Synthesis and screening of novel inositol phosphonate derivatives for anticancer functions in vitro

Article abstract of DOI:10.1016/j.cclet.2014.11.008

Phosphonates have been frequently used as suitable isosteric and isoelectronic replacements for biologically important phosphates in the development of drugs or drug candidates because of their stability toward the action of phosphatases and other enzymes. In this paper, 12 mono-phosphonate inositol compounds were prepared with phosphonate instead of phosphate by two kinds of strategies, nucleophilic substitution and Arbuzov rearrangement, respectively. All compounds were evaluated in vitro for their activity against non-small cell lung cancer (NSCLC) cell line A549. Two compounds (3ac and 3bb) exhibited good antitumor activity at 10 μg/mL.

Full text of DOI:10.1016/j.cclet.2014.11.008

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CCLET-3142; No. of Pages 5
Chinese Chemical Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
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Original article
Synthesis and screening of novel inositol phosphonate derivatives
for anticancer functions in vitro
a,
a
b
a
b
a,
Wen-Bin Chen *, Jian-Bing Liu , Dao-Lei Dou , Fan-Bo Song , Lu-Yuan Li , Zhen Xi *
a
State Key Laboratory of Elemento-Organic Chemistry, Department of Chemical Biology, 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:
Phosphonates have been frequently used as suitable isosteric and isoelectronic replacements for
biologically important phosphates in the development of drugs or drug candidates because of their
stability toward the action of phosphatases and other enzymes. In this paper, 12 mono-phosphonate
inositol compounds were prepared with phosphonate instead of phosphate by two kinds of strategies,
nucleophilic substitution and Arbuzov rearrangement, respectively. All compounds were evaluated in
vitro for their activity against non-small cell lung cancer (NSCLC) cell line A549. Two compounds (3ac
Received 5 September 2014
Received in revised form 9 October 2014
Accepted 17 October 2014
Available online xxx
Keywords:
and 3bb) exhibited good antitumor activity at 10
mg/mL.
Inositol phosphonates
Anticancer
ß 2014 Wen-Bin Chen and Zhen Xi. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All
rights reserved.
Non-small cell lung cancer
Nucleophilic substitution
Arbuzov rearrangement
1
. Introduction
Penicillium brevicompactum in 1993 [17] and were regarded as
functionally analogous to InsP3. With 10- to 100-fold greater
2
+
Inositol phosphates (IPs), as important second messengers of
potencies for receptor affinity [18–21] and Ca release [22] than
InsP3 itself, adenophostins A and B opened up a whole new chapter
in searching for antagonists or agonists of InsP3 receptors (InsP3R).
However, the difficulties in synthesizing these InsP3-like deriva-
tives hindered the step of using these compounds as probes to
signal transduction, regulate many biological functions in cellular
signaling [1–5], which are related to various physical actions such
as cell proliferation, muscle contraction, apoptosis, and gene
transcription [6]. IPs also regulate DNA editing and repair [7],
vesicle transport [8], and ion channel regulation [9], and thus have
been implicated in diseases such as cancer and diabetes [10].
Among the known IPs, D-myo-inositol 1,4,5-trisphosphate (InsP3,
IP3), which is generated by the intracellular phosphatidylinositol-
specific phospholipase C (PI-PLC)-mediated hydrolysis of phos-
phatidylinositol 4,5-bisphosphate (PtdInsP2), is a biologically
2
+
investigate intracellular Ca signal transduction and its mecha-
nism. The other main obstacle is the instability of inositol
phosphate analogues to kinase, phosphatases, and lipases, which
impedes further development of potentially beneficial drugs [23].
Although the generation, mobilization, release of Ca and
transduction, termination of signal mediated by inositol phos-
phates are well known to date through several decades of intense
studies by biologists and chemists, this area is far more complex
and difficult than that originally imagined as investigation became
more in-depth. It is therefore clear that the development of
selective, membrane-permeant and enzymatic stable InsP3R
2+
2+
important intracellular Ca -mobilizing second messenger. InsP3
interacts specifically with its receptors (InsP3R) on the endoplas-
mic or sarcoplasmic reticulum membrane to release intracellular
2+
Ca into the cytosol [11–15].
The biochemical importance of InsP3 has promoted many
syntheses of InsP3 and their derivatives. Although a lot of active
analogues attained similar potencies to that of InsP3, little has
surpassed it [16]. The only breakthrough in this area was the
discovery of adenophostins A and B, isolated from culture broths of
2+
analogues is vital to enable further studies of Ca and InsP3
signaling.
