Stereoselective synthesis of β-1-C-substituted 1,4-dideoxy-1,4-imino-d-galactitols and evaluation as UDP-galactopyranose mutase inhibitors

Article abstract of DOI:10.1016/j.bmc.2007.06.059

The synthesis of 1-C-substituted 1,4-dideoxy-1,4-imino-d-galactitols involving nitrone umpolung is described. The SmI₂-induced key coupling proved highly stereoselective in favor of the β-C-substituted products bearing a three-carbon chain at t

Full text of DOI:10.1016/j.bmc.2007.06.059

Bioorganic & Medicinal Chemistry 15 (2007) 6443–6449
Stereoselective synthesis of b-1-C-substituted
1,4-dideoxy-1,4-imino-D-galactitols and evaluation
as UDP-galactopyranose mutase inhibitors
a
b,
b
c
*
´
´
Stephanie Desvergnes, Valerie Desvergnes, Olivier R. Martin, Kenji Itoh,
Hung-wen Liuc and Sandrine Pya,*
aDe´partement de Chimie Mole´culaire (SERCO) UMR-5250, ICMG FR-2607, CNRS—Universite´ Joseph Fourier, BP 53,
F-38041 Grenoble Cedex 9, France
bInstitut de Chimie Organique et Analytique, UMR 6005, Universite´ d’Orle´ans—CNRS, Rue de Chartres, BP 6759,
F-45067 Orle´ans Cedex 2, France
cDivision of Medicinal Chemistry, College of Pharmacy and Department of Chemistry and Biochemistry,
University of Texas at Austin, Austin, TX 78712, USA
Received 25 April 2007; revised 19 June 2007; accepted 26 June 2007
Available online 4 July 2007
Abstract—The synthesis of 1-C-substituted 1,4-dideoxy-1,4-imino-D-galactitols involving nitrone umpolung is described. The SmI2-
induced key coupling proved highly stereoselective in favor of the b-C-substituted products bearing a three-carbon chain at the
pseudoanomeric position. Pyrrolidines 9 and 10, as well as the bicyclic compounds 8 and 11, exhibit weak inhibition of the activity
of the UDP-galactopyranose mutase from Escherichia coli.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
of new, selective therapeutic approaches against myco-
bacteria such as Mycobacterium tuberculosis.
Tuberculosis, leprosy, and other diseases caused by
mycobacterial infections are currently public health con-
cerns due to multiresistances appearing against most
existing treatments.1 The necessity to investigate new
therapeutic strategies against these pathologies has been
widely recognized,2 and the ones based on inhibition of
the mycobacterial cell wall biosynthesis have attracted
particular attention.3 One of the main components of
this rigid envelope is an arabinogalactan, that is essen-
tial to the survival of mycobacteria.4 Since the main con-
stituent of galactan (D-galactofuranose) is not found in
mammalian metabolism, inhibition of its biosynthesis5
constitutes an attractive approach for the development
In mycobacteria, galactan biopolymer arises from the
action of specific enzymes having uridine-diphospho-
galactofuranose (UDP-Galf) as substrate, such as
UDP-galactofuranose transferases6 and UDP-galacto-
pyranose mutase, which catalyzes the interconversion
of UDP-galactopyranose (UDP-Galp) and UDP-galac-
tofuranose (UDP-Galf) (Scheme 1).
UDP-Gal mutase
(UGM)
O
OH
O
HO
HO
OH
OUDP
OH
OH
OUDP
OH
OH
1: UDP-Gal
p
(92- 95%)
UDP-Gal
2: UDP-Gal
f (5-8%)
Keywords: Iminosugars; Pyrrolidines; UDP-galactose mutase; Nitrone;
Umpolung; Samarium diiodide; Reductive coupling; Enzyme inhibi-
tion; Tuberculosis; Mycobacterium; Arabinogalactan; Pyrrolizidines;
Iminogalactitols; Galactofuranose.
f
transferases
*
Corresponding authors. Tel.: +33 540 00 62 87; fax: +33 540 00
62 86 (V.D.); tel.: +33 476 63 59 83; fax: +33 476 51 48 03
(S.P.); e-mail addresses: v.desvergnes@ism.u-bordeaux1.fr; sandrine.
py@ujf-grenoble.fr
Bacteria cell-wall galactans
Scheme 1. Biosynthesis of the galactan polymer in mycobacteria cell
wall.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.06.059

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S. Desvergnes et al. / Bioorg. Med. Chem. 15 (2007) 6443–6449
O-
Lately, UDP-galactopyranose mutase (UGM, EC
5.4.99.9) has been the subject of extensive studies, dedi-
cated to its characterization,7 and to the understanding
of its intriguing mechanism of action.8 Synthetic work
has also been conducted with the aim of designing suit-
able inhibitors9 and/or mechanistic probes.10 Although
the details of UGM mechanism remain unclear, the
presence of an oxocarbenium-type intermediate in the
transition state of UDP-Galp to UDP-Galf isomeriza-
tion is widely admitted (Scheme 2).8f,11 Hence, strategies
similar to those sought for glycosidase and glycosyl-
transferase inhibition,12 i.e., the use of iminosugars as
transition state analogues (oxonium mimics) or sub-
strate analogues, have also been considered for inhibi-
tion of UGM.
