Synthesis and in vitro anticancer activity evaluation of novel bioreversible phosphate inositol derivatives

Article abstract of DOI:10.1016/j.ejmech.2015.01.064

The chemistry and biology of phosphorylated inositols have become intense areas of research during the last two decades due to their involvement in various cellular signaling processes. However, the metabolic instability by phosphatases or kinases and poo

Full text of DOI:10.1016/j.ejmech.2015.01.064

European Journal of Medicinal Chemistry 93 (2015) 172e181
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro anticancer activity evaluation of novel
bioreversible phosphate inositol derivatives
,
Wenbin Chen a *, Zhaohui Deng a, Kuangyu Chen a, Daolei Dou b, Fanbo Song a,
,
*
Luyuan Li b, Zhen Xi a
a State Key Laboratory of Elemento-Organic Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University,
Tianjin 300071, China
b State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Nankai University, Tianjin 300071, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The chemistry and biology of phosphorylated inositols have become intense areas of research during the
last two decades due to their involvement in various cellular signaling processes. However, the metabolic
instability by phosphatases or kinases and poor penetration make it difficult to become a drug used in
the clinic. The bioreversible protection technique can enhance membrane penetration characteristics and
increase the stability of phosphorylated inositols against enzymatic degradation and is applied widely in
drug discovery and development. In this paper, we described the design and synthesis of 22 bioreversible
phosphotriester inositols, along with the initial antitumor activity results. Most compounds exhibited
significant cytotoxic activity against human cancer cell lines A549, MDA-MB-231 and HeLa, but lower
cellular toxicity on normal cell MCF10A in comparison with Cisplatin. These compounds can be used as
probes to study the mechanism of intracellular signal transduction mediated by phosphate inositol or as
leads of phosphate inositol drugs in the clinic.
Received 24 September 2014
Received in revised form
29 December 2014
Accepted 31 January 2015
Available online 9 February 2015
Keywords:
Phosphate inositol
Bioreversible protection
4-Acyloxybenzyl
Anticancer
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
investigate directly the chemical biology of IP3-mediated cellular
signaling, so the development of a stable inositol phosphate probe
Inositol phosphates (IPs), such as D-myo-Inositol 1,4,5-
trisphosphate [Ins(1,4,5)P3, IP3], play important roles as second
messengers in intracellular signal transduction [1e6] and nuclear
processes such as transcription control, mRNA export and DNA
repair [7] and also have potential roles in cancer prevention and
therapy [8]. Ins(1,4,5)P3, along with the other important signaling
lipid diacylglycerol (DAG), is generated through the hydrolysis of
phosphatidylinositol 4,5-bisphosphate (PtdInsP2) by the intracel-
lular phosphatidylinositol-specific phospholipase C (PI-PLC). IP3
regulates cellular calcium flux by binding to IP3 receptors and
subsequently induces the release of Ca2þ from internal stores. After
triggering Ca2þ release from internal store, IP3 is rapidly deacti-
vated by stepwise dephosphorylation to Ins(1,4)P2, then to Ins-4-
P1 and finally myo-inositol by 5-phosphatase [9e11]. Alterna-
tively, phosphorylation by IP3 3-kinase produces Ins(1,3,4,5)P4
[8,12,13]. This metabolic instability of IP3 makes it difficult to
is very crucial in the pathway mediated by IP3/DAG. A number of
IP3 analogs have now been synthesized, such as deoxy analogs [8],
fluorinated analogs [14,15], ring-modified analogs [16e18], phos-
phorothioate analogs [19e22], phosphonate analogs [23], bicyclic
analogs [24,25], conformationally restricted analogs [26] and ade-
nophostin mimic analogs [27e29], many of which could behave as
IP3 receptor agonists or antagonists. Although some synthetic an-
alogs attained similar potencies to that of IP3 in terms of receptor
binding affinity or Ca2þ release, little has surpassed it [24]. Phos-
pholipase, phosphatase or kinase stability and cell membrane
penetration are synergistic to the activity in vitro or in vivo of these
analogs, sometimes the latter being more important, while most
work was focused on the enzymatic stability and little was noticed
regarding on the cell membrane penetration. We reasoned lower
activity of these “exogenous” phosphate inositol analogs are
ascribed to the cell membrane penetration to some extent, because
all these compounds are poly-phosphoric acid or poly-phosphate
sodium or other salt, which carry several negative charges at
physiological pH values making them very polar. This high polarity
is the basis for many deficiencies in terms of drug delivery.
* Corresponding authors.
E-mail addresses: wbchen@nankai.edu.cn (W. Chen), zhenxi@nankai.edu.cn
(Z. Xi).
http://dx.doi.org/10.1016/j.ejmech.2015.01.064
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

W. Chen et al. / European Journal of Medicinal Chemistry 93 (2015) 172e181
173
Specifically, ionized species do not readily undergo passive diffu-
sion across cellular membranes. Because of their high polarity,
these agents often exhibit a low volume of distribution and,
therefore, tend to be subject to efficient renal clearance as well as
possibly biliary excretion [30]. In previous work [31], we presented
a series of inositol phosphotriester analogs based on phosphati-
dylinositol with significant cytotoxic activity against human cancer
cell lines A549, HepG2, MDA-MB-231 and HeLa, while lower
cytotoxicity against normal cell. In this work, we reported other
bioreversible inositol phosphate derivatives to enhance the cell
membrane penetration and also to be used as pro-drugs.
Fig. 2. Design of the bioreversible target phosphates inositol analogs 41a-v.
Although acyloxymethyl esters were the most promising bio-
reversible prodrug modifications for phosphates and phospho-
nates, their instability prevented them from being suitable prodrug
forms. In contrast, the 4-acyloxybenzyl ester as an alternative
bioreversible protecting group was developed for the prodrugs of
antiviral phosphonoacetate [49,50]. When these prodrugs pene-
trated the cell, the triesters were esterase-catalyzed cleavage of one
acyloxymethyl ester to give the hydroxymethyl analog, which
would render it a better esterase substrate. The intermediate 4-
hydroxybenzyl ester was expected to undergo spontaneous frag-
mentation with loss of a quinone methide that presumably is hy-
drated to generate 4-hydroxymethylphenol and the electron-
donating 4-hydroxyl group also promotes this cleavage of the
benzyleoxygen bond. A repeat of this bio-activation step can
eventually release the drug in the cell (Fig. 3) [50e53].
myo-Inositol has a meso configurational structure and, therefore,
it possesses a symmetry plane with five equatorial and one axial
hydroxyl group. To investigate the effect of phosphorylation posi-
tion and phosphorylation degree on the biological activity, we need
a library of all kinds of bioreversible phosphate inositols, and the
key intermediates for the synthesis of these compounds are the
corresponding hydroxyl group-protected derivatives which having
free hydroxyl group(s) at desired positions. In this work, we ob-
tained the mono-hydroxyl compounds 4a-d, di-hydroxyl com-
pounds 4e-l and tri-hydroxyl compounds 4m-v at all the possible
positions of myo-inositol. The mono-hydroxyl inositols 4a-d and di-
hydroxyl inositol 4e were synthesized according to the reported
procedures [31] from myo-inositol (Scheme 1).
2. Results and discussion
2.1. Design principle of the target compounds
Phosphatidylinositol phosphates (PtdInsPs or PIPs) are phos-
phorylated derivatives of myo-inositol, which are an important
class of membrane phospholipids [32,33]. There are eight known
naturally occurring classes of PIPs differing only in the degree of
phosphorylation of the hydroxyl groups at the 3-, 4- and 5-
positions of the inositol rings, which can be metabolized to
inositol phosphates [34] (Fig. 1). A lot of PIPs analogs [35e41] were
synthesized to understand the roles of each of these PIPs in their
signaling pathways and their effect on downstream processes
because of the inability to isolate practically useful amounts of
these PIP compounds from cells. However, all these analogs are
poly-phosphoric acid or poly-phosphate salt, which have difficulty
in penetrating the cellular membranes. Our approach is to intro-
duce bioreversible phosphates to improve the cell membrane
penetration and hope to find a novel probe to investigate the
mechanism of these PIPs in their signaling pathways and a novel
inositol phosphate as anticancer reagent. To systematically inves-
tigate the effect of the position and degree of phosphorylation at
the inositol ring on their anticancer activities, we designed and
prepared a novel series of mono-phosphorylation, di-phosphory-
lation and tri-phosphorylation compounds 41a-v at the different
positions of the inositol ring based on the metabolism of phos-
phatidylinositol phosphate (Fig. 2).
