3-Fluoroazetidinecarboxylic Acids and trans,trans- 3,4-Difluoroproline as Peptide Scaffolds: Inhibition of Pancreatic Cancer Cell Growth by a Fluoroazetidine Iminosugar

Article abstract of DOI:10.1021/acs.joc.5b00463

Reverse aldol opening renders amides of 3-hydroxyazetidinecarboxylic acids (3-OH-Aze) unstable above pH 8. Aze, found in sugar beet, is mis-incorporated for proline in peptides in humans and is associated with multiple sclerosis and teratogenesis. Aze-con

Full text of DOI:10.1021/acs.joc.5b00463

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Featured Article
3-Fluoro-azetidine Carboxylic Acids and trans,trans-3,4-
Difluoroproline as Peptide Scaffolds: Inhibition of Pancreatic
Cancer Cell Growth by a Fluoroazetidine Iminosugar
Zilei Liu, Sarah F Jenkinson, Tom Vermaas, Isao Adachi, Mark R. Wormald, Yukako
Hata, Yukiko Kurashima, Akira Kaji, Chu-Yi Yu, Atsushi Kato, and George W. J. Fleet
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.5b00463 • Publication Date (Web): 10 Apr 2015
Downloaded from http://pubs.acs.org on April 16, 2015
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3-Fluoro-azetidine Carboxylic Acids and trans,trans-3,4-Difluoroproline as Peptide Scaffolds:
Inhibition of Pancreatic Cancer Cell Growth by a Fluoroazetidine Iminosugar
6
7
8
Zilei Liu,a Sarah F. Jenkinson,a Tom Vermaas,a Isao Adachi,b Mark R. Wormaldc, Yukako Hata,d
Yukiko Kurashima,d Akira Kaji,d Chu-Yi Yu,e,f Atsushi Kato,b* George W. J. Fleeta,f*
9
a Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, OX1 3TA,
UK
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b Department of Hospital Pharmacy, University of Toyama, Toyama 930-0194, Japan
c Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, UK
d Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa 920-1181, Japan
e
CAS Key Laboratory of Molecular Recognition and Function, Institute of Chemistry, Chinese
Academy of Sciences, Beijing 100190, PR China
f
National Engineering Research Center for Carbohydrate Synthesis, Jiangxi Normal University,
Nanchang 330022. PR China
*<george.fleet@chem.ox.ac.uk>; <kato@med.u-toyama.ac.jp>
Abstract Reverse aldol opening renders amides of 3-hydroxy azetidine carboxylic acids (3-OH-Aze)
unstable above pH 8. Aze, found in sugar beet, is mis-incorporated for proline in peptides in humans
and is associated with multiple sclerosis and teratogenesis. Aze-containing peptides may be
oxygenated by prolyl hydroxylases resulting in potential damage of the protein by a reverse aldol of
the hydroxyazetidine; this, rather than changes in conformation, may account for the deleterious
effects of Aze. This paper describes the synthesis of 3-fluoro-Aze amino acids as hydroxy-Aze
analogues which are not susceptible to aldol cleavage. 4-Azidomethyl-3-fluoro-Aze and 3,4-
difluoroproline are new peptide building blocks. trans,trans-2,4-Dihydroxy-3-fluoroazetidine, an
iminosugar, inhibits the growth of pancreatic cancer cells to a similar degree as gemcitabine.
Bn
N
6
F
F
F
OTBDMS
5
1
4
O
HOH2C
CO2Me
F
OMe
2
N
CO2Me
N
1
Bn
Bn
D-Glucose
numbering shows original
C atoms of glucose
Bn
N
F
F
5
1
5
4
O
OMe
HOH2C
CO2Me
N3H2C
CO2Me
F
N
N
2
Bn
1
Bn
Keywords: Amino acid; iminosugar; azetidine; pancreatic cancer; fluoroazetidine; difluoroproline;
fluoro compounds
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INTRODUCTION
Organofluorine compounds play a central role in the pharmaceutical industry.1 There is much current
interest in azetidine,2 including azetidine carboxylic acids, 3 as a pharmacophore in medicinal
chemistry;4 over 180 patents with azetidine in the title have been published since 2013.5 Azetidine
carboxylic acid (Aze) 1, a four membered ring analogue of proline 2, first isolated from Convallaria
majalis in 1955,6 occurs in many plants, including sugar beet7 and in supplements to many foods.8
Aze is mis-incorporated into proteins as a substitute for proline 2 in humans and causes numerous
toxic effects9 including teratogenesis and congenital malformations.10 There is a correlation between
areas of high sugar beet consumption and the occurrence of multiple sclerosis; dangers of Aze
entering the human food chain11 are increasingly recognized.12
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3-OH-Aze 3 has only been prepared by oxidation of Aze 1 by proline hydroxylases.13 The formation
of trans,trans-3-hydroxy-4-hydroxymethyl-L-Aze 4 from D-glucose was the first chemical synthesis
of an unprotected 3-hydroxy-Aze 3.14 3 is a ring contracted analogue of the dihydroxy proline 5.15
The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen
stabilization, hypoxia sensing, and translational regulation and are of considerable interest from
therapeutic perspectives; the RPS23 hydroxylases in S. cerevisiae (Tpa1p), Schizosaccharomyces
pombe and green algae have been shown to catalyze an unprecedented dihydroxylation modification
giving peptides containing 5. 16 Synthetic peptides containing 5 or its azetidine analogues as
constituents may provide interesting probes in biological investigations (Figure 1). However, although
the amide 6 is a potent inhibitor of hexosaminidases,17 6 is unstable above pH 8 due to a reverse aldol
opening of the azetidine ring and thus is not suitable for the preparation of stable peptidomimetics.