OH
N
*
OBn
BnO
OBn
N+
a
BnO
BnO
CO2Et
5
3
68%
d.r.>98:2
OBn
BnO
68%
OBn
3
4
b
H
N
*
OBn
BnO
CO2Et
BnO
71%
OBn
5
c
BnO
BnO
OBn
OBn
N
O
d
2. Results and discussion
N
3
1
BnO
BnO
7a
BnO
BnO
63%
In connection with previous work aimed at the design of
new UDP-Galf mimics,9d,e we decided to investigate the
utility of the SmI2-induced nitrone umpolung13 to access
a new class of potential UGM inhibitors, based on the
success of this synthetic methodology in the synthesis
of the polyhydroxylated alkaloid (+)-hyacinthacine
A2.14
H
7
H
6
Scheme 3. Synthetic route to C-substituted b-D-1,4-dideoxy-1,4-imino-
galactitol derivatives. Reagents and conditions: (a) ethyl acrylate,
SmI2/H2O, THF, ꢀ78 ꢁC, 3 h; (b) SmI2/H2O, THF, rt, 3 h; (c) K2CO3,
EtOH/H2O, 60 ꢁC, 5 h; (d) LiAlH4, THF, 66 ꢁC, 5 h.
Nitrone 3 was prepared as previously described9e,15 and
was used as a key intermediate for the present synthesis
of new 1-C-substituted 1,4-dideoxy-1,4-imino-D-galacti-
tol derivatives (Scheme 3). When treated with samarium
diiodide16 (3 equiv) at low temperature, 3 was reduced
to an a-amino radical species, that was trapped in situ
with ethyl acrylate (1.4 equiv). As noticed before, the
reaction was faster and better yielding when conducted
in the presence of water (8 equiv).13b,17 The role of this
additive could stand in the protonation of intermediates,
resulting in equilibrium displacement toward formation
of the product. A new C–C bond was thus formed in the
pseudoanomeric position, to afford N-hydroxypyrrolidine
4 in good yield (68%). Remarkably, the diastereoselec-
tivity of the reaction is excellent as the minor isomer,
if formed at all, could not be detected from NMR anal-
ysis of the crude material. The relative configuration at
the new stereogenic center in 4 could not be assigned
at this stage; however it was deduced from the structure
of a cyclized derivative (vide infra).
N-Hydroxypyrrolidine 4 was next deoxygenated using
samarium diiodide at room temperature to yield the cor-
responding pyrrolidine 5. The latter proved quite stable
and no spontaneous cyclization to the corresponding
lactame was observed on standing at room temperature.
Its cyclization was carried out by heating in basic med-
ium (K2CO3, ethanol/water, 60 ꢁC) to yield the pyrroliz-
idinone 6 in 71% yield. Reduction of 6 with lithium
aluminum hydride afforded the pyrrolizidine 7, on which
OH
O+
HO
FADH
OUDP
HO
OH
OH
HO
HO
HO
HO
OH
O
O
N
N R
OH
OH
OUDP
O
H
N
O
FADH
I
HN
O+
OH
H+
FADH
OUDP
OUDP
OH
OH
OH
OH
HO
HO
O
R
N
OH
OH
N+
N R
N
N
O
O
N
OH
O
OH
OH
OH
OH
FADH
OUDP
N
O
HN
OH
H
II
OH
OH
O
III
Scheme 2. Mechanistic hypotheses for UDP-Galp/UDP-Galf isomerization.

S. Desvergnes et al. / Bioorg. Med. Chem. 15 (2007) 6443–6449
6445
Table 1. Inhibition of UGM from Escherichia coli by compounds 8–11
NOE studies allowed unambiguous assignment of the
configuration at C-7a. Irradiation at the frequency of
signal H-7a resulted in enhancement of the H-2 signal,
in favor of a S configuration at C-7a. This result shows
that the acrylate addition, in the C–C bond formation,
occurred on the Si face of the nitrone, opposite to both
the C-3 benzyloxy group and the C-5 substituent (pyr-
rolidine numbering). This ‘matching’ double stereoin-
duction may be the reason for the very high
diastereoselectivity in favor of the trans adduct.
