Synthesis and Radioprotective Properties of Pulvinic Acid Derivatives

Article abstract of DOI:10.1002/cmdc.201000391

A high-throughput screening method has highlighted the marked antioxidant activity of some pulvinic acid derivatives (PADs) towards oxidation of thymidine, under γ and UV irradiation, and Fenton-like conditions. Here, we report the synthesis of a series of new hydrophilic PADs and the evaluation of their radioprotective efficacy in cell culture. Using a cell-based fluorescent assay, we show that some of these compounds have a pronounced ability to prevent cell death caused by radiation and to allow the subsequent resumption of proliferation. Thus, PADs may be considered as a novel class of radioprotective agents.

Full text of DOI:10.1002/cmdc.201000391

DOI: 10.1002/cmdc.201000391
Synthesis and Radioprotective Properties of Pulvinic Acid
Derivatives
Antoine Le Roux,[a] Stꢀphane Meunier,[a] Thierry Le Gall,[b] Jean-Marc Denis,[c]
Pierre Bischoff,*[d] and Alain Wagner[a]
Dedicated to Dr. Charles Mioskowski, deceased June 2007, who initiated this work.
A high-throughput screening method has highlighted the
marked antioxidant activity of some pulvinic acid derivatives
(PADs) towards oxidation of thymidine, under g and UV irradia-
tion, and Fenton-like conditions. Here, we report the synthesis
of a series of new hydrophilic PADs and the evaluation of their
radioprotective efficacy in cell culture. Using a cell-based fluo-
rescent assay, we show that some of these compounds have a
pronounced ability to prevent cell death caused by radiation
and to allow the subsequent resumption of proliferation. Thus,
PADs may be considered as a novel class of radioprotective
agents.
Introduction
The medical and industrial use of ionizing radiation has ex-
panded considerably during the last few decades, with applica-
tions ranging from the treatment and diagnosis of cancer to
power supply in nuclear plants. Consequently, the risk of acci-
dental exposure to ionizing radiation has appreciably in-
creased. Ionizing radiation consists of electromagnetic waves
(X-rays and g-rays), subatomic particles (a and b-particles, pro-
tons, neutrons) and atomic ions. When irradiated, water con-
tained in the cells generates reactive oxygen species (ROS) and
other free radicals. In turn, ROS react with DNA and additional
cellular material and components, including proteins and
membrane lipids. Living organisms have developed a number
of systems to cope with oxidative injuries (i.e., glutathione, thi-
oredoxin, superoxide dismutase). However, in the case of acute
or protracted irradiation, these mechanisms can be rapidly
overwhelmed. Whole-body irradiation results in acute radiation
syndrome (ARS).[1] Ionizing radiation can immediately affect all
organs (i.e., heart, brain, liver, lungs, kidneys, eyes) to varying
extents. The lympho-hematopoietic tissues and the gastroin-
testinal tract are the first tissues to be affected, due to the
acute sensitivity of lymphocytes and progenitor cells to radia-
tion. Death, cancers, or irreversible damage to the organism
can result from irradiation. The threat of accidental exposure
to ionizing radiation therefore represents a public health con-
cern, which fully justifies the need to develop novel radiopro-
tectants.[2] A radioprotectant is defined as a compound that,
when given before irradiation, may protect normal tissues
from the damaging effects of radiation by promoting cell
recovery and survival.
covered to date is amifostine.[7] This compound is currently
used as a cytoprotective agent in cancer patients undergoing
radiotherapy, but so far, its severe side-effects have hampered
its wider clinical applications. Thus, much remains to be done
to identify radioprotective agents without such drawbacks.
A high-throughput screening method that determines the
ability of a given chemical agent to protect thymidine from g
[a] Dr. A. Le Roux, Dr. S. Meunier, Dr. A. Wagner
Laboratory of Functional Chemo-Systems, UMR 7199
Universitꢀ de Strasbourg
74, route du Rhin 67401 Illkirch-Graffenstaden (France)
[b] Dr. T. Le Gall
Bioorganic Chemistry and Labeling Service
Le Commissariat ꢁ l’ꢂnergie Atomique (CEA)
Centre de Saclay, 91191 Gif-sur-Yvette Cedex (France)
[c] J.-M. Denis
UCL-IMRE, Facultꢀ de Mꢀdecine, Universitꢀ Catholique de Louvain
1200 Bruxelles (Belgium)
[d] Dr. P. Bischoff
There are currently several pharmacological approaches to
reduce the harmful consequences of irradiation.[3,4,5] Among
them, antioxidants such as polyphenols and thiols have been
broadly studied.[6] These compounds act as radical scavengers
by trapping free radicals before they attack DNA and other cel-
lular materials. One of the most efficient radioprotectants dis-
Laboratoire de Radiobiologie, EA 3430, Universitꢀ de Strasbourg
Centre rꢀgional de Lutte contre le Cancer Paul Strauss
3, rue de la Porte de l’Hꢃpital, BP 42, 67065 Strasbourg (France)
Fax: (+33)388-258-500
E-mail: pbischoff@strasbourg.fnclcc.fr
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201000391.
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irradiation has revealed that norbadione A (1), a polyphenol
present in mushrooms (Xerocomic badius and Pisolithus tinctor-
ius), exhibits strong antioxidant activity under g and UV expo-
sure.[8a,b] Subsequently, tests in cell culture and mice revealed
that norbadione A was also endowed with radioprotective
properties, but was too toxic to be used as a radioprotector.[9]
Norbadione A is constituted of two similar subunits derived
from xerocomic acid (2), a member of the family of the pulvin-
ic acid, which are well known as potent antioxidant com-
pounds.[8a,b,10] In order to develop new radioprotective agents,
we focused on the synthesis of hydrophilic pulvinic acid deriv-
atives (PADs). We describe here their synthesis and their radio-
protective activity on cultured lymphoid cells, evaluated using
a fluorometric assay. Results indicate that some of these com-
pounds can efficiently protect cells against radiation.
of sodium ethoxide to give 5, which exists in the cyclized
form. Acid hydrolysis gave access to 6, which was then dehy-
drated in refluxing acetic anhydride (Ac2O) to give bis-lactone
3 in 10% yield over three steps.
Bis-lactone 4 was synthesized from a tetronic acid according
to Mioskowski’s procedure.[13] Methyl 4-methoxyphenylacetate
and ethyl glycolate were refluxed in tetrahydrofuran (THF) in
the presence of sodium tert-butoxide to furnish the tetronic
acid 7 in 72% yield (Scheme 3). After protection by dimethyl
sulfate to give 8 in quantitative yield, deprotonation with
n-butyl lithium and condensation on methyl pyruvate gave
access to the tertiary alcohol 9, which was then dehydrated to
give 10 as a mixture of Z and E isomers (E/Z=70:30) with a
total yield of 88% over three steps.[14] The mixture of both iso-
mers was then reacted with magnesium bromide in N,N-dime-
thylformamide (DMF) to demethylate the enol and the ester in
one pot to give acid 11 as a single isomer in 78% yield. The
acid was then cyclized in refluxing Ac2O to give lactone 4 with
a total yield of 46% from the starting material, methyl 4-me-
thoxyphenylacetate. This procedure has been developed for
scale-up synthesis and using on a multiple gram scale.
With intermediates 3 and 4 in hand, we focused then on the
insertion of hydrophilic amines.
