Phosphoramidate protides of five flavones and their antiproliferative activity against HepG2 and L-O2 cell lines

Article abstract of DOI:10.1016/j.ejmech.2016.02.012

A series of flavone-7-phosphoramidate derivatives were synthesized and tested for their antiproliferative activity in vitro against human hepatoma cell line HepG2 and human normal hepatic cell line L-O2. Compound 8d, 16d and 17d, incorporating the amino acid alanine, exhibited high inhibitory activity on HepG2 cell line with IC₅₀ values of 9.0 μmol/L, 5.5 μmol/L and 6.6 μmol/L. The introduction of acyl groups played a pivotal role in the selective inhibition toward human hepatoma HepG2 cells, except for compound 8a, 9a and 16b. Compound 8d, 16d and 17d could significantly induce G2/M arrest in HepG2 cells. Specially, Compound 16d could lead early apoptosis in HepG2 cells.

Full text of DOI:10.1016/j.ejmech.2016.02.012

European Journal of Medicinal Chemistry 112 (2016) 196e208
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Phosphoramidate protides of five flavones and their antiproliferative
activity against HepG2 and L-O2 cell lines
Yue-qing Li, Fei Yang, Liu Wang, Zhi Cao, Tian-jiao Han, Zhe-ang Duan, Zhen Li,
Wei-jie Zhao*
School of Pharmaceutical Science and Technology, Dalian University of Technology, 2 Linggong Road, Dalian 116024, 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 flavone-7-phosphoramidate derivatives were synthesized and tested for their antiproliferative
activity in vitro against human hepatoma cell line HepG2 and human normal hepatic cell line L-O2.
Compound 8d, 16d and 17d, incorporating the amino acid alanine, exhibited high inhibitory activity on
Received 21 October 2015
Received in revised form
21 January 2016
Accepted 4 February 2016
Available online xxx
HepG2 cell line with IC50 values of 9.0 mmol/L, 5.5 mmol/L and 6.6 mmol/L. The introduction of acyl groups
played a pivotal role in the selective inhibition toward human hepatoma HepG2 cells, except for com-
pound 8a, 9a and 16b. Compound 8d, 16d and 17d could significantly induce G2/M arrest in HepG2 cells.
Specially, Compound 16d could lead early apoptosis in HepG2 cells.
Keywords:
Flavone
© 2016 Published by Elsevier Masson SAS.
Phosphoramidate
HepG2
G2/M arrest
Apoptosis
1. Introduction
mechanisms [16]. The potency of the compounds varies with the
individual components (aryl, ester, and amino acid) of the phos-
The study of the flavonoids dated back to the 17th century and
since then, various discoveries have been made regarding their
biological functions [1]. Chrysin, apigenin, luteolin, daidzein and
genistein (Fig. 1), polyphenolic compounds available in foods and
traditional Chinese medicines of plant origin, belong to the flavone
subclass of flavonoids usually occurring as glycosylated forms.
These five flavones demonstrate versatile biological activities, such
as antibacterial activities [2], antioxidant [3], anti-inflammatory [4],
antiviral [5], and antitumor [6e11]. Some reports have been dedi-
cated to the improvement of the anticancer activities and the
structure-activity relationships of these flavones' derivatives
[12,13].
phoramidate moiety. Our interest is focused on the application of
phosphoramidate chemistry to the five flavones and the change in
the antiproliferation in vitro. In this regard, herein we report the
synthesis of a series of amino acid-based flavone phosphor-
amidates. Subsequently, structurally related flavone phosphor-
amidates were evaluated in vitro against human hepatoma cell line
HepG2 and human normal hepatic cell line L-O2.
2. Results and discussion
2.1. Chemistry
A strategy known as ‘phosphoramidate protide’ was introduced
by McGuiganet al. as a means of improving the therapeutic po-
tential of a prototype drug for HIV-1 [14,15]. Traditionally, phos-
phoramidate groups are covalently linked to nucleosides in
‘phosphoramidate protide’. Phosphoramidate chemistry has also
been applied to nucleoside analogues to generate a new class of
anti-cancer agents, which overcome diverse resistance
To expand the structural diversity of title compounds, we pre-
pared
a series of flavone-7-phosphoramidate derivatives by
altering the amino acid and flavone moieties. The structures of
target compounds were confirmed on the basis of NMR and mass
spectrum. The target compounds were prepared using phosphor-
ochloridate chemistry [17,18]. The key reagent to prepare flavone
derivatives is the phenyl aminoacyl phosphorochloridate 2a ~ 2e
(Scheme 1). It was prepared using two reagents: an amino acid
ester and a phenyloxy phosphorodichloridate. The first reagent, an
amino acid ester, can be prepared by esterification of the appro-
priate amino acid via standard esterification methods [19]. Phenyl
* Corresponding author.
E-mail address: zyzhao@dlut.edu.cn (W.-j. Zhao).
http://dx.doi.org/10.1016/j.ejmech.2016.02.012
0223-5234/© 2016 Published by Elsevier Masson SAS.

Y.-q. Li et al. / European Journal of Medicinal Chemistry 112 (2016) 196e208
197
OH
phosphorochloridates 2a ~ 2e under microwave-assistant condi-
tion and turned to be successful (Scheme 3).
OH
OH
HO
O
O
HO
O
O
O
HO
O
O
2.2. Biological studies
OH
OH
OH
Among the screened compounds, compound 8d, 16d and 17d
from acetoxyflavone-7-phosphoramidate series were found to be
active in cell based cytotoxicity screening at less than 10 mM con-
centration. These three compounds were selected for detailed
mechanistic investigations.
Apigenin
Chrysin
HO
Luteolin
HO
O
O
OH
O
OH
OH
Daidzein
Genistein
2.2.1. Compounds inhibit cell proliferation in HepG2 and L-O2 cell
lines
Fig. 1. Structures of five flavones.
Cytotoxicity effects of compounds in HepG2 (hepatocellular
carcinoma) cell lines and L-O2 (human liver) cell lines were
analyzed by using MTT assay. Most of the compounds inhibited
cellular proliferation in HepG2 and L-O2 cell lines. The IC50 values
.HCl
COOCH3
NH2
O
P
NH2
i
ii
H
N
H
C
O
COOCH3
R1
C
H
R1
C
H
COOH
Cl
R1
(
m
M) for 48 h incubation were reported (Table 1). Among the 39
tested derivatives, 28 flavone-7-phosphoramidates showed inhi-
bition of HepG2 cells with IC50 values less than 100 mol/L. The
1a: R1 = PhCH2-
2a: R1 = PhCH2-
1b: R1 = (CH3)2-CH-CH2-
1c: R1 = H
2b: R1 = (CH3)2-CH-CH2-
2c: R1 = H
m
1d: R1 = CH3
2d: R1 = CH3
1e: R1 = (CH3)2-CH-
results revealed that most of the synthetic compounds exhibited
2e: R1 = (CH3)2-CH-
moderate inhibition of cell proliferation. Compound 8d (IC50
Scheme 1. Synthesis of phenyl aminoacyl phosphorochloridates (2aee). Reagents and
conditions: (i) SOCl2, CH3OH, ꢀ10 ꢁC; (ii) phenyl phosphorodichloridate, TEA, anhy-
drous CH2Cl2, ꢀ70 ꢁC.
9.0 1.7
displayed higher potency. Interestingly, these three compounds
were all -alanine phosphoramidates. This is consistent with the
mM)、16d (IC50 5.5 1.3 mM) and 17d (IC50 6.6 1.3 mM)
L
most potent gemcitabine Protides [16]. In addition, the acetyl group
plays an important role to increase the cytotoxicity toward tumor
cells HepG2 (Table 1), except for compounds 8a, 9a and 16b.
phosphorodichloridate is commercially available. The phenyl ami-
noacyl phosphorochloridates were formed by reaction of phenyl
phosphorodichloridates with appropriate amino acid esters in the
presence of triethylamine [17]. As noted in Table 1, we varied the
Especially,
acetylation
for
isoflavone-7-phosphoramidates
improved the selectivity between HepG2 and L-O2 more than six
fold.
amino acid from phenylalanine to leucine, glycine, L-alanine and
valine. Because of their limited stability, the crude product of
compounds 2a ~ 2e was directly used as materials in the ProTide
syntheses.
2.2.2. Compound 8d, 16d and 17d induces G2/M phase cell cycle
arrest in HepG2 cell line
Apigenin, daidzein and genistein were previously shown to
induce G2/M cell cycle arrest in human oesophageal adenocarci-
noma cells [7], breast cancer cells [28e30], colon cancer cells
[31,32], T24 human bladder cancer cells [33], and human malignant
glioma cells [34]. In view of the above-mentioned effects on cell
growth, it was needed to examine whether the flavone-7-
phosphoramidate derivatives 8d, 16d and 17d were able to induce
G2/M phase arrest in HepG2. Treatment of HepG2 cell line with
compound 8d, 16d and 17d at IC50 concentration induced cell cycle
arrest in G2/M phase (Fig. 2). Control cells showed 16.4% cells in G2/
M phase which was increased to 72.6%, 98.1% and 96.6% by the
treatment with compound 8d, 16d and 17d, respectively. Apigenin
could increase T24 human bladder cancer cells in G2/M phase to
Flavone-7-phosphoramidate derivatives were synthesized from
phenyl aminoacyl phosphorochloridate and different kinds of
flavone. The preparation of flavone-7-phosphoramidate derivatives
is illustrated in Scheme 2. The synthetic route for isoflavone-7-
phosphoramidate derivatives is outlined in Scheme 3. In order to
favor the 7-regioselectivity of the phosphorylation with the phenyl
aminoacyl phosphorochloridates, apigenin, luteolin, daidzein and
genistein were acetylated with Ac2O/pyridine and then selectively
deprotected on 7-OH group with PhSH/imidazole [20]. Selectively
deacylation on 7-OH group was optimized in mixture solvents of N-
methyl pyrrolidone/THF (1:3) with short reaction time and good
yield (74.8%e85.0%).
Each aminoacyl phosphorochloridate (2a ~ 2e) was reacted with
the 7-OH group of compounds chrysin, 6, 7, 14 and 15. The phos-
phorochloridates 2a ~ 2e were allowed to react with chrysin or
compound 7 in the presence of TEA to generate the desired com-
pounds 3a ~ 3e and 9a ~ 9e in THF. But to compounds 6, 14 and 15,
the reaction lasted more than 4 days when catalyzed by TEA in THF.
When the reaction system of K2CO3/acetone was used, the phos-
phoramidate derivatives (8a ~ 8e) syntheses were completed
within six hours [21]. The reaction between phosphorochloridates
2a ~ 2e and isoflavone derivatives 14 or 15 was very slow using
K2CO3/acetone. So microwave reaction was used to shorten the
reaction time. The synthetic conditions of the acetoxyflavones-7-
phosphoramidate derivatives were summarized (Table S1).
Finally, the deacylation conditions were screened [22e26] and
pyrrolidine was used to deacetylate [27] (Table S2). While deacy-
lating compounds 10a ~ 10e and 11a ~ 11e using pyrrolidine, the
product became complex and difficult to be purified. Therefore,
37.94% at the concentration of 160 mM and incubated for 24 h, in
contrast with control (14.45%) [33]. Daidzein could cause cell cycle
arrest at G2/M phase (21.8%e38.9%, 22.7% for control) in human
breast cancer MCF-7 at various concentration of 1e100
mM and
incubated for 72 h [30]. Genistein could also induce cell cycle arrest
at G2/M phase (58%, 34% for control) in human malignant glioma
LNT-229 cell line at the concentration of 100 mM and incubated for
24 h [34]. Compared with the literature, daidzein derivative 16d
and genistein derivative 17d almost completely arrested the cell
cycle at G2/M phase. We could infer confidently that the action of
apigenin, daidzein and genistein on cell cycle was increased by the
introduction of phosphoramidate groups dramatically.
2.2.3. Compound 16d leads early apoptosis in HepG2 cell line
The effects on cell growth and cell cycle progression of the
flavone-7-phosphoramidate derivatives 8d, 16d and 17d were
better than that of flavones. So the apoptosis was further measured
by double staining of cells with Annexin-V/PI and quantified by
daidzein
and
genistein
were
tried
to
react
with

