Synthesis and antitumor activities of novel α-aminophosphonate derivatives containing an alizarin moiety

Article abstract of DOI:10.1016/j.ejmech.2014.02.067

A series of novel α-aminophosphonate derivatives containing an alizarin moiety (6-7) was designed and synthesized as antitumor agents. MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay results indicated that most compounds exhibited moderate to high inhibitory activity against KB, NCI-H460, HepG 2, A549, MGC-803, Hct-116, CNE and Hela tumor cell lines. The action mechanism of representative compounds 7h, 7j and 7n were investigated by fluorescence staining assays, flow cytometric analysis and real-time polymerase chain reaction (PCR) assays, which indicated that these compounds induced apoptosis and involved G1 phase arrest by increasing the production of intracellular Ca²⁺ and reactive oxygen species (ROS) and affecting associated enzymes and genes. The results demonstrated that these compounds may induce apoptosis through a mitochondrion-dependent pathway.

Full text of DOI:10.1016/j.ejmech.2014.02.067

European Journal of Medicinal Chemistry 83 (2014) 116e128
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antitumor activities of novel
derivatives containing an alizarin moiety
a-aminophosphonate
,
,
, c, **
Man-Yi Ye a 1, Gui-Yang Yao b 1, Ying-Ming Pan a, Zhi-Xin Liao b, Ye Zhang a
,
Heng-Shan Wang a
,
*
a State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry & Pharmaceutical Science
of Guangxi Normal University, Yucai Road 15, Guilin 541004, Guangxi, PR China
b Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China
c Department of Chemistry & Pharmaceutical Science, Guilin Normal College, Xinyi Road 15, Guangxi 541001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel a-aminophosphonate derivatives containing an alizarin moiety (6e7) was designed and
Received 17 November 2013
Received in revised form
19 February 2014
Accepted 19 February 2014
Available online 12 June 2014
synthesized as antitumor agents. MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide)
assay results indicated that most compounds exhibited moderate to high inhibitory activity against KB,
NCI-H460, HepG 2, A549, MGC-803, Hct-116, CNE and Hela tumor cell lines. The action mechanism of
representative compounds 7h, 7j and 7n were investigated by fluorescence staining assays, flow cyto-
metric analysis and real-time polymerase chain reaction (PCR) assays, which indicated that these
compounds induced apoptosis and involved G1 phase arrest by increasing the production of intracellular
Ca2þ and reactive oxygen species (ROS) and affecting associated enzymes and genes. The results
demonstrated that these compounds may induce apoptosis through a mitochondrion-dependent
pathway.
Keywords:
Alizarin
a
-Aminophosphonate
Cytotoxicity
© 2014 Published by Elsevier Masson SAS.
Apoptosis
Mitochondrial pathway
1. Introduction
preferable biological activities such as antimicrobial, anti-leukemia,
antioxidant, anti-HIV and anticancer activities [11e19]. In addition,
The success of taxanes, anthracyclines, vinca alkaloids and their
related derivatives as antitumor drugs has demonstrated that
natural products is a good traditional source of new antitumor
agents [1e5]. So development of novel natural compounds and
their derivatives with better antitumor virtue have greatly gained
bioorganic chemists' interest, and a great deal of natural products,
as well as their synthetic derivatives have been exploited in the
hope of creating new and better antitumor agents [6].
the introduction of aminophosphonate group to pharmacy core is
able to increase the antitumor activity and many amino-
phosphonate derivatives have exhibited potent inhibition activities
against human tumors [20,21]. Owing to the synthetic and bio-
logical values of alizarin and aminophosphonate groups, it is thus to
expect that the condensation of alizarin and aminophosphonate
groups may contribute to good antitumor activity. However, the
studies on the synthesis, antitumor activities and apoptosis-
Natural anthraquinone compounds have been used widely in
the pharmaceutical industry because of their biological activities
[7e10]. Alizarin is a kind of anthraquinone which exists in rhubarb
and aloe plant. Moreover, some derivatives of alizarin have been
also synthesized and investigated, because they exhibit some
inducing effects of a-aminophosphonate derivatives containing
an alizarin moiety have not been described. Therefore, in this paper,
functional aminophosphonate group was rationally designed and
introduced to the alizarin skeleton to offer a series of novel
a-
aminophosphonate derivatives containing alizarin moiety. Their
antitumor activities in vitro against several selected tumor cell lines
were estimated. Results exhibited that the target products could
inhibit proliferation of these selected tumor cell lines at moderate
to high rates. Further investigation on the mode of action of
representative compounds 7h, 7j and 7n found that they could
* Corresponding author.
** Corresponding author. State Key Laboratory Cultivation Base for the Chemistry
and Molecular Engineering of Medicinal Resources, School of Chemistry & Phar-
maceutical Science of Guangxi Normal University, Yucai Road 15, Guilin 541004,
Guangxi, PR China.
induce cell apoptosis in NCI-H460 cells possibly through
a
mitochondrion-dependent pathway by affecting associated
enzymes.
E-mail addresses: zhangye81@126.com (Y. Zhang), whengshan@163.com
(H.-S. Wang).
1
Co-first author: These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2014.02.067
0223-5234/© 2014 Published by Elsevier Masson SAS.

M.-Y. Ye et al. / European Journal of Medicinal Chemistry 83 (2014) 116e128
117
2. Results and discussion
2.1. Chemistry
a
-Aminophosphonate derivatives bearing alizarin structure
(compounds 6ae6k and 7ae7q) were synthesized as outlined in
Scheme 1. Alizarin was synthesized by a single-step FriedeleCrafts
acylation reaction between o-phthalic anhydride and catechol [22].
Compounds 2 (or 3) were obtained by the condensation of com-
pound 1 and ethyl bromoacetate (or ethyl 2-bromopropionate), and
it was then treated with hydrochloric acid to offer compounds 4 (or
5).
cording to the literature [23], was treated with compound 4 (or 5)
to offer -aminophosphonate-alizarin derivatives 6 (or 7) in good
yields. The structures of -aminophosphonate-alizarin derivatives
a-Aminophosphonate derivatives, which were synthesized ac-
a
Fig. 1. ORTEP drawing of compound 7n.
a
6e7 were then confirmed by 1H NMR, 13C NMR, 31P NMR and high
resolution mass spectrum (HRMS). Single crystal of compound 7n
was obtained to further confirm the structure through recrystalli-
zation from methanol. The intensity data were collected in an
Agilent SuperNova CCD diffractometer equipped with a graphite-
In all human cancer cell lines, all the compounds exhibited
better inhibition than alizarin, and most of them even showed
preferable cytotoxic activities than 5-FU. The cytotoxic inhibition
screening results showed that the introduction of
a-amino-
phosphonate group on alizarin should markedly improve the
antitumor activity against most of human cancer cell line. For
example, in NCI-H460 assay, the IC50 of compounds 6 and 7 were in
monochromatized MoK
a
radiation (
l
¼ 0.71075 Å) by using a
j-
u
scan mode at 223(2) K. CCDC reference number was 960632. The
structure of compound 7n was shown in Fig. 1. The result was in
well agreement with the structure showed in Scheme 1.
the range of 11.93e47.88
was important to noting that all the compounds expect 6a, 6g
M).
mM and 3.80e28.92 mM, respectively. It
displayed better cytotoxic activities than 5-FU (IC50 ¼ 45.14
m
Especially, compounds 6i, 7e and 7h were found to be the most
2.2. In vitro cytotoxicity
active compounds on NCI-H460 cell line, with IC50 of
11.93 0.77 mM, 3.80 1.03 mM and 3.80 1.90 mM, respectively.
The in vitro antitumor activities of a-aminophosphonate-aliz-
NCI-H460 cells inhibiting activities of these tested compounds
decreased in the order of 6i > 6b > 6k > 6f > 6d > 6c >
6h > 6e > 6j > 6a > 6g; 7e ¼ 7h > 7b > 7d > 7n > 7a > 7m > 7g >
7f > 7p > 7j > 7c > 7o > 7l > 7k > 7i > 7q. On the basis of the above
observation, chloro, fluoro and bromo groups in benzene group of
arin derivatives 6e7 were respectively evaluated by MTT assay
against KB, NCI-H460, HepG 2, A549, MGC-803, Hct-116, CNE and
Hela tumor cell lines and HUVEC normal cells line, with 5-
fluorouracil (5-FU) as the positive control. The results were listed
in Table 1 and Table 1S (Supplementary).
As shown in Table 1, most of the compounds displayed much
higher inhibitory activity than alizarin against five tumor cell lines,
and even preferable cytotoxic activities than the commercial anti-
a-aminophosphonate moiety showed positive influence on anti-
tumor activities in NCI-H460 assay, while the methoxy, methyl,
group exhibited negative effect. Besides, the substituent position
on naphthalene also appeared to have important influences on the
antitumor activity in this assay.
In addition, the inhibition activities of compounds 6 and 7
against HUVEC normal cell lines were also estimated. The data of
MTT assay against HUVEC cell lines were listed in Table 1S
(Supplementary). The results indicated that the cytotoxicity of
most of compounds against cancer cells was much higher than
HUVEC normal cells, making them good candidates as antitumor
cancer drug 5-FU. The results suggested that the introduction of a-
aminophosphonate on alizarin could improve the antitumor ac-
tivity, which was consistent with our previous studies [20,21].
Moreover, by the comparison of IC50 values of compounds 6 with 7,
it could be concluded that the introduction of a methyl group at R1
was very important for improving the antitumor activity. In addi-
tion, these results also demonstrated that ortho-substituent at R2
will enhance cytotoxicity.
Scheme 1. Synthetic route to
DMSO, r.t.; (iii) NaOH, H2O, 80 ꢀC; HCl, H2O, r.t.; (iv) HOBt, EDAC, DMSO, r.t.
a
-aminophosphonate derivatives containing alizarin skeleton (6e7). Reagents and conditions: (i) AlCl3, NaCl, 165 ꢀC; HCl; H2O, 0e100 ꢀC; (ii) KOH,