Phosphonate, an isostere of phosphate, was extensively used to
replace phosphate and acted as phosphorylated substrates [24]
because of its stability toward the action of phosphatases [25,26]
and PI-specific phospholipases C [27]. Moreover, some inositol
2
phosphonate analogues with the core structure ‘‘inositol–CH –P’’
*
Corresponding authors.
have been synthesized to study their biological functions [28–32].
Studies with several cancer cell lines have shown that signal
E-mail addresses: wbchen@nankai.edu.cn (W.-B. Chen), zhenxi@nankai.edu.cn
(
Z. Xi).
http://dx.doi.org/10.1016/j.cclet.2014.11.008
001-8417/ß 2014 Wen-Bin Chen and Zhen Xi. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
1
Please cite this article in press as: W.-B. Chen, et al., Synthesis and screening of novel inositol phosphonate derivatives for anticancer
functions in vitro, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.11.008

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transduction mediated by inositol phosphates and phosphatidy-
linositol phosphates potentiated the effects of various cancer cell
lines, which suggested that they could be employed in combina-
tion drug therapy [33–35]. There are also other examples [36–39]
that phosphonates replaced phosphates in the drug development.
We previously reported a kind of inositol phosphonate analogues
using myo-inositol as the starting material, and found that two
phosphonate analogues exhibited relatively good cytotoxicity
against non-small cell lung cancer (NSCLC) cell line A549 [40].
Therefore, phosphonate moiety instead of the phosphate ester
group is a good alternative to resistant to enzymatic degradation
and has already achieved great success in the development of
antivirus drugs [39]. In continuation of our research program on
pursuing novel biologically active compounds in the fields of
human health, especially anticancer agents, we reported herein the
design and synthesis of a series of inositol phosphonate analogues
starting material was consumed completely. Then the mixture was
concentrated and the residue was separated by chromatography
on silica gel (PE/EA = 3:1–1:1) to give compound 5 as an oil.
2.3. Bioassay
NSCLC cell line A549 was obtained from ATCC (American type
2
culture collection) and were maintained in 5% CO at 37 8C. A549
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).
Approximately 1000 cells were seeded into individual wells of
96-well tissue culture plates and incubated for 12 h, medium was
0.2 mL/well. The compound was diluted to 10
mg/mL using DMEM
(final concentration in the well), to analyze the inhibition effect on
A549 roughly. The positive 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,
with the general structure ‘‘inositol–O–CH
CH CH –P’’ along with the initial antitumor activity evaluation
results.
2
–P’’ or ‘‘inositol–O–
2
2
DMSO. After 72 h incubation at 37 8C and 5% CO
assessed by MTT assay. Cells were replenished with fresh medium
0.1 mL/well) which contains 10% MTT. Culture medium was
removed after 4 h, and the formazan was dissolved in DMSO
200 L/well). Then OD570 were measured by Plate Reader
2
, cell viability was
(
2
. Experimental
Solvents were obtained from commercial sources and purified
(
m
(BIORAD).
according to the literature [41] if necessary. Nuclear magnetic
resonance spectra were recorded on Bruker AV 300 or 400 NMR
instrument in CDCl
3
. Chemical shifts (
d
) are reported in parts per
3. Results and discussion
million (ppm), relative to TMS as internal standard. High resolution
mass spectra (HR-MS) were carried out on IonSpec FTICR-MS
instrument from Varian using ESI as ionization mode. Flash-
column chromatography was performed using commercial grades
of silica gel 200–300 meshes. Analytical thin layer chromatography
In our previous work, we found mono-phosphate inositol
compounds had low anticancer activity (see Table 1) against non-
small cell lung cancer cell. We thus postulated that the instability
of the phosphates to most enzymes in the cell may compromise
their activities. Based on this hypothesis, we designed and
synthesized the phosphonate inositol derivatives as alternatives
of phosphate inositol compounds by introducing the known
(TLC) was performed on pre-coated silica gel 60 F254 plates, and
spot visualization was accomplished by UV light (254 nm) or
phosphomolybdic acid solution.