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Y.-q. Li et al. / European Journal of Medicinal Chemistry 112 (2016) 196e208
Table 1
IC50(mM) Values of 39 flavones derivatives against HepG2 and L-O2 cell lines and impact of acetoxy group on selective antiproliferation.
Compd.
Substituents
AA
IC50 SD(
HepG2
m
mol/L)a
L-O2/HepG2
Flavone
L-O2
chrysin
3a
3b
3c
3d
3e
apigenin
8a
10a
8a/10a
8b
10b
8b/10b
8c
10c
8c/10c
8d
10d
8d/10d
8e
e
chrysin
chrysin
chrysin
chrysin
chrysin
chrysin
apigenin
40, 5-diacetoxyapigenin
apigenin
e
40, 5-diacetoxyapigenin
apigenin
NAb
NAb
e
Phe
Leu
Gly
Ala
Val
e
NAb
NAb
e
NAb
42.6 3.7
35.3 3.6
21.1 2.6
70.18 2.1
38.5 0.8
36.6 3.8
1.052
22.1 0.5
37.2 1.0
0.594
47.8 1.4
59.7 0.7
0.801
9.0 1.7
34.7 3.2
0.259
18.4 0.8
27.3 3.2
0.674
34.7 5.9
27.1 0.6
15.7 3.3
1.726
21.0 0.3
22.4 2.4
0.938
26.4 2.9
47.7 5.4
0.553
27.3 2.9
16.6 1.5
NAb
60.2 3.8
52.1 3.9
1.155
NAb
e
96.5 4.8
62.3 4.3
NAb
2.265
1.765
>5
NAb
>1.425
1.192
>2.732
<0.436
2.706
1.419
1.907
>2.092
1.198
>1.746
2.467
1.017
2.429
1.590
0.952
1.678
>2.882
1.118
2.490
0.449
1.819
1.581
1.198
2.163
1
Phe
Phe
Phe
Leu
Leu
Leu
Gly
Gly
Gly
Ala
Ala
Ala
Val
Val
Val
e
45.9 2.9
NAb
<0.459
59.8 1.4
52.8 1.5
1.133
e
40, 5-diacetoxyapigenin
apigenin
NAb
71.5 1.0
>1.399
22.2 1.4
35.3 1.0
0.629
e
40, 5-diacetoxyapigenin
apigenin
e
40, 5-diacetoxyapigenin
apigenin
29.4 0.9
26.0 0.7
1.131
10e
8e/10e
luteolin
9a
11a
9a/11a
9b
11b
9b/11b
9c
11c
9c/11c
9d
11e
daidzein
16a
18a
16a/18a
16b
18b
16b/18b
16c
18c
16c/18c
16d
e
luteolin
30, 40, 5-triacetoxyluteolin
luteolin
e
30, 40, 5-triacetoxyluteolin
luteolin
NAb
Phe
Phe
Phe
Leu
Leu
Leu
Gly
Gly
Gly
Ala
Val
e
30.3 2.5
39.1 1.4
0.775
38.2 1.0
34.0 0.1
1.124
57.1 2.6
47.7 1.7
1.197
e
30, 40, 5-triacetoxyluteolin
luteolin
e
2.163
1.875
0.494
e
30, 40, 5-triacetoxyluteolin
luteolin
daidzein
40-acetoxydaidzein
daidzein
e
40-acetoxydaidzein
daidzein
51.2 0.6
8.2 0.2
NAb
Phe
Phe
Phe
Leu
Leu
Leu
Gly
Gly
Gly
Ala
Ala
Ala
e
NAb
>1.660
0.754
>2.203
0.747
0.723
1.033
e
39.3 2.5
>2.545
44.8 0.7
47.7 1.6
0.939
60.0 1.5
66.0 3.4
0.909
e
40-acetoxydaidzein
daidzein
NAb
NAb
48.8 2.0
>2.049
NAb
2.049
e
e
e
40-acetoxydaidzein
daidzein
5.5 1.3
28.8 2.4
0.191
24.3 3.0
20.5 3.0
1.185
4.418
0.712
6.204
e
18d
16d/18d
genistein
17a
19a
17a/19a
17b
19b
17b/19b
17c
19c
17c/19c
17d
e
genistein
40, 5-diacetoxygenistein
genistein
e
40, 5-diacetoxygenistein
genistein
NAb
NAb
Phe
Phe
Phe
Leu
Leu
Leu
Gly
Gly
Gly
Ala
Ala
Ala
e
63.2 2.5
NAb
1.582
e
NAb
NAb
<0.632
e
e
54.0 0.4
72.3 1.6
0.747
60.1 2.0
48.2 0.6
1.247
55.2 1.0
44.5 7.5
1.240
11.9 1.3
15.7 0.4
0.758
1.113
0.667
1.669
1.390
<0.445
>3.123
1.803
0.274
6.591
3.190
e
40, 5-diacetoxygenistein
genistein
39.7 1.1
NAb
e
<0.397
40, 5-diacetoxygenistein
genistein
6.6 1.3
57.2 4.8
0.115
19d
17d/19d
erlotinib
e
e
5.8 0.6
18.5 1.1
The shade indicates the impact of acetoxy group on the selective antiproliferation.
a
The IC50 value represents the concentration of each compound resulting in 50% inhibition in cell growth after 48 h incubation, and was the mean values of three repeated
experiments.
b
NA: Compounds having IC50 value > 100 mM.
flow cytometry to simultaneously differentiate viable, early
apoptotic, late apoptotic and necrotic cells. HepG2 cells were
treated with compound 8d, 16d and 17d at IC50 values and analyzed
by flow cytometry (Fig. 3 and Table S3). Compared with untreated
control, compound 8d and 17d mainly resulted in necrosis. Control
cells showed 6.7% necrosis cells which were increased to 9.88% and
18.62% by the treatment with compound 8d and 17d for 24 h in-
cubation, respectively. But 5.5
early apoptosis cells, compared with the 0.41% early apoptosis cells
for control. Daidzein induced cancer cells apoptosis [30,35]. 80
mM compound 16d resulted in 44.16%
m
M

Y.-q. Li et al. / European Journal of Medicinal Chemistry 112 (2016) 196e208
199
i
2-CH-
O
O
iii
OAc
2
1
3) -CH-, R = OH
Scheme 2. Synthetic method to obtain compounds 3aee, 8aee, 9aee, 10aee and 11aee. Reagents and conditions: (i) phosphorochloridates 2aee, TEA, anhydrous THF, r.t.; (ii) Ac2O,
Pyridine, reflux; (iii) PhSH, imidazole, NMP/THF, ꢀ10 ꢁC; (iv) a: phosphorochloridates 2aee, TEA, anhydrous THF, r.t.; b: phosphorochloridates 2aee, K2CO3, anhydrous acetone, r.t.;
(v) pyrrolidine, THF, r.t.
HO
O
O
HO
O
O
AcO
O
O
i
ii
R2
R2
R2
OH
OAc
OAc
daidzein, R2 = H
12: R2 = H
13: R2 = OAc
14: R2 = H
genistein, R2 = OH
15: R2 = OAc
iii
iii
O
P
O
P
H
C
H
N
H
C
H
N
H3COOC
O
O
O
H3COOC
O
O
R1
O
R1
O
Pyrrolidine, THF
r.t.
R2
O
R2
OAc
OH
16a: R1 = PhCH2-, R2 = H
16b: R1 = (CH3)2-CH-CH2-, R2 = H
16c: R1 = R2 = H
18a: R1 = PhCH2-, R2 = H
18b: R1 = (CH3)2-CH-CH2-, R2 = H
18c: R1 = R2 = H
18d: R1 = CH3, R2 = H
19a: R1 = PhCH2-, R2 = OH
19b: R1 = (CH3)2-CH-CH2-, R2 = OH
19c: R1 = H, R2 = OH
16d: R1 = CH3, R2 = H
17a: R1 = PhCH2-, R2 = OAc
17b: R1 = (CH3)2-CH-CH2-, R2 = OAc
17c: R1 = H, R2 = OAc
19d: R1 = CH3, R2 = OH
17d: R1 = CH3, R2 = OAc
Scheme 3. Synthetic method to obtain compounds 16aee, 17aee, 18aee and 19aee. Reagents and conditions: (i) Ac2O, Pyridine, reflux; (ii) PhSH, imidazole, NMP/THF, ꢀ10 ꢁC; (iii)
phosphorochloridates 2aee, K2CO3, anhydrous acetone, r.t., MW 600w.
daidzein leaded 32.5% BGC-823 cells in apoptosis (1.5% apoptosis
cells for control) [35]. We could conclude that the introduction of
phosphoramidate groups significantly improve the apoptosis in-
duction of compound 16d.
3. Conclusion
We herein report the successful application of ProTide tech-
nology to five flavones. Most of the series of flavone-7-
phosphoramidate derivatives were proved to be more potential
than original flavones in vitro. The studies revealed that derivatives

200
Y.-q. Li et al. / European Journal of Medicinal Chemistry 112 (2016) 196e208
Fig. 2. G2/M phase arrest in HepG2 cells after 24 h incubation with compound 8d, 16d and 17d.
derivatives without acetyl group, except for 8a, 9a and 16b. So the
protecting group on hydroxyl of flavones may be another key point
for structure modification and deserves more attention. Specif-
ically, compound 8d, 16d and 17d could almost thoroughly induce
G2/M phase arrest in HepG2 cells. One daidzein phosphoramidate
derivative in particular, 16d, showed significant induction of early
apoptosis in HepG2 cells. Further, studies related to the mecha-
nisms by which 16d induces early apoptosis in HepG2 cells can be
planned.
4. Experimental section
4.1. Chemistry
All chemicals and reagents used in current study were of
analytical grade. The reactions were monitored by thin layer
chromatography (TLC) on Merck pre-coated silica GF254 plates. 1H
and 13C NMR spectra were recorded on a Bruker AvanceⅡ400M
spectrometer (1H, 400 MHz; 13C, 100 MHz). All NMR spectra were
run at ambient temperature. Chemical shifts of 1H NMR spectra are
given in d values relative to tetramethylsilane (TMS) peak used as
Fig. 3. Induction of apoptosis/necrosis in HepG2 cells after 4 h and 24 h incubation
with compound 8d, 16d and 17d at IC50 value.
with acetyl group had lower IC50 concentration than corresponding

