ar-H), 7.66 (d, 1H, NH). 13C NMR
d
(ppm): 16.3, 49.0 (d, J ¼ 153.0 Hz, PCH), 55.2, 63.2 (d, J ¼ 65.0 Hz,
POCH2), 67.5 (OCH2), 114.0, 116.1, 126.6, 126.9, 129.2, 129.5, 155.8,
159.5, 167.2 (CO). MS (70 ev) m/z (%): 441 (Mþ). Anal. C20H25ClNO6P
(C, H, N).
(150 MHz CDCl3)
d
(ppm): 21.2, 49.1(d, J ¼ 154.4 Hz, PCH), 53.7 (d,
J ¼ 60.0 Hz, POCH3), 68.2 (OCH2), 114.7, 123.9, 127.5, 127.7, 128.0,
129.6, 130.2, 131.1, 138.4, 151.5, 166.4 (CO). MS (70 ev) m/z (%): 431
(Mþ). Anal. C18H20Cl2NO5P (C, H, N).
4.2.8. O,O0-Diethyl {[2-(3-trifluoromethylphenoxy)acetamido](4-
methoxyphenyl)methyl}phosphonate(5h)
White solid. m.p. 103e104 ꢂC. IR(KBr)
n
: 3212, 3051, 1692(C]O),
4.2.3. O,O0-Dimethyl {[2-(3-methyl-4-chlorophenoxy)acetamido](4-
1550, 1460, 1222 cmꢀ1 1H NMR (600 MH
Z
CDCl3) (ppm): 1.11e1.28
d
methylphenyl)methyl}phosphonate(5c)
(m, 6H, 2CH3), 3.80 (s, 3H, OCH3), 3.75e4.10 (m, 4H, OCH2),
4.53e4.61 (q, AB system, 2H, OCH2CO), 5.47 (dd, J ¼ 9.6, 20.4 Hz,1H,
PCH), 6.87e7.44 (m, 8H, Ar-H), 7.45 (m, 1H, NH). 13C NMR (150 MHz
White solid. m.p. 91e92 ꢂC. IR(KBr)
n
: 3255, 3062, 1689(C]O),
1554, 1480, 1234 cmꢀ1 1H NMR (400 MHz CDCl3)
d(ppm): 2.34 (s,

382
L. Ning et al. / European Journal of Medicinal Chemistry 48 (2012) 379e384
CDCl3)
d
(ppm): 16.0, 48.8 (d, J ¼ 154.2 Hz, PCH), 54.9, 63.0 (d,
111.9, 118.0, 118.8, 128.0, 128.3, 128.6, 130.3, 132.1 (d, J ¼ 31.8 Hz,
CF3), 134.6, 157.3, 166.9 (CO). MS (70 ev) m/z (%): 473 (Mþ). Anal.
C22H27F3NO6P (C, H, N).
J ¼ 62.4 Hz, POCH2), 67.0 (OCH2), 111.4, 113.8, 117.9, 118.4, 126.3,
129.1,130.0,131.7 (d, J ¼ 32.0 Hz, CF3),131.6,131.8,157.2,159.3,166.7
(CO). MS (70 ev) m/z (%): 475 (Mþ). Anal. C21H25F3NO6P (C, H, N).
