On the way to glycoprocessing inhibitors - Synthesis of an imidazolo-nectrisine-phosphono acid derivative: A potential glycosyltranferase inhibitor

Article abstract of DOI:10.1002/ejoc.200300190

Assuming the transition state of glycosyltransferase inhibitors to be similar to those encountered with potent glycosidase inhibitors - i.e. a flattened conformation with a positively charged anomeric centre - we worked out a synthesis of the D-arabino-configured phosphonic acid target molecule 2 derived from an imidazolo-sugar. The key synthetic intermediate is the linear imidazolo L-xylo compound 10 which could be obtained, either from L-threo precursor 6 by a coupling reaction with imidazole derivative 5, or from L-sorbose. A multi-step and site specific iodination of 10 gave the monoiodo-L-xylo derivative 14 which was cyclised to the D-arabino-configured bicyclic azasugar 15. Phosphorylation of the Grignard derivative of the latter, followed by mono-esterification with citronellol along with some protection-deprotection steps led to target molecule 2. The potential inhibitor 2 is supposed to be protonated at its most basic N atom by a carboxylic acid residue in the arabinosyl-transferase active site. Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003.

Full text of DOI:10.1002/ejoc.200300190

FULL PAPER
On the Way to Glycoprocessing Inhibitors ؊ Synthesis of an Imidazolo-
Nectrisine-Phosphono Acid Derivative: A Potential Glycosyltranferase Inhibitor
[a]
[a]
[b]
[c]
´
Theophile Tschamber,* Francois Gessier, Markus Neuburger, Sudagar S. Gurcha,
¸
Gurdyal S. Besra,[c] and Jacques Streith*[a]
Keywords: Azasugars / Imidazoles / Carbohydrates / Glycosyltransferase inhibitor / Nitrogen heterocycles
Assuming the transition state of glycosyltransferase inhib-
iodo-L-xylo derivative 14 which was cyclised to the D-arab-
itors to be similar to those encountered with potent glycosid- ino-configured bicyclic azasugar 15. Phosphorylation of the
ase inhibitors − i.e. a flattened conformation with a positively
charged anomeric centre − we worked out a synthesis of the
-arabino-configured phosphonic acid target molecule 2 de-
Grignard derivative of the latter, followed by mono-ester-
ification with citronellol along with some protection-depro-
tection steps led to target molecule 2. The potential inhibitor
D
rived from an imidazolo-sugar. The key synthetic interme- 2 is supposed to be protonated at its most basic N atom by a
diate is the linear imidazolo
be obtained, either from -threo precursor 6 by a coupling
reaction with imidazole derivative 5, or from -sorbose. A
L
-xylo compound 10 which could
carboxylic acid residue in the arabinosyl-transferase active
site.
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
L
L
multi-step and site specific iodination of 10 gave the mono-
Germany, 2003)
Introduction
progress so far and is of recent vintage, if only because the
lack of structural data for glycosyltransferases makes it dif-
Mycobacteria are widespread pathogens which are re- ficult to design structure-based inhibitors. Nevertheless, the
sponsible for diseases, such as tuberculosis and leprosy. Ar- mechanisms of the reactions which are catalysed by glycos-
abinogalactan (AG) and lipoarabinomannan (LAM) are es- yltransferases have been investigated to some extent, in par-
sential components of the mycobacterial cell wall. In view ticular for human α-1,3-fucosyltransferase-V by C.-H.
of their xenobiotic character in the human host, these arabi- Wong and his collaborators.[3,4] It is generally postulated
nose-polysaccharides provide attractive targets for drug re- that these enzymatic reactions proceed through a flattened
search. In 1994 β--arabinofuranosyl-1-monophosphoryl- (in the pyranose series it would be through a half-chair con-
decaprenol (1), a key mycobacterial arabinose, had been formed) transition state with substantial sp2 character at
identified in the lipid extracts of Mycobacterium smegmatis, the anomeric carbon.[5] Such a reaction mechanism is remi-
and considered to be an arabinosyl-donor in biogenetic niscent of the one which operates with glycosidases and is
pathways catalysed by glycosyltranferases.[1] To the best of believed to involve a positively charged transition state.
our knowledge, compound 1 is the only intermediate iso- With that mechanistic analogy in mind, we surmised that
lated so far which is most probably involved in arabinan properly phosphorylated derivatives of potent glycosidase
biosynthesis.
azasugar-inhibitors would be good candidates as potential,
Selective inhibition of arabinosyltransferases is of obvi- and hopefully potent, glycosyltransferase inhibitors. Pre-
ous interest, since it should impair the molecular recog- vious work along these lines from the Eustache group[6,7]
nition processes which occur in diseases such as tubercu- leads us to describe herein our own preliminary investi-
losis and leprosy, in which these enzymes play a key cata- gations.
lytic role.[2] Notwithstanding such ambitious a task, the de-
velopment of glycosyltransferase inhibitors has led to little
As reported by us in two previous publications, the eight
stereomers of type 4 imidazolo-pyrrolidinopentoses Ϫ
which are imidazolo analogues of nectrisine 3 Ϫ had been
synthesised and their inhibitory properties determined with
half a dozen glycosidases.[8,12] In these latter series the -
arabino stereomer 4 proved to be the most potent inhibitor
(Ki ϭ 5 µ  with α--mannosidase of Jack beans).[8] In view
of the assumed analogy between the transition states of gly-
cosidase and glycosyltransferase mechanisms (as alluded to
[a]
´
´
Ecole Nationale Superieure de Chimie, Universite de Haute-
Alsace,
3, rue Alfred Werner, 68093 Mulhouse, France
Fax: (internat.) ϩ33-(0)389336860
E-mail: j.streith@uha.fr
Institut für anorganische Chemie, Universität Basel,
Spitalstrasse 51, 4056 Basle, Switzerland
School of Biosciences, The University of Birmingham,
Birmingham, B15 2TT, England
[b]
[c]
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 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/ejoc.200300190
Eur. J. Org. Chem. 2003, 2792Ϫ2798

Synthesis of an Imidazolo-Nectrisine-Phosphono Acid Derivative
FULL PAPER
above), in view also of the -arabino configuration of the
-arabinosyl donor 1, we set out to synthesise the sugar-
derived phosphonic acid 2 as a likely candidate to inhibit
-arabinosyltranferases. The basic N(2) atom of 2 was sup-
posed to favour binding to the enzyme’s active site, while
the stable phosphonate C؊PO(OH)(OR) moiety had to re-
place the labile phosphate CO-PO(OH)(OR) (leaving
group). In other words, the task of the phosphonate moiety
is twofold: i) it permits optimal docking into the enzyme’s
active site; ii) it prevents the cleavage to take place inside
that active site. In the herein described first approach to a
potential glycosyltransferase inhibitor, we consider the ci-
tronellyl phosphonate moiety of 2 as a rough analogue of
the decaprenyl phosphate part of the natural arabinosyl-
donor compound 1 (Figure 1).
