Synthesis and antitumor activities of novel rhein α-aminophosphonates conjugates

Article abstract of DOI:10.1016/j.bmcl.2013.12.030

Several rhein α-aminophosphonates conjugates (5a-5q) were synthesized and evaluated for in vitro cytotoxicity against HepG-2, CNE, Spca-2, Hela and Hct-116 cell lines. Some compounds showed relatively high cytotoxicity. Especially, compound 5i exhibited t

Full text of DOI:10.1016/j.bmcl.2013.12.030

Bioorganic & Medicinal Chemistry Letters 24 (2014) 501–507
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antitumor activities of novel rhein
-aminophosphonates conjugates
a
Gui-yang Yao a,c, , Man-yi Ye , Ri-zhen Huang , Ya-jun Li , Ying-ming Pan , Qing Xu , Zhi-Xin Liao
⇑
,
Heng-shan Wang
a
a,
a
a
a
b
c,
a,
⇑
State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry & Pharmaceutical Science of Guangxi
Normal University, Guilin 541004, PR China
College of Pharmacy, Guilin Medical University, Guilin 541004, PR China
b
c
Department of Pharmaceutical Engineering, School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Several rhein a-aminophosphonates conjugates (5a–5q) were synthesized and evaluated for in vitro
cytotoxicity against HepG-2, CNE, Spca-2, Hela and Hct-116 cell lines. Some compounds showed rela-
tively high cytotoxicity. Especially, compound 5i exhibited the strongest cytotoxicity against Hct-116
cells (IC50 was 5.32 lM). All the synthesized compounds exhibited low cytotoxicity against HUVEC cells.
The mechanism of compound 5i was preliminarily investigated by Hoechst 33258 staining, JC-1 mito-
chondrial membrane potential staining and flow cytometry, which indicated that the compound 5i
induced apoptosis in Hct-116 cancer cells. Cell cycle analysis showed that these compound 5i mainly
arrested Hct-116 cells in G1 stage. The effects of 5i on the activation of caspases expression indicated that
Received 29 August 2013
Revised 25 November 2013
Accepted 10 December 2013
Available online 16 December 2013
Keywords:
Rhein
a-Aminophosphonates
5i might induce apoptosis via the membrane death receptor pathways. In addition, the binding properties
Synthesis
of a model analog 5i to DNA were investigated by methods (UV–vis, fluorescence, CD spectroscopy and
FRET-melting) in compare with that of rhein. Results indicated that 5i showed moderate ability to inter-
act ct-DNA.
Cytotoxicity
Apoptosis
DNA binding
Ó 2013 Elsevier Ltd. All rights reserved.
Improving the targeting of chemotherapeutic drugs to specific
IC50 were 135 lM and 62.5 l
M, respectively).10 Besides, it could
cells and organs is one of the emerging trends toward the develop-
induce apoptosis of Hct-116 cells via activation of the intrinsic
apoptotic pathway. Although various synthetic analogs of rhein
have been reported for the purpose of cytotoxicity evaluation,11
structure modifications aimed for cancer cells targeting colorectal
cancer has rarely been studied.
ment of new targeted anticancer therapies.1 Colorectal cancer
(
CRC) is the third most commonly diagnosed type of cancer and
2
the third leading cause of death from cancer. However, only a
few drugs are currently used in clinical for CRC treatments, includ-
ing oxaliplatin, leucovorin,4 5-FU and irinotecan. In addition,
the clinical application of traditional drugs was limited responsible
for the serious adverse effects or the drug resistance. Thus, there is
a need to develop more effective, less toxic and CRC cell-targeting
drugs for clinical use.
3
5,6
Recently, Berlicki and his co-workers suggested that the N–C–P
molecular fragment and its chemistry could offer many possibili-
1
2
ties for structural modifications, which have resulted in broad
biological relevance. As important classes of N–C–P contain molec-
ular,
class of compounds that exhibit intriguing biological activities,
especially anti-tumor and inhibitors of enzyme related to tumor-
a-aminophosphonates and their derivatives is an important
13
Rhein, a main constituent of rhubarb, is a well-characterized
anti-inflammatory agent which has been used for the treatment
of inflammatory diseases such as ostarthritis, diabetic nephropa-
1
4
genesis and invasions. However, there are far fewer reports about
improving synergistic effect by -aminophosphonates which was
7
thy, etc. It has also been reported to display inhibitory effects on
a
8
the proliferation of human breast, lung, NPC, CNS, glioma cancer
targeted certain cancer cells, especially colon cancer. Recently, it
was found that aminophosphonate groups could enhance the anti-
tumor activity of natural compound in our laboratory.15 As part of
cells and Ca Ski cells.9 Especially, it showed cytotoxic activity
against human colon cancer cells (Hct-116 and COLO 320DM;
our series of research work, rhein a-aminophosphonate conjugates
were designed in our resent study to establish new strategy toward
colon cancer therapy see Scheme 1.
⇑
Corresponding authors. Tel./fax: +86 0773 2120958.
E-mail addresses: zxliao@seu.edu.cn (Z.-X. Liao), whengshan@163.com (H.
Herein, we report (a) the synthesis of rhein
a-aminophosphonic
Wang).
Co-first author: These authors contributed equally to this work.
acids conjugates; (b) DNA binding of one potent conjugate 5i; (c)
0
960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.030

5
02
G. Yao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 501–507
OH
O
O
OH
O
O
P
R
OEt
OEt
OEt
i
O
ClH
3
N
P
iii
H
2
N
O
P
ii
iv
R
CHO
OEt
OEt
R
N
P
OEt
H
N
OEt
R
R
H
C
R
EtO
O
2
3
4
5
1
4 2 5 2
Scheme 1. General synthetic route for compounds 5a–5p. Reagents and conditions: (i) NH Ac, HPO(OC H ) ; (ii) (i) HCl(aq), (iii) NaOH(aq); (iv) rhein, EDAC, HOBt, DMSO, rt.
in vitro antitumor activity and cell selectivity of synthesized
conjugates; (d) the mechanism of the novel conjugate 5i killed
observation, it could be summarized that methoxy, methyl, fluoro
and chloro groups at meta position in benzene group of
Hct-116 cells.
a
-aminophosphonate moiety may have important influence on
the antitumor activity, while naphthalene and anthracene exhib-
ited negative effect. In addition, 5i exhibited similar activities
O,O’-dialky{[N-(phenylmethylene)-a-amino]-a-(substituted
phenyl)methyl}phosphonates 2, obtained by reacting substituted
benzaldehyde with ammonium acetate and O,O’-dialkyl
phosphite, were converted to O,O’-dialkyl -amino-[ -(substituted
phenyl)methyl] phosphonate 4 via hydrolysis. The -aminophos-
phonates 4 were then coupled with rhein to provide rhein
1
8
1
compared to 5-fluorouracil which is clinical drug. Moreover,
1
5b
a
a
a
compared our previous work,
-aminophosphonates which have the H at
lar to the rhein -aminophosphonates which have the methyl at
-position.
These results showed that rhein
the antitumor activities of rhein
16
a
a
-position were simi-
a
a-aminophosphonic acids conjugates 5 in satisfactory yields.
a
All of compounds 5 were obtained as yellowish solids after col-
a-aminophosphonate conju-
umn chromatography. Their structures were fully characterized by
gates had selective cytotoxicity toward Hct-116 among the cell
lines. In comparison, activities of compounds against normal cells
(HUVEC) were also examined (Table 1). The results indicated that
the anti-proliferative activity of some compounds against cancer
cells was much higher than against normal cells.