4.2.14. O,O0-Dipropyl {[2-(2,4-dichlorophenoxy)acetamido](4-
4.2.9. O,O0-Diethyl {[2-(4-nitrophenoxy)acetamido](4-
methoxyphenyl)methyl}phosphonate(5n)
methoxyphenyl)methyl}phosphonate(5i)
White solid. m.p. 101e102 ꢂC. IR(KBr)
n
: 3226, 3070, 1697(C]O),
White solid. m.p. 115e116 ꢂC. IR(KBr)
n
: 3223, 3134, 1696(C]O),
1554, 1477, 1222 cmꢀ1 1H NMR (600 MH
Z
CDCl3) (ppm): 0.82e0.92
d
1535, 1490, 1222 cmꢀ1 1H NMR (600 MH
Z
CDCl3) (ppm): 1.22e1.31
d
(m, 6H, 2CH3), 1.52e1.67 (m, 4H, CH2), 3.80 (s, 3H, OCH3), 3.71e4.00
(m, 4H, OCH2), 4.50e4.59 (q, AB system, 2H, OCH2CO), 5.49 (dd,
J ¼ 9.6, 20.4 Hz, 1H, PCH), 6.82 (d, J ¼ 9.0 Hz, 1H, Ar-H), 6.89 (d,
J ¼ 8.4 Hz, 2H, Ar-H), 7.20e7.44 (m, 4H, Ar-H), 7.65 (m, 1H, NH). 13C
(m, 6H, 2CH3), 3.80 (s, 3H, OCH3), 3.97e4.11 (m, 4H, OCH2),
4.61e4.73 (q, AB system, 2H, OCH2CO), 5.55 (dd, J ¼ 9.6, 19.8 Hz, 1H,
PCH), 6.90e8.09 (m, 8H, Ar-H, 1H, NH). 13C NMR (150 MHz CDCl3)
d
(ppm): 16.3, 49.0 (d, J ¼ 153.0 Hz, PCH), 55.4, 63.1 (d, J ¼ 65.0 Hz,
NMR (150 MHz CDCl3)
d
(ppm): 9.9, 10.9, 23.8, 49.0 (d, J ¼ 155.3 Hz,
POCH2), 67.7 (OCH2), 114.0, 116.1, 126.6, 126.9, 129.2, 140.7, 155.8,
159.5, 167.8 (CO). MS (70 ev) m/z (%): 452 (Mþ). Anal. C20H25N2O8P
(C, H, N).
PCH), 55.2, 68.2 (d, J ¼ 40.4 Hz, POCH2), 68.6 (OCH2), 114.0, 114.7,
123.8, 126.6, 127.3, 127.9, 129.0, 130.0, 151.5, 159.5, 166.3 (CO). MS
(70 ev) m/z (%): 504 (Mþ). Anal. C22H28Cl2NO6P (C, H, N).
4.2.10. O,O0-Diethyl {[2-(2-methyl-4-chlorophenoxy)acetamido](4-
4.2.15. O,O0-Dipropyl {[2-(3-trifluoromethylphenoxy)acetamido](4-
methoxyphenyl)methyl}phosphonate(5o)
methoxyphenyl)methyl}phosphonate(5j)
White solid. m.p. 113e114 ꢂC. IR(KBr)
n
: 3229, 3058, 1699(C]O),
White solid. m.p. 59e60 ꢂC. IR(KBr)
n
: 3216, 3055, 1690(C]O),
1556, 1490, 1223 cmꢀ1 1H NMR (600 MH
Z
CDCl3) (ppm): 1.12e1.30
d
1553,1459,1223 cmꢀ1 1H NMR (600 MH
Z
CDCl3) (ppm): 0.80e0.93
d
(m, 6H, 2CH3), 2.34 (s, 3H, Ar-CH3), 3.80 (s, 3H, OCH3), 3.76e4.10 (m,
4H, OCH2), 4.47e4.54 (q, AB system, 2H, OCH2CO), 5.44 (dd, J ¼ 9.6,
20.4 Hz, 1H, PCH), 6.67 (d, J ¼ 8.4 Hz, 1H, Ar-H), 6.88 (d, J ¼ 8.4 Hz,
2H, Ar-H), 7.11 (d, J ¼ 8.4 Hz, 1H, Ar-H), 7.18 (s, 1H, Ar-H), 7.32 (d,
J ¼ 7.8 Hz, 2H, Ar-H), 7.41e7.43 (m, 1H, NH). 13C NMR (150 MHz
(m, 6H, 2CH3),1.48e1.66 (m, 4H, CH2), 3.80 (s, 3H, OCH3), 3.64e4.00
(m, 4H, OCH2), 4.53e4.60(q, AB system, 2H, OCH2CO), 5.49 (dd,
J ¼ 9.6 Hz, 1H, PCH), 6.87 (d, J ¼ 9.0 Hz, 1H, Ar-H), 7.11 (d, J ¼ 7.8 Hz,
1H, Ar-H), 7.19 (s, 1H, Ar-H), 7.30e7.37 (m, 4H, Ar-H), 7.43e7.45 (m,
1H, NH). 13C NMR (150 MHz CDCl3)
d(ppm): 9.8, 23.8, 49.0 (d,
CDCl3)
d
(ppm):16.2, 16.3, 17.3, 49.1(d, J ¼ 155.5 Hz, PCH), 55.2
J ¼ 157.9 Hz, PCH), 55.1, 67.3 (OCH2), 68.6 (d, J ¼ 58.8 Hz, POCH2),
111.8, 114.0, 118.0, 118.7, 126.6, 129.2, 130.3, 132.0 (d, J ¼ 33.0 Hz,
CF3), 157.3, 159.5, 166.9 (CO).MS (70 ev) m/z (%): 503 (Mþ). Anal.