Results and Discussion
Chemistry
Pulvinic acids are the opened form of a bis-lactone (Scheme 1).
When reacted with an amine, one lactone is opened to furnish
This was achieved by the intro-
duction of amines bearing differ-
ent hydrophilic groups, such as
amines, alcohols, ethers, carbox-
ylic acid, or a glucose moiety
(Figure 1).
Scheme 1. Synthesis and reactivity toward amines of bis-lactones 3 and 4.
N-(2-Aminoethyl)morpholine
was successfully introduced on 3
the corresponding amide. We recently reported that unsym-
metrical mono-aromatic bis-lactones can selectively be ring-
opened by an amine in the presence of tetra-n-butylammoni-
um fluoride (TBAF).[11] This ability to be easily opened by
amines will be used to insert hydrophilic groups on both
mono-aromatic and di-aromatic bis-lactones 3 and 4.
and 4 to afford 12a and 13a in 95% and 88% yield, respec-
tively. The insertion of a glucose moiety required the prior syn-
thesis of amine 16, which was made according to the proce-
dure described by Osborne et al. (Scheme 4).[15] Glycosylation
of b-d-(+)-glucose pentaacetate with 2-bromoetanol in the
presence of boron trifluoride etherate (BF3·Et2O) gave access to
bromo derivative 14 in 51% yield. Substitution of the bromine
by sodium azide in DMF gave access to azide 15 in quantita-
tive yield, which was then reduced under a hydrogen atmos-
phere in the presence of Pd/C to
Bis-lactone
3 was synthesized according to Volhard’s
method (Scheme 2).[12] 4-Methoxyphenylacetonitrile was con-
densed on diethyl oxalate in refluxing ethanol in the presence
yield 16 in 98% yield. Lactone
opening of 3 and 4 gave access
to the protected pulvinamides
12h and 13 f in 98% and 57%
yield, respectively. These were
then deprotected using a cata-
lytic amount of sodium methox-
ide in methanol to give 12b and
13b, in 94% and 58%, respec-
tively.
A diethyleneglycol chain was
inserted giving compounds 12c
and 13c in 98% and 24% yield,
respectively. Compound 3 was
then opened by glycine, N,N-di-
Scheme 2. Reagents and conditions: a) EtONa, EtOH, reflux; b) H2SO4, H2O; c) Ac2O, reflux, 10% (three steps).
methylethylenediamine, ethyle-
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Synthesis and Radioprotective Properties of Pulvinic Acid Derivativess
nediamine and 2-aminoethanol to afford 12d (74%),
12e (92%), 12 f (74%) and 12g (61%). Finally 4 was
reacted with N-Boc-piperazine and 3-methoxypropyl-
amine to afford 13d and 13e in 88% and 92%, re-
spectively. Derivative 13d was obtained as the tri-
fluoroacetate salt after quantitative deprotection
using trifluoroacetic acid (TFA) (Scheme 5).
After synthesizing all of the derivatives, we turned
our attention to their aqueous solubility in phos-
phate-buffered saline (PBS; Figure 2). The most solu-
ble derivatives were those bearing a carboxylic acid
functionality or a glucose moiety, with solubility
values ranging from 4.02 mm (12d) to 15.10 mm (11).
Derivatives bearing an amine or an alcohol function-
ality were less soluble, with solubility values ranging
from 0.003 mm (12a) to 1.04 mm (13c).
Scheme 3. Reagents and conditions: a) tBuOK, THF, reflux, o/n, 72%; b) Me2SO4, K2CO3,
acetone, reflux, o/n, quant.; c) methyl pyruvate, LDA, THF, ꢀ788C, 1 h; d) TFAA, Et3N,
DMAP (cat), CH2Cl2, 08C 1 h; then 258C, o/n, 88% (two steps); e) MgBr2, DMF,
1208C, 5 h, 78%; f) Ac2O, reflux, 1 h, 94%.
Figure 2. Water solubility of compounds 6, 11, 12a-g and 13a-e.
Biology
The biological evaluation of radioprotectants relies predomi-
nantly on in vivo assays, which require the use of significant
numbers of animals, in most cases mice. In contrast, the as-
sessment of the antioxidant potency of compounds can be de-
termined using a variety of in vitro techniques, such as the te-
lomere repeat amplification protocol (TRAP),[16] oxygen radical
absorbance capacity (ORAC),[17] total oxyradical scavenging ca-
pacity (TOSC),[18] ferric reducing ability of plasma (FRAP),[19] and
2,2-diphenyl-l-picrylhydrazyl (DPPH) trapping assays. However,
the antioxidant potency and radioprotective activity of a mole-
cule do not always correlate. For this reason, we focused our
study on an in vitro assay based on X-ray irradiation of radio-
sensitive cells, and followed the effects of irradiation on their
viability and ability to resume division in the presence and
absence of varying concentrations of our PADs.
As the cellular assay requires sterile solutions, we were
forced to use the most soluble pulvinic acid derivatives. There-
Figure 1. Structures of the different synthesized hydrophilic PADs with yield
from corresponding bis-lactone 3 or 4.
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TK6 cells were irradiated by X-
rays at 8 Gy in the presence or
absence (control) of the respec-
tive PADs. Two days (48 h) after
irradiation, the culture medium
was removed by centrifugation
and the cell pellets were resus-
pended in the same volume of
fresh medium to prevent any
possible interference of the
PADs on cell proliferation. Cells
were then seeded in 96-well flat-
bottomed microplates and main-
tained for variable lengths of
time at 378C in a 5% CO2, hu-
midified atmosphere. For each
experiment group, five wells
Scheme 4. Reagents and conditions: a) 2-bromoethanol, BF3·Et2O, CH2Cl2, 08C!RT, o/n, 51%; b) NaN3, DMF, 608C,
4 h, quant.; c) H2, Pd/C 10%, THF, o/n, quant.; d) R=Me: TBAF, THF, see Ref. [11]; e) R=4-MeOC6H4: CH2Cl2, RT,
o/n; f) MeONa (cat), MeOH, RT, o/n, R=Me: 58%, R=4-MeOC6H4: 94%.
Scheme 5. Reagents and conditions: a) TBAF, THF, ꢀ788C!RT, 1 h (see
Ref. [11]); b) TFA, CH2Cl2, 1 h, 88%
fore, we chose to assess compounds 12b–d, 13b, 6 and 11 for
their toxicity on cultured cells. For this purpose, we selected
the TK6 cell line,[20] a human lymphoid, p53 wild-type, actively
dividing cell line. TK6 cells are frequently used in radiobiologi-
Figure 3. Viable cell numbers were recorded 24 h after incubation in the
presence of six pulvinic acid derivatives at 50 (&) and 100 mm (&), or PBS
(control). No significant cytotoxicity was detected. Data are the mean of
three independent determinations ꢁ standard error (SE). Cell number is
expressed in arbitrary fluorescence unit (AFU).
cal investigations due to their high radiosensitivity. Indeed, the
surviving fraction after irradiation by X-rays at 2 Gy (SF2) is
6.2%, and only 8.29ꢂ10ꢀ6 % after irradiation at 8 Gy (SF8), as
determined by clonogenic survival. They also have a pro-
nounced tendency to undergo apoptosis in response to radia-
tion and thus are well suited for the analysis of molecular
mechanisms underlying the induction of this type of cell
death.[21] Since the inhibition of apoptosis does not always re-
flect the ability of antioxidants to protect against radiation,[22]
we measured the number of viable cells using the fluorescent
reagent, alamar blue.[23] In radioprotection assays, compounds
are usually tested at relatively high concentrations, up to
1 mm;[24] however, we decided to tested PADs at 50 mm and
100 mm. First, the toxicity of PADs at those concentrations was
evaluated by incubating cells in the presence of the com-
pound for 24 h and comparing the number of living cells to
those of the PBS-treated controls (Figure 3). Using this proce-
dure, no loss of viability was observed. Under the same condi-
tions, amifostine appeared to be toxic toward TK6 cells, and
thus was not incorporated in our tests.
were prepared. Measurements were performed on day 2, 4, 7
and 9 post-irradiation. Results of experiments carried out with
compounds 6 and 12c are represented in Figure 4.