The two cis-hydroxyls of myo-inositol were selectively protected
to form 1,2 (or 2,3)-O-isopropylidene-myo-inositol 2 in DMSO,
which was treated with 2,2-dimethoxypropane in acetone to afford
the di-hydroxyl compound 4f as the major product [54,55] or
benzoylated and then hydrolyzed to give the di-hydroxyl com-
pound 4g [56]. (Scheme 1).
The di-hydroxyl inositols 4h-4l were prepared from the readily
available myo-inositol orthoformate 1 by judicial choice of protec-
tion, resolution and deprotection strategies. The 4-methoxybenzyl
2.2. Synthesis
Bioreversible protection is applied widely in nucleoside
diphosphate [42e45], phosphopeptide [46] and oligonucleotide
conjugates [47] with antiviral and antitumor therapy. The enzyme-
labile protecting technique can not only lead to an increase in
the intracellular levels of active metabolites, but also overcome
drug resistance in some cases and broaden the spectrum of appli-
cation [48].
Fig. 1. General structures of natural phosphatidylinositol phosphates and their metabolisms.

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W. Chen et al. / European Journal of Medicinal Chemistry 93 (2015) 172e181
Fig. 3. The proposed mechanism of enzymatic hydrolysis activation.
Scheme 1. Synthesis of mono-hydroxyl inositols 4a-4d and di-hydroxyl inositols 4e-4g. Reagents and conditions: (i) HC(OEt)3, PTSA monohydrate, DMF, 110 ꢀC, 28 h, 83%; (ii) NaH,
BnBr, DMF, 0 ꢀC-RT, overnight, 68% for 4a, 48% for 4e, 82% for 3, 90% for 5; (iii) TBDMSCl, imidazole, DMF, RT, overnight, 90%; (iv) DIBAL, CH2Cl2, 0ꢀC-RT, 4 h, 95%; (v) AlMe3, CH2Cl2,
0
ꢀC-RT, 5 h, 68%; (vi) 2,2-dimethoxypropane, TEA, DMSO, 41%; (vii) 2,2-dimethoxypropane, TEA, acetone, 26%; (viii) 0.1 M HCl, MeOH, reflux, 90%.
group can be removed selectively in the presence of ketal under the
condition of DDQ to give the intermediates 4h, 4i and 4j, (Scheme 2,
Scheme 3). The di-hydroxyl inositols 4k and 4l were obtained based
on the standard procedures of benzylation, propargylation or hy-
drolysis. The introduction of propargyl group in compound 4k was
ready to be further functionalized such as click chemistry in the
future.
The three free hydroxyl intermediates 4m-4q were prepared by
the same strategies, where the 4-methoxybenzyl group or t-butyl
dimethylsilyl (TBDMS) group can be total removed under the dra-
matic conditions (HCl and reflux) (Scheme 4).
give the mixture of 23 and 26, isolated in 26% and 33% yield,
respectively. Then the same procedures were conducted to give the
tri-hydroxyl inositol 4r and 4s. The desired isomer of compounds
29 and 34 were also isolated from the reaction mixture in 18% and
34% yield. Then the selective hydrolysis of 5,6-isopropylidene
group, followed by benzylation or allylation and deprotection to
give the desired tri-hydroxyl inositol intermediates 4t, 4u (Scheme
5, Scheme 6 and Scheme 7).
Several methods were reported to synthesize phosphoric acid
triester inositol 41, such as phosphoric acid diester chloride derived
from 4-acyloxybenzyl alcohol reacted with protected inositol. But
the phosphoramidite method is a versatile method to introduce
phosphates as diesters into alcohols to establish phosphoric acid
triesters via intermediate phosphites [57]. The synthesis of starting
The tri-hydroxyl inositols 4r-4v were synthesized from the di-
hydroxyl inositol 4f. 2,3-O-Isopropylidene-1,4-di-O-allyl-myo-
inositol 22 was benzylated with 1.0 equivalent benzyl bromide to
Scheme 2. Synthesis of di-hydroxyl inositol 4h and 4i. Reagents and conditions: (i) NaH, Imidazole, PMBCl, DMF, 57%; (ii) NaH, PMBCl, DMF, 40%; (iii) NaH, BnBr, DMF, 0 ꢀC-RT,
overnight, 99% for 8, 90% for 9; (iv) DDQ, DCM/H2O, 40% for 4h, 86% for 4i; (v) DIBAL, CH2Cl2, 0ꢀC-RT, 4 h, 92%.

W. Chen et al. / European Journal of Medicinal Chemistry 93 (2015) 172e181
175
Scheme 3. Synthesis of di-hydroxyl inositol 4j-4l. Reagents and conditions: (i) NaH, PMBCl, DMF, 96%; (ii) DIBAL, CH2Cl2, 0ꢀC-RT, 4 h, 99%; (iii) DDQ, DCM/H2O, 62% for 4j; (iv) NaH,
propargyl bromide, DMF, 0 ꢀC-RT, overnight, 85%; (v) 0.1 M HCl, MeOH, reflux, 90% for 4k, 99% for 4l; (vi) NaH, BnBr, DMF, 0 ꢀC-RT, overnight, 99%.
phosphoramidite (i-Pr2NP(OCH2C6H4OCOCH3-4)2) was approached
by adopting a procedure where N, N-diisopropylphosphoramidic
dichloride derived from PCl3 was treated with the 4-acyloxybenzyl
alcohol in the presence of triethylamine in dry THF. After the
treatment of phosphoramidite with protected inositols in
dichloromethane in the presence of tetrazole, the resulting phos-
phites were oxidized in situ with m-chloroperoxybenzoic acid to
afford the desired phosphates 41 in acceptable yields after purifi-
cation by silica gel column chromatography (Fig. 4).
among all the lung cancer types, non-small cell lung cancer is the
most common type of lung cancer. About 85% of lung cancers are
non-small cell lung cancers [58]. Although chemotherapy is the
mainstay of cancer therapy, the use of available chemotherapeutics
is often limited mainly due to undesirable side effects and a limited
choice of available anticancer drugs. There is an urgent need of
developing novel chemotherapeutic agents with more potent
antitumor activities. Having the above bioreversible phosphate
inositols in hands, we first evaluated the inhibitory effects of these
compounds against NSCLC cell line A549 by the MTT method [59]
with Cisplatin as positive control at 10 m
g mLꢁ1 (final concentra-
2.3. In vitro anticancer screening
tion in the test well). The inhibition percent result was outlined in
Table 1 and represented graphically in Fig. 5. The obtained data
Lung cancer has become the first killer of human death, and
Scheme 4. Synthesis of tri-hydroxyl inositol 4m-4q. Reagents and conditions: (i) HC(OEt)3, PTSA monohydrate, DMF, 110 ꢀC, 28 h, 83%; (ii) NaH, BnBr, DMF, 0 ꢀC-RT, overnight, 82%
for 3, 92% for 18, 30% for 20; (iii) 0.1 M HCl, MeOH, reflux, 90% for 4n, 80% for 4p, 85% for 4q; (iv) NaH, PMBCl, DMF, 92% for 15, 96% for 10; (v) DIBAL, CH2Cl2, 0ꢀC-RT, 99% for 12, 95%
for 16; (vi) DDQ, DCM/H2O, 48%; (vii) NaH, propargyl bromide, DMF, 0 ꢀC-RT, 91%; (viii)AlMe3, CH2Cl2, 0 ꢀC-RT, 63%.