C23H29F3NO6P (C, H, N).
(CH3OAr), 63.2 (d, J ¼ 62.4 Hz, POCH2), 67.5 (OCH2), 112.6, 114.1,
123.8, 126.5, 126.6, 128.5, 129.0, 130.8, 153.9, 159.5, 167.2 (CO). MS
(70 ev) m/z (%): 455 (Mþ). Anal. C21H27ClNO6P (C, H, N).
4.2.11. O,O0-Dipropyl {[2-(2,4-dichlorophenoxy)acetamido](phenyl)
4.2.16. O,O0-Dibutyl {[2-(2,3-dimethylphenoxy)acetamido](phenyl)
methyl}phosphonate(5k)
methyl}phosphonate(5p)
White solid. m.p. 111e112 ꢂC. IR(KBr)
n
: 3230, 2972, 1701(C]O),
Yellowish oil. IR(KBr)
n
: 3225, 3061, 1694(C]O), 1550, 1471,
(ppm): 0.82e0.90 (m, 6H,
1556, 1478, 1218 cmꢀ1 1H NMR (600 MH
Z
CDCl3) (ppm): 0.80e0.92
d
1222 cmꢀ1 1H NMR (600 MHz CDCl3)
d
(m, 6H, 2CH3), 1.51e1.68 (m, 4H, CH2), 3.70e4.01 (m, 4H, OCH2),
4.51e4.60(q, AB system, 2H, OCH2CO), 5.52e5.57 (dd, J ¼ 9.6,
20.4 Hz, 1H, PCH), 6.82 (d, J ¼ 9.0 Hz, 1H, Ar-H), 7.20e7.44 (m, 7H,
2CH3),1.20e1.37 (m, 4H, 2CH2),1.43e1.61 (m, 4H, 2CH2), 2.29 (s, 3H,
Ar-CH3), 2.32 (s, 3H, Ar-CH3), 3.68e4.04 (m, 4H, 2CH2), 4.49e4.56
(q, AB system, 2H, OCH2CO), 5.53 (dd, J ¼ 9.6, 20.4 Hz,1H, PCH), 6.65
(d, J ¼ 9.0 Hz, 1H, Ar-H), 6.87 (d, J ¼ 8.4 Hz, 1H, Ar-H), 7.04e7.41 (m,
Ar-H), 7.71e7.73 (m, 1H, NH). 13C NMR (150 MHz CDCl3)
d(ppm):
9.9, 11.0, 23.8, 49.8 (d, J ¼ 154.2 Hz, PCH), 68.3 (d, J ¼ 50.3 Hz,
POCH2), 68.7 (OCH2), 114.7, 123.9, 127.4, 127.8, 127.9, 128.2, 128.7,
130.1, 134.6, 151.5, 166.4 (CO). MS (70 ev) m/z (%): 473 (Mþ). Anal.
C21H26Cl2NO6P (C, H, N).
