Synthesis and antitumor activities of novel α-aminophosphonates dehydroabietic acid derivatives

Article abstract of DOI:10.1016/j.bmcl.2013.08.005

A series of novel α-aminophosphonate derivatives containing DHA structure were designed and synthesized as antitumor agents. In vitro antitumor activities of these compounds against the NCI-H460 (human lung cancer cell), A549 (human lung adenocarcinoma ce

Full text of DOI:10.1016/j.bmcl.2013.08.005

Accepted Manuscript
Synthesis and antitumor activities of novel α-aminophosphonates dehydroa‐
bietic acid derivatives
Xiao-Chao Huang, Meng Wang, Ying-Ming Pan, Xiao-Yan Tian, Heng-Shan
Wang, Ye Zhang
PII:
S0960-894X(13)00934-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.08.005
BMCL 20743
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
7 May 2013
19 July 2013
2 August 2013
Please cite this article as: Huang, X-C., Wang, M., Pan, Y-M., Tian, X-Y., Wang, H-S., Zhang, Y., Synthesis and
antitumor activities of novel α-aminophosphonates dehydroabietic acid derivatives, Bioorganic & Medicinal
Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/j.bmcl.2013.08.005
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Graphical Abstract
Synthesis and antitumor activities of novel α-
aminophosphonates dehydroabietic acid
derivatives
Xiao-Chao Huang, Meng-Wang, Ying-Ming Pan, Xiao-Yan Tian, Heng-Shan Wang and Ye Zhang
O
NH
S
P
OC2H5
NH
OC2H5
R
O
7 (7a-7q)
1 / 1

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Synthesis and antitumor activities of novel α-aminophosphonates dehydroabietic
acid derivatives
Xiao-Chao Huanga,†, Meng-Wanga,†, Ying-Ming Pana, Xiao-Yan Tiana, Heng-Shan Wanga,* and Ye
Zhanga,b,
aState Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry & Chemical Engineering of
Guangxi Normal University, Yucai Road 15, Guilin 541004, Guangxi, PR China
bDepartment of Chemistry, Guilin Normal College, Xinyi Road 15, Guangxi 541001, PR China
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of novel α-aminophosphonate derivatives containing DHA structure were designed and
synthesized as antitumor agents. In vitro antitumor activities of these compounds against the
NCI-H460 (human lung cancer cell), A549 (human lung adenocarcinoma cell), HepG2 (human
liver cancer cell) and SKOV3 (human ovarian cancer cell) human cancer cell lines were
evaluated and compared with commercial anticancer drug 5-fluorouracil (5-FU), employing
standard MTT assay. The pharmacological screening results revealed that many compounds
exhibited moderate to high levels of antitumor activities against the tested cancer cell lines and
that most demonstrated more potent inhibitory activities compared with the commercial
anticancer drug 5-FU. The action mechanism of representative compound 7c was preliminarily
investigated by acridine orange/ethidium bromide staining, Hoechst 33258 staining, JC-1
mitochondrial membrane potential staining and flow cytometry, which indicated that the
compound can induce cell apoptosis in NCI-H460 cells. Cell cycle analysis showed that
compound 7c mainly arrested NCI-H460 cells in G1 stage.
keywords:
Dehydroabietic acid
Aminophosphonate
Cytotoxicity
Apoptosis
Cell cycle
2009 Elsevier Ltd. All rights reserved.
Since the success of taxol, vinblastine and their related
derivatives as anticancer agents, developing other novel natural
compounds and their derivatives with better efficiency have
greatly attracted bioorganic chemists’ interest and become a
central research theme in bioorganic chemistry.1-5 So natural
products have been always a good traditional source of new
medicinal leads, and they play a significant role in drug
discovery, especially in the area of cancer pharmacology, where
the fraction of the drugs derived from natural products amounts
to 60%.6
activity by introducing to
a
pharmacy core.12 Many
aminophosphonates derivatives have shown good inhibition
activities against human tumors.13−16 As natural amino acid
analogues, α-aminophosphonate compounds have thus attracted
the current interest in organic and medicinal chemistry due to
biological and pharmacological properties of these
organophosphorus compounds.17−19
Owing to the synthetic and biological values of DHA and
aminophosphonate groups, it is thus to expect that the
condensation of DHA and aminophosphonate groups may
contribute to good antitumor activity. In addition, literature
Dehydroabietic acid (DHA, 4) is a natural occurring diterpenic
resin acid. Previous studies have revealed that DHA has
properties of increasing the inhibition activity of an anticancer
drug in various cells, e.g. cervical carcinoma cells, hepatocellular
carcinoma cells, and breast cancer cells.7 So DHA skeleton was
then chosen as active pharmacal core to screen for new potential
antitumor agents by the introduction of various functional groups
on ring B and C, as well as in carboxylic acid group of DHA.8-11
survey
has
proved
that
compounds
containing
aminophosphonates with DHA moiety have not been reported so
far. Therefore, aminophosphonate group was rationally designed
and introduced to the DHA structure. Our present work in this
paper is to design and synthesize α-aminophosphonates
derivatives containing DHA skeleton, and to evaluate their in
vitro antitumor activities. Preliminary investigation on the mode
of action of representative compound 7c is also investigated.
Aminophosphonate group, which is considered as potent
antitumor agents, is able to effectively improve the antitumor
† Co-first author: These authors contributed equally to this work.
*Corresponding author. Tel./fax: +86 0773 2120958; e-mail addresses: whengshan@163.com (H. -S. Wang), zhangye81@126.com (Y.
Zhang).
1 / 7

O
P
OC2H5
O
P
OC2H5
OC2H5
OC2H5
R
a
b
H2N
N
R
CHO
R
R
1
3
2
e
O
P
OC2H5
NH
S
OC2H5
NH
c
O
d
R
7 (7a-7q)
COOH
COCl
COSCN
6
5
4
Scheme 1. Synthetic route to aminophosphonate DHA derivatives. Reagents and conditions: (a) NH4Ac, HPO(OC2H5)2, Al(OTf)3; (b) (i) HCl(aq), (ii) NaOH(aq);
(c) (COCl)2, CH2Cl2, r.t.; (d) C6H5CH3, KSCN, 110 oC; (e) CH2Cl2, r.t.
Table 1. Effect of aminophosphonate DHA derivatives 7 against cell viability of different cell lines.
Compounds
IC50(μM)
R
Ph
NCI-H460
28.05±1.23
25.00±2.12
3.33±0.58
15.83±1.24
26.45±1.62
17.82±2.09
4.39±0.86
17.81±2.58
27.51±2.67
19.68±2.53
30.72±3.26
36.37±3.48
18.74±1.83
18.57±2.36
14.84±1.60
32.61±2.85
22.74±2.19
84.53±1.11
44.04±0.54
A549
HepG2
SKOV3
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
24.23±0.78
15.35±1.32
18.37±1.04
29.58±1.46
21.95±2.34
18.75±2.46
23.42±1.86
14.03±1.78
24.44±3.12
12.66±1.76
19.60±2.75
26.40±2.88
20.19±2.57
22.19±2.86
19.48±2.14
28.34±3.11
45.74±3.87
79.46±1.09
34.33±0.23
15.18±0.69
32.08±3.24
7.70±1.06
17.99±2.21
29.96±2.46
37.30±3.21
10.34±1.66
11.38±2.46
31.63±2.34
26.46±2.46
24.87±3.62
>50
24.03±2.83
27.05±2.67
15.19±1.23
>50
>50
85.00±1.16
29.98±0.37
12.14±1.02
21.35±2.01
18.92±2.23
9.96±1.08
14.12±1.58
16.64±1.68
13.50±2.46
10.03±1.56
13.66±1.54
14.54±1.34
15.30±1.89
19.69±1.79
13.97±1.73
17.41±1.95
20.67±2.48
19.09±1.11
>50
p-Ph-OCH3
o-Ph-OCH3
m-Ph-OCH3
p-Ph-CH3
m-Ph-CH3
p-Ph-F
o-Ph-F
m-Ph-F
p-Ph-Cl
o-Ph-Cl
m-Ph-Cl
p-Ph-Br
o-Ph-Br
m-Ph-Br
1-Naphthalene
2-Naphthalene
DHA
5-FU
84.00±1.21
24.43±0.41
(a)
(b)
(c)
Figure 1. AO/EB staining of compound 7c in NCI-H460 cells. (a) Not treated with the 7c was used as control for 24h and (b, c) treatment with compound 7c (5
μM) for 12 h and 24 h, respectively.