Microbial 2-oxoglutarate-dependent dioxygenases also cause oxidation of Aze containing peptides 7
in which oxygen modified hydroxyl-Aze peptides 8 will be susceptible to aldol ring opening, thus
severely damaging the protein structure.18 Modification of the secondary structure of proteins
containing Aze instead of Pro may affect their biological function; however, the damage in protein
structure inherent in the introduction of oxygen into C3 of an azetidine amide may be the cause of
much of the pathology of the Aze-Pro substitution. Access to stable functionalized Aze analogues
may allow investigation of peptides with interesting biological and structural features.
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OH
OH
HO
OH
OH
4
5
6
7
8
9
CONH
CO2H
CONH
F
CO2H HOH2C
CO2H
HOH2C
CONHMe
N
N
H
CO2H
N
H
CO2H
N
N
H
N
H
N
H
H
CO
1
2
3
5
6
4
7
H
O
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F
F
F
F
F
F
N
CO2H
HOH2C
CO2H HOH2C
CONHMe
H2NH2C
CO2H
CO2H
N
H
N
H
N
H
N
H
N
H
CO
13
8
10L
11
12
9
NH2
N
O
N
O
HO
OH
F
F
F
F
HOH2C
NH
O
CH2OH
OH
HOH2C
CH2OH
F
HOH2C
N
H
O
CH2OH
N
H
CH2OH
N
H
N
H
N
H
OH
19
F
18
16
15
14
17
Figure 1: Azetidine and pyrrolidine structures: synthetic targets
Since unprotected 3-hydroxy-Aze derivatives cannot give stable peptides, 3-fluoro-Aze 9 where
fluorine replaces the hydroxyl group, may be an alternative peptidomimetic. In 1970 9 was formed by
the direct photofluorination of Aze. 19 In spite of there being considerable interest in 3-
fluoroazetidines,20 there are no reports of the use of 9 or its derivatives as a template for the
introduction of 3-fluoro-Aze components into peptides.
This paper describes the synthesis of protected building blocks for the incorporation of the
enantiomeric 3-fluoro-Aze 10L and 10D into peptides; both the amide 11 and a dimer derived from
10L were stable, showing 3-fluoro-Aze to be suitable as a peptide mimetic. Building blocks for
inclusion of trans,trans-difluoro proline 12 and the δ-amino-Aze 13 were also prepared; oligomers of
δ-amino -oxetane21 and -THF22 analogues of 13 show a predisposition towards adopting a wide range
of secondary structures. None of the fluoro iminosugars - including the fluoroazetidines 14 and 15,
and the difluoro pyrrolidine analogue 16 of the α-glucosidase inhibitor DAB 1723 - showed any
inhibition of any glycosidase. The meso-fluoroazetidine diol 15 was compared to 5-fluorouracil 18
and gemcitabine 19 and demonstrated significant inhibition of pancreatic cancer cell growth; the anti-
cancer activity of fluoroazetidine 15 provides a further example of biological activity of iminosugars
which does not depend on glycosidase inhibition.
RESULTS AND DISCUSSION
Synthesis
Treatment of 1,3-di-O-triflates derived from carbohydrates with benzylamine provides a strategy for
the efficient synthesis of many azetidines.24 In particular, the cyclization of 2,4-triflates of pyranoses,
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such as 22 and 25, to form bicyclic azetidines in high yields is reliable provided all the substituents in
the pyranose ring are equatorial [trans to each other].25 No other stereochemistry allows any
cyclization and it has not been possible to adjust the stereochemistry after the bicyclic azetidine has
been formed.26
The diacetonide of 3-fluoroglucose 21, the starting material for all the 3-fluoroazetidines prepared, is
formed in 97% yield from diacetone allose 2027 by triflation and nucleophilic displacement with
caesium fluoride in tert-butanol28 on a large scale [over 100 g].29 21 was transformed by standard
carbohydrate manipulations into the pyranose ditriflates 22 and 25 which underwent high yield
reactions with benzylamine to give the bicyclic azetidines 23 and 26, respectively. Ring opening of
23, followed by periodate cleavage of the C5-C6 bond of glucose, allowed access to the fluoro-D-Aze
24D. The bicyclic azetidine 26, in which the C5-C6 bond of glucose had been cleaved prior to
cyclization, led to the L-enantiomer 24L. Activation of the primary hydroxyl group in 24L towards
nucleophilic substitution can lead to the synthesis of the azidomethyl-Aze 27 or of the ring-expanded
difluoroproline 28 as building blocks for the incorporation of novel amino acids into peptides.
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6
F
O
Bn
N
O
6
F
OTBDMS
1
O
O
F3CO2SO
6
OSO2CF3
OMe
5
1
BnNH2
84%
O
4
O
5
O
4
2
O
O
HOH2C
CO2Me
F
OMe
2
N
O
O
1
O
F
Bn
HO
OTBDMS
20
21
22
23
24D
Bn
F
N
F
F
F
F
F3CO2SO
OSO2CF3
OMe
5
BnNH2
97%
1
5
4
O
4
2
and
CO2Me
HOH2C
CO2Me
N3H2C
F
OMe
2
N
N
5
O
CO2Me
N
1
Bn
Bn
Bn
26
25
24L
27
28
Scheme 1: Synthetic strategy and targets [numbering refers to original C in glucose]
The synthesis of the D-trans,trans-Aze ester 24D from the diacetonide 21 required cleavage of the
C5-C6 bond after the formation of azetidine ring (Scheme 2). Hydrolysis of the acetonide 21 by
Dowex H+, followed by peracetylation in pyridine, afforded the tetraacetate 29 as a 1:1 anomeric
mixture (97%).30 Treatment of the tetraacetate 29 with HBr in acetic acid gave the anomeric bromide
which, with methanol in the presence of silver carbonate, gave the β-methyl pyranoside 3031 (87%).
