Synthesis of stable analogues of thiamine di- and triphosphate as tools for probing a new phosphorylation pathway

Article abstract of DOI:10.1002/1521-3765(20021018)8:20<4649::AID-CHEM4649>3.0.CO;2-M

Thiamine (vitamin B1) is an essential nutritional factor metabolized inside the body in its mono-, di-, and triphosphate forms. Although the action of thiamine and thiamine diphosphate have been intensely investigated, many questions remain unanswered and

Full text of DOI:10.1002/1521-3765(20021018)8:20<4649::AID-CHEM4649>3.0.CO;2-M

FULL PAPER
Synthesis of Stable Analogues of Thiamine Di- and Triphosphate as Tools for
Probing a New Phosphorylation Pathway
Emmanuel Klein,[a] Hoa¡ng-Oanh Nghie√m,[c] Alain Valleix,[b] Charles Mioskowski,[a, b] and
Luc Lebeau*[a]
Abstract: Thiamine (vitamin B1) is an
essential nutritional factor metabolized
inside the body in its mono-, di-, and
triphosphate forms. Although the action
of thiamine and thiamine diphosphate
have been intensely investigated, many
questions remain unanswered and the
role of thiamine triphosphate is still
especially unknown. To probe recent
hypotheses on the implication of thi-
amine triphosphate in a newphosphor-
ylation pathway involving synaptic pro-
teins, we synthesized a series of thiamine
di- and triphosphate analogues that are
resistant to both enzymatic and chemical
hydrolyses. The key step in the prepara-
tion of the title compounds is the
coupling of thiamine propyl disulfide
with adequately protected methylenebis-
phosphonic acid, the corresponding tri-
phosphate analogue, and difluorometh-
ylenebisphosphonic acid.
Keywords: difluoromethylene-
phosphonate
¥ phosphorylation ¥
triphosphates ¥ vitamins
Introduction
Cl
OH
O
Thiamine (1) was first isolated in rice bran in 1926[1] and its
structure elucidated in 1936.[2] In 1937, thiamine diphosphate
(ThDP) 3 was identified as cocarboxylase, the essential
cofactor of a number of key enzymes.[3] Thiamine is a vitamin
(vitamin B1) required by the human body. The recommended
daily intake is approximately 1.5 mg and about 30 mg are
stored in the body with 80% as the diphosphate 3, 10% as
triphosphate (ThTP) 4, and the rest as thiamine and its
monophosphate (ThMP) 2. Thiamine is produced on an
industrial scale (around 4000 tons annually) and is routinely
added to bread in the western world to prevent deficiency
diseases.
N
N
P
OH
S
O
n
N
NH2
1: n = 0
2: n = 1
3: n = 2
4: n = 3
pounds in various medical conditions (beri beri, Wernicke×s
encephalopathy, Korsakoff psychosis, Leigh syndrome, mega-
loblastic anaemia, and others) have been documented.[4]
However, these studies mainly focused on thiamine and its
pyrophosphate (90 95% of the results published in the
literature), and very few investigations were directed toward
ThTP. Very recently the involvement of ThTP in the specific
phosphorylation of Torpedo 43K Rapsyn has been report-
ed.[5, 6] 43K Rapsyn is a peripheral protein specifically
associated with the nicotinic acetylcholine receptor (nAChR)
present in the post-synaptic membrane of the neuromuscular
junction (NMJ) and of the electrocyte.[7] 43K Rapsyn is
essential for a functional NMJ.[8] The protein phosphorylation
results from transfer of the g-phosphate from ThTP onto a
histidine residue by endogenous kinases that have not yet
been identified.[5] The use of a phosphate donor (ThTP)
belonging to the thiamine family represents a novel phos-
phorylation pathway possibly important for synaptic proteins
and cell signaling.
Since the early times, investigations on thiamine and
thiamine derivatives have continued (ꢀ200 papers in year
2001) and the implications of the presence of these com-
[a] Dr. L. Lebeau, Dr. E. Klein, Dr. C. Mioskowski
¬
Laboratoire de Chimie Bioorganique associe au CNRS
¬
Faculte de Pharmacie
¬
Universite Louis Pasteur de Strasbourg
74, route du Rhin, B.P.24, 67401 Illkirch (France)
Fax : (‡33)3-90-24-43-06
E-mail: lebeau@aspirine.u-strasbg.fr
[b] A. Valleix, Dr. C. Mioskowski
¬
¬
√
CEA-CE Saclay, Service des Molecules Marquees, Bat. 547
¬
¬
Departement de Biologie Moleculaire et Cellulaire
91191 Gif sur Yvette (France)
√
[c] Dr. H.-O. Nghiem
¬
Institut Pasteur, Recepteurs et Cognition
CNRS URA, 2182, 25, rue du Docteur Roux
75724 Paris Cedex 15 (France)
To investigate the scope of that ThTP-dependent phos-
phorylation, analogues of the phosphate donor and its
Chem. Eur. J. 2002, 8, No. 20
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L. Lebeau et al.
FULL PAPER
metabolites are required. Thus, we became interested in the
design of enzymatically and chemically non-hydrolyzable
analogues of thiamine di- and triphosphate. Herein we
describe the synthesis of a series of ThDP and ThTP
analogues.
Bn
HO OBn
OH
PPh3, DEAD
N
N
+
P
CHO S
N
NH2
O
2
8
9
Bn
O
OBn
PPh3, H2O
N
N
N
P
CHO S
NH2
O
N
N
Results and Discussion
2
10
Though many thiamine and thiamine polyphosphate analogues
have been described in the literature (for a reviewsee [9]), only
a very small number incorporate modifications of the poly-
phosphate chain (see below). To the best of our knowledge,
three compounds are documented: thiamine a:b-methylene-
diphosphate (5),[10] thiamine a,b-dithiodiphosphate (6),[11] and
thiamine b:g-methylenetriphosphate (7).[12] Whereas the
Cl
Bn
O
OBn
H2, Pd/C
N
P
S
O
NH2
11
Bn
O
O
N
N
P
S
O
N
NH2
12
OH OH
O
Scheme 1. Synthesis of ThMP analogue 12.
P
P
5 : R =
6 : R =
O
O
SH SH
OR
O
O
N
N
P
P
in the molecule did not cause any problem in that last
transformation.
That same reaction sequence starting from methylenebi-
sphosphonic tribenzyl ester 13a[20] turned out differently
(Scheme 2). Whereas the double phosphonylation of thiamine
S
O
O
N
NH2
OH
O
OH OH
O
P
P
P
7 : R =
O
O
O
dithio derivative and triphosphate analogue do not fulfill the
stability requirements toward hydrolytic conditions, com-
pound 5 appears to be a good candidate for probing part of the
biological activity of the ThTP-dependent kinases. The
original synthesis of that compound was achieved starting
from thiamine disulfide and methylenebisphosphonic acid,
but very fewexperimental details if any were reported.
OR OR
PPh3, DEAD
HO
OR
8
+
P
P
O
O
13a : R = Bn
13b : R = Me
Reducing
agent
OR OR
O
OR
N
N
P
P
During the past fewyears we have been involved in a
CHO S
NH2
O
O
N
2
program focused on the synthesis of stable analogues of
14a : R = Bn
14b : R = Me
18]
nucleotides[13
and dinucleoside polyphosphates.[19] Our
strategy consists of preparing a polyphosphonate building
block that can be regioselectively functionalized with ade-
quately protected nucleosides in organic solvents and in high
yields. Due to the lipophilicity of the compounds, separation
problems are largely overcome and purification can be
performed by flash chromatography over silica gel. The final
compounds are obtained after a quasi-quantitative polyde-
protection of both phosphonyl and nucleoside moieties.