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W. Chen et al. / European Journal of Medicinal Chemistry 93 (2015) 172e181
Scheme 5. Synthesis of tri-hydroxyl inositol 4r and 4s. Reagents and conditions: (i) NaH, Allyl bromide, DMF, 0 ꢀC-RT, 67% for 21, 97% for 24 and 27; (ii) acetone, PTSA$H2O,
TEA,NaHCO3, 55%; (iii) NaH, BnBr, DMF, 0 ꢀC-RT, overnight, 26% for 23, 33% for 26; (iv) 0.1 M HCl, MeOH, reflux, then NaH, BnBr, DMF, 0 ꢀC-RT, 91% for 25, 92% for 28; (v) EtOH/H2O,
Pd/C, PTSA$H2O, 93% for 4r, 83% for 4s.
Scheme 6. Synthesis of tri-hydroxyl inositol 4t. Reagents and conditions: (i) NaH, Allyl bromide, DMF, 0 ꢀC-RT, 18% for 29, 95% for 32; (ii) NaH, BnBr, DMF, 0 ꢀC-RT, overnight, 99%;
(iii) acetone, PTSA$H2O, TEA,NaHCO3, 51%; (iv) 0.1 M HCl, MeOH, reflux, then NaH, BnBr, DMF, 0 ꢀC-RT, 97%; (v) EtOH/H2O, Pd/C, PTSA$H2O, 84%.
revealed that compounds 41a-41f, 41h, 41i, 41m and 41o were
more potent than others with inhibition rate 75%e100%. Among
them, compound 41b, 41d, 41h, 41i and 41o showed the best
inhibitory activity and exhibited good or similar in vitro activity
with the positive control, Cisplatin. The SAR study of these de-
rivatives demonstrated that the higher the degree of phosphory-
lation, the lower inhibitory activity against NSCLC cell line A549.
For example, most tri-phosphate inositol compounds did not
display any appreciable inhibition activity except for compound
41m and 41o, while all mono-phosphate inositol and most di-
phosphate inositol compounds exhibited good in vitro anticancer
activity. The phosphorylation position is also an important factor in
the inhibition of di-phosphate inositol compounds. The compounds
that phosphorylated at the 4-position have the best activity, which
indicate that phosphorylation at the 4-position of inositol is
favorable for their anticancer activity.
The interesting results above encouraged us to screen the other
two important human cancer cell lines MDA-MB-231 and HeLa, in
order to determine the selectivity and universality between
different cancer cell lines. Similar results (Table 1 and Fig. 5) were
Scheme 7. Synthesis of tri-hydroxyl inositol 4u and 4v. Reagents and conditions: (i) NaH, BnBr, DMF, 0 ꢀC-RT, overnight, 34% for 34 and 38, 98% for 37, 28% for 40; (ii) NaH, Allyl
bromide, DMF, 0 ꢀC-RT, 99%; (iii) acetone, PTSA$H2O, TEA,NaHCO3, 52% for 36, 57% for 39; (iv) EtOH/H2O, Pd/C, PTSA$H2O, 88%; (v) 0.1 M HCl, MeOH, reflux, 99%.

W. Chen et al. / European Journal of Medicinal Chemistry 93 (2015) 172e181
177
Fig. 4. Synthetic strategy of the target compounds 41.
obtained with most synthesized compounds, and especially, most
3. Conclusion
compounds have the best selective inhibition against Hela cell with
100% inhibition rate. To our delight, most compounds having good
inhibitory activity have lower cellular cytotoxicity on the non-
cancerous cell line MCF10A than Cisplatin (Table 1 and Fig. 5).
To better assess the activity of the synthesized compounds, all
compounds having excellent inhibitory activities were further
tested for their antitumor potency indicated by IC50 values, which
were calculated by linear regression analysis of the concen-
trationeresponse curves obtained for each compound. We found
that most of the compounds are more potent than the positive
control Cisplatin in inhibiting three human cancer cell lines with
In summary, we have synthesized a novel family of bioreversible
phosphate derivatives. Most compounds showed significant anti-
cancer activity with broad-spectrum against several human cancer
cell lines and lower cellular toxicity on normal cell in comparison
with Cisplatin. These findings have encouraged us to continue the
development the novel phosphate inositol derivatives and to
conduct further studies to investigate their mechanisms of action
or as a useful tool in drug discovery and development.
4. Experimental section
IC50 less than 5.0 mM or 1.0 mM, while lower toxicity on the normal
cells with IC50 value of more than 20.0 mM(Table 2 and Fig. 6).
4.1. Chemistry
All water-sensitive syntheses were performed under an inert
atmosphere of argon using standard Schlenk techniques. Nuclear
magnetic resonance spectra were recorded on Bruker AVANCE-300
or AVANCE-400 NMR instrument in CDCl3 or (CD3)2SO. Chemical
shifts (dH) are reported in parts per million (ppm), relative toTMS as
internal standard. High resolution mass (HRMS) were carried out
Table 1
Results of in vitro inhibition percentage of the synthesized compounds on different
cell lines.
Compound
Growth inhibition percentage of cancer cell line (%) (10
m
g mLꢁ1
MCF10A
)
A549
HELA
MB-231
41a
41b
41c
41d
41e
41f
41g
41h
41i
41j
41k
41l
41m
41n
41o
41p
41q
41r
88.9 3%
100.4 1%
75.4 3.6%
96.6 1.4%
85.7 1.9%
90.1 6.5%
14.9 1.7%
98.5 0.8%
92.1 0.6%
43.7 17%
9.9 5.5%
27.1 9.9%
85.8 2.3%
12.8 7%
100 0.8%
100 1.4%
98.1 1.1%
100 0.8%
100 0.8%
100 1.1%
15.8 5.6%
100 0.5%
100 1.2%
100 0.6%
100 1.1%
27.2 2.8%
94.2 1.4%
69.3 3.9%
94.3 1%
100 0.7%
ꢁ6.2 8.3%
80 2%
84.5 3.7%
49.5 8.5%
68.5 7.3%
ꢁ1.2 10%
95.5 0.3%
95.8 2.1%
84.3 3.2%
12.4 5.3%
9.5 10.8%
98.8 1.2%
91.8 2.4%
ꢁ17.2 14%
99.7 1%
20.1 16.4%
100 1.4%
48.9 9.2%
98.1 0.8%
ꢁ8.2 15.3%
ꢁ10.7 13.4%
42.3 6.1%
38.8 3%
86.8 0.9%
99.4 0.2%
8.7 6.4%
90.4 0.7%
48 4.2%
38.6 10.4%
10.5 20.3%
ꢁ2.9 10.6%
34.9 7.8%
2.5 6.3%
15.4 16.4%
15.6 23.1%
9.2 21.2%
37.4 14.3%
45.1 6.6%
55.3 0.6%
5.1 13.9%
72.3 3.4%
88.4 0.9%
92.4 0.7%
ꢁ22.3 5.2%
94.4 0.4%
88.1 3.8%
ꢁ2.1 13.6
21.3 7.6%
11 13.6%
19.2 5.7%
52.8 4.1%
ꢁ11 5.5%
82.1 4.3%
94 0.7%
2.5 7.5%
ꢁ1.2 1.2%
7.8 0.5%
6.4 1.8%
4.7 4.9
34.9 3.3%
9.1 1.4%
95 4.5%
41s
41t
41u
41v
Cisplatin
Fig. 5. The inhibition results of the synthesized compounds on different cell lines.

178
W. Chen et al. / European Journal of Medicinal Chemistry 93 (2015) 172e181
Table 2
acid was quenched with a 10% aqueous solution of sodium
hydrogen sulfite. The layers were separated, and the aqueous layer
was extracted with dichloromethane. The combined organic layers
IC50 values of the selected compounds against cancer cells and normal cell.
Compound
IC50
A549
(m
M)a
were washed with
a saturated aqueous solution of sodium
HELA
MB-231
MCF10A
hydrogen carbonate and brine, dried (sodium sulfate), filtered, and
concentrated under reduced pressure. Purification by silica gel
column chromatography, eluting with ethyl acetate and petroleum
ether yielded 41 as a colorless oil.