Y.-q. Li et al. / European Journal of Medicinal Chemistry 112 (2016) 196e208
201
the internal reference. High-resolution mass spectra were obtained
2.33 (s, 3H, eOCOCH3).
using TOF-ESI-MS spectrometer. Microwave reactions were con-
ducted using a XH-100B Initiator reactor (Xiang Hao Corp., Beijing,
China). Separation of the compounds by column chromatography
was carried out with silica gel 60.
4.1.3.3. 40, 7- O-diacetyldaidzein (12). Compound 12 was recrys-
tallized from ethyl acetate. Yield 74.9%; White solid. 1H NMR
(400 MHz, CDCl3)
d
8.32 (d, 1H, J ¼ 8.0 Hz, H-5), 8.01 (s, 1H, H-2),
7.58 (d, 2H, J ¼ 8.0 Hz, H-20, H-60), 7.31 (d, 1H, J ¼ 2.0 Hz, H-8),
7.19e7.16 (m, 3H, H-6, H-30, H-50), 2.36 (s, 3H, eOCOCH3), 2.32 (s,
3H, eOCOCH3).
4.1.1. General procedure for synthesis of the amino acid methyl ester
hydrochlorides 1aee
A suspension of L-amino acid (50 mmol) in methanol (50 mL)
was stirred under ice cold conditions. Thionyl chloride (5 mL) was
slowly dropped to the solution at ꢀ5 ꢁC. Then, the mixture was
allowed to slowly warm to room temperature while being stirred.
The reaction was monitored by TLC (N-butanol: water: acetic
acid ¼ 4:1:1). When the reaction was completed, the solvent was
evaporated under reduced pressure to afford the crude product,
which was recrystallized from methanol/ether to give a white solid.
4.1.3.4. 40, 5, 7-O-triacetylgenistein (13). Compound 13 was
recrystallized from ethyl acetate. Yield 98.0%; White solid. 1H NMR
(400 MHz, CDCl3)
d
7.89 (s, 1H, H-2), 7.49 (d, 2H, J ¼ 8.0 Hz, H-20, H-
60), 7.25 (d, 1H, J ¼ 2.0 Hz, H-8), 7.15 (d, 2H, J ¼ 8.0 Hz, H-30, H-50),
6.86 (d, 1H, J ¼ 2.0 Hz, H-6), 2.41 (s, 3H, eOCOCH3), 2.34 (s, 3H,
eOCOCH3), 2.31 (s, 3H, eOCOCH3).
L
-Phenylalanine methyl ester hydrochloride (1a) Yield 98.1%
from -Phenylalanine.
-Leucine methyl ester hydrochloride (1b) Yield 95% from
Leucine.
4.1.4. Deprotection of 7-actyl group
L
Compound 4, or 5, or 12, or 13 (2.5 mmol) was dissolved in N-
methylpyrrolidone (12.5 mL) and tetrahydrofuran (37.5 mL)
at ꢀ10 ꢁC, followed by the addition of imidazole (0.06 g) and thi-
ophenol (0.3 mL). After that, the mixture was allowed to slowly
warm to room temperature, and monitored by TLC. The reaction
solution was evaporated under reduced pressure distillation. Then,
the residue was diluted with ethyl acetate (100 mL) and washed
with 1 M HCl aq (30 mL ꢂ 5). The organic phase was dried over
anhydrous Na2SO4overnight. After filtration and concentration, the
crude was washed with ethanol to afford product 6, 7, 14 and 15.
L
L-
L-
L-
L
-
L-Glycine methyl ester hydrochloride (1c) Yield 98% from
Glycine.
L-Alanine methyl ester hydrochloride (1d) Yield 96.4% from
Alanine.
L-Valine methyl ester hydrochloride (1e) Yield 70.2% from
Valine.
4.1.2. General procedure for synthesis of phosphochloridate 2aꢀe
Amino acid methyl ester hydrochloride (3 mmol) and dichloro-
phenyl phosphate (0.45 mL, 3 mmol) were dissolved in dichloro-
methane (24 mL) and cooled at ꢀ70 ꢁC in a dry ice-acetone bath.
Then, triethylamine (0.9 mL) was slowly dropped to the solution.
After one hour, the mixture was stirred at room temperature until
the reaction was deemed complete by TLC (chloroform:
methanol ¼ 30: 1). The solvent was evaporated under reduced
pressure. The crude product was filtered and washed with ether for
three times. Finally, the filtrate was concentrated to give clear oily
liquid 2aee. The clear oily liquid was used for the next reaction
without separation.
4.1.4.1. 40, 5-diacetylapigenin (6). Yield 74.8%; White solid. 1H NMR
(400 MHz, DMSO-d6)
d
11.13 (s, 1H, 7-OH), 8.09 (d, 2H, J ¼ 8.8 Hz, H-
20, H-60), 7.33 (d, 2H, J ¼ 8.8 Hz, H-30, H-50), 6.94 (d, 1H, J ¼ 2.2 Hz, H-
8), 6.75 (s, 1H, H-3), 6.57 (d, 1H, J ¼ 2.2 Hz, H-6), 2.31 (s, 3H,
eOCOCH3), 2.30 (s, 3H, eOCOCH3).
4.1.4.2. 30, 40, 5-triacetylluteolin (7). Yield 85.0%; White solid. 1H
NMR (400 MHz, DMSO-d6)
d 11.22 (s, 1H, 7-OH), 8.01e7.99 (m, 2H,
H-20, H-60), 7.49e7.47 (m,1H, H-50), 6.96 (d,1H, J ¼ 1.6 Hz, H-8), 6.78
(s, 1H, H-3), 6.58 (d, 1H, J ¼ 1.6 Hz, H-6), 2.33 (s, 3H, eOCOCH3), 2.32
(s, 3H, eOCOCH3), 2.30 (s, 3H, eOCOCH3). 13C NMR (100 MHz,
Phenylalanine phosphochloridate (2a) was purified by column
DMSO-d6) d 175.7, 169.3, 168.6, 168.5, 162.8, 159.9, 158.7, 150.5,
chromatography in the yield of 96.7%. 1H NMR (400 MHz, CDCl3)
d
145.0, 142.9, 129.9, 125.2, 124.9, 122.1, 109.9, 109.3, 108.4, 101.5, 21.4,
20.8, 20.8. HRMS (ESI, m/z) for C21H15O9 ([M ꢀ H]ˉ) Calcd: 411.0716;
Found: 411.0720.
7.38e7.10 (m, 10H, Ph-H), 4.49e4.37 (m, 1H, eNH), 4.24e4.10 (m,
1H, eCH), 3.75 (s, 3H, eOCH3), 3.19e3.10 (m, 2H, eCH2).
4.1.3. Acylation of apigenin, luteolin, daidzein and genistein
To solution of apigenin (3 mmol) in acetic anhydride (6 mL) was
added pyridine (0.6 mL). Then, the mixture was heated to reflux
until apigenin consumption and was poured into ice-cold water
(70 mL) to afford 4 as a white solid. The crude product was
recrystallized to get pure compound. Compounds 5, 12 and 13 were
synthesized according to the method for 4.
4.1.4.3. 40-Acetyldaidzein (14). Yield 80.4%; White solid. 1H NMR
(400 MHz, DMSO-d6) d 10.87 (s, 1H, 7-OH), 8.43 (s, 1H, H-2), 8.00 (d,
1H, J ¼ 8.8 Hz, H-5), 7.60 (d, 2H, J ¼ 8.8 Hz, H-20, H-60), 7.18 (d, 2H,
J ¼ 8.8 Hz, H-30, H-50), 6.95 (dd, 1H, J ¼ 8.8, 2.4 Hz, H-6), 6.89 (d, 1H,
J ¼ 2.4 Hz, H-8), 2.32 (s, 3H, eOCOCH3).
4.1.4.4. 40, 5-diacetylgenistein (15). Yield 77.0%; White solid. 1H
NMR (400 MHz, DMSO-d6)
d 11.17 (s, 1H, 7-OH), 8.35 (s, 1H, H-2),
4.1.3.1. 40, 5, 7- O-triacetylapigenin (4). Compound 4 was recrys-
7.50 (d, 2H, J ¼ 8.4 Hz, H-20, H-60), 7.17 (d, 2H, J ¼ 8.4 Hz, H-30, H-50),
6.81 (d, 1H, J ¼ 2.4 Hz, H-8), 6.59 (d, 1H, J ¼ 2.4 Hz, H-6), 2.29 (s, 3H,
eOCOCH3), 2.28 (s, 3H, eOCOCH3).
tallized from ethyl acetate. Yield 87.2%; White solid. 1H NMR
(400 MHz, CDCl3)
d
7.88 (d, 2H, J ¼ 8.8 Hz, H-20, H-60), 7.35 (d, 1H,
J ¼ 2.0 Hz, H-8), 7.27 (d, 2H, J ¼ 8.8 Hz, H-30, H-50), 6.85 (d, 1H,
J ¼ 2.0 Hz, H-6), 6.62 (s, 1H, H-3), 2.44 (s, 3H, eOCOCH3), 2.35 (s, 3H,
eOCOCH3), 2.34 (s, 3H, eOCOCH3).
4.1.5. Synthesis of chrysin-7-yl phosphoramidate derivatives 3aee
To a solution of chrysin (0.381 g, 1.5 mmol) in THF (60 mL) was
added phosphochloridate 2a-e (2 mmol) and stirred at room
temperature. Then, the mixed solution of TEA (0.5 mL) and THF
(5 mL) was added dropwise to the mixture. The resulting mixture
was stirred at room temperature until complete by TLC. The solvent
was removed under reduced pressure and the crude product was
purified by column chromatography or crystallized from the
appropriate solvent.
4.1.3.2. 30, 40, 5, 7- O-tetraacetylluteolin (5). Compound 5 was
recrystallized from chloroform/methanol. Yield 92.0%; White solid.
1H NMR (400 MHz, CDCl3)
d
7.74 (dd, 1H, J ¼ 8.8, 2.0 Hz, H-60), 7.71
(d, 1H, J ¼ 2.0 Hz, H-20), 7.37 (d, 1H, J ¼ 8.8 Hz, H-50), 7.36 (d, 1H,
J ¼ 2.0 Hz, H-8), 6.85 (d, 1H, J ¼ 2.0 Hz, H-6), 6.61 (s, 1H, H-3), 2.44
(s, 3H, eOCOCH3), 2.35 (s, 3H, eOCOCH3), 2.35 (s, 3H, eOCOCH3),