Removal of the acetate groups in 30 with sodium methoxide in methanol gave the triol 31 (96%) in
which the primary hydroxyl group selectively reacted with tert-butyldimethylsilyl (TBDMS) chloride
in the presence of imidazole in DMF to give the silyl ether 32 (98%). Esterification of the diol 32 with
trifluoromethanesulfonic (triflic) anhydride formed the ditriflate 22. Treatment of 22 with
benzylamine in the presence of N,N-diisopropylethylamine (DIPEA) in acetonitrile afforded the
bicyclic azetidine 23 (84% over two steps); only the β-anomer of the ditriflate 22 allowed cyclization
to the azetidine ring.
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O
F
F
O
AcO
OAc
OAc
RO
OR
(i)
(ii)
O
O
7
8
O
F
AcOH2C
O
ROH2C
O
OMe
30 R = Ac
31 R = H
9
21
29
(iii)
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(iv)
F
F
Bn
OTBDMS
N
CH2OAc
OAc
(vii)
RO
TBDMSO
OR
AcO
O
OMe
CH
MeO
F
N
O
OMe
Bn
(vi)
33
23
32 R = H
(v)
22 R = SO2CF3
(viii)
F
F
F
(x)
5
1
(xi)
CH2OH
OH
HOH2C
CO2Me
HOH2C
CO2H
HOH2C
(ix)
N
N
R
N
R
Bn
34 R = Bn
14 R = H
35D R = Bn
10D R = H
(xii)
24D
Scheme 2: Synthesis of D-trans,trans-Aze 24D Reagents and conditions (i) Dowex® (50W X8, H+);
then Ac2O, pyridine, 97% (ii) HBr, AcOH; then AgCO3, MeOH, 87% (iii) NaOMe, MeOH, 96% (iv)
TBDMSCl, imidazole, 98% (v) (CF3SO2)2O, pyridine (vi) BnNH2, DIPEA, MeCN, 84% (vii)
Et2O:BF3, Ac2O, 100% (viii) DIBALH, toluene; then NaBH4, MeOH, 97% (ix) Pd/C, H2, H2O,
dioxane, 88% (x) NaIO4, H2O, dioxane; then I2, K2CO3, MeOH, 69% (xi) K2CO3, H2O, dioxane, 57%
(xii) Pd/C, H2, H2O, dioxane, 45% [numbering refers to original C in glucose]
The acid hydrolysis of the bicycle 23 under a wide range of conditions, followed by treatment of the
crude product with sodium borohydride, only gave traces of the triol 34. However, acetolysis of 23 by
acetic anhydride in the presence of boron trifluoride etherate gave the mixed acetal 33 in 100% yield.
All attempts to directly reduce 33 with sodium borohydride under a variety of conditions afforded
only traces of 34. Accordingly sequential reduction of the triacetate acetal 33 with DIBALH in
toluene, followed by sodium borohydride in methanol, formed the azetidine iminosugar 34 in 97%
yield. These conditions were also required in the hydrolysis of other bicyclic nitrogen heterocycles.32
Oxidative cleavage of the 1,2-diol moiety in 34 by sodium periodate in aqueous dioxane, followed by
further oxidation of the crude aldehyde by iodine in methanol gave the peptidomimetic building block
24D in 69% yield (45% overall yield from the diacetone fluoroglucose 21).
Unprotected azetidine derivatives were prepared from 24D for assaying their biological properties.
Hydrogenolysis of the benzyl group in 34 in the presence of 10% palladium on charcoal in aqueous
dioxane gave the azetidine iminosugars 14 (88%). Hydrolysis of the methyl ester 24D by potassium
carbonate in aqueous dioxane gave the N-benzyl Aze 35D (57%) from which the benzyl group was
removed by hydrogenolysis in the presence of palladium to give the fluoro D-Aze 10D (45%).
A similar sequence of reaction with cleavage of the C5-C6 bond before the formation of azetidine ring
gave the L-trans,trans-Aze ester 24L (Scheme 3). Mild acid hydrolysis of the diacetonide 21 gave the
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monoacetonide 36 (100%). Oxidation of the diol in 36 by sodium periodate, followed by sodium
borohydride reduction of the resulting aldehyde, afforded 37 (92%). Hydrolysis of 37 by Dowex resin
in aqueous dioxane gave 3-fluoro xylose 3833 (88%) in an α:β ratio of 3:2. 38 with acetic anhydride in
pyridine yielded an anomeric mixture of the triacetate 39 (89%). Treatment of 39 with HBr in acetic
acid followed by reaction of the resulting bromide with silver carbonate in methanol gave the
diacetate 40 (60%); removal of the acetate protecting groups by sodium methoxide in methanol gave
the β-methyl xylopyranoside 41 (97%). Esterification of the diol 41 with triflic anhydride in
dichloromethane in the presence of pyridine gave the ditriflate 25; treatment of 25 with benzylamine
in acetonitrile in the presence of DIPEA afforded the bicyclic azetidine 26 (97%). Hydrolysis of the
acetal in 26 with hydrochloric acid in aqueous dioxane gave the corresponding lactol which was
oxidised by iodine in methanol to give the L-Aze ester building block 24L (74%) in an overall yield of
30% from the diacetonide 21.