Compound [I] = 25 mM
Residual activitya
Method 1 Method 2
8
9
65
64
78
64
57
57
81
57
10
11
a See Section 3.
converted to UDP-Galp using two HPLC methods
adapted from the work by Lee and co-workers.18
The transformation of compounds 5–7 into iminogalactitol
derivatives was then undertaken (Scheme 4). Although
classical hydrogenolytic conditions (H2, Pd/C) allowed
the preparation of 8 from lactam 6 in 57% yield, deben-
zylation of 5 under the same conditions only led to a
complex mixture of products. The cleavage of benzylic
ethers in 5 and 7 was thus performed using BCl3. In
the case of the debenzylation of 5, initial attempt to neu-
tralize the reaction mixture using a water suspension
of Dowex 1X8 resin afforded substantial amount
(51%) of the carboxylic acid 10 resulting from acid
hydrolysis of the corresponding ethyl ester 9. Finally
the polyhydroxypyrrolizidine 11 could also be obtained
in good yield (75%) using the same method.
The residual activities determined by method 1 are in
good agreement with those of method 2. All iminogal-
actitol derivatives tested were found to exhibit moderate
inhibition of UGM, including lactam 8, in which the
nitrogen atom cannot be protonated under the
conditions of the assay. In this respect, and because of
its constitution (the lactam carbonyl is not in the
pseudoanomeric position), compound 8 cannot be in-
cluded in the family of ‘transition state analogue’ inhib-
itors. Noticeably, compound 10 which carries
a
carboxylic acid function is the least active of this series.
This is somewhat surprizing since the carboxylate could
have been considered as a simple surrogate of the polar
pyrophosphate group of the natural substrate, and a
favorable interaction with a cationic residue at the
UGM putative active site could have been expected.
In this work, because of the stereoselectivity observed in
the key C–C bond forming step, 1-C-substituted 1,4-
dideoxy-1,4-imino-D-galactitols were obtained with a b
configuration at the pseudoanomeric center. Until
now, a single report presents the synthesis of such
D-galactofuranose mimics via a 1,3-dipolar cycloaddition
of the nitrone 3 with an allylphosphonate partner.9e
Compounds 8, 9, and 11 exhibit modest activities on
UGM in the same range (less than 50% inhibition at
25 mM) as other 1-C-substituted iminogalactitols exhib-
iting opposite configuration at the pseudoanomeric cen-
ter.19 This observation is in agreement with recent work
suggesting that modifications of the galactose moiety in
UDP-Galf analogues are well tolerated while the UDP-
core of the substrate seems necessary for better bind-
ing.11c Introduction of UDP-mimicking chains in b-1-
C-substituted iminogalactitols is currently being studied
in our laboratories.
The inhibitory activity of compounds 8–11 against
UGM from Escherichia coli was then evaluated (Table
1). Inhibition assays on the purified enzyme were con-
ducted in the reverse direction in which UDP-Galf is
BnO
HO
HO
OBn
N
OH
N
O
O
a
BnO
57%
3. Experimental
H
6
H
8
BnO
HO
3.1. General experimental section
H
N
H
N
OBn
BnO
OH
Reactions were performed under positive pressure of dry
argon in oven-dried or flame-dried glassware equipped
with a magnetic stirring bar. Standard inert atmosphere
techniques were used in handling all air and moisture
sensitive reagents. THF and CH2Cl2 were freshly dis-
tilled, respectively, from sodium and CaH2. Reactions
were monitored by thin layer chromatography (TLC)
using aluminum-backed silica gel plates. Product purifi-
cation by gravity column chromatography was per-
formed using silica gel (70–230 mesh). Infrared (IR)
spectra were obtained with a Fourier transform infrared
spectrometer (FTIR) as neat films on sodium chloride
plates. NMR chemical shifts d are given in ppm, using
tetramethylsilane as the reference. Coupling constants
J are given with 0.5 Hz accuracy. In pseudo multiplets,
CO2Et
b
CO2R
BnO
HO
b,c
HO
OBn
OH
5
9 (R=Et): 49%
87%
10 (R=H): 51%
9.HCl
BnO
BnO
HO
OH
OBn
N
b
N
HO
75%
H
7
H
11
BnO
HO
Scheme 4. Cleavage of benzyl ethers. Reagents and conditions: (a) H2,
Pd/C, HCl 6 M, MeOH, THF, rt, 4 days, then Dowex 1X8; (b) BCl3,
CH2Cl2, ꢀ78 to 0 ꢁC, 15 h; (c) Dowex 1X8, H2O.

6446
S. Desvergnes et al. / Bioorg. Med. Chem. 15 (2007) 6443–6449
the reported J values are average values of measured
constants. Mass spectra (MS) were obtained using
DCI (ammonia/isobutane, 63/37).