1
1
H NMR. For example, the corresponding H NMR spectrum
showed the two methyleneoxy (OCH CH ) groups attached with
phosphorus appears as three multiplets at 3.30–4.30. The chemical
shifts of the two methyl (OCH CH ) hydrogens were different due
2
3
2
3
to the low rate of environmental exchange caused by the slow
rotation of the P–C bond. The carbonyl group leads to a downfield
shift of the two phenolic hydroxyls. The structures of all the com-
DNA is generally the primary intracellular target of anticancer
1
9
drugs, and the interaction of small molecules with DNA plays
2
0
an important role in many biological processes. Recently, there
is a great interest on the binding of small molecules with DNA ow-
ing to their possible applications as new cancer therapeutic
13
31
pounds were further confirmed by C NMR, P NMR, EI–MS and
1
7
elemental analysis.
2
1
The in vitro antitumor activities of all of these compounds were
evaluated by MTT assay against HepG-2, CNE, Spca-2, Hela and
Hct-116 cell lines, using 5-fluorouracil (5-FU) as the positive con-
trol. The tested results were shown in Table 1. As shown in Table 1,
the IC50 values of most of the conjugates were lower than that of
rhein. 5i exhibited better cytotoxic effects than other compounds.
These results demonstrated that large aromatic ring (e.g., 5l and
agents. In addition, DNA appears to be the major cellular target
for most anthraquinone-based structures which is generally ac-
2
2
cepted by DNA-interactive drugs. Furthermore, anticancer drugs
targeting DNA can inhibit cell cycle progression and/or induce
apoptosis by activation of cell cycle checkpoint in response to
2
3
DNA damage. However, to our best knowledge, little research
has been devoted to the mechanism of rhein derivative and their
DNA-binding properties. Because compounds 5 are isostructural,
5i was selected as a representative to investigate their binding
properties to DNA. To this end, several analytical methods, includ-
ing UV–vis, fluorescence, circular dichroism spectroscopies and
FRET-melting were performed.
5
q) as R substituent showed low cytotoxicity and meta-substituent
as R is favorable to the cytotoxicity. As for Hct-116 cells, in the ser-
ies of compounds (5a, 5b and 5c) substituted at the R position with
bromo group derivatives, compound 5c showed the strongest
inhibitory activity with IC50 40.18 ± 3.56 lM. Based on the above
Table 1
a
IC50 values (
l
M) of rhein and derivatives 5a–5q towards five selected tumor cell lines and normal cell lines for 48 h
M)
Compd
IC50(l
R
HepG-2
CNE
Spca-2
Hela
Hct-116
HUVEC
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
p-Ph-Br
o-Ph-Br
m-Ph-Br
p-Ph-F
94.24 ± 5.69
88.32 ± 7.14
62.37 ± 2.17
59.38 ± 4.29
71.25 ± 3.69
87.34 ± 6.29
71.95 ± 2.37
39.19 ± 1.02
22.08 ± 1.30
30.01 ± 2.31
39.25 ± 2.09
>100
44.36 ± 2.16
62.22 ± 5.22
77.86 ± 2.22
56.83 ± 2.34
>100
42.47 ± 3.23
55.68 ± 4.25
40.12 ± 3.14
34.57 ± 2.31
67.59 ± 4.28
55.79 ± 3.18
45.56 ± 2.17
67.98 ± 5.71
24.14 ± 1.28
28.56 ± 1.87
34.28 ± 2.97
>100
42.85 ± 3.47
74.25 ± 2.37
79.38 ± 6.24
49.22 ± 3.41
>100
88.17 ± 7.12
85.47 ± 6.23
75.36 ± 4.89
24.31 ± 2.17
79.89 ± 6.89
89.47 ± 8.73
76.98 ± 8.27
28.27 ± 2.14
28.27 ± 3.44
14.97 ± 3.26
27.36 ± 1.89
>100
38.94 ± 1.87
67.68 ± 1.65
74.36 ± 6.87
55.41 ± 4.32
>100
98.24 ± 7.21
79.63 ± 5.14
78.23 ± 6.32
95.36 ± 4.56
94.25 ± 4.34
87.47 ± 5.13
73.24 ± 6.14
35.23 ± 2.23
19.34 ± 1.58
25.64 ± 2.59
38.19 ± 2.57
>100
40.12 ± 3.47
73.34 ± 6.89
88.95 ± 4.68
64.83 ± 5.14
>100
65.45 ± 6.51
48.93 ± 4.12
40.18 ± 3.56
57.82 ± 4.56
36.56 ± 5.67
61.19 ± 3.12
22.36 ± 2.12
48.20 ± 2.34
5.32 ± 0.75
8.37 ± 0.88
17.79 ± 1.23
>100
22.06 ± 2.35
45.35 ± 3.36
54.32 ± 4.54
16.66 ± 3.40
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
o-Ph-F
p-Ph-Cl
m-Ph-Cl
o-Ph-Cl
m-Ph-OCH
o-Ph-OCH
Ph
2-Naphthalene
p-Ph-OCH
m-Ph-CH
p-Ph-CH
m-Ph-F
3
3
3
3
3
9-Anthracene
Rhein
-FU
>100
29.98 ± 0.37
>100
40.21 ± 1.98
28.31 ± 1.40
42.20 ± 2.14
>100
35.34 ± 2.72
>100
5.78 ± 0.85
89.74 ± 6.40
56 ± 4.40
5
a
IC50 values are presented as the mean ± SD (standard error of the mean) from three separated experiments.

G. Yao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 501–507
503
The UV–vis absorption spectroscopy was primarily employed to
probe the binding modes of 5i to the calf thymus DNA (ct-DNA).
Rhein belongs to the same class of coplanar anthraquinones as
daunorubicin and mitoxantrone, which have been in clinical use
over 30 years for the treatment of various tumors and were used
as DNA targeting agents.24 Their function as non-covalent DNA
binders is generally believed to be essential for their activity. The
inherent absorbance of 5i allowed us to investigate its interaction
with ct-DNA by ultraviolet absorption spectrum. As shown in
Figure 1, although the absorbance of 5i changed upon addition of
DNA, the mother compound (rhein) did not seem to have any
change (Fig. b, ESI). This indicated that DNA was one potential
target of 5 owing to the dianionic character of N–C–P molecular
fragment. The DNA binding constant
b
K was calculated by
Figure 2. Fluorescence emission spectra of GelRed bound with ct-DNA
non-linear fitting according to the Equations (1a, ESI), the value
À4
À5
(
[DNA] = 2.0 Â 10
M, [GelRed] = 2.0 Â 10 M) in the absence (dash line) and
4
À1
of K
b
for 5i was found to be 1.03 Â 10 M
.
presence (solid lines) of 5i with [5i]/[GelRed] ratios range from 1:1 to 9:1. Inset:
The binding ability of 5i to ct-DNA was primarily investigated
by competitive binding in which they served as an intercalative
binding probe in competition with GelRed. GelRed, which is envi-
ronmentally safe and ultra-sensitive for DNA staining, is a newly
developed DNA intercalator to replace the classic DNA intercalator
EB. Furthermore, both GelRed and EB bound with ct-DNA emit
characteristic fluorescence at 590 nm upon 350 nm UV light
linear fitting for quenching constant K
q
based on Stern–Volmer equation.
2
5
excitation.
In competitive binding experiments, GelRed and ct-DNA solu-
tions were pre-incubated for 30 min to ensure sufficient interac-
tions. The concentration ratio of GelRed to DNA was set at
[
GelRed]/[DNA] = 1:10 to ensure sufficient binding sites of DNA
for GelRed. The emission spectras of the GelRed-ctDNA system in
the absence and presence of 5i were shown in Figure 2. GelRed-
DNA binary solution system gave a characteristic fluorescence
emission at around 590 nm when excited at 350 nm, indicating
that GelRed molecules intercalated between the adjacent base
pairs of DNA and sufficiently prevented fluorescence quenching
by polar solvent molecules. The presence of 5i considerably
quenched the fluorescence emission of GelRed with the saturation
state achieved at the [5i]/[GelRed] ratio of 8:1.