118
M.-Y. Ye et al. / European Journal of Medicinal Chemistry 83 (2014) 116e128
Table 1
Effect of alizarin derivatives 6 and 7 against different tumor cell lines.
a
Compound
IC50
R2
(mM)
Hct-116
MGC-803
NCI
A549
KB
3.26 0.010
6a
6b
6c
6d
6e
4-Br
2-Br
2-OCH3
4-OCH3
3-OCH3
2-F
4-F
2-Cl
Ph
3-CH3
4-CH3
4-Br
2-Br
3-Br
4-Cl
2-Cl
3-Cl
4-F
45.77 1.12
17.15 0.77
30.18 1.19
44.89 2.31
49.80 2.52
14.70 0.46
36.27 1.20
16.28 0.68
17.39 0.70
15.99 0.52
13.71 0.55
12.36 4.42
7.31 1.12
15.73 1.34
15.57 0.45
14.79 2.27
36.33 2.08
17.92 1.18
13.35 1.52
47.12 3.01
10.53 0.25
16.97 4.93
13.71 0.55
8.07 0.73
39.98 2.03
23.01 0.90
36.17 2.14
36.99 1.50
>50
35.22 1.45
18.74 0.23
29.19 2.32
36.09 2.11
38.01 1.03
27.09 2.51
30.91 2.54
39.20 3.56
12.97 2.49
37.87 1.54
12.64 1.20
18.54 1.36
15.07 2.96
27.52 1.97
25.06 3.55
11.02 1.29
28.97 9.61
22.89 1.90
18.83 8.69
28.90 1.78
10.09 7.90
27.60 1.03
22.51 3.37
6.66 .0.02
3.77 0.70
5.60 1.72
8.02 4.01
27.89 2.32
39.35 2.39
46.93 2.09
46.19 2.91
12.03 1.00
20.78 2.12
19.99 0.16
27.92 0.92
12.93 1.26
47.88 1.98
21.24 1.63
11.93 0.77
33.93 3.34
12.24 0.55
7.83 4.54
4.11 0.66
10.65 1.85
5.11 0.33
3.80 1.03
9.10 0.11
8.92 0.53
3.80 1.90
25.89 1.65
9.80 0.81
14.92 2.57
14.32 4.64
8.11 3.52
6.83 0.15
14.14 1.89
9.31 0.94
28.92 2.55
>50
45.10 4.01
35.78 2.11
39.01 2.54
42.21 1.00
44.13 0.99
37.76 2.62
49.01 1.97
38.02 2.19
12.06 0.97
46.98 3.77
33.80 1.09
30.28 1.07
11.43 3.09
32.75 2.26
10.70 1.98
10.14 1.23
18.72 4.54
19.39 6.35
6.76 1.48
35.11 0.99
12.27 6.03
36.01 2.45
33.73 1.33
34.55 1.29
15.92 7.34
25.20 0.63
25.60 1.92
37.99 3.66
>50
6.64 0.51
1.24 0.15
7.32 0.17
15.47 1.96
2.60 0.14
6.45 0.54
1.55 0.26
6.70 0.37
12.03 1.27
9.37 0.94
0.26 0.020
0.13 0.021
2.29 0.09
0.20 0.010
0.14 0.012
0.22 0.027
0.87 0.018
0.70 0.011
9.12 1.03
0.88 0.046
5.78 0.43
1.00 0.028
0.65 0.081
1.02 0.098
1.21 0.066
9.68 0.79
0.99 0.037
15.65 0.98
1.23 0.16
6f
6g
6h
6i
6j
6k
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
Alizarin
5-Fu
2-F
3-F
2-OCH3
3-OCH3
4-OCH3
Ph
1-Nap.
2-Nap.
3-CH3
4-CH3
e
e
10.05 6.34
45.14 2.14
34.34 3.90
a
Each data represents mean S.D. from three different experiments performed in triplicate.
drugs. These results showed that these conjugates have selective
and significant effect on the cell lines.
changes in the morphological character of NCI-H460 cells were
determined using JC-1 mitochondrial membrane potential staining
and Hoechst 33258 staining under fluorescence microscopy.
From the above results, some interesting structureeactivity re-
lationships could be concluded: (1) the introduction of a-amino-
phosphonate was crucial for improving their activity; (2) In KB,
NCI-H460, MGC-803, A549 and Hct-116 assays, chloro, fluoro and
bromo groups as R2 showed different positive influence on anti-
tumor activities, and the antitumor activities were found to be in
the order of ortho- > para- > meta-; (3) Compared the antitumor
activity of compound 6 with 7, it could be found that the antitumor
activity of compound 7 were better than that of 6. It was of interest
that the introduction of a methyl group at R1 was important for
improving antitumor activities.
2.3.1.1. Mitochondrial membrane potential staining. To determine
the apoptosis-inducing effect of the representative compounds (7h,
7j and 7n), mitochondrial membrane potential changes were
assessed using the JC-1 assay. JC-1 is a lipophilic cationic dye and it
can easily pass through the plasma membrane into cells and
accumulate in actively respiring mitochondria. The JC-1 monomer
and J-aggregates were excited at 514 nm and 585 nm, respectively,
and light emissions were collected at 515e545 nm (green) and
570e600 nm (red) for fluorescence microscopy [21,26].
NCI-H460 cells treated with compound 7h at 5
mM, 10 mM,
2.3. Preliminary investigation of the apoptosis-inducing effect of
target compound 7h, 7j and 7n
20
m
M for 24 h and at 8 M for 12 h, 24 h and 48 h were stained with
m
JC-1. The results in Fig. 2 showed that cells not treated with the
compound 7h were normally red (in the web version), while for 7h
treatment, cells showed strong green fluorescence and indicated
typical apoptotic morphology. NCI-H460 cells treated with 7h from
Apoptosis plays a central role in cancer, since its induction in
cancer cells is critical to a successful therapy [24,25]. It is thus
believed that apoptosis assay may provide important information
to preliminary investigation of the mode of action. In order to
12 to 48 h, 5 to 20 mM had obviously changed. Therefore, it could be
concluded that compound 7h induced apoptosis against NCI-
H460 cell line in concentration-dependent and time-dependent
manners. Compounds 7j and 7n had a similar effect as 7h when
assessed under the same conditions (data shown in Fig. 1S,
Supplementary).
clarify the mechanism of alizarin a-aminophosphonate derivatives
inducing cell death in cells, both early and late apoptosis were
determined using fluorescence staining and annexin V-FITC and PI
(Propidium iodide) labeling of live cells. Compounds 7h, 7j and 7n
exhibited potent cytotoxic inhibition in NCI-H460 cell lines, and
thus could be used as representative compounds 6e7 for further
investigation of their apoptosis-inducing effect.
2.3.1.2. Hoechst 33258 staining. Hoechst 33258 is a membrane
permeable blue fluorescent dye that stains the cell nucleus. Live
cells with uniformly light blue nuclei were observed under fluo-
rescence microscope after treatment with Hoechst 33258, whereas
apoptotic cells had bright blue nuclei because of karyopyknosis and
2.3.1. Fluorescence staining
In order to investigate whether the growth inhibitory activity of
the selected compound was related to the induction of apoptosis,

M.-Y. Ye et al. / European Journal of Medicinal Chemistry 83 (2014) 116e128
119
Fig. 2. JC-1 mitochondrial membrane potential staining of compound 7h in NCI-H460 cells.
chromatin condensation, and the nuclei of dead cells could not be
stained [27].
2.3.2.1. Apoptosis analysis. In the present study, NCI-H460 cells
were stimulated for 24 h with 5 M, 10 M, 20 M compounds and
for 12 h, 24 h and 48 h with 8 M, respectively. The results (Fig. 3S,
Supplementary) showed that the apoptosis ratios of compound 7h
measured at 8 M were found to be 30.3% (12 h), 38.8% (24 h), 56.4%
(48 h), and measured at different concentration points were found
to be 28.9% (5 M), 56.3% (10 M), 55.1% (20 M), respectively.
Compound 7j measured at 8 M were found to be 21.9% (12 h),
38.9% (24 h), 50.2% (48 h), and measured at different concentration
points were shown to be 26.6% (5 M), 32.4% (10 M), and 34.7%
(20 M), respectively. Compound 7n measured at 8 M were found
to be 19.9% (12 h), 36.6% (24 h), 54.9% (48 h), and measured at
different concentration points were found to be 10.9% (5 M), 35.7%
(10 M), 38.8% (20 M), respectively, while that of control was 6.8%.
m
m
m
NCI-H460 cells treated with compounds 7h at 5
m
M, 10
m
M,
m
20
m
M for 24 h and at 8 M for 12 h, 24 h and 48 h, respectively,
m
were stained with Hoechst 33258, and the cells not treated with the
compounds 7h were used as control. Fig. 3 shows that cells not
treated with the compounds 7h were normally blue (in the web
version). For 7h treatment, the cells exhibited stronger blue fluo-
rescence and revealed typical apoptotic morphology. These find-
ings demonstrated that compounds 7h induced apoptosis against
NCI-H460 cell lines. Moreover, the fluorescence intensity gradually
increased as the prolongation of the time and the increase of the
concentration, which was consistent with the findings from mito-
chondrial membrane potential staining. Compounds 7j and 7n had
a similar but inferior effect in NCI-H460 cells compared with 7h
(data shown in Fig. 2S, Supplementary).
m
m
m
m
m
m
m
m
m
m
m
m
Notably, the percentage of cells deemed to be in early apoptosis for
the compounds (7h, 7j, 7n) was much higher than that of the
control. Moreover, the results showed that compounds were acted
in concentration-dependent and time-dependent manners.
The apoptosis ratio of compounds 7b, 7e, 7h, 7j, 7m, 7n and 7p
2.3.2. Flow cytometry
Flow cytometry was used to investigate analogue-induced
apoptosis and cell death. The representative compounds (7h, 7j
and 7n) were investigated for the effects on the apoptosis of NCI-
H460 cells. Annexin V binds to phosphatidylserine, which is
exposed on the cell membrane and is one of the earliest indicators
of cellular apoptosis. PI is used as a DNA stain for both flow
cytometry to evaluate cell viability or DNA content in cell cycle
analysis and microscopy to visualize the nucleus and other DNA
containing organelles. It can be used to differentiate necrotic,
apoptotic and normal cells.
at 5 mM, 10 mM, 20 mM for 24 h, respectively, was shown in Fig. 4.
The results evidently illustrated that compounds 7b, 7e, 7h, 7j, 7m,
7n and 7p were of special interest as these compounds seem to
drive NCI-H460 cells directly into early apoptosis.
2.3.2.2. Cell cycle analysis. The cell cycle is a series of events that
takes place in a cell leading to its division and duplication (repli-
cation). The cell cycle consists of four distinct phases: G1 phase, S
phase (synthesis), G2 phase (collectively known as inter phase) and

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M.-Y. Ye et al. / European Journal of Medicinal Chemistry 83 (2014) 116e128
Fig. 3. Hoechst 33258 staining of compound 7h in NCI-H460 cells.
M phase (mitosis). The G1 stage is the stage when preparation of
energy and material for DNA replication occurs. The S stage is the
stage when DNA replication occurs. The G2 stage is the stage when
preparation for the M stage occurs. The M stage is “mitosis”, and is
when nuclear and cytoplasmic division occurs.
48 h and 55.78%, 60.05%, 65.52% at 8
60.79%, 65.10% for 7j at 5 M, 10 M, 20
64.92%, 68.16% at 8 M for 12 h, 24 h, 48 h; 57.20%, 59.86%, 61.60%
M, 20 M for 48 h and 65.57%, 66.33%, 68.09% at
m
M for 12 h, 24 h, 48 h; 56.85%,
m
m
mM for 48 h and 56.84%,
m
for 7n at 5
mM, 10
m
m
The effect of compounds (7h, 7j and 7n) on the cell cycle of NCI-
H460 cell lines was investigated by flow cytometry and the data
was shown in Table 2 (Fig. 4S, Supplementary). NCI-H460 cells were
Table 2
The percentage of cells in the different phases of the cell-cycle analysis by flow
cytometry of NCI-H460 cells.
stimulated for 24 h with 5
M compounds from 12 to 48 h, respectively. G1-phase pop-
ulations of 64.52%, 65.81%, 68.89% for 7h at 5 M, 10 M, 20 M for
mM, 10 mM, 20 mM compounds and with
Groups
Cell cycle phase
G1 phase
8
m
S phase
G2 phase
m
m
m
A.
B.
C.
D.
E.
F.
G.
H.
I.
J.
K.
L.
M.
N.
O.
P.
Q.
R.
S.
Control
46.69%
55.78%
60.05%
65.52%
64.52%
65.81%
68.89%
56.84%
64.92%
68.16%
56.85%
60.79%
65.10%
65.57%
66.33%
68.09%
57.20%
59.86%
61.60%
19.41%
14.65%
16.30%
10.87%
12.34%
13.42%
11.25%
15.62%
13.09%
8.87%
8.74%
9.84%
11.81%
0.11%
1.11%
33.9%
8
8
8
5
10
20
8
8
8
5
10
20
m
m
m
m
M 7ha
M 7hb
M 7hc
M 7hc
29.57%
23.05%
23.61%
23.14%
20.77%
20.06%
27.54%
21.99%
22.97%
34.41%
29.38%
23.09%
34.32%
32.56%
31.50%
25.78%
24.74%
21.34%
m
m
M 7hc
M 7hc
m
M 7ja
M 7jb
M 7jc
M 7jc
m
m
m
m
m
M 7jc
M 7jc
8
8
8
5
m
M 7na
M 7nb
M 7nc
M 7nc
m
m
m
0.41%
17.02%
15.41%
17.06%
10
20
m
m
M 7nc
M 7nc
a
Treatment with 7h, 7j and 7n for 12 h.
Treatment with 7h, 7j and 7n for 24 h.
Treatment with 7h, 7j and 7n for 48 h.
b
c
Fig. 4. Early stages of apoptosis ratio detection of compounds 7 by Annexin V/PI assay.