(a)
(b)
(c)
Figure 2. Hoechst 33258 staining of compound 7c in NCI-H460 cells. (a) Not treated with compound 7c was used as control for 24h and (b, c) treatment with
compound 7c (5 μM) for 12 h and 24 h, respectively.
2 / 7

(a)
(b)
(c)
Figure 3. JC-1 mitochondrial membrane potential staining of compound 7c in NCI-H460 cells. (a) Not treated with the 7c was used as control for 24 h and (b, c)
treatment with compound 7c (5 μM) for 12 h and 24 h, respectively .
(a)
(b)
(c)
Figure 4. Apoptosis ratio detection of compound 7c by Annexin V/PI assay. (a) Not treated with the 7c was used as control for 24 h and (b, c) treatment with
compound 7c (5 μM) for 12 h and 24 h, respectively .
Figure 5. Cell-cycle analysis by flow cytometry of NCI-H460 cells treated with compound 7c at IC50 concentration.
Aminophosphonate DHA derivatives 7 were synthesized as
outlined in Scheme 1. Compound 2 was synthesized by the
As shown in Table 1, all the compounds 7 showed antitumor
activities in NCI-H460 assay. It was important to note that in
NCI-H460 assay all the compounds 7 exhibited better inhibition
than DHA (IC50=84.53 μM), and even better than the commercial
anticancer drug 5-FU (IC50=44.04 μM), with IC50 in the range of
3.33−36.37 μM, indicating good antitumor activities of these
compounds on NCI-H460 cell line. This cytotoxic inhibition
screening results showed that the introduction of α-
aminophosphonate group on DHA should markedly improve the
antitumor activity against NCI-H460 cell line. Among all the
compounds, compound 7c exhibited the best antitumor activity,
with IC50 of 3.33 μM, while compound 7l showed the lowest,
with IC50 of 36.37 μM, respectively. The antitumor activities of
tested were found to be in the order of 7c > 7g > 7o > 7d > 7h >
7f > 7n > 7m > 7j > 7q > 7b > 7e > 7i > 7a > 7k > 7p > 7l > 5-
FU > DHA. On the basis of the above observation, methoxy,
methyl, fluoro and bromo groups in benzene group of α-
aminophosphonate moiety showed positive influence on
antitumor activities in NCI-H460 assay, while the chloro group
exhibited negative effect. Besides, the substituent position on
treatment of aromatic aldehydes
1 with the mixture of
ammonium acetate and diethyl phosphate in the presence of
Al(OTf)3, and it was then treated with HCl (aq) and NaOH (aq),
respectively, to offer α-aminophosphonate 3, according to
literature.20 Dehydroabietic chloride 5 was obtained by the
treatment of DHA 4 with oxalyl chloride, and it was then treated
with KSCN to offer dehydroabietic isothiocyanate 6. α-
Aminophosphonate DHA derivatives 7 were finally obtained by
the condensation of compound 6 with α-aminophosphonate 3 in
good yields. The structures of aminophosphonate DHA
1
derivatives 7 were confirmed by H NMR, 13CNMR, 31P NMR
and high resolution mass spectrum (HRMS).21
The in vitro antitumor activities of aminophosphonate DHA
derivatives 7 were evaluated by MTT assay22 against NCI-H460,
HepG2, A549 and SKOV3 tumor cell lines, using 5-fluorouracil
(5-FU) as the positive control. The tested results were shown in
Table 1.
3 / 7

naphthalene also appeared to have important influences on the
antitumor activity in this assay.
33258 staining,24 JC-1 mitochondrial membrane potential
staining25 and flow cytometry.26
In A549 assay, all the compounds 7 exhibited much better
inhibition than DHA (IC50=79.46 μM), with IC50 in the range of
12.66−45.74 μM. It was worth noting that except 7q all these
compounds 7 also showed further more cytotoxic inhibition than
5-FU (34.33 μM), indicating potent cytotoxicity of these
compounds on A549 cell line. This cytotoxicity screening results
implied that the introduction of α-aminophosphonate on DHA
obviously improve the antitumor activity against A549 cell line.
Compound 7j exhibited the best cytotoxicity among all the
compounds, with IC50 of 12.66 μM, while compound 7q showed
lowest, with IC50 of 45.74 μM. Clearly, the order of antitumor
activity of tested in this assay was: 7j > 7h > 7b > 7c > 7f > 7o >
7k > 7m > 7e > 7n > 7g > 7a > 7i > 7l > 7p > 7d > 5-FU > 7q >
DHA. Based on the above observation, it could be summarized
that methoxy, methyl, fluoro and chloro groups at ortho and para
position in benzene group of α-aminophosphonate moiety may
have important influence on the antitumor activity, while
naphthalene exhibited negative effect.
Both of AO and EB are important dyes, and AO can stain
nuclear DNA across an intact cell membrane, while EB only
stains cells that had lost their membrane integrity. Therefore,
after synchronous treating with AO and EB, live cells will be
evenly stained as green (in the web version) and early apoptotic
cells will be thickly stained as green yellow or show green
yellow fragments (in the web version), while late apoptotic cells
will be densely stained as orange or display orange fragments and
necrotic cells will be stained as orange with no condensed
chromatin. The cytotoxicity of compound 7c at the concentration
of 5 μM against NCI-H460 cells from 12 to 24 h was assayed by
AO/EB staining, and NCI-H460 cells not treated with the 7c
were used as control for 24 h. The results were shown in Fig. 1.
As shown in Fig.1, the NCI-H460 cells treated with 7c from 12 to
24 h had obviously changed. The nuclei markedly stained as
yellow green or orange, and the morphology displayed pycnosis,
membrane blebbing and cell budding. These phenomena were
associated with cell apoptosis. Based on the observation, the cells
represented with an apoptotic morphology. These findings
indicated that compounds 7c could induce apoptosis with low
cytotoxicity.
In HepG2 assay, except compounds 7l, 7p and 7q, other
compounds displayed better cytotoxicity than DHA (IC50=85.00
μM), with IC50 in the range of 7.70−37.30 μM, indicating that the
introduction of α-aminophosphonate on DHA should improve the
antitumor activity against HepG2 cell line. Moreover,
compounds 7a, 7c, 7d, 7e, 7g, 7h, 7j, 7k, 7m, 7n and 7o even
demonstrated better cytotoxic inhibition than 5-FU, implying
favorable inhibition activities of these compounds on HepG2 cell
line. Compounds 7c and 7g showed better inhibition on HepG2
cell line than other compounds, with IC50 of 7.70 μM and 10.34
μM, respectively. HepG2 cells inhibiting activities of these tested
compounds decreased in the order of 7c, 7g, 7h , 7a , 7o, 7d, 7m,
7k, 7j, 7n, 7e, 5-FU, 7i, 7b, 7f, DHA. Evidently, methoxy at
ortho, as well as fluoro at ortho and para position in benzene
group of α-aminophosphonate moiety may result in the
enhancement of antitumor activity, while the presence of methyl,
chloro and bromo in benzene lead to the decrease of antitumor
activity. In addition, displacement of benzene with naphthalene
also appeared to have negative influences on the antitumor
activity in this assay.