Scheme 1. Retrosynthetic pathway for the synthesis of the citro-
nellylphosphonate imidazolo--arabinose 2
Synthesis of the L-Xylo Intermediate 10 (Scheme 2): In a
first approach, we made use of the 4-iodo-1-trityl derivative
5 which had already been described by Kirk.[10] The reac-
tion of the Grignard derivative of 5 with the known -threo
compound 6[11] gave a mixture of the two diastereomers 7
and 8 (in a 4:6 ratio) which were not separated. Since only
the minor -xylo diastereomer 7 was of interest, the preced-
ing mixture was oxidised (MnO2) to the crystalline ketone
9 in 69% overall yield (from 6); its enantiomer ent-9 had
already been described in a recent publication.[8]
Figure 1. β--Arabinofuranosyl-1-monophosphoryldecaprenol (1),
S-(Ϫ)-β-citronellylphosphonate imidazolo--arabinose (2), nectri-
sine 3 and imidazolo--arabino derivative 4
Results and Discussion
Retrosynthetic Analysis (Scheme 1): Retrosynthetic analy-
sis of the target molecule 2 is reproduced in an abridged
form in Scheme 1. The key intermediate is a linear 4Ј(5Ј)-
iodo--xylo imidazole derivative which can be obtained,
either from -threose by a nucleophilic addition of a C-
metallated imidazole to the aldehyde function, or from -
sorbose via a double condensation with formamidine, al-
most all reacting species having to be used with the appro-
priate protecting groups. Site specific iodination of the said
-xylo derivative, followed by intramolecular cyclisation,
was expected to lead to a bicyclic -arabino iodoimidazole
intermediate. The Grignard derivative of the latter product
had to be phosphorylated, followed by mono-esterification
to the target molecule 2. The experimental results (see be-
low) did confirm the above retrosynthetic expectations in-
deed.[9]
Scheme 2. Reagents and conditions: a) CH2Cl2, EtMgBr/Et2O,
room temp., 30 min; b) room temp., 2 h; c) CH2Cl2, MnO2, room
temp., 1.5 h (69% from 6); d) THF, -selectride, Ϫ78 °C, 30
min (84%); e) 1) THF, NaH, nBu4N cat., 30 min, 2) BnBr, room
temp., 2 h, 3) THF, 4  HCl, reflux (77%)
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T. Tschamber, F. Gessier, M. Neuburger, S. S. Gurcha, G. S. Besra, J. Streith
FULL PAPER
-Selectride reduction of 9 at low temperature led in 84% silylation of the primary alcohol of 12 was achieved in high
yield to the desired -xylo derivative 7 as the major com- yield (93%) with TBDPSCl in the presence of imidazole and
pound (95%), the minor diastereomer 8 being formed in Et3N and led to 13. That latter compound 13 was reduced
[14]
trace amounts only (5%). O-Benzylation of 7 followed by with Na2SO3
in high yield to give selectively the mono-
removal of the acid labile protecting groups gave the target iodo derivative 14. The position of the iodine of 14 was
-xylo molecule 10 in 77% overall yield (from 7). The syn- demonstrated unambiguously by a single crystal X-ray dif-
thesis of ent-10 has already been described;[8] not surpris- fraction analysis (Figure 2). Overall, the three-step conver-
1
ingly, H and 13C NMR spectroscopic data of 10 and ent- sion of 10 into the desired mono-iodo intermediate 14 was
10 turned out to be identical (see Exp. Sect.).
achieved in 78% overall yield.
The second approach was quite simple: the imidazolo--
Synthesis of the -arabino Citronellyl-Phosphono Ester
D
xylo derivative 11, which we had obtained previously from Target Molecule 2 (Scheme 4): O-Mesylation of the key -
-sorbose,[12] was partly deprotected in acidic medium to xylo intermediate 14 in pyridine was at once followed by
give the target -xylo key intermediate 10.
intramolecular cyclisation (via a Walden inversion) to the
Selective Iodination of the Imidazole Ring at C-4Ј,5Ј) bicyclic -arabino compound 15 in 96% overall yield. Reac-
(Scheme 3): By applying a procedure which had been devel- tion of the Grignard reagent of 15 with diethyl chloro-
oped with histidine derivatives by Jain and his collabor- phosphite at low temperature gave the diethyl phosphinite
ators,[13] reaction of 10 with NIS in excess led to the bis- which, without isolation, was at once oxidised with tert-
iodo derivative 12 in close to quantitative yield. Selective butyl hydroperoxide to the corresponding diethylphosphon-
ate 16 (88% overall yield from 15). Cleavage of the SiϪO
bond of 16 with TBAF led to 17 (72%), and hydrogenolysis
of the two OϪBn bonds (H2/Pd(OH)2/C) of the latter gave
18 (92%). The diethylphosphonate ester moiety of com-
pound 18 was cleaved quantitatively (Me3SiBr) to the corre-
sponding phosphonic acid 19, whose triethylammonium
Scheme 3. Reagents and conditions: a) EtOH/6  HCl, 65 °C, 1.5 h
(71%); b) acetonitrile, NIS, room temp., 12 h (93%); b) CH2Cl2,
NEt3, TBDPSCl, imidazole, room temp., 12 h; d) EtOH, H2O,
Na2SO3, reflux, 2 h [84% for step c) and d)]
Scheme 4. a) 1) pyridine, MsCl, room temp., 1 h, 2) ϩ Ac2O, 80
°C, 18 h (96%); b) 1) CH2Cl2, EtMgBr/Et2O, room temp., 30 min,
2), ϩ (EtO)2PCl, Ϫ78 °C to room temp., 3) ϩ tert-butyl hydroper-
oxide, Ϫ15 °C to room temp. (88%); c) THF, nBu4NF, room temp.
(72%); d) EtOH, AcOH, H2, Pd(OH)2/C, room temp., 12 h; e)
CH2Cl2, Me3SiBr, room temp., 60 h (quant.); f) 1) NEt3, H2O,
room temp. 15 min, 2) pyridine, Ac2O, room temp., 16 h; g) aceto-
Figure 2. ORTEP plot of the structure of 14 (50% probability ellip-
soids; H atoms were removed for clarity)
nitrile, pyridine, citronellol, 70 °C, 16 h; h) MeOH, Amberlyst A-
26 (OHϪ), room temp., 1.5 h
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Synthesis of an Imidazolo-Nectrisine-Phosphono Acid Derivative
FULL PAPER
(2.33 g in CH2Cl2, 40 mL). After 90 min, the reaction mixture was
filtered, evaporated to dryness and the residue purified by chroma-
tography (EtOAc/cyclohexane 1:1), to provide ketone 9 as a colour-
less foam (1.69 g, 69%) which was recrystallised (EtOAc/cyclohex-
ane). M.p. 125 °C (ent-9: m.p. 126 °C[8]). [α]2D0 ϭ ϩ43 (c ϭ 2,
salt was treated with acetic anhydride to give the triacetate
phosphono acid salt 20. Compound 20 was not purified
and reacted at once with citronellol and CCl3CN in pyri-
dine to give a mixture containing 21. That mixture was
treated with a basic Amberlyst A-26 (OH Ϫ) resin to give
the fully characterised target molecule 2, in poor yield
though (20% overall from 20).
1
CHCl3) (ent-9: [α]2D0 ϭ Ϫ41[8]). H NMR (CDCl3, 250 MHz): δ ϭ
1.27 (s, 3 H, CH3 acetonide], 1.30 (s, 3 H, CH3 acetonide], 3.96
(dd, 1 H, 4-Hb], 4.01 (dd, 1 H, 4-Ha), 4.48 and 4.70 (AB, J ϭ 11.7,
2 H, OCH2Ph), 4.56 (td, 1 H, 3-H), 4.70 (d, 1 H, 2-H), 7.06Ϫ7.38
(m, 20 H, H arom.), 7.47 (d, 1 H, 5Ј-H), 7.85 (d, 1 H, 2Ј-H) ppm,
Arabinosyltransferase Assay: Based on the previous use
of specific neoglycolipid acceptors,[15] assays performed in
the presence of membranes and the potential inhibitor 2 J2Ј,5Ј ϭ 1.2, J2,3 ϭ 4.7, J3,4a ϭ 6.6, J3,4b ϭ 6.8, J4a,4b ϭ 8.3 Hz.