202
Y.-q. Li et al. / European Journal of Medicinal Chemistry 112 (2016) 196e208
4.1.5.1. ((Phenoxy)(5-hydroxy-2-phenyl-4-oxo-4H-chromene-7-yl)
phosphoryl) phenylalanine methyl ester (3a). The residue was
recrystallized from ethyl acetate/diethyl ether. Yield 51.9%; Yellow
4.1.6. Synthesis of 40,5-diacetylapigenin-7-yl phosphoramidate
derivatives 8aee
Method A: A solution of a compound 6 (0.354 g, 1 mmol) dis-
solved in acetone (60 mL) was added phosphoramidate 2a-
d (2 mmol), anhydrous potassium carbonate (0.27 g, 2 mmol) and
stirred at room temperature. The reaction was monitored by TLC.
After filtration and concentration, the crude product was washed
with ethanol to afford product 8aed. Method B: A solution of a
compound 6 (0.354 g, 1 mmol) dissolved in acetone (60 mL) was
added phosphoramidate 2e (2 mmol), anhydrous potassium car-
bonate (0.27 g, 2 mmol). The mixture was stirred at 25 ꢁC (600 W)
under microwave irradiation for 40 min. After filtration and con-
centration, the crude product was washed with ethanol to afford
product 8e.
solid; mp: 152e153 ꢁC. 1H NMR (400 MHz, Acetone-d6)
d 12.90 (s,
1H, 5-OH), 8.14e8.11 (m, 2H, H-20, H-60), 7.66e7.62 (m, 3H, H-30, H-
40, H-50), 7.40e7.35 (m, 2H, H-300, H-500), 7.23e7.16 (m, 7H, Ph-H),
7.13e7.08 (m, 1H, H-4000), 6.94(2dd, 1H, J ¼ 2.0, 1.0 Hz, H-8), 6.92
(s, 1H, H-3), 6.59 (dd, 1H, J ¼ 2.0, 1.0 Hz, H-6), 5.59e5.50 (m, 1H,
eNH), 4.31e4.27 (m, 1H, eCH), 3.61 (s, 3H, eOCH3), 3.12e2.90 (m,
2H, eCH2). 13C NMR (100 MHz, Acetone-d6)
d 182.8, 172.3, 164.7,
162.0,157.0,156.4,150.8,136.7,132.2,131.1,129.7,129.7,129.4,129.4,
129.2, 129.2, 128.2, 128.2, 126.6, 126.6, 126.6, 125.0, 120.3, 120.2,
107.9, 105.7, 103.5, 99.2, 56.6, 51.5, 39.7. HRMS (EI, m/z) for
C
31H26NO8P([MþH]þ) Calcd: 571.1396; Found: 571.1384.
4.1.6.1. ((Phenoxy)(5-acetoxy-2-(4-acetoxyphenyl)-4-oxo-4H-chro-
mene-7-yl)phosphoryl) phenylalanine methyl ester (8a). Yield
73.2%; White solid; mp: 139e140 ꢁC. 1H NMR (400 MHz, Acetone-
4.1.5.2. ((Phenoxy)(5-hydroxy-2-phenyl-4-oxo-4H-chromene-7-yl)
phosphoryl)) leucine methyl ester (3b). The residue was recrystal-
lized from ethanol. Yield 41.7%; Yellow solid; mp: 146e147 ꢁC. 1H
d6)
d
8.13 (d, 2H, J ¼ 8.6 Hz, H-20, H-60), 7.40e7.35 (m, 5H, Ph-H),
NMR (400 MHz, Acetone-d6)
d 12.96 (s, 1H, 5-OH), 8.16e8.13 (m,
7.23e7.16 (m, 7H, Ph-H), 7.12e7.08 (m, 1H, H-400), 6.91 (dd, 1H,
J ¼ 2.8, 0.6 Hz, H-8), 6.74 (d, 1H, J ¼ 2.8 Hz, H-6), 5.65e5.57 (m, 1H,
eNH), 4.37e4.27 (m, 1H, eCH), 3.62 (s, 3H, eOCH3), 3.13e3.07 (m,
2H, eCH2), 2.34 (s, 3H, eOCOCH3), 2.31 (s, 3H, eOCOCH3). 13C NMR
2H, H-20, H-60), 7.67e7.62 (m, 3H, H-30, H-40, H-50), 7.46e7.42 (m,
2H, H-300, H-500), 7.38e7.34 (m, 2H, H-200, H-600), 7.26 (t, 1H,
J ¼ 7.2 Hz, H-400), 7.15 (dd, 1H, J ¼ 2.0, 1.0 Hz, H-8), 6.95 (s, 1H, H-3),
6.75 (dd, 1H, J ¼ 2.0, 1.0 Hz, H-6), 5.53e5.45 (m, 1H, eNH),
4.14e4.05 (m, 1H, eCHeNH), 3.63 (s, 3H, eOCH3), 1.74e1.62 (m, 1H,
eCH), 1.60e1.52 (m, 2H, eCH2), 0.89e0.85 (m, 6H, eCH(CH3)2).
(100 MHz, Acetone-d6) d 175.3,172.2,168.5,161.5,161.4,157.7, 154.3,
153.7, 150.8, 150.5, 136.6, 129.7, 129.4, 129.4, 128.6, 128.2, 128.2,
127.7, 127.7, 126.6,125.1, 122.6,122.6,120.3, 120.2, 114.3,112.3, 107.9,
106.9, 78.3, 56.6, 51.5, 39.6, 20.2, 20.1.HRMS (EI, m/z) for
4.1.5.3. ((Phenoxy)(5-hydroxy-2-phenyl-4-oxo-4H-chromene-7-yl)
phosphoryl)) glycine methyl ester (3c). The residue was recrystal-
lized from ethanol. Yield 61.9%; Yellow solid; mp: 160e161 ꢁC. 1H
C
35H30NO11P ([MþH]þ) Calcd: 671.1556; Found: 671.1605.
4.1.6.2. ((Phenoxy)(5-acetoxy-2-(4-acetoxyphenyl)-4-oxo-4H-chro-
NMR (400 MHz, Acetone-d6)
d 12.93 (s, 1H, 5-OH), 8.13e8.10 (m,
mene-7-yl)phosphoryl) leucine methyl ester (8b). Yield 62.9%; White
2H, H-20, H-60), 7.67e7.59 (m, 3H, H-30, H-40, H-50), 7.42 (t, 2H,
J ¼ 8.0 Hz, H-300, H-500), 7.37e7.34 (m, 2H, H-200, H-600), 7.22 (t, 1H,
J ¼ 8.0 Hz, H-400), 7.15e7.14 (m, 1H, H-8), 6.92 (s, 1H, H-3), 6.75e6.74
(m, 1H, H-6), 5.47e5.40 (m, 1H, eNH), 3.96 (dd, 2H, J ¼ 13.2, 7.0 Hz,
eCH2), 3.66 (s, 3H, eOCH3).
solid; mp: 149e150 ꢁC. 1H NMR (400 MHz, Acetone-d6)
d 8.14e8.11
(m, 2H, H-20, H-60), 7.60 (dd, 1H, J ¼ 2.4, 1.0 Hz, H-8), 7.43e7.40 (m,
2H, H-300, H-500), 7.38e7.34 (m, 2H, H-30, H-50), 7.34e7.31 (m, 2H, H-
200, H-600), 7.26e7.22 (m, 1H, H-400), 7.06 (dd, 1H, J ¼ 2.4, 1.0 Hz, H-6),
6.74 (s, 1H, H-3), 5.52e5.46 (m, 1H, eNH), 4.13e4.05 (m, 1H,
eCHeNH), 3.61 (s, 3H, eOCH3), 2.34 (s, 3H, eOCOCH3), 2.31 (s, 3H,
eOCOCH3), 1.71e1.61 (m, 1H, (CH3)2eCHeCH2), 1.57e1.52 (m, 2H,
eCH2), 0.88e0.83 (m, 6H, (CH3)2eCH). 13C NMR (100 MHz,
4.1.5.4. ((Phenoxy)(5-hydroxy-2-phenyl-4-oxo-4H-chromene-7-yl)
phosphoryl)) alanine methyl ester (3d). The residue was purified by
column chromatography using chloroform/methanol (130: 1) as an
eluent. Yield 41.8%; Pale yellow solid; mp: 134e135 ꢁC. 1H NMR
Acetone-d6)
d 176.2, 174.3, 169.4, 169.4, 162.4, 158.8, 155.5, 154.7,
151.7,151.6,130.6,130.6,129.5,128.6,128.6,126.1,123.5,123.5,121.3,
121.3, 115.0, 113.4, 108.9, 108.1, 54.3, 52.4, 43.6, 24.9, 23.1, 21.6, 21.1,
21.0. HRMS (EI, m/z) for C32H32NO11P ([MþH]þ) Calcd:637.1713;
Found:637.1741.
(400 MHz, DMSO-d6)
d
12.89 (s, 1H, 5-OH), 8.13 (d, 2H, J ¼ 8.4 Hz, H-
20, H-60), 7.67e7.58 (m, 3H, Ph-H), 7.46e7.41 (m, 2H, Ph-H),
7.32e7.22 (m, 3H, Ph-H), 7.16 (s, 1H, H-3), 7.11e7.10 (m, 1H, H-8),
6.77e6.75 (m, 1H, H-6), 5.59e5.37 (m, 1H, eNH), 4.07e3.98 (m, 1H,
eCH), 3.57 (s, 3H, eOCH3), 1.26e1.23 (m, 3H, eCH3). 13C NMR
4.1.6.3. ((Phenoxy)(5-acetoxy-2-(4-acetoxyphenyl)-4-oxo-4H-chro-
(100 MHz, Acetone-d6) d 182.7, 173.1, 164.7, 162.1, 161.8, 157.1, 156.6,
150.9, 132.2, 131.1, 129.7, 129.7, 129.2, 129.2, 126.6, 126.6, 125.1,
mene-7-yl)phosphoryl) glycine methyl ester (8c). Yield 60.6%; White
solid; mp: 143e144 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d 8.15 (d, 2H,
J ¼ 8.0 Hz, H-20, H-60), 7.64 (dd, 1H, J ¼ 2.2, 0.8 Hz, H-8), 7.42 (t, 2H,
J ¼ 8.0 Hz, H-300, H-500), 7.37 (d, 2H, J ¼ 8.0 Hz, H-30, H-50), 7.27 (d, 2H,
J ¼ 8.0 Hz, H-200, H-600), 7.24 (t, 1H, J ¼ 8.0 Hz, H-400), 7.10 (d, 1H,
J ¼ 2.0 Hz, H-6), 6.92 (s, 1H, H-3), 6.70e6.65 (m, 1H, eNH), 3.85 (dd,
2H, J ¼ 13.2, 7.0 Hz, -eCH2), 3.60 (s, 3H, eOCH3), 2.33 (s, 3H,
eOCOCH3), 2.31 (s, 3H, eOCOCH3). 13C NMR (100 MHz, DMSO-d6)
120.3, 107.4, 105.7, 103.6, 99.4, 51.5, 50.5, 23.7. HRMS (ESI, m/z) for
C
25H22NO8NaP ([MþNa]þ) Calcd: 518.4076; Found: 518.1467.
4.1.5.5. ((Phenoxy)(5-hydroxy-2-phenyl-4-oxo-4H-chromene-7-yl)
phosphoryl)) valine methyl ester (3e). The residue was purified by
column chromatography using chloroform/methanol (130: 1) as an
eluent. Yield 41.8%; Pale yellow solid; mp: 136e137 ꢁC. 1H NMR
d
175.8, 171.3, 169.4, 169.2, 161.5, 157.8, 154.4, 153.7, 150.6, 150.2,
(400 MHz, DMSO-d6)
d
12.86 (s, 1H, 5-OH), 8.12 (d, 2H, J ¼ 8.0 Hz, H-
130.4, 130.4, 128.5, 128.4, 128.4, 125.7, 123.2, 123.2, 120.8, 120.7,
20, H-60), 7.62e7.57 (m, 3H, Ph-H), 7.43e7.39 (m, 2H, Ph-H),
7.29e7.20 (m, 3H, Ph-H), 7.14 (s, 1H, H-3), 7.12 (dd, 1H, J ¼ 8.0,
0.8 Hz, H-8), 6.71 (d, 1H, J ¼ 8.0 Hz, H-6), 6.71e6.57 (m, 1H, eNH),
3.68e3.63 (m, 1H, -CH-NH), 3.54 (s, 3H, eOCH3), 1.96e1.89 (m, 1H,
eCH), 0.82e0.76 (m, 3H, eCH(CH3)2). 13C NMR (100 MHz, DMSO-
114.1, 112.9, 108.4, 107.9, 52.3, 42.9, 21.4, 21.3. HRMS (EI, m/z) for
C
28H24NO11P([MþH]þ) Calcd:581.1087; Found:581.1071.
4.1.6.4. ((Phenoxy)(5-acetoxy-2-(4-acetoxyphenyl)-4-oxo-4H-chro-
mene-7-yl)phosphoryl) alanine methyl ester (8d). Yield 54.4%;
d6)
d
182.9, 172.9, 164.6, 161.6, 157.0, 156.4, 150.7, 132.9, 130.8, 130.3,
White solid; mp: 144e145 ꢁC. 1H NMR (400 MHz, Acetone-d6)
d 8.14
130.3, 129.7, 129.7, 127.1, 127.1, 125.6, 120.7, 120.6, 107.9, 106.2, 103.9,
99.9, 60.9, 52.1, 31.7, 19.3, 18.4. HRMS (ESI, m/z) for C27H26NO8NaP
([MþNa]þ) Calcd:546.1294; Found:546.1287.
(d, 2H, J ¼ 8.0 Hz, H-20, H-60), 7.62e7.56 (m, 1H, H-8), 7.42 (t, 2H,
J ¼ 8.0 Hz, H-300, H-500), 7.33 (d, 2H, J ¼ 8.0 Hz, H-30, H-50), 7.32 (d,
2H, J ¼ 8.0 Hz, H-200, H-600), 7.23 (t, 1H, J ¼ 8.0 Hz, H-400), 7.09e7.04