M.-Y. Ye et al. / European Journal of Medicinal Chemistry 83 (2014) 116e128
121
Fig. 5. Effects of compounds 7h (a), 7j (b) and 7n (c) after treatment for 24 h on the apoptosis cascade by qRT-PCR.
8
m
M for 12 h, 24 h, 48 h, respectively, were observed compared
down-regulation of Bcl-2 expression. Along with that, upregulating
expressions of TP53 were also observed in these cells.
with the G1-phase population of 46.69% for untreated cells. These
results suggested that the cell-cycle of NCI-H460 cells were mainly
stopped in the G1 stage.
The upregulating expressions of CAMK 1, CAMK 2 were also
consistent with the upregulating expressions of cytochrome c,
because the increased mitochondrial Ca2þ accumulation was a
trigger for the release of cytochrome c from the mitochondrial
intermembrane space into the cytosol where it can activate cas-
pases and lead to apoptosis [31]. The result told us that compound
7h induced apoptosis in NCI-H460 lung cancer cells by stimulating
the expressions of TP53, CAMK 1, CAMK 2, AIF, cytochrome c, and
2.4. Compounds 7h, 7j and 7n induces caspase-dependent
apoptosis in NCI-H460 cells
The alizarin a-aminophosphonate derivatives induced apoptotic
pathway was then investigated by real-time quantitative reverse
transcriptase polymerase chain reaction (qRT-PCR) analysis of
caspase expressions. Caspase activation was examined in NCI-
H460 cells, which were treated with or without compounds (7h, 7j
and 7n) (Fig. 5). These caspases play important roles in the
apoptosis pathway. Data in Fig. 5a indicated that caspases-3, -4, and
-9 were activated in concentration-dependent manners after
activating caspases. Previous reports have revealed that a-amino-
phosphonate derivatives may induce apoptosis by affecting asso-
ciated enzymes in mitochondria-dependent pathways [32e34], it is
reasonable to believe that compound 7h may induce apoptosis in
the same mechanism. Similarly, compounds 7j and 7n acted in an
analogous way to 7h (see Fig. 5b and c). In summary, compounds
7h, 7j and 7n induced apoptosis in NCI-H460 lung cancer cells via a
mitochondrion-dependent pathway.
treatment with compound 7h for 24 h at 5 mM,10 mM, and 20 mM, as
assessed by qRT-PCR. In addition, compound 7h treatment showed
concentration-dependent effects in downregulating the expres-
sions of Bcl-2 and upregulating TP53, CAMK 1, CAMK 2, as well as
AIF and cytochrome C. And the data in Fig. 5b and c showed that
compounds 7j and 7n displayed a similar effect as that of 7h.
It has been reported that caspases activation, AIF and cyto-
chrome c release are important signals of apoptosis [28]. Especially
the release of cytochrome c, which was the key matter in apoptosis,
also the main routes that mitochondrion took part in apoptosis.
Moreover, expression of TP53 induces either a stable growth arrest
or programmed cell death (apoptosis) [29]. Overexpression of BAX
is known to promote programmed or apoptotic cell death, and
conversely Bcl-2 inhibits this type of cell death and promotes cell
survival. It is speculated that the ratio of BAX to Bcl-2 plays a critical
part in regulating the relative sensitivity of cells towards apoptosis
[30]. In this study, BAX expression was elevated with a concomitant
2.5. Effect of compounds 7h, 7j and 7n on intracellular reactive
oxygen species (ROS)
Reactive oxygen species (ROS) play an important role in
apoptosis induction under both physiologic and pathologic condi-
tions. Interestingly, mitochondrion is a target of ROS. The genera-
tion of ROS may contribute to mitochondrial damage, reduction of
mitochondrial transmembrane potential. Cytochrome c release
from mitochondria, that triggers caspase activation, appears to be
largely mediated by direct or indirect ROS action [35]. Natural
anthraquinone compounds have been shown to induce apoptosis in
different cancer cell types. Several studies indicated that natural
anthraquinone compounds may induce apoptosis which was
caused and influenced by ROS [36]. Therefore, we supposed that

122
M.-Y. Ye et al. / European Journal of Medicinal Chemistry 83 (2014) 116e128
alizarin
a-aminophosphonate derivatives could significantly
increased the intracellular level of ROS in a dose-dependent
manner. In this article, in vivo estimation of intracellular ROS
levels was measured by 20, 70-dichlorofluorescein (DCF) fluores-
cence. 20, 70-dichlorofluorescin diacetate (DCFH-DA) passively dif-
fuses into cells and is hydrolyzed by cellular esterases to 20, 70-
dichlorofluorescin (DCFH), a nonfluorescent molecule that can be
oxidized to the fluorescent DCF in the presence of peroxides.
Cellular fluorescence was quantified using fluorescence microplate
reader (Nikon ECLIPSETE2000-S) at an excitation of 485 nm and an
emission of 538 nm. Intracellular ROS production was expressed in
comparison with control cells. The result was showed in Fig. 6 and
Fig. 5S (Supplementary). As showed in Fig. 6, NCI-H460 treated for
24 h with increasing concentrations (5 mM, 10 mM and 20 mM) of
compounds 7h, 7j and 7n produced an increase in the DCFH-DA
probe fluorescence emission that correlated well with increasing
concentrations. Fig. 6S shows that cells not treated with the com-
pounds 7h, 7j and 7n were normally green (in the web version). For
7h, 7j and 7n treatment, the cells exhibited strong green fluores-
cence. Therefore, it could be concluded that compounds 7h, 7j and
7n significantly increased the intracellular level of ROS.
Fig. 7. The increase of Fluo-3 fluorescence intensity (arbitrary units) induced by
compounds 7h, 7j and 7n. Values are means S.D. of three determinations. P < 0.01
compared to Blank group.
2.6. Effect of compounds 7h, 7j and 7n on calcium release
the compounds 7h, 7j and 7n were normally green (in the web
version). While for 7h, 7j and 7n treatment, the cells exhibited
strong green fluorescence. Therefore, these findings indicated that
compounds 7h, 7j and 7n significantly increased the intracellular
level of calcium release, which was well consistent with the results
for qRT-PCR.
Calcium has long been recognized as a significant participant in
cell apoptosis. Alizarin and its derivatives are capable of forming
complexes with metal ions. It has been reported that while being
weak complex-forming agents with respect to the free Ca2þ ions,
alizarin derivatives exhibit affinity to calcium in biological struc-
tures [37]. Therefore, we supposed that alizarin
a-amino-
3. Conclusion
phosphonate derivatives may induce calcium signaling linked
apoptosis in NCI-H460 cell line. To monitor the effect of compounds
7h, 7j and 7n on calcium release, NCI-H460 treated for 24 h with
In summary, we have described the design, synthesis and in vitro
antitumor activities of hybrids of aminophosphonate and alizarin.
The in vitro antitumor activities screening showed that many target
compounds exhibited moderate to high levels of antitumor activ-
ities against these tested cancer cell lines. Most of the compounds 7
displayed preferable cytotoxic activities than the commercial
anticancer drug 5-FU, which suggested that the introduction of
aminophosphonate into the parent alizarin scaffold can success-
fully improve the antitumor activity. The action mechanism of
compounds 7h, 7j and 7n were investigated by fluorescence
staining assays, flow cytometric analysis and real-time PCR assays,
which indicated that these compounds induced apoptosis and
involved G1 phase arrest by increasing the production of intracel-
lular Ca2þ and ROS and affecting associated enzymes and genes.
The results indicate that the target compounds may induce
apoptosis through a mitochondrion-dependent pathway.
increasing concentrations (5
nophosphonate derivatives were loaded with the Ca2þ indicator
Fluo-3-acetoxymethyl-ester (Fluo-3/AM, 5 M; Beyotime, Jiangsu,
China). Fluorescence measurements were performed using fluo-
rescence microplate reader (Nikon ECLIPSETE2000-S), Fluo-3 was
excited by argon laser light at 488 nm and fluorescence was
measured at wavelengths of 515 nm. As showed in Fig. 7 and Fig. 6S
(Supplementary), compounds 7h, 7j and 7n produced an increase
in the Fluo-3 probe fluorescence emission that correlated well with
increasing concentrations. Fig. 7S shows that cells not treated with
mM, 10 mM, 20 mM) of alizarin a-ami-
m
4. Experimental
4.1. General
All the chemical reagents and solvents used were of analytical
grade. Compound alizarin (1) was synthesized according the liter-
ature [22]. a-Aminophosphonate derivatives were synthesized ac-
cording to the literature [23].
4.2. Instrumentation
Melting points were measured on an XT-4 apparatus (Taike
Corp., Beijing, China). NMR spectra were recorded on a Bruker AV-
500 NMR spectrometer. Mass spectra were determined on a FTMS
ESI spectrometer. All compounds were determined by PE2400 Type
II elemental analyzer (U.S. PE firm). Apoptosis and cell cycle were
Fig. 6. The increase of DCF-fluorescence intensity (arbitrary units) induced by com-
pounds 7h, 7j and 7n. Values are means
S.D. of three determinations. P < 0.01
compared to Blank group.