Hoechst 33258 is a membrane permeable dye with blue
fluorescence. 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
contrary, the nuclei of dead cells could not be stained. NCI-H460
cells treated with compound 7c at 5 μM from 12 to 24 h were
stained with Hoechst 33258. NCI-H460 cells not treated with the
7c were used as control at for 24 h. The results were given in Fig.
2. As shown in Fig. 2, cells not treated with compound 7c were
normally blue (a). It was worth noting that, for 7c treatment, the
cells displayed strong blue fluorescence and indicated typical
apoptotic morphology after 12 (b) and 24 h (c). The observation
revealed that compounds 7c induced apoptosis against NCI-H460
cells, consistent with the results for AO/EB double staining.
Mitochondrial membrane potential changes were also designed
and detected to further investigate the apoptosis-inducing effect
of target compound 7c, 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
mitochondria.27 NCI-H460 cells treated with compound 7c at 5
μM from 12 to 24 h were stained with JC-1 and not treated with
the compound 7c were used as control at for 24 h. The results
were shown in Fig. 3. The JC-1 monomer and J-aggregates were
excited at 514 nm and 585 nm, respectively, and light emissions
were collected at 515−545 nm (green) and 570−600 nm (red).
For fluorescence microscopy, Fig. 3 displayed that cells not
treated with the compound 7c were normally red (in the web
version a), while for 7c treatment (in the web version b and c),
cells exhibited strong green fluorescence as that for 5-FU which
is known to induce the apoptosis26c (Fig. s1) and indicated typical
apoptotic morphology after 12 h and 24 h. Therefore, it could be
concluded that compound 7c induced apoptosis against NCI-
H460 cell line. The results were identical with that of previous
experiment of AO/EB double staining and Hoechst 33258
staining.
In SKOV3 assay, all the compounds except 7q displayed better
cytotoxicity than DHA (IC50=84.00 μM), with IC50 in the range
of 9.96−21.35 μM, exhibiting that the introduction of α-
aminophosphonate on DHA should improve the antitumor
activity against SKOV3 cell line. It was important to point out
that these compounds even exhibited better cytotoxic inhibition
than 5-FU, showing good inhibition activities of these
compounds on SKOV3 cell line. Among all these compounds,
compound 7d exhibited the best inhibition activity on SKOV3
cells, with IC50 of 9.96 μM, while compound 7b displayed the
lowest, with IC50 of 21.35 μM. The antitumor activity order in
this assay was listed as follow: 7d > 7h > 7a > 7g > 7i > 7m > 7e
> 7j > 7k > 7f > 7n > 7c > 7p > 7l > 7o > 7b > 5-FU > DHA.
The order obviously indicated that the benzene group was
important to their antitumor activity, and methoxy at meta-
position and fluoro at ortho-position in benzene group of α-
aminophosphonate moiety were important contributors the
antitumor activity.
In the present study, compound 7c, which exhibited good
cytotoxic inhibition in four cell lines and could be used as a good
representative of compounds 7, was selected and its mechanism
of growth inhibition of NCI-H460 cells was evaluated by
acridine orange(AO)/ethidium bromide(EB) staining23, Hoechst
The apoptosis ratios induced by compound 7c in NCI-H460
tumor cells were then quantitatively determined by flow
cytometry. Four quadrant images were observed by flow
cytometric analysis: the Q1 area represented damaged cells
appearing in the process of cell collection, the Q2 region showed
4 / 7

necrotic cells and later period apoptotic cells, the Q3 area showed
normal cells, and the early apoptotic cells were located in the Q4
area. The results were given in Fig. 4. Fig. 4 revealed that
compound 7c could induce apoptosis in NCI-H460 cells.
Apoptosis ratios (including the early and late apoptosis ratios) for
compound 7c were obtained after 12 h of treatment at the
concentration of 3 μM and 5 μM, respectively. The apoptosis of
NCI-H460 cells treated with compound 7c increased gradually in
a concentration manner. The apoptosis ratios of compound 7c
measured at different concentration points were found to be
19.5% (3 μM) and 51.5% (5 μM), respectively, while that of
control was 5.7%. In addition, the apoptosis ratio of 5-FU was
assayed to be 23.3% (Fig. s2) under the similar condition. The
results evidently illustrated that compound 7c suppressed cell
proliferation by inducing apoptosis. Previous reports28 have
revealed that α-aminophosphonates derivatives may induce
apoptosis by affecting associated enzymes in mitochondria-
dependent and-independent pathways, it is reasonable to believe
that compound 7c may induce apoptosis in the same mechanism.
And the investigation on precise mechanism is in process.
References and notes
1. Jordan, M. A.; Wilson, L. Nat. Rev. Cancer. 2004, 4, 253.
2.
Vennila, R.; Kamalraj, S.; Muthumary, J. Biomedicine & Aging
Pathology. 2012, 2, 16.
3. Noble, R. L.; Beer, C. T.; Cutts, J. H.; Ann. N.Y. Acad. Sci. 1958,
76, 882.
4. Moncrief, J. W.; Lipscomb, W. N.; J. Am. Chem. Soc. 1965, 87,
4963.
5. Neuss, N.; Neuss, M. N. In: The Alkaloids; Brossi, A.; Suffness,
M. (Eds.), Academic: San Diego, 1990, Vol. 37, p 229.
6. Newman, D. J.; Cragg, G. M.; Snader, K. M. J. Nat. Prod. 2003,
66, 1022.
7. Lin, C. -H.; Chuang, H. -S. U.S. Patent 5, 248, 696, 2003.
8. Cui, Y. –M.; Yasutomi, E.; Otani, Y.; Yoshinaga, T.; Ido, K.;
Sawada, K.; Ohwada, T. Bioorg. Med. Chem. Lett. 2008, 18, 5201.
9. Rao, X.; Song, Z.; He, L.; Jia, W. Chem. Pharm. Bull. 2008, 56,
1575.
10. Tanaka, R.; Tokuda, H.; Ezaki, Y. Phytomedicine. 2008, 15, 985.
11. Li, F.; He, L.; Song, Z. -Q.; Yao, J. -C.; Rao, X. –P.; Li, H. -T.; J.
Pharm. Pharmacol. 2008, 60, 205.
12. Gnant, M.; Clézardin, P. Cancer Treat. Rev. 2012, 38, 407.
13. Clézardin, P. Bone. 2011, 48, 71.
14. Ning, L.; Wang, W.; Liang, Y. -J.; Peng, H.; Fu, L. -W.; He, H. -
W. Eur. J. Med. Chem. 2012, 48, 379.
The above studies have proved that compound 7c can induce
apoptosis, therefore the effect of compounds 7c on the cell cycle
of NCI-H460 cell lines was investigated by flow cytometry at its
IC50 concentrations and showed in Fig. 5. And the effect of
compound 5-FU on the cell cycle of NCI-H460 cell was also
studied (Fig. s3). As shown in Fig. 5 and Fig. s3, the treatment of
NCI-H460 cells with compounds 7c and 5-FU enhanced cell-
cycle arrest at the G1 phase, resulting in concomitant population
increase in the G1 phase (62.80% and 71.93%) compared with
the control cells (55.64% and 68.60%) and the population of the
G2 phase decrease at a certain extent (13.70% and 0) compared
with the control cells (21.84% and 8.14%). These results
suggested that compound 7c mainly arrest the cell-cycle of NCI-
H460 cells in the G1 stage. It also indicated that 5-FU arrest
NCI-H460 cells in the G1 and S stage, consistent with that of it
was treated with oral cancer cell.26c
15. Ali, N. S.; Zakir, S.; Patel, M.; Farooqui, M. Eur. J. Med. Chem.
2012, 50, 39.