In our first attempt to prepare compound 5 we tried to use
the same strategy. Thiamine chloride hydrochloride being
insoluble in organic solvents, we used thiamine disulfide 8 as
starting material (Scheme 1). First, we set up the reaction
sequence with benzylphosphonic monobenzyl ester 9[20] as a
model phosphonate. Phosphonylation of the dimeric precur-
sor of thiamine was carried out under the Mitsunobu
conditions.[21] Bis-phosphonylated compound 10 was obtained
in 81% yield. The well-documented reduction of the disulfide
bridge with rapid cyclization and thiazolium formation[22] was
realized in 97% yield using triphenylphosphine in the
presence of water. The resulting mixed benzyl thiamine
phosphonate 11 was debenzylated by catalytic hydrogenolysis
to afford thiamine phosphonate 12 in 78% yield. The
presence of a benzyliminium moiety and of a cyclic sulfide
Cl
N
OR OR
O
OR
N
P
P
S
O
O
N
NH2
15a : R = Bn
15b : R = Me
Scheme 2. Tentative preparation of protected ThDP analogues starting
from thiamine disulfide 8.
disulfide could be carried out in 62% yield to give 14a, further
reduction of the disulfide bridge proved ineffective. Whatever
the experimental conditions (PPh3/H2O, PBu3/H2O, PBu3/
H2O/HBF4, b-mercaptoethanol, nBuSH, iPrSH, tBuSH) we
invariably failed in forming the reduced compound, and the
starting material remained unchanged. A plausible explan-
ation for this inert behavior toward reduction could be some
compact folding of the dimer, possibly due to cumulative p-
stacking interactions between the six phenyl and two pyrimi-
dine rings in the molecule. To test the hypothesis we
conducted parallel experiments in the methyl ester series.
Phosphonylation of 8 with methylenebisphosphonic trimethyl
ester 13b yielded dimeric compound 14b, which, however,
proved equally resistant to disulfide reduction. Additional
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Thiamine Analogues
4649 4655
experiments were carried out in which 14a was first submitted
to hydrogenolysis before attempting disulfide reduction. We
could neither identify nor detect the expected products in the
crude reaction mixtures.
These negative results prompted us to revise our strategy. If
the problem indeed resulted from a tight fit of the disulfide
bridge within the dimer, breaking the symmetry of the
molecule should restore disulfide accessibility toward reduc-
ing agents. Thus, phosphonylation of thiamine propyl disulfide
16[23] with 13a under the Mitsunobu conditions afforded 17 in
63% yield (Scheme 3). The latter compound was then treated
OBn
OBn OBn
HO
OBn
16
PPh3
DEAD
P
P
P
OBn
OBn OBn
O
O
+
O
DABCO
BnO
OBn
19
P
P
P
OBn
OBn OH
O
O
O
BnO
OBn
P
P
P
18
O
O
O
20
OBn
OBn OBn
O
OBn
N
N
P
P
P
CHO S
NH2
O
O
O
N
N
S-n-Pr
21
PPh3, H2O
O
+
OBn
P
O
OBn
N
P
O
O
OBn
CHO S
NH2
P
N
S-n-Pr
OBn
22
13a
PPh3, DEAD
OH
N
N
CHO S
OBn OBn OBn
N
NH2
S-nPr
Cl
O
OBn
N
N
P
P
P
16
S
O
O
O
N
N
NH2
Cl
23
TMSBr
H2O
OBn OBn
OBn
O
+
O
OBn
OBn
N
N
P
P
P
OBn
O
O
CHO S
NH2
O
O
N
P
N
S-nPr
17
O
S
P
N
NH2
OBn
24
PPh3, H2O
TMSBr, H2O
15a
5
OH OH OH
O
O
Scheme 3. Synthesis of ThDP analogue 5.
N
N
P
P
P
S
O
O
O
N
NH2
25
26
O
+
O
P
with triphenylphosphine in aqueous 1,2-dimethoxyethane
(DME) and thiamine phosphonate 15a was obtained in
96% yield. This validates hypothesis of a compact conforma-
tion of dimers 14a and 14b and preventing access of any
reducing agent close to the disulfide bridge. Hydrogenolysis of
15a, however, proved troublesome. Deprotection of com-
pound 11 could be achieved within 30 minutes without
formation of byproducts, whereas the full debenzylation of
15a required several hours. This is not compatible with the
preservation of the thiazolium and benzyliminium moieties
and by-products rapidly become the major products. Accord-
ing to previous results obtained with AZT derivatives by some
of us[16, 18] debenzylation of 15a could be achieved in a very
clean manner using bromotrimethylsilane, and 5 was obtained
in 96% yield.
O
OH
N
N
N
P
O
O
S
OH
P
NH2
OH
Scheme 4. Synthesis of ThTP analogues 25 and 26.
To obtain a ThDP analogue with pKa values closer to those
of ThDP than compound 5, we studied fluorinated compound
27.
F
OH
OH
O
O
N
N
P
P
F
S
O
O
N
NH2
27
We then focused our attention on the synthesis of the
thiamine triphosphate analogue 25 (Scheme 4). According to
the previous strategy, disulfide 16 was condensed with
phosphonic acid 19 which results from the monodeprotection
of pentabenzyl ester 18.[20, 24]
All the difluoromethylenebisphosphonic acid monoesters
described so far in the literature result from nucleophilic
displacement of tosylates by a quaternary ammonium salt of
28]
difluoromethylenebisphosphonic acid (Scheme 5).[25
Phosphonic acid 19 was obtained as a mixture with
phosphinic acid 20 (19:20 8:2). As it could not be purified
by flash chromatography over silica gel, the mixture was
directly used in the coupling reaction with 16. The two isomers
21 and 22 were obtained in 42% yield (21:22 8:2). Compound
21 was present in the crude mixture as two diastereomers and
was not separated from 22. Reduction of the mixture with
triphenylphosphine in the presence of water afforded com-
pounds 23 and 24 that were directly involved in the
debenzylation step using bromotrimethylsilane. Compounds
25 and 26 were finally separated by reversed-phase HPLC.
F
F
O
O
O
O
R'-OTs
R'-O
O
O
O
P
P
P
P
4 NR4
3 NR4
F
F
O
O
O
O
Scheme 5. General scheme for the preparation of difluoromethylenebi-
sphosphonic monoesters described in the literature.[25
28]
Due to the high electronegativity of the fluorine atom,
difluoromethylenebisphosphonic acid is a very poor nucleo-
phile. Consequently tosylate substitution is very slowand
yields remain low. To adapt our overall strategy to the
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L. Lebeau et al.
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synthesis of the difluoromethylene analogue of ThDP 27, w e
prepared difluoromethylenebisphosphonic tetrabenzyl ester
by electrophilic fluorination[29] of 28 (Scheme 6).
further purified butdirectly treated with bromotrimethylsi-
lane to convert the remaining methyl esters to their corre-
sponding phosphonic acids. Finally, compound 27 was then
obtained in 38% yield after purification by reversed-phase
HPLC.
F
"F
"
BnO
OBn
OBn
OBn OBn
NaH
BnO
OBn
BnO
F
P
P
P
P
MeO
OMe
F
16
OMe
O
O
O
O
O
PPh3, DEAD
N
N
P
P
F
32b
28
29
CHO S
O
O
N
NH2
S-nPr
Scheme 6. Electrophilic fluorination of 28.
33
F
MeO
OMe
OMe
PPh3
H2O
Cl
TMSBr
NH4OH
Different fluorinating agents have been tested (N-fluoro-N-
methyl-p-toluenesulfonamide, Selectfluor, N-fluorobenzene-
sulfonimide (NFSI)); NFSI gave the best results and afforded
29 in 68% yield. The latter compound, however, proved
labile. Whereas selective monodebenzylation of 28 can be
achieved in refluxing toluene with 1 equiv DABCO to afford
13a,[20] compound 29 led to a non-separable complex mixture
of acids, even under milder experimental conditions. Con-
sequently we prepared the mixed ester 30a from dibenzyl
methanephosphonate and benzyl methyl chlorophosphate
(Scheme 7).