6H, Ar-H), 7.58 (m, 1H, NH). 13C NMR (150 MHz CDCl3)
d(ppm): 11.8,
13.4, 18.4, 18.5, 19.9, 32.4, 49.8 (d, J ¼ 155.4 Hz, PCH), 66.7 (d,
J ¼ 35.4 Hz, POCH2), 67.6 (OCH2), 109.3, 123.5, 124.9, 126.0, 127.8,
128.0, 128.5, 134.9, 138.1, 155.2, 167.7 (CO). MS (70 ev) m/z (%): 461
(Mþ). Anal. C25H36NO5P (C, H, N).
4.2.12. O,O0-Dipropyl {[2-(4-chlorophenoxy)acetamido](phenyl)
methyl}phosphonate(5l)
4.2.17. O,O0-Dibutyl {[2-(2-chlorophenoxy)acetamido](phenyl)
methyl}phosphonate(5q)
White solid. m.p. 102e103 ꢂC. IR(KBr)
n
: 3215, 3052, 1694(C]O),
1550, 1484, 1221 cmꢀ1 1H NMR (600 MH
Z
CDCl3) (ppm): 0.78e0.92
d
Yellowish oil. IR(KBr)
n
: 3215, 3052, 1694(C]O), 1550, 1484,
(m, 6H, 2CH3), 1.46e1.65 (m, 4H, CH2), 3.59e4.00 (m, 4H, OCH2),
4.48e4.55 (q, AB system, 2H, OCH2CO), 5.51e5.56 (dd, J ¼ 9.6,
20.4 Hz, 1H, PCH), 6.82 (d, J ¼ 8.4 Hz, 2H, Ar-H), 7.27e7.43 (m, 7H,
1221 cmꢀ1 1H NMR (600 MHz CDCl3)
d(ppm): 0.83e0.90 (m, 6H,
2CH3), 1.21e1.37 (m, 4H, 2CH2), 1.47e1.62 (m, 4H, 2CH2), 3.77e4.05
(m, 4H, 2CH2), 4.05e4.63 (q, AB system, 2H, OCH2CO), 5.56 (dd,
J ¼ 9.6, 20.4 Hz, 1H, PCH), 6.90 (d, 1H, Ar-H, J ¼ 8.4 Hz), 6.99e7.45
Ar-H, 1H, NH). 13C NMR (150 MHz CDCl3)
d(ppm): 9.9, 11.0, 23.6,
23.8, 49.7 (d, J ¼ 155.4 Hz, PCH), 67.5(OCH2), 68.6(d, J ¼ 50.1 Hz,
POCH2), 116.1, 127.0, 128.0, 128.2, 128.6, 129.5, 134.7, 155.8, 167.2
(CO). MS (70 ev) m/z (%): 439 (Mþ). Anal. C21H27ClNO6P (C, H, N).
(m, 8H, Ar-H), 7.82 (d, 1H, NH). 13C NMR (150 MHz CDCl3)
d(ppm):
13.5, 18.5, 32.4, 49.8 (d, J ¼ 154.2 Hz, PCH), 66.9 (d, J ¼ 35.4 Hz,
POCH2), 68.0 (OCH2), 113.9, 123.0, 123.1, 127.8, 128.0, 128.2, 128.7,
130.4, 134.6, 152.7, 166.8 (CO). MS (70 ev) m/z (%): 467 (Mþ). Anal.
C18H20Cl2NO5P (C, H, N).