M.-Y. Ye et al. / European Journal of Medicinal Chemistry 83 (2014) 116e128
123
analyzed by flow cytometry (BD FACSCalibur Flow Cytometer). An
applied Biosysterms 7500 real-time polymerase chain reaction (RT-
PCR) system was used to carry out the PCR amplification.
124.45, 124.38, 120.49, 119.29, 116.63, 68.64, 63.57, 63.30, 49.76,
16.33,16.10; HRMS (m/z) (ESI): calcd for C27H25BrNO8P [MþHþ]:
601.05012; found:601.04703; Anal. Calc. (for C27H25BrNO8P): C,
53.84; H, 4.18; N, 2.33; Found: C, 53.73; H, 4.11; N, 2.36.
4.3. Synthesis
4.3.1.3. O, O0-diethyl (2-methoxyphenyl) (2-(1-hydroxy-9, 10-dioxo-
9, 10-dihydroanthracen-2-yloxy) acetamido) methylphosphonate
(6c). Yields 88.14%; m.p. 146e148 ꢀC; 1H NMR (500 MHz, CDCl3)
4.3.1. General procedure for compounds 6 (7)
A solution of alizarin (1 mmol) in acetone (200 mL) at room
temperature was treated with bromoacetate (1 mmol) and K2CO3
(1 mmol). The reagents were mixed. The solution was refluxed
vigorously and stirred. The course of the reaction was monitored by
TLC. After the reaction was finished, a part of the solvent was
distilled off. The mixture was treated with water acidified with HCl.
The resulting precipitate was filtered off and dried. The product (2)
was isolated by column chromatography and elution with cyclo-
hexane/EtOAc (V:V, 5:1). According to the same procedure, alizarin
was treated with 2-bromo-ethyl propionate and then purified on
silica gel column using cyclohexane/EtOAc (V:V, 5:1) to obtain
compound 3. To the solution of compound 2 (1 mmol), sodium
hydroxide (2.5 mmol) in water was added. After the solution was
stirred at 70 ꢀC for 10 h, 10% hydrochloric acid (50 mL) was added
dropwise with stirring to adjust the pH value to 4, the resulting
mixture was filtered. Upon recrystallization in hot ethanol, pale
yellowish crystal of compound 4 were obtained. According to the
same procedure, compound 3 was respectively treated with so-
dium hydroxide and hydrochloric acid to offer compound 5. To a
mixture of 4 and HOBt in DMSO (10 mL), the appropriate EDAC was
d
12.98 (s, eOH, 1H), 8.30e8.26 (m, AreH, 2H), 8.22 (d, AreH,
J ¼ 9.8 Hz, 1H), 7.83e7.75 (m, AreH, 3H), 7.35 (d, AreH, J ¼ 7.6 Hz,
1H), 7.26 (dd, AreH, J ¼ 11.3, 4.4 Hz, 1H), 7.12 (d, NH, J ¼ 8.4 Hz, 1H),
6.94 (t, AreH, J ¼ 7.5 Hz, 1H), 6.89 (d, AreH, J ¼ 8.3 Hz,1H), 5.95 (dd,
PCH, J ¼ 20.9, 9.8 Hz, 1H), 4.65 (dd, OCH2CO, J ¼ 49.9,14.6 Hz, 2H),
4.16e4.04 (m, OCH2, 2H), 4.00e3.96 (m, OCH2, 1H), 3.90 (s, OCH3,
3H), 3.87e3.82 (m, OCH2, 1H), 1.28 (t, OCH3, J ¼ 7.1 Hz, 3H), 1.14 (t,
OCH3, J ¼ 7.1 Hz, 3H); 13C NMR (125 MHz, CDCl3)
d 189.02, 181.17,
166.09, 166.07, 157.30, 152.86, 151.35, 134.90, 133.89, 133.83, 133.12,
129.55, 127.41, 126.87, 126.64, 122.52, 120.84, 120.57, 118.50, 116.51,
111.16, 68.25, 63.13, 62.86, 55.65, 46.87, 16.34, 16.17; HRMS (m/z)
(ESI): calcd for C28H28NO9P [MþHþ]: 553.15017; found:553.14617;
Anal. Calc. (for C28H28NO9P): C, 60.76; H, 5.10; N, 2.53; Found: C,
60.70; H, 5.13; N, 2.56.
4.3.1.4. O, O0-diethyl (4-methoxyphenyl) (2-(1-hydroxy-9, 10-dioxo-
9, 10-dihydroanthracen-2-yloxy) acetamido) methylphosphonate
(6d). Yields 86.12%; m.p. 201e203 ꢀC; 1H NMR (500 MHz, CDCl3)
d
12.95 (s, eOH, 1H), 8.32e8.25 (m, AreH, 2H), 7.88e7.76 (m, AreH,
stirred for 15 min at 0 ꢀC. Then
a
-aminophosphonate was added
4H), 7.38 (d, AreH, J ¼ 7.0 Hz, 2H), 7.16 (d, NH, J ¼ 8.4 Hz, 1H), 6.86
(d, AreH, J ¼ 8.6 Hz, 2H), 5.50 (dd, PCH, J ¼ 20.3, 9.6 Hz, 1H), 4.68
(dd, OCH2CO, J ¼ 33.2, 14.8 Hz, 2H), 4.10e3.75 (m, OCH2, 4H), 3.76
(s, OCH3, 3H), 1.28 (t, OCH3, J ¼ 7.0 Hz, 3H), 1.15 (t, OCH3, J ¼ 7.1 Hz,
dropwise with constant stirring at 0 ꢀC, the mixture was stirred at
ambient temperature for another 5 h, then washed with brine,
dried with anhydrous sodium sulfate and evaporated. The residue
was purified by column chromatography on silica gel with petro-
leum ether/ethyl acetate (V:V, 3:1) to give the pure title compounds
6 as yellowish solids. According to the same procedure, compound
5 was respectively treated with sodium hydroxide and hydrochloric
acid to offer compound 7. Their structure identification data of
compounds 6e7 were listed as follow.
3H); 13C NMR (125 MHz, CDCl3)
d 188.96, 181.23, 166.58, 166.52,
159.51, 152.95, 151.43, 134.93, 133.94, 133.81, 133.13, 129.20, 129.15,
127.43, 127.00, 126.93, 126.49, 120.52, 119.38, 116.69, 114.12, 68.75,
63.37, 62.99, 55.21, 49.78, 16.36, 16.23; HRMS (m/z) (ESI): calcd for
C
C
28H28NO9P [MþHþ]: 553.15017; found:553.14697; Anal. Calc. (for
28H28NO9P): C, 60.76; H, 5.10; N, 2.53; Found: C, 60.71; H, 5.14; N,
2.55.
4.3.1.1. O, O0-diethyl (4-bromophenyl) (2-(1-hydroxy-9, 10-dioxo-9,
10-dihydroanthracen-2-yloxy) acetamido) methylphosphonate (6a).
4.3.1.5. O, O0-diethyl (3-methoxyphenyl) (2-(1-hydroxy-9, 10-dioxo-
9, 10-dihydroanthracen-2-yloxy) acetamido) methylphosphonate
(6e). Yields 86.19%; m.p. 183e185 ꢀC; 1H NMR (500 MHz, CDCl3)
Yields 88.12%; m.p. 216e218 ꢀC; 1H NMR (500 MHz, CDCl3)
d 12.98
(s, eOH, 1H), 8.36e8.28 (m, AreH, 2H), 7.90e7.79 (m, AreH, 4H),
7.48 (d, AreH, J ¼ 8.2 Hz, 2H), 7.36e7.31 (m, AreH, 2H), 7.21 (d, NH,
J ¼ 8.4 Hz, 1H), 5.49 (dd, PCH, J ¼ 20.9, 9.5 Hz, 1H), 4.71 (dd,
OCH2CO, J ¼ 39.7, 14.1 Hz, 2H), 4.14e3.99 (m, OCH2, 3H), 3.95e3.86
(m, OCH2, 1H), 1.29 (t, OCH3, J ¼ 7.6 Hz, 3H), 1.19 (t, OCH3, J ¼ 7.1 Hz,
d
12.96 (s, eOH, 1H), 8.32e8.27 (m, AreH, 2H), 7.89e7.78 (m, AreH,
4H), 7.24 (d, AreH, J ¼ 8.0 Hz, 1H), 7.17 (d, NH, J ¼ 8.4 Hz, 1H), 7.03
(d, AreH, J ¼ 7.5 Hz, 1H), 6.97e6.81 (m, AreH, 2H), 5.52 (dd, PCH,
J ¼ 20.6, 9.6 Hz, 1H), 4.70 (dd, OCH2CO, J ¼ 33.8, 14.9 Hz, 2H),
4.11e3.77 (m, OCH2, 4H), 3.76 (s, OCH3, 3H), 1.29 (t, OCH3, J ¼ 7.0 Hz,
3H), 1.17 (t, OCH3, J ¼ 7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3)
3H); 13C NMR (125 MHz, CDCl3)
d 189.08, 181.30, 166.83, 166.77,
153.03, 151.40, 135.04, 134.04, 133.88, 133.76, 133.17, 131.86, 129.58,
129.54, 127.55, 127.26, 127.03, 122.44, 120.57, 119.70, 116.85, 68.91,
63.54, 63.27, 50.17, 16.38, 16.27; HRMS (m/z) (ESI): calcd for
d
189.00, 181.24, 166.66, 159.73, 152.99, 151.43, 135.96, 134.94,
133.96, 133.85, 133.16, 129.68, 127.46, 127.07, 126.94, 120.54, 120.22,
119.46,116.75,113.90,113.51, 68.80, 63.48, 63.14, 55.20, 50.56,16.32,
16.23; HRMS (m/z) (ESI): calcd for C28H28NO9P [MþHþ]: 553.15017;
found:553.14694; Anal. Calc. (for C28H28NO9P): C, 60.76; H, 5.10; N,
2.53; Found: C, 60.73; H, 5.12; N, 2.51.
C
27H25BrNO8P [MþHþ]: 601.05012; found:601.04705; Anal. Calc.
(for C27H25BrNO8P): C, 53.84; H, 4.18; N, 2.33; Found: C, 53.71; H,
4.12; N, 2.38.
4.3.1.2. O, O0-diethyl (2-bromophenyl) (2-(1-hydroxy-9, 10-dioxo-9,
10-dihydroanthracen-2-yloxy) acetamido) methylphosphonate (6b).
4.3.1.6. O, O0-diethyl (2-fluorophenyl) (2-(1-hydroxy-9, 10-dioxo-9,
10-dihydroanthracen-2-yloxy) acetamido) methylphosphonate (6f).
Yields 88.02%; m.p. 213e215 ꢀC; 1H NMR (500 MHz, CDCl3)
d 12.93
(s, eOH, 1H), 8.31e8.23 (m, AreH, 2H), 8.00 (dd, AreH, J ¼ 8.9,
4.9 Hz, 1H), 7.82e7.76 (m, AreH, 3H), 7.55 (dd, AreH, J ¼ 11.2,
4.9 Hz, 2H), 7.31 (t, AreH, J ¼ 7.4 Hz, 1H), 7.18e7.11 (m, AreH, 1H,
NH, 1H), 6.11 (dd, PCH, J ¼ 20.8, 9.2 Hz, 1H), 4.70 (dd, OCH2CO,
J ¼ 38.3, 14.5 Hz, 2H), 4.19e3.76 (m, OCH2, 4H), 1.32 (t, OCH3,
J ¼ 7.1 Hz, 3H), 1.12 (t, OCH3, J ¼ 7.1 Hz, 3H); 13C NMR (125 MHz,
Yields 78.62%; m.p. 193e195 ꢀC; 1H NMR (500 MHz, CDCl3)
d 12.97
(s, eOH, 1H), 8.34e8.29 (m, AreH, 2H), 7.96 (d, AreH, J ¼ 5.3 Hz,
1H), 7.85e7.79 (m, AreH, 3H), 7.46 (t, AreH, J ¼ 7.2 Hz, 1H),
7.31e7.27 (m, AreH, 1H), 7.20e7.12 (m, AreH, 1H, NH, 1H),
7.09e7.05 (m, AreH, 1H), 5.89 (dd, PCH, J ¼ 21.0, 9.4 Hz, 1H), 4.70
(dd, OCH2CO, J ¼ 39.3, 14.6 Hz, 2H), 4.17e3.90 (m, OCH2, 4H), 1.31 (t,
OCH3, J ¼ 7.0 Hz, 3H), 1.18 (t, OCH3, J ¼ 6.9 Hz, 3H); 13C NMR
CDCl3)
d 188.89, 181.16, 166.54, 166.48, 152.89, 151.36, 134.89,
134.78, 133.91, 133.76, 133.08, 129.64, 129.16, 127.77, 127.39, 126.89,
(125 MHz, CDCl3) d 189.03, 181.31, 166.62, 166.56, 153.04, 151.41,