13C NMR (CDCl3, 62.9 MHz): δ ϭ 25.4 and 26.1 ppm (2 ϫ CH3
acetonide), 65.7 (C-4), 72.7 (CH2Ph), 76.2 [C(Ph)3), 76.4 (C-3), 82.1
(C-2), 109.8 [C(CH3)2 acetonide), 127.7Ϫ129.6 (C arom. and C-5Ј),
137.5 (Cs of Ph), 138.7 (C-4Ј), 139.6 (C-2Ј), 141.6 (Cs of CPh3),
193.3 (C-1) ppm. C36H34N2O4 (558.65): calcd. C 77.39, H 6.13, N
5.01; found C 77.0, H 6.2, N 5.1. Both NMR spectra were identical
with those of ent-9.[8]
were examined for [14C]Araf incorporation from DP-[14C]A
onto the synthetic acceptors. Scintillation counting followed
by TLC/autoradiography of the reaction products demon-
strated that 2 possessed no inhibitory activity, i.e. control
acceptor activity corresponded to a duplicate set of assays
determined to be 3,280 and 3,450 cpm; with the inhibitor
they were within experimental error and corresponded to
values between 3,190 and 3,650 cpm.
Imidazolo-L-xylo Derivative 7: A solution of -Selectride (1 ,
1.2 mL, 1.2 mmol) in THF was added dropwise at Ϫ78 °C to a
stirred solution of 9 (455 mg, 0.81 mmol) in anhydrous THF
(8 mL). The reaction was monitored by TLC (EtOAc/cyclohexane,
1:1). After 1 h at Ϫ78 °C, the solution was quenched with MeOH
(2 mL), warmed up to room temp., diluted with a saturated solu-
tion of NH4Cl (10 mL) and the aq. phase was extracted with
CH2Cl2 (3 ϫ 10 mL). The combined organic phases were dried
(MgSO4), filtered, concentrated to dryness, and purified by chro-
matography (EtOAc/cyclohexane, 1:1) to give a mixture of 7 ϩ 8
(381 mg, 84%) in a 95:5 ratio, as determined by reverse phase
HPLC (Chiracel OD-R column, eluent: MeOH/H2O, 95:5). The
two diastereomers were not separated. 1H NMR (CDCl3,
250 MHz) of 7: delta ϭ 1.34 and 1.41 (2s, 6 H, 2 ϫ CH3 acetonide),
3.14 (d large, OH), 3.73 (t, 1 H, 2-H), 3.94 (m, 2 H, 4-Hb), 4.26 (q,
1 H, 4-Ha), 4.50 and 4.68 (AB, J ϭ 11.3, 2 H, OCH2Ph), 4.72 (m,
1 H, 1-H), 6.90 (s, 1 H, 5Ј-H), 7.10Ϫ7.35 (m, 20 H, H arom.), 7.43
(d, 1 H, 2Ј-H) ppm.
Experimental Section
General: Flash chromatography (FC): silica gel (Merck 60;
230Ϫ400 mesh). TLC: silica gel on aluminium sheets (Merck 60
HF254); the spots were viewed under UV or by heating with a ther-
mogun after spraying with a solution of KMnO4 (20 g) and
Na2CO3 (40 g) in H2O (1 L) or a solution of phosphomolybdic acid
(5% in 96% EtOH). M.p. Kofler hot-bench or Büchi SMP appar-
atus; corrected values. Optical rotations were all measured at ϩ20
1
°C: SchmidtϪHaensch Polartronic Universal polarimeter. H and
13C NMR spectra: 250 or 400 MHz and 62.9 or 100.6 MHz, respec-
tively; Bruker ACF-250 and Bruker DSX-400 spectrometers at
300 K. 31P NMR spectra: Bruker DSX-400 at 161.9 MHz. Internal
1
references for H NMR: SiMe4 (δ ϭ 0.00 ppm), CDCl3 (δ ϭ 7.26
ppm), CD3OD (δ ϭ 3.30 ppm), dioxane for spectra in D2O (δ ϭ
3.7 ppm); for 13C NMR: CDCl3 (δ ϭ 77.03 ppm), CD3OD (δ ϭ
49.02 ppm), dioxane for spectra in D2O (δ ϭ 67.34 ppm); external
reference for 31P: 85% H3PO4 (δ ϭ 0.0 ppm); δ in ppm and J
in Hz. HR-MS were measured with ESI mode in the departments
of spectroscopy of Hoffmann-La Roche and Novartis in Basle.
Microanalyses were carried out by the Service Central de Microan-
alyses of the CNRS, 69390 Vernaison, France, and by the Service
de Microanalyses of the ICSN/CNRS, 91198 Gif-sur-Yvette,
France. ‘‘MeOH ϩ NH3’’ stands for a solution of pure MeOH
saturated at room temp. with NH3 (ex gas form). CH2Cl2 was dried
by distilling over P2O5.
Imidazolo-L-xylo Derivative 10 (First Approach): A suspension of
NaH in oil (50%, 50 mg, ca. 1.0 mmol) was added at 0 °C to a
stirred solution of almost pure 7 (as obtained above: 381 mg,
0.68 mmol) in anhydrous THF (10 mL) under argon. When the
evolution of H2 had ceased, Bu4NI (ca. 5 mg) and BnBr (100 µL,
0.82 mmol) were added, the vigorously stirred solution was heated
at 40 °C for 12 h, cooled to room temp., and MeOH (1 mL) was
added slowly. The solution was concentrated to dryness, the residue
taken up in CH2Cl2 (15 mL), and the resulting solution was washed
with H2O, then with brine and concentrated to dryness, to afford
the dibenzyl intermediate which was neither purified nor character-
ised. The latter crude product was dissolved in a mixture of THF
(10 mL) and 4  HCl (6 mL) and heated at reflux for 4 h. THF
was evaporated in vacuo, the aqueous phase was diluted with H2O
(30 mL) and extracted with Et2O (2 ϫ 50 mL), basified with some
solid Na2CO3, and the heterogeneous mixture was extracted with
CH2Cl2 (3 ϫ 15 mL). The combined organic fractions were dried
(MgSO4), filtered, and concentrated to dryness, and the crude resi-
due was purified by chromatography (CH2Cl2/MeOH ϩ NH3, 9:1)
to afford 10 as a crystalline compound (193 mg, 77%). M.p.