41a
41b
41c
41d
41e
41f
41h
41i
6.75 0.57
9.51 1.00
8.15 1.45
11.38 1.15
1.76 0.38
2.34 0.46
1.37 0.31
2.01 0.81
5.55 0.42
26.71 1.90
7.09 0.42
4.27 0.41
>30
1.26 0.25
1.35 0.31
1.52 0.21
2.79 0.29
1.23 0.23
0.62 0.07
0.76 0.11
0.62 0.19
0.95 0.05
12.17 1.90
1.41 0.37
1.08 0.10
19.03 13.21
>20
2.77 0.71
2.35 0.18
1.58 0.35
2.85 0.22
0.91 0.22
0.83 0.15
1.53 0.48
0.43 0.18
2.37 0.58
28.62 0.87
1.89 0.10
1.68 0.13
>30
45.36 2.62
38.09 5.81
18.86 4.67
8.19 1.56
>50
25.07 0.42
>50
0.85 0.34
23.24 5.67
>40
41a: yield 55% (Rf ¼ 0.4, petroleum ether: ethyl acetate ¼ 3: 2, v/
v). 31P NMR (162 MHz, DMSO)
d
ꢁ1.67; 1H NMR (400 MHz, DMSO)
d
7.38 (d, J ¼ 8.4 Hz, 4H, HeAr), 7.35e7.20 (m, 10H, HeAr), 7.03 (d,
J ¼ 8.4 Hz, 4H, HeAr), 5.67 (s, 1H, CH(O)3), 5.07 (d, J ¼ 8.0 Hz, 4H,
2 ꢂ CH2O), 4.79 (d, J ¼ 7.5 Hz, 1H, H-ring), 4.67 (s, 1H, H-ring), 4.63,
4.57 (AB system, Dn ¼ 0.06, JAB ¼ 12 Hz, 4H, 2 ꢂ CH2O), 4.40e4.28
(m, 4H, H-ring), 2.27 (s, 6H, 2 ꢂ CH3); 13C NMR (101 MHz, DMSO)
41j
41l
41m
41o
41p
41u
Cisplatin
>20
>20
>30
>20
d
169.7, 150.9, 138.2，133.8，133.7, 129.7, 128.7, 128.1, 122.4102.7,
3.17 1.18
6.82 2.56
3.28 1.07
7.3 3 0.56
73.8, 71.2, 70.6, 68.8, 67.7, 67.9, 67.2, 21.3; HRMS (MALDI): m/z
769.2024[MþNa]þ (calcd for C39H39O13PNa, 769.2026).
1.73 0.33
3.30 0.1
a
IC50: concentration of the tested compound that inhibits 50% of cell growth.
41b: yield 59% (Rf ¼ 0.4, petroleum ether: ethylacetate ¼ 2: 1, v/
v). 31P NMR (162 MHz)
d
ꢁ2.21; 1H NMR (400 MHz, DMSO)
d
7.33e7.22 (m, 9H, HeAr), 7.11 (d, J ¼ 7.2 Hz, 4H, HeAr), 5.63 (s, 1H,
on IonSpec FTICR-MS instrument from Varian using MALDI as
ionization mode. Flash-column chromatography was performed
using commercial grades of silica gel 200e300 meshes. Analytical
thin layer chromatography (TLC) was performed on precoated silica
gel 60 F254 plates, and spot visualization was accomplished by UV
light (254 nm) or phosphomolybdic acid solution.
CH(O)3), 4.96 (t, J ¼ 8.0 Hz, 4H, 2 ꢂ CH2O), 4.65, 4.59 (AB system,
Dn ¼ 0.06, JAB ¼ 12 Hz, 2H, CH2O), 4.50 (s, 1H, H-ring), 4.29 (s, 1H, H-
ring), 4.24e4.20 (m, 2H, H-ring), 4.16e4.10 (m, 2H, H-ring), 2.27 (s,
6H, 2 ꢂ CH3), 0.86 (s, 9H, 3 ꢂ CH3), 0.04 (d, J ¼ 1.6 Hz, 6H, 2 ꢂ CH3);
13C NMR (101 MHz, DMSO)
d 169.1, 166.9, 150.6, 150.5, 139.9, 137.8,
133.2, 133.1, 131.7, 131.5, 129.2, 129.1, 129.0, 128.7, 128.2, 127.6, 127.4,
127.2, 122.1, 121.9, 121.6, 121.4, 101.9, 73.2, 72.1, 71.9, 71.2, 71.1, 70.8,
68.4, 67.1, 65.0, 62.3, 60.5, 59.7, 29.9, 25.6, 20.8, 20.7, 18.6, 17.9, 14.1,
13.5, ꢁ5.1; HRMS (MALDI): m/z 793.2420 [MþNa]þ (calcd for
Dichloromethane was distilled from CaH2 and all other chem-
icals were obtained from commercial sources and used without
further purification.
For the synthesis of the protected inositol intermediates 4a-4v
see the reference [31] and Supporting Information.
C38H47O13PSiNa, 793.2421).
41c: yield 49% (Rf ¼ 0.4, petroleum ether: ethyl acetate ¼ 2: 1, v/
v). 31P NMR (162 MHz, DMSO)
d
ꢁ2.11; 1H NMR (400 MHz, DMSO)
4.1.1. Typical procedure for the synthesis of target compounds 41a-
41v
d
7.42e7.33 (m, 9H, HeAr), 7.31e7.23 (m, 10H, HeAr), 7.11 (d,
J ¼ 8.4 Hz, 4H, HeAr), 5.27 (d, J ¼ 5.0 Hz, 1H, OCH2O), 5.02 (d,
J ¼ 7.7 Hz, 4H, 2 ꢂ CH2O), 4.71 (d, J ¼ 5.0 Hz, 1H, CH2O2), 4.65 (s, 2H,
CH2), 4.59e4.54 (m, 5H, 2 ꢂ CH2O, H-ring), 4.24 (t, J ¼ 6.5 Hz,1H, H-
ring), 4.07e3.98 (m, 4H, H-ring), 2.28 (s, 6H, 2 ꢂ CH3); 13C NMR
Di-(4-acyloxybenzyl) N, N-diisopropylphosphoramidite (1.5 eq
for per hydroxyl in compound 4) was stirred with 1H-tetrazole
(equal equivalent of phosphoramidite) in dry CH2Cl2 under argon
for 30 min at room temperature. Compound 4 was added and the
resulting mixture stirred overnight. The mixture was cooled to 0 ꢀC,
and 3-chloroperoxybenzoic acid (equal equivalent of phosphor-
amidite) was added. The resulting mixture was allowed to warm to
room temperature and stirred for 2 h. The 3-chloroperoxybenzoic
(101 MHz, DMSO)
d 169.3, 150.4, 137.5, 133.4, 133.3, 131.6, 131.5,
129.1, 128.6, 128.3, 128.2, 127.8, 127.7, 127.6, 127.5, 127.4, 121.9, 121.4,
84.4, 80.1, 80.0, 75.0, 74.9, 70.9, 70.1, 69.5, 68.8, 68.2, 65.1, 20.7;
HRMS (MALDI): m/z 861.2650 [MþNa]þ (calcd for C46H47O13PNa,
861.2652).
41d: yield 52% (Rf ¼ 0.2, petroleum ether: ethyl acetate ¼ 2: 1, v/
v). 31P NMR (162 MHz, DMSO)
d
ꢁ1.91; 1H NMR (400 MHz, DMSO)
d
7.37e7.32 (m, 4H, HeAr), 7.30e7.18 (m, 15H, HeAr), 7.06e7.01 (m,
4H, HeAr), 5.26 (q, J ¼ 4.5 Hz, 1H, OCH(CH3)), 5.15 (dd, J ¼ 16.7,
8.5 Hz, 1H, CH2O), 4.99e4.88 (m, 4H, 2 ꢂ CH2O), 4.76 (d, J ¼ 11.6 Hz,
1H, CH2O), 4.70e4.55 (m, 5H, 2 ꢂ CH2O, H-ring), 4.47e4.40 (m, 2H,
H-ring), 4.33 (t, J ¼ 7.6 Hz, 1H, H-ring), 4.06 (d, J ¼ 8.8 Hz, 1H, H-
ring), 3.90 (t, J ¼ 3.5 Hz, 1H, H-ring), 2.26, 2.27 (ds, 6H, 2 ꢂ CH3), 1.17
(d, J ¼ 4.7 Hz, 3H, CH3); 13C NMR (101 MHz, DMSO)
d 169.1, 150.3,
138.0,137.8, 137.7, 133.5, 133.4,128.8,128.7, 128.3,128.2, 128.1, 127.7,
127.5, 121.8, 121.7, 90.4, 78.8, 74.9, 72.4, 72.2, 71.8, 71.4, 70.3, 67.9,
67.8, 67.7, 67.3, 20.8, 20.6; HRMS (MALDI): m/z 875.2808 [MþNa]þ
(calcd for C47H49O13PNa, 875.2808).