O
P
P
N
N
27
F
S
O
O
N
NH2
34
Scheme 8. Synthesis of fluorinated ThDP analogue 27.
Conclusion
Four different analogues of polyphosphorylated thiamine
species (compounds 5, 25, 26, and 27) have been prepared.
Whereas compounds 5 and 27 are clearly ThDP analogues,
and 25 a ThTP analogue, 26 can be considered either as a
ThTP analogue or as a ™supercharged∫ ThDP analogue as
earlier defined in the nucleotide series by Blackburn.[31]
Compounds 5, 25, 26, and 27 are currently under biological
evaluation. Extensive description of the biological properties
of the compounds will be reported elsewhere.
RO
RO
OR
RO
RO
OR
OR'
nBuLi
CH3
Cl
OR'
P
P
P
P
+
O
O
O
O
30a : R = Bn, R' = Me
30b : R = Me, R' = Bn
KCN
or
F
F
NFSI
NaH
RO
RO
OR
OH
RO
OR
H2, Pd/C
RO
OR'
P
P
P
P
F
F
O
O
O
O
Experimental Section
31a : R = Bn, R' = Me
31b : R = Me, R' = Bn
32a : R = Bn
32b : R = Me
General: H, 13C, 31P, and 19F NMR chemical shifts d are reported in ppm
1
Scheme 7. Synthesis of difluoromethylenebisphosphonic acid triesters 32a
and 32b.
relative to their standard reference (1H: CHCl3 at 7.27 ppm, HDO at
4.63 ppm, CD2HOD at 3.31 ppm; 13C: CDCl3 at 77.0 ppm, CD3OD at
49.0 ppm; 31P: H3PO4 external at 0.00 ppm; 19F: CFCl3 external at
0.00 ppm). IR spectra were recorded in wavenumbers (cmÀ1). Mass spectra
(MS) were recorded as chemical ionization (CI) or in the electrospray (ES)
mode. Mass data are reported in mass units (m/z). Analytical HPLC studies
were carried out in the isocratic mode using a reversed-phase column
Electrophilic fluorination of compound 30a was achieved
with 72% yield and removal of methyl ester in 31a using
1 equiv KCN in hot DMF[30] was quantitative. Our attempts to
perform the condensation of thiamine propyl disulfide 16 with
32a under the Mitsunobu conditions however failed, presum-
ably due to a rapid displacement of some benzyl groups by
N,N'-dicarboxyethyl hydrazine or its deprotonated precursor.
Considering the high sensitivity of benzylic positions toward
nucleophiles, we transposed the reaction sequence in the
methyl series assuming that methyl esters are more resistant
and should prevent degradation. Trimethyl benzyl ester 30b
was fluorinated under the same conditions as for 30a. The
resulting monobenzyl ester 31b was hydrogenolyzed to yield
phosphonic acid 32b quantitatively.
Esterification of the latter compound with 16 afforded 33 in
66% yield (Scheme 8). Although more stable than in the
benzyl series, the compound slowly decomposes after a few
hours, even at À208C. Consequently the reduction of the
disulfide bridge was carried out immediately after purifica-
tion. Compound 34 was purified by dissolving the crude
reaction mixture in water and washing it with diethyl ether. It
was obtained along with some compounds resulting from the
partial deprotection of methyl esters. That mixture was not
À1
(Zorbax SBC18, 250 Â 4.6 mm, 5 mm; flowrate 1 mLmin at 258C) and a
photodiode array detector (LKB2410, detection at 230 nm). Preparative
HPLC was carried out in the isocratic mode (Zorbax SBC18, 250 Â
À1
21.2 mm, 7 mm; flowrate 5 mLmin
for 3 min then 20 mLminÀ1 at
258C). Aqueous triethylamine was acidified using CO2. Abbreviations: s,
singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet; br, broad.
Thiamine a:b-methylenediphosphate (5): Bromotrimethylsilane (250 mL,
1.93 mmol) was added to
a suspension of compound 15a (199 mg,
260 mmol) in anhydrous methylene chloride (25 mL) at room temperature.
The reaction mixture was stirred for 6 h before the solvent was removed
under vacuum and water (5 mL) added to the crude residue. The aqueous
solution was washed with AcOEt (2 Â 5 mL) and methylene chloride (2 Â
5 mL), and was lyophylized to yield 5 (105 mg, 96%) as a white hygroscopic
powder. Analytical HPLC (aqueous Et3N 62 mm, pH 7.3/CH3CN 99.9:0.1):
tR ˆ 30.3 min; 1H NMR (D2O, 300 MHz): d ˆ 9.67 (s, 1H), 7.95 (s, 1H), 5.55
(s, 2H), 4.19 (dt, J ˆ 6.0, 5.3 Hz, 2H), 3.22 (t, J ˆ 5.3 Hz, 2H), 2.61 (s, 3H),
2.54 (s, 3H), 2.36 (dd, J ˆ 20.2, 20.3 Hz, 2H); 13C NMR (D2O, 75 MHz): d ˆ
163.8, 163.6 (m), 155.6, 144.9, 144.0, 136.4, 107.0, 64.3 (d, J ˆ 5.8 Hz), 50.5,
28.1 (d, J ˆ 7.2 Hz), 26.8 (t, J ˆ 127.1 Hz), 21.6, 11.8; 31P NMR (D2O,
121 MHz): d ˆ 24.46 (d, J ˆ 8.4 Hz, 1P), 20.96 (d, J ˆ 8.4 Hz, 1P); IR
‡
(KBr): nÄ ˆ 3332 (b), 1644, 1221, 1009; MS (ES): m/z (%): 422 [M] (100),
‡
444 [M À H‡Na] (4).
Thiamine disulfide bis(benzyl benzylphosphonate) (10): Triphenylphos-
phine (629 mg, 2.4 mmol) in anhydrous THF (3 mL) was added dropwise to
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Chem. Eur. J. 2002, 8, No. 20

Thiamine Analogues
4649 4655
a mixture of thiamine disulfide (225 mg, 0.4 mmol), phosphonic acid
monobenzyl ester 9[20] (214 mg, 0.8 mmol), and DEAD (0.4 mL, 2.6 mmol)
in THF (15 mL) at room temperature. The initially heterogeneous reaction
mixture rapidly became clear and was stirred for 30 min before the solvent
was removed under vacuum. The oily residue was purified by flash
chromatography over silica gel (EtOAc/MeOH 10:0 to 6:4) and 10 (340 mg,
81%) was obtained as a slightly yellow solid (mixture of two diaster-
eomers). TLC: Rf ˆ 0.35 (EtOAc/MeOH 6:4); 1H NMR (CDCl3,
200 MHz): d ˆ 7.76 (s, 2H), 7.65 (s, 2H), 7.39 7.25 (m, 20H), 6.25 (s, 4H),
5.02 (A part of ABX syst., J ˆ À11.7, 8.7 Hz, 2H), 4.94 (B part of ABX
syst., J ˆ À11.7, 9.2 Hz, 2H), 3.92 3.70 (m, 4H), 3.15 (d, J ˆ 21.6 Hz, 4H),
2.47 (s, 6H), 2.44 2.32 (m, 4H), 1.75 (s, 6H); 13C NMR (CDCl3, 50 MHz):
d ˆ 168.0, 163.1, 162.1, 156.2, 136.1 (d, J ˆ 5.8 Hz), 130.9 (d, J ˆ 8.7 Hz),
129.8, 129.6, 128.7, 128.6, 128.1, 127.0 (m), 107.8, 68.2 (d, J ˆ 5.8 Hz), 62.6 (d,