114Ϫ115 °C (EtOAc/MeOH). [α]2D0 ϭ Ϫ42 (c ϭ 1, MeOH) (ent-10:
m.p. 114 °C, [α]2D0 ϭ ϩ47 [8]). 1H NMR (CDCl3, 250 MHz): δ ϭ
3.33 (td, 1 H, 3-H), 3.49 (dd, 1 H, 4-Hb), 3.51 (dd, 1 H, 4-Ha), 3.96
(dd, 1 H, 2-H), 4.36 and 4.47 (AB, J ϭ 11.6, 2 H, OCH2Ph), 4.67
and 4.94 (AB, J ϭ 11.0, 2 H, OCH2Ph), 4.83 (d, 1 H, 1-H), 7.10
(s, 1 H, 5Ј-H), 7.22Ϫ7.36 (m, 10 H, H arom.), 7.71 (s, 1 H, 2Ј-H)
Synthesis of Ketone 9: A solution of EtMgBr in Et2O (3 , 1.9 mL,
5.70 mmol) was added dropwise under argon at room temp. to a
stirred solution of 4-iodo-1-tritylimidazole (5, 2.30 g,
5.27 mmol)[16] in anhydrous CH2Cl2. After 30 min, a solution of
aldehyde 6 (1.10 g, 4.39 mmol)[11] in anhydrous CH2Cl2 (6 mL) was
added to the stirred mixture and left to react at room temp., the
reaction being monitored by TLC (EtOAc/cyclohexane, 7:3). After
2 h, the reaction was quenched with a saturated aq. solution of
NH4Cl (40 mL) and the organic phase was separated, dried
(MgSO4), filtered, and concentrated to dryness in vacuo. The crude
residue was purified by chromatography (EtOAc/cyclohexane, 1:1,
then 8:2), the two diastereomers 7 and 8 (in a 4:6 ratio) as well as
trace amounts of imidazole not being separated (all together:
2.33 g).
Precipitated activated MnO2 (9.0 g) was added under argon at
room temp. to a stirred solution of the above mixture of 7 ؉8
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T. Tschamber, F. Gessier, M. Neuburger, S. S. Gurcha, G. S. Besra, J. Streith
Characterisation of 13 by NMR Spectroscopy: 1H NMR (CDCl3,
FULL PAPER
ppm, J1,2 ϭ 8.3, J2,3 ϭ 1.6, J3,4a ϭ J3,4b ϭ 6.3, J4a,4b ϭ 10.2 Hz.
13C NMR (CDCl3, 62.9 MHz): δ ϭ 64.5 (C-4), 71.9 (OCH2Ph), 250 MHz): δ ϭ 1.06 [s, 9 H, SiC(CH3)3], 2.82 (slarge, OH), 3.62 (dd,
72.8 (C-3), 76.6 (OCH2Ph), 78.0 (C-1), 82.5 (C-2), 128.5Ϫ129.4 (C 1 H, 4-Hb), 3.74 (dd, 1 H, 4-Ha), 3.85 (dd, 1 H, 2-H), 4.16 (m, 1
arom. and C-5Ј), 137.1 (C-2Ј), 139.9 (C-4Ј), 140.3 (2 Cs phenyl) H, 3-H), 4.30 and 4.44 (AB, J ϭ 11.8, 2 H, OCH2Ph), 4.41 and
ppm. C21H24N2O4·1/2H2O (377.44): calcd. C 66.83, H 6.68, N 7.42;
found C 66.8, H 6.6, N 7.5.
4.47 (AB, J ϭ 11.0, 2 H, OCH2Ph), 4.73 (d, 1 H, 1-H), 7.10Ϫ7.65
(m, 20 H, H arom. phenyl), 10.56 (slarge, 1 H, NH) ppm, J1,2 ϭ 5.5,
J
62.9 MHz): δ ϭ 19.0 [SiC(CH3)3], 26.7 [SiC(CH3)3], 64.3 (C-4),
70.6 (C-3), 70.8 (OCH2Ph), 72.1 (C-1), 73.9 (OCH2Ph), 77.0 (C-
2), 84.3 (C-5Ј), 86.4 (C-2Ј), 127.6Ϫ128.4 (CH phenyl), 129.7 (CH
phenyl), 132.6 and 132.7 (Cs phenyl), 134.9 (C(4Ј)), 135.3 (CH phe-
nyl), 136.7 and 136.8 (Cs phenyl) ppm.
2,3 ϭ 2.6, J3,4a ϭ 6.4, J3,4b ϭ 6.2, J4a,4b ϭ 10.2. 13C NMR (CDCl3,
Imidazolo-L-xylo Derivative 10 (Second Approach): A stirred solu-
tion of the known imidazole derivative 11 (4.24 g, 4.97 mmol)[12] in
EtOH/6  HCl (70:30, 70 mL) was heated to 65 °C for 90 min.
After cooling to room temp. the solution was evaporated to near
dryness, then diluted with H2O (10 mL) and extracted with Et2O
(10 mL). The aq. phase was basified with some NaOH pellets and
the resulting heterogeneous mixture was extracted with EtOAc (3
ϫ 50 mL). The combined organic fractions were dried (MgSO4),
filtered, evaporated to dryness and the crude residue was purified
by chromatography (CH2Cl2/MeOH ϩ NH3, 9:1) to give 10 (1.30 g,
Characterisation of Compound 14: [α]2D0 ϭ Ϫ9.5 (c ϭ 2, CHCl3). 1H
NMR (CDCl3, 250 MHz): δ ϭ 1.05 [s, 9 H, SiC(CH3)3], 2.85 (d, 1
H, OH), 3.63 (dd, 1 H, 4-Hb), 3.72 (dd, 1 H, 4-Ha), 3.91 (dd, 1 H,
2-H), 4.12 (m, 1 H, 3-H), 4.30 and 4.43 (AB, J ϭ 11.8, 2 H,
OCH2Ph), 4.45 (s, 2 H, OCH2Ph), 4.82 (d, 1 H, 1-H), 7.10Ϫ7.65
(m, 20 H, H arom. phenyl), 7.56 (s, 1 H, C-2Ј-H), 10.55 (slarge, NH)
ppm, J1,2 ϭ 5.7, J2,3 ϭ 2.5, J3,4a ϭ 6.3, J3,OH ϭ 5.6, J3,4b ϭ 6.6,
J4a,4b ϭ 10.2 Hz. 13C NMR (CDCl3, 100.6 MHz): δ ϭ 19.2
[SiC(CH3)3], 26.9 [SiC(CH3)3], 64.6 (C-4), 70.9 (C-3), 71.0
(OCH2Ph), 72.8 (C-1), 74.1 (OCH2Ph), 77.6 (C-2), 86.2 (C-5Ј),
127.8Ϫ128.5 (CH phenyl), 129.3 (C-4Ј), 129.9 (CH phenyl), 133.0
and 133.1 (Cs phenyl), 135.6 (CH phenyl), 137.3 (Cs phenyl), 137.8
(C-2Ј) ppm. C37H41IN2O4Si (732.73): calcd. C 60.65, H 5.64, N
3.82, I 17.32; found C 60.7, H 5.7, N 3.8, I 17.2.
1
71%) as a crystalline compound (EtOAc/MeOH). The H and 13C
NMR spectroscopic data of the latter compound were identical and
superimposable with those of 10 as obtained according to the first
approach (see above).