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M.-Y. Ye et al. / European Journal of Medicinal Chemistry 83 (2014) 116e128
134.97, 133.98, 133.88, 133.20, 130.04, 129.36, 127.49, 127.10,
126.98, 124.47, 120.55, 119.38, 116.77, 115.87, 115.70, 68.76,
63.50, 63.31, 44.88, 16.35, 16.19; HRMS (m/z) (ESI): calcd for
133.98, 133.89, 133.20, 129.05, 128.67, 128.57, 127.48, 126.97, 124.95,
120.55, 119.66, 119.55, 116.80, 68.99, 63.45, 50.60, 49.37, 21.38,
16.33, 16.17; HRMS (m/z) (ESI): calcd for C28H28NO8P [MþHþ]:
537.15525; found:537.15228; Anal. Calc. (for C28H28NO8P): C, 62.57;
H, 5.25; N, 2.61; Found: C, 62.63; H, 5.22; N, 2.59.
C
C
27H25FNO8P [MþHþ]: 541.13018; found:541.12697; Anal. Calc. (for
27H25FNO8P): C, 59.89; H, 4.65; N, 2.59; Found: C, 59.93; H, 4.62;
N, 2.56.
4.3.1.11. O, O0-diethyl (4-tolyl) (2-(1-hydroxy-9, 10-dioxo-9, 10-
4.3.1.7. O, O0-diethyl (4-fluorophenyl) (2- (1-hydroxy-9, 10-dioxo-9,
10-dihydroanthracen-2-yloxy) acetamido) methylphosphonate (6g).
dihydroanthracen-2-yloxy) acetamido) methylphosphonate (6k).
Yields 87.57%; m.p. 219e221 ꢀC; 1H NMR (500 MHz, CDCl3)
d 12.97
Yields 82.54%; m.p. 215e217 ꢀC; 1H NMR (500 MHz, CDCl3)
d
12.99
(s, eOH, 1H), 8.34e8.29 (m, AreH, 2H), 7.88e7.78 (m, AreH, 4H),
7.34 (d, AreH, J ¼ 6.7 Hz, 2H), 7.26e7.14 (m, AreH, 2H, NH, 1H), 5.52
(dd, PCH, J ¼ 20.4, 9.6 Hz, 1H), 4.69 (dd, OCH2CO, J ¼ 35.9, 14.7 Hz,
2H), 4.12e3.81 (m, OCH2, 4H), 2.31 (s, CH3, 3H), 1.29 (t, OCH3,
J ¼ 7.0 Hz, 3H), 1.16 (t, OCH3, J ¼ 7.0 Hz, 3H); 13C NMR (125 MHz,
(s, eOH, 1H), 8.34e8.29 (m, AreH, 2H), 7.91e7.80 (m, AreH, 4H),
7.50e7.40 (m, AreH, 2H), 7.20 (d, NH, J ¼ 8.4 Hz, 1H), 7.04 (t, AreH,
J ¼ 8.6 Hz, 2H), 5.52 (dd, PCH, J ¼ 20.7, 9.5 Hz, 1H), 4.70 (dd,
OCH2CO, J ¼ 29.6, 14.8 Hz, 2H), 4.13e3.86 (m, OCH2, 4H), 1.29 (t,
OCH3, J ¼ 7.1 Hz, 3H), 1.16 (d, OCH3, J ¼ 7.1 Hz, 3H); 13C NMR
CDCl3) d 189.02, 181.30, 166.52, 166.51, 153.01, 151.46, 138.10, 134.97,
(125 MHz, CDCl3)
d
189.06, 181.28, 166.76, 166.70, 153.01, 151.41,
133.98, 133.18, 131.44, 129.41, 127.83, 127.78, 127.49, 127.07, 126.98,
120.56, 119.42, 116.75, 68.81, 63.43, 63.07, 50.31, 21.09, 16.37, 16.24;
HRMS (m/z) (ESI): calcd for C28H28NO8P [MþHþ]: 537.15525;
found:537.15208; Anal. Calc. (for C28H28NO8P): C, 62.57; H, 5.25; N,
2.61; Found: C, 62.59; H, 5.27; N, 2.65.
135.02, 134.02, 133.86, 133.16, 130.51, 129.74, 129.69, 127.52,
127.21, 127.00, 120.56, 119.61, 116.82, 115.78, 115.60, 68.87,
63.51, 63.22, 49.94, 16.37,16.24; HRMS (m/z) (ESI): calcd for
C
C
27H25FNO8P [MþHþ]: 541.13018; found:541.12688; Anal. Calc. (for
27H25FNO8P): C, 59.89; H, 4.65; N, 2.59; Found: C, 59.94; H, 4.70;
N, 2.64.
4.3.1.12. O, O0-diethyl (4-bromophenyl) (2-(1-hydroxy-9, 10-dioxo-9,
10-dihydroanthracen-2-yloxy) propanamido) methylphosphonate
(7a). Yields 79.12%; m.p. 166e168 ꢀC; 1H NMR (500 MHz, CDCl3)
4.3.1.8. O, O0-diethyl (2-chlorophenyl) (2-(1-hydroxy-9, 10-dioxo-9,
10-dihydroanthracen-2-yloxy) acetamido) methylphosphonate (6h).
d 13.05 (s, eOH, 1H), 8.33e8.28 (m, AreH, 2H), 8.03e7.90 (m, AreH,
Yields 82.14%; m.p. 210e212 ꢀC; 1H NMR (500 MHz, CDCl3)
d
12.92
1H), 7.86e7.76 (m, AreH, 3H), 7.51 (d, AreH, J ¼ 8.3 Hz, 1H), 7.35
(dd, AreH, J ¼ 8.5, 2.1 Hz, 2H), 7.23 (m, AreH, 1H, NH, 1H), 5.40 (dd,
PCH, J ¼ 20.8, 9.1 Hz, 1H), 4.85 (dd, OCHCO, J ¼ 27.8, 6.9 Hz, 1H),
4.20e3.77 (m, OCH2, 4H), 1.65 (d, CH3, J ¼ 6.8 Hz, 3H), 1.15 (t, OCH3,
J ¼ 7.1 Hz, 3H), 1.09 (t, OCH3, J ¼ 7.1 Hz, 3H); 13C NMR (125 MHz,
(s, eOH, 1H), 8.28e8.23 (m, AreH, 2H), 8.01 (dd, AreH, J ¼ 9.1,
4.8 Hz, 1H), 7.81e7.75 (m, AreH, 3H), 7.55 (dt, AreH, J ¼ 7.6, 1.9 Hz,
1H), 7.37 (d, AreH, J ¼ 7.8 Hz, 1H), 7.27e7.21 (m, AreH, 2H), 7.14 (d,
NH, J ¼ 8.3 Hz, 1H), 6.12 (dd, PCH, J ¼ 20.9, 9.3 Hz, 1H), 4.68 (dd,
OCH2CO, J ¼ 20.1, 8.9 Hz, 2H), 4.21e4.12 (m, OCH2, 2H), 4.00e3.79
(m, OCH2, 2H), 1.31 (t, OCH3, J ¼ 7.1 Hz, 3H), 1.12 (t, OCH3, J ¼ 7.1 Hz,
CDCl3)
d 189.03, 181.30, 170.61, 168.40, 153.32, 151.20, 135.03,
134.04, 133.76, 133.33, 131.87, 131.68, 129.50, 129.45, 127.50, 127.21,
127.00, 121.10, 121.02, 120.53, 116.93, 77.32, 63.50, 63.08, 50.06,
3H); 13C NMR (125 MHz, CDCl3)
d 188.89, 181.15, 166.55, 166.49,
152.88, 151.35, 134.89, 133.98, 133.90, 133.76, 133.07, 132.99, 129.75,
129.38, 129.08, 127.38, 127.15, 126.89, 120.49, 119.25, 116.62, 68.62,
63.59, 63.34, 47.20, 16.32, 16.08; HRMS (m/z) (ESI): calcd for
18.65, 16.25, 16.20; 31P NMR (200 MHz, CDCl3)
d 20.29; ESI-MS m/z:
616.1 (MþH)þ; Anal. Calc. (for C28H27BrNO8P): C, 54.56; H, 4.42; N,
2.27; Found: C, 54.60; H, 4.52; N, 2.29.
C
27H25ClNO8P [MþHþ]: 557.10063; found:557.09725; Anal. Calc.
(for C27H25ClNO8P): C, 58.13; H, 4.52; N, 2.51; Found: C, 58.43; H,
4.62; N, 2.56.
4.3.1.13. O, O0-diethyl (2-bromophenyl) (2-(1-hydroxy-9, 10-dioxo-9,
10-dihydroanthracen-2-yloxy) propanamido) methylphosphonate
(7b). Yields 88.44%; m.p. 156e158 ꢀC; 1H NMR (500 MHz, CDCl3)
4.3.1.9. O, O0-diethyl (phenyl) (2-(1-hydroxy-9, 10-dioxo-9, 10-
d 12.98 (s, eOH, 1H), 8.34e8.27 (m, AreH, 2H), 8.01 (dd, AreH,
dihydroanthracen-2-yloxy) acetamido) methylphosphonate (6i).
J ¼ 9.1, 4.7 Hz, 1H), 7.85e7.76 (m, AreH, 3H), 7.74 (s, AreH, 1H),
7.42e7.33 (m, AreH, 2H), 7.16e7.02 (m, AreH,1H, NH,1H), 6.06 (dd,
PCH, J ¼ 20.9, 9.2 Hz, 1H), 4.83 (dd, OCHCO, J ¼ 28.0, 6.8 Hz, 1H),
4.23e3.67 (m, OCH2, 4H), 1.73 (d, CH3, J ¼ 6.7 Hz, 3H), 1.36 (t, OCH3,
J ¼ 7.1 Hz, 3H), 1.11 (t, OCH3, J ¼ 7.0 Hz, 3H); 13C NMR (125 MHz,
Yields 75.12%; m.p. 171e173 ꢀC; 1H NMR (500 MHz, CDCl3)
d 12.93
(s, eOH, 1H), 8.27 (m, AreH, 2H), 7.98e7.90 (m, AreH, 1H),
7.81e7.74 (m, AreH, 3H), 7.45 (d, AreH, J ¼ 7.6 Hz, 2H), 7.35e7.27
(m, AreH, 3H), 7.15 (d, NH, J ¼ 8.3 Hz, 1H), 5.55 (dd, PCH, J ¼ 20.6,
9.6 Hz, 1H), 4.67 (dd, OCH2CO, J ¼ 33.1, 14.7 Hz, 2H), 4.12e3.80 (m,
OCH2, 4H),1.27 (t, OCH3, J ¼ 7.0 Hz, 3H),1.13 (t, OCH3, J ¼ 7.0 Hz, 3H);
CDCl3) d 188.99, 181.27, 170.60, 167.36, 153.15, 151.15, 134.97, 134.44,
134.00, 133.76, 133.01, 129.63, 129.09, 127.66, 127.46, 126.94, 126.57,
124.46, 120.86, 120.57, 116.76, 77.25, 63.81, 63.55, 49.75, 18.64,
13C NMR (125 MHz, CDCl3)
d 188.89, 181.14, 166.62, 166.56, 152.90,
151.38, 134.89, 134.48, 133.90, 133.75, 133.07, 128.65, 128.64,
128.22, 127.90, 127.86, 127.38, 126.88, 120.48, 119.34, 116.63, 68.69,
63.43, 63.09, 50.54, 16.31, 16.16; HRMS (m/z) (ESI): calcd for
16.34, 16.04; 31P NMR (200 MHz, CDCl3)
d 20.32; ESI-MS m/z: 616.1
(MþH)þ; Anal. Calc. (for C28H27BrNO8P): C, 54.56; H, 4.42; N, 2.27;
Found: C, 54.51; H, 4.50; N, 2.31.
C
C
27H26NO8P [MþHþ]: 523.13960; found:557.13657; Anal. Calc. (for
27H26NO8P): C, 61.95; H, 5.01; N, 2.68; Found: C, 61.93; H, 5.12; N,
4.3.1.14. O, O0-diethyl (3-bromophenyl) (2-(1-hydroxy-9, 10-dioxo-9,
10-dihydroanthracen-2-yloxy) propanamido) methylphosphonate
(7c). Yields 86.12%; m.p. 155e157 ꢀC; 1H NMR (500 MHz, CDCl3)
2.56.
4.3.1.10. O, O0-diethyl (3-tolyl) (2-(1-hydroxy-9, 10-dioxo-9, 10-
d 13.02 (s, eOH, 1H), 8.34e8.25 (m, AreH, 2H), 8.00e7.92 (m, AreH,
dihydroanthracen-2-yloxy) acetamido) methylphosphonate (6j).
1H), 7.84e7.73 (m, AreH, 3H), 7.47e7.26 (m, AreH, 4H), 7.22e7.10
(m, NH, 1H), 5.45 (dd, PCH, J ¼ 20.13, 8.3 Hz, 1H), 4.89 (q, OCHCO,
J ¼ 30.6, 13.6 Hz, 1H), 4.19e3.93 (m, OCH2, 4H), 1.66 (d, CH3,
J ¼ 6.7 Hz, 3H), 1.17 (t, OCH3, J ¼ 7.2 Hz, 3H), 1.09 (t, OCH3, J ¼ 7.1 Hz,
Yields 79.13%; m.p. 156e158 ꢀC; 1H NMR (500 MHz, CDCl3)
d 12.98
(s, eOH, 1H), 8.34e8.29 (m, AreH, 2H), 7.89e7.78 (m, AreH, 4H),
7.25e7.16 (m, AreH, 4H), 7.10 (d, NH, J ¼ 6.7 Hz, 1H), 5.51 (dd, PCH,
J ¼ 20.4, 9.2 Hz, 1H), 4.70 (dd, OCH2CO, J ¼ 35.6, 14.7 Hz, 2H),
4.12e3.82 (m, OCH2, 4H), 2.33 (s, CH3, 3H), 1.29 (t, OCH3, J ¼ 6.9 Hz,
3H), 1.15 (t, OCH3, J ¼ 6.8 Hz, 3H); 13C NMR (125 MHz, CDCl3)
3H); 13C NMR (125 MHz, CDCl3)
d 188.99, 181.29, 170.66, 153.40,
151.22, 134.97, 134.01, 133.78, 133.11, 131.29, 131.18, 130.97, 130.86,
130.23, 129.99, 127.46, 126.98, 126.63, 122.68, 121.31, 116.92, 77.41,
68.27, 63.15, 48.89, 18.56, 16.18, 16.07; ESI-MS m/z: 616.1 (MþH)þ;
d
189.02, 181.28, 166.57, 153.09, 151.50, 138.37, 134.96, 134.37,