4.2.13. O,O0-Dipropyl {[2-(3-trifluoromethylphenoxy)
acetamido](phenyl)methyl}phosphonate(5m)
White solid. m.p. 63e64 ꢂC. IR(KBr)
n
: 3229, 3066, 1695(C]O),
CDCl3) (ppm): 0.78e0.92
4.2.18. O,O0-Dibutyl {[2-(2,4-dichlorophenoxy)acetamido](phenyl)
methyl}phosphonate(5r)
1552, 1492, 1218 cmꢀ1 1H NMR (600 MH
Z
d
(m, 6H, 2CH3), 1.47e1.64 (m, 4H, CH2), 3.61e4.00 (m, 4H, OCH2),
4.54e4.61 (q, AB system, 2H, OCH2CO), 5.54 (dd, J ¼ 9.6, 21.0 Hz,1H,
PCH), 7.12 (d,J ¼ 8.4 Hz, 1H, Ar-H), 7.20 (s, 1H, Ar-H), 7.30e7.45 (m,
Yellowish oil. IR(KBr)
n
: 3222, 3065, 1694(C]O), 1558, 1480,
(ppm): 0.82e0.90 (m, 6H,
1228 cmꢀ1 1H NMR (400 MHz CDCl3)
d
2CH3), 1.21e1.37 (m, 4H, 2CH2), 1.44e1.63 (m, 4H, 2CH2), 3.73e4.06
(m, 4H, 2CH2), 4.50e4.61 (q, AB system, 2H, OCH2CO), 5.53 (dd,
J ¼ 9.6, 20.8 Hz, 1H, PCH), 6.82 (d, J ¼ 8.8 Hz, 1H, Ar-H), 7.20e7.45
7H, Ar-H,1H, NH). 13C NMR (150 MHz CDCl3)
d(ppm): 9.8, 23.6, 23.8,
49.8 (d, J ¼ 155.4 Hz, PCH), 67.4 (OCH2), 68.7 (d, J ¼ 58.8 Hz, POCH2),

L. Ning et al. / European Journal of Medicinal Chemistry 48 (2012) 379e384
383
(m, 7H, Ar-H), 7.70e7.73 (m, 1H, NH). 13C NMR (150 MHz CDCl3)
NMR (150 MHz CDCl3)
d
(ppm): 13.4, 18.5, 32.3, 49.8 (d, J ¼ 158.7 Hz,
d
(ppm): 13.4, 18.5, 32.4, 49.8 (d, J ¼ 154.2 Hz, PCH), 66.8 (d,
PCH), 66.8 (d, J ¼ 59.7 Hz, POCH2), 68.5 (OCH2), 114.6, 117.8, 123.8,
127.7, 128.2, 128.6, 134.5, 149.3, 156.5, 158.1, 166.6 (CO). MS (70 ev)
m/z (%): 485 (Mþ). Anal. C23H30ClFNO5P (C, H, N).
J ¼ 33.0 Hz, POCH2), 68.1 (OCH2), 114.7, 123.8, 127.2, 127.8, 128.1,
128.6, 130.0, 134.6, 151.6, 166.4 (CO). MS (70 ev) m/z (%): 502 (Mþ).
Anal. C23H30Cl2NO5P (C, H, N).
4.2.24. O,O0-Dibutyl {[2-(2,3-dimethylphenoxy)acetamido](4-
methoxyphenyl)methyl}phosphonate(5x)
4.2.19. O,O0-Dibutyl {[2-(2-methyl-4-chlorophenoxy)
acetamido](phenyl)methyl}phosphonate(5s)
Yellowish oil. IR(KBr)
n
: 3220, 3048, 1695(C]O), 1552, 1482,
(ppm): 0.83e0.90 (m, 6H,
Yellowish oil. IR(KBr)
n
: 3221, 3061, 1691(C]O), 1555, 1491,
1246 cmꢀ1 1H NMR (600 MHz CDCl3)
d
1227 cmꢀ1 1H NMR (600 MHz CDCl3)
d(ppm): 0.81e0.90 (m, 6H,
2CH3), 1.23e1.36 (m, 4H, 2CH2), 1.44e1.61 (m, 4H, 2CH2), 2.27e2.31
(d, 3H, Ar-CH3), 3.70e4.02 (m, 4H, 2CH2), 3.80 (s, 3H, OCH3),
4.48e4.54 (q, AB system, 2H, OCH2CO), 5.48 (dd, J ¼ 9.6, 20.4 Hz,1H,