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O
HOH2C
O
O
O
(ii)
HOH2C
(i)
O
O
O
O
HO
O
O
O
F
F
F
21
36
37
(iii)
F
F
Bn
N
RO
OR
RO
OR
OR
O
F
(vii)
(v)
OMe
O
OMe
(vi)
O
26
(xi)
40 R = Ac
41 R = H
38 R = H
(iv)
39 R = Ac
(viii)
F
F
F
(xii)
1
5
(ix)
HOH2C
(xiii)
CH2OH
HOH2C
CO2Me
HOH2C
CO2H
N
N
N
R
R
Bn
42 R = Bn
15 R = H
35L R = Bn
10L R = H
24L
(x)
Scheme 3: Synthesis of L-trans,trans-Aze 24L: Reagents and conditions (i) MeOH, 1% H2SO4, 100%
(ii) NaIO4, H2O, dioxane; then NaBH4, 92% (iii) Dowex® (50W X8, H+), H2O, dioxane, 88% (iv)
Ac2O, pyridine, 89% (v) HBr, AcOH; then AgCO3, MeOH, 60% (vi) MeONa, MeOH, 97% (vii)
(CF3SO2)2O, pyridine, CH2Cl2; then BnNH2, MeCN, 97% (viii) HCl, H2O, dioxane; then I2, K2CO3,
MeOH, 74% (ix) K2CO3, H2O, dioxane, 43% (x) Pd/C, H2, H2O, dioxane, 82% (xi) HCl, H2O,
dioxane; then NaBH4, MeOH, 40% (xii) NaBH4, MeOH, 90% (xiii) Pd/C, H2, H2O, dioxane, 100%
[numbering refers to original C in glucose]
Hydrolysis of the methyl ester 24L by potassium carbonate in aqueous dioxane gave the N-benzyl
acid 35L (43%) from which the benzyl group was removed by hydrogenolysis in the presence of
palladium on charcoal to produce the azetidine carboxylic acid 10L (82%). The lactol derived from
hydrolysis of 26 was reduced by sodium borohydride in methanol to give the meso-diol 42 (40%);
alternatively, sodium borohydride reduction of the ester 24L gave 42 in 90% yield. Hydrogenolysis of
the N-benzyl group in 42 gave the meso-azetidine 15 (100%).
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Other peptide building blocks were prepared from the L-ester 24L (Scheme 4). The mesylate 43,
formed by treatment of 24L with mesyl chloride in pyridine (100%), with sodium azide in DMF gave
the azide 27 (80%) as a precursor to δ-amino Aze analogues. A variety of reagents for attempted
displacement of the mesylate 43 by fluoride nucleophiles gave complex mixtures, which gave very
low yields of the difluoroazetidine 46. However, reaction of 24L with XtalFluor-M 34 and
triethylamine:HF in dichloromethane afforded the difluoro-proline ester 28 (84%). Neighbouring
group participation by the ring nitrogen on the initial complex 44 with the azetidine nitrogen would
give the bicyclic aziridinium ion 45 to afford the ring expanded proline 28. 28 would allow fluoro
analogues of dihydroxyproline 5 to be incorporated into peptides. Examples of ring expansions of
azetidines to pyrrolidines35 and opening of aziridinium ions by fluoride36 have been reported.
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F
F
F
(i)
(ii)
MsOH2C
CO2Me
N3H2C
CO2Me
HOH2C
CO2Me
N
N
N
Bn
Bn
Bn
43
24L
27
(iii)
F
F
F
CO2Me
FH2C
CO2Me
O
CO2Me
N
N+
Bn
N
H+
N
Bn
SF2
Bn
O
46
45
44
F
F
F
F
F
F
(vi)
(iv)
CH2OH
N
CO2H
N
CO2Me
N
R
R
Bn
47 R = Bn
16 R = H
48 R = Bn
12 R = H
(vii)
(v)
28
Scheme 4: Peptide building blocks Reagents and conditions (i) MsCl, pyridine, 100% (ii) NaN3,
DMF, 80% (iii) XtalFluor-M, Et3N:3HF, CH2Cl2, 84% (iv) NaOH, H2O, dioxane, 64% (v) Pd/C, H2,
H2O, dioxane, 86% (vi) LiAlH4, THF, 66% (vii) Pd/C, H2, H2O, dioxane, 82%
Hydrolysis of 28 with aqueous sodium hydroxide gave the N-benzyl proline 48 (64%) from which the
benzyl group was removed by palladium-catalysed hydrogenolysis to give trans,trans-3,4-difluoro-L-
proline 12 (86%). Reduction of 28 with lithium aluminium hydride in THF gave 47 (66%).
Hydrogenolysis of the benzyl group in 47 afforded 16 (82%), the difluoro analogue of DAB 17, an
iminosugar which has been isolated from many plant sources.
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2
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F
F
4
5
6
(iii)
tBuPh2SiOH2C
CO2R
HOH2C
CO2Me
N
N
Bn
Bn
F
5
24L
(i)
1
7
8
9
50 R = Me
51 R = H
3
tBuPh2SiOH2C
CONHMe
(v)
(iv)
N
Bn
6
10
N
(vii)
t
O
CH2OSiPh2 Bu
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
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42
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45
46
47
48
49
50
51
52
53
54
55
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60
8
F
F
F
54
HOH2C
(ii)
CONHMe
tBuPh2SiOH2C
CONHMe
N
N
(numbering in 54 shows NMR
assignments in experimental section)
R
R
52 R = Bn
53 R = H
49 R = Bn
11 R = H
(vi)
Scheme 5: Amide formation Reagents and conditions (i) MeNH2, CaCl2, MeOH, 74% (ii) Pd/C, H2,
H2O, dioxane, 90% (iii) TBDMSCl, imidazole, DMF, 95% (iv) MeNH2, CaCl2, MeOH, 100% (v)
K2CO3, H2O, dioxane (vi) Pd/C, H2, H2O, dioxane (vii) 51, 53, HBTU, DMF, 45% from 50
The stability of the 3-fluoro Aze motif in peptides was firmly established (Scheme 5). Reaction of the
ester 24L with methylamine in methanol in the presence of calcium chloride formed the methylamide
49 (74%). Palladium-catalysed hydrogenolysis of 49 in aqueous dioxane formed 11 (90%); unlike the
corresponding 3-OH azetidine amide 6, the 3-fluoro amide 11 was stable to a wide range of pH and
vulnerable to neither hydrolysis nor ring fragmentation on treatment with acid or base. A dipeptide 54
was prepared from 24L. Protection of the primary hydroxyl in 24L with TBDMS chloride in DMF in
the presence of imidazole gave the fully protected silyl ether 50 (95%); hydrolysis of 50 with
potassium carbonate in aqueous dioxane afforded the free acid 51. The ester 50 was converted to the
corresponding methylamide 52 on reaction with methylamine in methanol in the presence of calcium
chloride (100%); the benzyl group in 52 was removed by hydrogenation in the presence of palladium
to give the free amine 53. The acid 51 and the amine 53 were coupled with N,N,N’,N’-tetramethyl-O-
(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) in DMF to give the fully protected
dipeptide 54 (45% from 50). The dipeptide was stable and indicated that such Aze analogues could be
useful as components for peptides and for study of the predisposition of substituted Aze units to
induce secondary structure.