16. Gu, L.; Jin, C. Org. Biomol. Chem. 2012, 10, 7098.
17. Kafarski, P.; Lejczak, B. In Aminophosphonic and
Aminophosphinic Acids. Chemistry and Biological Activity;
Kukhar, V.P.; Hudson, H.R., Eds.; John Wiley
Chichester, 2000; Vol. 12, pp 407-442.
& Sons:
18. Atherton, F. R.; Hassal, C. H.; Lambert, R.W. J. Med. Chem. 1986,
29, 29.
19. Du, S.; Faiger, H.; Belakhov, V.; Baasov, T. Bioorg. Med. Chem.
1999, 7, 2671.
20. Sobhani, S.; Tashrifi, Z. Synthetic Communications. 2008, 39,
120.
21. (a) General procedure for the preparation of compounds 7:
Al(OTf)3 (0.01 mmol) was added to a stirring mixture of aldehyde
(1) (2 mmol), NH4OAc (2 mmol), and diethyl phosphite (2 mmol).
The resulting mixture was heated in an oil bath at 100 oC and
stirred for 30 min. The reaction mixture was cooled and acidified
to pH 1 by HCl (aq). The solution was washed with Et2O (2 × 10
mL). The aqueous phase was then made alkaline with NaOH (aq),
and the product was extracted with Et2O (2 × 10 mL). The
combined organic phase was dried over Na2SO4 and concentrated
under reduced pressure to offer α-aminophosphonates 3.
Compound 4 (1mmol) added to CH2Cl2 (15mL) was stirred at 0oC
and oxalyl chloride (1.5mmol) was dripped into the mixture and
stirred at room temperature for 6 h. After the reaction, the solvent
and excess oxalyl chloride was evaporated under reduced
pressure. Toluene (15mL) and KSCN (1.2mmol) were added to
In this paper, we have described the design, synthesis and in
vitro antitumor activities of α-aminophosphonates derivatives,
derived from the DHA pharmacy core. The in vitro antitumor
activities screening showed that almost all the compounds 7
exhibited better inhibition than the commercial anticancer drug 5-
FU in both NCI-H460 and A549 assays, and compounds 7a, 7c,
7d, 7e, 7h, 7j, 7k, 7m, 7n and 7o demonstrated better cytotoxic
inhibition than 5-FU in HepG2 assay, while all the compounds
except 7q displayed better cytotoxicity than 5-FU in SKOV3
assay, respectively. The apoptosis-inducing activity of
representative compound 7c in NCI-H460 cells was studied and
the results revealed that this compound showed clear cell
apoptosis inducing effects. Cell cycle analysis indicated that
compound 7c could arrest NCI-H460 cell in G1 stage. The above
o
the mixture and stirred at 110 C for 12 h. After the reaction, the
solvent was evaporated under reduced pressure, and the crude
product was purified by chromatography on silica gel eluted with
petroleum ether/ethyl acetate (V:V = 10:1) to offer compound 6.
Compound 6 (1mmol) and α-aminophosphonate 3 (1.2mmol) were
added to CH2Cl2 (15mL) the mixture was stirred at room
temperature for 3 h. After the reaction was completed, the solvent
was evaporated under reduced pressure, and the crude product was
purified by chromatography on silica gel eluted with petroleum
ether/ethyl acetate (V:V = 3:1) to offer compounds 7 (7a−7q); (b)
results
demonstrate
that
the
rational
design
of
aminophosphonates DHA derivatives as novel antitumor leads is
feasible. The precise mechanism of action is currently under way.
Experimental: NMR spectra were recorded on
a BRUKER
AVANCE 500 NMR spectrometer in CDCl3. Mass spectra were
determined on a FTMS ESI spectrometer. 7a: Yields 85%; 1H
NMR (500 MHz, CDCl3): δ 11.52 (s, 1H, NH), 8.63 (s, 1H, NH),
7.45 (d, J = 6.9 Hz, 2H), 7.40−7.35 (m, 2H), 7.32−7.30 (m, 1H),
7.16 (d, J = 8.2 Hz, 1H), 7.01 (dd, J = 8.2, 1.9 Hz, 1H), 6.90−6.86
(m, 1H), 6.12−6.05 (m, 1H, N-CH-P), 4.14−3.94 (m, 4H, 2 ×
OCH2CH3), 2.93−2.79 (m, 3H), 2.35 (d, J = 13.7 Hz, 1H),
2.13−2.06 (m, 1H), 1.76−1.52 (m, 7H), 1.35 (d, J = 3.3 Hz, 3H,
CH3), 1.30−1.26 (m, 3H, CH3), 1.23 (dd, J = 3.5, 1.9 Hz, 6H, 2 ×
CH3), 1.23−1.19 (m, 6H, 2 × CH3). 13C NMR (125 MHz, CDCl3) δ
180.64, 179.52, 146.26, 146.09, 134.19, 133.69, 128.71, 128.29,
128.25, 128.21, 127.96, 126.93, 124.14, 124.03, 63.52, 63.32,
63.60, 63.57, 63.42, 57.32, 56.11, 48.65, 45.36, 37.69, 37.08,
36.93, 33.49, 29.73, 29.67, 25.10, 25.06, 23.97, 21.45, 18.49,
16.14. 31P NMR (200 MHz, CDCl3) δ 19.61. HRMS (m/z) (ESI):
Acknowledgments
This study was supported by 973 project (No.2011CB512005,
2012CB723501), the National Natural Science Foundation of
China (No. 81260472), Guangxi Natural Science Foundation of
China (2011GXNSFD018010 and No. 2010GXNSFF013001),
the Guilin Scientific Research and Technological Development
Project (No. 20110106-2; 20120108-6), Bagui Scholar project
and the Foundation of Ministry of Education Innovation Team
(NO. IRT1225).