41e: yield 69% (Rf ¼ 0.3, petroleum ether: ethyl acetate ¼ 1.5: 1,
v/v). 31P NMR (162 MHz, CDCl3)
d
ꢁ1.81, ꢁ2.04; 1H NMR (400 MHz,
CDCl3)
d 7.37e7.34 (m, 4H, HeAr), 7.30e7.21 (m, 9H, HeAr),
7.07e7.03 (m, 8H, HeAr), 5.51 (d,1H, J ¼ 0.8 Hz, CH(O)3), 5.32 (s, 2H,
CH2O), 5.08e5.06 (m, 5H, 2 ꢂ CH2O, ring-H), 4.95e4.93 (m, 2H,
CH2O), 4.92e4.90 (m, 1H, ring-H), 4.87 (dd, J ¼ 8.3, 3.1 Hz, 2H,
CH2O), 4.43, 4.58(AB, JAB ¼ 12 Hz, 2H, CH2O), 4.9e4.47 (m, 1H, ring-
H), 4.37e4.35 (m, 3H, ring-H), 2.32e2.31 (m, 12H, 4 ꢂ CH3); 13C
Fig. 6. IC50 values of the selected compounds against cancer cells and normal cell.
NMR (101 MHz, CDCl3) d 169.3, 150.9, 150.8, 129.3, 128.6, 128.1,

W. Chen et al. / European Journal of Medicinal Chemistry 93 (2015) 172e181
179
127.7, 121.8, 73.1, 71.8, 69.2, 69.1, 21.1; HRMS (MALDI): m/z
1055.2268 [MþNa]þ (calcd for C50H50O20P2Na, 1055.2268).
41f: yield 91% (Rf ¼ 0.2, petroleum ether: ethyl acetate ¼ 3: 4, v/
(t, J ¼ 16.8 Hz, 2H, H-ring), 3.41 (t, J ¼ 18.4 Hz, 1H, H-ring), 2.45 (s,
1H, CH≡), 2.34 (s, 3H, CH3), 2.31 (s, 9H, 3 ꢂ CH3); 13C NMR
(101 MHz, CDCl3)
d 169.9, 151.3, 139.0, 138.8, 133.8, 130.3, 130.0,
v). 31P NMR (162 MHz, CDCl3)
d
ꢁ0.11, ꢁ1.32, ꢁ1.83, ꢁ2.20; 1H NMR
129.7, 129.0, 128.6, 128.3, 128.2, 127.8, 122.7, 122.4, 83.0, 80.4, 80.2,
80.1, 78.7, 78.4, 78.3, 78.2, 76.3, 75.3, 69.6, 69.5, 69.4, 69.3, 61.4, 51.6,
21.8; HRMS (MALDI): m/z 1263.3518 [MþNa]þ (calcd for
(400 MHz, CDCl3)
d
7.41 (d, J ¼ 7.8 Hz, 8H, HeAr), 7.10 (d, J ¼ 8.1 Hz,
8H, HeAr), 5.11 (d, J ¼ 6.3 Hz, 8H, 4 ꢂ CH2O), 4.82e4.77 (m, 1H, H-
ring), 4.73e4.66 (m, 1H, H-ring), 4.62 (t, J ¼ 4.4 Hz, 1H, H-ring),
4.19e4.10 (m, 2H, H-ring), 3.49 (t, J ¼ 10.2 Hz, 1H, H-ring), 2.33 (s,
12H, 4 ꢂ CH3), 1.57 (s, 3H, CH3), 1.45 (s, 3H, CH3), 1.41 (s, 3H, CH3),
C66H66O20P2Na, 1263.3520).
41l: yield 81% (Rf ¼ 0.2, petroleum ether: ethyl acetate ¼ 1: 1, v/
v).31P NMR (162 MHz, CDCl3)
CDCl3) d7.31e7.22 (m, 8H, HeAr), 7.20e7.08 (m, 14H, HeAr),
d
ꢁ1.35, ꢁ1.91; 1H NMR (400 MHz,
1.30 (s, 3H, CH3); 13C NMR (101 MHz, CDCl3)
d
169.4, 150.7, 129.4,
129.3, 129.1, 121.8, 121.7, 121.6, 69.3, 68.8, 68.6, 27.8, 26.9, 25.9, 21.2;
HRMS (MALDI): m/z 1035.2578 [MþNa]þ (calcd for C48H54O20P2Na,
1035.2581).
7.03e6.98 (m, 3H, HeAr), 6.93e6.87 (m, 7H, HeAr), 6.83e6.80 (m,
4H, HeAr), 4.88e4.48 (m, 16H, 8 ꢂ CH2O), 4.42e4.35 (m, 1H, H-
ring), 4.28 (s, 1H, H-ring), 4.08e3.97 (m, 2H, H-ring), 3.41 (s, 2H, H-
ring), 2.23e2.20 (m, 12H, 4 ꢂ CH3); 13C NMR (101 MHz, CDCl3)
41g: yield 63% (Rf ¼ 0.2, petroleum ether: ethyl acetate ¼ 4: 1, v/
v). 31P NMR (162 MHz, CDCl3)
d
ꢁ0.95, ꢁ2.01; 1H NMR (400 MHz,
d 169.3, 168.2, 150.7, 150.6, 150.4, 138.5, 138.1, 137.6, 133.5, 133.1,
CDCl3)
d
7.40e7.39 (m, 5H, HeAr), 7.32e7.24 (m, 20H, HeAr),
133.0, 130.9, 129.3, 129.2, 129.0, 128.9, 128.8, 128.4, 128.3, 128.2,
128.1, 127.7, 127.6, 127.5, 127.3, 127.2, 127.1, 121.9, 121.8, 121.7, 121.5,
80.4, 79.9, 78.9, 78.4, 75.9, 75.1, 74.7, 74.6, 72.7, 68.8, 68.7, 68.6, 68.4,
21.1; HRMS (MALDI): m/z 1315.3830 [MþNa]þ (calcd for
7.15e7.11 (m, 5H, HeAr), 7.05e6.97 (m, 6H, HeAr), 5.40 (d,
J ¼ 8.4 Hz, 1H, H-ring), 5.11e4.71 (m, 16H, 8 ꢂ CH2O), 4.57 (d,
J ¼ 10.8 Hz, 1H, H-ring), 4.46 (t, J ¼ 9.2 Hz, 1H, H-ring), 3.97 (t,
J ¼ 9.2 Hz, 1H, H-ring), 3.91 (t, J ¼ 9.2 Hz, 1H, H-ring), 3.58 (t,
J ¼ 8.0 Hz, 2H, H-ring), 2.31 (s, 12H, 4 ꢂ CH3); 13C NMR (101 MHz,
C70H70O20P2Na, 1315.3833).
41m: yield 74% (Rf ¼ 0.3, petroleum ether: ethyl acetate ¼ 1: 2,
CDCl3)
d
169.2, 150.7, 150.4, 138.2, 138.0, 133.4, 133.3, 133.1, 133.0,
v/v). 31P NMR (162 MHz, CDCl3)
CDCl3) d7.44e7.26 (m, 12H, HeAr), 7.10e7.02 (m, 12H, HeAr), 5.46
d
ꢁ2.04, ꢁ2.16; 1H NMR (400 MHz,
132.9, 129.3, 129.0, 128.8, 128.7, 128.6, 128.4, 128.3, 128.2, 127.8,
127.7, 127.6, 127.5, 127.4, 127.2, 121.8, 121.7, 121.6, 121.5, 121.4, 79.7,
78.1, 77.8, 75.7, 74.6, 68.9, 68.7, 68.6, 21.1; HRMS (MALDI): m/z
1315.3828 [MþNa]þ (calcd for C70H70O20P2Na, 1315.3833).