PCH), 6.64 (d, J ¼ 7.8 Hz, 1H, Ar-H), 6.88 (d, J ¼ 7.8 Hz, 3H, Ar-H),
7.04e7.06 (t, 1H, Ar-H), 7.33 (d, J ¼ 7.8 Hz, 1H, Ar-H), 7.51 (d, 1H,
2CH3), 1.20e1.36 (m, 4H, 2CH2), 1.42e1.63 (m, 4H, 2CH2), 2.34 (s, 3H,
Ar-CH3), 3.65e4.02 (m, 4H, 2CH2), 4.48e4.54 (q, AB system, 2H,
OCH2CO), 5.49 (dd, J ¼ 9.6, 20.4Hz,1H, PCH), 6.67 (d, J ¼ 8.4 Hz,1H, Ar-
H), 7.11 (d, J ¼ 8.4 Hz,1H, Ar-H), 7.19 (s,1H, Ar-H), 7.31e7.40 (m, 5H, Ar-
H), 7.48(m,1H, NH).13C NMR (150MHz CDCl3)
d(ppm):13.5,17.3,18.6,
NH). 13C NMR (150 MHz CDCl3)
d(ppm): 11.8, 13.6, 18.5, 19.0, 32.4,
32.4, 49.7 (d, J ¼ 153.0 Hz, PCH), 66.9 (d, J ¼ 58.8 Hz, POCH2) 67.5
(OCH2), 113.9, 123.0, 123.1, 127.8, 128.0, 128.2, 128.7, 130.4, 134.6, 152.7,
167.2 (CO).MS (70 ev) m/z (%): 481 (Mþ). Anal. C24H33ClNO5P (C, H, N).
49.5 (d, J ¼ 155.4 Hz, PCH), 55.2 (CH3OAr), 66.7 (d, J ¼ 50.6 Hz,
POCH2), 67.5 (OCH2), 109.3, 114.1, 123.7, 125.0, 126.1, 126.7, 128.9,
138.4, 155.1, 159.4, 167.7 (CO). MS (70 ev) m/z (%): 532 (Mþ). Anal.
C26H38NO6P (C, H, N).
4.2.20. O,O0-Dibutyl {[2-(3-methyl-4-chlorophenoxy)
acetamido](phenyl)methyl}phosphonate(5t)
4.2.25. O,O0-Dibutyl {[2-(2-chlorophenoxy)acetamido](4-
Yellowish oil. IR(KBr)
n
: 3223, 3065, 1696(C]O), 1555, 1479,
methoxyphenyl)methyl}phosphonate(5y)
1224 cmꢀ1 1H NMR (600 MHz CDCl3)
d(ppm): 0.81e0.91 (m, 6H,
Yellowish oil. IR(KBr)
n
: 3234, 3075, 1698(C]O), 1567, 1513,
(ppm): 0.84e0.90 (m, 6H,
2CH3),1.20e1.36 (m, 4H, 2CH2),1.40e1.61 (m, 4H, 2CH2), 2.33 (s, 3H,
Ar-CH3), 3.64e4.03 (m, 4H, 2CH2), 4.46e4.54 (q, AB system, 2H,
OCH2CO), 5.51 (dd, J ¼ 9.6, 20.8 Hz, 1H, PCH), 6.70e6.72 (q, 1H, Ar-
H), 6.82 (d, J ¼ 3.0 Hz, 1H, Ar-H), 7.25 (d, J ¼ 9.0 Hz, 1H, Ar-H),
7.30e7.39 (m, 5H, Ar-H), 7.42e7.44 (m, 1H, NH). 13C NMR
1246 cmꢀ1 1H NMR (600 MHz CDCl3)
d
2CH3), 1.26e1.37 (m, 4H, 2CH2), 1.49e1.62 (m, 4H, 2CH2), 3.80 (s,
3H, OCH3), 3.80e4.03 (m, 4H, 2CH2), 4.53e4.61 (q, AB system, 2H,
OCH2CO), 5.50 (dd, J ¼ 9.6, 20.4 Hz, 1H, PCH), 6.88e7.44 (m, 8H, Ar-
H), 7.74 (d, 1H, NH). 13C NMR (150 MHz CDCl3)
d(ppm): 13.6, 18.6,
(150 MHz CDCl3)
d
(ppm): 13.5, 18.6, 20.2, 32.2, 49.7 (d, J ¼ 151.8 Hz,
32.4, 49.2 (d, J ¼ 158.7 Hz, PCH), 55.1, 66.9 (d, J ¼ 50.1 Hz, POCH2),
67.4 (OCH2), 113.9, 114.1, 122.9, 123.0, 126.6, 128.1, 129.0, 130.4,
152.6, 159.4, 166.7 (CO). MS (70 ev) m/z (%): 566 (Mþ). Anal.