J ˆ 7.2 Hz), 40.0, 33.7 (d, J ˆ 135.7 Hz), 31.0 (d, J ˆ 5.8 Hz), 25.6, 18.2; 31P
NMR (CDCl3, 121 MHz): d ˆ 28.89; IR (film): nÄ ˆ 3366, 1660, 1458,
3.43 (m, 4H), 3.08 2.91 (m, 4H), 2.40 (s, 6H), 2.00 (s, 6H), 1.90 (t, J ˆ
‡
20.1 Hz, 4H); MS (CI/NH3): m/z (%): 964 [M‡H] (100).
Thiamine a:b-methylenediphosphate tribenzyl ester (15a): Compound 15a
(293 mg, 97%) was obtained from 17 as a yellowsolid by following the
same procedure as described for 11. TLC: Rf ˆ 0.20 (CHCl3/MeOH/H2O
1
10:6:1); m.p. 155 1568C; H NMR (CD3OD, 200 MHz): d ˆ 9.85 (s, 1H),
8.31 (s, 1H), 7.43 7.28 (m, 15H), 5.55 (s, 2H), 5.18 5.00 (m, 6H), 4.39
4.15 (m, 2H), 3.27 (t, J ˆ 5.5 Hz, 2H), 2.97 (t, J ˆ 21.2 Hz, 2H), 2.71 (s, 3H),
2.64 (s, 3H); 13C NMR (CD3OD, 50 MHz): d ˆ 165.3, 164.0 (m), 156.2,
147.4, 145.5, 137.2 (d, J ˆ 4.3 Hz), 136.0, 129.7, 129.4, 129.2 (m), 106.5, 70.0
(d, J ˆ 5.8 Hz), 69.7 (d, J ˆ 5.8 Hz), 69.6 (d, J ˆ 5.8 Hz), 66.5 (d, J ˆ 5.8 Hz),
51.8, 28.7 (d, J ˆ 7.2 Hz), 25.1 (t, J ˆ 135.7 Hz), 21.9, 12.3; 31P NMR
(CD3OD, 121 MHz): d ˆ 22.28 (d, J ˆ 6.7 Hz, 1P), 21.69 (d, J ˆ 6.7 Hz, 1P);
IR (film): nÄ ˆ 3376, 3064, 1653, 1244, 1016 cmÀ1
.
Thiamine propyl disulfide [benzyl (dibenzylphosphonomethane)phospho-
nate] (17): Compound 17 (650 mg, 63%) was obtained as a yellow
hygroscopic solid starting from thiamine propyl disulfide 16[23] and
phosphonic acid 13a[20], and following the same procedure as described
for 14a. TLC: Rf ˆ 0.40 (EtOAc/MeOH 9:1); 1H NMR (CDCl3, 200 MHz):
d ˆ 7.89 (s, 1H), 7.80 (s, 1H), 7.36 7.28 (m, 15H), 6.02 (brs, 2H), 5.14 5.02
(m, 6H), 4.75, 4.15 (2brs, 2H), 4.12 3.97 (m, 2H), 2.85 (t, J ˆ 6.4 Hz, 2H),
2.53 (t, J ˆ 21.2 Hz, 2H), 2.43 (s, 3H), 2.28 (t, J ˆ 6.9 Hz, 2H), 1.90 (s, 3H),
1.45 (tq, J ˆ 6.9, 7.3 Hz, 2H), 0.90 (t, J ˆ 7.3 Hz, 3H); 13C NMR (CDCl3,
75 MHz): d ˆ 168.0, 163.7, 162.1, 156.4, 135.9 (d, J ˆ 5.8 Hz), 134.0, 133.2,
128.6 128.1 (m), 108.2, 68.4, 68.1, 68.0 (3d, J ˆ 5.8 Hz), 63.7 (d, J ˆ 6.5 Hz),
41.5, 40.1, 30.5 (d, J ˆ 6.5 Hz), 26.0 (t, J ˆ 135.8 Hz), 25.6, 22.0, 18.9, 13.0;
31P NMR (CDCl3, 121 MHz): d ˆ 20.55 (d, J ˆ 5.9 Hz, 1P), 20.36 (d, J ˆ
5.9 Hz, 1P); IR (film): nÄ ˆ 3331, 2961, 1660, 1258, 998 cmÀ1; MS (CI/NH3):
‡
1008 cmÀ1; MS (CI/NH3): m/z (%): 1052 [M‡H] (100).
Thiamine chloride (benzyl benzylphosphonate) (11): Compound 10
(42 mg, 40 mmol) and triphenylphosphine (21 mg, 80 mmol) were dissolved
in DME/water (4:1, 2 mL) and the pH brought to 1 with aqueous HCl. The
reaction mixture was stirred at room temperature for 30 min and the
solvent removed under vacuum. Water (5 mL) was added to the crude
residue and the resulting solution washed with methylene chloride (3 Â
5 mL) and diethyl ether (5 mL). The aqueous layer was evaporated to yield
compound 11 (45 mg, 97%) as a slightly yellow solid that was used without
further purification. TLC: Rf ˆ 0.40 (CHCl3/MeOH/H2O 20:8:1); 1H NMR
(CD3OD, 200 MHz): d ˆ 9.85 (s, 1H), 8.32 (s, 1H), 7.51 7.26 (m, 10H), 5.58
(s, 2H), 5.07 (A part of ABX syst., J ˆ À11.5, 8.0 Hz, 1H), 5.04 (B part of
ABX syst., J ˆ À11.5, 9.4 Hz, 1H), 4.19 (dt, J ˆ 6.9, 5.5 Hz, 2H), 3.35 (d,
J ˆ 21.2 Hz, 2H), 3.24 (t, J ˆ 5.5 Hz, 2H), 2.66 (s, 3H), 2.56 (s, 3H).
‡
‡
m/z (%): 786 [M‡H] (100), 803 [M‡NH4] (22).
Compounds 21 and 22: These two isomers are obtained as a mixture
(140 mg, 42%; 21 (two diastereomers):22 78:22) starting from thiamine
propyl disulfide 16 and a mixture of phosphonic and phosphinic acids 19
and 20, and by following the same procedure as described for 14a. TLC:
Rf ˆ 0.45 (CHCl3/EtOH 9:1); 1H NMR (CDCl3, 300 MHz): d ˆ 7.96, 7.92,
7.89 (3s, 1H), 7.82, 7.79 (2s, 1H), 7.37 7.25 (m, 20H), 6.08 (brs, 2H), 5.20
4.93 (m, 8H), 4.17 3.94 (m, 2H), 3.05 2.70 (m, 6H), 2.43 (s, 3H), 2.29,
2.28 (2t, J ˆ 6.8 Hz, 2H), 2.04, 1.94, 1.88 (3s, 3H), 1.52 1.40 (m, 2H), 0.90,
0.89 (2t, J ˆ 7.2 Hz, 3H); 13C NMR (CDCl3, 75 MHz): d ˆ 167.7, 163.8,
163.7, 162.2, 156.1, 136.0 135.7 (m), 134.0, 133.9, 133.4, 133.3, 128.6 127.9
(m), 108.4, 108.3, 68.6 67.0 (m), 63.8, 63.5 (2d, J ˆ 6.3, 6.8 Hz), 41.5, 40.0,
30.5, 28.6 (d, J ˆ 88.3 Hz), 28.5 (d, J ˆ 132.1 Hz), 25.3, 21.9, 19.0, 18.9 (2s),
12.9; 31P NMR (CDCl3, 81 MHz): d ˆ 38.83 (dd, J ˆ 2.5, 4.5 Hz, 0.4P), 38.69
(dd, J ˆ 3.5, 5.0 Hz, 0.4P), 38.48 (t, J ˆ 4.0 Hz, 0.2P), 21.07 (d, J ˆ 2.5 Hz,
0.4P), 20.97 (d, J ˆ 3.5 Hz, 0.4P), 20.91 (d, J ˆ 4.5 Hz, 0.4P), 20.83 (d, J ˆ
5.0 Hz, 0.4P), 20.68 (d, J ˆ 4.0 Hz, 0.4P); IR (film): nÄ ˆ 3336 (b), 2962, 1655,
Thiamine benzylphosphonic acid (12): Compound 11 (49 mg, 84 mmol) and
Pd/C 10% (45 mg) in MeOH/water (1:1, 3 mL) were vigorously stirred
under a hydrogen atmosphere (1 bar) at room temperature for 30 min. The
mixture was filtered over a Celite pad and the filtrate lyophylized to yield
12 (30 mg, 78%) as a white hygroscopic powder. TLC: Rf ˆ 0.10 (CHCl3/
MeOH/H2O 10:6:1); 1H NMR (CD3OD/D2O 1:1, 200 MHz): d ˆ 9.83 (s,
1H), 9.28 (s, 1H), 7.37 7.28 (m, 5H), 5.59 (s, 2H), 4.25 4.16 (m, 2H), 3.29
(t, J ˆ 4.1 Hz, 2H), 3.23 (d, J ˆ 21.6 Hz, 2H), 2.65 (s, 3H), 2.57 (s, 3H).