Y.-q. Li et al. / European Journal of Medicinal Chemistry 112 (2016) 196e208
203
(m, 1H, H-6), 6.75 (s, 1H, H-3), 5.62e5.56 (m, 1H, eNH), 4.22e4.16
(m, 1H, eCH), 3.63 (s, 3H, eOCH3), 2.34 (s, 3H, eOCOCH3), 2.32 (s,
3H, eOCOCH3), 1.36 (d, 3H, J ¼ 7.0 Hz, eCH3). 13C NMR (100 MHz,
Acetone-d6)
d
8.02 (dd, 1H, J ¼ 8.4, 2.2 Hz, H-60), 7.99 (d, 1H,
J ¼ 2.2 Hz, H-20), 7.62 (dd, 1H, J ¼ 2.2, 1.0 Hz, H-8), 7.48 (d, 1H,
J ¼ 8.4 Hz, H-50), 7.41 (t, 2H, J ¼ 8.0 Hz, H-300, H-500), 7.34 (d, 2H,
J ¼ 8.0 Hz, H-200, H-600), 7.24 (t, 1H, J ¼ 8.0 Hz, H-400), 7.07 (d, 1H,
J ¼ 2.2 Hz, H-6), 6.77 (s, 1H, H-3), 5.51e5.48 (m, 1H, eNH), 3.95 (dd,
2H, J ¼ 13.4, 7.0 Hz, eCH2), 3.65 (s, 3H, eOCH3), 2.34 (s, 3H,
eOCOCH3), 2.33 (s, 3H, eOCOCH3), 2.32 (s, 3H, eOCOCH3). 13C NMR
Acetone-d6)
d 175.3, 173.1, 168.5, 168.5, 161.5, 157.9, 154.5, 153.7,
150.7, 150.6, 129.7, 129.7, 128.6, 127.7, 127.7, 125.2, 122.6, 122.6,
120.4, 120.3, 114.4, 112.5, 107.9, 107.3, 51.6, 50.4, 50.3, 20.2, 19.6.
HRMS (EI, m/z) for
Found:595.1262.
C
29H26NO11P([MþH]þ) Calcd:595.1243;
(100 MHz, DMSO-d6) d 182.1, 175.8, 171.3, 169.2, 168.7, 164.7, 160.6,
157.8, 154.5, 150.2, 145.3, 143.0, 130.4, 129.7, 125.7, 121.9, 120.7,
120.5, 119.4, 115.7, 114.2, 113.0, 108.8, 104.1, 94.3, 52.3, 43.0, 22.7,
4.1.6.5. ((Phenoxy)(5-acetoxy-2-(4-acetoxyphenyl)-4-oxo-4H-chro-
mene-7-yl)phosphoryl) valine methyl ester (8e). Yield 63.2%; White
21.3, 20.8. HRMS (ESI, m/z) for
Calcd:662.1039; Found:662.1027.
C30H26NO13NaP ([MþNa]þ)
solid; mp: 157e158 ꢁC. 1H NMR (400 MHz, Acetone-d6)
d
8.13 (dd,
2H, J ¼ 8.0, 1.6 Hz, H-20, H-60), 7.59 (dd, 1H, J ¼ 2.2, 0.9 Hz, H-8),
7.44e7.40 (m, 6H, Ph-H), 7.24 (t, 1H, J ¼ 8.0 Hz, H-400), 7.06 (d, 1H,
J ¼ 2.2 Hz, H-6), 6.75 (s, 1H, H-3), 5.53e5.45 (m, 1H, eNH),
3.93e3.84 (m, 1H, eCHeNH), 3.62 (s, 3H, eOCH3), 2.34 (s, 3H,
eOCOCH3), 2.32 (s, 3H, eOCOCH3), 2.09e2.04 (m, 1H, eCH),
0.92e0.88 (m, 6H, (CH3)2eCH). 13C NMR (100 MHz, Acetone-d6)
4.1.9.1. (((Phenoxy)(5-acetoxy-2-(3,4-diacetoxyphenyl)-4-oxo-4H-
chromene-7-yl)phosphoryl) alanine methyl ester (9d). Yield 44.6%;
White solid; mp: 109e110 ꢁC. 1H NMR (400 MHz, Acetone-d6)
d
8.02 (dd,1H, J ¼ 8.4, 2.0 Hz, H-60), 7.98 (d,1H, J ¼ 2.0 Hz, H-20), 7.62
(d, 1H, J ¼ 2.0 Hz, H-8), 7.48 (d, 1H, J ¼ 8.0 Hz, H-50), 7.41 (t, 2H,
J ¼ 8.0 Hz, H-300, H-500), 7.34 (d, 2H, J ¼ 8.0 Hz, H-200, H-600), 7.24 (t,
1H, J ¼ 8.0 Hz, H-400), 7.08 (d, 1H, J ¼ 2.0 Hz, H-6), 6.77 (s, 1H, H-3),
5.60e5.55 (m, 1H, eNH), 4.24e4.13 (m, 1H, -eCH), 3.63 (s, 3H,
eOCH3), 2.34 (s, 3H, eOCOCH3), 2.33 (s, 3H, eOCOCH3), 2.32 (s, 3H,
eOCOCH3), 1.37 (d, 3H, J ¼ 7.0 Hz, eCH3). 13C NMR (100 MHz,
d
175.3, 171.1, 168.5, 168.5, 161.5, 157.8, 154.6, 153.7, 153.1, 150.6,
129.7, 128.6, 127.7, 127.7, 125.2, 122.6, 122.6, 120.4, 120.3, 112.5,
112.4, 108.0, 107.2, 107.1, 60.6, 51.3, 31.8, 20.2, 18.4, 17.3, 17.1. HRMS
(ESI, m/z) for C31H30NO11NaP ([MþNa]þ) Calcd: 646.5335; Found:
646.2009.
DMSO-d6) d 182.1, 174.8, 172.5, 169.5, 168.7, 164.9, 164.3, 161.5, 157.7,
4.1.7. Synthesis of 30, 40, 5-triacetylluteolin -7-yl phosphoramidate
derivatives 9aee
Compound 9aee was prepared from 7 according to the same
procedure described for 3a.
150.3, 146.3, 130.4, 129.8, 129.4, 129.2, 121.9, 120.7, 119.3, 116.5,
115.7, 113.9, 104.0, 103.2, 99.3, 94.4, 53.2, 50.3, 47.9, 45.8, 22.8, 17.7.
HRMS (ESI, m/z) for C31H28NO13NaP ([MþNa]þ) Calcd: 676.1196;
Found: 676.1207.
4.1.7.1. ((Phenoxy)(5-acetoxy-2-(3,4-diacetoxyphenyl)-4-oxo-4H-
chromene-7-yl)phosphoryl) phenylalanine methyl ester (9a).
Yield 88.0%; White solid; mp:145e146 ꢁC. 1H NMR (400 MHz,
4.1.10. ((Phenoxy)(5-acetoxy-2-(3,4-diacetoxyphenyl)-4-oxo-4H-
chromene-7-yl)phosphoryl) valine methyl ester (9e)
Yield 65.9%; White solid; 1H NMR (400 MHz, Acetone-d6)
d 8.01
Acetone-d6)
d
8.02 (dd, 1H, J ¼ 8.4, 2.2 Hz, H-60), 7.98 (d, 1H,
(dd,1H, J ¼ 8.6,1.8 Hz, H-60), 7.98 (d,1H, J ¼ 1.8 Hz, H-20), 7.60 (d,1H,
J ¼ 2.2 Hz, H-8), 7.48 (d, 1H, J ¼ 8.6 Hz, H-50), 7.41 (t, 2H, J ¼ 8.0 Hz,
H-300, H-500), 7.33 (d, 2H, J ¼ 8.0 Hz, H-200, H-600), 7.23 (t, 1H,
J ¼ 8.0 Hz, H-400), 7.06 (d, 1H, J ¼ 2.2 Hz, H-6), 6.77 (s, 1H, H-3),
5.54e5.46 (m, 1H, eNH), 3.93e3.83 (m, 1H, eCH-eNH), 3.62 (s, 3H,
-eOCH3), 2.34 (s, 3H, eOCOCH3), 2.33 (s, 3H, eOCOCH3), 2.32 (s, 3H,
eOCOCH3), 2.10e2.06 (m, 1H, eCH), 0.92e0.88 (m, 6H, (CH3)2eCH).
J ¼ 2.2 Hz, H-20), 7.49 (d, 1H, J ¼ 8.4 Hz, H-50), 7.39e7.35 (m, 3H, Ph-
H), 7.22e7.16 (m, 7H, Ph-H), 7.11e7.07 (m, 1H, H-400), 6.90 (d, 1H,
J ¼ 2.4 Hz, H-6), 6.79e6.77 (m, 1H, H-3), 5.65e5.57 (m, 1H, eNH),
4.34e4.25 (m, 1H, eCH), 3.62 (s, 3H, eOCH3), 3.12e2.90 (m, 2H,
eCH2), 2.33 (s, 3H, eOCOCH3), 2.32 (s, 3H, eOCOCH3), 2.30 (s, 3H,
eOCOCH3). 13C NMR (100 MHz, Acetone-d6)
d 175.7, 172.9, 169.2,
168.7, 168.5, 160.5, 157.6, 150.6, 150.5, 145.3, 143.0, 137.2, 130.3,
130.3, 129.7, 129.7, 129.5, 129.4, 128.5, 128.5, 127.7, 125.6, 125.4,
125.1, 122.3, 120.5, 120.4, 114.0, 113.5, 108.8, 107.5, 56.8, 56.5, 52.4,
21.3, 20.9, 20.8. HRMS (ESI, m/z) for C37H32NO13NaP ([MþNa]þ)
Calcd: 752.1509; Found: 752.0060.
13C NMR (100 MHz, Acetone-d6)
d 175.2, 172.4, 168.4, 167.8, 167.6,
160.6, 157.8, 154.6, 154.6, 150.6, 145.3, 143.2, 129.7, 129.7, 129.6,
125.2, 124.6, 124.4, 121.8, 120.4, 120.4, 114.1, 112.6, 108.4, 107.2, 60.6,
51.3, 31.9, 20.2, 19.6, 19.6, 18.4, 17.1. HRMS (ESI, m/z) for
C
33H32NO13NaP ([MþNa]þ) Calcd: 704.1509; Found: 704.1519.
4.1.8. ((Phenoxy)(5-acetoxy-2-(3,4-diacetoxyphenyl)-4-oxo-4H-
chromene-7-yl)phosphoryl) leucine methyl ester (9b)
4.1.11. Synthesis of apigenin-7-yl phosphoramidate derivatives
10aee
Yield 86.0%; White solid; mp: 121e122 ꢁC. 1H NMR (400 MHz,
To a solution of 8aee (0.05 mmol) in tetrahydrofuran (30 mL)
was added dropwise the mixed solution of pyrrolidine (0.4 mL) and
tetrahydrofuran (3 mL). Then, the mixture was stirred at room
temperature until the reaction was deemed complete by TLC. The
solution was neutralized with 0.5 M HCl and the solvent was
removed under reduced pressure. The residue was diluted in ethyl
acetate (120 mL) and washed with water (40 mL ꢂ 4). The organic
layer was dried over anhydrous Na2SO4 and concentrated to give a
crude product which was purified by column chromatography or
crystallized from the appropriate solvent to give 10aee.
Acetone-d6)
d
8.02 (dd, 1H, J ¼ 8.4, 1.8 Hz, H-60), 7.98 (d, 1H,
J ¼ 1.8 Hz, H-20), 7.60 (d, 1H, J ¼ 1.8 Hz, H-8), 7.48 (d, 1H, J ¼ 8.4 Hz,
H-50), 7.41 (t, 2H, J ¼ 8.0 Hz, H-300, H-500), 7.33 (t, 2H, J ¼ 8.0 Hz, H-200,
H-600), 7.24 (t, 1H, J ¼ 8.0 Hz, H-400), 7.06 (d, 1H, J ¼ 1.8 Hz, H-6), 6.77
(s, 1H, H-3), 5.59e5.50 (m, 1H, -NH), 4.13e4.05 (m, 1H, eCHeNH),
3.60 (s, 3H, eOCH3), 2.34 (s, 3H,eOCOCH3), 2.33 (s, 3H, eOCOCH3),
2.32 (s, 3H, eOCOCH3), 1.72e1.60 (m, 1H, eCH), 1.58e1.53 (m, 2H,
eCH2), 0.87e0.83 (m, 6H, eCH(CH3)2). 13C NMR (100 MHz, DMSO-
d6)
d 182.5, 175.7, 173.8, 169.2, 168.7, 164.7, 161.9, 157.8, 154.4, 150.7,
150.2,146.3,145.3,130.3,125.7, 122.3,120.7, 119.8,116.5,115.7,113.8,
108.8, 103.8, 99.3, 94.3, 53.4, 52.3, 42.3, 24.1, 23.1, 22.7, 22.4, 21.3,
4.1.11.1. ((Phenoxy)(5-hydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chro-
20.8. HRMS (ESI, m/z) for
C
34H34NO13NaP ([MþNa]þ)
mene-7-yl)phosphoryl)
phenylalanine
methyl
ester
(10a).
Calcd:718.1665; Found:718.1659.
Compound 10a was purified by column chromatography using
chloroform/methanol (40:1) as an eluent. Yield 80.5%; Yellow solid;
4.1.9. ((Phenoxy)(5-acetoxy-2-(3,4-diacetoxyphenyl)-4-oxo-4H-
chromene-7-yl)phosphoryl) glycine methyl ester (9c)
mp: 104e105 ꢁC$1H NMR (400 MHz, DMSO-d6)
d 13.02 (s, 1H, 5-
OH), 10.46 (s, 1H, 40-OH), 8.00 (dd, 2H, J ¼ 8.0, 1.7 Hz, H-20, H-60),
Yield 68.5%; White solid; mp: 133e134 ꢁC. 1H NMR (400 MHz,
7.36 (t, 2H, J ¼ 8.0 Hz, H-300, H-500), 7.22e7.13 (m, 5H, Ph-H), 7.09 (d,