Bioassays
Glycosidase Inhibition
The fluoro iminosugars prepared in this paper were compared with the analogous 3-OH azetidines
,
[Figure 2] as inhibitors of the following glycosidases [Table 1 in SI]: 37 38 α-glucosidases (EC
3.2.1.20, rice, yeast, Aspergillus niger), β-glucosidases (EC 3.2.1.21, almond, bovine liver,
Aspergillus niger), α-galactosidase (EC 3.2.1.22, coffee beans), β-galactosidase (EC 3.2.1.23, bovine
liver), α-mannosidase (EC 3.2.1.24, Jack bean), β-mannosidase (EC 3.2.1.25, snail), α-L-
rhamnosidase (EC 3.2.1.40, Penicillium decumbens), α-L-fucosidase (EC 3.2.1.44, bovine kidney), β-
glucuronidases (EC 3.2.1.31, Escherichia coli, bovine liver), trehalase (EC 3.2.1.28, porcine kidney),
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and amyloglucosidases (EC 3.2.1.3, Aspergillus niger, Rhizopus sp.), β-N-acetylglucosaminidases
(EC 3.2.1.52, human placenta, bovine kidney, Jack bean, HL 60, Aspergillus oryzae), α-N-
acetylgalactosaminidase (EC 3.2.1.49, chicken liver, Charonia lampas), and β-N-
acetylgalactosaminidases, (EC 3.2.1.53, HL 60, Aspergillus oryzae).
O
F
F
F
F
F
F
10
11
12
13
14
15
16
17
18
19
20
21
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50
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F
CH2OH
OH
NH
HOH2C
COOH HOH2C
CONHMe
HOH2C
CH2OH
HOH2C
N
R
N
R
N
R
N
R
CH2OH
N
N
H
O
R
10L R = H
35L R = Bn
11 R = H
49 R = Bn
15 R = H
42 R = Bn
14 R = H
34 R = Bn
16 R = H
47 R = Bn
18
NH2
N
HO
OH
OH
OH
OH
OH
N
O
CH2OH
OH
HOH2C
COOH HOH2C
CONHMe
HOH2C
CH2OH
HOH2C
HOH2C
CH2OH
O
N
H
N
N
H
N
H
N
H
R
F
OH
19
F
6
55 R = H
56 R = (CH2)8CH3
4
57
17
Figure 2: Compounds assayed for inhibition of glycosidases and/or pancreatic cancer cell growth
Several azetidine iminosugars show specific inhibition of non-mammalian glycosidases.24 Although
no significant inhibition of any glycosidase is shown either by the free acid 4 or by the iminohexitol
57, meso-azetidine triol 55 is a potent inhibitor of yeast α-glucosidase [IC50 9.5 µM] and a good
inhibitor of trehalase [IC50 30 µM] and rice α-glucosidase [IC50 83 µM].25 Potent inhibition of the
yeast enzyme is rare; DAB 17 and DMDP are only natural iminosugars to do so.39 The N-nonyl
derivative 56 is an inhibitor of some ceramide-specific glucosyl transferases and glucosidases.40 The
amide 6 is a potent inhibitor of a number of β-hexosaminidases but shows no inhibition of any other
glycosidase; however it decomposes rapidly above pH 8.14 Assays of the fluoro azetidines 10L, 11, 14
and 15 showed there was no inhibition of any glycosidase (Table 1 in SI). The fluoro prolinol 16, the
fluorine analogue of DAB 17, similar showed no inhibition of any glucosidase. The N-benzyl
analogues 34, 35L, 42, 47, and 49 also showed no glycosidase inhibition (Table 2 in SI). Replacement
of hydroxyl groups in iminosugars by fluorine substituents almost invariably reduces or eliminates
glycosidase inhibition;41 the only exception so far reported is increased glucosidase inhibition in
fluorinated australines.42
Inhibition of various human cancer cell growth
In the first screening, the fluoro compounds (10L, 10D, 11, 15, and 14) and their oxygen equivalents
(4, 6, 55, and 57) were tested for growth inhibition effects against human pancreatic carcinoma cell
line (PANC-1)43 and were compared with 5-fluorouracil 18 and gemcitabine 19 as positive controls.44
The PANC-1 cells were incubated in an atmosphere of 5% CO2 at 37 °C and sub-cultured every 3
days. In all experiments, cells were grown to 80–90% confluence. The growth inhibition activities
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were elucidated by MTT assay. PANC-1 (1×104 cells/mL) was seeded in each well of 96-well plates.