À4
Figure 3. CD spectra of ct-DNA (2 mL solution, 1.5 Â 10 M) in the absence and
À5
presence of 5i (1.5 Â 10 M).
À4
(
1.5 Â 10 M) showed a positive absorption peak at 279 nm and
a negative absorption peak at 248 nm due to
p–p base stacking
The quenching ability of 5i to GelRed fluorescence can be
quantitively estimated by their respective quenching constants,
and right-hand helicity, respectively. This was consistent with
2
8
the characteristic B conformation of DNA.
q
K , which were derived from the Stern–Volmer quenching equa-
3
Upon the addition of 5i at a [5i]/[DNA] ratio of 1:10, the CD
absorption of ct-DNA showed an obvious decrease in the intensi-
ties of both the negative and positive absorption bands. The
percentage decreases in the maximal DNA positive and negative
absorption by 5i were 8.5% and 28.0%, respectively. It suggested
that 5i might intercalate between the neighbouring base pairs of
ct-DNA mainly due to the aromatic planarity of the anthraquinone
skeleton. The decrease in the intensities of both positive and neg-
ative bands can usually be observed in the intercalative binding of
tion. The K
q
value for 5i was 1.66 Â 10 , exhibiting similar inten-
sive intercalation to DNA as rhein. Meanwhile, it can also be
confirmed that the intercalative binding mode of 5i to DNA was
2
6
similar to GelRed.
The circular dichroism (CD) is a useful technique to assess
whether the nucleic acids undergo conformational changes as a
result of complex formation or changes in environmental
2
7
conditions. As indicated in Figure 3, the CD spectra of ct-DNA
2
9
small molecules to DNA
.
Fluorescence signals may be created by various means to detect
DNA hybridization during genotyping and similar assays.30 The
simplest method is to use an intercalating dye (e.g., SYBR Green
I) that is highly specific for double-stranded DNA. Fluorescence
resonance energy transfer (FRET) is a powerful tool for detecting
spatial relationships between macromolecules, one use of which
3
1
is the tracking of DNA hybridization status.
Melting of the duplex structures was followed by fluorescence
method (FRET measurements) and the FRET experiments are re-
ported here (Fig. 4). The FRET results indicated that the rhein deriv-
ative (
D
T
m
values was 3.1 °C) had higher stabilizing ability than the
values was 2.0 °C) (Fig. c, ESI),
mother compound (rhein) (
DT
m
which suggested that the dianionic character of N–C–P molecular
fragment provided an important contribution of the rhein conju-
gates to interact with double-stranded (duplex) DNA. Based on
the UV–vis, fluorescence, circular dichroism spectroscopies and
Figure 1. UV–vis absorption spectra of complex 5i in the absence (—) and presence
(
—) of ct-DNA with increasing [DNA]/[5i] ratios in the range from 1:1 to 6:1.

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04
G. Yao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 501–507
apoptosis against Hct-116 cell line. The results were identical with
that of previous experiment of Hoechst 33258 staining.
It was reported that many natural anthraquinones such as rhein
3
2
and emodin could induce apoptosis. Several studies have con-
cluded that rhein induced apoptosis in HL-60 cells through the
mitochondrial death pathway by causing the loss of mitochondrial
membrane potential, cytochrome c release, and cleavage of Bid
protein. In addition, it inhibited invasion in NPC cells by reducing
the expression of matrix metalloproteinase-9 (MMP-9) and vascu-
8
lar endothelial growth factor (VEGF). Furthermore, rhein can
3
3
affect mitochondrial membrane permeability. So, when the mito-
chondrial membrane potential is lost, ATP production ceases and
3
4
the cell dies. Recently, some
a-aminophosphonic acid derivatives
Figure 4. Representative FRET-melting profiles of F32T (0.5
increasing concentration of compound 5i.
lM) without and with
have been evaluated as inhibitors of matrix metalloproteinases
3
5
MMP-1, MMP-2, MMP-3 and MMP-8. Some derivatives were
3
6
found to trigger apoptotic cell death in DOHH-2 cells. Based on
these facts, we postulated that these conjugates could induce
apoptosis via reducing the expression of apoptosis-related
proteins.
FRET-melting, it can be concluded that DNA is one potential target
of 5i as expected.
Hoechst 33258 is a membrane permeable dye with blue fluores-
cence. Live cells with uniformly light blue nuclei were obviously
detected under fluorescence microscope after treatment with
Hoechst 33258, whereas apoptotic cells had bright blue nuclei
due to karyopyknosis and chromatin condensation. On the con-
trary, the nuclei of dead cells could not be stained. Hct-116 cells
which were treated with compound 5i for 24 h at different concen-
trations were stained with Hoechst 33258. Hct-116 cells not trea-
ted with 5i were used as control at for 24 h. The results were given
in Figure 5. As shown in Figure 5, cells not treated with compound
In the present study, the apoptotic cell rates were determined
for the Hct-116 cells after stimulation for 24 h by the compound
5i at different concentrations. The graphical values of the results
were given in Figure 7. From the results, it was found that these
compounds stimulated apoptosis for Hct-116 cells compared to
negative (unstimulated cells by compounds) controls. Compound
5i induced apoptosis in 33.5% of the Hct-116 cells in 5 lM. In addi-
tion, the apoptosis ratio of 5-FU was assayed to be 17.9% (Fig. e,
ESI) under the similar condition. Meanwhile, we investigated the
effects on the apoptosis of HUVEC cells. The results showed that
the compounds we synthesized had no significant apoptotic effects
on HUVEC cells (Fig. a, ESI).
Flow-cytometric analysis further confirmed tumor cell apopto-
sis as shown in Figure 8. Cytometric profiles of the PI-stained DNA
showed cell cycle arrest of Hct-116 cells treated with 5i for 48 h at
different concentrations. As shown in Figure 8, the treatment of
Hct-116 cells with compound 5i enhanced cell cycle arrest at the
G1 phase at different concentrations, resulting in concomitant pop-
ulation increase in the G1 phase (45.26% and 49.67%) compared
with the control cells (39.08%) and the population of the G2 phase
decrease at a certain extent (20.09% and 17.90%) compared with
the control cells (24.09%). These results suggested that compound
5i mainly arrest the cell-cycle of Hct-116 cells in the G1 stage. And
the effect of compound 5-FU on the cell cycle of Hct-116 cell was
5
i were normally blue. It was worth noting that, for 5i treatment,
the cells displayed strong blue fluorescence and indicated typical
apoptotic morphology at 3 and 5 M. The observation revealed
l
that compound 5i induced apoptosis against Hct-116 cells.
Mitochondrial membrane potential changes were also designed
and detected to further investigate the apoptosis-inducing effect of
target compound 5i, employing a fluorescent probe JC-1. JC-1 is a
lipophilic cationic dye and it can easily pass through the plasma
membrane into cells and accumulate in actively respiring mito-
chondria. Hct-116 cells treated with 5i for 24 h at different concen-
trations were stained with JC-1 and not treated with 5i were used
as control at for 24 h. The results were shown in Figure 6. The JC-1
monomer and J-aggregates were excited at 514 and 585 nm,
respectively, and light emissions were collected at 515–545 nm
(
green) and 570–600 nm (red). For fluorescence microscopy,
also studied in 5 lM (Fig. f, ESI). It indicated that 5-FU arrest
Figure 6 displayed that cells not treated with the compounds were
normally red, while for 5i treatment, cells exhibited strong green
fluorescence as that for 5-FU which is known to induce the apop-
tosis (Fig. d, ESI) and indicated typical apoptotic morphology.