We observed that the cell numbers progressively decreased
during the first week after irradiation, irrespective of the treat-
ment they had received (Figure 4). This is due to the massive
loss of viability of TK6 cells following an irradiation at this
dose. However, on day 7, in the presence of 12c, the rate of
cell loss was reduced. By day 9, treated and nontreated cells
had resumed proliferation. In the control cells, the proliferation
levels were low, but in cells treated with 6 and 12c prior to ir-
radiation, the rate of proliferation recovered faster. Five times
more living cells were observed in cells treated with the pulvi-
namide 12c.
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Synthesis and Radioprotective Properties of Pulvinic Acid Derivativess
be devoid of cytotoxicity and afforded moderate (11,
12b, 12d) to pronounced (12c, 13b) protection to
TK6 cells against radiation. No loss of viability was
observed with 12c, even at 400 mm, the highest con-
centration used. Among the six derivatives, 13b dis-
played the best protection, with greater than sixfold
more living cells at 100 mm than untreated cells. It is
also interesting to note that 6 was weakly active, that
11 was less active at 100 mm than at 50 mm, and that
12d gave the same protection at 50 and 100 mm. To-
gether, these observations lead us to suggest that
the presence of a carboxylic acid moiety could de-
crease the activity by developing a pro-oxidant activi-
ty. On the other hand, it remains to be established
whether aqueous insolubility of the agent and the ra-
dioprotective effect are related. Forthcoming work
will consist of verifying the ability of PADs to protect
animals from the harmful consequences of a whole-
body irradiation. We also intend to provide a better
understanding of the mechanisms underlying the ra-
dioprotective activity of PADs, in an attempt to im-
prove their efficiency as radioprotectants.
Figure 4. Viable cell number of TK6 treated with 6, 12c or untreated and irradiated at
8 Gy, as a function of the time post-irradiation.
Experimental Section
The other derivatives were tested using the same protocol.
Figure 5 shows the normalized data recorded at day 9 (non-
Chemistry
General method: Commercial reagents were used without addi-
tional purification. Anhydrous tetrahydrofuran (THF) was obtained
by distillation over sodium and benzophenone. Analytical thin-
layer chromatography (TLC) was performed using plates cut from
glass sheets (silica gel 60F-254; Merck). Visualization was achieved
treated cells value set at 100). Surprisingly, compound 6 was
found to be less active than expected, with only 1.5 and 1.7
times more cells surviving at 50 and 100 mm, respectively, as
compared to nontreated cells. The other derivatives also pro-
vided good levels of protection, with 3.5–5.1 times more living
cells observed at 50 mm than untreated cells, and 3.1–6.2 times
more living cells at 100 mm. Interestingly, compound 11 was
less active at 100 mm than at 50 mm, suggesting the presence
of a pro-oxidant activity at higher concentrations. Indeed,
some polyphenols, such as resveratrol, are known to be both
pro-oxidant and antioxidants,[25] and this possibility cannot be
ruled out.
Next, we selected 12c for a concentration effect study. This
derivative was assessed for its radioprotective activity at con-
centrations ranging from 12.5 mm to 400 mm, and cell number
measurements were carried out on day 9 after irradiation by X-
rays at 8 Gy (Figure 6).[26] We observed a steady concentration-
dependent effect. At the highest tested concentration
(400 mm), no loss of viability was recorded. However, the rate
of cell growth recovered more slowly at lower concentrations
than at higher ones. Indeed, from 100 mm to 400 mm, the
number of viable cells only increased by a factor of 1.5.
Conclusions
Figure 5. Effect of pulvinic acid derivatives upon TK6-cell survival following
exposure to 8 Gy radiation. Compound concentrations were 50 (&) or
100 mm (&); control cells were incubated in PBS. Alamar blue test was per-
formed nine days after irradiation. Results are from a single representative
experiment. Values are the mean of five determinations ꢁ standard error
(SE). Cell number is expressed in normalized arbitrary fluorescence unit
(AFU).
In conclusion, we have synthesized several hydrophilic PADs
and assessed their capacity to protect lymphoid cells from ra-
diation. We described the scalable synthesis of a common pre-
cursor, the bis-lactone 4, and the easy insertion of several hy-
drophilic groups on 3 and 4. These compounds were found to
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ed with concd HCl to pH 1 and the mixture was extracted with
EtOAc. The combined organic phases were dried (MgSO4), filtered
and concentrated. Compound 7 crystallized out of solution during
evaporation and was collected and washed with Et2O. The filtrate
was concentrated, and the operation repeated until no further
crystals were obtained (total yield: 78%); 1H NMR (300 MHz,
[D6]DMSO): d=3.75 (s, 3H), 4.74 (s, 3H), 6.94 (d, J=9.0 Hz, 2H),
7.84 (d, J=9.0 Hz, 2H), 12.58 ppm (s, 1H); 13C NMR (75 MHz,
[D6]DMSO): d=55.0, 66.0, 97.3, 113.5, 122.9, 127.6, 157.6, 173.1,
173.3 ppm; MS (ESI): m/z: 207.1 [M+H]+.
4-Methoxy-3-(4-methoxyphenyl)furan-2(5H)-one
(8):
K2CO3
(20 mmol) and dimethyl sulfate (20 mmol) were added to a sus-
pension of 7 (20 mmol) in acetone (85 mL). The mixture was re-
fluxed for 4 h. After cooling to RT, the mixture is filtered through
celite, and the filtrate was concentrated to give 8 as a white solid
(yield: 99%); 1H NMR (200 MHz, CDCl3): d=3.76 (s, 3H), 3.86 (s,
3H), 4.72 (s, 2H), 6.86 (d, J=8.8 Hz, 2H), 7.77 ppm (d, J=8.8 Hz,
2H); 13C NMR (50 MHz, CDCl3): d=55.3, 58.0, 64.6, 102.1, 113.7,
121.9, 128.9, 158.9, 172.3, 173.1 ppm; MS (ESI): m/z: 221.1 [M+H]+.
Figure 6. The dose-dependent radioprotective efficacy of derivative 12c on
TK6-cell survival measured nine days after exposure to 8 Gy radiation.