204
Y.-q. Li et al. / European Journal of Medicinal Chemistry 112 (2016) 196e208
2H, J ¼ 8.0 Hz, H-200, H-600), 7.08 (t, 1H, J ¼ 8.0 Hz, H-400), 6.96 (s, 1H,
H-3), 6.95 (d, 2H, J ¼ 8.0 Hz, H-30, H-50), 6.85e6.84 (m, 1H, H-8),
6.48 (d, 1H, J ¼ 2.0 Hz, H-6), 4.10e4.04 (m, 1H, eCH), 3.54 (s, 3H,
eOCH3), 2.98e2.75 (m, 2H, eCH2). 13C NMR (100 MHz, DMSO-d6)
J ¼ 2.0 Hz, H-8), 6.97 (s, 1H, H-3), 6.94 (d, 2H, J ¼ 8.0 Hz, H-30, H-50),
6.69 (d, 1H, J ¼ 2.0 Hz, H-6), 6.69e6.60 (m, 1H, eNH), 3.70e3.61 (m,
1H, eCHeNH), 3.55 (s, 3H, eOCH3), 1.96e1.89 (m, 1H, eCH),
0.82e0.76 (m, 6H, (CH3)2eCH). 13C NMR (100 MHz, DMSO-d6)
d
182.6, 172.9, 165.2, 162.0, 161.4, 156.7, 155.9, 150.6, 137.2, 130.3,
d 182.6, 175.9, 172.9, 165.2, 162.0, 161.5, 156.8, 156.1, 130.3, 130.3,
130.3, 129.7, 129.7, 129.3, 128.5, 128.5, 126.8, 125.5, 121.2, 120.5,
120.4, 116.5, 116.5, 107.7, 103.8, 103.5, 99.4, 79.6, 56.8, 52.3, 31.2.
129.3,129.3,125.5,121.2,120.7,120.6,116.5,116.5,107.7,103.8,103.7,
99.8, 60.9, 52.1, 49.1, 18.4, 18.3. HRMS (ESI, m/z) for C25H21NO9P
([M ꢀ H]ˉ) Calcd:538.1267; Found:538.1273.
HRMS (EI, m/z) for
C
31H26NO9P([MþH]þ) Calcd: 587.1345;
Found:587.1353.
4.1.16. Synthesis of luteolin-7-yl phosphoramidate derivatives
11aee
Compound 11aee was prepared from 9aee according to the
same procedure described for 10ae11e.
4.1.12. ((Phenoxy)(5-hydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-
chromene-7-yl)phosphoryl) leucine methyl ester (10b)
Compound 10b was purified by column chromatography using
chloroform/methanol (40:1) as an eluent. Yield 85.5%; Yellow solid;
mp: 130e131 ꢁC$1H NMR (400 MHz, DMSO-d6)
d
13.05 (s, 1H, 5-
4.1.16.1. ((Phenoxy)(5-hydroxy-2-(3,4-dihydroxyphenyl)-4-oxo-4H-
chromene-7-yl)phosphoryl) phenylalanine methyl ester (11a).
Compound 11a was purified by preparative TLC. Yield 16.0%; Pale
OH), 10.44 (s, 1H, 40-OH), 8.00 (dd, 2H, J ¼ 8.8, 2.0 Hz, H-20, H-60),
7.45e7.40 (m, 2H, H-300, H-500), 7.29e7.22 (m, 3H, H-200, H-400, H-600),
7.07 (d, 1H, J ¼ 1.6 Hz, H-8), 6.96 (s, 1H, H-3), 6.95 (d, 2H, J ¼ 8.8 Hz,
H-30, H-50), 6.68 (d, 1H, J ¼ 1.6 Hz, H-6), 6.65e6.62 (m, 1H, eNH),
3.91e3.84 (m, 1H, -eCH), 3.53 (s, 3H, eOCH3), 1.89e1.74 (m, 1H,
(CH3)2eCHeCH2), 1.53e1.38 (m, 2H, eCH2), 0.80e0.72 (m, 6H,
yellow solid; mp: 181e182 ꢁC$1H NMR (400 MHz, DMSO-d6)
d 13.01
(s, 1H, 5-OH), 10.01 (s, 1H, 40-OH), 9.45 (s, 1H, 30-OH), 7.48 (dd, 1H,
J ¼ 8.4, 1.6 Hz, H-60), 7.45 (d, 1H, J ¼ 1.6 Hz, H-20), 7.37 (t, 2H,
J ¼ 8.0 Hz, H-300, H-500), 7.22e7.06 (m, 8H, Ph-H), 6.90 (d, 1H,
J ¼ 8.0 Hz, H-50), 6.84e6.80 (m, 2H, Ph-H), 6.49 (d, 1H, J ¼ 1.6 Hz, H-
6), 4.10e4.04 (m, 1H, eCH), 3.54 (s, 3H, eOCH3), 2.97e2.74 (m, 2H,
(CH3)2eCH). 13C NMR (100 MHz, DMSO-d6)
d 182.6, 173.7, 165.2,
162.0, 161.6, 156.9, 156.1, 150.7, 130.4, 130.3, 129.3, 129.3, 125.6,
121.2, 120.8, 120.5, 116.5, 116.5, 107.7, 103.8, 103.7, 99.7, 53.4, 52.3,
42.3, 29.0, 18.9, 18.9. HRMS (ESI, m/z) for C28H27NO9P ([M ꢀ H]ˉ)
Calcd:552.1423; Found:552.1432.
eCH2). 13C NMR (100 MHz, DMSO-d6)
d 182.5, 172.9, 165.2, 161.5,
156.5, 155.9, 150.7, 150.6, 146.3, 137.2, 130.3, 130.3, 129.7, 129.7,
128.6, 128.5, 126.8, 125.5, 121.6, 120.5, 120.4, 120.3, 119.9, 116.6,
114.0, 107.7, 103.8, 99.2, 56.8, 52.3, 45.6. HRMS (ESI, m/z) for
4.1.13. ((Phenoxy)(5-hydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-
chromene-7-yl)phosphoryl) glycine methyl ester (10c)
C31H25NO10P ([M ꢀ H]ˉ) Calcd:602.1216; Found:602.1102.
Compound 10c was recrystallized from ethyl acetate/diethyl
4.1.17. ((Phenoxy)(5-hydroxy-2-(3,4-dihydroxyphenyl)-4-oxo-4H-
chromene-7-yl)phosphoryl) leucine methyl ester (11b)
ether. Yield 84.0%; Yellow solid; mp: 77e78 ꢁC. 1H NMR (400 MHz,
DMSO-d6)
d
13.05 (s, 1H, 5-OH), 10.44 (s, 1H, 40-OH), 8.01 (d, 2H,
Compound 11b was purified by column chromatography using
chloroform/methanol (40:1) as eluent. Yield 20.0%; Pale yellow
J ¼ 8.8 Hz, H-20, H-60), 7.43 (t, 2H, J ¼ 8.0 Hz, H-300, H-500), 7.28 (d, 2H,
J ¼ 8.0 Hz, H-200, H-600), 7.24 (t, 1H, J ¼ 8.0 Hz, H-400), 7.10 (d, 1H,
J ¼ 1.6 Hz, H-8), 6.96 (s, 1H, H-3), 6.94 (d, 2H, J ¼ 8.8 Hz, H-30, H-50),
6.69 (d, 1H, J ¼ 1.6 Hz, H-6), 6.61e6.54 (m, 1H, eNH), 3.80 (dd, 2H,
J ¼ 16.0, 7.6 Hz, eCH2), 3.60 (s, 3H, eOCH3). 13C NMR (100 MHz,
solid; mp: 193e194 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d 13.07 (s, 1H,
5-OH), 10.03 (s, 1H, 40-OH), 9.45 (s, 1H, 30-OH), 7.49 (dd, 1H, J ¼ 8.0,
2.0 Hz, H-60), 7.47 (d, 1H, J ¼ 2.0 Hz, H-20), 7.45e7.41 (m, 2H, H-300,
H-500), 7.30e7.23 (m, 3H, H-200, H-600, H-400), 7.05 (d,1H, J ¼ 1.8 Hz, H-
8), 6.92 (d, 1H, J ¼ 8.0 Hz, H-50), 6.85 (s, 1H, H-3), 6.67 (d, 1H,
J ¼ 1.8 Hz, H-6), 6.68e6.67 (m, 1H, eNH), 3.91e3.84 (m, 1H, eCH),
3.54 (s, 3H, eOCH3), 1.50e1.42 (m, 3H, e(CH3)2CHCH2), 0.80 (d, 6H,
DMSO-d6) d 182.7, 171.2, 165.2, 162.0, 161.5, 156.9, 156.1, 150.7, 130.4,
130.4, 129.3, 129.3, 125.6, 121.2, 120.7, 120.7, 116.5, 116.5, 107.8,
103.8, 99.8, 79.6, 52.2, 42.9. HRMS (ESI, m/z) for C24H19NO9P
([M ꢀ H]ˉ) Calcd:496.0797; Found:496.0785.
J ¼ 6.4 Hz, eCH(CH3)2). 13C NMR (100 MHz, DMSO-d6)
d 182.5,173.8,
165.3, 161.6, 156.8, 156.1, 156.1, 150.7, 146.3,130.4, 130.3, 125.7, 121.5,
120.8, 120.5, 119.8, 116.5, 114.0, 107.7, 103.8, 99.5, 99.5, 53.3, 52.3,
41.8, 24.1, 23.2, 21.4. HRMS (ESI, m/z) for C28H27NO10P ([M ꢀ H]ˉ)
Calcd: 568.1378; Found:568.1108.
4.1.14. ((Phenoxy)(5-hydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-
chromene-7-yl)phosphoryl) alanine methyl ester (10d)
Compound 10d was purified by column chromatography using
chloroform/methanol (40:1) as an eluent. Yield 58.0%; Yellow solid;
mp: 81e82 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
13.06 (s, 1H, 5-OH),
4.1.18. ((Phenoxy)(5-hydroxy-2-(3,4-dihydroxyphenyl)-4-oxo-4H-
chromene-7-yl)phosphoryl) glycine methyl ester (11c)
10.46 (s, 1H, 40-OH), 8.00 (d, 2H, J ¼ 8.0 Hz, H-20, H-60), 7.46e7.41
(m, 2H, H-300, H-500), 7.31e7.22 (m, 1H, H-400), 7.26 (d, 2H, J ¼ 8.0 Hz,
H-200, H-600), 7.11e7.05 (m, 1H, H-8), 6.96 (s, 1H, H-3), 6.94 (d, 2H,
J ¼ 8.0 Hz, H-30, H-50), 6.75e6.71 (m, 1H, H-6), 6.70e6.65 (m, 1H,
eNH), 4.04e3.97 (m, 1H, -eCH), 3.56 (s, 3H, eOCH3), 1.25e1.22 (m,
Compound 11c was purified by column chromatography using
chloroform/methanol (30:1) as eluent. Yield 52.0%; Pale yellow
solid; mp: 156e157 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d 12.96 (s, 1H,
5-OH), 9.66 (s, 1H, -OH), 7.49 (dd, 1H, J ¼ 8.0, 2.0 Hz, H-60), 7.45 (d,
1H, J ¼ 2.0 Hz, H-20), 7.42 (t, 2H, J ¼ 8.0 Hz, H-300, H-500), 7.28 (d, 2H,
J ¼ 8.0 Hz, H-200, H-600), 7.23 (t,1H, J ¼ 8.0 Hz, H-400), 7.06 (s,1H, H-8),
6.91 (d, 2H, J ¼ 8.0 Hz, H-50), 6.84 (s, 1H, H-3), 6.68 (s, 1H, H-6),
6.59e6.55 (m, 1H, eNH), 3.80 (dd, 2H, J ¼ 4.5, 14.7 Hz, eCH2), 3.61
3H, eCH3). 13C NMR (100 MHz, DMSO-d6)
d 182.7,173.7,165.2,162.0,
161.6, 156.9, 156.1, 150.7, 130.4, 130.4, 129.3, 129.3, 125.6, 121.2,
120.7, 120.7, 116.5, 116.5, 107.8, 103.8, 103.7, 99.8, 52.36, 50.3, 50.3.
HRMS (EI, m/z) for
Found:511.1031.
C
25H22NO9P([MþH]þ) Calcd:511.1032;
(s, 3H, eOCH3). 13C NMR (100 MHz, DMSO-d6)
d 182.6, 171.3, 165.3,
161.6, 156.8, 156.1, 150.8, 150.7, 146.3, 130.4, 130.4, 125.6, 121.5,
120.7, 120.7, 119.9, 116.5, 114.0, 107.8, 103.8, 99.7, 99.6, 52.3, 42.9. 31
NMR (162 MHz, DMSO-d6):
ꢀ0.39. HRMS (ESI, m/z) for
24H19NO10P ([M ꢀ H]ˉ) Calcd:512.0747; Found:512.0432.
4.1.15. ((Phenoxy)(5-hydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-
chromene-7-yl)phosphoryl) valine methyl ester (10e)
P
d
Compound 10e was recrystallized from ethyl acetate/diethyl
C
ether. Yield 53.4%; Yellow solid; mp: 87e88 ꢁC. 1H NMR (400 MHz,
DMSO-d6)
d
13.05 (s, 1H, 5-OH), 10.47 (s, 1H, 40-OH), 8.00 (d, 2H,
4.1.19. ((Phenoxy)(5-hydroxy-2-(3,4-dihydroxyphenyl)-4-oxo-4H-
chromene-7-yl)phosphoryl) alanine methyl ester (11d)
J ¼ 8.0 Hz, H-20, H-60), 7.42 (t, 2H, J ¼ 8.0 Hz, H-300, H-500), 7.26 (t, 2H,
J ¼ 8.0 Hz, H-200, H-600), 7.23 (t, 1H, J ¼ 8.0 Hz, H-400), 7.08 (d, 1H,
Compound 11d was purified by column chromatography using