Therefore, it could be concluded that compound 5i induced
Hct-116 cells in the G1 and S stage, resulting in concomitant
population increase in the G1 phase (65.60%) compared with the
control cells (39.08%) and the population of the S phase decrease
at a certain extent (28.00%) compared with the control cells
(23.29%).
Control (-)
5i: 3 µM
5i: 5 µM
Figure 5. Hoechst 33258 staining of compound 5i in Hct-116 cells.

G. Yao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 501–507
505
Control (-)
5i: 3 μM
5i: 5 μM
Figure 6. JC-1 mitochondrial membrane potential staining of compound 5i in Hct-116 cells.
Control (-)
5i: 3 µM
5i: 5 µM
Figure 7. Effect of compound 5i on apoptosis of Hct-116 cells. Apoptotic cells were analyzed by flow cytometry, after being stained with Annexin V-FITC together with PI. The
percentage of cells positive for PI and/or Annexin V-FITC is reported inside the quadrants.
Control (-)
5i:3 µM
5i:5 µM
Figure 8. Fluorescence-activated cell sorting analyses based on DNA content of Hct-116 cells treated with 5i for 48 h and stained by propidium iodide.
Apoptosis can be triggered by several stimuli and is controlled
by two major pathways, namely the mitochondrial pathway and
membrane death receptor pathway. The membrane death receptor
pathway is characterized by the binding between cell death ligands
and cell death receptors and the subsequent activation of caspase-
Therefore, we preliminarily concluded that 5i induced the apopto-
sis of Hct-116 cells via the membrane death receptor pathways.
In summary, a series of rhein a-aminophosphonates conjugates
were synthesized and the cytotoxic of the rhein derivatives on five
cancer cell lines were determined. It was detected that the
compound 5i had selective and significant cytotoxic effects on
Hct-116 cells and the toxicity of all conjugates were lower than
rhein. Moreover, 5i exhibited similar activities compared to fluoro-
uracil which was used as clinical drugs. The apoptosis-inducing
activity of representative compound 5i in Hct-116 cells was
8
and caspase-3. The rhein a-aminophosphonate derivatives
induced apoptotic pathway was investigated by qRT–PCR analysis
of caspase expressions. As showed in Figure 9, compound 5i treat-
ment showed concentration-dependent effects in caspases-3 and
caspases-8, and these effects were concentration-dependent.

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G. Yao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 501–507
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Hu, D.-Y.; Yang, S. Bioorg. Med. Chem. Lett. 2006, 16, 1537; (f) Kudzin, Z. H.;
Kudzin, M. H.; Drabowicz, J.; Stevens, C. V. Curr. Org. Chem. 2011, 15, 2015; (g)
Orsini, F.; Sello, G.; Sisti, M. Curr. Med. Chem. 2010, 17, 264; (h) Naydenova, E.;
Troev, K.; Topashka-Ancheva, M.; Hägele, G.; Ivanov, I.; Kril, A. Amino Acids
2
2
007, 33, 695; (i) Naydenova, E. D.; Todorov, P. T.; Troev, K. D. Amino Acids
010, 33, 23.
1
1
4. (a) Flors, V.; Miralles, C.; Gonzalez-Bosch, C.; Carda, M.; Garcia-Agustin, P.
Physiol. Mol. Plant P. 2003, 63, 151; (b) Lamberth, C.; Kempf, H. J.; Kri zˇ , M. Pest
Manag. Sci. 2007, 63, 57; (c) Jennings, L. D.; Rayner, D. R.; Jordan, D. B.; Okonya,
J. F.; Basarab, G. S.; Amorose, D. E.; Anaclerio, B. M.; Lee, J. K.; Schwartz, D. B.;
Whitmore, K. A. Bioorg. Med. Chem. 2000, 8, 897; (d) Navickiene, H. M. D.;
Miranda, J. E.; Bortoli, S. A.; Kato, M. J.; Bolzani, V. S.; Furlan, M. Pest Manag. Sci.
Figure 9. 5i affected the activities of caspase-4, -9, -8, and -3 of Hct-116 cells.
studied and the results revealed that this compound showed clear
cell apoptosis inducing effects. Cell cycle analysis indicated that
compound 5i could arrest Hct-116 cell in G1 stage. The effects of
2007, 63, 399.
5. (a) Huang, X.-C.; Wang, M.; Pan, Y.-M.; Tian, X.-Y.; Wang, H.-S.; Zhang, Y.
Bioorg. Med. Chem. 2013, Lett. 23, 5289; (b) Ye, M.-Y.; Yao, G.-Y.; Wei, J.-C.; Pan,
Y.-M.; Liao, Z.-X.; Wang, H.-S. Int. J. Mol. Sci. 2013, 14, 9424.
6. (a) Kaboudin, B.; Moradi, K. Tetrahedron Lett. 2005, 46, 2989; (b) Sara, S.; Zahra,
T. Synth. Commun. 2008, 39, 120.
5
i on the activation of caspases expression indicated that 5i might
induce apoptosis via the membrane death receptor pathways. The
binding properties of 5i to DNA examined by various methods indi-
cated that 5i interacted with DNA. Therefore, these compounds
may be considered as the agents with high potential anti-cancer
activity and good candidates for more advanced screening.
1
1
7. (a) General procedure for the preparation of rhein conjugates 5: A suspension of
aromatic aldehyde (10 mmol), diethyl phosphite (5 mmol) and ammonium
acetate (11 mmol) was stirred for 6 h at 70 °C to give the 2. After that,
hydrochloric acid (4 mmol) in 30 mL ether was added to the reaction mixture,
which was stirred for 2 h at 0 °C to give the precipitate 3. The precipitate was
filtered and washed with ether (15 mL), and then it was added to 10 mL
sodium hydroxide solution (15%) and stirred for 30 min at room temperature.
Extraction with dichloromethane (3 Â 30 mL) and evaporation of the solvent
gave the oils of 4, which was purified by column chromatography on silica gel
with ethyl acetate. To a mixture of rhein and HOBt in DMSO (10 mL), the
appropriate EDAC was stirred for 15 min at 0 °C. Then 4 was added 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 petroleum ether/ethyl acetate (3:1, V: V) to give the pure title
compounds 5 as yellowish solids.
Acknowledgments
This study was supported by 973 project (No. 2011CB512005,
012CB723501), the National Natural Science Foundation of
China (No. 81260472, 21101035, 21362002), Guangxi Natural
Science Foundation of China (2011GXNSFD018010 and No.
010GXNSFF013001), Bagui Scholar project and the Foundation
2
2
of Ministry of Education Innovation Team (No. IRT1225).
(
b) Experimental: NMR spectra were recorded on a BRUKER AVANCE 500 NMR
spectrometer in CDCl . Mass spectra were determined on a BRUKER ESQUIRE
HCT spectrometer. 5a: Yield 81%. mp 221–222 °C. H NMR (500 MHz CDCl
ppm): 1.17 (t, J = 7.0 Hz, 3H, CH ), 1.31 (t, J = 6.9 Hz, 3H, CH ), 3.86–4.21 (m,
H, OCH ), 5.77 (dd, J = 9.2, 21.9 Hz 1H, PCH), 7.29–8.62 (m, 9H, Ar-H, 1H, NH),
11.93 (s, 1H, OH), 11.96 (s, 1H, OH). C NMR (125 MHz, DMSO) d (ppm):
Supplementary data
3
1
3
) d
(
4
3
3
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
2
13
1
1
1
91.41, 181.04, 164.60, 161.36, 160.91, 140.89, 137.52, 134.68, 133.49, 133.28,
31.12, 130.70, 130.66, 124.49, 123.03, 121.13, 121.10, 119.38, 118.09, 117.84,
1
2.030.