Formation of the 2,5-Diiodoimidazole L-xylo Derivative 12: A solu-
tion of 10 (2.46 g, 6.68 mmol) and NIS (3.6 g, 16.0 mmol) in
CH3CN (50 mL) was stirred at room temp. in the dark. After 12 h
the solution was evaporated to near dryness, and the residue dis-
solved in EtOAc (60 mL). To the resulting solution a saturated aq.
solution of Na2S (15 mL) was added and the biphasic mixture was
vigorously stirred at room temp. for 15 min. The organic phase was
Synthesis of 1-Iodo-imidazolo-D-arabino-pyrrolidinose Derivative 15:
separated, washed with brine (2 ϫ 50 mL), dried (MgSO4), filtered To a stirred solution of 14 (500 mg, 0.682 mmol) in anhydrous pyri-
and the solvents evaporated to dryness. The residue was purified
dine (8 mL) at 0 °C, MsCl (160 µL, 2.05 mmol, 3.0 equiv.uiv.) was
added. After 1 h at room temp., Ac2O (500 µL; excess) was added,
the reaction mixture was heated at 80 °C for 18 h, then cooled to
by chromatography (CH2Cl2/MeOH ϩ NH3, 9:1) to give 12 (3.87 g,
1
93%) as a pale yellow foam. [α]2D0 ϭ Ϫ2 (c ϭ 2, CHCl3). H NMR
(CDCl3, 400 MHz): δ ϭ 3.56 (dd, 1 H, 4-Hb), 3.62 (dd, 1 H, 4-Ha), room temp., treated with EtOH (500 µL) and the solvents evapo-
3.77 (dd, 1 H, 2-H), 3.94 (m, 1 H, 3-H), 4.32 and 4.46 (AB, J ϭ rated to dryness. The oily residue was taken up in a stirred mixture
11.8, 2 H, OCH2Ph), 4.50 and 4.56 (AB, J ϭ 10.8, OCH2Ph, 2 H),
of EtOAc (40 mL) and H2O (40 mL). The resulting organic phase
4.74 (d, 1 H, 1-H), 7.20Ϫ7.34 (m, 10 H, H arom.) ppm. 13C NMR was separated, washed with a saturated solution of NaHCO3
(CDCl3, 100.6 MHz): δ ϭ 64.1 (C-4), 70.9 (C-3), 71.2 (OCH2Ph), (20 mL) and with brine (20 mL), dried (MgSO4), filtered and the
73.1 (C-1), 74.5 (OCH2Ph), 79.4 (C-2), 85.2 (C-2Ј), 128.1Ϫ128.7 (C
arom. phenyl), 136.9 (Cs arom. phenyl); the (C-4Ј) and (C-5Ј) bands
could not be detected on the recorded spectrum.
solvents evaporated to dryness. The residue was purified by chro-
matography (EtOAc/cyclohexane, 3:7) to give 15 (467 mg, 96%) as
a pale yellow oil. [α]2D0 ϭ ϩ9 (c ϭ 2, CHCl3). 1H NMR (CDCl3,
400 MHz): δ ϭ 1.05 [s, 9 H, SiC(CH3)3], 3.79 (dd, 1 H, 8-Hb), 3.82
(dd, 1 H, 8-Ha), 4.27 (slarge, 1 H, 6-H), 4.35 (ddd, 1 H, 5-H), 4.46
and 4.51 (AB, J ϭ 11.9, 2 H, OCH2Ph), 4.56 and 4.67 (AB, J ϭ
11.5, 2 H, OCH2Ph), 4.71 (d, 1 H, 7-H), 7.19Ϫ7.60 (m, 20 H, H
arom. phenyl), 7.51 (s, 1 H, 3-H) ppm, J7,6 ϭ 0.6, J6.5 ϭ 1.1, J5,Ha ϭ
5.5, J,Hb ϭ 8.4, JHa,Hb ϭ 10.5 Hz. 13C NMR (CDCl3, 100.6 MHz):
δ ϭ 19.1 [SiC(CH3)3], 26.8 [SiC(CH3)3], 64.3 (CH2OSi), 65.0 (C-5),
71.7 (OCH2Ph), 71.8 (OCH2Ph), 75.6 (C-7), 76.8 (C-1), 89.3 (C-6),
127.6Ϫ128.5 (CH arom. phenyl), 129.9 and 130.0 (CH arom. phe-
nyl), 132.3 and 132.5 (Cs phenyl), 134.5 (C-3), 135.4 and 135.5 (CH
arom. phenyl), 136.8 and 137.4 (Cs phenyl), 138.6 (C-7a) ppm. HR-
MS: [M ϩ H]ϩ ion 715.1852 (C37H40IN2O3Si, calcd. 715.1853).
Formation of 4-O-TBDPS Derivative 13 and of 4Ј-Imidazolyl-5Ј-
iodo-1,2-di-O-TBDPS-L-xylo Derivative 14: A solution of imidazole
(637 mg, 9.36 mmol), which had been dried by azeotropic distil-
lation with toluene, anhydrous Et3N (1.1 mL, 8.1 mmol), TBDPSCl
(1.8 mL, 6.9 mmol) and a catalytic amount of DMAP in anhydrous
CH2Cl2 (30 mL) was stirred at room temp. for 30 min. To that solu-
tion was added a solution of di-iodo derivative 12 (3.87 g,
6.24 mmol) in CH2Cl2 (20 mL) and the resulting mixture was
stirred in the dark at room temp. for 12 h. The reaction medium
was washed with a saturated aq. solution of NH4Cl (30 mL), and
the organic phase was evaporated to dryness to give crude com-
pound 13 which was not purified any further and used as such in
the next reaction step. To a solution of that crude compound 13 in
EtOH (50 mL), Na2SO3 (1.57 g, 12.48 mmol) and H2O (50 mL)
were added. The resulting heterogeneous mixture was vigorously
stirred at 80 °C for 2 h, cooled to room temp. and EtOH was evapo-
rated in vacuo. The resulting aqueous mixture was extracted with
EtOAc (2 ϫ 50 mL), the combined organic phases were dried
(MgSO4), filtered and the solvents evaporated. The residue was
purified by chromatography (EtOAc/cyclohexane, 1:1) to give 14
(3.85 g, 84%) as colourless crystals. M.p. 162 °C (EtOH/H2O); one
of the monocrystals was used for X-ray diffraction analysis (Fig-
ure 1).
Synthesis of 1-Diethylphosphonate-imidazolo-D-arabino -Pyrrolidi-
nose Derivative 16: To a stirred solution of 15 (1.14 g, 1.59 mmol)
in anhydrous CH2Cl2 (40 mL) was added dropwise a 3.0  solution
of EtMgBr in Et2O (640 µL, 1.92 mmol) at room temp. After
30 min the solution was cooled to Ϫ78 °C, diethylphosphite chlo-
ride (345 µL, 2.38 mmol) was added and the reaction mixture was
slowly heated to room temp. After 1 h at room temp. it was cooled
to Ϫ15 °C, a solution of ca. 5  tBuOOH in n-decane (480 µL, ca.
2.4 mmol) was added, the reaction mixture was stirred at Ϫ15 °C
for 15 min, then at room temp. for 60 min, and finally hydrolysed
with a saturated aqueous solution of NaHCO3 (40 mL). The or-
2796
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Eur. J. Org. Chem. 2003, 2792Ϫ2798

Synthesis of an Imidazolo-Nectrisine-Phosphono Acid Derivative
FULL PAPER
ganic phase was dried (MgSO4), filtered and the solvents evapo-
rated to dryness. The residue was purified by chromatography
(CH2Cl2/MeOH, 98:2) to give 16 (1.02 g, 88%) as a pale yellow oil.
1H NMR (CDCl3, 250 MHz): δ ϭ 1.05 [s, 9 H, SiC(CH3)3], [M
1.25Ϫ1.41 (m, 6 H, 2 ϫ OCH2CH3), 3.81 (dd, 1 H, 8-Hb), 3.86
2JC,P ϭ 4, CH2CH3), 68.2 (C-5), 73.4 (C-7), 85.1 (C-6), 121.7 (d,
J1C,P ϭ 251, C-1), 135.6 (d, J3C,P ϭ 21, C-3), 148.7 (d, J2C,P ϭ 37, C-
7a) ppm. 31P NMR (CDCl3, 101.2 MHz): δ ϭ 21.4 ppm. HR-MS:
ϩ
H]ϩ ion 307.1061 (C11H20N2O6P calcd. 307.1059).