Y.-q. Li et al. / European Journal of Medicinal Chemistry 112 (2016) 196e208
205
chloroform/methanol (40:1) as eluent. Yield 42.4%; Pale yellow
21.8. HRMS (ESI, m/z) for
602.1556; Found: 602.0697.
C
30H30NO9NaP ([MþNa]þ) Calcd:
solid; mp: 165e166 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
13.07 (s, 1H,
5-OH), 10.05 (s, 1H, 40-OH), 9.45 (s, 1H, 30-OH), 7.45 (s, 4H, Ph-H),
7.25 (s, 3H, Ph-H), 7.07 (s, 1H, Ph-H), 6.90e6.69 (m, 3H, Ph-H),
6.69(1H, eNH), 4.02 (s, 1H, eCH), 3.56 (s, 3H, eOCH3), 1.23 (s, 3H,
4.1.24. ((Phenoxy)(2-(4-acetoxyphenyl)-4-oxo-4H-chromene-7-yl)
phosphoryl) glycine methyl ester (16c)
eCH3). 13C NMR (100 MHz, DMSO-d6)
d
182.6, 173.7, 165.3, 161.6,
156.8, 156.1, 150.8, 150.7, 146.3, 130.3, 130.3, 125.6, 121.5, 120.7,
120.7, 119.8, 116.5, 114.0, 107.7, 103.8, 103.7, 99.6, 52.4, 50.3, 20.1. 31
NMR (162 MHz, DMSO-d6):
ꢀ1.53, ꢀ1.77. HRMS (ESI, m/z) for
25H21NO10P ([M ꢀ H]ˉ) Calcd:526.0903; Found:526.0549.
Yield 53.5%; Colorless oil. 1H NMR (400 MHz, Acetone-d6)
d 8.34
(s, 1H, H-2), 8.20 (d, 1H, J ¼ 8.0 Hz, H-5), 7.66 (d, 2H, J ¼ 8.6 Hz, H-20,
H-60), 7.56e7.55 (m, 1H, H-8), 7.43e7.32 (m, 5H, Ph-H), 7.27e7.22
(m, 1H, Ph-H), 7.18 (d, 2H, J ¼ 8.6 Hz, H-30, H-50), 5.59e5.52 (m, 1H,
eNH), 3.97e3.92 (m, 2H, eCH2), 3.65 (s, 3H, eOCH3), 2.28 (s, 3H,
P
d
C
eOCOCH3). 13C NMR (100 MHz, Acetone-d6)
d 174.6, 170.7, 168.9,
4.1.20. ((Phenoxy)(5-hydroxy-2-(3,4-dihydroxyphenyl)-4-oxo-4H-
chromene-7-yl)phosphoryl) valine methyl ester (11e)
156.8, 155.0, 154.1, 150.9, 150.8, 130.0, 130.0, 129.8, 129.8, 129.5,
127.7, 125.2, 124.4, 124.0, 121.6, 121.6, 121.4, 120.4, 118.6, 109.3, 51.5,
42.7, 20.2. HRMS (ESI, m/z) for C26H22NO9NaP ([MþNa]þ) Calcd:
546.0930; Found: 546.0288.
Compound 11e was purified by column chromatography using
chloroform/methanol (40:1) as eluent. Yield 62.4%; Pale yellow
solid; mp: 178e179 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d 13.05 (s, 1H,
5-OH), 10.02 (s, 1H, eOH), 9.43 (s, 1H, eOH), 7.48 (dd, 1H, J ¼ 8.4,
2.0 Hz, H-60), 7.45 (d, 1H, J ¼ 2.0 Hz, H-20), 7.50e7.40 (m, 2H, H-300,
H-500), 7.28 (d, 2H, J ¼ 8.0 Hz, H-200, H-600), 7.22 (t, 1H, J ¼ 8.0 Hz, H-
400), 7.04 (d, 1H, J ¼ 1.6 Hz, H-8), 6.91 (d, 1H, J ¼ 8.4 Hz, H-50), 6.84 (s,
1H, H-2), 6.67 (d, 1H, J ¼ 1.6 Hz, H-6), 3.70e3.61 (m, 1H, eCHeNH),
3.54 (s, 3H, eOCH3), 1.96e1.88 (m, 1H, eCH), 0.83e0.77 (m, 6H,
4.1.25. ((Phenoxy)(2-(4-acetoxyphenyl)-4-oxo-4H-chromene-7-yl)
phosphoryl) alanine methyl ester (16d)
Yield 84.3%; Colorless oil. 1H NMR (400 MHz, Acetone-d6)
d 8.36
(s, 1H, H-2), 8.22 (d, 1H, J ¼ 8.0 Hz, H-5), 7.67 (d, 2H, J ¼ 8.6 Hz, H-20,
H-60), 7.57 (dd, 1H, J ¼ 2.0, 1.2 Hz, H-8), 7.44e7.32 (m, 5H, Ph-H),
7.27e7.23 (m, 1H, Ph-H), 7.18 (d, 2H, J ¼ 8.6 Hz, H-30, H-50),
5.67e5.60 (m, 1H, eH), 4.22e4.15 (m, 1H, eCH), 3.62 (s, 3H,
eOCH3), 2.28 (s, 3H, eOCOCH3), 1.37e1.34 (m, 3H, CH3). 13C NMR
eCH(CH3)2). 13C NMR (100 MHz, DMSO-d6)
d 182.5, 172.9, 165.3,
161.6, 156.8, 156.1, 150.7, 150.6, 146.3, 130.3, 130.3, 125.6, 121.6,
120.8, 120.5, 119.8, 116.5, 114.0, 107.7, 103.9, 103.7, 99.6, 60.9, 52.1,
31.7, 19.3, 18.4. HRMS (ESI, m/z) for C27H25NO10P ([M ꢀ H]ˉ)
Calcd:554.1216; Found:554.1215.
(100 MHz, Acetone-d6) d 174.5,173.2,168.8,156.8,155.1,154.1,150.9,
150.8, 130.0, 130.0, 129.8, 129.8, 129.5, 127.7, 125.2, 124.4, 124.0,
121.6,121.6,121.4,120.4,118.6,109.3, 51.5, 51.4, 20.1,19.8.HRMS (ESI,
m/z) for
560.0339.
C
27H24NO9NaP ([MþNa]þ) Calcd: 560.1086; Found:
4.1.21. Synthesis of 40-acetyldaidzein-7-yl phosphoramidate
derivatives 16aed
A 250 mL, three-necked flask was changed with compound 14
4.1.26. Synthesis of 40, 5-diacetylgenistein-7-yl phosphoramidate
derivatives 17aed
(0.296 g,
1 mmol), a solution of phosphoramidate 2aed
(2e5 mmol) in acetone (60 mL), anhydrous potassium carbonate
(2e5 mmol). The mixture was stirred at 25 ꢁC (600 W) under mi-
crowave irradiation for 4e5 h (TLC). The solvent was removed
under reduced pressure and the filtrate was concentrated to give a
crude residue as an oil, which was purified by column chroma-
tography on silica gel to afford the product 16aee.
Compound 17aed was prepared from 15 according to the same
procedure described for 16aee.
4.1.26.1. ((Phenoxy)(5-acetoxy-2-(4-acetoxyphenyl)-4-oxo-4H-chro-
mene-7-yl)phosphoryl) phenylalanine methyl ester (17a). Yield
69.3%; White solid; mp: 144e145 ꢁC.1H NMR (400 MHz, DMSO-d6)
d
8.51 (s, 1H, H-2), 7.55 (d, 2H, J ¼ 8.0 Hz, H-20, H-60), 7.37 (t, 2H,
4.1.22. ((Phenoxy)(2-(4-acetoxyphenyl)-4-oxo-4H-chromene-7-yl)
phosphoryl) phenylalanine methyl ester (16a)
J ¼ 8.0 Hz, H-300, H-500), 7.22e7.19 (m, 8H, Ph-H), 7.14e7.08 (m, 3H,
Ph-H), 7.01e6.95 (m, 1H, Ph-H), 6.93-e6.91 (m, 1H, eNH),
4.13e4.08 (m, 1H, eCH), 3.53 (s, 3H, eOCH3), 3.04e2.75 (m, 2H,
eCH2), 2.32 (s, 3H, eOCOCH3), 2.30 (s, 3H, eOCOCH3). 13C NMR
Yield 69.6%; White solid; mp: 142e143 ꢁC. 1H NMR (400 MHz,
DMSO-d6)
d
8.59 (s, 1H, H-2), 8.09 (d, 1H, J ¼ 8.8 Hz, H-5), 7.65 (d,
2H, J ¼ 8.0 Hz, H-20, H-60), 7.37 (t, 2H, J ¼ 8.0 Hz, H-300, H-500), 7.30 (d,
1H, J ¼ 2.2 Hz, H-8), 7.23e7.16 (m, 8H, Ph-H), 7.15e7.09 (m, 3H, Ph-
H), 6.94e6.85 (m, 1H, eNH), 4.13e4.07 (m, 1H, eCH), 3.55 (s, 3H,
eOCH3), 3.02e2.75 (m, 2H, eCH2), 2.30 (s, 3H, eOCOCH3). 13C NMR
(100 MHz, DMSO-d6) d 173.9, 172.8, 169.7, 169.2, 157.7, 154.3, 154.2,
150.8, 150.7, 150.7, 137.2, 130.8, 130.8, 130.3, 130.3, 129.7, 129.6,
129.2, 128.6, 126.9, 125.6, 124.6, 122.1, 122.1, 122.0, 120.5, 120.4,
114.6, 112.8, 107.1, 56.8, 56.7, 52.4, 21.3, 21.3. HRMS (ESI, m/z) for
(100 MHz, DMSO-d6)
d
174.9, 172.8, 169.7, 156.6, 155.3, 154.8, 150.8,
C
35H30NO11NaP ([MþNa]þ) Calcd: 694.1454; Found: 694.0739.
150.6, 137.2, 130.5, 130.5, 130.3, 130.3, 129.7, 129.7, 129.6, 128.6,
128.6,127.9, 126.9,125.5,123.7, 122.1, 122.1,121.2, 120.5,120.4,118.7,
109.1, 57.0, 56.7, 52.4, 21.3. HRMS (ESI, m/z) for C33H28NO9NaP
([MþNa]þ) Calcd: 636.1399; Found: 636.0933.
4.1.26.2. ((Phenoxy)(5-acetoxy-2-(4-acetoxyphenyl)-4-oxo-4H-chro-
mene-7-yl)phosphoryl) leucine methyl ester (17b). Yield 80.3%;
White solid; mp: 150e151 ꢁC. 1H NMR (400 MHz, Acetone-d6)
d
8.31 (s, 1H, H-2), 7.58 (d, 2H, J ¼ 8.0 Hz, H-20, H-60), 7.47 (s, 1H, H-
4.1.23. ((Phenoxy)(2-(4-acetoxyphenyl)-4-oxo-4H-chromene-7-yl)
phosphoryl) leucine methyl ester (16b)
8), 7.42 (t, 2H, J ¼ 8.0 Hz, H-300, H-500), 7.32 (d, 2H, J ¼ 8.0 Hz, H-200, H-
600), 7.24 (t, 1H, J ¼ 8.0 Hz, H-400), 7.18 (d, 2H, J ¼ 8.0 Hz, H-30, H-50),
7.08 (s, 1H, H-6), 4.11e4.05 (m, 1H, eNHeCH), 3.62 (s, 3H, eOCH3),
2.32 (s, 3H, eOCOCH3), 2.28 (s, 3H, eOCOCH3), 1.67e1.62 (m, 1H,
eCH2eCH), 1.59e1.53 (m, 2H, eCH2eCH), 0.89e0.83 (m, 6H,
Yield 65.9%; White solid; mp: 37e38 ꢁC. 1H NMR (400 MHz,
CDCl3)
d
8.28 (d, 1H, J ¼ 8.8 Hz, H-5), 7.99 (s, 1H, H-2), 7.58 (d, 2H,
J ¼ 8.6 Hz, H-20, H-60), 7.45 (dd, 1H, J ¼ 2.2, 1.0 Hz, H-8), 7.38e7.33
(m, 2H, Ph-H), 7.30e7.20 (m, 4H, Ph-H), 7.17 (d, 2H, J ¼ 8.6 Hz, H-30,
H-50), 4.13-e4.08 (m, 1H, eNH), 3.89e3.79 (m, 1H, eNHeCH), 3.66
(s, 3H, eOCH3), 2.32 (s, 3H, eOCOCH3), 1.68e1.48 (m, 3H,
eCH2eCHe(CH3)2), 0.91e0.87 (m, 6H, eCHe(CH3)2). 13C NMR
eCHe(CH3)2). 13C NMR (100 MHz, Acetone-d6)
d 173.6, 173.3, 168.7,
168.5, 157.9, 154.5, 153.1, 151.2, 151.0, 150.8, 130.2, 130.2, 129.7, 129.1,
125.2, 125.0, 121.5, 121.5, 120.4, 120.4, 114.7. HRMS (ESI, m/z) for
C
32H32NO11NaP([MþNa]þ) Calcd: 660.1611; Found: 660.1012.
(100 MHz, CDCl3)
d 175.4, 173.7, 169.5, 156.9, 154.7, 153.2, 150.7,
150.4, 130.0, 130.0, 129.8, 129.2, 128.2, 128.2, 125.4, 124.7, 121.7,
121.7, 121.6, 120.2, 120.1, 118.4, 109.1, 53.4, 52.4, 43.7, 30.9, 24.4, 22.7,
4.1.26.3. ((Phenoxy)(5-acetoxy-2-(4-acetoxyphenyl)-4-oxo-4H-chro-
mene-7-yl)phosphoryl) glycine methyl ester (17c). Yield 50.2%;