5 / 7

calcd for C32H45N2O4PS [M+H+]: 585.28377; found: 585.28766;
7b: Yields 86.25%; 1H NMR (500 MHz, CDCl3): δ 11.61 (s, H,
NH), 8.63 (s, 1H, NH), 7.40−7.36 (m, 2H), 7.18 (d, J = 8.2 Hz,
1H), 7.03 (dd, J = 8.2, 1.8 Hz, 1H), 6.97−6.87 (m, 3H), 6.04−5.98
(m, 1H, N-CH-P), 4.21−3.94 (m, 4H, 2 × OCH2CH3), 3.82 (s, 3H,
OCH3), 3.04−2.64 (m, 3H), 2.36 (d, J = 13.5 Hz, 1H), 2.12−2.18
(m, 1H), 1.86−1.51 (m, 7H), 1.36 (d, J = 3.4 Hz, 3H, CH3),
1.31−1.28 (m, 3H, CH3), 1.25 (dd, J = 6.5, 4.5 Hz, 9H, 3 × CH3),
1.23 (d, J = 5.3 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3) δ
180.16, 179.41, 159.58, 146.29, 146.09, 134.20, 129.56, 126.52,
125.71, 124.14, 124.03, 123.99, 114.19, 63.50, 63.29, 56.66,
55.36, 48.63, 45.34, 37.69, 37.07, 36.96, 36.93, 33.49, 29.70,
25.08, 23.97, 21.44, 18.49, 16.35, 16.14. 31P NMR (200 MHz,
CDCl3) δ 19.91. HRMS (m/z) (ESI): calcd for C33H47N2O5PS
[M+H+]: 615.29433; found: 615.29822; 7c: Yields 88.55%; 1H
NMR (500 MHz, CDCl3): δ 11.81 (s, 1H, NH), 8.59 (s, 1H, NH),
7.33−7.27 (m, 2H), 7.16 (d, J = 8.2 Hz, 1H), 7.01 (d, J = 8.2 Hz,
1H), 6.97−6.95 (m, 1H), 6.94−6.90 (m, 1H), 6.88 (s, 1H),
6.48−6.44 (m, 1H, N-CH-P), 4.16−3.95 (m, 4H, 2 × OCH2CH3),
3.92 (s, 3H, OCH3), 2.86−2.80 (m, 3H), 2.33 (d, J = 13.2 Hz, 1H),
2.13 (dd, J = 12.5, 2.1 Hz, 1H), 1.88−1.63 (m, 7H), 1.34 (d, J =
2.1 Hz, 3H, CH3), 1.29−1.28 (m, 3H, CH3), 1.23 (d, J = 1.2 Hz,
6H, 2 × CH3), 1.22 (d, J = 1.1 Hz, 3H, CH3), 1.20 (dd, J = 7.1, 4.4
Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3) δ 180.00, 178.87,
157.40, 146.37, 146.04, 134.28, 129.60, 126.92, 124.13, 124.11,
124.03, 120.89, 111.32, 77.30, 77.05, 76.79, 63.20, 55.82, 53.97,
52.74, 52.55, 48.51, 45.19, 37.68, 37.13, 36.58, 33.48, 29.77,
25.12, 23.97, 21.43, 18.51, 16.38, 16.20. 31P NMR (200 MHz,
CDCl3) δ 19.87. HRMS (m/z) (ESI): calcd for C33H47N2O5PS
[M+H+]: 615.29433; found: 615.29938; 7d: Yields 88.25%; 1H
NMR (500 MHz, CDCl3): δ 11.67 (s, 1H, NH), 8.72 (s, 1H, NH),
7.33−7.26 (m, 1H), 7.17 (d, J = 8.2 Hz, 1H), 7.08−7.00 (m, 3H),
6.93−6.85 (m, 2H), 6.15−6.06 (m, 1H, N-CH-P), 4.15−4.09 (m,
4H, 2×OCH2CH3), 3.81 (s, 3H, OCH3), 2.97−2.79 (m, 3H), 2.35
(d, J = 13.1 Hz, 1H), 2.13−2.11 (m, 1H), 1.81−1.50 (m, 7H), 1.36
(d, J = 3.5 Hz, 3H, CH3), 1.30−1.29 (m, 3H, CH3), 1.28−1.26 (m,
3H, CH3), 1.24 (dd, J = 5.9, 3.4 Hz, 9H, 3×CH3). 13C NMR (125
MHz, CDCl3) δ 180.60, 179.5, 159.73, 146.29, 146.03, 135.13,
134.18, 129.69, 126.91, 124.13, 124.03, 120.46, 120.41, 113.90,
63.58, 63.46, 60.36, 57.24, 56.03, 55.25, 48.63, 45.29, 37.67,
37.06, 36.86, 33.48, 29.72, 25.09, 23.97, 21.46, 21.02, 18.49,
16.53, 14.20. 31P NMR (200 MHz, CDCl3) δ 19.51. HRMS (m/z)
(ESI): calcd for C33H47N2O5PS [M+H+]: 615.29433; found:
615.29913; 7e: Yields 86.25%; 1H NMR (500 MHz, CDCl3):δ
130.08, 130.03, 130.02, 129.97, 129.65, 126.95, 124.16, 123.98,
63.50, 56.57, 55.35, 48.67, 45.37, 37.69, 37.18, 36.83, 33.48,
29.69, 25.07, 25.03, 23.94, 21.47, 18.48, 16.57, 16.05. 31P NMR
(200 MHz, CDCl3) δ 19.39. HRMS (m/z) (ESI): calcd for
C33H44FN2O4PS [M+H+]: 603.27374; found: 603.27887.; 7h:
Yields 89.56%; 1H NMR (500 MHz, CDCl3): δ 11.36 (s, 1H, NH),
8.66 (s, 1H, NH), 7.41 (t, J = 7.5 Hz, 1H), 7.32 (dd, J = 10.7, 4.8
Hz, 1H), 7.19 (dd, J = 12.8, 5.6 Hz, 2H), 7.11 (d, J = 9.2 Hz, 1H),
7.03 (d, J = 6.2 Hz, 1H), 6.90 (d, J = 2.3 Hz, 1H), 6.39−6.34 (m,
1H, N-CH-P), 4.20−4.01 (m, 4H, 2×OCH2CH3), 3.02−2.74 (m,
3H), 2.36 (d, J = 13.6 Hz, 1H), 2.18−2.07 (m, 1H), 1.70 (dd, J =
90.7, 47.2 Hz, 7H), 1.38−1.35 (m, 3H, CH3), 1.35−1.32 (m, 3H,
CH3), 1.27−1.25 (m, 6H, 2×CH3), 1.25−1.22 (m, 6H, 2×CH3). 13C
NMR (125 MHz, CDCl3) δ 180.83, 179.38, 146.09, 134.21,
129.96, 129.47, 126.92 , 124.47, 124.13, 124.01, 115.77, 115.63,
115.33, 63.58, 51.69, 50.46, 48.64, 45.35, 45.31, 37.69, 37.07,
36.95, 33.49, 29.69, 25.07, 23.96, 21.49, 21.41, 18.49, 16.41,
16.02. 31P NMR (200 MHz, CDCl3) δ 18.66. HRMS (m/z) (ESI):
calcd for C33H44FN2O4PS [M+H+]: 603.27374; found: 603.27850;
1
7i: Yields 89.46%; H NMR (500 MHz, CDCl3) δ 11.66 (dd, J =
14.5, 9.1 Hz, 1H), 8.67 (s, 1H), 7.40−7.33 (m, 1H), 7.26 (d, J =
7.7 Hz, 1H), 7.18 (dd, J = 8.9, 4.3 Hz, 2H), 7.08−6.99 (m, 2H),
6.91 (d, J = 6.1 Hz, 1H), 6.07-5.98 (m, 1H, N-CH-P), 4.17−3.97
(m, 4H, 2×OCH2CH3), 2.97−2.79 (m, 3H), 2.37 (d, J = 13.4 Hz,
1H), 2.13 (m, 1H), 1.84−1.52 (m, 7H), 1.38 (d, J = 3.2 Hz, 3H,
CH3), 1.33−1.29 (m, 3H, CH3), 1.29−1.26 (m, 6H, 2×CH3), 1.24
(dd, J = 6.9, 2.5 Hz, 6H, 2×CH3). 13C NMR (125 MHz, CDCl3) δ