Methyl 2-(3-methoxy-4-(4-methoxyphenyl)-5-oxofuran-2(5H)-yli-
dene)propanoate (10): A solution of diisopropylamine (30 mmol)
in anhydrous THF (125 mL) was cooled to ꢀ208C, then nBuLi
(30 mmol) was added dropwise. The mixture was stirred for 30 min
at that temperature and then cooled further to ꢀ788C. A solution
of 8 (20 mmol) in anhydrous THF (65 mL) was added dropwise,
and the mixture was stirred for 30 min at that temperature. Methyl
pyruvate (60 mmol) was then added dropwise. The mixture was
stirred for 30 min at ꢀ788C and then warmed to RT. Saturated aq
NH4Cl was added, the aqueous layer was extracted with EtOAc,
and the combined organic phases were dried (MgSO4), filtered and
concentrated. The resulting oil was diluted in CH2Cl2 (200 mL) and
cooled to 08C. Et3N (120 mmol) and DMAP (0.2 mmol) were then
added, followed by the addition of trifluoroacetic anhydride (TFAA;
60 mmol) dropwise. The mixture was stirred overnight at RT, and
then hydrolyzed by a 1m aq HCl. The aqueous phase was extract-
ed with CH2Cl2, and the combined organic phases were dried
(MgSO4), filtered and concentrated. The crude was then purified by
column chromatography (cyclohexane/EtOAc, 90:10!50:50) to
give 10 (yellow solid) as a mixture of E and Z isomers (E/Z=70:30;
yield: 88%); 1H NMR (300 MHz, CDCl3): E isomer: d=2.15 (s, 3H),
3.72 (s, 3H), 3.82 (s, 3H), 3.83 (s, 3H), 6.92–6.95 (m, 2H), 7.41–
7.43 ppm (m, 2H); Z isomer: d=2.27 (s, 3H), 3.80 (s, 3H), 3.83 (s,
3H), 3.86 (s, 3H), 6.93–6.96 (m, 2H), 7.40–7.43 ppm (m, 2H);
13C NMR (75 MHz, CDCl3): E isomer: d=15.2, 52.8, 55.6, 61.1, 109.0,
113.4, 114.2, 120.8, 131.4, 143.3, 160.3, 161.3, 168.2, 168.7 ppm; MS
(ESI): m/z: 305.1 [M+H]+.
under a 254 or 365 nm ultraviolet (UV) light and by immersion in
an EtOH solution of Ce(SO4)2, followed by treatment with a heat
gun. Purification of compounds was performed by column chroma-
tography using silica gel 60 (Merck). Infrared (IR) spectra were re-
corded on a Nicolet 380 FT-IR spectrometer (Thermo Electron Cor-
poration) with samples prepared as a CH2Cl2 solution or neat solid
on a diamond plate. 1H and 13C NMR spectra were recorded at
238C on a Bruker 400 MHz, Bruker 300 MHz or Brucker 200 MHz
spectrometer. Chemical shifts (d) are reported in parts per million
(ppm) and calibrated using the residual nondeuterated solvent
peak. Mutiplicity is given as follows: s=singlet, d=doublet, t=
triplet, q=quartet, quint=quintet, m=multiplet, br s=broad sin-
glet; coupling constants (J) are given in Hz. High-resolution mass
spectra (HRMS) were obtained using a Agilent Q-TOF 6520 and
mass spectra (MS) using a Agilent MSD 1200 SL (ESI/APCI) with a
Agilent HPLC 1200 SL. Melting points were measured on a Stuart
Scientific SMP3 apparatus (Bibby) and are uncorrected.
3,6-Bis(4-methoxyphenyl)fuoro[3,2-b]furan-2,5-dione (3): Sodium
(3.6 g, 156 mmol, 2 equiv) were dissolved in cooled absolute EtOH
(50 mL). After complete consumption of sodium, diethyl oxalate
(10.8 mL, 80 mmol, 1 equiv) and 4-methoxyphenylacetonitrile
(22.6 mL, 156 mmol, 2 equiv) were added. The mixture was then
stirred at 708C for 90 min, and then cooled to RT. Water (8 mL) was
then added, followed by acidification to pH 5 using acetic acid
(AcOH). The precipitate was then removed by filtration to give an
orange solid (9.55 g). This solid was then dissolved in AcOH
(20 mL) and water (10 mL), and concd H2SO4 (8.5 mL) was added
dropwise. After a red solid is formed, the mixture was heated at
reflux for 30 min, then cooled in an ice bath. The solid was collect-
ed by filtration and resuspended in Ac2O (15 mL). The suspension
was heated at reflux for 30 min then cooled to RT. The bis-lactone
3 was collected by filtration and washed with heptane (yield:
2-(3-Hydroxy-4-(4-methoxyphenyl)-5-oxofuran-2(5H)-ylidene)-
propanoic acid (11): MgBr2 (1 mmol) was added to a solution of
10 (1 mmol) in DMF (10 mL). The mixture was stirred at 1208C
until no more starting material remained. The solvent was removed
in vacuo, and the residue was diluted with water. The aqueous
phase was washed with CH2Cl2, and acidified with 1m aq HCl. The
aqueous phase was then extracted with EtOAc. Combined organic
phases were washed with brine, dried (Na2SO4), filtered and con-
centrated to give 11 as an orange solid (yield: 78%); 1H NMR
(200 MHz, CD3OD): d=2.11 (s, 3H), 3.81 (s, 3H), 6.92 (d, J=9.0 Hz,
2H), 8.02 ppm (d, J=9.0 Hz, 2H); 13C NMR (50 MHz, CD3OD): d=
14.5, 55.6, 103.9, 114.1, 114.6, 123.4, 129.9, 154.6, 160.6, 160.9,
168.3, 174.7 ppm; MS (ESI): m/z (%): 231.0 (100%) [MꢀCO2]ꢀ.
1
10%); H NMR (400 MHz, CDCl3): d=3.89 (s, 6H), 7.01 (d, J=8.6 Hz,
4H), 8.01 ppm (d, J=8.6 Hz, 2H); 13C NMR (100 MHz, CDCl3): d=
54.8, 96.4, 100.3, 115.3, 117.6, 130.0, 134.4, 159.5 ppm; IR (neat):
n˜ =595, 869, 1019, 1157, 1257, 1354, 1506, 1600, 1657, 1782,
1909 cmꢀ1
.
4-Hydroxy-3-(4-methoxyphenyl)furan-2(5H)-one (7): Ethyl glyco-
late (28 mmol) tBuOK (56 mmol) was added to a solution of methyl
4-methoxyphenylacetate (28 mmol) in THF (180 mL). The mixture
was refluxed overnight. After cooling to RT, the reaction was treat-
3-(4-Methoxyphenyl)-6-methylfuro[3,2-b]furan-2,5-dione
(4):
Compound 13 (3.6 mmol) was suspended in Ac2O (17 mL) and
566
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ChemMedChem 2011, 6, 561 – 569

Synthesis and Radioprotective Properties of Pulvinic Acid Derivativess
heated to reflux for 1 h. After cooling to RT, the crystallized com-
pound was collected by filtration and washed with pentane to
give 4 as a yellow crystalline solid (yield: 94%); H NMR (300 MHz,
CDCl3). 2.06 (s, 3H); 3.86 (s, 3H); 6.98 (d, J=9.1 Hz, 2H); 7.94 (d,
J=9.1 Hz, 2H)2.06 (s, 3H); 3.86 (s, 3H); 6.98 (d, J=9.1 Hz, 2H);
7.94 ppm (d, J=9.1 Hz, 2H); 13C NMR (75 MHz, CDCl3): d=7.64,
55.5, 98.8, 101.1, 114.8, 119.0, 129.9, 155.6, 159.3, 161.0, 166.1,
168.8 ppm; MS (ESI+APCI): m/z (%): 258.0 (100%) [M]+.