CDCl3): d 1.22 (t, 3H, J = 7.5 Hz); 1.71–1.88 (m, 2H);
2.19 (br s, 1H); 2.25–2.44 (m, 2H); 3.05–3.11 (m, 1H);
3.20 (ps t, 1H, J = 5.0 Hz,); 3.65–3.74 (m, 4H); 3.88
(dd, 1H, J = 3.0, 5.5 Hz); 4.09 (q, 2H, J = 7.0 Hz); 4.39
(d, 1H, J = 12.0 Hz); 4.36–4.54 (m, 6H); 4.74 (d, 1H,
J = 11.5 Hz); 7.26–7.32 (m, 20H). 13C NMR (75 MHz,
CDCl3): d 14.2 (CH3); 28.8 (CH2); 31.5 (CH2); 60.2
(CH2); 61.3 (CH); 63.4 (CH); 71.4 (CH2); 71.9 (CH2);
72.0 (CH2); 73.0 (CH2); 73.3 (CH2); 76.8 (CH); 86.0
(CH); 89.6 (CH); 127.5–128.3 (CH); 138.2 (Cq); 138.3
(Cq); 173.5 (C@O). Anal. Calcd for C39H45NO6: C,
75.09; H, 7.27; N, 2.25. Found: C, 75.10; H, 7.14; N,
2.19.
3.2. 3-((2S,3S,4S,5S)-5-((S)-1,2-Bis(benzyloxy)ethyl)-
3,4-bis(benzyloxy)-1-hydroxy-pyrrolidin-2-yl) propionic
acid ethyl ester (4)
A stirred and carefully deoxygenated solution of nitrone
39e (102 mg, 0.19 mmol) in dry THF (4 mL) was cooled
to ꢀ78 ꢁC under argon. Freshly distilled ethyl acrylate
(28 lL, 0.26 mmol), degassed water (27 lL, 1.50 mmol),
and a 0.1 M solution of SmI2 in THF (5.6 mL,
0.56 mmol) were then added. The temperature was kept
at ꢀ78 ꢁC during 3 h, then air was introduced in the
reaction mixture until disappearance of its blue color.
A saturated aqueous solution of Na2S2O3 (10 mL) and
ethyl acetate (30 mL) were then added; the yellow mix-
ture was extracted with AcOEt (3· 30 mL), and the
combined organic layers were washed with brine
(30 mL), dried over MgSO4, filtered, and concentrated
under vacuum. Purification of the resulting residue by
chromatography on silica gel (pentane/AcOEt, 4/1, then
1/1) afforded the expected N-hydroxyamino ester as a
colorless oil (81 mg, 68%). LRMS (DCI) m/z (%): 640
(100) [M+H]+. IR: mmax (cmꢀ1) 3412, 3090, 3063, 3034,
3.4. (5S,6S,7S,7aS)-5-((S)-1,2-Bis(benzyloxy)ethyl)-6,7-
bis(benzyloxy)-hexahydropyrrolizin-3-one (6)
Compound 5 (46 mg, 0.07 mmol) was dissolved in etha-
nol (8 mL) and treated with a solution of potassium car-
bonate (12 mg, 0.11 mmol) in water (1 mL) at 60 ꢁC
temperature during 5 h. The mixture was then concen-
trated under vacuum, and the residue was extracted with
ethyl acetate (3 · 20 mL). The combined organic layers
were washed with brine, dried over MgSO4, and concen-
trated under vacuum. Purification of the resulting resi-
due by chromatography on silica gel (pentane/AcOEt,
1
2981, 2869, 1737. H NMR (300 MHz, CDCl3): d 1.21
4/1, then 1/1) afforded the expected pyrrolizidinone 6
20
(t, 3H, J = 7.0 Hz); 1.76–1.87 (m, 1H); 2.11–2.23 (m,
1H); 2.40 (ps t, 2H, J = 7.0 Hz); 3.20–3.27 (m, 1H);
3.49 (ps t, 1H, J = 4.5 Hz); 3.67–3.78 (m, 3H); 3.96–
4.19 (m, 4H); 4.30–4.54 (m, 7H), 4.74 (d, 1H,
J = 12.0 Hz); 5.63 (br s, 1H); 7.11–7.53 (m, 20H). 13C
NMR (75 MHz, CDCl3): d 14.2 (CH3); 22.8 (CH2);
32.0 (CH2); 60.4 (CH2); 70.0 (CH); 71.4 (CH2); 71.7
(CH2); 71.9 (CH2); 72.8 (CH); 72.9 (CH2); 73.3 (CH2);
78.4 (CH); 85.5 (CH); 86.7 (CH); 127.5–128.3 (CH);
138.1 (Cq); 138.3 (Cq); 138.4 (Cq); 174.2 (C@O). Anal.