181.06, 179.58, 164.08, 161.90, 146.24, 146.12, 136.35, 134.15,
130.20, 126.93, 124.17, 124.04, 124.01, 123.69, 115.36, 115.19,
63.80, 63.11, 56.88, 55.67, 48.70, 45.38, 37.68 , 37.20, 36.56,
33.49, 29.70, 25.08, 23.96, 21.48, 18.48, 16.41, 16.01. 31P NMR
(200 MHz, CDCl3) δ19.40. HRMS (m/z) (ESI): calcd for
C33H44FN2O4PS [M+H+]: 603.27374; found: 603.27887; 7j: Yields
1
88.58%; H NMR (500 MHz, CDCl3): δ 11.65 (s, 1H, NH), 8.67
(s, 1H, NH), 7.41−7.39 (m, 2H), 7.37 (dd, J = 8.6, 2.5 Hz, 2H),
7.18 (d, J = 8.2 Hz, 1H), 7.04 (dd, J = 8.2, 1.8 Hz, 1H), 6.91 (dd, J
= 6.0, 1.5 Hz, 1H), 6.04−5.99 (m, 1H, N-CH-P), 4.16−3.99 (m,
4H, 2×OCH2CH3), 3.06−2.55 (m, 3H), 2.37 (d, J = 13.4 Hz, 1H),
2.12−2.08 (m, 1H), 1.86−1.47 (m, 7H), 1.37 (d, J = 3.4 Hz, 3H,
CH3), 1.31−1.30 (m, 3H, CH3), 1.29−1.25 (m, 6H, 2×CH3),
1.25−1.23 (m, 6H, 2×CH3). 13C NMR (125 MHz, CDCl3) δ
180.86, 179.67, 146.24, 146.13, 134.29, 134.14, 132.40, 129.61,
129.57, 128.91, 126.93, 124.18, 124.04, 124.00, 123.87, 63.45,
56.68, 55.47, 48.69, 45.36, 37.68, 37.20, 36.57, 33.49, 29.70,
25.08, 23.97, 21.48, 18.48, 16.53, 16.18, 16.13. 31P NMR (200
11.78 (s, 1H ， NH), 8.64 (s, 1H, NH), 7.38−7.33 (m, 2H),
MHz, CDCl3)
δ 19.10. HRMS (m/z) (ESI): calcd for
7.23−7.14 (m, 3H), 7.03 (dd, J = 8.2, 1.9 Hz, 1H), 6.92−6.87 (m,
1H), 6.07−5.98 (m, 1H, N-CH-P), 4.17−3.94 (m, 4H,
2×OCH2CH3), 2.94−2.81 (m, 3H), 2.36 (s, 3H, p-CH3-C6H5),
2.12−2.10 (m, 1H), 1.88 (dd, J = 13.8, 8.0 Hz, 1H), 1.81−1.48 (m,
7H), 1.36 (d, J = 3.2 Hz, 3H, CH3), 1.30−1.28 (m, 3H, CH3), 1.26
(dd, J = 4.4, 3.0 Hz, 6H, 2×CH3), 1.25−1.22 (m, 6H, 2×CH3). 13C
NMR (125 MHz, CDCl3): δ 180.43, 179.46, 146.27, 146.08,
138.11, 134.20, 130.62, 129.44, 128.16, 128.12, 126.93, 124.13,
124.02, 120.01, 63.49, 63.27, 57.05, 55.84, 48.63, 45.35, 37.69,
37.07, 36.93, 33.49, 29.70, 25.08, 23.97, 21.44, 21.20, 18.49,
16.45, 16.02. 31P NMR (200 MHz, CDCl3) δ 19.81. HRMS (m/z)
(ESI): calcd for C33H47N2O4PS [M+H+]: 599.29942; found:
C33H44ClN2O4PS [M+H+]: 619.24479; found: 619.24951; 7k:
Yields 88.65%; 1H NMR (500 MHz, CDCl3) δ 11.74 (s, 1H, NH),
8.66 (s, 1H, NH), 7.43 (dd, J = 4.9, 2.7 Hz, 2H), 7.34−7.27 (m,
2H), 7.18 (d, J = 8.2 Hz, 1H), 7.03 (d, J = 7.8 Hz, 1H), 6.90 (dd, J
= 5.1, 1.5 Hz, 1H), 6.57−6.55 (m, 1H, N-CH-P), 4.23−3.89 (m,
4H, 2×OCH2CH3), 3.05−2.71 (m, 3H), 2.36 (d, J = 13.5 Hz, 1H),
2.13−2.07 (m, 1H), 1.86−1.45 (m, 7H), 1.37 (d, J = 2.4 Hz, 3H,
CH3), 1.35 (dd, J = 7.1, 1.9 Hz, 3H, CH3), 1.25 (t, J = 2.6 Hz, 6H,
2×CH3), 1.24−1.20 (m, 6H, 2×CH3). 13C NMR (125 MHz, CDCl3)
δ 180.82, 179.47, 146.18, 145.96, 145.74, 134.21, 132.43, 129.81,
129.33, 129.03, 127.21, 126.92, 124.13, 124.00, 63.82, 63.16,
54.24, 53.02, 48.64, 45.31, 37.69, 37.07, 36.95, 33.49, 29.72,
29.67, 25.08, 23.98, 21.46, 18.49, 16.45, 16.20.. 31P NMR (200
1
599.30377; 7f: Yields 87%; H NMR (500 MHz, CDCl3): δ 11.60
(s, 1H, NH), 8.76 (s, 1H, NH), 7.29 (dd, J = 5.9, 2.0 Hz, 1H),
7.22−7.15 (m, 2H), 7.04 (dd, J = 10.6, 4.3 Hz, 2H), 6.92 (d, J =
0.9 Hz, 2H), 6.11−6.02 (m, 1H, N-CH-P), 4.18−3.99 (m, 4H,
2×OCH2CH3), 2.94−2.87 (m, 3H), 2.40 (s, 3H, m-CH3-C6H5), 2.28
(dd, J = 12.4, 2.0 Hz, 2H), 2.16 (m, 1H), 1.86−1.75 (m, 7H), 1.39
(d, J = 3.6 Hz, 3H, CH3), 1.35−1.31 (m, 3H, CH3), 1.27 (d, J = 1.2
Hz, 6H, 2×CH3), 1.25 (d, J = 0.9 Hz, 6H, 2×CH3). 13C NMR (125
MHz, CDCl3) δ 180.73, 179.44, 146.26, 146.09, 134.21, 129.96,
129.47, 126.92, 124.47, 124.07, 115.77, 115.63, 115.33, 63.58,
51.69, 50.46, 48.64, 45.33, 37.69, 37.01, 33.49, 29.69, 25.10,
25.03, 23.96, 21.45, 18.49, 16.41, 16.02.31P NMR (200 MHz,
CDCl3) δ 19.41. HRMS (m/z) (ESI): calcd for C33H47N2O4PS
[M+H+]: 599.29942; found: 599.30377; 7g: Yields 83.58%; 1H
NMR (500 MHz, CDCl3) δ 11.66 (s, 1H, NH), 8.65 (s, 1H, NH),
7.48–7.41 (m, 2H), 7.18 (d, J = 8.2 Hz, 1H), 7.09 (d, J = 8.6, 2.6
Hz, 2H), 7.04 (dd, J = 8.2, 1.7 Hz, 1H), 6.93 – 6.89 (m, 1H), 6.06-
5.98 (m, 1H, N-CH-P), 4.16–3.98 (m, 4H), 2.97–2.81 (m, 3H),
2.37 (d, J = 13.5 Hz, 1H), 2.18–2.08 (m, 1H), 1.78-165 (m, 7H),
1.37 (s, 3H, CH3), 1.31 (d, J = 7.0, 3H, CH3), 1.26 (t, J = 4.5 Hz,
6H, 2×CH3), 1.24 (d, J = 4.4 Hz, 6H, 2×CH3). 13C NMR (125
MHz, CDCl3) δ 180.73, 179.63, 163.71, 161.68, 145.89, 133.86,
MHz, CDCl3)
δ 18.86. HRMS (m/z) (ESI): calcd for
C33H44ClN2O4PS [M+H+]: 619.24479; found: 619.25000; 7l:
Yields 89.15%; 1H NMR (500 MHz, CDCl3): δ 11.65 (s, 1H, NH),
8.68 (s, 1H, NH), 7.43 (s, 1H), 7.39−7.30 (m, 3H), 7.18 (d, J = 8.2
Hz, 1H), 7.04 (dd, J = 8.2, 1.8 Hz, 1H), 6.91 (dd, J = 5.4, 1.6 Hz,