After incubation with 200 µM test compounds for 72 h, the MTT solution (1 mg/mL in PBS : pH 7.4)
was added to each well (10 µL/well). The plate was incubated for an additional 4 h at 37 °C, and
resulting MTT-formazan crystals produced were dissolved with 200 µL of DMSO. Absorbance was
measured by FLUOstar OPTIMA (BMG LABTECH) at 540 nm. The given values were counted in
triplicate. Growth inhibition was estimated as the reduction in values from a DMSO control.
Preliminary results indicate that only meso-azetidine 15 showed significant inhibition of growth
PANC-1 cells; different samples of 15 synthesized by the alternative pathways in Scheme 3 showed
the same activity. Of all the fluoroiminosugars tested, only 15 showed activity; the N-benzyl
derivative 42 gave no inhibition. It is noteworthy that the fluoroazetidine diol 15 did not inhibit any
glycosidases; in contrast the meso-triol 55 was a good inhibitor of several glucosidases but had no
effect on growth of cancer cells.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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28
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37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
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Table 1. Concentration of fluoroazetidine 15, 5-fluorouracil 18, gemcitabine 19 against human cancer
cell lines
IC50 (µM)
PANC-1
Hep G2
SW480
MCF-7
Fluoroazetidine 15
5-Fluorouracil 18
Gemcitabine 19
165.3 ± 9.1
33.7 ± 11.6
122.9 ± 66.4
80.3 ± 6.2
22.5 ± 8.9
2.6 ± 0.1
194.7 ± 1.2
9.4 ± 4.1
2.8 ± 0.1
332.4 ± 50.2
3.1 ± 0.4
3.1 ± 0.2
Thus, we next investigated the effect of the fluoro meso-azetidine 15 on growth inhibition effects
against various human cancer cells (Table 1). The IC50 values of meso-azetidine 15, fluorouracil 18
and gemcitabine 19 toward PANC-1 were 165.3 ± 9.1, 33.7 ± 11.6, and 122.9 ± 66.4 µM,
respectively. It is noteworthy that growth inhibition potency of 15 against PANC-1 was equivalent to
gemcitabine 19. Furthermore, 15 also showed superior inhibition spectrum widely against human liver
carcinoma (Hep G2), human colon adenocarcinoma (SW480), and human breast adenocarcinoma cell
line (MCF-7), with IC50 values of 80.3 ± 6.2, 194.7 ± 1.2, and 332.4 ± 50.2 µM, respectively. The
inhibition mechanism of 15 is now under investigation but the inhibition is similar to that of 18 and 19
as it had the same induction profile of apoptosis for the cancer cells and the effects depended on the
incubation time.
CONCLUSION
Due to the instability of amides of 3-hydroxy-Aze such as 6, it is not possible to incorporate such
moieties into peptides. Oxygenation of Aze-containing peptides by prolyl hydroxylases may account
for the pathologies associated with human consumption of Aze. In contrast, the fluoroanalogues are
stable and the enantiomeric fluoro-Aze esters 24D and 24L and the difluoroproline ester 28 provide
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suitable building blocks for modified azetidine and proline peptides. The azidomethyl azetidine 27 is
a dipeptide isostere, which may form oligomers with disposition to form novel secondary structures.
None of the fluoroazetidines show any significant inhibition of glycosidases. The inhibition of
pancreatic cell growth by the meso-fluoroazetidine diol 15 is a further example of biological activity
of an iminosugar which is not due to glycosidase inhibition; other analogues will be studied in the
hope of elucidating the mechanism.
10
11
12
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14
15
16
17
18
19
20
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50
51
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60
EXPERIMENTAL SECTION
General Experimental
All commercial reagents were used as supplied. Thin layer chromatography (TLC) was performed on
aluminium sheets coated with 60 F254 silica. Plates were visualized using a spray of 0.2% w/v cerium
(IV) sulfate and 5% ammonium molybdate solution in 2 M aqueous sulfuric acid. Flash
chromatography was performed on Sorbsil C60 40/60 silica. Melting points were recorded on a
Kofler hot block and are uncorrected. Optical rotations are quoted in 103.deg.cm2.g-1 at concentrations
(c) in g.100 mL-1. 1H and 13C NMR spectra were assigned by utilizing 2D COSY, HSQC and HMBC
spectra. All chemical shifts (δ) are quoted in ppm and coupling constants (J) in Hz. Residual signals
from the solvents were used as an internal reference.45 For solutions in D2O acetonitrile was used as
an internal reference. HRMS measurements were made using a microTOF mass analyzer.
3-Deoxy-1,2;5,6-di-O-isopropylidene-3-fluoro-α-D-glucofuranose, 21
Triflic anhydride (11.0 mL, 38.1 mmol) was added dropwise to a solution of 1,2;5,6-di-O-
isopropylidene-D-allose (5.30 g, 20.3 mmol) and anhydrous pyridine (10 mL, 76.7 mmol) in
dichloromethane (25 mL) at -20 °C. After 1.5 h, TLC (cyclohexane/ethyl acetate, 1:1) indicated the
consumption of the starting material (Rf 0.30) and the formation of one major product (Rf 0.64). The
reaction mixture was diluted with DCM (40 mL) and washed with HCl (2 M, aq. 3 x 40 mL). The
organic layer was dried (MgSO4) and the solvent was removed in vacuo to give the crude triflate (7.5
g) as yellow crystalline solid.