Calcd for C39H45NO7: C, 73.22; H, 7.09; N, 2.19.
Found: C, 73.16; H, 7.01; N, 2.15.
as a colorless oil (30 mg, 71%). ½aꢁ þ 3 (c 0.98, CHCl3);
D
LRMS (DCI) m/z (%): 578 (100) [M+H]+. IR: mmax
(cmꢀ1), 3057, 3028, 2920, 2866, 1691. 1H NMR
(300 MHz, CDCl3): d 1.76–1.89 (m, 1H); 2.17–2.39 (m,
2H); 2.56 (td, 1H, J = 10.0, 17.0 Hz); 3.65 (dd, 1H,
J = 5.5, 8.0 Hz,); 3.69–3.78 (m, 3H); 3.96 (ps q, 1H,
J = 8.0 Hz); 4.01 (dd, 1H, J = 2.0, 4.0 Hz); 4.29 (dd,
1H, J = 4.0, 5.0 Hz); 4.39 (d, 1H, J = 11.5 Hz); 4.46 (d,
1H, J = 11.5 Hz); 4.48 (d, 1H, J = 11.5 Hz); 4.52 (d,
2H, J = 11.5 Hz); 4.59 (d, 1H, J = 12.0 Hz); 4.60 (d,
1H, J = 11.5 Hz); 4.79 (d, 1H, J = 11.5 Hz); 7.17–7.39
(m, 20H). 13C NMR (75 MHz, CDCl3): d 24.8 (CH2);
32.5 (CH2); 60.6 (CH); 64.2 (CH); 72.1 (CH2); 72.2
(CH2); 72.2 (CH2); 73.4 (CH2); 73.6 (CH2); 79.1 (CH);
87.8 (CH); 89.6 (CH); 127.5–128.4 (CH); 137.7 (Cq);
137.9 (Cq); 138.2 (Cq); 138.4 (Cq); 175.3 (C@O). Anal.
Calcd for C37H39NO5: C, 76.92; H, 6.80; N, 2.42.
Found: C, 77.31; H, 7.07; N, 2.61.
3.3. 3-((2S,3S,4S,5S)-5-((S)-1,2-Bis(benzyloxy)ethyl)-
3,4-bis(benzyloxy)pyrrolidin-2-yl) propionic acid ethyl
ester (5)
To a stirred and carefully deoxygenated solution of
N-hydroxypyrrolidine ester 4 (105 mg, 0.16 mmol) in dry
THF (3 mL), degassed water (24 lL, 1.31 mmol) and a
0.1 M solution of SmI2 in THF (4.9 mL, 0.49 mmol)
were added under argon. The solution was stirred at
room temperature during 30 min. Then air was intro-
duced in the reaction mixture until disappearance of
its blue color, whereupon a saturated aqueous solution
of Na2S2O3 (10 mL) and ethyl acetate (30 mL) were
added. The yellow mixture was extracted with AcOEt
(3· 30 mL) and the combined organic layers were
washed with brine (30 mL), dried over MgSO4, filtered,
and concentrated under vacuum. Purification of the
resulting residue by chromatography on silica gel (pen-
3.5. (1S,2S,3S,7aS)-3-((S)-1,2-Bis(benzyloxy)ethyl)-1,2-
bis(benzyloxy)-hexahydro-1H-pyrrolizine (7)
A solution of pyrrolizidinone 6 (90 mg, 0.16 mmol) in
THF (5 mL) was cooled to 0 ꢁC under argon, then lith-
ium aluminum hydride (10 mg, 0.26 mmol) was added.
The reaction mixture was stirred at 66 ꢁC during 5 h
then it was quenched with water (10 lL), an aqueous
15% solution of NaOH (10 lL), and water (40 lL) and
stirred during 1 h. Then sodium sulfate was added, the
mixture was stirred during 1 h and filtered through Cel-
ite. The filtrate was concentrated under vacuum to give a
residue, which upon column chromatography over basic
alumina (pentane/AcOEt, 9/1, 4/1, 1/1, then 0/1) yielded
tane/AcOEt, 4/1, then 1/1) afforded the expected pyrrol-
20
D
(c 1.00, CHCl3); LRMS (DCI) m/z (%): 624 (100)
idine ester 5 as a colorless oil (70 mg, 68%). ½aꢁ ꢀ 25
the pyrrolizidine 7 as a colorless oil (55 mg; 63%).
20
D
[M+H]+; 578 (85) [M+HꢀEtOH]+. IR: mmax (cmꢀ1
)
½aꢁ ꢀ 11 (c 1.00, CHCl3); LRMS (DCI) m/z (%): 564
3061, 3028, 2920, 2862, 1728. 1H NMR (300 MHz,
(100) [M+H]+. IR: mmax (cmꢀ1) 3060, 3031, 2926, 2866.