Methylenebisphosphonic acid trimethyl ester 13b: Compound 30b
(560 mg, 1.82 mmol) and Pd/C 10% (56 mg) in MeOH (15 mL) were
vigorously stirred under a hydrogen atmosphere (1 bar) at room temper-
ature for 15 min. The mixture was filtered over a Celite pad and the solvent
removed under vacuum to yield 13b (396 mg, 99%) as a colorless oil that
was used without further purification. 1H NMR (CDCl3, 200 MHz): d ˆ
3.64 (d, J ˆ 11.3 Hz, 6H), 3.61 (d, J ˆ 11.3 Hz, 3H), 2.40 (t, J ˆ 21.2 Hz,
2H); 13C NMR (CDCl3, 50 MHz): d ˆ 53.2 (d, J ˆ 6.4 Hz), 52.4 (d, J ˆ
6.4 Hz), 23.8 (t, J ˆ 135.2 Hz); 31P NMR (CDCl3, 121 MHz): d ˆ 24.97 (s,
1P), 19.43 (s, 1P); IR (film): nÄ ˆ 3412 (b), 2961, 1462, 1236, 1039 cmÀ1; MS
‡
‡
1248, 1012; MS (CI/NH3): m/z (%): 954 [M‡H] (100), 971 [M‡NH4]
(43).
Compounds 23 and 24: These compounds were obtained as a mixture of
three non-separated isomers (106 mg, 90%) starting from the previous
mixture of compounds 21 and 22, and following the same procedure as
described for 11. M.p. 170 1718C; 1H NMR (CD3OD, 300 MHz): d ˆ 9.86,
9.85, 9.84 (3s, 1H), 8.27 (s, 1H), 7.41 7.32 (m, 20H), 5.48 (s, 2H), 5.20 4.75
(m, 8H), 4.43 4.15 (m, 2H), 3.39 3.22 (m, 2H), 3.21 2.60 (m, 4H), 2.62,
2.60 (2s, 3H), 2.56, 2.55 (2s, 3H); 13C NMR (CD3OD, 50 MHz): d ˆ 165.0,
164.0, 156.4, 156.3, 147.3, 147.1, 147.0, 145.7, 145.5, 138.0 137.0 (m), 136.4,
136.0, 129.7 128.2 (m), 106.7, 106.6, 106.5, 69.9 68.4 (m), 66.6 (d, J ˆ
4.8 Hz), 51.7, 29.5 (dd, J ˆ 85.9, 115.9 Hz), 28.6 (dd, J ˆ 85.9, 115.9 Hz), 28.8,
28.5 (2d, J ˆ 7.7 Hz), 21.8, 12.3; 31P NMR (CD3OD, 121 MHz): d ˆ 42.9
32.9 (m, 1P), 23.4 21.5 (m, 1P), 20.0 18.1 (m, 1P); IR (film): nÄ ˆ 3040 (b),
‡
(CI/NH3): m/z (%): 219 [M‡H] (100).
Thiamine disulfide bis[benzyl (dibenzylphosphonomethane)phosphonate]
(14a): DEAD (1.04 mL, 6.6 mmol) was added dropwise to triphenylphos-
phine (1.73 g, 6.6 mmol) in anhydrous THF (5 mL) at 08C. The mixture was
stirred for 15 min before being added to thiamine disulfide 8 (0.50 g,
0.89 mmol) and phosphonic acid 13a (0.84 g, 1.88 mmol) in THF (5 mL).
The resulting solution was stirred at room temperature for 1 h and the
solvent removed under vacuum. The crude residue was chromatographed
over silica gel (EtOAc/EtOH 10:0 to 7:3) to yield compound 14a (0.78 g,
62%) as a yellowsolid (mixture of two diastereomers). TLC: Rf ˆ 0.30
(EtOAc/EtOH 7:3); 1H NMR (CD3OD, 200 MHz): d ˆ 8.18 (s, 2H), 7.86 (s,
2H), 7.45 7.11 (m, 30H), 5.52 4.87 (m, 12H), 4.63 4.06 (m, 8H), 3.11
2.93 (m, 4H), 2.33 (s, 3H), 2.29 (s, 3H), 1.98 (s, 6H), 2.09 1.90 (m, 4H); IR
(KBr): nÄ ˆ 3030, 1660, 1466, 1420, 1012 cmÀ1; MS (CI/NH3): m/z (%): 1421
1652, 1245, 999 cmÀ1
.
Thiamine a:b,b:g-bismethylenetriphosphate (25): Compound 25 (41 mg,
57%) was obtained as a white, very hygroscopic powder from the previous
mixture of compounds 23 and 24 by following the same procedure as
described for 5. Purification was achieved by preparative reversed-phase
HPLC and compound 25 was obtained as its triethyl ammonium salt.
Analytical HPLC (aqueous Et3N 62 mm, pH 7.67/CH3CN 99.9:0.1): tR ˆ
‡
‡
[M‡H] (100), 1438 [M‡NH4] (17).
Thiamine disulfide bis[methyl (dimethylphosphonomethane)phospho-
nate] (14b): Compound 14b (0.19 g, 56%) was obtained as a yellow
hygroscopic solid (mixture of two diastereomers) starting from compounds
8 and 13b by following the same procedure as described for 14a. TLC: Rf ˆ
0.30 (EtOAc/MeOH 7:3); 1H NMR (CDCl3/CD3OD 1:1, 200 MHz): d ˆ
8.13 (s, 2H), 7.80 (s, 2H), 4.42 4.10 (m, 4H), 3.89 3.70 (m, 18H), 3.65
1
31.8 min; H NMR (D2O, 300 MHz): d ˆ 9.41 (s, 1H), 7.82 (s, 1H), 5.77 (s,
2H), 4.02 (dd, J ˆ 2.9, 4.5 Hz, 2H), 3.19 (t, J ˆ 2.9 Hz, 2H), 3.10 2.85 (m,
6H), 2.41 (s, 6H), 2.22 1.97 (m, 4H), 1.18 1.08 (m, 9H); 13C NMR (D2O,
75 MHz): d ˆ 163.8, 163.5, 155.6, 145.0, 144.0, 136.2, 106.8, 64.4 (d, J ˆ
Chem. Eur. J. 2002, 8, No. 20
¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0947-6539/02/0820-4653 $ 20.00+.50/0
4653

L. Lebeau et al.
FULL PAPER
5.8 Hz), 50.6, 30.9 (t, J ˆ 127.1 Hz), 30.0 (dd, J ˆ 51;3, 74.4 Hz), 28.4 (d, J ˆ
6.5 Hz), 21.6, 11.9; 31P NMR (D2O, 121 MHz): d ˆ 28.76 (brs, 1P), 19.09
(brs, 1P), 15.39 (brs, 1P); IR (KBr): nÄ ˆ 3412 (b), 2935, 2672, 1662, 1622,
50 MHz): d ˆ 136.1 (d, J ˆ 6.4 Hz), 128.6, 128.5, 128.1, 68.2 (d, J ˆ 5.5 Hz),
68.1 (d, J ˆ 6.6 Hz), 53.0 (d, J ˆ 6.6 Hz), 25.8 (t, J ˆ 136.1 Hz); 31P NMR
(CDCl3, 121 MHz): d ˆ 22.23 (d, J ˆ 6.7 Hz, 1P), 21.38 (d, J ˆ 6.7 Hz, 1P);
IR (film): nÄ ˆ 3090, 3030, 2955, 1465, 1250, 1010 cmÀ1; MS (CI/NH3): m/z
‡
‡
1192, 1035 cmÀ1; MS (ES): m/z (%): 500 [M] (100), 522 [M À H‡Na] (36).