J=9.2 Hz, 2H), 10.87 ppm (br s, 1H); 13C NMR (100 MHz,
[D6]DMSO): d=33.9, 42.8, 54.8, 55.1, 57.1, 91.1, 112.1, 113.0, 113.8,
125.2, 126.1, 127.4, 129.8, 144.1, 155.5, 158.5, 168.3, 170.8,
174.7 ppm; IR (neat): n˜ =428, 518, 592, 666, 768, 809, 833, 901.3,
978, 1031, 1102, 1146, 1181, 1245, 1271, 1289, 1508, 1562, 1603,
1632, 1719, 2710, 2836, 3040, 3217 cmꢀ1; HRMS (ESI): m/z [M+H]+
calcd for C24H27N2O6: 439.1866, found: 439.1864.
1
N-(2-Aminoethyl)-2-(3-hydroxy-4-(4-methoxyphenyl)-5-oxofuran-
2(5H)-ylidene)-2-(4-methoxyphenyl)acetamide (12 f): Compound
12 f was obtained by opening 3 with ethylenediamine according
to the general procedure. The compound was purified by tritura-
General procedure for the synthesis of compounds 12a and
12c–h: The bis-lactone 3 (200 mg, 0.6 mmol) was suspended in
CH2Cl2 (10 mL), and the amine was added (1 equiv). The mixture
was stirred at RT for 15 min before concentration in vacuo and pu-
rification by column chromatography (CH2Cl2/MeOH, 100:0!
90:10) to furnish 12a, 12c–h (yield: 61–98%).
1
tion in CH2Cl2 and Et2O. Yield: 74%; H NMR (400 MHz, [D6]DMSO):
d=3.01 (m, 2H), 3.48 (m, 2H), 3.72 (s, 3H), 3.78 (s, 3H), 6.81 (d, J=
9.2 Hz, 2H), 6.98 (d, J=9.2 Hz, 2H), 7.58 (d, J=9.2 Hz, 2H), 8.10 (br
s, 2H), 8.19 (d, J=9.2 Hz, 2H), 8.62 ppm (t, J=6.0 Hz, 1H); 13C NMR
(100 MHz, [D6]DMSO): d=36.2, 54.8, 55.1, 90.1, 112.0, 112.9, 113.7,
125.2, 126.3, 127.8, 129.8, 144.3, 155.4, 158.4, 168.4, 171.1,
175.1 ppm; IR (neat): n˜ =434, 522, 667, 682, 832, 929, 988, 1030,
1092, 1152, 1179, 1259, 1298, 1411, 1509, 1547, 1604, 1639, 1712,
2836, 2934, 3065, 3227, 3371, 3501 cmꢀ1; HRMS (ESI): m/z [M+H]+
calcd for C22H23N2O6: 411.1557, found: 411.1551.
2-(3-Hydroxy-4-(4-methoxyphenyl)-5-oxofuran-2(5H)-ylidene)-2-
(4-methoxyphenyl)-N-(2-morpholinoethyl)acetamide (12a): Com-
pound 12a was obtained by opening 3 with N-(2-aminoethyl)mor-
pholine according to the general procedure. Yield: 95%; 1H NMR
(400 MHz, [D6]DMSO): d=3.32 (m, 6H), 3.60 (m, 6H), 3.73 (s, 3H),
3.78 (s, 3H), 4.01 (br s, 4H), 6.85 (d, J=9.2 Hz, 2H), 6.98 (d, J=
9.2 Hz, 2H), 7.57 (d, J=9.2 Hz, 2H), 8.07 ppm (d, J=9.2 Hz, 2H);
13C NMR (100 MHz, [D6]DMSO): d=32.7, 51.6, 54.9, 55.2, 56.7, 63.2,
91.5, 112.1, 113.0, 113.8, 125.6, 125.9, 127.1, 129.8, 144.1, 155.7,
158.6, 168.4, 170.8, 174.3 ppm; IR (neat): n˜ =431, 592, 630, 659,
829, 925, 950, 1035, 1103, 1148, 1179, 1249, 1293, 1509, 1552, 1603,
1659, 1698, 2358, 2832, 2946, 3279 cmꢀ1; HRMS (ESI): m/z [M+H]+
calcd for C26H29N2O7: 481.1976, found: 481.1969.
2-(3-Hydroxy-4-(4-methoxyphenyl)-5-oxofuran-2(5H)-ylidene)-N-
(2-hydroxyethyl)-2-(4-methoxyphenyl)acetamide (12g): Com-
pound 12g was obtained by opening 3 with 2-aminoethanol ac-
cording to the general procedure. Yield: 61%; 1H NMR (400 MHz,
[D6]DMSO): d=3.31 (q, J=6.0 Hz, 2H), 3.46 (t, J=6.0 Hz, 2H), 3.78
(s, 3H), 3.83 (s, 3H), 7.01 (d, J=9.2 Hz, 2H), 7.05 (d, J=9.2 Hz, 2H),
7.32 (d, J=9.2 Hz, 2H), 7.95–7.98 ppm (m, 3H); 13C NMR (100 MHz,
[D6]DMSO): d=43.1, 55.1, 55.2, 58.7, 113.8, 114.1, 117.8, 122.2,
123.6, 128.1, 131.6, 150.5, 158.4, 159.6, 166.9, 168.4 ppm; IR (neat):
n˜ =420, 443, 457, 496, 539, 581, 700, 750, 797, 831, 840, 959, 993,
1027, 1055, 1160, 1178, 1218, 1237, 1289, 1301, 1320, 1421, 1462,
2-(3-Hydroxy-4-(4-methoxyphenyl)-5-oxofuran-2(5H)-ylidene)-N-
(2-(2-hydroxyethoxy)ethyl)-2-(4-methoxyphenyl)acetamide
(12c): Compound 12c was obtained by opening 3 with 2-(2-ami-
noethoxy)ethanol according to the general procedure. Yield: 98%;
1H NMR (400 MHz, [D6]DMSO): d=3.39–3.51 5 (m, 8H), 3.78 (s, 3H),
3.83 (s, 3H), 7.01 (d, J=9.2 Hz, 2H), 7.05 (d, J=9.2 Hz, 2H), 7.32 (d,
J=9.2 Hz, 2H), 7.96 (d, J=9.2 Hz, 2H), 8.04 ppm (t, J=5.6 Hz, 1H);
13C NMR (100 MHz, [D6]DMSO): d=40.2, 55.1, 55.2, 60.2, 67.8, 72.0,
113.9, 114.1, 117.9, 122.1, 123.6, 128.2, 131.6, 150.4, 158.4, 159.6,
166.8, 168.4 ppm; IR (neat): n˜ =457, 483, 524, 580, 697, 832, 915,
957, 1025, 1062, 1116, 1176, 1242, 1290, 1506, 1548, 1594, 1659,
1747, 2838, 2933, 3354 cmꢀ1; HRMS (ESI): m/z [M+H]+ calcd for
C24H26NO8: 456.1659, found: 456.1653.
1486, 1509, 1552, 1575, 1594, 1656, 1731, 2949, 3258, 3488 cmꢀ1
;
HRMS (ESI): m/z [M+H]+ calcd for C22H22N2O7: 412.1394, found:
412.1391.