S. Desvergnes et al. / Bioorg. Med. Chem. 15 (2007) 6443–6449
6447
1H NMR (300 MHz, CDCl3): d 1.61–1.69 (m, 1H); 1.74–
1.83 (m, 2H); 1.93–2.04 (m, 1H); 2.58–2.66 (m, 1H);
2.93–3.04 (m, 2H); 3.46 (ps q, 1H, J = 7.5 Hz); 3.66–
3.72 (m, 3H); 3.75 (t, 1H, J = 7.0 Hz); 4.15 (ps t, 1H,
J = 6.5 Hz); 4.33 (d, 1H, J = 11.5 Hz); 4.45–4.64 (m,
6H); 4.74 (d, 1H, J = 12.0 Hz); 7.21–7.50 (m, 20H).
13C NMR (75 MHz, CDCl3): d 26.2 (CH2); 31.5
(CH2); 56.7 (CH2); 67.1 (CH); 69.8 (CH); 71.4 (CH2);
71.8 (CH2); 72.3 (CH2); 73.0 (CH2); 73.3 (CH2); 78.2
(CH); 85.2 (CH); 88.9 (CH); 127.4–128.4 (CH); 138.3
(Cq); 138.3 (Cq); 138.5 (Cq); 138.7 (Cq). Anal. Calcd
for C37H41NO4: C, 78.83; H, 7.34; N, 2.48. Found: C,
78.49; H, 7.74; N, 2.62.
(CH3); 27.1 (CH2); 31.4 (CH2); 61.9 (CH2); 62.9 (CH);
64.9 (CH2); 65.1 (CH); 69.3 (CH); 77.2 (CH); 80.1
(CH); 174.3 (C@O).
3.8. 3-((2S,3S,4S,5S)-3,4-Dihydroxy-5-((S)-1,2- dihydr-
oxyethyl)pyrrolidin-2-yl)propanoic acid (10)
To a stirred solution of pyrrolidine ester 5 (92 mg,
0.15 mmol) in CH2Cl2 (15 mL) cooled to ꢀ78 ꢁC, under
argon, a solution of BCl3 (1 M in hexanes, 1.8 mL,
1.80 mmol) was added at ꢀ78 ꢁC. The temperature
was slowly raised to 0 ꢁC overnight. After 15 h, ethanol
was added and the reaction mixture was concentrated
under vacuum. The residue was dissolved in a minimum
of water and neutralized with DOWEX 1X8 (OHꢀ
form). After filtration, the filtrate was concentrated un-
der vacuum and purification of the resulting residue by
chromatography on silica gel (CH2Cl2/MeOH: 90/10,
80/20, then 70/30) afforded the deprotected pyrrolidine
acid 10 (18 mg, 51%) and the pyrrolidine ester 9
3.6. (5S,6S,7S,7aS)-Hexahydro-6,7-dihydroxy-5-((S)-
1,2-dihydroxyethyl)pyrrolizin-3-one (8)
To a solution of pyrrolizidinone 6 (103 mg, 0.14 mmol)
in a 4:1 mixture of methanol and THF (12.5 mL) was
added Pd/C 10% (31 mg). After the reaction flask was
purged with hydrogen, 13 drops of HCl 6 M were added
and the reaction mixture was stirred for 4 days at room
temperature under hydrogen (1 atm). The mixture was
then filtered through Celite, and the filtrate was concen-
trated under vacuum. The residue was dissolved in a
minimum of water and neutralized with DOWEX 1X8
resin (OHꢀ form). After filtration, the filtrate was con-
centrated under vacuum to give a residue, which upon
column chromatography over silica gel (CH2Cl2/
MeOH/NH4OH: 98/2/0, 95/5/0, 90/10/0, 80/20/0, then
(16 mg, 49%), both as colorless oils. Compound 10:
20
½aꢁ ꢀ 20 (c 1.01, MeOH). LRMS (DCI) m/z (%): 236
D
(20) [M+H]+; 218 (100) [M+HꢀH2O]+. IR: mmax
(cmꢀ1) 3333, 2921, 1722. 1H NMR (300 MHz, CD3OD):
d 2.06 (ps t, 2H, J = 6.0 Hz); 2.39–2.62 (m, 2H); 3.30–
3.39 (m, 1H); 3.44 (dd, 1H, J = 4.0, 8.0 Hz); 3.64–3.66
(m, 2H); 3.87 (t, 1H, J = 6.5 Hz); 3.92 (dd, 1H,
J = 5.0, 9.0 Hz); 4.04 (dd, 1H, J = 6.5, 8.0 Hz). 13C
NMR (75 MHz, CD3OD): d 27.0 (CH2); 34.5 (CH2);
63.9 (CH); 64.0 (CH); 64.8 (CH2); 69.2 (CH); 77.2
(CH); 79.7 (CH).