‡
‡
(%): 461 [M‡H] (19), 478 [M‡NH4] (100).
Compound 26: Compound 26 (15 mg, 21%) was isolated as a white, very
hygroscopic powder during the HPLC purification of 25. Analytical HPLC
(aqueous Et3N 62 mm, pH 7.67/CH3CN 99.9:0.1): tR ˆ 26.2 min; 1H NMR
(D2O, 300 MHz): d ˆ 7.85 (s, 1H), 5.26 (s, 2H), 4.20 4.09 (m, 2H), 3.20 (t,
J ˆ 5.7 Hz, 2H), 2.40 (s, 3H), 2.32 (s, 3H), 2.32 (t, J ˆ 19.1 Hz, 4H);
13C NMR (D2O, 75 MHz): d ˆ 163.8, 163.5, 155.7, 145.1, 144.4, 135.7, 106.8,
65.1 (d, J ˆ 6.5 Hz), 50.7, 30.7 (dd, J ˆ 72.2, 125.6 Hz), 28.1 (d, J ˆ 8.7 Hz),
21.6, 11.8; 31P NMR (D2O, 121 MHz): d ˆ 52.78 (brs, 1P), 10.56 (d, J ˆ
6.7 Hz, 2P); IR (KBr): nÄ ˆ 3417 (b), 2968, 1661, 1183, 1043 cmÀ1; MS (ES):
Methylenebisphosphonic acid benzyl trimethyl ester (30b): Compound
30b (2.52 g, 60%) was obtained as a colorless oil starting from dimethyl
methanephosphonate and benzyl methyl chlorophosphate by following the
same procedure as described for 28. TLC: Rf ˆ 0.40 (EtOAc/MeOH 9:1);
1H NMR (CDCl3, 200 MHz): d ˆ 7.41 7.32 (m, 5H), 5.14 (d, J ˆ 8.8 Hz,
2H), 3.79 (d, J ˆ 12.1 Hz, 3H), 3.77 (d, J ˆ 11.3 Hz, 3H), 3.73 (d, J ˆ
11.7 Hz, 3H), 2.46 (t, J ˆ 21.2 Hz, 2H); 13C NMR (CDCl3, 50 MHz): d ˆ
135.5 (d, J ˆ 5.8 Hz), 128.1, 128.0, 127.6, 67.7 (d, J ˆ 5.8 Hz), 52.7 (d, J ˆ
5.8 Hz), 52.5 (d, J ˆ 5.8 Hz), 23.8 (t, J ˆ 136.4 Hz); 31P NMR (CDCl3,
121 MHz): d ˆ 23.06 (d, J ˆ 5.9 Hz, 1P), 22.28 (d, J ˆ 5.9 Hz, 1P); IR (film):
‡
‡
m/z (%): 500 [M] (100), 522 [M À H‡Na] (42).
Thiamine a:b-difluoromethylenediphosphate (27): A suspension of com-
pound 34 (91 mg, 158 mmol) and bromotrimethylsilane (1 mL, 7.5 mmol) in
anhydrous chloroform (7 mL) was sonicated for 4 h at room temperature.
Solvent and excess reagent were removed under vacuum. The crude
residue was decomposed by the addition of water (5 mL) and the mixture
purified by preparative HPLC to yield the triethylammonium salt of 27
(25 mg, 38%) as a white hygroscopic solid. Analytical HPLC (aqueous
Et3N 62 mm, pH 7.67/CH3CN 99.9:0.1): tR ˆ 59.3 min; 1H NMR (D2O,
300 MHz): d ˆ 7.89 (s, 1H), 5.38 (s, 2H), 4.20 (dd, J ˆ 6.0, 6.4 Hz, 2H), 3.23
(t, J ˆ 6.4 Hz, 2H), 3.10 (q, J ˆ 7.1 Hz, 6H), 2.49 (s, 3H), 2.43 (s, 3H), 1.21
(t, J ˆ 7.1 Hz, 9H); 13C NMR (D2O, 75 MHz): d ˆ 164.1, 162.6, 162.5, 155.1,
145.7, 143.5, 136.0, 106.7, 65.4, 50.1, 46.8, 28.1, 21.6, 11.2, 8.3; 31P NMR (D2O,
121 MHz): d ˆ 6.02 3.09 (m, 2P); 19F NMR (D2O, 188 MHz): d ˆ 120.55 (t,
‡
nÄ ˆ 3504, 2957, 1457, 1259, 1037; MS (CI/NH3): m/z (%): 309 [M‡H] (10),
‡
326 [M‡NH4] (100).
Difluoromethylenebisphosphonic acid tribenzyl methyl ester (31a): Com-
pound 31a (0.96 g, 72%) was obtained as a slightly yellow oil starting from
compound 30a, and by following the same procedure as described for 29.
TLC: Rf ˆ 0.50 (EtOAc/nC6H14 5:5); 1H NMR (CDCl3, 200 MHz): d ˆ
7.39 7.32 (m, 15H), 5.37 5.12 (m, 6H), 3.87 (d, J ˆ 11.0 Hz, 3H);
13C NMR (CDCl3, 75 MHz): d ˆ 135.0 (d, J ˆ 4.3 Hz), 128.7, 128.5, 128.1,
116.0 (tt, J ˆ 186.5, 277.0 Hz), 70.3 (d, J ˆ 5.8 Hz), 55.2 (d, J ˆ 5.8 Hz); 31P
NMR (CDCl3, 121 MHz): d ˆ 6.35 (A part of ABX2 syst., J ˆ À71.9,
86.5 Hz, 1P), 5.21 (B part of ABX2 syst., J ˆ À71.9, 86.5 Hz, 1P); 19F NMR
(CDCl3, 188 MHz): d ˆ À121.10 (t, J ˆ 86.5 Hz); IR (film): nÄ ˆ 2962, 1457,
‡
‡
‡
1381, 1282, 1055, 1013; MS (CI/NH3): m/z (%): 498 [M‡H] (16), 515
J ˆ 82.1 Hz); MS (ES): m/z (%): 458 [M] (100), 480 [M À H‡Na] (59).
‡
[M‡NH4] (100).
Methylenebisphosphonic acid tetrabenzyl ester 28: Dibenzyl methane-
phosphonate (7.82 g, 28.3 mmol) in anhydrous THF (125 mL) was treated
dropwise with n-butyllithium (1.6m in hexane, 17.7 mL, 28.3 mmol) at
À788C. The mixture was stirred for 30 min and added in one portion to
dibenzyl chlorophosphate (4.20 g, 14.2 mmol) in THF (20 mL) at À788C.