31
16.12, 62.69, 62.45, 49.36, 16.22, 16.02. P NMR (200 MHz, CDCl
3
) d (ppm):
References and notes
21.31. ESI–MS m/z: 610.1 (M+Na)
.94; N, 2.38. Found: C, 53.11; H, 4.17; N, 2.38. Compound 5b: Yield 87%. mp
+
. Anal. Calcd (for C26H23BrNO P): C, 53.08; H,
8
3
1
1
.
(a) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.; Langer, R. Nat.
Nanotechnol. 2007, 2, 751; (b) Szakács, G.; Paterson, J. K.; Ludwig, J. A.; Booth-
Genthe, C.; Gottesman, M. M. Nat. Rev. Drug Disc. 2006, 5, 219; (c) Zutphen, S.
V.; Reedijk, J. Coord. Chem. Rev. 2005, 249, 2845; (d) Schrama, D.; Reisfeld, R. A.;
Becker, J. C. Nat. Rev. Drug Disc. 2006, 5, 147; (e) Storr, T.; Thompson, K. H.;
Orvig, C. Chem. Soc. Rev. 2006, 35, 534.
197–199 °C. H NMR (500 MHz CDCl
1.35 (t, J = 7.2 Hz, 3H, CH ), 3.72–4.26 (m, 4H, OCH
1H, PCH), 7.17–8.22 (m, 9H, Ar-H, 1H, NH), 11.95 (s, 1H, OH),12.00 (s, 1H, OH).
3
) d (ppm): 1.09 (t, J = 7.1 Hz, 3H, CH
3
),
3
2
), 6.31 (dd, J = 9.0, 21.4 Hz
13
3
C NMR (125 MHz, CDCl ) d (ppm): 192.63, 180.91, 164.40, 162.75, 162.52,
141.54, 137.69, 134.90, 133.96, 133.41, 133.20, 129.76, 129.61, 127.91, 124.93,
124.68, 123.51, 120.36, 119.97, 118.13, 115.74, 63.91, 63.50, 49.62, 16.39,
16.03. 31P NMR (200 MHz, CDCl
) d (ppm): 21.33. ESI–MS m/z: 610.1 (M+Na) .
+
2
3
.
.
(a) Jemal, A.; Siegel, R.; Ward, E.; Hao, Y.; Xu, J.; Thun, M. J. CA-Cancer J. Clin.
3
2
009, 59, 225; (b) Yan, F.; Cao, X.-X.; Jiang, H.-X.; Zhao, X.-L.; Wang, J.-Y.; Lin,
Anal. Calcd (for C26
4.18; N, 2.39. Compound 5c: Yield 82%. mp 208–209 °C. H NMR (500 MHz
CDCl ) d (ppm): 1.17 (t, J = 7.3 Hz, 3H, CH ), 1.32 (t, J = 7.2 Hz, 3H, CH ), 3.76–
4.19 (m, 4H, OCH ), 6.12 (dd, J = 9.8, 21.2 Hz 1H, PCH), 6.97–8.42 (m, 9H, Ar-H,
1H, NH), 11.94 (s, 1H, OH), 11.99 (s, 1H, OH). C NMR (125 MHz, CDCl
H23BrNO P): C, 53.08; H, 3.94; N, 2.38. Found: C, 53.10; H,
8
1
Y.-H.; Liu, Q.-L.; Zhang, C.; Jiang, B.; Guo, F. J. Med. Chem. 2010, 53, 5502.
Argyriou, A. A.; Polychronopoulos, P.; Iconomou, G.; Chroni, E.; Kalofonos, H. P.
Cancer Treat. Rev. 2008, 34, 368.
3
3
3
2
13
4
5
.
.
Hoff, P. M. Invest. New Drugs 2000, 18, 331.
3
) d
Vita, F. D.; Orditura, M.; Matano, E.; Bianco, R.; Carlomagno, C.; Infusino, S.;
Damiano, V.; Simeone, E.; Diadema, M. R.; Lieto, E.; Castellano, P.; Pepe, S.;
Placido, S. D.; Galizia, G.; Martino, N. D.; Ciardiello, F.; Catalano, G.; Bianco, A. R.
Br. J. Cancer 2005, 92, 1644.
(ppm): 192.63, 180.88, 164.81, 162.74, 162.43, 141.56, 137.68, 137.07, 133.85,
133.41, 131.35, 131.25, 130.15, 126.94, 124.89, 123.48, 122.69, 120.33, 118.51,
3
1
117.44, 115.74, 63.79, 63.57, 49.81, 16.40, 16.12. P NMR (200 MHz, CDCl
3
) d
P): C,
53.08; H, 3.94; N, 2.38. Found: C, 53.12; H, 4.16; N, 2.38. Compound 5d: Yield
+
(ppm): 21.31. ESI–MS m/z: 610.1 (M+Na) . Anal. Calcd (for C26H23BrNO
8
6
7
.
.
Sargenta, D.; Shi, Q.; Yothers, G.; Cutsem, E. V.; Cassidy, J.; Saltz, L.; Wolmark,
N.; Bot, B.; Grothey, A.; Buyse, M.; Gramont, A. D. Eur. J. Cancer 2011, 47, 990.
(a) Cai, Y.; Sun, M.; Xing, J.; Corke, H. J. Agric. Food Chem. 2004, 52, 7884; (b)
Gao, Q.; Qin, W.-S.; Jia, Z.-H.; Zheng, J.-M.; Zeng, C.-H.; Li, L.-S.; Liu, Z.-H. Planta
Med. 2010, 76, 27; (c) Tamura, T.; Shirai, T.; Kosaka, N.; Ohmori, K.; Takafumi, N.
Eur. J. Pharmacol. 2002, 448, 81; (d) Cai, J.; Duan, Y.-B.; Yu, J.; Chen, J.-Q.; Chao,
M.; Ji, M. Eur. J. Med. Chem. 2012, 55, 409.
1
85%. mp 197–199 °C. H NMR (500 MHz CDCl
CH ), 1.31 (t, J = 7.1 Hz, 3H, CH ), 3.80–4.21 (m, 4H, OCH
21.3 Hz 1H, PCH), 7.01–8.22 (m, 9H, Ar-H, 1H, NH), 11.94 (s, 1H, OH), 11.99 (s,
1H, OH). 13C NMR (125 MHz, CDCl
) d (ppm): 192.61, 180.89, 164.68, 162.74,
3
) d (ppm): 1.14 (t, J = 7.1 Hz, 3H,
3
3
2
), 5.77 (dd, J = 9.2,
3
162.46, 161.61, 141.67, 137.69, 133.89, 133.39, 130.61, 130.22, 130.16, 130.11,
124.91, 123.43, 120.33, 118.34, 117.43, 115.72, 115.56, 63.66, 63.40, 49.58,
3
1
8
9
.
.
Lin, M.-L.; Chung, J.-G.; Lu, Y.-C.; Yang, C.-Y.; Chen, S.-S. Oral Oncol. 2009, 45,
16.42, 16.12. P NMR (200 MHz, CDCl
3
) d (ppm): 21.29. ESI–MS m/z: 550.1
+
5
31.