(s, 1H, CH(O)3), 5.19e4.91 (m, 12H, 6 ꢂ CH2), 4.48e4.41 (m, 3H, H-
ring), 4.31 (s, 1H, H-ring), 3.97 (s, 1H, H-ring), 3.50 (s, 1H, H-ring),
2.34e2.30 (m, 18H, 6 ꢂ CH3); 13C NMR (101 MHz, CDCl3)
d 169.5,
41h: yield 63% (Rf ¼ 0.2, petroleum ether: ethyl acetate ¼ 1: 2, v/
169.4, 169.2, 150.9, 150.8, 150.7, 133.1, 132.9, 132.8, 132.7, 129.7,
129.6, 129.5, 129.4, 129.3, 122.0, 121.9, 121.8, 117.9, 70.3, 69.6, 69.5,
69.4, 69.2, 66.7, 21.1; HRMS (MALDI): m/z 1341.2510 [MþH]þ (calcd
for C61H61O27P3Na 1341.2511).
v). 31P NMR (162 MHz, CDCl3)
d
ꢁ2.13; 1H NMR (400 MHz, CDCl3)
d
7.26e7.18 (m, 13H, HeAr), 6.97 (d, J ¼ 7.2 Hz, 8H, HeAr), 5.43 (s,
1H, CH(O)3), 4.96e4.94 (m, 2H, CH2O), 4.86 (s, 2H, CH2O), 4.84 (s,
2H, CH2O), 4.83 (s, 2H, CH2O), 4.81 (s, 2H, CH2O), 4.50 (s, 2H, H-
ring), 4.27 (s, 3H, H-ring), 3.76 (s,1H, H-ring), 2.23 (s, 12H); 13C NMR
41n: yield 78% (Rf ¼ 0.2, petroleum ether: ethyl acetate ¼ 1.5: 1,
v/v). 31P NMR (162 MHz, CDCl3)
CDCl3) d7.32e7.29 (m, 5H, HeAr), 7.24e7.13 (m, 16H, HeAr),
d
ꢁ1.42, ꢁ1.80; 1H NMR (400 MHz,
(101 MHz, CDCl3)
d
169.2, 151.3, 137.2, 132.7, 129.4, 128.4, 127.96,
122.0, 121.9, 77.2, 71.2, 70.6, 70.5, 69.8, 69.7, 69.3, 69.2, 65.2, 53.4,
7.06e6.98 (m, 8H, HeAr), 6.95e6.87 (m, 10H, HeAr), 5.02e4.61 (m,
18H, 9 ꢂ CH2), 4.87 (d, 1H, J ¼ 8.0 Hz, H-ring), 4.37 (t, 1H, J ¼ 9.2 Hz,
H-ring), 3.87 (t, 1H, J ¼ 8.0 Hz, H-ring), 3.81 (t, 1H, J ¼ 8.0 Hz, H-
ring), 3.48 (t, 2H, J ¼ 9.2 Hz, H-ring), 2.23e2.20 (m, 18H, 6 ꢂ CH3);
50.8, 21.1; HRMS (MALDI): m/z 1055.2270 [MþNa]þ (calcd for
C
50H50O20P2Na, 1055.2268).
41i: yield 81% (Rf ¼ 0.2, petroleum ether: ethyl acetate ¼ 1: 1, v/
v). 31P NMR (162 MHz, CDCl3)
d
ꢁ2.27, ꢁ2.40; 1H NMR (400 MHz,
13C NMR (101 MHz, CDCl3)
d 169.3, 169.2, 150.8, 150.7, 150.6, 150.4,
CDCl3)
d
7.41e7.31 (m, 9H, HeAr), 7.28e7.23 (m, 9H, HeAr),
138.2,138.1, 137.2,133.5,133.4,133.3,133.2, 133.1,133.0, 129.3,129.1,
128.9,128.6,128.4,128.3,128.1,128.0,127.9,127.7,127.5,121.9,121.8,
121.7, 121.6, 121.5, 82.7, 80.6, 79.5, 78.4, 76.2, 75.9, 75.8, 75.5, 72.8,
68.9, 68.8, 68.6, 66.7, 66.6, 64.7, 21.1; HRMS (MALDI): m/z 1601.4071
[MþNa]þ (calcd for C81H81O27P3Na, 1601.4076).
7.08e7.03 (m, 8H, HeAr), 5.51 (d, J ¼ 4.4 Hz, 1H, OCH2O), 5.10e4.83
(m, 10H, 5 ꢂ CH2O), 4.64 (d, J ¼ 4.4 Hz, 1H, OCH2O), 4.62 (s, 2H,
CH2O), 4.60e4.57 (m, 1H, H-ring), 4.52e4.49 (m, 2H, H-ring), 4.38
(s, 1H, H-ring), 4.15e4.13 (m, 2H, H-ring), 2.32 (s, 12H, 4 ꢂ CH3); 13
C
NMR (101 MHz, CDCl3)
d
169.2, 150.8, 137.3, 133.3, 129.3, 129.1,
41o: yield 80% (Rf ¼ 0.3, petroleum ether: ethyl acetate ¼ 1: 2, v/
128.5, 128.4, 127.9, 127.8, 127.5, 121.8, 87.5, 72.0, 70.5, 70.3, 69.1,
68.9, 68.8, 68.7, 21.1; HRMS (MALDI): m/z 1147.2888 [MþNa]þ
(calcd for C57H58O20P2Na, 1147.2894).
v). 31P NMR (162 MHz, CDCl3)
d
ꢁ2.04, ꢁ2.37, ꢁ2.46; 1H NMR
(400 MHz, CDCl3)
d 7.26e7.19 (m, 10H, HeAr), 7.17-7.13 (m, 8H,
HeAr), 6.97e6.91 (m, 11H, HeAr), 5.08 (d, 1H, J ¼ 4.8 Hz, OCH2O),
5.03e4.75 (m, 15H, 7 ꢂ CH2, OCH2O), 4.53e4.49 (m, 2H, H-ring),
4.44 (s, 1H, H-ring), 4.41 (s, 1H, H-ring), 4.29 (s, 1H, H-ring), 3.41 (s,
1H, H-ring), 2.22e2.21 (m, 18H, 6 ꢂ CH3); 13C NMR (101 MHz,
41j: yield 71% (Rf ¼ 0.3, petroleum ether: ethyl acetate ¼ 1: 1.5,
v/v). 31P NMR (162 MHz, CDCl3)
CDCl3) d 7.23e7.17 (m, 18H, HeAr), 6.95 (m, 4H, HeAr), 6.90 (m, 4H,
d
ꢁ2.16, ꢁ2.18; 1H NMR (400 MHz,
HeAr), 5.07 (d, J ¼ 4.8 Hz, 1H, OCH2O), 5.02e4.90 (m, 6H,
3 ꢂ CH2O), 4.87e4.84 (m, 3H, CH2O, OCH2O), 4.59e4.55 (m, 2H, H-
ring), 4.52e4.44 (dd, 4H, J ¼ 12.0, 8.0 Hz, 2 ꢂ CH2O), 4.29e4.24 (m,
2H, H-ring), 3.99e3.94 (m, 2H, ring-H), 2.24e2.22 (m, 12H); 13C
CDCl3) d 169.2, 169.1, 150.9, 150.8, 150.7, 146.1, 137.1, 133.2, 132.9,
129.2, 129.1, 128.4, 127.9, 127.6, 121.9, 121.7, 85.1, 79.3, 77.4, 77.2,
72.1, 69.1, 68.9, 68.8, 21.1; HRMS (MALDI): m/z 1433.3132 [MþNa]þ
(calcd for C68H69O27P3Na, 1433.3137).