C11H19N2O6P (306.26): calcd. C 43.14, H 6.25, N 9.15, P 10.11;
(dd, 1 H, 8-Ha), 4.07Ϫ4.34 (m, 6 H, 5-H, 6-H and 2 ϫ OCH2CH3), found C 43.0, H 6.2, N 9.1, P 9.8.
4.39 and 4.50 (AB, J ϭ 11.8, 2 H, OCH2Ph), 4.72 and 4.78 (AB,
Synthesis of the 1-Phosphonic Imidazole-D-arabino-Pyrrolidinose
J ϭ 11.6, 2 H, OCH2Ph), 5.04 (s, 1 H, 7-H), 7.19Ϫ7.63 (m, 20 H,
Acid Derivative 19: To a stirred suspension of 18 (200 mg,
0.65 mmol) in CH2Cl2 at 0 °C was added dropwise Me3SiBr
(2.1 mL, 16 mmol, 25 equiv.). After 15 min the reaction mixture
was heated to room temp. for 60 h, then evaporated to dryness. The
residue was co-distilled with toluene (2 ϫ 10 mL) and dissolved in
MeOH (18 mL) and H2O (2 mL). The resulting solution was stirred
vigorously for 10 min and the solvents evaporated to dryness. The
residue was again co-distilled with toluene leading thereby to an
orange coloured foam which was dissolved in H2O (10 mL), the
resulting solution was filtered and submitted to lyophilisation to
give 19 (165 mg, quantitative) as a hygroscopic orange resin, whose
H arom. phenyl), 7.72 (d, 1 H, 3-H) ppm, J3,P ϭ 1.6, J5,Ha ϭ 5.9,
J
5,Hb ϭ 8.6, JHa,Hb ϭ 10.5 Hz. 13C NMR (CDCl3, 62.9 MHz): δ ϭ
16.0Ϫ16.4 (CH2CH3), 19.1 [SiC(CH3)3], 26.8 [SiC(CH3)3],
62.3Ϫ62.4 (CH2CH3), 64.3 (CH2OSi), 65.3 (C-5), 71.7 (OCH2Ph),
71.8 (OCH2Ph), 75.3 (C-7), 90.0 (C-6), 123.9 (d, J1C,P ϭ 248, C-1),
127.6Ϫ128.5 (CH phenyl), 130.0 and 130.1 (CH phenyl), 132.4 and
132.6 (Cs phenyl), 134.2 (d, J3C,P ϭ 21, C-3), 135.4 and 135.5 (CH
phenyl), 136.8 and 137.9 (Cs phenyl), 144.4 (d, J2C,P ϭ 38, C-7a)
ppm. 31P NMR (CDCl3, 101.2 MHz): δ ϭ 12.1 ppm.
Synthesis of the 1-Diethylphosphonate-Imidazole-D-arabino-Pyrroli-
1
purity was sufficient for the next step. H NMR (D2O, 250 MHz):
δ ϭ 3.86 (dd, 1 H, 8-Hb), 4.14 (dd, 1 H, 8-Ha), 4.48Ϫ4.57 (m, 2 H,
dinose Derivative 17: To a stirred solution of 16 (1.00 g, 1.38 mmol)
in THF (30 mL) at room temp. was added dropwise a solution of
1.0  TBAF in THF (2.0 mL, 2.0 mmol). After 1 h the reaction
medium was evaporated to near dryness, taken up in EtOAc
(50 mL) and the resulting solution washed with a saturated aq.
solution of NH4Cl (30 mL) and with brine (30 mL). The organic
phase was dried (MgSO4), filtered and the solvents evaporated to
dryness. The residue was purified by chromatography (CH2Cl2/
5-H and 6-H), 5.14 (d, 1 H, 7-H), 8.85 (d, 1 H, 3-H) ppm, J7,6
ϭ
3.1, J5,Ha ϭ 3.4, J5,Hb ϭ 7.0, JHa,Hb ϭ 12.2, J43,P ϭ 1.8 Hz. 13C
NMR (D2O, 62.9 MHz): δ ϭ 60.6 (C-8), 68.2 (C-5), 72.5 (C-7),
82.4 (C-6), 124.4 (d, J1C,P ϭ 203, C-1), 132.3 (d, J3C,P ϭ 8, C-3),
141.1 (d, J2C,P ϭ 21, C-7a) ppm. 31P NMR (D2O, 101.2 MHz): δ ϭ
Ϫ0.6 ppm.
MeOH, 95:5) to give 17 (481 mg, 72%) as a pale yellow oil. [α]2D0
ϭ
Triethylamino Salt of the D-arabino Phosphonic Acid 19: To a stirred
ϩ1 (c ϭ 2, CHCl3). 1H NMR (CDCl3, 250 MHz): δ ϭ 1.33 and
1.29 (2t, J ϭ 7.1, 6 H, 2 ϫ CH2ϪCH3), 3.80 (dd, 1 H, 8-Hb), 3.91
(dd, 1 H, 8-Ha), 4.06Ϫ4.24 (m, 4 H, 2 ϫ CH2CH3), 4.33 (s, 1 H,
6-H), 4.38 (dd, 1 H, 5-H), 4.48 and 4.58 (AB, J ϭ 11.8, 2 H,
OCH2Ph), 4.77 and 4.83 (AB, J ϭ 11.5, 2 H, OCH2Ph), 5.06 (s, 1
H, 7-H), 7.23Ϫ7.35 (m, 10 H, H arom. phenyl), 7.65 (d, 1 H, 3-H)
ppm, J3,P ϭ 1.6, J5,Ha ϭ 4.8, J5,Hb ϭ 7.9, JHa,Hb ϭ 11.3) Hz. 13C
solution of 19 (165 mg, 0.65 mmol) in H2O (10 mL) at room temp.
Et3N (270 µL, 1.95 mmol) was added. After 15 min the solution
was evaporated in vacuo to give a colourless solid which was dis-
solved again in H2O. The resulting solution was filtered and sub-
mitted 3 times to lyophilisation to give the triethylammonium salt
of acid 19 as a colourless powder, which was neither purified nor
characterised. It was used as such in the next reaction step.
3
NMR (CDCl3, 62.9 MHz]: δ ϭ 16.2 (d, JC,P ϭ 1, CH2CH3), 16.3
3
2
(d, JC,P ϭ 1, CH2CH3), 62.4 (d, JC,P ϭ 6, CH2CH3), 62.5 (d,
2JC,P ϭ 6, CH2CH3), 62.6 (CH2OH), 65.5 (C-5), 71.8 (OCH2Ph),
71.9 (OCH2Ph), 75.5 (C-7), 90.0 (C-6), 123.3 (d, J1C,P ϭ 249, C-1),
127.7Ϫ128.5 (CH arom phenyl), 134.3 (d, J3C,P ϭ 21, C-3), 136.7
and 137.6 (2Cs, phenyl), 144.1 (d, J2C,P ϭ 37, C-7a) ppm. 31P NMR
(CDCl3, 101.2 MHz): δ ϭ 12.3 ppm. HR-MS: [M ϩ ]ϩ ion
487.1996 (C25H32N2O6P, calcd. 487.1998).