(2S,3S,4R,5S,6S)-2-(Acetoxymethyl)-6-(2-(2-(3-hydroxy-4-(4-meth-
oxyphenyl)-5-oxofuran-2(5H)-ylidene)-2-(4-methoxyphenyl)acet-
amido)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (12 h):
Compound 12h was obtained by opening 3 with 16 (1.5 equiv) ac-
cording to the general procedure (reaction run overnight). Yield:
1
99%; H NMR (400 MHz, CDCl3): d=1.99 (s, 3H), 2.00 (s, 3H), 2.02
2-(2-(3-Hydroxy-4-(4-methoxyphenyl)-5-oxofuran-2(5H)-ylidene)-
2-(4-methoxyphenyl)acetamido)acetic acid (12d): Compound
12d was obtained by opening 3 with glycine according to the
general procedure, and subsequent purification by column chro-
matography (CH2Cl2/MeOH/AcOH, 90:9:1). Yield: 74%; 1H NMR
(400 MHz, [D6]DMSO): d=3.77 (s, 3H), 3. 82 (s, 3H), 3.90 (d, J=
6.0 Hz, 2H), 7.00 (d, J=9.2 Hz, 2H), 7.06 (d, J=9.2 Hz, 2H), 7.36 (d,
J=9.2 Hz, 2H), 7.98 ppm (d, J=9.2 Hz, 2H); 13C NMR (100 MHz,
[D6]DMSO): d=42.4, 55.1, 55.2, 113.8, 114.2, 116.9, 122.5, 123.8,
128.0, 131.2, 150.3, 158.3, 159.6, 167.1, 168.7, 169.8 ppm; IR (neat):
n˜ =541, 583, 651, 836, 953, 993, 1033, 1108, 1168, 1178, 1250, 1291,
1303, 1341,1417, 1454, 1484, 1505, 1547, 1589, 1658, 1715, 1750,
2839, 2934, 3290, 3397 cmꢀ1; HRMS (ESI): m/z [M+H]+ calcd for
C22H20NO8: 426.1178, found: 426.1183.
(s, 3H), 2.03 (s, 3H), 3.48–3.78 (m, 5H), 3.83 (s, 3H), 3.88 (s, 3H),
4.03–4.14 (m, 2H), 4.46 (d, J=7.6 Hz, 1H), 4.84 (dd, J=7.6, 9.6 Hz,
1H), 4.99 (t, J=9.6 Hz, 1H), 5.16 (t, J=9.6 Hz, 1H), 6.46 (t, J=
5.6 Hz, 1H), 6.94 (d, J=8.8 Hz, 2H), 7.03 (d, J=8.8 Hz, 2H), 7.24 (d,
J=8.8 Hz, 2H), 8.11 (d, J=8.8 Hz, 2H), 15.70 ppm (s, 1H); 13C NMR
(100 MHz, CDCl3): d=20.6, 20.7, 20.8, 40.6, 55.4, 55.5, 62.1, 67.2,
68.4, 71.3, 72.1, 72.8, 100.5, 103.5, 113.9, 114.9, 116.8, 122.5, 123.8,
129.1, 131.6, 152.9, 159.2, 160.5, 160.6, 167.3, 169.2, 169.3, 169.5,
170.2, 170.5 ppm; MS (ESI): m/z (%): 650.21 (100%) [M+H]+.
General procedure for the synthesis of compounds 13a, 13c
and 13e–g: Bis-lactone 4 (200 mg, 0.8 mmol) was suspended in
dry THF (10 mL) and cooled to ꢀ358C. TBAF in THF (1m) was then
added dropwise (1.6 mL, 2 equiv) and the solution was stirred for
15 min at ꢀ358C, and then cooled to ꢀ788C. A solution of amine
(2.5 equiv) in dry THF (5 mL) was added dropwise, and the reaction
was stirred for 15 min before warming to RT. EtOAc was added,
and the solution was washed with a 1m aq HCl and brine, dried
(MgSO4), filtered and concentrated. The crude was purified by
column chromatography (CH2Cl2/MeOH, 100:0!90:10) to give
13a, 13c and 13e–g (yield: 24–88%).
N-(2-(Dimethylamino)ethyl)-2-(3-hydroxy-4-(4-methoxyphenyl)-
5-oxofuran-2(5H)-ylidene)-2-(4-methoxyphenyl)acetamide (12e):
Compound 12e was obtained by opening 3 with N,N-dimethyle-
thylenediamine according to the general procedure. Yield: 92%;
1H NMR (400 MHz, [D6]DMSO): d=2.97 (s, 3H), 2.98 (s, 3H), 3.29 (t,
J=4.8 Hz, 2H), 3.58 (m, 2H), 3.72 (s, 3H), 3.78 (s, 3H), 6.85 (d, J=
9.2 Hz, 2H), 7.57 (d, J=9.2 Hz, 2H), 8.19 (d, J=9.2 Hz, 2H), 8.70 (d,
ChemMedChem 2011, 6, 561 – 569
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567

P. Bischoff et al.
MED
2-(3-Hydroxy-4-(4-methoxyphenyl)-5-oxofuran-2(5H)-ylidene)-N-
(2-morpholinoethyl)propanamide (13a): Compound 13a was ob-
tained by opening 4 with N-(2-aminoethyl)morpholine according
to the general procedure. Yield: 88%; 1H NMR (300 MHz, CDCl3):
d=2.07 (s, 3H), 2.64 (t, J=4.2 Hz, 4H), 2.72 (t, J=5.7 Hz, 2H), 3.47
(q, 5.7 Hz, 2H), 3.76 (t, J=4.2 Hz, 4H), 3.80 (s, 3H), 6.88 (d, J=
9.0 Hz, 2H), 7.18 (t, J=5.4 Hz, 1H), 8.04 ppm (d, J=9.0 Hz, 2H);
13C NMR (75 MHz, CDCl3): d=13.7, 36.4, 53.2, 55.4, 55.9, 66.6, 66.9,
111.2, 113.9, 112.9, 128.8, 158.9, 161.6, 167.8, 168.3, 169.0 ppm; IR
(neat): n˜ =534, 587, 661, 841, 910, 1074, 1102, 1237, 1294, 1441,
1511, 1548, 1658, 2340, 2361, 3268 cmꢀ1; HRMS (ESI): m/z [M+H]+
calcd for C20H25N2O6: 389.1707, found: 389.1692.
(2S,3S,4R,5S,6S)-2-(Acetoxymethyl)-6-(2-bromoethoxy)tetrahy-
dro-2H-pyran-3,4,5-triyl triacetate (14): A solution of b-d-glucose
pentaacetate (500 mg, 1.3 mmol) and 2-bromoethanol (91 mL,
1.3 mmol) in CH2Cl2 (10 mL) was cooled to 08C, and BF3·Et2O
(552 mL, 4.3 mmol) was added dropwise. The mixture was stirred
for 30 min at 08C and then allowed to stand at RT overnight. The
mixture was quenched with saturated aq NaHCO3. After separation,
the aqueous phase was extracted with CH2Cl2, and the combined
organic phases were washed with water, dried (MgSO4), filtered
and concentrated. The crude was purified by column chromatogra-
phy (cyclohexane/EtOAc, 80:20!70:30) to give 14 as a white solid
(yield: 51%); 1H NMR (400 MHz, CDCl3): d=2.00 (s, 3H), 2.02 (s,
3H), 2.06 (s, 3H), 2.08 (s, 3H), 3.44–3.48 (m, 2H), 3.69–3.73 (m, 1H),
3.79–3.85 (m, 1H), 4.12–4.16 (m, 2H), 4.23–4.27 (dd, J=4.8, 12.3 Hz,
1H), 4.57 (d, J=7.9 Hz, 1H), 4.98–5.10 (m, 2H), 5.21 ppm (t, J=
9.5 Hz, 1H); 13C NMR (100 MHz, CDCl3): d=20.7, 21.0, 30.3, 61.7,
62.1, 68.6, 70.0, 71.3, 72.1, 72.8, 89.3, 101.2, 169.1, 169.2, 169.9,
170.4 ppm; MS (ESI): m/z (%): 455.05 [M+H]+; 472.08 (50%)
[M+NH4]+.