3/2/0.5) yielded the deprotected pyrrolizidinone
8
20
(22 mg, 57%) as a colorless oil. ½aꢁ þ 18 (c 0.65,
D
MeOH). LRMS (DCI) m/z (%): 218 (100) [M+H]+.
IR: mmax (cmꢀ1) 3326, 2925, 2883, 1659. 1H NMR
(300 MHz, CD3OD): d 1.93–2.04 (m, 1H); 2.31–2.46
(m, 2H); 2.70 (td, 1H, J = 10.0, 17.0 Hz); 3.53–3.58 (m,
3H); 3.62 (dd, 1H, J = 3.0, 6.0 Hz); 3.77–3.84 (m, 2H);
4.30 (ps t, 1H, J = 6.5 Hz). 13C NMR (75 MHz,
CD3OD): d 25.0 (CH2); 33.4 (CH2); 63.6 (CH); 65.0
(CH2); 66.3 (CH); 72.9 (CH); 81.0 (CH); 82.9 (CH);
179.9 (C@O).
3.9. (1S,2S,3S,7aS)-Hexahydro-3-((S)-1,2-dihydroxyeth-
yl)-1H-pyrrolizine-1,2-diol (11)
To a stirred solution of pyrrolizidine 7 (38.3 mg,
0.068 mmol) in CH2Cl2 (7 mL) cooled to ꢀ78 ꢁC, under
argon, a solution of BCl3 (1 M in hexanes, 820 lL,
0.820 mmol) was added at ꢀ78 ꢁC. The temperature
was slowly raised to 0 ꢁC overnight. After 15 h, metha-
nol was added and the reaction mixture was concen-
trated under vacuum. The residue was dissolved in a
minimum of water and neutralized with DOWEX 1X8
(OHꢀ form). After filtration, the filtrate was concen-
trated under vacuum and purification of the resulting
residue by chromatography on silica gel (CH2Cl2/
MeOH: 90/10, then 80/20) afforded the deprotected
3.7. 3-((2S,3S,4S,5S)-3,4-dihydroxy-5-((S)-1,2-dihydr-
oxyethyl)pyrrolidin-2-yl) propanoic acid ethyl ester (9)
To a stirred solution of pyrrolidine ester 5 (61 mg,
0.10 mmol) in CH2Cl2 (10 mL) cooled to ꢀ78 ꢁC, under
argon, a solution of BCl3 (1 M in hexanes, 1.20 mL,
1.2 mmol) was added at ꢀ78 ꢁC. The temperature was
slowly raised to 0 ꢁC overnight. After 15 h, ethanol
was added and the reaction mixture was concentrated
under vacuum. The residue was purified by chromatog-
raphy on silica gel (CH2Cl2/MeOH: 90/10, then 80/20) to
polyhydroxypyrrolizidine 11 (10.3 mg, 75%) as a color-
20
less oil. ½aꢁ ꢀ 11 (c 1.54, MeOH). LRMS (DCI) m/z
D
(%): 204 (100) [M+H]+. IR: mmax (cmꢀ1) 3300, 2962.
1H NMR (300 MHz, CD3OD): d 2.03–2.21 (m, 4H);
3.40–3.46 (m, 2H); 3.54–3.61 (m, 1H); 3.66 (dd, 1H,
J = 5.5, 11.5 Hz); 3.72 (dd, 1H, J = 5.0, 11.5 Hz); 3.84–
3.95 (m, 2H); 3.98 (ps q, 1H, J = 5.0 Hz); 4.07 (ps t,
1H, J = 7.0 Hz). 13C NMR (75 MHz, CD3OD): d 25.7
(CH2); 29.8 (CH2); 59.1 (CH2); 64.6 (CH2); 69.9 (CH);
72.8 (CH); 74.6 (CH); 78.2 (CH); 79.7 (CH).