The reaction mixture was stirred for 1 h at that temperature before being
decomposed by the addition of aqueous ammonium chloride. The resulting
solution was allowed to warm to room temperature and extracted with
EtOAc. The organic layer was dried over MgSO4, reduced under vacuum
and purified over silica gel (EtOAc/nC6H14 4:6) to yield 28 (5.2 g, 68%) as a
white solid. TLC: Rf ˆ 0.50 (EtOAc/nC6H14 8:2); 1H NMR (CDCl3,
200 MHz): d ˆ 7.37 7.28 (m, 20H), 5.06 (A part of ABX syst., J ˆ À11.7,
8.9 Hz, 4H), 5.03 (B part of ABX syst., J ˆ À11.7, 8.1 Hz, 4H), 2.54 (t, J ˆ
21.1 Hz, 2H); 13C NMR (CDCl3, 50 MHz): d ˆ 135.8 (d, J ˆ 5.8 Hz), 128.5,
128.4, 128.0, 67.9 (d, J ˆ 5.8 Hz), 26.1 (t, J ˆ 136.1 Hz); 31P NMR (CDCl3,
Difluoromethylenebisphosphonic acid benzyl trimethyl ester (31b): Com-
pound 31b (0.94 g, 78%) was obtained as a colorless oil starting from
compound 30b, and following the same procedure as described for 29.
TLC: Rf ˆ 0.70 (EtOAc/MeOH 9:1); 1H NMR (CDCl3, 200 MHz): d ˆ
7.42 7.30 (m, 5H), 5.28 (A part of ABX syst., J ˆ À10.2, 7.2 Hz, 1H),
5.21 (B part of ABX syst., J ˆ À10.2, 8.5 Hz, 1H), 3.92 (d, J ˆ 9.9 Hz, 3H),
3.89 (d, J ˆ 9.1 Hz, 3H), 3.87 (d, J ˆ 9.1 Hz, 3H); 13C NMR (CDCl3,
50 MHz): d ˆ 134.8 (d, J ˆ 4.3 Hz), 128.6, 128.4, 127.9, 116.0 (tt, J ˆ 187.0,
278 Hz), 70.1 (d, J ˆ 5.1 Hz), 55.1 (d, J ˆ 4.3 Hz), 55.0 (d, J ˆ 4.3 Hz); 31P
NMR (CDCl3, 121 MHz): d ˆ 6.11 (A part of ABX2 syst., J ˆ À71.0,
85.1 Hz, 1P), 5.05 (B part of ABX2 syst., J ˆ À71.0, 85.1 Hz, 1P); 19F NMR
(CDCl3, 188 MHz): d ˆ À121.42 (t, J ˆ 85.1 Hz); IR (film): nÄ ˆ 2967, 1284,
‡
‡
1051 cmÀ1; MS (CI/NH3): m/z (%): 345 [M‡H] (13), 362 [M‡NH4] (100).
Difluoromethylenebisphosphonic acid tribenzyl ester (32a): Compound
31a (0.55 g, 1.11 mmol) and potassium cyanide (0.08 g, 1.11 mmol) were
stirred in anhydrous DMF (7 mL) for 3 h at 808C. The solvent was removed
under vacuum, the residue dissolved in aqueous 10% HCl (10 mL), and the
solution extracted with EtOAc and methylene chloride. The organic layer
was dried over magnesium sulfate and reduced under vacuum to yield 32a
(0.53 g, 99%) as a colorless oil that was used without further purification.
1H NMR (CDCl3/CD3OD 1:1, 200 MHz): d ˆ 7.28 7.21 (m, 15H), 5.12
4.98 (m, 6H); 13C NMR (CDCl3/CD3OD 1:1, 50 MHz): d ˆ 135.9 (d, J ˆ
6.5 Hz), 134.8 (d, J ˆ 5.1 Hz), 128.6, 128.4, 128.2, 128.1, 127.9, 127.5 (m),
116.4 (tt, J ˆ 180.1, 277.3 Hz), 70.2 (d, J ˆ 6.5 Hz), 69.4 (d, J ˆ 6.5 Hz); 31P
NMR (CDCl3/CD3OD 1:1, 121 MHz): d ˆ 5.64 (dt, J ˆ 68.0, 90.2 Hz, 1P),
1.76 (dt, J ˆ 68.0, 90.2 Hz, 1P); 19F NMR (CDCl3/CD3OD 1:1, 188 MHz):
d ˆ À121.86 (dd, J ˆ 84.0, 90.2 Hz); IR (film): nÄ ˆ 2924 (b), 1456, 1261,
121 MHz): d ˆ 20.53; IR (film): nÄ ˆ 3070, 3033, 2953, 1468, 1250, 998 cmÀ1
;
‡
‡
MS (CI/NH3): m/z (%): 537 [M‡H] (100), 554 [M‡NH4] (29).
Difluoromethylenebisphosphonic acid tetrabenzyl ester 29: Sodium hy-
dride (60% in oil, 166 mg, 4.15 mmol) was added to compound 28 (1.11 g,
2.07 mmol) in anhydrous THF (40 mL) at À108C and the resulting mixture
allowed to warm to room temperature for 10 min. It was then cooled to
À108C again and N-fluorobenzenesulfonimide (1.31 g, 4.15 mmol) in THF
(15 mL) was added dropwise. The temperature was maintained at À10
08C and a white precipitate appeared after a few minutes. The reaction
mixture was decomposed with aqueous NH4Cl, extracted with diethyl
ether, dried over magnesium sulfate, and reduced under vacuum. The
residue was chromatographed over silica gel (EtOAc/nC6H14 2:8) to yield
29 (0.81 g, 68%) as a white solid. TLC: Rf ˆ 0.35 (EtOAc/nC6H14 3:7); m.p.
69 708C; 1H NMR (CDCl3, 200 MHz): d ˆ 7.38 7.30 (m, 20H), 5.26
(A part of ABX syst., J ˆ À11.7, 7.3 Hz, 4H), 5.19 (B part of ABX syst., J ˆ
À11.7, 9.1 Hz, 4H); 13C NMR (CDCl3, 75 MHz): d ˆ 134.6 (d, J ˆ 8.9 Hz),
128.3, 128.1, 127.7, 115.6 (tt, J ˆ 188.0, 280.2 Hz,), 69.9 (d, J ˆ 2.9 Hz); 31P
NMR (CDCl3, 121 MHz): d ˆ 4.86 (t, J ˆ 87.6 Hz); 19F NMR (CDCl3,
188 MHz): d ˆ À121.01 (t, J ˆ 87.6 Hz); IR (film): nÄ ˆ 3066, 1457, 1281,
‡
‡
1052; MS (CI/NH3): m/z (%): 483 [M‡H] (5), 500 [M‡NH4] (100).
Difluoromethylenebisphosphonic acid trimethyl ester (32b): Compound
32b (141 mg, 99%) was obtained as a colorless oil starting from 31b, and by
following the same procedure as described for 13b. 1H NMR (CDCl3,
300 MHz): d ˆ 11.98 (brs, 1H), 3.95 (d, J ˆ 10.9 Hz, 6H), 3.89 (d, J ˆ
10.9 Hz, 3H); 13C NMR (CDCl3, 50 MHz): d ˆ 116.3 (tt, J ˆ 187.7,
275.8 Hz), 55.5 (d, J ˆ 6.5 Hz), 55.2 (d, J ˆ 6.5 Hz); 31P NMR
(CDCl3:CD3OD 1:1, 121 MHz): d ˆ 7.99 (dt, J ˆ 66.2, 89.8 Hz, 1P), 2.72
(dt, J ˆ 66.2, 73.2 Hz, 1P); 19F NMR (CDCl3, 188 MHz): d ˆ À122.20 (dd,
J ˆ 88.9, 83.9 Hz); IR (film): nÄ ˆ 3966 (b), 1454, 1256, 1048 cmÀ1; MS (CI/
‡
1054, 1010, 998 cmÀ1; MS (CI/NH3): m/z (%): 573 [M‡H] (9), 590
‡
[M‡NH4] (100).