(M+Na) . Anal. Calcd (for C26
59.12; H, 4.50; N, 2.59. Compound 5e: Yield 87%. mp 234–236 °C. H NMR
(500 MHz CDCl ) d (ppm): 1.13 (t, J = 7.1 Hz, 3H, CH ), 1.35 (t, J = 7.0 Hz, 3H,
CH ), 3.84–4.22 (m, 4H, OCH ), 6.03 (dd, J = 9.2, 21.7 Hz 1H, PCH), 7.10–8.21
(m, 9H, Ar-H, 1H, NH), 11.97 (s, 1H, OH), 12.05 (s, 1H, OH). C NMR (125 MHz,
DMSO) (ppm): 191.41, 181.08, 164.62, 161.36, 160.90, 140.76, 137.52,
133.53, 133.30, 130.52, 130.08, 124.50, 124.43, 123.06, 122.46, 119.38, 118.07,
H23FNO P): C, 59.21; H, 4.40; N, 2.66. Found: C,
8
1
(a) Raimondi, F.; Santoro, P.; Maiuri, L.; Londei, M.; Annunziata, S.; Ciccimarra,
F.; Rubino, A. J. Pediatr. Gastroenterol. Nutr. 2002, 34, 529; (b) Lin, S.; Fujii, M.;
Hou, D.-X. Arch. Biochem. Biophys. 2003, 418, 99; (c) Ip, S.-W.; Weng, Y.-S.; Lin,
S.-Y.; Yang, M.-D.; Tang, N.-Y.; Su, C.-C.; Chung, J.-G. Anticancer Res. 2007, 27,
3
3
3
2
13
3
79; (d) Lin, M.-L.; Chen, S.-S.; Lu, Y.-C.; Liang, R.-Y.; Ho, Y.-T.; Yang, C.-Y.;
d
Chung, J.-G. Anticancer Res. 2007, 27, 3313.

G. Yao et al. / Bioorg. Med. Chem. Lett. 24 (2014) 501–507
507
1
17.91, 116.16, 115.16, 114.98, 62.75, 62.51, 42.29, 16.21, 15.90. 31P NMR
118.16, 117.76, 116.10, 62.50, 62.33, 49.86, 20.95, 16.23, 15.98. 31P NMR
+
+
(
200 MHz, CDCl
3
) d (ppm): 21.24. ESI–MS m/z: 550.1 (M+Na) . Anal. Calcd (for
(200 MHz, CDCl
26NO
Compound 5o: Yield 85%. mp 223–225 °C. H NMR (500 MHz CDCl
1.11 (t, J = 7.1 Hz, 3H, CH ), 1.29 (t, J = 7.1 Hz, 3H, CH ), 2.29 (s, 3H, Ar-CH
3.75–4.20 (m, 4H, OCH ), 5.78 (dd, J = 9.4, 20.6 Hz 1H, PCH), 7.09–8.66 (m, 10H,
3
) d (ppm): 20.93. ESI–MS m/z: 546.1 (M+Na) . Anal. Calcd (for
C
26
H
23FNO
8
P): C, 59.21; H, 4.40; N, 2.66. Found: C, 59.18; H, 4.42; N, 2.57.
C
27
H
8
P): C, 61.95; H, 5.01; N, 2.68. Found: C, 61.89; H, 5.11; N, 2.59.
1
1
Compound 5f: Yield 84%. mp 224–226 °C. H NMR (500 MHz CDCl
.16 (t, J = 7.0 Hz, 3H, CH ), 1.32 (t, J = 7.1 Hz, 3H, CH ), 3.82–4.21 (m, 4H,
OCH ), 5.77 (dd, J = 9.3, 22.4 Hz 1H, PCH),7.26–8.41 (m, 9H, Ar-H, 1H, NH),
3
) d (ppm):
3
) d (ppm):
),
1
3
3
3
3
3
2
2
1
3
13
1
1
1
1
2
4
1
1
1
1.94 (s, 1H, OH), 11.98 (s, 1H, OH). C NMR (125 MHz, DMSO) d (ppm):
91.43, 181.08, 164.60, 161.36, 160.91, 140.90, 137.53, 134.24, 133.52, 133.31,
32.53, 130.38, 130.33, 128.19, 124.50, 123.02, 119.39, 118.08, 117.88, 116.16,
Ar-H, 1H, NH), 11.86 (s, 1H, OH), 11.89 (s, 1H, OH). C NMR (125 MHz, DMSO)
d (ppm): 191.43, 181.04, 164.49, 161.36, 160.92, 141.12, 137.51, 137.02,
137.00, 133.45, 133.29, 132.09, 128.73, 128.48, 128.43, 124.47, 123.01, 119.37,
3
1
31
15.92, 62.73, 62.50, 50.51, 16.26, 16.01. P NMR (200 MHz, CDCl
3
) d (ppm):
P): C, 57.42; H,
.26; N, 2.58. Found: C, 57.32; H, 4.36; N, 2.60. Compound 5g: Yield 80%. mp
118.14, 117.75, 116.11, 62.46, 62.28, 49.59, 20.63, 16.24, 16.02. P NMR
+
+
1.34. ESI–MS m/z: 566.1 (M+Na) . Anal. Calcd (for C26
H23ClNO
8
(200 MHz, CDCl
26NO
Compound 5p: Yield 88%. mp 163–165 °C. H NMR (500 MHz CDCl
1.15 (t, J = 7.0 Hz, 3H, CH ), 1.31 (t, J = 7.1 Hz, 3H, CH ), 3.85–4.22 (m, 4H,
OCH ), 5.81 (dd, J = 9.4, 22.0 Hz 1H, PCH), 6.95–8.60 (m, 10H, Ar-H, 1H, NH),
3
) d (ppm): 21.02. ESI–MS m/z: 524.2 (M+H) . Anal. Calcd (for
C
27
H
8
P): C, 61.95; H, 5.01; N, 2.68. Found: C, 61.90; H, 5.06; N, 2.75.
1
1
94–196 °C. H NMR (500 MHz CDCl
3
) d (ppm): 1.16 (t, J = 7.0 Hz, 3H, CH
3
),
3
) d (ppm):
.33 (t, J = 7.1 Hz, 3H, CH ), 3.82–4.21 (m, 4H, OCH
3
2
), 5.72 (dd, J = 8.5, 21.7 Hz
3
3
H, PCH), 7.28–8.24 (m, 9H, Ar-H, 1H, NH), 11.96 (s, 1H, OH), 12.03 (s, 1H, OH).
C NMR (125 MHz, DMSO) d (ppm): 191.40, 181.04, 164.59, 161.37, 160.92,
40.83, 137.60, 137.52, 133.49, 133.27, 132.94, 130.00, 128.31, 127.79, 127.33,
2
13
13
11.93 (s, 1H, OH), 11.97 (s, 1H, OH). C NMR (125 MHz, DMSO) d (ppm):
1
1
1
191.40, 181.03, 164.58, 161.37, 160.92, 140.87, 137.85, 137.52, 133.49, 133.27,
24.49, 123.04, 119.38, 118.10, 117.86, 116.11, 62.74, 62.52, 49.44, 16.21,
130.12, 130.06, 124.74, 124.49, 123.03, 119.38, 118.09, 117.84, 116.10, 115.38,
5.97. 31P NMR (200 MHz, CDCl
) d (ppm): 21.30. ESI–MS m/z: 566.1 (M+Na) .