Synthesis of the
D-arabino-Triacetate Derivative 20: A solution of
19 (103 mg, 0.20 mmol) in Ac2O (1.5 mL) and pyridine (3 mL) was
stirred overnight (ca. 16 h) and thence distilled in vacuo to near
dryness. The residue was co-distilled 3 times with toluene, dissolved
in MeOH and filtered. The filtrate was evaporated to dryness to
give 20 (137 mg) as a pale yellow solid which, without any purifi-
cation, was used as such for the next reaction step. 1H NMR
(CD3OD, 250 MHz): δ ϭ 1.31 [t, 9 H, J ϭ 6.9, (CH3CH2)3N], 2.08,
2.10, 2.12 (3s, 3 ϫ 3 H, 3 COCH3), 3.20 [q, J ϭ 7.0, 6 H,
(CH3CH2)3N], 4.33 (dd, 1 H, 8-Ha), 4.70 (m, 2 H, 8-Hb and 5-H),
5.50 (s, 1 H, 6-H), 6.18 (s, 1 H, 7-H), 8.16 (s, 1 H, 3-H) ppm,
J5,Hb ϭ 6.3, JHa,Hb ϭ 11.4 Hz.
Synthesis of 1-Diethylphosphonate-Imidazole-D-arabino-Pyrrolidi-
nose Derivative 18: A solution of 17 (480 mg, 0.98 mmol) in EtOH
(7 mL) and AcOH (7 mL) which contained 20% Pd(OH)2/C (Pearl-
man catalyst) was placed under H2 (2 at). The heterogeneous reac-
tion mixture was vigorously stirred for 12 h at room temp., then
submitted to centrifugation. The catalyst was washed several times
Synthesis of the
D-arabino-Citronellyl-Phosphonate Triacetate De-
with hot MeOH, the combined organic portions were evaporated rivative 21: To a stirred solution of crude 20 (137 mg), as obtained
in vacuo to dryness, the residue was dissolved in MeOH (3 mL)
and percolated over some IRA 400 (OHϪ form) resin beads with
MeOH. The fractions which contained the product were combined,
then evaporated to dryness and the resulting residue was purified
above, in pyridine (3 mL) were added Cl3CCN (300 µL) and (S)-
(Ϫ)-β-citronellol (40 µL, 0.22 mmol). That reaction mixture was
heated at 70 °C for 16 h, thence evaporated to near dryness, co-
distilled 3 times with toluene and dissolved in EtOAc (10 mL). The
by chromatography (CHCl3/MeOH 8:2) to give 18 (280 mg, 92%) organic solution was washed with H2O, and the aq. phase extracted
as a crystalline material. M.p. 122 °C. [α]2D0 ϭ ϩ42 (c ϭ 1, MeOH).
with EtOAc (4 ϫ 10 mL). The combined organic portions were
1H NMR (CD3OD, 400 MHz): δ ϭ 1.31 and 1.32 (2t, J ϭ 7.1, 6 dried (MgSO4), filtered and the solvents evaporated to dryness. The
H, 2 ϫ CH2CH3), 3.77 (dd, 1 H, 8-Hb), 4.02 (dd, 1 H, 8-Ha), residue was purified by chromatography (CHCl3/MeOH/H2O,
4.09Ϫ4.16 (m, 4 H, 2 ϫ CH2CH3), 4.19 (td, 1 H, 5-H), 4.42 (t, 1 7:2.6:0.4) to give 21 (237 mg) as a beige solid which was not crystal-
H, 6-H), 4.92 (dd, 1 H, 7-H), 7.83 (d, 1 H, 3-H) ppm, J7,P ϭ 1.2, lised. 1H NMR (CD3OD, 250 MHz): δ ϭ 0.80 (d, 3 H, J ϭ 6.2,
J7,6 ϭ J6,5 ϭ 3.0, J5,Ha ϭ 3.9, J5,Hb ϭ 7.2, JHa,Hb ϭ 11.6, J3,P
ϭ
CHCH3), 1.06 (m, 1 H, CHCH3), 1.24Ϫ1.57 (m, 4 H, 2 ϫ CH3),
2.0 Hz. 13C NMR (CD3OD, 100.6 MHz): δ ϭ 16.5 and 16.6 (2s, 1.57 and 1.65 (2s, 2 ϫ 3 H, C(CH3)2], 1.93 (m, 2 H, CH2), 2.07,
2
CH2CH3), 62.6 (CH2OH), 63.9 (d, JC,P ϭ 4, CH2CH3), 64.0 (d, 2.09 and 2.11 (3s, 3 ϫ 3 H, 3COCH3), 3.83 (m, 2 H, CH2OP), 4.30
Eur. J. Org. Chem. 2003, 2792Ϫ2798
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 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2797

T. Tschamber, F. Gessier, M. Neuburger, S. S. Gurcha, G. S. Besra, J. Streith
(dd, 1 H, 8-Hb), 4.63 (m, 2 H, 8-Ha and 5-H), 5.05 [m, 1 H, CHϭ butanol saturated water, the pooled extracts were back-washed
FULL PAPER
C(CH3)2], 5.49 (s, 1 H, 6-H), 6.12 (s, 1 H, 7-H), 7.92 (s, 1 H, 3-H) twice with water saturated with 1-butanol (3 mL). The 1-butanol-
ppm, J5,Hb ϭ 7.8, JHa,Hb ϭ 12.8 Hz. 31P NMR (CD3OD, saturated water fraction was dried and re-suspended in 200 µL of
101.2 MHz): δ ϭ 11.5 ppm.
1-butanol. The total cpm of radiolabeled material extractable into
the 1-butanol phase was measured by scintillation counting using
10% of the labeled material and 10 mL of EcoScintA (National
Diagnostics, Atlanta). The incorporation of [14C]Araf was deter-
mined by substracting counts present in control assays (incubation
of the reaction components in the absence of the compounds). An-
other 10% of the labeled material was subjected to thin-layer chro-
matography (TLC) in CHCl3/CH3OH/NH4OH/H2O (65:25:0.5:3.6)
on aluminium backed Silica Gel 60 F254 plates (E. Merck, Darm-
stadt, Germany). Autoradiograms were obtained by exposing TLCs
to X-ray films (Kodak X-Omat) for 3 d. Competition based experi-
ments were performed by mixing compounds together, followed by
thin-layer chromatography/autoradiography as described earlier to
determine the extent of product formation.