NMR (101 MHz, CDCl3)
d
170.0, 169.9,151.5, 151.4, 138.0,133.9, 133.7,
41p: yield 93% (Rf ¼ 0.5, petroleum ether: ethyl acetate ¼ 1: 4, v/
133.6, 130.2, 129.9, 129.8, 129.1, 128.4, 128.3, 122.7, 122.6, 122.5,
122.4, 85.8, 80.6, 77.9, 74.7, 72.6, 72.3, 70.8, 70.7, 69.7, 69.4, 61.1, 54.1,
21.8; HRMS (MALDI): m/z 1147.2890 [MþNa]þ (calcd for
v). 31P NMR (162 MHz, CDCl3)
d
ꢁ1.33, ꢁ2.09; 1H NMR (400 MHz,
CDCl3)
d 7.33e7.28 (m, 5H, HeAr), 7.22e7.15 (m, 14H, HeAr),
7.11e7.00 (m, 5H, HeAr), 6.93e6.86 (m, 10H, HeAr), 5.01e4.65 (m,
16H, 8ХCH2), 4.50 (s,1H, H-ring), 4.34e4.27 (m, 4H, 2H-ring, OCH2),
3.81 (t, 2H, J ¼ 9.2 Hz, H-ring), 3.38 (t,1H, J ¼ 8.8 Hz, H-ring), 2.34 (s,
1H, CH≡), 2.21e2.19 (m, 18H, 6 ꢂ CH3); 13C NMR (101 MHz, CDCl3)
C
57H58O20P2Na, 1147.2894).
41k: yield 76% (Rf ¼ 0.5, petroleum ether: ethyl acetate ¼ 1: 2 v/
v). 31P NMR (162 MHz, CDCl3)
d
ꢁ1.74; 1H NMR (400 MHz, CDCl3)
d
7.41e7.39 (m, 5H, HeAr), 7.33e7.29 (m, 12H, HeAr), 7.22e7.11 (m,
d 169.2, 150.6, 137.8, 133.3, 133.2, 133.1, 133.0, 129.2, 129.1, 129.0,
9H, HeAr), 7.04e7.00 (m, 5H, HeAr), 5.08 (m, 2H, CH2O), 4.92e4.88
(m, 8H, 4 ꢂ CH2O), 4.87e4.76 (m, 4H, 2 ꢂ CH2O), 4.53 (s,1H, H-ring),
4.47 (d, J ¼ 2.0 Hz, 2H, CH2O), 4.29 (t, J ¼ 12.8 Hz, 2H, H-ring), 4.03
128.9, 128.3, 128.1, 127.9, 127.7, 121.8, 121.7, 121.6, 81.9, 79.5, 79.1,
79.0, 77.2, 75.5, 74.8, 69.1, 69.0, 68.9, 68.8, 68.6, 60.8, 21.1; HRMS
(MALDI): m/z 1549.3760 [MþNa]þ (calcd for C77H77O27P3Na,

180
W. Chen et al. / European Journal of Medicinal Chemistry 93 (2015) 172e181
1549.3763).
129.5, 129.3, 129.1, 129.0, 128.9, 128.6, 128.5, 128.4, 128.3, 128.2,
41q: yield 55% (Rf ¼ 0.4, petroleum ether: ethyl acetate ¼ 1: 2, v/
127.9, 127.7, 127.5, 127.3, 127.2, 121.8, 121.7, 121.6, 121.5, 78.2, 77.4,
77.2, 72.7, 72.5, 69.2, 69.0, 68.8, 68.6, 21.1; HRMS (MALDI): m/z
1601.4075 [MþNa]þ (calcd for C81H81O27P3Na, 1601.4076).
41v: yield 65% (Rf ¼ 0.3, petroleum ether: ethyl acetate ¼ 1: 2, v/
v). 31P NMR (162 MHz, CDCl3)
(400 MHz, CDCl3)
d
ꢁ1.11, ꢁ1.36, ꢁ1.73; 1H NMR
d
7.40e7.20 (m, 19H, HeAr), 7.14e6.91 (m, 20H,
HeAr), 5.11e5.04 (m, 5H, 1HeCH2O, 2 ꢂ CH2O), 4.97 (d, J ¼ 8.0 Hz,
2H, CH2O), 4.93e4.90 (m, 1H, 1HeCH2O), 4.87e4.73 (m, 8H,
4 ꢂ CH2O), 4.69e4.62 (m, 2H, CH2O), 4.55e4.42 (m, 3H, H-ring),
3.97 (t,1H, J ¼ 9.6 Hz, H-ring), 3.88 (t,1H, J ¼ 9.6 Hz, H-ring), 3.58 (d,
1H, J ¼ 9.6 Hz, H-ring), 2.34e2.30 (m, 18H, 6 ꢂ CH3); 13C NMR
v). 31P NMR (162 MHz, CDCl3)
d
ꢁ0.88, ꢁ1.04, ꢁ2.40; 1H NMR
(400 MHz, CDCl3)
d 7.34e7.29 (m, 3H, HeAr), 7.22e7.05 (m, 21H,
HeAr), 6.93e6.80 (m, 15H, HeAr), 5.09e4.70 (m, 18H, 9 ꢂ CH2O),
4.58 (t, 1H, J ¼ 8.0 Hz, H-ring), 4.55 (t, 1H, J ¼ 8.0 Hz, H-ring),
4.44e4.39 (m, 2H, H-ring), 3.90 (t, 1H, J ¼ 9.6 Hz, H-ring), 3.41 (m,
1H, H-ring), 2.22-2.20 (m, 18H, 6 ꢂ CH3); 13C NMR (101 MHz, CDCl3)
(101 MHz, CDCl3)
d 169.4, 169.3, 169.2, 169.0, 150.8, 150.7, 150.6,
150.5, 150.4, 138.2, 137.9, 136.9, 133.5, 133.4, 129.3, 129.1, 128.8,
128,7, 128.6, 128.4, 128.2, 128.0, 127.3, 127.2, 127.1, 121.8, 121.7, 121.6,
121.5, 78.3, 77.9, 74.8, 74.6, 72.9, 69.0, 68.9, 68.7, 68.6, 68.5, 21.1;
HRMS (MALDI): m/z 1601.4068 [MþNa]þ (calcd for C81H81O27P3Na,
1601.4076).
d
169.2, 150.5, 137.9, 137.7, 133.4, 130.1, 129.7, 129.3, 129.2, 128.9,
128.8,128.7,128.6,128.5,128.3,128.2,127.6,127.3,121.8,121.7,121.6,
121.5, 77.4, 77.2, 68.8, 68.7, 68.6, 68.5, 68.2, 60.4, 21.1; HRMS
(MALDI): m/z 1601.4068 [MþNa]þ (calcd for C81H81O27P3Na,
1601.4076).
41r: 57% (Rf ¼ 0.3, petroleum ether: ethyl acetate ¼ 1: 2, v/v). 31
P
NMR (162 MHz, CDCl3)
d
ꢁ1.51, ꢁ1.74, ꢁ2.07; 1H NMR (400 MHz,
CDCl3)
d
7.32 (d, 2H, J ¼ 7.2 Hz, HeAr), 7.26e7.07 (m, 18H, HeAr),
7.02 (d, 2H, J ¼ 8.4 Hz, HeAr), 6.94e6.78 (m, 17H, HeAr), 5.02e4.90
(m, 4H, 2 ꢂ CH2O), 4.87e4.69 (m, 10H, 5 ꢂ CH2O), 4.63 (d, 2H,
J ¼ 8.0 Hz, CH2O), 4.60e4.53 (m, 2H, CH2O), 4.45 (s, 2H, H-ring),
4.37 (s, 1H, H-ring), 4.16 (t, 1H, J ¼ 7.6 Hz, H-ring), 3.50 (t, 1H,
J ¼ 9.2 Hz, H-ring), 3.42 (t, 1H, J ¼ 9.2 Hz, H-ring), 2.22e2.21 (m,
4.2. In vitro cytotoxicity of the synthesized compounds against
cancer and normal cells
Non-small cell lung cancer cell line A549, breast cancer cell line
MDA-MB-231, cervical carcinoma cell line HeLa and normal breast
epithelial cell line MCF10A were obtained from ATCC (American
type culture collection) and were maintained in 5% CO2 at 37 ꢀC.