M.-Y. Ye et al. / European Journal of Medicinal Chemistry 83 (2014) 116e128
125
Anal. Calc. (for C28H27BrNO8P): C, 54.56; H, 4.42; N, 2.27; Found: C,
54.59; H, 4.40; N, 2.31.
4.3.1.19. O, O0-diethyl (2-fluorophenyl) (2-(1-hydroxy-9, 10-dioxo-9,
10-dihydroanthracen-2-yloxy) propanamido) methylphosphonate
(7h). Yields 76.90%; m.p. 166e168 ꢀC; 1H NMR (500 MHz, CDCl3)
4.3.1.15. O, O0-diethyl (4-chlorophenyl) (2-(1-hydroxy-9, 10-dioxo-9,
10-dihydroanthracen-2-yloxy) propanamido) methylphosphonate
(7d). Yields 83.72%; m.p. 178e180 ꢀC; 1H NMR (500 MHz, CDCl3)
d 13.05 (s, eOH, 1H), 8.35e8.28 (m, AreH, 2H), 8.06e7.90 (m, AreH,
1H), 7.87e7.73 (m, AreH, 3H), 7.52e7.29 (m, AreH, 2H), 7.21e6.94
(m, AreH, 2H, NH, 1H), 5.82 (dd, PCH, J ¼ 21.0, 9.5 Hz, 1H), 4.88 (dd,
OCHCO, J ¼ 32.4, 6.5 Hz, 1H), 4.21e3.81 (m, OCH2, 4H), 1.65 (d, CH3,
J ¼ 6.4 Hz, 3H), 1.16 (m, OCH3, J ¼ 6.9 Hz, 3H), 1.09 (t, OCH3,
d
12.99 (s, eOH, 1H), 8.30e8.22 (m, AreH, 2H), 8.11 (dd, AreH,
J ¼ 9.3, 3.9 Hz, 1H), 7.99e7.75 (m, AreH, 4H), 7.41e7.32 (m, AreH,
3H), 7.26e7.20 (m, NH, 1H), 5.46 (dd, PCH, J ¼ 21.1, 9.5 Hz, 1H), 4.88
(dd, OCHCO, J ¼ 31.6, 6.4 Hz, 1H), 4.08e3.71 (m, OCH2, 4H), 1.63 (d,
CH3, J ¼ 6.8 Hz, 3H), 1.14 (t, OCH3, J ¼ 7.1 Hz, 3H), 1.06 (t, OCH3,
J ¼ 6.8 Hz, 3H); 13C NMR (125 MHz, CDCl3)
d 189.00, 181.32, 170.57,
163.18, 153.31, 151.28, 135.01, 134.05, 133.79, 133.13, 132.80, 129.89,
129.75, 129.58, 129.48, 129.07, 127.48, 127.37, 127.01, 120.78, 120.58,
77.13, 63.85, 63.41, 47.19, 18.55, 16.29, 16.10; 31P NMR (200 MHz,
J ¼ 7.1 Hz, 3H); 13C NMR (125 MHz, CDCl3)
d 188.96, 181.25, 170.89,
170.83, 163.11, 153.28, 151.21, 134.95, 133.98, 133.72, 133.05, 132.81,
129.24, 129.19, 128.92, 128.75, 127.43, 126.93, 121.06, 120.48, 116.82,
77.11, 63.80, 63.43, 49.92, 18.54, 16.13, 16.02; 31P NMR (200 MHz,
CDCl3)
d
20.25; ESI-MS m/z: 554.1 (MꢁH)d; Anal. Calc. (for
C28H27FNO8P): C, 60.54; H, 4.90; N, 2.52; Found: C, 60.61; H, 4.93;
N, 2.53.
CDCl3)
d
20.45; ESI-MS m/z: 570.1 (MꢁH)d; Anal. Calc. (for
C
28H27ClNO8P): C, 58.80; H, 4.76; N, 2.45; Found: C, 58.70; H, 4.72;
4.3.1.20. O, O0-diethyl (3-fluorophenyl) (2-(1-hydroxy-9, 10-dioxo-9,
10-dihydroanthracen-2-yloxy) propanamido) methylphosphonate
(7i). Yields 80.93%; m.p. 178e180 ꢀC; 1H NMR (500 MHz, CDCl3)
N, 2.49.
4.3.1.16. O, O0-diethyl (2-chlorophenyl) (2-(1-hydroxy-9, 10-dioxo-9,
10-dihydroanthracen-2-yloxy) propanamido) methylphosphonate
(7e). Yields 83.18%; m.p. 164e166 ꢀC; 1H NMR (500 MHz, CDCl3)
d 13.02 (s, eOH, 1H), 8.34e8.28 (m, AreH, 2H), 7.96 (dd, AreH,
J ¼ 9.3, 4.7 Hz, 1H), 7.85e7.74 (m, AreH, 3H), 7.38e7.24 (m, AreH,
2H), 7.21e7.11 (m, AreH, 2H), 7.03e6.94 (m, NH, 1H), 5.49 (dd, PCH,
J ¼ 9.5, 5.1 Hz, 1H), 4.90 (dd, OCHCO, J ¼ 32.1, 6.3 Hz, 1H), 4.16e3.80
(m, OCH2, 4H), 1.74 (d, CH3, J ¼ 6.8 Hz, 3H), 1.32 (t, OCH3, J ¼ 7.1 Hz,
3H), 1.09 (t, OCH3, J ¼ 7.1 Hz, 3H); 13C NMR (125 MHz, CDCl3)
d
13.00 (s, eOH, 1H), 8.30e8.21 (m, AreH, 2H), 8.13 (dd, AreH,
J ¼ 9.3, 4.6 Hz, 1H), 7.83e7.70 (m, AreH, 3H), 7.57 (dd, AreH, J ¼ 7.7,
1.9 Hz, 1H), 7.39e7.24 (m, AreH, 3H), 7.16e7.08 (m, NH, 1H), 6.06
(dd, PCH, J ¼ 21.0, 9.3 Hz, 1H), 4.96e4.87 (dd, OCHCO, J ¼ 20.8,
6.6 Hz, 1H), 4.23e3.69 (m, OCH2, 4H), 1.62 (d, CH3, J ¼ 6.8 Hz, 3H),
1.17 (t, OCH3, J ¼ 7.1 Hz, 3H), 1.04 (t, OCH3, J ¼ 7.1 Hz, 3H); 13C NMR
d
189.07,189.01, 181.24,170.62, 153.19,151.23,137.02,134.98,134.02,
133.99, 133.81, 133.13, 130.28, 127.48, 126.98, 123.56, 121.34, 120.54,
116.95, 115.17, 114.98, 77.26, 63.50, 63.12, 50.18, 18.66, 16.39, 16.21;
(125 MHz, CDCl3)
d
188.99, 181.31, 170.62, 163.17, 153.29, 151.27,
31P NMR (200 MHz, CDCl3)
d 20.93; HRMS (m/z) (ESI): calcd for
135.00, 134.04, 133.77, 133.11, 132.78, 129.88, 129.73, 129.57, 129.47,
129.05, 127.46, 127.37, 126.99, 120.76, 120.57, 77.12, 63.83, 63.40,
C
28H27FNO8P [MþHþ]: 555.14583; found: 555.14386; Anal. Calc.
(for C28H27FNO8P): C, 60.54; H, 4.90; N, 2.52; Found: C, 60.56; H,
4.92; N, 2.54.
47.18,18.70,16.23,16.04; 31P NMR (200 MHz, CDCl3)
d 20.31; ESI-MS
m/z: 570.1 (MꢁH)d; Anal. Calc. (for C28H27ClNO8P): C, 58.80; H,
4.76; N, 2.45; Found: C, 58.84; H, 4.79; N, 2.50.
4.3.1.21. O, O0-diethyl (2-methoxyphenyl) (2-(1-hydroxy-9, 10-dioxo-
9, 10-dihydroanthracen-2-yloxy) propanamido) methylphosphonate
(7j). Yields 86.52%; m.p. 180e182 ꢀC; 1H NMR (500 MHz, CDCl3)
4.3.1.17. O, O0-diethyl (3-chlorophenyl) (2-(1-hydroxy-9, 10-dioxo-9,
10-dihydroanthracen-2-yloxy) propanamido) methylphosphonate
(7f). Yields 89.42%; m.p. 148e150 ꢀC; 1H NMR (500 MHz, CDCl3)
d
13.08 (s, eOH, 1H), 8.36e8.25 (m, AreH, 3H), 8.02e7.84 (m, AreH,
4H), 7.40 (d, AreH, J ¼ 7.2 Hz, 1H), 7.33 (t, AreH, J ¼ 7.8 Hz, 1H), 7.00
(t, AreH, J ¼ 7.4 Hz, 1H), 6.94 (d, NH, J ¼ 8.2 Hz, 1H), 5.96 (dd, PCH,
J ¼ 20.8, 9.8 Hz, 1H), 4.96 (dd, OCHCO, J ¼ 27.6, 6.8 Hz, 1H),
4.03e3.96 (m, OCH2, 2H), 3.97e3.76 (m, OCH2, 2H), 3.91 (s, OCH3,
3H), 1.67 (d, CH3, J ¼ 6.6 Hz, 3H), 1.18 (t, OCH3, J ¼ 7.0 Hz, 3H), 1.09 (t,
d
13.06 (s, eOH, 1H), 8.36e8.26 (m, AreH, 2H), 7.97 (dd, AreH,
J ¼ 9.3, 4.8 Hz, 1H), 7.85e7.74 (m, AreH, 3H), 7.46e7.27 (m, AreH,
4H), 7.17 (d, NH, J ¼ 8.0 Hz, 1H), 5.43 (dd, PCH, J ¼ 21.1, 9.4 Hz, 1H),
4.90 (dd, OCHCO, J ¼ 6.8, 1.2 Hz, 1H), 4.09e3.77 (m, OCH2, 4H), 1.67
(d, CH3, J ¼ 6.8 Hz, 3H), 1.16 (t, OCH3, J ¼ 7.1 Hz, 3H), 1.09 (t, OCH3,
OCH3, J ¼ 7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3)
d 188.96, 181.28,
J ¼ 7.1 Hz, 3H); 13C NMR (125 MHz, CDCl3)
d
189.03, 181.34, 170.63,
170.52, 163.26, 151.25, 135.67, 134.93, 133.97, 133.72, 133.05, 129.72,
129.61, 127.41, 127.01, 126.92, 120.84, 120.48, 120.06, 120.02, 119.77,
116.78, 77.06, 63.46, 63.02, 50.43, 18.55, 16.16, 16.05; 31P NMR
153.38, 151.22, 136.61, 135.03, 134.58, 134.06, 133.77, 133.10, 129.98,
128.45, 127.96, 127.51, 127.26, 127.01, 126.01, 121.21, 120.52, 116.92,
77.32, 63.46, 63.10, 48.93, 18.60, 16.20, 16.10; ESI-MS m/z: 570.1
(MꢁH)d; Anal. Calc. (for C28H27ClNO8P): C, 58.80; H, 4.76; N, 2.45;
Found: C, 58.82; H, 4.78; N, 2.51.
(200 MHz, CDCl3)
d
21.19; ESI-MS m/z: 566.2 (MꢁH)d; Anal. Calc.
(for C29H30NO9P): C, 61.37; H, 5.33; N, 2.47; Found: C, 61.43; H, 5.37;
N, 2.50.
4.3.1.18. O, O0-diethyl (4-fluorophenyl) (2-(1-hydroxy-9, 10-dioxo-9,
10-dihydroanthracen-2-yloxy) propanamido) methylphosphonate
(7g). Yields 84.44%; m.p. 176e178 ꢀC; 1H NMR (500 MHz, CDCl3)
4.3.1.22. O, O0-diethyl (3-methoxyphenyl) (2-(1-hydroxy-9, 10-dioxo-
9, 10-dihydroanthracen-2-yloxy) propanamido) methylphosphonate
(7k). Yields 83.16%; m.p. 157e159 ꢀC; 1H NMR (500 MHz, CDCl3)
d
13.01 (s, eOH, 1H), 8.31e8.26 (m, AreH, 2H), 7.90 (dd, AreH,
d 13.03 (s, eOH, 1H), 8.33e8.23 (m, AreH, 2H), 8.05e7.99 (m, AreH,
J ¼ 9.3, 4.5 Hz, 1H), 7.83e7.72 (m, AreH, 3H), 7.48e7.39 (m, AreH,
2H), 7.24 (d, NH, J ¼ 5.5 Hz, 1H), 7.05 (t, AreH, J ¼ 8.6 Hz, 2H), 5.43
(dd, PCH, J ¼ 20.7, 9.5 Hz, 1H), 4.89e4.84 (dd, OCHCO, J ¼ 31.4,
6.5 Hz, 1H), 4.16e3.72 (m, OCH2, 4H), 1.63 (d, CH3, J ¼ 6.8 Hz, 3H),
1.13 (t, OCH3, J ¼ 7.1 Hz, 3H), 1.05 (t, OCH3, J ¼ 7.1 Hz, 3H); 13C NMR
1H), 7.83e7.71 (m, AreH, 3H), 7.29e7.22 (m, AreH, 2H), 7.04 (d, NH,
J ¼ 7.6 Hz, 1H), 6.84 (dd, AreH, J ¼ 16.7, 8.0 Hz, 2H), 5.46 (dd, PCH,
J ¼ 20.7, 9.6 Hz, 1H), 4.89 (dd, OCHCO, J ¼ 6.7, 0.9 Hz, 1H), 3.96e3.83
(m, OCH2, 4H), 3.81 (s, OCH3, 3H),1.66 (d, CH3, J ¼ 6.8 Hz, 3H),1.15 (t,
OCH3, J ¼ 7.1 Hz, 3H), 1.07 (t, OCH3, J ¼ 7.1 Hz, 3H); 13C NMR
(125 MHz, CDCl3)
d
189.07, 181.35, 170.58, 153.46, 151.29, 135.04,
(125 MHz, CDCl3) d 188.85, 181.23, 170.21, 153.21, 151.15, 134.81,
134.07, 133.85, 133.17, 130.49, 129.69, 129.58, 127.54, 127.34, 127.04,
121.31, 120.54, 116.99, 115.85, 115.68, 115.50, 77.40, 63.41, 63.04,
133.87, 133.65, 133.01, 130.65, 128.99, 129.78, 128.66, 127.32, 126.87,
126.60, 125.88, 125.14, 123.12, 120.81, 120.38, 116.72, 77.13, 63.39,
49.92, 18.60, 16.24, 16.14; 31P NMR (200 MHz, CDCl3)
d
20.70; ESI-
62.87, 45.89, 18.44, 16.12, 15.77; 31P NMR (200 MHz, CDCl3)
d 20.86;
MS m/z: 554.1 (MꢁH)d; Anal. Calc. (for C28H27FNO8P): C, 60.54; H,
ESI-MS m/z: 566.2 (MꢁH)d; Anal. Calc. (for C29H30NO9P): C, 61.37;
4.90; N, 2.52; Found: C, 60.63; H, 4.89; N, 2.50.
H, 5.33; N, 2.47; Found: C, 61.40; H, 5.34; N, 2.51.