+
114.72, 62.70, 62.49, 49.49, 16.21, 15.98. P NMR (200 MHz, CDCl
31
) d (ppm):
P+H O): C,
59.21; H, 4.40; N, 2.66. Found: C, 59.15; H, 4.62; N, 2.57. Compound 5q: Yield
3
3
+
Anal. Calcd (for C26
4
CDCl
4
H
23ClNO
8
P): C, 57.42; H, 4.26; N, 2.58. Found: C, 57.37; H,
20.24. ESI–MS m/z: 550.1 (M+Na) . Anal. Calc. (for C26
H
23FNO
8
2
1
.38; N, 2.51. Compound 5h: Yield 82%. mp 242–244 °C. H NMR (500 MHz
1
3
3
) d (ppm): 1.09 (t, J = 7.0 Hz, 3H, CH ), 1.35 (t, J = 7.1 Hz, 3H, CH
3
), 3.72–
83%. mp 205–207 °C. H NMR (500 MHz CDCl
CH ), 1.42 (t, J = 7.1 Hz, 3H, CH ), 2.30 (s, 3H, Ar-CH
3
) d (ppm): 0.63 (t, J = 7.1 Hz, 3H,
), 3.31–4.35 (m, 4H, OCH ),
.26 (m, 4H, OCH
H, NH), 11.95 (s, 1H, OH), 12.00 (s, 1H, OH). C NMR (125 MHz, CDCl
2
), 6.31 (dd, J = 9.1, 21.4 Hz 1H, PCH), 7.17–8.22 (m, 9H, Ar-H,
3
3
3
2
1
3
1
3
) d
7.25–7.27 (m, 2H, Ar-H), 7.35 (dd, J = 8.5, 25.7 Hz 1H, PCH), 7.48–8.89 (m, 12H,
Ar-H, 1H, NH), 11.88 (s, 1H, OH), 11.91 (s, 1H, OH). 13C NMR (125 MHz, DMSO)
d (ppm): 191.37, 180.87, 165.43, 161.32, 160.79, 140.92, 137.46, 133.21,
131.30, 131.28, 130.90, 130.38, 129.70, 128.70, 128.67, 127.55, 127.35, 126.63,
126.53, 125.31, 124.98, 124.94, 124.41, 123.85, 123.16, 119.32, 118.31, 117.60,
(
ppm): 192.65, 180.94, 164.40, 162.78, 162.56, 161.20, 141.51, 137.71, 134.02,
1
1
33.42, 133.09, 129.90, 129.55, 129.37, 127.32, 124.96, 123.48, 120.39, 117.97,
31
17.57, 115.76, 63.88, 63.46, 47.07, 16.40, 16.03. P NMR (200 MHz, CDCl
3
) d
P): C,
7.42; H, 4.26; N, 2.58. Found: C, 57.31; H, 4.43; N, 2.53. Compound 5i: Yield
+
(
ppm): 21.33. ESI–MS m/z: 566.1 (M+Na) . Anal. Calcd (for C26H23ClNO
8
3
1
5
8
CH
5
1
1
1
6
116.00, 62.58, 62.40, 46.38, 16.26, 15.54. P NMR (200 MHz, CDCl
3
) d (ppm):
1
+
7%. mp 176–178 °C. H NMR (500 MHz CDCl
), 1.31 (t, J = 6.6 Hz, 3H, CH ), 3.76 (s, 3H, OCH
.77(dd, J = 7.6, 21.7 Hz 1H, PCH), 6.80–8.25 (m, 9H, Ar-H, 1H, NH), 11.94 (s,
3
) d (ppm): 1.13 (t, J = 6.6 Hz, 3H,
22.34. ESI–MS m/z: 632.2 (M+Na) . Anal. Calcd (for C34H28NO P): C, 66.99; H,
8
3
3
3
), 3.80–4.18 (m, 4H, OCH ),
2
4.64; N, 2.30. Found: C, 66.92; H, 4.71; N, 2.23.
18. Sharma, R. I.; Smith, T. A. D. J. Nucl. Med. 2008, 49, 1386.
1
3
H, OH), 11.99 (s, 1H, OH). C NMR (125 MHz, DMSO) d (ppm): 191.40, 181.01,
64.55, 161.37, 160.93, 159.15, 141.10, 137.50, 136.56, 133.44, 133.25, 129.18,
24.47, 123.03, 120.84, 119.37, 118.14, 117.74, 116.07, 114.28, 113.26, 62.55,
19. (a) Hecht, S. M. J. Nat. Prod. 2000, 63, 158; (b) Metcalfe, C.; Thomas, J. A. Chem.
Soc. Rev. 2003, 32, 215; (c) Tan, J.; Wang, B.-C.; Zhu, L. C. Bioorg. Med. Chem.
2009, 17, 614.
20. Tang, H.; Wang, X.-D.; Wei, Y.-B.; Huang, S.-L.; Huang, Z.-S.; Tan, J.-H.; An, L.-K.;
Wu, J.-Y.; Chan, A. S. C.; Gu, L.-Q. Eur. J. Med. Chem. 2008, 43, 973.
21. (a) Basu, A.; Jaisankar, P.; Kumar, G. S. Bioorg. Med. Chem. 2012, 20, 2498; (b)
Abe, Y.; Nakagawa, O.; Yamaguchi, R.; Sasaki, S. Bioorg. Med. Chem. 2012, 20,
3470.
22. (a) Cashman, D. J.; Kellogg, G. E. J. Med. Chem. 2004, 47, 1360; (b) Chaudhuri, P.;
Majumder, H. K.; Bhattacharya, S. J. Med. Chem. 2007, 50, 2536.
23. (a) Zhou, B.-B. S.; Bartek, J. Nat. Rev. Cancer 2004, 4, 1; (b) Kastan, M. B.; Bartek,
J. Nature 2004, 432, 316; (c) Tao, Z. F.; Lin, N. H. Anti-Cancer Agents Med. 2006, 6,
377; (d) Xie, L.-J.; Xiao, Y.; Wang, F.; Xu, Y.-F.; Qian, X.-H.; Zhang, R.; Cui, J.-N.;
Liu, J.-W. Bioorg. Med. Chem. 2009, 17, 7615.
3
1
2.39, 55.12, 49.86, 16.29, 16.02. P NMR (200 MHz, CDCl
3
) d (ppm): 20.92.
P): C, 60.11; H, 4.86; N,
.60. Found: C, 60.17; H, 4.96; N, 2.51. Compound 5j: Yield 90%. mp 228–
+
ESI–MS m/z: 562.2 (M+Na) . Anal. Calcd (for C27H26NO
9
2
2
1
30 °C. H NMR (500 MHz CDCl
), 3.76–4.19 (m, 4H, OCH
J = 9.8, 21.2 Hz 1H, PCH), 6.97–8.15 (m, 9H, Ar-H, 1H, NH), 11.93 (s, 1H, OH),
3
) d (ppm): 1.10 (t, J = 7.3 Hz, 3H, CH
3
), 1.32 (t,
J = 7.2 Hz, 3H, CH
3
2
), 3.98 (s, 3H, OCH ), 6.12 (dd,
3
1
1
1
5
2.01 (s, 1H, OH). 13C NMR (125 MHz, CDCl
3
) d (ppm): 192.57, 180.89, 163.86,
62.69, 162.55, 157.35, 142.01, 137.62, 133.86, 133.35, 129.74, 129.70, 129.66,
24.89, 123.41, 122.63, 121.06, 120.29, 117.75, 117.34, 111.36, 63.35, 62.96,
3
1
5.96, 46.30, 16.41, 16.09. P NMR (200 MHz, CDCl
3
) d (ppm): 21.31. ESI–MS
+
m/z: 562.2 (M+Na) . Anal. Calcd (for C27
Found: C, 60.01; H, 4.96; N, 2.51. Compound 5k: Yield 79%. mp 223–225 °C.
NMR (500 MHz CDCl ) d (ppm): 1.11 (t, J = 7.0 Hz, 3H, CH ), 1.32 (t, J = 7.2 Hz,
), 3.85–4.22 (m, 4H, OCH ), 5.76 (dd, J = 9.3, 22.0 Hz 1H, PCH), 7.17–
.21 (m, 10H, Ar-H, 1H, NH), 11.96 (s, 1H, OH), 12.03 (s, 1H, OH). C NMR
H26NO P): C, 60.11; H, 4.86; N, 2.60.
9
1
H
24. Monneret, C. Eur. J. Med. Chem. 2001, 36, 483.
25. Liu, M.; Zhao, H.-M.; Chen, S.; Yu, H.-T.; Zhang, Y.-B.; Quan, X. Chem. Commun.
2011, 7749.
26. Zhang, G. W.; Guo, J. B.; Zhao, N.; Wang, J. R. Sensor. Actuat. B-Chem. 2010, 144,
239.