Caesium fluoride (9.10 g, 60.0 mmol) was added in one portion to a solution of the crude triflate (7.5
g) in tert-butanol (30 mL). The reaction mixture was stirred at 80 °C for 26 h until TLC
(cyclohexane/ethyl acetate, 2:1) indicated consumption of the triflate (Rf 0.54) and formation of a new
product (Rf 0.61). The reaction mixture was diluted with ethyl acetate (50 mL), washed with 1:1
H2O/NaHCO3 (sat. aq., 50 mL) and brine (50 mL) sequentially and the aqueous layer was back
extracted with DCM (3 x 50 mL). The combined organics were dried (MgSO4), filtered and
concentrated under reduced pressure. The residue was purified by flash chromatography
(cyclohexane/ethyl acetate, 6:1 → 4:1) to give the title compound 21 as clear oil (4.70 g, 89%).
+
20
HRMS (ESI+ve): found 285.1110 [M+Na]+; C12H19FNaO5 requires 285.1109; [α]D -19.7 (c 1.06,
20
CHCl3), {lit.46 [α]D -37.0 (c 1.00, CHCl3)}; vmax (thin film): fingerprint region only; δH (CDCl3, 400
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MHz): 1.32 (3H, s, CH3), 1.36 (3H, s, CH3), 1.44 (3H, s, CH3), 1.50 (3H, s, CH3), 4.03 (1H, dd, H6,
J6,5 4.8, Jgem 8.8), 4.10 (1H, ddd, H4, J4,3 2.2, J4,5 8.3, J4,F 29.1), 4.12 (1H, dd, H6', J6',5 6.1, Jgem 8.8),
4.28 (1H, ddd, H5, J5,6 4.9, J5,6' 6.1, J5,4 8.3), 4.69 (1H, dd, H2, J2,1 3.8, J2,F 10.6), 5.01 (1H, dd, H3, J4,3
2.2, J3,F 49.9), 5.95 (1H, d, H1, J1,2 3.7); δC (CDCl3, 100 MHz): 25.1 (CH3), 26.2 (CH3), 26.7 (CH3),
27.0 (CH3), 67.2 (C6), 72.0 (d, C5, J5,F 7.0), 80.6 (d, C4, J4,F 19.1), 82.5 (d, C2, J2,F 33.4), 93.8 (d, C3,
J3,F 184.4), 105.2 (C1), 109.5 (C(CH3)2), 112.4 (C(CH3)2); δF (CDCl3, 376 MHz): -207.6 (ddd, JF,2
10.8, JF,4 29.2, JF,3 49.8); m/z (ESI+ve): 263 ([M+H]+, 100%), 285 ([M+Na]+, 72%).
10
11
12
13
14
15
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17
18
19
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50
51
52
53
54
55
56
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3-Deoxy-3-fluoro-1,2-O-isopropylidene-α-D-glucofuranose, 36
A solution of the diacetonide 21 (4.20 g, 16.0 mmol) in methanol (20 mL) and 1% aqueous sulphuric
acid (20 mL) was stirred at rt for 18 h after which TLC (ethyl acetate) indicated that the disappearance
of starting material (Rf 0.88) and the formation of a product (Rf 0.65). The reaction mixture was
neutralized with triethylamine and the solvent was removed in vacuo to give a residue that was
purified by column chromatography (cyclohexane/ethyl acetate, 3:1 → 0:1) to form the
monoacetonide 36 (3.55 g, 100%).
+
20
HRMS m/z (ESI+ve): found 245.0794 [M+Na]+, C9H15FNaO5 requires 245.0796; [α]D -18.5 (c
0.80, CHCl3); νmax (thin film): 3411 (br, m, O-H); δH (CDCl3, 400 MHz): 1.33 (3H, s, CH3), 1.50 (3H,
s, CH3), 2.68 (2H, s, OH), 3.75 (1H, ddd, H6, J 0.5, J6,5 5.1, Jgem 11.5), 3.86 (1H, dd, H6', J6',5 3.2, Jgem
11.5) 3.96 (1H, ddd, H5, J5,6' 3.3, J5,6 5.3, J5,4 8.7), 4.16 (1H, ddd, H4, J4,3 2.2, J4,5 8.8, J4,F 29.3), 4.70
(1H, dd, H2, J2,1 3.9, J2,F 10.8), 5.09 (1H, dd, H3, J3,4 2.2, J3,F 49.9), 5.96 (1H, d, H1, J1,2 3.7); δC
(CDCl3, 100 MHz): 26.2 (CH3), 26.6 (CH3), 64.1 (C6), 68.3 (d, C5, J5,F 6.4), 79.7 (d, C4, J4,F 19.1),
82.4 (d, C2, J2,F 31.8), 94.1 (d, C3, J3,F 182.8), 105.1 (C1), 112.4 (C(CH3)2); δF (CDCl3, 376 MHz): -
208.0 (ddd, JF,2 10.8, JF,4 29.2, JF,3 49.8); m/z (ESI+ve): 245 ([M+Na]+, 100%).
3-Deoxy-3-fluoro-1,2-O-isopropylidene-α-D-xylofuranose, 37
Sodium periodate (14.5 g, 67.6 mmol) was added in portions to a solution of diol 36 (12.5 g, 56.3
mmol) in 1,4-dioxane/H2O (2:1, 60 mL). The reaction mixture was stirred at rt for 3 h until TLC
(cyclohexane/ethyl acetate, 1:1) showed the consumption of starting material (Rf 0.15) and the
formation of a new major product (Rf 0.31) after which time ethanol (50 mL) was added and stirred
for a further 20 min. The white solid formed was removed by filtration and sodium borohydride (2.13
g, 56.3 mmol) was added to the stirred reaction mixture. After 2 h, the formation of the desired
product 37 and the consumption of the intermediate aldehyde was confirmed by mass spectrometry
(aldehyde: m/z 245 [M+MeOH+Na+]). The reaction mixture was adjusted to pH 7 by addition of
acetic acid. The mixture was filtered and concentrated under reduced pressure to give a residue that
was purified by column chromatography (cyclohexane/ethyl acetate, 6:1 → 1:1) to afford the title
compound 37 (9.90 g, 92%).