126
M.-Y. Ye et al. / European Journal of Medicinal Chemistry 83 (2014) 116e128
4.3.1.23. O, O0-diethyl (4-methoxyphenyl) (2-(1-hydroxy-9, 10-dioxo-
9, 10-dihydroanthracen-2-yloxy) propanamido) methylphosphonate
(7l). Yields 84.82%; m.p. 160e162 ꢀC; 1H NMR (500 MHz, CDCl3)
4.3.1.27. O, O0-diethyl (3-tolyl) (2-(1-hydroxy-9, 10-dioxo-9, 10-
dihydroanthracen-2-yloxy) propanamido) methylphosphonate (7p).
Yields 87.32%; m.p. 101e103 ꢀC; 1H NMR (500 MHz, CDCl3)
d 12.97
d
12.99 (s, eOH, 1H), 8.30e8.21 (m, AreH, 2H), 7.89e7.68 (m, AreH,
(s, eOH, 1H), 8.31e8.23 (m, AreH, 2H), 7.90 (dd, AreH, J ¼ 9.6,
4.1 Hz, 1H), 7.81e7.69 (m, AreH, 3H), 7.27e7.23 (m, AreH, 2H),
7.13e6.96 (m, AreH, 2H, NH, 1H), 5.46 (dd, PCH, J ¼ 9.7, 5.3 Hz, 1H),
4.82 (dd, OCHCO, J ¼ 12.2, 5.5 Hz,1H), 4.17e3.70 (m, OCH2, 4H), 2.20
(s, CH3, 3H), 1.73 (d, CH3, J ¼ 6.8 Hz, 3H), 1.32 (t, OCH3, J ¼ 7.1 Hz,
3H), 1.05 (t, OCH3, J ¼ 7.1 Hz, 3H); 13C NMR (125 MHz, CDCl3)
4H), 7.42e7.20 (m, AreH, 2H, NH, 1H), 6.80 (dd, AreH, J ¼ 89.3,
8.4 Hz, 2H), 5.41 (dd, PCH, J ¼ 20.2, 9.6 Hz, 1H), 4.87 (dd, OCHCO,
J ¼ 12.3, 5.3 Hz, 1H), 4.15e3.80 (m, OCH2, 4H), 3.70 (s, OCH3, 3H),
1.63 (d, CH3, J ¼ 6.7 Hz, 3H), 1.14 (t, OCH3, J ¼ 6.9 Hz, 3H), 1.06 (t,
OCH3, J ¼ 7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3)
d 188.91, 181.20,
170.26, 159.46, 153.33, 151.25, 134.88, 133.92, 133.73, 133.06, 129.07,
127.38, 127.07, 126.90, 126.47, 120.99, 120.43, 116.80, 114.11, 113.90,
77.25, 63.21, 62.77, 55.20, 49.84, 18.52, 16.18, 16.13; HRMS (m/z)
(ESI): calcd for C29H30NO9P [MþHþ]: 567.16852; found:567.16357;
Anal. Calc. (for C29H30NO9P): C, 61.37; H, 5.33; N, 2.47; Found: C,
61.40; H, 5.35; N, 2.45.
d 188.94, 181.22, 170.45, 153.38, 151.28, 138.35, 134.90, 134.28,
133.92, 133.08, 129.02, 128.87, 128.43, 127.41, 127.10, 126.92, 124.88,
121.05, 120.84, 120.49, 116.83, 63.35, 63.12, 50.46, 49.23, 21.24,
18.70, 16.33, 16.16; 31P NMR (200 MHz, CDCl3)
d 21.02; HRMS (m/z)
(ESI): calcd for C29H30NO8P [MþHþ]: 551.17090; found: 551.16870;
Anal. Calc. (for C29H30NO8P): C, 63.15; H, 5.48; N, 2.54; Found: C,
63.20; H, 5.47; N, 2.50.
4.3.1.24. O, O0-diethyl (phenyl) (2-(1-hydroxy-9, 10-dioxo-9, 10-
4.3.1.28. O, O0-diethyl (4-tolyl) (2-(1-hydroxy-9, 10-dioxo-9, 10-
dihydroanthracen-2-yloxy) propanamido) methylphosphonate (7m).
Yields 88.16%; m.p. 167e169 ꢀC; 1H NMR (500 MHz, CDCl3)
d 13.04
dihydroanthracen-2-yloxy) propanamido) methylphosphonate (7q).
Yields 83.98%; m.p. 155e157 ꢀC; 1H NMR (500 MHz, CDCl3)
d 13.00
(s, eOH, 1H), 8.33e8.28 (m, AreH, 2H), 8.06e8.01 (m, AreH, 1H),
7.85e7.78 (m, AreH, 3H), 7.47e7.03 (m, AreH, 5H, NH, 1H), 5.50
(dd, PCH, J ¼ 20.7, 9.6 Hz,1H), 4.91 (dd, OCHCO, J ¼ 32.3, 6.8 Hz, 1H),
3.97e3.69 (m, OCH2, 4H), 1.66 (d, CH3, J ¼ 6.8 Hz, 3H), 1.14 (t, OCH3,
J ¼ 7.0 Hz, 3H), 1.05 (t, OCH3, J ¼ 7.1 Hz, 3H); 13C NMR (125 MHz,
(s, eOH, 1H), 8.33e8.25 (m, AreH, 2H), 7.87e7.69 (m, AreH, 4H),
7.36e7.09 (m, AreH, 4H, NH, 1H), 5.44 (dd, PCH, J ¼ 20.3, 9.6 Hz,
1H), 4.88 (dd, OCHCO, J ¼ 6.8, 1.2 Hz, 1H), 4.14e3.69 (m, OCH2, 4H),
2.33 (s, CH3, 3H),1.64 (d, CH3, J ¼ 6.8 Hz, 3H),1.14 (t, OCH3, J ¼ 7.1 Hz,
3H), 1.07 (t, OCH3, J ¼ 7.1 Hz, 3H); 13C NMR (125 MHz, CDCl3)
CDCl3) d 189.04, 181.19,170.48, 153.43, 151.36, 135.01, 134.03, 133.81,
d
188.95, 181.25, 170.27, 170.21, 153.42, 151.29, 138.00, 134.91,
133.14, 131.80, 130.45, 129.66, 129.61, 129.55, 127.51, 127.30, 127.01,
123.97, 121.28, 120.51, 116.95, 77.36, 63.42, 63.06, 49.77, 18.56, 16.21,
133.95, 133.78, 133.12, 131.42, 129.39, 127.71, 127.67, 127.42, 127.14,
126.94, 121.09, 120.45, 116.86, 63.23, 62.77, 50.24, 49.01, 21.09,
16.15; 31P NMR (200 MHz, CDCl3)
d 20.99; ESI-MS m/z: 538.2
18.55, 16.19, 16.13; 31P NMR (200 MHz, CDCl3)
d 20.23; HRMS (m/z)
(MþH)þ; Anal. Calc. (for C28H28NO8P): C, 62.57; H, 5.25; N, 2.61;
(ESI): calcd for C29H30NO8P [MþHþ]: 551.17090; found: 551.16852;
Anal. Calc. (for C29H30NO8P): C, 63.15; H, 5.48; N, 2.54; Found: C,
63.21; H, 5.53; N, 2.58.
Found: C, 62.53; H, 5.27; N, 2.60.
4.3.1.25. O, O0-diethyl (naphthalen-1-yl) (2-(1-hydroxy-9, 10-dioxo-
9, 10-dihydroanthracen-2-yloxy) propanamido) methylphosphonate
(7n). Yields 89.14%; m.p. 186e188 ꢀC; 1H NMR (500 MHz, CDCl3)
4.4. In vitro cytotoxicity
d
13.05 (s, eOH, 1H), 8.33e8.20 (m, AreH, 3H), 8.08 (dd, AreH,
Cell Lines. The following in vitro human cancer cell lines were
used: NCI-H460 (human large cell carcinoma cells), KB (human
mouth epidermal carcinoma cells), MGC-803 (human gastric can-
cer cells), HepG 2 (human epidermoid larynx carcinoma cells), Hela
(henrietta lacks strain of cancer cells), Hct-116 (human colorectal
cells), CNE (human nasopharyngeal carcinoma cells), A549 (human
lung adenocarcinoma cells), HUVEC (human umbilical vein endo-
thelial cells). The cell lines (NCI-H460, KB, MGC-803, HepG 2, Hela,
Hct-116, CNE, A549 and HUVEC) were purchased from the Cell
Resource Center of Shanghai Institutes for Biological Sciences, the
Academy of Sciences of China.
J ¼ 9.4, 3.1 Hz, 1H), 7.83e7.56 (m, AreH, 6H), 7.57 (dd, AreH,
J ¼ 14.5, 7.1 Hz,1H), 7.53e7.45 (m, AreH, 2H), 7.27 (d, NH, J ¼ 9.3 Hz,
1H), 6.39 (dd, PCH, J ¼ 20.8, 9.6 Hz, 1H), 4.91 (dd, OCHCO, J ¼ 20.4,
6.7 Hz, 1H), 4.01e3.52 (m, OCH2, 4H), 1.62 (d, CH3, J ¼ 6.7 Hz, 3H),
1.18 (t, OCH3, J ¼ 7.0 Hz, 3H), 0.85 (t, OCH3, J ¼ 7.1 Hz, 3H); 13C NMR
(125 MHz, CDCl3)
d 188.97, 181.35, 170.39, 170.34, 153.34, 151.27,
134.94, 134.87, 133.99, 133.77, 130.77, 129.11, 129.03, 128.78, 128.54,
127.44, 126.99, 126.73, 126.00, 125.26, 123.24, 122.96, 120.93,
120.50, 116.84, 77.12, 63.51, 62.99, 46.01, 18.56, 16.24, 15.89; 31P
NMR (200 MHz, CDCl3)
Calc. (for C32H30NO8P): C, 65.41; H, 5.15; N, 2.38; Found: C, 65.50; H,
5.17; N, 2.40.
d
21.33; ESI-MS m/z: 586.2 (MꢁH)d; Anal.
Cell Culture. NCI-H460, KB, MGC-803, HepG-2, Hela, Hct-116,
CNE, A549, HUVEC cells were cultured in Dulbecco Modified Ea-
gle Medium (DMEM) (HyClone, USA), containing 4.0 mM L-Gluta-
4.3.1.26. O, O0-diethyl (naphthalen-2-yl) (2-(1-hydroxy-9, 10-dioxo-
9, 10-dihydroanthracen-2-yloxy) propanamido) methylphosphonate
(7o). Yields 85.62%; m.p. 177e179 ꢀC; 1H NMR (500 MHz, CDCl3)
mine and 4500 mg/L Glucose, supplemented with 10% (v/v) fetal
bovine serum (FBS; HyClone, USA). The cell culture media was
supplemented with penicillin/streptomycin at 100 Units/mL as
adherent monolayers. Cell cultures were kept in a humidified
incubator with 5% CO2 at 37 ꢀC. Stock solutions were prepared in
dimethyl sulfoxide (DMSO) and further dilutions were made with
fresh culture medium. The concentration of DMSO in the final
culture medium was 1%, which had no effect on the cell viability.
MTT assay. Chemosensitivity was assessed using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
assay. Briefly, exponentially growing HepG-2 (2000e3000 cells/
well), NCI-H460 (2000e3000 cells/well), KB (2000e3000 cells/
well), MGC-803 (2000e3000 cells/well), Hela (2000e3000 cells/
well), Hct-116 (3000e4000 cells/well), CNE (2000e3000 cells/
well), HUVEC(2000e3000 cells/well) and A549 (2000e3000 cells/
well), were seeded into 96-well plates and treated with indicated
d
13.09 (s, eOH, 1H), 8.32 (dd, AreH, J ¼ 6.6, 4.4 Hz, 2H), 8.11 (dd,
AreH, J ¼ 9.5, 4.0 Hz, 1H), 7.93e7.81 (m, AreH, 6H), 7.65 (d, AreH,
J ¼ 8.3 Hz, 1H), 7.58 (d, AreH, J ¼ 8.6 Hz, 1H), 7.51e7.47 (m, AreH,
2H), 7.30 (d, NH, J ¼ 8.5 Hz, 1H), 5.66 (dd, PCH, J ¼ 20.7, 9.5 Hz, 1H),
4.90 (dd, OCHCO, J ¼ 28.2, 6.7 Hz,1H), 4.21e3.62 (m, OCH2, 4H),1.66
(d, CH3, J ¼ 6.7 Hz, 3H), 1.16 (t, OCH3, J ¼ 7.0 Hz, 3H), 1.03 (t, OCH3,
J ¼ 7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3)
d 189.09, 183.36, 181.40,
157.80, 153.43, 135.05, 134.09, 133.18, 132.95, 131.78, 128.66, 128.19,
128.11, 127.96, 127.71, 127.55, 127.40, 127.06, 126.47, 125.48, 123.84,
121.15, 120.87, 120.61, 81.21, 77.32, 63.62, 63.30, 49.52, 18.67, 17.83,
16.27; 31P NMR (200 MHz, CDCl3)
d 20.90; ESI-MS m/z: 586.2
(MꢁH)d; Anal. Calc. (for C32H30NO8P): C, 65.41; H, 5.15; N, 2.38;
Found: C, 65.45; H, 5.20; N, 2.42.