2-(3-Hydroxy-4-(4-methoxyphenyl)-5-oxofuran-2(5H)-ylidene)-N-
(2-(2-hydroxyethoxy)ethyl)propanamide (13c): Compound 13c
was obtained by opening 4 with 2-(2-aminoethyl)ethanol accord-
ing to the general procedure. Yield: 24%; 1H NMR (400 MHz,
[D6]DMSO): d=2.12 (s, 3H), 3.44–3.60 (m, 9H), 3.77 (s, 3H), 7.00 (d,
J=9.2 Hz, 2H), 7.97 ppm (d, J=9.2 Hz, 2H); 13C NMR (100 MHz,
[D6]DMSO): 13.5, 40.4, 55.1, 60.2, 67.8, 72.1, 113.5, 113.8, 122.2,
128.0, 151.0, 158.3, 161.0, 166.6, 168.6 ppm; IR (neat): n˜ =418, 457,
513, 554, 577, 633, 653, 698, 719, 745, 806, 833, 888, 910, 936,
1022, 1055, 1087, 1110, 1158, 1246, 1270, 1416, 1454, 1510, 1557,
1605, 1747, 2879, 2934, 3079, 3307, 3458 cmꢀ1; HRMS (ESI): m/z
[M+H]+ calcd for C18H22NO7: 364.1389, found: 364.1391.
(2S,3S,4R,5S,6S)-2-(Acetoxymethyl)-6-(2-azidoethoxy)tetrahydro-
2H-pyran-3,4,5-triyl triacetate (15): NaN3 (860 mg, 13 mmol) was
added to a solution of 14 (1 g, 2.2 mmol) in DMF (15 mL), and the
solution was heated to 608C for 5 h. After cooling to RT, the sol-
vent was partially removed in vacuo before water was added. The
aqueous phase was extracted with EtOAc, and the combined or-
ganic phases were washed with a 1m aq HCl, brine, and then
dried (MgSO4), filtered and concentrated to give 15 as a white
2-(3-Hydroxy-4-(4-methoxyphenyl)-5-oxofuran-2(5H)-ylidene)-N-
(3-methoxypropyl)propanamide (13e): Compound 13e was ob-
tained by opening 4 with 3-methoxypropylamine according to the
1
solid (yield: quant); H NMR (400 MHz, CDCl3): d=1.98 (s, 3H), 1.99
1
general procedure. Yield: 92%; H NMR (400 MHz, CDCl3): d=1.84
(s, 3H), 2.00 (s, 3H), 2.06 (s, 3H), 3.24–3.29 (ddd, J=3.5, 4.8,
13.4 Hz, 1H), 3.44–3.50 (ddd, J=3.4, 8.3, 13.3 Hz, 1H), 3.64–3.72 (m,
2H), 3.98–4.03 (ddd, J=3.5, 4.9, 0.7 Hz, 1H), 4.12–4.15 (dd, J=2.5,
12.3 Hz, 1H), 4.21–4.25 (dd, J=4.6, 12.3 Hz, 1H), 4.58 (d, J=7.9 Hz,
1H), 4.97–5.02 (dd, J=8.0, 9.7 Hz, 1H), 5.07 (t, J=9.8 Hz, 1H),
5.19 ppm (t, J=9.5 Hz, 1H); 13C NMR (100 MHz, CDCl3): d=20.3,
20.4, 50.3, 61.6, 68.1, 68.3, 70.9, 71.6, 72.5, 100.4, 169.0, 169.1,
169.8, 170.2 ppm; MS (ESI): m/z (%): 418.15 [M+H]+; 435.17 (96%)
[M+NH4]+.
(m, 2H), 2.08 (s, 3H), 3.36 (s, 3H), 3.49 (q, J=6.8 Hz, 2H), 3.56 (t,
J=6.8 Hz, 2H), 3.80 (s, 3H), 6.92 (d, J=9.0 Hz, 2H), 7.73 (br s, 1H),
8.10 ppm (d, J=9.0 Hz, 2H); 13C NMR (100 MHz, CDCl3): d=13.3,
27.7, 40.8, 55.3, 59.1, 73.0, 102.2, 111.9, 113.7, 122.7, 128.7, 152.4,
158.8, 161.2, 167.6, 168.4 ppm; IR (neat): n˜ =418, 464, 498, 514,
558, 585, 670, 831, 923, 1027, 1088, 1121, 1154, 1180, 1248, 1289,
1381, 1449, 1511, 1552, 1605, 1746, 2819, 2841, 2879, 2930,
3343 cmꢀ1; HRMS (ESI): m/z [M+H]+ calcd for C19H25N2O5: 361.1758,
found: 361.1764.
(2S,3S,4R,5S,6S)-2-(Acetoxymethyl)-6-(2-aminoethoxy)tetrahy-
dro-2H-pyran-3,4,5-triyl triacetate (16): 10% Pd/C (20 mg) was
added to a solution of 15 (200 mg, 0.5 mmol) in THF (7 mL). The
suspension was then stirred under H2 overnight. The suspension
was filtered through celite and concentrated to give 16 as a white
(2S,3S,4R,5S,6S)-2-(Acetoxymethyl)-6-(2-(2-(3-hydroxy-4-(4-meth-
oxyphenyl)-5-oxofuran-2(5H)-ylidene)propanamido)ethoxy)tetra-
hydro-2H-pyran-3,4,5-triyl triacetate (13 f): Compound 13 f was
obtained by opening 4 with 16 (1.1 equiv) according to the gener-
al procedure. Yield: 57%; 1H NMR (200 MHz, CDCl3): d=1.98 (s,
3H); 2.00 (s, 3H), 2.01 (s, 3H), 2.04 (s, 3H), 2.14 (s, 3H), 3.5–3.8 (m,
9H), 4.0–4.2 (m, 3H), 4.51 (d, J=7.8 Hz, 1H), 4.9–5.3 (m, 3H), 6.91
(d, J=9.0 Hz, 2H), 8.06 ppm (d, J=9.0 Hz, 2H); 13C NMR (50 MHz,
CDCl3): d=13.4, 20.6, 20.7, 40.5, 55.3, 61.8, 67.5, 68.2, 71.3, 72.2,
72.5, 100.8, 102.8, 111.5, 113.8, 122.5, 128.8, 152.8, 159.0, 160.5,
169.1, 169.5, 170.1, 170.6 ppm; HRMS (ESI): m/z [M+Li]+ calcd for
C30H35LiNO15: 656.2162, found: 656.2153.