1H), 6.08−6.02 (m, 1H, N-CH-P), 4.18−4.03 (m, 4H,
2×OCH2CH3), 3.04−2.76 (m, 3H), 2.37 (d, J = 13.3 Hz, 1H),
2.13−2.10 (m, 1H), 1.86−1.50 (m, 7H), 1.38 (d, J = 3.2 Hz, 3H,
CH3), 1.33−1.31 (m, 3H, CH3), 1.30−1.26 (m, 6H, 2×CH3), 1.24
(dd, J = 6.9, 2.4 Hz, 6H, 2×CH3). 13C NMR (125 MHz, CDCl3) δ
180.96, 179.65, 146.25, 146.21, 135.89, 134.56, 134.15, 129.92,
128.51, 128.27, 126.94, 126.45, 124.17, 124.04, 63.84, 63.42,
56.85, 55.64, 48.70, 45.39, 37.69 , 37.32, 36.77, 33.49, 29.71,
25.09, 23.97, 21.49, 18.48, 16.38, 16.24. 31P NMR (200 MHz,
CDCl3) δ 18.91. HRMS (m/z) (ESI): calcd for C33H44ClN2O4PS
[M+H+]: 619.24479; found: 619.25085; 7m: Yields 90.25%; 1H
NMR (500 MHz, CDCl3): δ 11.47 (s, 1H, NH), 8.67 (s, 1H, NH),
7.52 (dd, J = 8.4, 2.5 Hz, 2H), 7.37−7.32 (m, 2H), 7.18 (d, J = 8.2
Hz, 1H), 7.04 (dd, J = 8.2, 1.9 Hz, 1H), 6.91 (dd, J = 6.2, 1.7 Hz,
1H), 6.05−5.98 (m, 1H, N-CH-P), 4.16−3.97 (m, 4H,
6 / 7

2×OCH2CH3), 3.05−2.73 (m, 3H), 2.37 (d, J = 13.5 Hz, 1H),
2.12−2.08 (m, 1H), 1.63−1.48 (m, 7H), 1.37 (d, J = 3.3 Hz, 3H,
CH3), 1.33−1.29 (m, 3H, CH3), 1.29−1.26 (m, 6H, 2×CH3), 1.24
(dd, J = 6.9, 2.9 Hz, 6H, 2×CH3). 13C NMR (125 MHz, CDCl3) δ
180.86, 179.67, 146.26, 146.13, 134.14, 131.86, 129.92, 129.88,
126.93, 124.18, 124.04, 124.00, 63.63, 63.52, 56.75, 55.54, 48.69,
45.38, 45.33, 37.71, 37.17, 33.49, 29.73, 29.67, 25.08, 23.97,
21.48, 18.48, 16.41, 16.25. 31P NMR (200 MHz, CDCl3) δ 18.93.
HRMS (m/z) (ESI): calcd for C33H44BrN2O4PS [M+H+]:
were exposed to compounds at concentrations from 100 to 0.01
μM for 48 h, medium was aspirated and replenished with complete
medium. IC50 was evaluated by MTT tetrazolium dye assay. Each
experiment was performed three times; (c) Statistical analysis: All
statistical analysis was performed with SPSS Version 10. Data
was analyzed by one-way ANOVA. Mean separations were
performed using the least significant difference method. Each
experiment was replicated thrice, and all experiments yielded
similar results. Measurements from all the replicates were
combined, and treatment effects were analyzed.
1
665.19423; found: 665.19678; 7n: Yields 88.75%; H NMR (500
MHz, CDCl3) δ 11.75 (s, 1H, NH), 8.65(s, 1H, NH), 7.61 (d, J =
8.0 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.38−7.32 (m, 1H),
7.22−7.13 (m, 2H), 6.90 (d, J = 5.0 Hz, 1H), 6.53-6.48 (m, 1H, N-
CH-P), 4.25−3.90 (m, 4H, 2×OCH2CH3), 3.02−2.74 (m, 3H), 2.36
(d, J = 13.2 Hz, 1H), 2.13−2.06 (m, 1H), 1.82−1.47 (m, 7H), 1.37
(d, J = 2.5 Hz, 3H, CH3), 1.35 (dd, J = 7.1, 1.7 Hz, 3H, CH3), 1.25
(t, J = 2.6 Hz, 6H, 2×CH3), 1.24−1.15 (m, 6H, 2×CH3). 13C NMR
(125 MHz, CDCl3) δ 180.81, 179.45, 146.27, 146.08, 134.21,
133.15, 129.56, 129.16, 127.82, 126.92, 124.13, 124.03, 120.00,
63.68, 63.55, 56.69, 55.47, 48.64, 45.32, 37.69, 37.07, 36.95,
33.49, 29.72, 29.67, 25.08 , 23.97, 21.46, 18.49, 16.43, 16.27. 31P
NMR (200 MHz, CDCl3) δ 18.86. HRMS (m/z) (ESI): calcd for
C33H44BrN2O4PS [M+H+]: 665.19423; found: 665.19647; 7o:
Yields 88.45%; 1H NMR (500 MHz, CDCl3): δ 11.67(s, 1H, NH),
8.68 (s, 1H, NH), 7.57−7.54 (m, 1H), 7.45 (d, J = 7.9 Hz, 1H),
7.39 (d, J = 7.8 Hz, 1H), 7.24 (dd, J = 7.8, 2.5 Hz, 1H), 7.16 (d, J
= 8.2 Hz, 1H), 7.01 (dd, J = 8.1, 1.8 Hz, 1H), 6.88 (dd, J = 5.0, 1.6
Hz, 1H), 6.06−6.01 (m, 1H, N-CH-P), 4.16−3.98 (m, 4H,
2×OCH2CH3), 3.01−2.77 (m, 3H), 2.35 (d, J = 13.4 Hz, 1H),
2.16−2.07 (m, 1H), 1.82−1.48 (m, 7H), 1.35 (d, J = 3.1 Hz, 3H,
CH3), 1.31−1.28 (m, 3H, CH3), 1.28−1.24 (m, 6H, 2×CH3), 1.22
(dd, J = 6.9, 2.3 Hz, 6H, 2×CH3). 13C NMR (125 MHz, CDCl3)
180.86, 179.65, 146.24, 146.12, 136.11, 134.15, 131.29, 131.14,
130.44, 130.19, 126.93, 124.15, 124.03, 124.00, 122.67, 63.66,
63.51, 56.76, 55.57, 48.69, 45.41, 45.37, 37.68, 37.08,
36.96,,36.42, 33.49, 29.71, 25.09, 23.97, 21.48, 18.48, 16.33,
16.14. 31P NMR (200 MHz, CDCl3) δ 18.92. HRMS (m/z) (ESI):
calcd for C33H44BrN2O4PS [M+H+]: 665.19423; found: 665.19696;
23. General procedure for AO/EB staining: cells were seeded at a
concentration of 5×104 cell/ mL in a volume of 2 mL on a sterile
cover slip in six-well tissue culture plates. Following incubation,
the medium was removed and replaced with fresh medium plus
10% fetal bovine serum and supplemented with compound 7c (5
μM). After the treatment period, the cover slip with monolayer
cells was inverted on a glass slide with 20 μL of AO/EB stain (100
mg/mL). Fluorescence was read on an Nikon ECLIPSETE2000-S
fluorescence microscope (OLYMPUS Co., Japan).