206
Y.-q. Li et al. / European Journal of Medicinal Chemistry 112 (2016) 196e208
White solid; mp: 152e153 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d
8.53
J ¼ 2.0 Hz, H-8), 7.43 (t, 2H, J ¼ 8.0 Hz, H-300, H-500), 7.42 (d, 2H,
J ¼ 8.0 Hz, H-20, H-60), 7.38 (dd, 1H, J ¼ 8.0, 2.0 Hz, H-6), 7.30 (d, 2H,
J ¼ 8.0 Hz, H-200, H-600), 7.25 (t, 1H, J ¼ 8.0 Hz, H-400), 6.84 (d, 2H,
J ¼ 8.0 Hz, H-30, H-50), 6.66e6.55 (m, 1H, eNH), 3.86e3.80 (m, 2H,
(s, 1H, H-2), 7.53 (d, 2H, J ¼ 8.0 Hz, H-20, H-60), 7.53 (d, 1H, J ¼ 1.4 Hz,
H-8), 7.43 (t, 2H, J ¼ 8.0 Hz, H-300, H-500), 7.29 (d, 2H, J ¼ 8.0 Hz, H-200,
H-600), 7.25 (t, 1H, J ¼ 8.0 Hz, H-400), 7.20 (d, 2H, J ¼ 8.0 Hz, H-30, H-
50), 7.13 (d, 1H, J ¼ 1.4 Hz, H-6), 6.73e6.69 (m, 1H, eNH), 3.83 (dd,
2H, J ¼ 15.0, 7.0 Hz, eCH2), 3.60 (s, 3H, eOCH3), 2.32 (s, 3H,
eOCOCH3), 2.30 (s, 3H, eOCOCH3). 13C NMR (100 MHz, DMSO-d6)
eCH2), 3.61 (s, 3H, eOCH3). 13C NMR (100 MHz, DMSO-d6)
d 175.2,
171.3, 157.8, 156.7, 154.8, 154.2, 150.7, 130.6, 130.6, 130.4, 130.4,
128.0, 125.6, 124.4, 122.5, 121.3, 120.7, 120.7, 118.9, 115.5, 115.5,
109.5, 52.3, 42.9. HRMS (ESI, m/z) for C24H19NO8P ([M ꢀ H]ˉ) Calcd:
480.0848; Found: 480.1865.
d
173.9, 171.2, 169.7, 169.2, 157.8, 154.4, 154.3, 154.3, 150.8, 150.6,
130.8, 130.8, 130.4, 130.4, 129.2, 125.7, 124.6, 122.1, 122.1, 120.7,
120.7, 114.7, 113.2, 107.7, 52.3, 42.9, 21.3, 21.3. 31P NMR (162 MHz,
DMSO-d6):
Calcd: 604.0985; Found: 604.0985.
d
ꢀ0.26. HRMS (ESI, m/z) for C28H24NO11NaP ([MþNa]þ)
4.1.31. ((Phenoxy)(2-(4-hydroxyphenyl)-4-oxo-4H-chromene-7-yl)
phosphoryl) alanine methyl ester (18d)
Yield 44.6%; Colorless oil. 1H NMR (400 MHz, Acetone-d6)
d 8.56
4.1.26.4. ((Phenoxy)(5-acetoxy-2-(4-acetoxyphenyl)-4-oxo-4H-chro-
(s, 1H, 40-OH), 8.24 (s, 1H, H-2), 8.21 (d, 1H, J ¼ 8.0 Hz, H-5), 7.55 (s,
1H, H-8), 7.47 (d, 2H, J ¼ 8.0 Hz, H-20, H-60), 7.44e7.33 (m, 5H, Ph-H),
7.23 (t, 1H, J ¼ 8.0 Hz, H-400), 6.88 (d, 2H, J ¼ 8.0 Hz, H-30, H-50),
5.66e5.63 (m, 1H, -eNH), 4.24e4.17 (m, 1H, eCH), 3.63 (s, 3H,
eOCH3), 1.37 (t, 3H, J ¼ 6.4 Hz, -CH3). 13C NMR (100 MHz, Acetone-
mene-7-yl)phosphoryl) alanine methyl ester (17d). Yield 47.5%;
White solid; mp: 142e143 ꢁC. 1H NMR (400 MHz, DMSO-d6)
d 8.53
(s, 1H, H-2), 7.54 (d, 2H, J ¼ 8.0 Hz, H-20, H-60), 7.44 (d, 1H, 2.4 Hz, H-
8), 7.46e7.41 (m, 2H, H-300, H-500), 7.32 (d, 1H, J ¼ 8.0 Hz, H-400), 7.25
(d, 2H, J ¼ 8.0 Hz, H-200, H-600), 7.20 (d, 2H, J ¼ 8.0 Hz, H-30, H-50), 7.16
(d, 1H, 2.4 Hz, H-6), 6.84e6.78 (m, 1H, eNH), 4.06e4.04 (m, 1H,
eCH), 3.57 (s, 3H, eOCH3), 2.32 (s, 3H, eOCOCH3), 2.30 (s, 3H,
eOCOCH3), 1.25e1.22 (m, 3H, eCH3). 13C NMR (100 MHz, DMSO-d6)
d6) d 174.9, 173.1, 157.5, 156.7, 154.8, 153.2, 150.8, 130.2, 130.2, 129.8,
129.8, 129.7, 127.7, 125.2, 124.7, 122.9, 121.5, 120.4, 118.3, 115.1, 115.1,
109.2, 51.6, 50.4, 19.7. HRMS (ESI, m/z) for C25H21NO8P ([M ꢀ H]ˉ)
Calcd: 494.1005; Found: 494.2001.
d
173.9, 173.6, 169.7, 169.2, 157.8, 154.4, 154.3, 150.8, 150.6, 150.5,
130.8, 130.8, 130.4, 130.4, 129.2, 125.7, 124.6, 122.1, 122.1, 120.7,
4.1.32. Synthesis of genistein-7-yl phosphoramidate derivatives
Compound 19aee was prepared from genistein according to the
same procedure described for 16a-16e.
120.7, 114.7, 113.1, 107.5, 52.4, 50.3, 21.3, 21.3, 20.0. 31P NMR
(162 MHz, DMSO-d6):
d
ꢀ1.41, -1.63. HRMS (ESI, m/z) for
C
29H26NO11NaP ([MþNa]þ) Calcd: 618.1141; Found: 618.0414.
4.1.32.1. ((Phenoxy)(5-hydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-
4.1.27. Synthesis of daidzein-7-yl phosphoramidate derivatives
Compound 18aee was prepared from daidzein according to the
same procedure described for 16a-16e.
chromene-7-yl)phosphoryl) phenylalanine methyl ester (19a).
Yield 68.0%; Colorless oil. 1H NMR (400 MHz, DMSO-d6)
d 12.91 (s,
1H, 5-OH), 9.66 (s, 1H, 40-OH), 8.49 (s, 1H, H-2), 7.42 (d, 2H,
J ¼ 8.0 Hz, H-20, H-60), 7.37 (t, 2H, J ¼ 8.0 Hz, H-300, H-500), 7.30e7.26
(m, 4H, Ph-H), 7.13e7.05 (m, 4H, Ph-H), 6.85 (d, 2H, J ¼ 8.0 Hz, H-30,
H-50), 6.75e6.73 (m, 1H, Ph-H), 6.55 (d, 1H, J ¼ 2.0 Hz, H-6),
4.10e3.99 (m, 1H, eCH), 3.55 (s, 3H, eOCH3), 3.01e2.74 (m, 2H,
4.1.28. ((Phenoxy)(2-(4-hydroxyphenyl)-4-oxo-4H-chromene-7-yl)
phosphoryl) phenylalanine methyl ester (18a)
Yield 84.9%; Colorless oil. 1H NMR (400 MHz, DMSO-d6)
d 9.61 (s,
1H, 40-OH), 8.45 (s,1H, H-2), 8.07 (d, 1H, J ¼ 8.0 Hz, H-5), 7.43 (d, 2H,
J ¼ 8.0 Hz, H-20, H-60), 7.37 (t, 2H, J ¼ 8.0 Hz, H-300, H-500), 7.27 (s, 1H,
Ph-H), 7.22e7.08 (m, 9H, Ph-H), 6.93e6.90 (m, 1H, Ph-H), 6.84 (d,
2H, J ¼ 8.0 Hz, H-30, H-50), 4.11e4.07 (m, 1H, eCH), 3.55 (s, 3H,
eOCH3), 3.03e2.75 (m, 2H, eCH2). 13C NMR (100 MHz, DMSO-d6)
eCH2). 13C NMR (100 MHz, DMSO-d6)
d 180.6, 171.3, 165.4, 162.4,
158.0, 154.6, 137.8, 137.1, 136.0, 130.6, 129.9, 129.8, 129.6, 129.2,
128.9,128.8,128.5,127.5,127.1,126.7,122.7,121.6,122.0,120.6,115.6,
104.7, 99.6, 94.3, 57.0, 52.6, 37.6. HRMS (ESI, m/z) for C31H25NO9P
([M ꢀ H]ˉ) Calcd:586.1267; Found: 586.1258.
d
175.2, 172.9, 157.8, 156.6, 154.6, 154.2, 150.6, 137.2, 130.6, 130.6,
130.3, 130.3, 129.7, 129.7, 128.6, 128.6, 127.9, 126.9, 125.5, 124.4,
122.5, 121.2, 120.5, 120.4, 118.5, 115.5, 115.4, 109.0, 56.8, 52.36, 31.15.
HRMS (ESI, m/z) for C31H25NO8P ([M ꢀ H]ˉ) Calcd: 570.1318; Found:
570.1325.
4.1.33. ((Phenoxy)(5-hydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-
chromene-7-yl)phosphoryl) leucine methyl ester (19b)
Yield 46.8%; Colorless oil. 1H NMR (400 MHz, DMSO-d6)
d 13.04
(s, 1H, 5-OH), 9.71 (s, 1H, 40-OH), 8.56 (s, 1H, H-2), 7.49e7.44 (m, 4H,
Ph-H), 7.33e7.26 (m, 3H, Ph-H), 7.02 (d, 1H, J ¼ 1.6 Hz, H-8), 6.88 (d,
2H, J ¼ 8.4 Hz, H-30, H-50), 6.80e6.75 (m, 1H, H-6), 6.73e6.70 (m,
1H, eNH), 3.95e3.85 (m, 1H, eCHeNH), 3.59 (s, 3H, eOCH3),
1.53e1.47 (m, 3H, eCH2eCHe(CH3)2), 0.89e0.75 (m, 6H,
4.1.29. ((Phenoxy)(2-(4-hydroxyphenyl)-4-oxo-4H-chromene-7-yl)
phosphoryl) leucine methyl ester (18b)
Yield 46.8%; Colorless oil. 1H NMR (400 MHz, DMSO-d6)
d 9.59 (s,
1H, 40-OH), 8.45 (s, 1H, H-2), 8.17 (d, 1H, J ¼ 8.0 Hz, H-5), 7.52 (s, 1H,
H-8), 7.42 (d, 2H, J ¼ 8.0 Hz, H-20, H-60), 7.41 (t, 2H, J ¼ 8.0 Hz, H-300,
H-500), 7.36 (dd, 1H, J ¼ 8.8, 1.2 Hz, H-6), 7.29e7.21 (m, 3H, H-200, H-
400, H-600), 6.83 (d, 2H, J ¼ 8.0 Hz, H-30, H-50), 6.69e6.63 (m, 1H,
eNH), 3.90e3.86(m, eCHeNH), 3.54 (s, 3H, eOCH3), 1.49e1.36 (m,
3H, eCH2eCHe(CH3)2), 0.79 (d, 6H, J ¼ 6.2 Hz, eCHe(CH3)2). 13C
eCHe(CH3)2). 13C NMR (100 MHz, DMSO-d6)
d 181.2, 173.8, 162.1,
158.1, 157.1, 156.3, 155.6, 150.6, 130.6, 130.6, 130.4, 130.3, 125.7,
123.4, 121.2, 120.7, 120.4, 115.6, 115.6, 108.4, 103.6, 99.4, 53.3, 52.3,
42.3, 24.1, 23.1, 21.4. HRMS (ESI, m/z) for C28H28NO9NaP ([MþNa]þ)
Calcd: 576.1399; Found: 576.1766.
NMR (100 MHz, DMSO-d6)
d
175.2, 173.8, 157.9, 156.7, 154.9, 154.2,
4.1.33.1. ((Phenoxy)(5-hydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-
150.7, 130.6, 130.6, 130.3, 130.3, 128.0, 125.7, 124.5, 122.5, 121.3,
120.8,120.8,118.8,115.5,115.5,109.3, 53.4, 52.3, 42.3, 24.1, 23.1, 21.4.
HRMS (ESI, m/z) for C28H27NO8P ([M ꢀ H]ˉ) Calcd: 536.1474; Found:
536.1479.
chromene-7-yl)phosphoryl) glycine methyl ester (19c). Yield 68.3%;
Colorless oil. 1H NMR (400 MHz, DMSO-d6)
d 13.00 (s, 1H, 5-OH),
9.67 (s, 1H, 40-OH), 8.50 (s, 1H, H-2), 7.45e7.37 (m, 4H, Ph-H), 7.29
(d, 2H, J ¼ 8.0 Hz, H-200, H-600), 7.24 (t, 1H, J ¼ 8.0 Hz, H-400), 7.00 (d,
1H, J ¼ 2.0 Hz, H-8), 6.85 (d, 2H, J ¼ 8.0 Hz, H-30, H-50), 6.73 (d, 1H,
J ¼ 2.0 Hz, H-6), 6.66e6.59 (m, 1H, eNH), 3.83 (dd, 2H, J ¼ 15.0,
7.0 Hz, eCH2), 3.61 (s, 3H, eOCH3). 13C NMR (100 MHz, DMSO-d6)
4.1.30. ((Phenoxy)(2-(4-hydroxyphenyl)-4-oxo-4H-chromene-7-yl)
phosphoryl) glycine methyl ester (18c)
Yield 68.2%; Colorless oil. 1H NMR (400 MHz, DMSO-d6)
d
9.63 (s,
d
181.2, 171.3, 162.1, 158.1, 157.1, 156.2, 155.6, 150.6, 130.7, 130.7,
1H, 40-OH), 8.46 (s, 1H, H-2), 8.17 (d, 1H, J ¼ 8.0 Hz, H-5), 7.56 (d, 1H,
130.4, 130.4, 125.7, 123.4, 121.2, 120.7, 120.7, 115.6, 115.6, 108.5,

Y.-q. Li et al. / European Journal of Medicinal Chemistry 112 (2016) 196e208
207
103.8, 99.6, 52.3, 42.9. HRMS (ESI, m/z) for C24H20NO9NaP
([MþNa]þ) Calcd: 520.0773; Found: 520.0952.
the Central Universities of China (DUT11LK26). We thank the free
trial of Attune NxT flow cytometry from Thermo Fisher. We also
thank Dr. Wenfeng Sun for the technology support on cell cycle
analysis and assessment of apoptosis. We appreciate erlotinib
proved by Prof. Qingwei Meng in our college.
4.1.33.2. ((Phenoxy)(5-hydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-
chromene-7-yl)phosphoryl) alanine methyl ester (19d). Yield 78.3%;
Colorless oil. 1H NMR (400 MHz, DMSO-d6)
d 13.00 (s, 1H, 5-OH),
9.65 (s, 1H, 40-OH), 8.50 (s, 1H, H-2), 7.46e7.41 (m, 4H, Ph-H),
7.36e7.22 (m, 3H, Ph-H), 7.01 (d, 1H, J ¼ 2.0 Hz, H-8), 6.85 (d, 2H,
J ¼ 8.0 Hz, H-30, H-50), 6.75 (d,1H, J ¼ 2.0 Hz, H-6), 6.75e6.69 (m,1H,
eNH), 4.04e4.02 (m, 1H, eCH), 3.58 (s, 3H, eOCH3), 1.27e1.23 (m,
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.02.012
3H, eCH3). 13C NMR (100 MHz, DMSO-d6)
d 181.3, 173.6, 162.1, 158.1,
157.1, 156.2, 155.5, 150.6, 130.6, 130.6, 130.3, 130.3, 129.8, 125.6,
121.2, 120.8, 120.7, 115.6, 115.6, 108.5, 103.7, 99.5, 52.4, 50.3, 20.1.
HRMS (ESI, m/z) for C25H21O9P ([M ꢀ H]ˉ) Calcd:510.0954; Found:
510.0943.
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