afford the deprotected polyhydroxylated pyrrolidine
20
D
(c 0.98, MeOH). LRMS (DCI) m/z (%): 264 (100)
ester 9 as its hydrochloride salt (23 mg, 87%): ½aꢁ ꢀ 33
1
[M+H]+. IR: mmax (cmꢀ1) 3446, 2985, 2925, 1720. H
NMR (300 MHz, CD3OD): d 1.26 (t, 3H, J = 7.0 Hz);
2.01–2.2 (m, 2H); 2.58 (td, 2H, J = 1.5, 7.5 Hz); 3.33–
3.38 (m, 1H); 3.46 (dd, 1H, J = 4.5, 7.5 Hz); 3.63–3.73
(m, 2H); 3.82 (dd, 1H, J = 6.5, 8.0 Hz); 3.92 (q, 1H,
J = 4.5 Hz); 4.04 (ps t, 1H, J = 6.5 Hz,); 4.16 (q, 2H,
J = 7.0 Hz). 13C NMR (75 MHz, CD3OD): d 14.5
3.10. Inhibition assay
UDP-galactopyranose mutase (UGM) was purified
according to the previously reported procedure.8c Imi-
nogalactitol derivatives (25 mM) were individually pre-

6448
S. Desvergnes et al. / Bioorg. Med. Chem. 15 (2007) 6443–6449
2. (a) O’Brien, R. J.; Nunn, P. P. Am. J. Respir. Crit. Care
Med. 2001, 163, 1055–1058; (b) Zhang, Y.; Amzel, L. M.
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incubated with UGM (8.2 nm) for 10 min at room tem-
perature in 50 lL of 100 mM potassium phosphate buf-
fer (pH 7.5). After the pre-incubation, 10 lL of freshly
prepared sodium dithionite (100 mM) was added to
the solution to reduce UGM and then 20 lL of UDP-
galactofuranose (UDP-Galf, 0.2 mM) was added as a
substrate. The concentrations of sodium dithionite and
UDP-Galf in the reaction mixture were 12.5 mM and
50 lM, respectively. The reaction was carried out at
37 ꢁC for 2 min, and the resulting mixture was immedi-
ately frozen by liquid nitrogen to terminate the reaction.
The reaction mixture was analyzed by HPLC using a
C18 column (Microsorb-MV, Varian, 4.6 · 250 mm)
with the detector set at 262 nm. Two eluting methods
were examined to determine the conversion of UDP-
Galf to the product UDP-galactopyranose (UDP-Galp).
3. See for example: (a) Takayama, K.; Kilburn, J. O.
Antimicrob. Agents Chemother. 1989, 33, 1493–1499; (b)
Wen, X.; Crick, D. C.; Brennan, P. J.; Hultin, P. G.
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3.10.1. Method 1. Mobile phase was 0.5% acetonitrile in
50 mM triethylammonium acetate buffer, pH 6.8. Flow
rate was at 1.0 mL/min. Under these conditions, UDP-
Galp and UDP-Galf were eluted at 6.1 and 7.6 min,
respectively.
3.10.2. Method 2. Mobile phase A: 50 mM potassium
phosphate buffer (pH 7.0) containing 2.5 mM tetrabuty-
lammonium hydrogen sulfate (TBAHS). Mobile phase
B: 50% acetonitrile in 50 mM potassium phosphate buf-
fer (pH 7.0) containing 2.5 mM TBAHS.20 The sample
was loaded on the column and eluted isocratically with
mobile phase A containing 4% mobile phase B. The elu-
tion rate was 0.55 mL/min. Under these conditions,
UDP-Galp and UDP-Galf were eluted at 10.9 and
12.6 min, respectively.
7. Escherichia coli UGM: (a) Sanders, D. A. R.; Staines, A.
G.; McMahon, S. A.; McNeil, M.; Whitfield, C.; Nai-
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tuberculosis
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(b)
Beis,
K.;
Srikannathasan, V.; Liu, H.; Fullerton, S. W. B.; Bam-
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Fullerton, S. W. B.; Daff, S.; Sanders, D. A. R.; Ingledew,
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The residual activity of UGM in the presence of individ-
ual iminogalactitol derivative was evaluated by dividing
the conversion in the presence of each derivative with
that in the absence of the derivatives. The results are
summarized in Table 1.
Acknowledgment
9. (a) Lee, R. E.; Smith, M. D.; Nash, R. J.; Griffiths, R.
C.; McNeil, M.; Grewal, R. K.; Yan, W.; Besra, G. S.;
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38, 6733–6736; (b) Lee, R. E.; Smith, M. D.; Pickering,
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B. M. Carbohydr. Res. 2004, 339, 2205–2217; (d) Liau-
tard, V.; Desvergnes, V.; Martin, O. R. Org. Lett. 2006,
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7345.
´
This work was supported by the CNRS, the Universite
Joseph Fourier, and the Agence Nationale pour la
Recherche (Grant No. ANR-05-JCJC-0130-01). H-w.L.
is grateful for the grant support from Welch Foundation
(F-1511). S.D. also thanks the LEDSS for financial
support.
Supplementary data
10. (a) Caravano, A.; Mengin-Lecreulx, D.; Brondello, J.-M.;
Vincent, S. P.; Sinay, P. Chem. Eur. J. 2003, 9, 5888–5898;
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
2007.06.059.
¨
(b) Caravano, A.; Vincent, S. P.; Sinay, P. Chem.
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