A549, MDA-MB-231 and HeLa cells were grown in Dulbecco's
modified Eagle's medium (DMEM, Gibco) supplemented with 10%
fetal bovine serum (FBS, Omega Scientific) and 1% penicillin/
streptomycin (Omega Scientific). MCF10A cells were grown in
DMEM/F12 (Gibco) supplemented with 5% horse serum,
18H, 6 ꢂ CH3); 13C NMR (101 MHz, CDCl3)
d 169.3, 169.2, 150.8,
150.5, 141.5, 137.9, 137.2, 129.4, 129.3, 129.0, 128.9, 128.8, 128.4,
128.3, 128.2, 128.1, 127.8, 127.7, 127.6, 127.5, 127.2, 121.9, 121.8, 121.6,
121.5, 121.4, 77.2, 76.9, 76.8, 76.6, 76.4, 76.3, 76.2, 75.2, 74.7, 74.3,
73.7, 73.4, 72.8, 69.2, 68.7, 68.6, 68.4, 68.2, 21.1; HRMS (MALDI): m/z
1601.4073 [MþNa]þ (calcd for C81H81O27P3Na, 1601.4076).
41s: yield 77% (Rf ¼ 0.4, petroleum ether: ethyl acetate ¼ 1: 2, v/
v). 31P NMR (162 MHz, CDCl3)
d
ꢁ1.70, ꢁ1.91, ꢁ2.13; 1H NMR
20 ng mLꢁ1 EGF (Peprotech), 0.5
m
g mLꢁ1 hydrocortisone (Sigma),
(400 MHz, CDCl3)
d
7.27 (t, 4H, J ¼ 8.0 Hz, HeAr), 7.20e7.12 (m, 10H,
100 ng mLꢁ1 cholera toxin (Sigma), 10
m
g mLꢁ1 insulin (Sigma), 1%
HeAr), 7.09e7.03 (m, 7H, HeAr), 6.99e6.90 (m, 8H, HeAr),
6.86e6.84 (m, 7H, HeAr), 6.80 (t, 3H, J ¼ 8.0 Hz, HeAr), 4.97e4.84
(m, 4H, 2 ꢂ CH2O), 4.82e4.61 (m,12H, 6 ꢂ CH2O), 4.54e4.44 (m, 4H,
CH2O, 2H-ring), 4.29 (s, 1H, H-ring), 4.24 (t, 1H, J ¼ 8.0 Hz, H-ring),
4.08e4.01 (m, 1H, H-ring), 3.45e3.41 (m, 1H, H-ring), 2.21 (s, 18H,
Pen/Strep (Invitrogen). Cisplatin (Sigma) was dissolved in 1% NaCl
(Sigma) solution (pH 7). Approximately 1000 cells were seeded into
individual wells of 96-well tissue culture plates and incubated for
12 h, medium was 0.2 mL per well. The compound was diluted to
10 m
g mLꢁ1 using DMEM (final concentration in the test well), to
6 ꢂ CH3); 13C NMR (101 MHz, CDCl3)
d
169.3, 169.2, 169.1, 133.6,
analyze the inhibition effect on A549 roughly. After that, cells were
exposed to triplicates of different concentration solutions (from
132.9, 129.4, 129.3, 129.2, 129.0, 128.9, 128.4, 128.3, 128.2, 127.6,
121.8, 121.7, 121.6, 121.5, 77.2, 76.9, 76.8, 76.6, 76.5, 76.4, 68.9, 68.6,
21.14; HRMS (MALDI): m/z 1601.4071 [MþNa]þ (calcd for calcd for
0.39 to 50 m
g mLꢁ1) of test compounds to determine their potency.
The analyzed inhibitors were dissolved in DMSO reaching a final
DMSO concentration of 0.5%. Viability was normalized to control
cells which were treated with the vehicle, DMSO. After 72 h incu-
bation at 37 ꢀC and 5% CO2, cell viability was assessed by MTT assay.
Cells were replenished with fresh medium (0.1 mL per well) which
contains 10% MTT. Culture medium was removed after 4 h, and the
formazan was dissolved in DMSO (200 mL per well). Then OD570
were measured by Plate Reader (BIORAD). The IC50 values were
calculated by Origin 6.0.
C
81H81O27P3Na, 1601.4076).
41t: yield 68% (Rf ¼ 0.4, petroleum ether: ethyl acetate ¼ 1: 2, v/
v). 31P NMR (162 MHz, CDCl3)
d
ꢁ1.47, ꢁ1.63, ꢁ2.20; 1H NMR
(400 MHz, CDCl3)
d 7.38e7.36 (m, 6H, HeAr), 7.32e7.19 (m, 16H,
HeAr), 7.16 (t, 4H, J ¼ 8.0 Hz, HeAr), 7.03e7.00 (m, 6H, HeAr),
6.97e6.90 (m, 7H, HeAr), 5.11e4.83 (m, 14H, 7 ꢂ CH2O), 4.78e4.75
(m, 2H, CH2O), 4.65 (t, 2H, J ¼ 8.0 Hz, CH2O), 4.59e4.47 (m, 4H, H-
ring), 4.25 (t, 1H, J ¼ 8.0 Hz, H-ring), 3.51 (d, 1H, J ¼ 9.6 Hz, H-ring),
2.31 (s, 18H, 6 ꢂ CH3); 13C NMR (101 MHz, CDCl3)
d 169.4, 169.3,
169.2, 169.1, 150.9, 150.8, 150.7, 150.6, 150.5, 150.4, 129.4, 129.3,
129.2, 129.1, 129.0, 128.4, 128.3, 128.1, 127.9, 127.7, 127.6, 127.1, 121.8,
121.7, 121.6, 121.5, 121.4, 77.5, 77.2, 76.8, 72.7, 69.1, 68.9, 68.8, 68.6,
21.1; HRMS (MALDI): m/z 1601.4072 [MþNa]þ (calcd for
Acknowledgments
We thank Tianjin Municipal Natural Science Foundation (Key
Program No. 12JCZDJC22000), Ministry of Science and Technology
of China (2010CB126102, 2011BAE06B05) and National Science
Foundation for Fostering Talents in Basic Research of the National
Natural Science Foundation of China (NFFTBS, No. J1103306) for
financial support in this work.
C
81H81O27P3Na, 1601.4076).
41u: yield 80% (Rf ¼ 0.2, petroleum ether: ethyl acetate ¼ 1: 2, v/
v). 31P NMR (162 MHz, CDCl3)
d
ꢁ0.88, ꢁ1.41, ꢁ2.36; 1H NMR
(400 MHz, CDCl3)
d 7.32e7.30 (m, 3H, HeAr), 7.27e7.23 (m, 6H,
HeAr), 7.19-7.15 (m, 9H, HeAr), 7.12e7.03 (m, 8H, HeAr), 6.94e6.86
(m, 10H, HeAr), 6.82 (t, 3H, J ¼ 8.0 Hz, HeAr), 5.09e4.69 (m, 18H,
9 ꢂ CH2O), 4.61 (t, 1H, J ¼ 4.0 Hz, H-ring), 4.58 (t, 1H, J ¼ 4.0 Hz, H-
ring), 4.44 (d,1H, J ¼ 12.0 Hz, H-ring), 4.32 (t,1H, J ¼ 8.0 Hz, H-ring),
3.83 (t, 1H, J ¼ 12.0 Hz, H-ring), 3.41 (s, 1H, H-ring), 2.22-2.21 (m,
Appendix A. Supplementary data
18H, 6 ꢂ CH3); 13C NMR (101 MHz, CDCl3)
d
169.7,169.3,169.1,166.9,
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.01.064.
151.3, 150.8, 150.7, 150.5, 150.4, 139.9, 139.5, 138.1, 137.9, 133.3,

W. Chen et al. / European Journal of Medicinal Chemistry 93 (2015) 172e181
181
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