2 2 2
structural features such as P–CH –O and P–CH CH –O fragments
2
.1. General procedure for synthesis of methylenephosphonate
at the different positions of the inositol ring. In fact, the use of
phosphonates or phosphonic acids as analogues of natural
phosphates represents a more systematic approach to metabolic
regulation and enhancement or inhibition, than is commonly
attributed to ‘‘analogue’’ study [39]. The synthetic strategies of
these novel phosphonate inositol derivatives were depicted in
Scheme 1. A nucleophilic substitution of trifluoromethanesulfo-
nate-activated phosphonates with the protected inositols afforded
inositol compounds 3aa–3ad and 3ba–3bd
To
0.25 mmol) in dry THF (5 mL) at À60 8C under N
butyllithium (2.5 mol/L in THF, 0.3 mmol) by syringe. After string
0 min, the resulting mixture was added to (dialkoxyphosphinyl)
a
stirred solution of mono-hydroxy inositol 2a–d
(
2
was added n-
3
methyl triflate 1a or 1b (0.325 mmol) in 1 mL of dry THF by
syringe. Then the reactant was allowed to warm up to 0 8C over a
period of 1 h and stirred for 1 h at 0 8C. After the completion of
starting material by thin layer chromatography, the reactant was
compounds 3 containing P–CH
rearrangement of triethylphosphite with bromoethylinositols
furnished compounds 5 with P–CH CH –O fragment.
2
–O fragment, whereas an Arbuzov
2
2
quenched by saturated NH
with DCM (15 mL Â 4), dried over Na
DCM, the residue was purified by chromatography on silica gel
PE/EA = 2:1–1:1) to give compound 3 as an oil.
4
Cl aqueous (5 mL), and extracted
The starting (dialkyloxyphosphinyl)methyltriflate 1a and 1b
were prepared according to the reported procedures in three steps
2
SO . After concentration of
4
3
from PCl [42–44]. Reaction of paraformaldehyde with diethylpho-
(
sphite or dibenzylphosphite gave the dialkyl (hydroxymethyl)pho-
sphonate, which was converted to triflate 1a, 1b using 2,6-lutidine
as the base in total yield of 38% and 30%, respectively (Scheme 2).
2.2. General procedure for synthesis of ethylenephosphonate inositol
compounds 5a–d
Mono-hydroxyl inositol compounds 2 were obtained by
selective protection or ring-opening from commercially available
myo-inositol in two to three steps with desired yields [23].
Bromoethyl inositol compounds 4a–d were generated from the
A mixture of bromoethyl inositol 4a–d (0.03 mmol)and triethyl
phosphite (3 mL) was heated to 140 8C for 10 h, TLC showed the
Table 1
The inhibition rate against A549 (10 mg/mL).
Compd.
Inhibition rate (%)
Compd.
Inhibition rate (%)
Compd.
Inhibition rate (%)
Compd.
Inhibition rate (%)
Compd.
Inhibition rate (%)
3
3
3
3
aa
ab
ac
ad
<15
3ba
<15
5a
5b
5c
5d
<15
6a
6b
6c
6d
19.5Æ 4.2
26.1Æ 2.1
30.4 Æ 2.3
24.1Æ 4.6
7a
7b
7c
7d
35.6 Æ 4.4
57.7 Æ 0.9
23.1 Æ 1.3
20.8 Æ 9.0
<15
3bb
59.3 Æ 3.0
39.8 Æ 5.9
32.6 Æ 11.7
61.5 Æ 2.6
19.1 Æ 4.5
43.9 Æ 6.0
16.2 Æ 12.0
49.0 Æ 6.6
<15
3bc
3bd
DMSO
0.0 Æ 1.9
Cisplatin
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3
which were then treated with nucleophiles 2a–d under basic
condition to afford the title compounds 3 in good yields.
With respect to compound 5, we attempted to prepare them
according to the procedure of compounds 3 with 2-(diethoxypho-
sphinyl)ethyltriflate ((EtO)
yphosphinyl)methyl triflate 1a, but were not successful. The
reason of the unsuccessful attempts could be the -elimination
reaction of (EtO) P(O)CH CH OTf under basic condition to give the
-unsaturated phosphonate, diethyl vinylphosphonate, whose
2 2 2
P(O)CH CH OTf) instead of (diethox-
b
2
2
2
a, b
1
31
1
Scheme 1. Synthetic route of titled phosphonate inositol compounds 3 and 5.
structure was determined by H NMR and P NMR. H NMR
400 MHz, CDCl ): 6.27–5.94 (m, 3H, CH 55 CH), 4.03–3.99 (m, 4H,
2 Â CH CH O), 1.25 (t, 6H, J = 7.0 Hz, 2 Â CH
101 MHz, CDCl ): 135.36, 125.89 (d, J = 184.83 Hz), 61.76 (d,
J = 5.05 Hz), 16.25 (d, J = 6.06 Hz); P NMR (162 MHz, CDCl
7.18. Numerous methods have been reported in the literature for
(
3
d
2
13
mono-hydroxyl inositol compounds with 2-bromoethyl trifluor-
omethanesulfonate using 2,6-lutidine as the base. Although we
could not achieve satisfactory results by direct substitution of
protected inositol 2a–d with dibromoethane in basic condition
such as NaH, n-BuLi or DBU due to the low reactivity of the
reactants, we used the more reactive 2-bromoethyl trifluoro-
methanesulfonate instead of dibromoethane to make the desired
bromoethyl inositol 4a–d under lower temperature in acceptable
yields (Scheme 3).