C24H33ClNO6P (C, H, N).
PCH), 66.8 (d, J ¼ 51.5 Hz, POCH2), 67.5 (OCH2), 113.3, 117.3, 127.2,
128.0, 128.1, 128.5, 129.7, 134.7, 137.3, 154.4, 167.4 (CO). MS (70 ev)
m/z (%): 481 (Mþ). Anal. C24H33ClNO5P (C, H, N).
4.2.21. O,O0-Dibutyl {[2-(2,4,5-trichlorophenoxy)
4.2.26. O,O0-Dibutyl {[2-(4-t-butylphenoxy)acetamido](4-
acetamido](phenyl)methyl}phosphonate(5u)
methoxyphenyl)methyl}phosphonate(5z)
Yellowish oil. IR(KBr)
n
: 3235, 3067, 1691(C]O), 1555, 1477,
(ppm): 0.82e0.91 (m, 6H,
Yellowish oil. IR(KBr)
n
: 3216, 3046, 1691(C]O), 1552, 1410,
1227 cmꢀ1 1H NMR (600 MHz CDCl3)
d
1243 cmꢀ1 1H NMR (600 MHz CDCl3)
d(ppm): 0.83e0.91 (m, 6H,
2CH3), 1.22e1.37 (m, 4H, 2CH2), 1.44e1.63 (m, 4H, 2CH2), 3.72e4.06
(m, 4H, 2CH2), 4.51e4.60 (q, AB system, 2H, OCH2CO), 5.53 (dd,
J ¼ 9.6, 21.0 Hz, 1H, PCH), 6.99 (s, 1H, Ar-H), 7.31e7.54 (m, 6H, Ar-H),
2CH3),1.22e1.61 (m, 8H, 4CH2),1.30 (s, 9H, Ar-C(CH3)3), 3.70e4.03 (m,
4H, 2CH2), 3.79 (s, 3H, OCH3), 4.48e4.54 (q, AB system, 2H, OCH2CO),
5.49 (dd, J ¼ 9.6, 20.4 Hz,1H, PCH), 6.85e7.33 (m, 8H, Ar-H), 7.41 (d,1H,
7.65 (d, 1H, NH). 13C NMR (150 MHz CDCl3)
d(ppm): 13.5, 18.5, 32.4,
NH).13C NMR (150 MHz CDCl3)
d(ppm): 13.5,18.5, 31.4, 31.5, 31.9, 32.4,
49.7 (d, J ¼ 155.2 Hz, PCH), 66.8 (d, J ¼ 55.5 Hz, POCH2), 67.5 (OCH2),
114.8,115.8, 127.9,128.2, 128.6, 134.7,151.3, 154.8, 167.9 (CO). MS (70
ev) m/z (%): 536 (Mþ). Anal. C23H29Cl3NO5P (C, H, N).
34.1, 49.0 (d, J ¼ 155.6 Hz, PCH), 55.1, 66.8 (d, J ¼ 50.1 Hz, POCH2), 67.4
(OCH2), 114.0, 114.3, 126.4, 126.7, 129.2, 144.8, 154.9, 159.4, 167.8 (CO).
MS (70 ev) m/z (%): 493 (Mþ). Anal. C28H43NO6P (C, H, N).
4.2.22. O,O0-Dibutyl {[2-(4-methoxyphenoxy)acetamido](phenyl)
4.3. Cytotoxicity assay
methyl}phosphonate(5v)
Yellowish oil. IR(KBr)
n
: 3228, 3066, 1694(C]O), 1554, 1506,
(ppm): 0.82e0.91 (m, 6H,
Cytotoxic activities were evaluated by using standard MTT assay
[17] after exposure of cells to the tested compounds for 72 h. Each
experiment was performed at least 3 times. There was a good repro-
ducibility between replicate wells with standard errors below 10%.