Synthesis of the -arabino-Citronellyl-Phosphonate Derivative 2: A
D
solution of the above compound 21 (237 mg) in MeOH (10 mL)
was vigorously stirred for 90 min at room temp. in the presence of
Amberlyst A-26 (OHϪ form) beads. After filtration, the beads were
rinsed several times with MeOH, and the combined organic por-
tions were evaporated to dryness. The residue was purified by chro-
matography (CHCl3/MeOH/H2O, 6:3.5:0.5) to give compound 2
(15 mg, 20%) as a faintly beige solid. 1H NMR (CD3OD,
250 MHz): δ ϭ 0.81 (d, J ϭ 6.2, 3 H, CHCH3), 1.09 (m, 1 H,
CHCH3), 1.19Ϫ1.59 (m, 4 H, 2 ϫ CH2), 1.57 and 1.65 [2s, 2 ϫ 3
H, C(CH3)2), 1.92 (m, 2 H, CH2), 3.75 (dd, 1 H, 8-Hb), 3.83 (m, 2
H, CH2OP), 4.05 (m, 2 H, 8-Ha), 4.11 (td, 1 H, 5-H), 4.37 (t, 1 H,
6-H), 4.94 (dd, 1 H, 7-H), 5.05 [m, 1 H, CHϭC(CH3)2], 7.75 (s, 1
H, 3-H) ppm, J47,P ϭ 1.0, J7,6 ϭ J6,5 ϭ J5,Ha ϭ 4.0, J5,Hb ϭ 7.1,
JHa,Hb ϭ 11.4 Hz. 13C NMR (CD3OD, 62.9 MHz): δ ϭ 17.8
(CH3CϭCH), 19.7 (CH3CH), 25.9 (CH3CϭCH), 26.5 (CH2), 30.3
(CHCH3), 38.3 (CH2), 38.9 (d, J3C,P ϭ 7.5, CH2CH2OP), 62.8
(CH2OH), 64.1 (d, J2C,P ϭ 5.2, CH2OP), 67.2 (C-5), 73.5 (C-7), 84.8
(C-6), 125.9 [CHϭC(CH3)2], 131.9 [CHϭC(CH3)2] ppm. HR-MS:
[M Ϫ H]Ϫ ion 387.1687 (C17H28N2O6P, calcd. 387.1685).
Acknowledgments
We thank the Fondation pour l’Ecole de Chimie de Mulhouse for
a PhD grant to one of us (F.G.), and our colleague J. Eustache for
having pointed out to us the possibility of applying the principle
of glycosyltransferase inhibition to the treatment of mycobacterial
infections. G.S.B. who is currently a Lister Institute Jenner research
fellow acknowledges support from the Medical Research Council
(49343 and 49342). Last but not least, the support of the Centre
National de la Recherche Scientifique (UMR-7015) is gratefully
acknowledged.
X-ray Diffraction Analysis of 14: Single crystals of 14, suitable for
X-ray crystallography, were grown by crystallisation from meth-
anol. Data were collected at 293 K. The usual corrections were ap-
plied. The computing calculations were made with the programs
CRYSTALS[17,18]. Scattering factors were taken from the Inter-
national Tables Vol. IV Table 2.2B.The compound has a chemical
formula of C37H40IN2O4Si. The dimensions of the unit-cell are:
a ϭ 9.5364(2), b ϭ 18.1656(8), c ϭ 20.954(1) A, α ϭ β ϭ γ ϭ 90°.
The crystal system is orthorhombic and the space group P212121.
The radiation used was Mo-Kα (λ ϭ 0.71073).
[1]
B. A. Wolucka, M. R. McNeil, E. de Hoffman, T. Chojnacki,
P. J. Brennan, J. Biol. Chem. 1994, 269, 23328Ϫ23335.
˚
[2]
M. M. Palcic, Methods in Enzymology 1994, 230, 300Ϫ316.
[3]
B. W. Murray, S. Takayama, J. Schultz, C.-H. Wong, Biochemis-
try 1996, 35, 11183Ϫ11195.
B. W. Murray, V. Wittmann, M. D. Burkart, S.-C. Hung, C.-
[4]
CCDC-186927 contains the supplementary crystallographic data
for this paper. These data can be obtained free of charge at
www.ccdc.cam.ac.uk/conts/retrieving.htlm [or from the Cambridge
Crystallographic Data Centre, 12, Union Road, Cambridge
CB2 1EZ, UK; fax: (internat.) ϩ44-1223/336-033; E-mail:
deposit@ccdc.cam.ac.uk].
H. Wong, Biochemistry 1997, 36, 823Ϫ831.
R. Wang, D. H. Steensma, Y. Takaoka, J. W. Yun, T. Kajimoto,
[5]
C.-H. Wong, Bioorg. Med. Chem. 1997, 5, 661Ϫ672.
[6]
C. Bouix, P. Bisseret, J. Eustache, Tetrahedron Lett. 1998, 39,
825Ϫ828.
[7]
M. Bosco, P. Bisseret, C. Bouix-Peter, J. Eustache, Tetrahedron
Lett. 2001, 42, 7949Ϫ7952.
T. Tschamber, H. Siendt, C. Tarnus, D. Deredas, A. Frankow-
Arabinosyltransferase Assay:[15] DP[14C]A (20,000 cpm, 9 µ , 10
µL (stored in chloroform/methanol, 2:1) including inhibitor 2 (100
µM) were dried under a stream of argon in a microcentrifuge tube
(1.5 mL) and placed in a vacuum dessicator for 15 min to remove
any residual solvent. The dried constituents of the assay were then
suspended in 8 µL of a 1% aqueous solution of Igepal. The remain-
ing constituents of the arabinosyltransferase assay containing
50 m MOPS (adjusted to pH 8.0 with KOH), 5 m β-mercapto-
ethanol, 10 m MgCl2, 1 m ATP, membranes (250 µL) were ad-
ded to a final reaction volume of 80 µL. The reaction mixtures
were then incubated at 37 °C for 1 h. A CHCl3:CH3OH (1:1, 533
µL) solution was then added to the incubation tubes and the entire
contents centrifuged at 18,000 ϫ g. The supernatant was recovered
and dried under a stream of argon and re-suspended in C2H5OH/
H2O (1:1, 1 mL) and loaded onto a pre-equilibrated (C2H5OH/H2O
[1:1] 1 mL Whatmann strong anion exchange (SAX) cartridge
which was washed with 3 mL of ethanol. The eluate was dried and
the resulting products partitioned between the two phases arising
from a mixture of 1-butanol (3 mL) and H2O (3 mL). The resulting
organic phase was recovered following centrifugation at 3,500 ϫ g
and the aqueous phase was again extracted twice with 3 mL of 1-
[8]
ski, J. Kohler, J. Streith, Eur. J. Org. Chem. 2002, 702Ϫ712.
[9]
This paper describes part of the PhD thesis by Franc¸ois Gess-
´
ier, Universite de Haute-Alsace, Mulhouse, 2001.
[10]
[11]
K. L. Kirk, J. Heterocyclic Chem. 1985, 22, 57Ϫ59.
T. Tschamber, H. Siendt, A. Boiron, F. Gessier, D. Deredas, A.
Frankowski, S. Picasso, H. Steiner, A.-M. Aubertin, J. Streith,
Eur. J. Org. Chem. 2001, 1335Ϫ1347.
[12]
[13]
[14]
[15]
J. Streith, H. Rudyk, T. Tschamber, C. Tarnus, C. Strehler, D.
Deredas, A. Frankowski, Eur. J. Org. Chem. 1999, 893Ϫ898.
R. Jain, B. Avramovitch, L. A. Cohen, Tetrahedron 1998, 54,
3235Ϫ3242.
H. B. Bensusan, M. S. Rao Naidu, Biochemistry 1967, 6,
12Ϫ15.
R.E. Lee, P.J. Brennan, G.S. Besra, Glycobiology 1997, 7,
1121Ϫ1128.
R. M. Turner, S. D. Lidell, J. Org. Chem. 1991, 56, 5739Ϫ5740.
D. J. Watkin, C. K. Prout, J. R. Carruthers, P. W. Betteridge,
R. I. Cooper, CRYSTALS Issue 11. Chemical Crystallography
Laboratory, Oxford, UK, 2001.
D. J. Watkin, C. K. Prout, L. J. Pearce, CAMERON. Chemical
Crystallography Laboratory, Oxford, UK, 1996.
Received March 21, 2003
[16]
[17]
[18]
2798
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2003, 2792Ϫ2798