Methylenebisphosphonic acid tribenzyl methyl ester (30a): Compound 30a
(2.49 g, 78%) was obtained as a slightly yellow oil starting from benzyl
methyl methanephosphonate and dibenzyl chlorophosphate and by
following the same procedure as described for 28. TLC: Rf ˆ 0.40 (EtOAc);
1H NMR (CDCl3, 300 MHz): d ˆ 7.41 7.33 (m, 15H), 5.17 4.99 (m, 6H),
3.68 (d, J ˆ 11.3 Hz, 3H), 2.50 (t, J ˆ 21.1 Hz, 2H); 13C NMR (CDCl3,
‡
‡
‡
NH3): m/z (%): 255 [M‡H] (2), 272 [M‡NH4] (100), 526 [2M‡NH4]
(6).
Thiamine propyl disulfide [methyl (dimethylphosphonodifluoromethane)-
phosphonate] (33): Compound 33 (155 mg, 66%) was prepared starting
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Thiamine Analogues
4649 4655
from thiamine propyl disulfide 16 and phosphonic acid 32b and by
following the same procedure as described for 14a. TLC: Rf ˆ 0.30 (CHCl3/
EtOH 9:1); 1H NMR (CDCl3, 200 MHz): d ˆ 7.95 (s, 1H), 7.82 (s, 1H), 6.13
(brs, 2H), 4.72 (brs, 1H), 4.10 (brs, 1H), 4.37 (dd, J ˆ 5.9, 6.5 Hz, 2H), 3.99
(d, J ˆ 8.8 Hz, 3H), 3.98 (d, J ˆ 8.4 Hz, 3H), 3.97 (d, J ˆ 8.0 Hz, 3H), 3.02 (t,
J ˆ 5.9 Hz, 2H), 2.44 (s, 3H), 2.30 (t, J ˆ 7.3 Hz, 2H), 2.05 (s, 3H), 1.47 (tq,
J ˆ 7.3, 6.9 Hz, 2H), 0.91 (t, J ˆ 6.9 Hz, 3H); 31P NMR (CDCl3, 121 MHz):
d ˆ 6.72 (A part of ABX syst., J ˆ À72.5, 84.3 Hz, 1P), 5.84 (B part of ABX
syst., J ˆ À72.5, 84.7 Hz, 1P).
Thiamine a:b-difluoromethylenediphosphate trimethyl ester (34): Com-
pound 34 (133 mg, 91%) was obtained as a sligthly yellow solid starting
from 33 and by following the same procedure as described for 15a. It slowly
decomposed to give phosphonic acids. 1H NMR (D2O, 200 MHz): d ˆ 9.49
(s, 1H), 7.72 (s, 1H), 5.36 (s, 2H), 4.37 (dt, J ˆ 5.8, 5.5 Hz, 2H), 3.72 (d, J ˆ
10.6 Hz, 6H), 3.66 (d, J ˆ 11.3 Hz, 3H), 3.24 (t, J ˆ 5.5 Hz, 2H), 2.40 (s,
3H), 2.33 (s, 3H).
[10] G. M. Yakoleva, Y. M. Ostrovskii, Bioorg. Khim. 1985, 11, 1279 1282.
[11] D. Kusewicz, T. Galamon, Chem. Spozyw. 1966, 35 43.
[12] R. L. Barchi, P. E. Braun, J. Biol. Chem. 1972, 247, 7668 7673.
[13] M. Saady, L. Lebeau, C. Mioskowski, Synlett 1995, 643 644.
[14] S. Vincent, C. Mioskowski, L. Lebeau, Tetrahedron Lett. 1998, 39,
2321 2324.
[15] S. Vincent, S. Grenier, A. Valleix, C. Salesse, L. Lebeau, C.
Mioskowski, J. Org. Chem. 1998, 63, 7244 7257.
¬
[16] T. Brossette, A. Valleix, L. Goujon, C. Creminon, J. Grassi, C.
Mioskowski, L. Lebeau, Tetrahedron Lett. 1999, 40, 3391 3394.
¬
[17] L. Lebeau, T. Brossette, L. Goujon, C. Creminon, J. Grassi, C.
Mioskowski, Tetrahedron Lett. 1999, 40, 4323 4326.
¬
[18] T. Brossette, A. LeFaou, L. Goujon, A. Valleix, C. Creminon, J. Grassi,
C. Mioskowski, L. Lebeau, J. Org. Chem. 1999, 64, 5083 5090.
[19] E. Klein, S. Mons, A. Valleix, C. Mioskowski, L. Lebeau, J. Org. Chem.
2002, 67, 146 153.
[20] M. Saady, L. Lebeau, C. Mioskowski, J. Org. Chem. 1995, 60, 2946
2947.
[21] O. Mitsunobu, Synthesis 1981, 1 28.
Acknowledgement
[22] P. Karrer, M. Viscontini, Helv. Chim. Acta 1946, 29, 711 718.
[23] T. Matsukawa, H. Kawasaki, Yakugaku Zasshi 1953, 73, 216 219.
[24] M. Saady, L. Lebeau, C. Mioskowski, Helv. Chim. Acta 1995, 78, 670
678.
¡
This work was supported by MENRT (Ministere de l×Education Nationale,
de la Recherche et de la Technologie, France).
[25] V. J. Davisson, A. B. Woodside, T. R. Neal, K. E. Stremler, M.
Muehlbacher, C. D. Poulter, J. Org. Chem. 1986, 51, 4768 4779.
[26] V. J. Davisson, D. R. Davies, V. M. Dixit, C. D. Poulter, J. Org. Chem.
1987, 52, 1794 1801.
[27] L. Arabshahi, N. N. Khan, M. Butler, T. Noona, N. C. Brown, G. E.
Wright, Biochemistry 1990, 29, 6820 6826.
[1] B. C. P. Jansen, W. F. Donath, Chem. Weekblad 1926, 23, 201 203.
[2] R. R. Williams, J. Am. Chem. Soc. 1936, 58, 1063 1064.
[3] K. Lohmann, P. Schuster, Biochem. Z. 1937, 294, 188 214.
[4] L. Bettendorff, P. Wins, Recent Res. Devel. Neurochem. 1999, 2, 37
62.
[28] G. M. Blackburn, S. P. Langston, Tetrahedron Lett. 1991, 32, 6425
6428.
√
[5] H. O. Nghiem, L. Bettendorff, J. P. Changeux, Faseb J. 2000, 14, 543
[29] S. D. Taylor, C. C. Kotoris, G. Hum, Tetrahedron 1999, 55, 12431
12477.
554.
√
[6] H. O. Nghiem, Vol. DI 99/53. Licensing opportunity, Patent applica-
[30] P. Savignac, G. Lavielle, Bull. Soc. Chim. Fr. 1974, 1506 1508.
[31] X. H. Liu, C. Brenner, A. Guranowski, E. Starzynska, G. M. Black-
burn, Angew. Chem. 1999, 111, 1324 1347; Angew. Chem. Int. Ed.
1999, 38, 1244 1247.
tion US1473888, 2000; [Chem. Abstr. 2000, 134, 157593].
√
[7] H. O. Nghiem, J. Cartaud, C. Dubreuil, C. Kardeli, G. Buttin, J. P.
Changeux, Proc. Natl. Acad. Sci. USA 1983, 80, 6403 6407.
[8] M. Gautam, P. G. Noakes, J. Mudd, M. Nichol, G. C. Chu, J. R. Sanes,
J. P. Merlie, Nature 1995, 377, 232 236.
[9] A. Schellenberger, Biochim. Biophys. Acta 1998, 1385, 177 186.
Received: April 23, 2002 [F4036]
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