3
3
3
8
H, CH
3
2
13
(
125 MHz, DMSO) d (ppm): 191.43, 181.05, 164.62, 161.36, 160.92, 141.06,
1
1
37.51, 135.13, 133.46, 133.29, 128.55, 128.50, 128.17, 127.75, 127.65, 127.31,
27. (a) Palchaudhuri, R.; Hergenrother, P. J. Curr. Opin. Biotechnol. 2007, 18, 497; (b)
Wheate, N. J.; Brodie, C. R.; Collins, J. G.; Kemp, S.; Aldrich-Wright, J. R. Mini-
Rev. Med. Chem. 2007, 7, 627.
28. Zhang, Z. C.; Yang, Y. Y.; Zhang, D. N.; Wang, Y. Y.; Qian, X. H.; Liu, F. Y. Bioorg.
Med. Chem. 2006, 14, 6962.
31
24.48, 123.03, 119.37, 118.14, 116.12, 62.52, 62.33, 49.91, 16.23, 15.98.
P
+
NMR (200 MHz, CDCl
for C26 24NO P): C, 61.30; H, 4.75; N, 2.75. Found: C, 61.20; H, 4.87; N, 2.69.
Compound 5l: Yield 84%. mp 248–250 °C. H NMR (500 MHz CDCl
.85 (t, J = 7.0 Hz, 3H, CH ), 1.37 (t, J = 7.1 Hz, 3H, CH ), 3.50–4.30 (m, 4H,
OCH ), 6.60 (dd, J = 9.3, 22.3 Hz 1H, PCH), 7.26–8.33 (m, 12H, Ar-H, 1H, NH),
3
) d (ppm): 21.30. ESI–MS m/z: 532.1 (M+Na) . Anal. Calcd
(
H
8
1
3
) d (ppm):
0
3
3
29. Eriksson, M.; Nordén, B. Methods Enzymol. 2001, 340, 68.
30. (a) Rosi, N. L.; Mirkin, C. A. Chem. Rev. 2005, 105, 1547; (b) Tian, S.-J.; Liu, J.-Y.;
Zhu, T.; Knoll, W. Chem. Mater. 2004, 16, 4103; (c) Jin, R.-C.; Wu, G.-S.; Li, Z.;
Mirkin, C. A.; Schatz, G. C. J. Am. Chem. Soc. 2003, 125, 1643.
31. Tan, J.-H.; Ou, T.-M.; Hou, J.-Q.; Lu, Y.-J.; Huang, S.-L.; Luo, H.-B.; Wu, J.-Y.;
Huang, Z.-S.; Wong, K.-Y.; Gu, L.-Q. J. Med. Chem. 2009, 52, 2825.
32. (a) Kuo, P.-L.; Hsu, Y.-L.; Ng, L.-T.; Lin, C.-C. Planta Med. 2004, 70, 12; (b)
Kagedal, K.; Bironaite, D.; Öllinger, K. Free Radical Res. 1999, 31, 419.
33. Bironaite, D.; Öllinger, K. Chem.-Biol. Interact. 1997, 103, 35.
34. Huang, Q.; Lu, G.-D.; Shen, H.-M.; Chung, M. C. M.; Ong, C. N. Med. Res. Rev.
2007, 27, 609.
35. (a) Caldwell, C. G.; Sahoo, S. P.; Polo, S. A.; Eversole, R. R.; Lanza, T. J.; Mills, S.
G.; Niedzwiecki, L. M.; Izquierdo-Martin, M.; Chang, B. C.; Harrison, R. K.; Kuo,
D. W.; Lin, T.-Y.; Stein, R. L.; Durette, P. L.; Hagmann, W. K. Bioorg. Med. Chem.
Lett. 1996, 6, 323; (b) Bianchini, G.; Aschi, M.; Cavicchio, G.; Crucianelli, M.;
Preziuso, S.; Gallina, C.; Nastari, A.; Gavuzzo, E.; Mazza, F. Bioorg. Med. Chem.
2005, 13, 4740; (c) Pochetti, G.; Gavuzzo, E.; Campestre, C.; Agamennone, M.;
Tortorella, P.; Consalvi, V.; Callina, C.; Hiller, O.; Tschesche, H.; Tucker, P. A.;
Mazza, F. J. Med. Chem. 2006, 49, 923.
2
1
3
1
1
1
1
1.94 (s, 1H, OH), 11.98 (s, 1H, OH). C NMR (125 MHz, DMSO) d (ppm):
91.41, 181.05, 164.49, 161.35, 160.91, 140.86, 137.50, 133.50, 133.28, 133.22,
31.37, 130.84, 128.68, 128.52, 127.42, 126.58, 125.78, 125.18, 124.47, 123.13,
3
1
23.03, 119.36, 118.10, 117.84, 116.12, 62.67, 62.33, 45.07, 16.29, 15.82.
P
+
NMR (200 MHz, CDCl
for C30 26NO P): C, 64.40; H, 4.68; N, 2.50. Found: C, 64.27; H, 4.68; N, 2.43.
Compound 5m: Yield 89%. mp 213–215 °C. H NMR (500 MHz CDCl
3
) d (ppm): 21.33. ESI–MS m/z: 582.2 (M+Na) . Anal. Calcd
(
H
8
1
3
) d (ppm):
), 3.77 (s, 3H, OCH ),
), 5.72 (dd, J = 9.5, 20.1 Hz 1H, PCH), 6.84–8.20 (m, 9H,
1
3
.13 (t, J = 7.2 Hz, 3H, CH
.77–4.22 (m, 4H, OCH
3
), 1.32 (t, J = 7.0 Hz, 3H, CH
3
3
2
1
3
Ar-H, 1H, NH), 11.94 (s, 1H, OH), 11.97 (s, 1H, OH). C NMR (125 MHz, DMSO)
d (ppm): 191.42, 181.06, 164.47, 161.36, 160.93, 158.88, 141.14, 137.50,
1
1
33.46, 133.29, 129.90, 129.85, 126.96, 124.47, 123.00, 119.36, 118.12, 117.76,
31
16.12, 114.46, 113.62, 62.41, 62.26, 55.06, 49.19, 16.25, 16.03. P NMR
+
(
200 MHz, CDCl
3
) d (ppm): 21.98. ESI–MS m/z: 562.2 (M+Na) . Anal. Calcd (for
C
27 9
H26NO P): C, 60.11; H, 4.86; N, 2.60. Found: C, 60.02; H, 5.06; N, 2.51.
1
Compound 5n: Yield 86%. mp 194–196 °C. H NMR (500 MHz CDCl
1
3
Ar-H, 1H, NH), 11.88 (s, 1H, OH), 11.91 (s, 1H, OH). C NMR (125 MHz, DMSO)
d (ppm): 191.42, 181.04, 164.57, 161.36, 160.92, 141.09, 137.51, 137.32,
1
3
) d (ppm):
), 2.30 (s, 3H, Ar-CH ),
), 5.77 (dd, J = 9.4, 20.7 Hz 1H, PCH), 7.06–8.53 (m, 10H,
.11 (t, J = 7.1 Hz, 3H, CH
.75–4.20 (m, 4H, OCH
3
), 1.29 (t, J = 7.0 Hz, 3H, CH
3
3
2
36. (a) Naydenova, E.; Topashka-Ancheva, M.; Todorov, P.; Yordanova, T.; Troev, K.
Bioorg. Med. Chem. 2006, 14, 2190; (b) Naydenova, E. D.; Todorov, P. T.;
Topashka-Ancheva, M. N.; Momekov, G. T.; Yordanova, T. Z.; Konstantinov, S.
M.; Troev, K. D. Eur. J. Med. Chem. 2008, 43, 1199.
1
3
34.99, 133.44, 133.28, 129.15, 128.37, 128.06, 125.68, 124.47, 123.03, 119.38,