M.-Y. Ye et al. / European Journal of Medicinal Chemistry 83 (2014) 116e128
127
concentrations of samples for 48 h, and then 10
mL of MTT (10 mg/
30 min. Analysis was performed with the system software (Cell
Quest; BD Biosciences).
mL) (Sigma, USA) was added. After incubation for 4 h at 37 ꢀC, the
purple formazan crystals (i.e. a reduced form of MTT) generated
from viable cells were dissolved by adding 100 mL DMSO in each
4.8. Immunoblotting and real-time quantitative reverse-
transcription polymerase chain reaction (qRT-PCR)
well. The plates were swirled gently for 10 min to dissolve the
precipitate, and quantified by measuring the optical density (OD) of
the plates at a wavelength of 490 nm on plate reader (TECAN,
infinite, M1000). Each concentration was repeated in three wells
and the same experimental conditions were provided for all com-
pounds and MTT analysis was repeated three times for each cell
line.
Total RNA was extracted from the NCI-H460 cells after treatment
with 5 mM, 10 mM, 20 mM alizarin a-aminophosphonate derivatives
for 24 h using the RNApure Kit (Aidlab, RN0302, China) as described
previously [38]. RNA samples were reverse-transcribed for 30 min
at 42 ꢀC with the High Capacity cDNA Reverse Transcription Kit
(TaKaRa, Biotechnology, Dalian). The SYBR® Green PCR Master Mix
(Fermentas, K0251, Lithuania) and specific primer pairs were used
for selected genes, and the primer pair for actin was used as the
reference gene. RT-PCR was performed according to the following
conditions: 2 min at 50 ꢀC, 10 min at 95 ꢀC, and 40 cycles of 15 s at
4.5. Hoechst 333258 staining
Cells grown on a sterile cover slip in six-well plates were treated
with compounds for a certain range of time. The culture medium
containing compounds was removed, and the cells were fixed in 4%
paraformaldehyde for 10 min. After being washed twice with PBS,
the cells were stained with 0.5 mL of Hoechst 33258 (Beyotime,
Haimen, China) for 5 min and then again washed twice with PBS.
The stained nuclei were observed under a Nikon ECLIPSETE2000-S
fluorescence microscope using 350 nm excitation and 460 nm
emission.
95 ꢀC and 1 min at 60 ꢀC using 0.5
ml of complementary (c) DNA,
2 ꢂ SYBR Green PCR Master Mix, and 500 nM of the forward and
reverse primers on a 7500 real-time PCR System (Applied Bio-
systerms). The threshold cycle number (Ct) was calculated with the
7500 ABI software. Relative transcript quantities were calculated
using the △Ct method with actin as the reference gene amplified
from the same samples. Ct is the difference in the threshold cycles
of messenger (m)RNA for selected genes relative to those of actin
mRNA. The real-time RT-PCR was performed in triplicate for each
experimental group. The PCR primers used here are given in
Table 2S, ESI.
4.6. Mitochondrial membrane potential staining
JC-1 (Beyotime, Haimen, China) probe was employed to measure
mitochondrial depolarization in NCI-H460 cells. Briefly, cells
cultured in six-well plates after indicated treatments were incu-
4.9. ROS assay
bated with an equal volume of JC-1 staining solution (5 mg/ml) at
37 ꢀC for 20 min and rinsed twice with PBS. Mitochondrial mem-
brane potentials were monitored by determining the relative
amounts of dual emissions from mitochondrial JC-1 monomers or
aggregates using an Nikon ECLIPSETE2000-S fluorescent micro-
scope. Mitochondrial depolarization is indicated by an increase in
the green/red fluorescence intensity ratio.
NCI-H460 cells were seeded into six-well plates and subjected
to various treatments. Following treatment, cells were incubated
with 10 mM DCFH-DA (Beyotime, Haimen, China) dissolved in cell-
free medium at 37 ꢀC for 30 min in dark, and then washed three
times with PBS. Cellular fluorescence was quantified using Nikon
ECLIPSETE2000-S fluorescence microscope at an excitation of
485 nm and an emission of 538 nm.
4.7. Flow cytometry
4.10. Calcium analysis
4.7.1. Apoptosis analysis
Apoptosis was discriminated with the annexin V-FITC/propi-
dium iodide test. Cells were seeded at 2 ꢂ 106/well in 10% FBS-
DMEM into 6-well plates, and treated with compounds for 24 h.
The cells were washed twice with cold Phosphate Buffered Saline
(PBS) and then resuspend cells in 1 ꢂ Binding Buffer (0.1 M Hepes/
NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl2) at a concentration of
To monitor the effect of alizarin
rivatives on calcium release, NCI cells were seeded into 6-well
plates, and loaded with 5 M of the membrane-permeable cal-
cium indicator Fluo-3 acetoxymethyl (AM) ester (Beyotime, Hai-
men, China) for 40 min at 37 ꢀC in PBS buffer. After loading with the
Fluo-3 dye, cells were washed with PBS solution and suspended in
PBS that lacked calcium and contained 5 mM EGTA. Fluo-3 was
excited by argon laser light at 488 nm and fluorescence was
measured at wavelengths of 515 nm, and cellular fluorescence was
quantified using Nikon ECLIPSETE2000-S fluorescence microscope.
a-aminophosphonate de-
m
1 ꢂ 106 cells/ml. Transfer 100
m
L of the solution (1 ꢂ 105 cells) to a
5 mL culture tube, and add 5 mL of FITC Annexin V (BD, Pharmingen)
and 5
m
L propidium iodide (PI) to each tube. Gently vortex the cells
and incubate for 30 min at RT (25 ꢀC) in the dark. Add 200
mL PBS to
each tube. Analysis was performed with the system software
(CellQuest; BD Biosciences). Lower left quadrant, viable cells
(annexin Vꢁ/PIꢁ); lower right quadrant, early apoptotic cells
(annexin Vþ/PIꢁ); upper right quadrant, late apoptotic cells
(annexin Vþ/PIþ); upper left quadrant, necrotic cells (annexin Vꢁ/
PIþ). The percentage of cells positive for PI and/or Annexin V-FITC
was reported inside the quadrants.
Acknowledgments
This study was supported by 973 project (Nos. 2011CB512005
and 2012CB723501), the National Natural Science Foundation of
China (Nos. 81260472, 21101035 and 21362002), Guangxi Natural
Science Foundation of China (2011GXNSFD018010 and No.
2010GXNSFF013001), Bagui Scholar project and the Foundation of
Ministry of Education Innovation Team (No. IRT1225).
4.7.2. Cell cycle analysis
The cells lines were treated with indicated concentrations of
compounds. After incubated for 48 h, cells were washed twice with
ice-cold PBS, fixed and permeabilized with ice-cold 70% ethanol
Appendix A. Supplementary data
at ꢁ20 ꢀC overnight. The cells were treated with 100
mg/ml RNase A
at 37 ꢀC for 30 min after washed with ice-cold PBS, and finally
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.02.067.
stained with 1 mg/ml propidium iodide (PI) in the dark at 4 ꢀC for

128
M.-Y. Ye et al. / European Journal of Medicinal Chemistry 83 (2014) 116e128
[19] J.H. Lee, N.W. Kim, E. Her, B.K. Kim, W.S. Choi, K.H. Hwang, D.K. Choi, B.O. Lim,
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