The in vitro cytotoxicity of the synthesized compounds against
different cancer cell lines was performed with the MTT assay
according to the Mosmann’s method [23]. The MTT assay is based on
the reduction of the soluble 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-2H-tetrazolium bromide (MTT) into a blueepurple for-
mazan product, mainly by mitochondrial reductase activity inside
living cells. The cells used in cytotoxicity assay were cultured in RPMI
1640 medium supplemented with 10% fetal calf serum. Cells sus-
pended in the medium (2Y0 104/mL) were plated in 96-well culture
plates and incubated at 37 ꢂC in a 5% CO2 incubator. After 12 h, the test
sample (2 mL) was added to the cells (2Y0 104/mL) in 96-well plates
and cultured at 37 ꢂC for 3 days. The cultured cells were mixed with
20 mL of MTTsolution and incubated for 4 h at 37 ꢂC. The supernatant
was carefully removed from each well and 100 mL of DMSO was
1227 cmꢀ1 1H NMR (600 MHz CDCl3)
d
2CH3), 1.20e1.37 (m, 4H, 2CH2), 1.42e1.65 (m, 4H, 2CH2), 3.67e4.02
(m, 4H, 2CH2), 3.78 (s, 3H, OCH3), 4.46e4.52 (q, AB system, 2H,
OCH2CO), 5.53 (dd, J ¼ 9.6, 21.0 Hz, 1H, PCH), 6.84e7.47 (m, 9H, Ar-
H), 7.49 (d, 1H, NH). 13C NMR (150 MHz CDCl3)
d(ppm): 13.5, 18.5,
32.4, 49.7 (d, J ¼ 156.3 Hz, PCH), 55.6, 66.8 (d, J ¼ 47.7 Hz, POCH2),
68.2 (OCH2), 114.8, 115.8, 127.9, 128.2, 128.6, 134.7, 151.3, 154.8, 167.9
(CO). MS (70 ev) m/z (%): 463 (Mþ). Anal. C24H34NO6P (C, H, N).
4.2.23. O,O0-Dibutyl {[2-(2-chloro-4-fluorophenoxy)
acetamido](phenyl)methyl}phosphonate(5w)
Yellowish oil. IR(KBr)
n
: 3224, 3063, 1693(C]O), 1556, 1492,
(ppm): 0.82e0.90 (m, 6H,
1229 cmꢀ1 1H NMR (600 MHz CDCl3)
d
2CH3), 1.23e1.37 (m, 4H, 2CH2), 1.46e1.61 (m, 4H, 2CH2), 3.74e4.04
(m, 4H, 2CH2), 4.50e4.59 (q, AB system, 2H, OCH2CO), 5.55 (dd,
J ¼ 9.6, 21.0 Hz, 1H, PCH), 6.85 (d, J ¼ 4.8 Hz, 1H, Ar-H), 6.87 (d,
J ¼ 4.8 Hz, 1H, Ar-H), 6.95e7.45 (m, 6H, Ar-H), 7.75 (m, 1H, NH). 13C

384
L. Ning et al. / European Journal of Medicinal Chemistry 48 (2012) 379e384
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added to each well to dissolve the formazan crystals which were
formed by the cellular reduction of MTT. After mixing with
a mechanical plate mixer, the absorbance of each well was measured
by a microplate reader using a test wavelength of 570 nm. The results
were expressed as the IC50, which is the concentration of the drugs
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Acknowledgments
We gratefully acknowledge financial support of this work by the
National Basic Research Program of China (2010CB126100), the
National Natural Science Foundation of China (No. 21172090 and No.
21002037). The research was supported in part by the PCSIRT (No.
IRT0953) and the self-determined research funds of CCNU from the
colleges’ basic research and operation of MOE (No. CCNU10A01007).
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