3
2
3 2
CH O); C NMR
(
3
d
3
1
3
): d
1
the synthesis of phosphonate besides the above nucleophilic
substitution, the best known of which are the Arbuzov and
Michaelis–Becker reactions [47]. Because of the
b-elimination
reaction of (EtO) P(O)CH CH OTf, we thus tried the Arbuzov
2
2
2
rearrangement to prepare the title compounds 5. As shown in
Scheme 1, the mixture of triethylphosphite and brominated
inositol compounds 4a–d was refluxed for 10 h to obtain the final
products in good yields.
After protected mono-hydroxyl inositol derivatives 2a–d, our
first attempt to synthesize the final compounds 3aa used
(
RO)
2 2 2 2 2 2 2
P(O)CH Br, (RO) P(O)CH I, (RO) P(O)CH OTs or (RO) P(O)-
CH OMs as the phosphorylation reagents to react with 2a under
2
Having obtained the inositol mono-phosphonates, we then
assessed the antitumor activity against non-small cell lung cancer
(NSCLC) cell line A549. The results show most compounds have
low inhibition against A549, except compounds 3ac and 3bb,
which have about 50%–60% inhibition against A549, similar to that
of the positive control, Cisplatin. The structure-activity relation-
ship (SAR) shows benzyl phosphonates 3bb and 3bd exhibit more
potential anticancer activity than ethyl phosphonates 3ab and 3ad.
However, adding one methylene between inositol ring and
phosphorus atom (compounds 5) seems no improvement on the
anticancer activity in comparison with compounds 3aa–3ad. The
basic condition (NaH or n-BuLi), but no expected product was
obtained (Scheme 4). The reason may be the less reactive reactants
because of the steric hindrance of the protected inositol and the
weak electrophilicity of the phosphorylation reagents. So the
effective method was to enhance the reactivity of the phosphor-
ylation reagent or the protected inositol. Trifluoromethanesulfo-
nate (triflate) is an excellent leaving group and has been widely
used in organic synthesis [45]. Meanwhile (dialkyloxyphosphi-
nyl)methyltriflate is also easy to be obtained [46]. We therefore
synthesized the (dialkyloxyphosphinyl)methyltriflate 1a and 1b,
Scheme 2. Synthesis of (dialkyloxyphosphinyl)methyltriflate 1a and 1b.
Scheme 3. Synthesis of mono-hydroxyl inositol compounds 2a–d and bromoethyl inositol 4a–d. Reagents and conditions: (i) HC(OEt)
3%; (ii) NaH, BnBr, DMF, 0 8C–r.t., overnight, 68% for 2a, 48% for 2e, 74% for 2f; (iii) TBDMSCl, imidazole, DMF, r.t., overnight, 90%; (iv) DIBAL, CH
AlMe , CH Cl , 0 8C–r.t., 5 h, 68%; (vi) BrCH CH
OTf, THF, À60–08C, 2 h, 20% for 4a, 44% for 4b, 32% for 4c, 40% for 4d.
3
, PTSA monohydrate, DMF, 110 8C, 28 h,
8
2
Cl , 0 8C–r.t., 4 h, 95%; (v)
2
3
2
2
2
2
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Scheme 4. Synthesis of methylenephosphonateinositol compounds 3aa. Reagents and conditions: (i) TsCl, TEA, DCM; NaI or NaBr, acetone, reflux; (ii) TsCl or MsCl, TEA, DCM;
iii) Tf
O, 2,6-lutidine, DCM, À50 8C, 71%; (iv) NaH, DMF, 0 8C–r.t., overnight; and (v) n-BuLi, THF, À65–0 8C.
(
2
Scheme 5. The structures of compounds 6 and 7.
[
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3
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Please cite this article in press as: W.-B. Chen, et al., Synthesis and screening of novel inositol phosphonate derivatives for anticancer
functions in vitro, Chin. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.cclet.2014.11.008