24. General procedure for Hoechst 33258 staining: cells grown on a
sterile cover slip in six-well tissue culture 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) for 5 min and then again washed twice with PBS. The
stained nuclei were observed under an Nikon ECLIPSETE2000-S
fluorescence microscope using 350 nm excitation and 460 nm
emission.
25. General procedure for mitochondrial membrane potential staining:
JC-1 probe was employed to measure mitochondrial
depolarization in NCI-H460 cells. Briefly, cells cultured in six-
well plates after indicated treatments were incubated with an equal
volume of JC-1 staining solution (5μg/ml) at 37oC for 20 min and
rinsed twice with PBS. Mitochondrial membrane potentials were
monitored by determining the relative amounts of dual emissions
from mitochondrial JC-1 monomers or aggregates using an Nikon
ECLIPSETE2000-S fluorescent microscope. Mitochondrial
depolarization is indicated by an increase in the green/red
fluorescence intensity ratio.
1
7p: Yields 88.85%; H NMR (500 MHz, CDCl3): δ11.86 (s, 1H,
NH), 8.66 (s, 1H, NH), 8.36 (d, J = 8.5 Hz, 1H), 7.87 (dd, J =
13.6, 8.4 Hz, 2H), 7.69 (d, J = 6.9 Hz, 1H), 7.63 (t, J = 7.7 Hz,
1H), 7.55−7.50 (m, 2H), 7.18 (d, J = 8.2 Hz, 1H), 7.03 (d, J = 9.7
Hz, 1H), 7.01−6.93 (m, 1H), 6.90−6.88 (m, 1H, N-CH-P),
4.17−3.68 (m, 4H, 2×OCH2CH3), 3.01−2.80 (m, 3H), 2.36 (d, J =
13.2 Hz, 1H), 2.13 (dd, J = 11.5, 5.4 Hz, 1H), 1.83−1.51 (m, 7H),
1.37 (d, J = 4.0 Hz, 3H, CH3), 1.32 (t, J = 7.0 Hz, 3H, CH3), 1.24
(dd, J = 7.0, 4.6 Hz, 9H, 3×CH3), 1.01 (t, J = 6.8 Hz, 3H, CH3).
13C NMR (125 MHz, CDCl3) δ 180.51, 179.46, 146.26, 146.08,
134.21, 133.81, 130.50, 129.02, 128.72, 126.93, 126.69, 126.08,
126.04, 125.98, 125.32, 124.12, 124.01, 123.75, 63.54, 63.38,
53.38, 52.16, 48.65, 45.36, 37.69, 37.08, 36.94, 33.48, 29.69,
25.08, 23.97, 21.44, 18.49, 16.40, 16.14. 31P NMR (200 MHz,
CDCl3) δ 19.86. HRMS (m/z) (ESI): calcd for C36H47N2O4PS
[M+H+]: 635.29942; found: 635.30341; 7q: Yields 89.42%; 1H
NMR (500 MHz, CDCl3): δ 11.65 (s, 1H, NH), 8.68 (s, 1H, NH),
7.96−7.82 (m, 4H), 7.61 (dd, J = 8.5, 1.4 Hz, 1H), 7.51−7.46 (m,
2H), 7.19 (d, J = 8.2 Hz, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.94−6.85
(m, 1H), 6.30−6.25 (m, 1H, N-CH-P), 4.18−3.96 (m, 4H,
2×OCH2CH3), 2.89−2.81 (m, 3H), 2.37 (d, J = 11.4 Hz, 1H),
2.15−2.10 (m, 1H), 1.85−1.47 (m, 7H), 1.38 (d, J = 3.4 Hz, 3H,
CH3), 1.33−1.30 (m, 3H, CH3), 1.27−1.25 (m, 6H, 2×CH3),
1.25−1.21 (m, 6H, 2×CH3). 13C NMR (125 MHz, CDCl3) 180.67,
179.58, 146.27, 146.10, 134.20, 133.20, 133.12, 128.54, 128.16,
127.82, 127.69, 127.56, 126.94, 126.38, 126.34, 125.79, 124.15,
124.04, 63.61, 63.39, 57.50, 56.30, 48.68, 45.39, 37.70, 37.09,
36.93, 33.49, 29.75, 29.69, 25.09, 23.98, 21.47, 18.50, 16.56,
16.03. 31P NMR (200 MHz, CDCl3) δ 19.57. HRMS (m/z) (ESI):
calcd for C36H47N2O4PS [M+H+]: 635.29942; found: 635.30347.
22. (a) Chen, Z. -F.; Gu, Y. -Q.; Song, X. -Y.; Liu, Y. -C.; Peng, Y.;
Liang, H. Eur. J. Med. Chem. 2013, 59, 194; (b) General
procedure for cytotoxic evaluation in vitro: NCI-H460, A549,
HepG2 and SKOV3 cells were seeded into 96-well microculture
plates and allowed to adhere for 24 h, respectively. After cells
26. (a) General procedure apoptosis ratios: Prepared NCI-H460 cells
(1×106 cells/mL) were washed twice with cold PBS and then
resuspended gently in 500 μL of binding buffer. Thereafter, cells
were stained in 5 μL of Annexin V-FITC and shaken well. Finally,
the cells were mixed with 5 μL of PI, incubated for 20 min in the
dark and subsequently analyzed using FACSCali-bur (Becton
Dickinson).; (b) General procedure cell cycle: NCI-H460 cell lines
were maintained in Dulbecco’s modified Eagle’s medium with
o
10% foetal calf serum in 5% CO2 at 37 C. Cells were harvested
by trypsinization and rinsed with PBS. After centrifugation, the
pellet (105−106 cells) was suspended in 1 mL of PBS and kept on
ice for 5 min. The cell suspensionwas then fixed by the dropwise
addition of 9 mL precooled (4 oC) 75% ethanol with violent
o
shaking. Fixed samples were kept at 4 C until use. For staining,
cells were centrifuged, resuspended in PBS, digested with 500 μL
of RNase A (250 mg/mL), and treated with 25 μL of propidium
iodide (PI) (0.15 mM), then incubated for 30 min at 4 oC. PI-
positive cells were counted with
a FACScan Fluorescence-
activated cell sorter (FACS). The population of cells in each cell-
cycle phase was determined using Cell Modi FIT software
(Becton Dickinson); (c) Tong, G.; Poot, M.; Hu, D.; Oda, D. Oral
Oncology. 2000, 36(2), 236.
27. Wang, P.; Ownby, S.; Zhang, Z.; Yuan, W.; Li, S. Y. Bioorg.
Med. Chem. Lett. 2010, 20, 2790.
28. (a) Mucha, A.; Drag, M.; Dalton, J. P.; Kafarski, P. Biochimie.
2010, 92, 1509; (b) Mucha, A.; Kafarski, P.; Berlicki, Ł. J. Med.
Chem. 2011, 54, 5955;(c) Culcasi, M.; Casano, G.; Lucchesi, C.;
Mercier, A.; Clement, J. L.; Pique, V.; Michelet, L.; Krieger-
Liszkay, A.; Robin, M.; Pietri, S. J. Med. Chem. 2013, 56, 2487.
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