1
solid (yield: quant); H NMR (400 MHz, CDCl3): d=1.98 (s, 3H), 2.00
(s, 3H), 2.03 (s, 3H), 2.06 (s, 3H), 3.56–3.62 (m, 1H), 3.67–3.72 (m,
1H), 3.84–3.89 (m, 1H), 4.12–4.16 (dd, J=2.5, 12.3 Hz, 1H), 4.20–
4.25 (dd, J=4.8, 12.3 Hz, 1H), 4.52 (d, J=7.9 Hz, 1H), 4.95–4.99 (dd,
J=8.0, 9.7 Hz, 1H), 5.06 (t, J=9.8 Hz, 1H), 5.19 ppm (t, J=9.5 Hz,
1H); 13C NMR (100 MHz, CDCl3): d=20.5, 20.6, 20.7, 20.8, 41.6, 61.9,
68.4, 71.3, 71.8, 72.3, 72.7, 100.9, 169.3, 169.4, 170.6 ppm; MS (ESI):
m/z (%): 392.16 (100%) [M+H]+.
tert-Butyl
4-(2-(3-hydroxy-4-(4-methoxyphenyl)-5-oxofuran-
2-(3-Hydroxy-4-(4-methoxyphenyl)-5-oxofuran-2(5H)-ylidene)-2-
(4-methoxyphenyl)-N-(2-((2S,3S,4R,5R,6S)-3,4,5-trihydroxy-6-(hy-
droxymethyl)tetrahydro-2H-pyran-2-yloxy)ethyl)acetamide
(12b): Sodium methoxide (16 mg, 3.1 equiv) was added to a solu-
tion of 12h (70 mg, 0.09 mmol) in MeOH (3 mL), and the mixture
was stirred overnight at RT. After addition of DOWEX 50WX8–200
cation-exchange resin, the mixture was filtered over celite and con-
centrated to give 12b as an orange solid (yield: 94%); 1H NMR
(400 MHz, CD3OD): d=3.11 (dd, J=7.8, 9.3 Hz, 1H), 3.19–3.26 (m,
3H), 3.33 (t, J=8.8 Hz, 2H), 3.50–3.63 (m, 3H), 3.68–3.76 (m, 1H),
3.81 (s, 3H), 3.86 (s, 3H), 3.89–3.95 (m, 1H), 4.24 (d, J=7.8 Hz, 1H),
6.95 (d, J=9.0 Hz, 2H), 7.04 (d, J=8.8 Hz, 2H), 7.31 (d, J=8.8 Hz,
2(5H)-ylidene)propanoyl)piperazine-1-carboxylate (13g). Com-
pound 13g is obtained by opening 4 with N-Boc-piperazine ac-
cording to the general procedure. Yield: 88%; 1H NMR (400 MHz,
CDCl3): d=1.47 (s, 9H), 2.13 (s, 3H), 3.51 (br s, 4H), 3.59 (br s, 4H),
3.81 (s, 3H), 6.93 (d, J=8.9 Hz, 2H), 8.01 ppm (d, J=8.9 Hz, 2H);
13C NMR (100 MHz, CDCl3): d=16.2, 28.4, 43.9, 45.8, 55.3, 80.9,
104.6, 112.7, 113.9, 121.8, 129.1, 149.6, 154.4, 158.7, 159.3, 166.8,
171.4 ppm; IR (neat): n˜ =538, 836, 1155, 1245, 1360, 1420, 1593,
1687, 1764, 2361, 2974 cmꢀ1; HRMS (ESI): m/z [M+H]+ calcd for
C23H29N2O7: 445.1969, found: 445.1966.
568
www.chemmedchem.org
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 561 – 569

Synthesis and Radioprotective Properties of Pulvinic Acid Derivativess
2H), 8.05 ppm (d, J=9.0 Hz, 2H); 13C NMR (100 MHz, CD3OD): d=
41.2, 55.6, 55.7, 62.7, 69.4, 71.6, 75.2, 77.9, 94.6, 104.5, 114.2, 114.7,
115.4, 127.8, 127.9, 131.8, 147.3, 158.0, 160.7, 171.1, 175.7,
177.7 ppm; IR (neat): n˜ =458, 524, 580, 698, 833, 915, 957, 1024,
1072, 1161, 1178, 1244, 1294, 1338, 1507, 1548, 1595, 1748, 2838,
3363 cmꢀ1; HRMS (ESI): m/z [M+H]+ calcd for C28H32NO12: 574.919,
found: 574.1920.
Keywords: ionizing radiation · pulvinic acids · radioprotective
agents · synthesis · TK6 cells
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2-(3-Hydroxy-4-(4-methoxyphenyl)-5-oxofuran-2(5H)-ylidene)-N-
(2-((2S,3S,4R,5R,6S)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahy-
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1H NMR: (400 MHz, CD3OD): d=2.18 (s, 3H), 3.19–3.24 (dd, J=7.8,
9.0 Hz, 1H), 3.28–3.33 (m, 3H), 3.37 (t, J=9.0 Hz, 1H), 3.52–3.59
(ddd, J=4.5, 6.9, 13.9 Hz, 1H), 3.64–3.71 (m, 2H), 3.81 (s, 3H), 3.86–
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1H), 6.93 (d, J=9.0 Hz, 2H), 8.04 ppm (d, J=9.0 Hz, 2H); 13C NMR
(100 MHz, CD3OD): d=13.6, 41.8, 55.7, 62.7, 69.0, 71.6, 75.1, 78.1,
103.1, 104.5, 114.6, 114.5, 123.8, 129.7, 152.7, 160.4, 162.1, 169.0,
170.4 ppm; IR (neat): n˜ =458, 512, 581, 633, 699, 746, 833, 919,
1025, 1160, 1180, 1247, 1298, 1417, 1511, 1552, 1602, 1744, 2930,
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found: 482.1657.
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Cell line and culture: The human B-lymphoblastoid cell line TK6 was
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inactivated fetal calf serum (Invitrogen), 1 mm sodium pyruvate,
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Irradiation and treatment schedule: Irradiations were carried out at
RT by 6 MV X-rays produced by a medical linear accelerator. Cells
were contained in 25 cm2 or 12.5 cm2 culture flasks filled with
10 mL or 5 mL cell suspension, respectively. Cell concentrations
were adjusted to 2ꢂ105 cellsmLꢀ1. Dose rate was 0.8 Gyminꢀ1, and
doses were 2–16 Gy, but most commonly 8 Gy.
Cell viability assay: Cell growth was determined using the alamar
blue assay (UptiBlue, Interchim; MontluÅon, France), according to
the manufacturer’s instructions. Flasks were centrifuged 24 h after
irradiation, and the medium was replaced by an identical volume
of fresh medium. Aliquots of the suspension (200 mL) were trans-
ferred into 96-well flat-bottomed microplates (Falcon F-3072). Five
replicates were prepared for each concentration. At various time
intervals, 20 mL of UptiBlue solution were then added to the wells.
After incubation at 378C for 4 h, microplates were read at 590 nm
(excitation at 560 nm), using a Perkin–Elmer Victor X2 2030–0020
microplate reader.
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[26] The toxicity at each concentration after incubation for 24 h is given in
the Supporting Information. Toxicity at higher doses was not assessed,
thus the therapeutic index was not determined.
Acknowledgements
A. Le Roux is supported by a fellowship from the Direction Gꢀnꢀr-
Received: September 13, 2010
ale de l’Armement (DGA; France).
Published online on January 18, 2011
ChemMedChem 2011, 6, 561 – 569
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
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