+
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HRMS m/z (ESI+ve): found 215.0686 [M+Na]+; C8H13FNaO4 requires 215.0690; [α]D -25.1 (c
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0.95, CHCl3), {lit.47 [α]D -17.1 (c 1.06, DCM)}; νmax (thin film): 3437 (br, w, O-H); δH (CDCl3, 400
MHz): 1.32 (3H, s, CH3), 1.50 (3H, s, CH3), 1.98 (1H, s, OH), 3.88 (1H, dd, H5, J5,4 5.5, Jgem 11.7),
3.93 (1H, ddd, H5', J 1.2, J5',4 6.6, Jgem 11.7), 4.35 (1H, dddd, H4, J4,3 2.3, J4,5 5.5, J4,5' 6.6, J4,F 30.2),
4.70 (1H, dd, H2, J2,1 3.9, J2,F 11.2), 4.97 (1H, dd, H3, J3,4 2.4, J3,F 50.4), 5.99 (1H, d, H1, J1,2 3.7); δC
(CDCl3, 100 MHz): 26.2 (CH3), 26.6 (CH3), 59.8 (d, C5, J5,F 9.5), 80.2 (d, C4, J4,F 19.1), 82.8 (d, C2,
J2,F 33.4), 94.1 (d, C3, J3,F 184.4), 104.8 (C1), 112.3 (C(CH3)2); δF (CDCl3, 376 MHz): -208.7 (ddd,
JF,2 11.4, JF,4 30.2, JF,3 50.4); m/z (ESI+ve): 215 ([M+Na]+, 100%).
3
4
5
6
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8
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49
50
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3-Deoxy-3-fluoro-D-xylose, 38
Dowex® (50W X8, H+) (2.00 g) was added to a solution of monoacetonide 37 (9.90 g, 51.6 mmol) in
1,4-dioxane/H2O (1:1, 60 mL). The reaction mixture was stirred at 80 °C for 18 h, after which TLC
analysis (ethyl acetate) indicated the disappearance of starting material (Rf 0.74) and the formation of
a single product (Rf 0.29). The reaction mixture was filtered and the solvent removed in vacuo to give
a residue that was purified by column chromatography (cyclohexane/ethyl acetate, 4:1 → 10%
methanol in ethyl acetate) to give the unprotected xylose 38 (6.90 g, 88%) as a white solid in a 3:2,
α:β ratio.
+
HRMS m/z (ESI+ve): found 175.0383 [M+Na]+; C5H9FNaO4 requires 175.0377; mp. 106 - 108 °C,
{lit.48 mp. 127 °C}; νmax (thin film): 3284 (br, s, O-H); δH (CD3OD, 400 MHz): 3.20 (1H, ddd, H5β, J
1.2, J 10.8, Jgem 11.6), 3.28-3.36 (1H, m, H2β), 3.51-3.61 (2H, m, H2α, H5α), 3.65-3.78 (3H, m,
H4α, H4β, H5'α), 3.84 (1H, dt, H5'β, J5',4 6.1, Jgem 11.5), 4.16 (1H, dt, H3β, J3,2 = J3,4 8.8, J3,F 53.1),
4.42 (1H, d, H1β, J1,2 7.8), 4.44 (1H, dt, H3α, J3,2 = J3,4 8.4, J3,F 54.4), 5.07 (1H, t, H1α, J1,2 = J1,F 3.7);
δC (CD3OD, 100 MHz): 61.8 (d, C5α, J5,F 8.0), 65.7 (d, C5β, J5,F 9.5), 69.6 (d, C4α, J4,F 19.1), 69.9 (d,
C4β, J4,F 17.5), 72.2 (d, C2α, J2,F 15.9), 74.5 (d, C2β, J2,F 17.5), 94.4 (d, C1α, J1,F 11.1) 96.5 (d, C3α,
J3,F 179.6) 98.2 (d, C3β, J3,F 182.8) 98.4 (d, C1β, J1,F 12.7); δF (CD3OD, 376 MHz) -195.8 (ddt, Fβ, J
6.7, JF,2 = JF,4 13.6, JF,3 52.8), -201.3 – -201.5 (m, Fα); m/z (ESI+ve): 175 ([M+Na]+, 100%).
3-Deoxy-3-fluoro-1,2,4-tri-O-acetyl-D-xylopyranose, 39
A solution of xylose 38 (6.90 g, 45.4 mmol) in acetic anhydride/pyridine (1:1, 60 mL) was stirred at rt
for 14 h until TLC (cyclohexane/ethyl acetate, 1:1) indicated the consumption of the starting material
(Rf 0.00) and the formation of a single major product (Rf 0.71). The solvent was concentrated under
reduced pressure, the residue dissolved in ethyl acetate (60 mL) and washed sequentially with HCl (2
M, aq., 2 x 60 mL), NaHCO3 (sat. aq., 60 mL) and brine (60 mL). The organic layer was dried
(MgSO4), filtered and the solvent was removed in vacuo to afford a residue which was purified by
column chromatography (cyclohexane/ethyl acetate, 7:1 → 5:1) to give triacetate xylopyranose 39
(11.3 g, 89%) as a white solid in an 8:5, α:β ratio of anomers.
+
HRMS m/z (ESI+ve): found 301.0696 ([M+Na]+), C11H15FNaO7 requires 301.0696; mp. 44 – 48 °C;
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νmax (thin film): 1751 (s, C=O); δH (CDCl3, 400 MHz): 2.09 (3H, s, CH3), 2.11 (12H, s, 4 x CH3), 2.15
(3H, s, CH3), 3.46 (1H, dd, H5β, J5,4 7.8, Jgem 12.2), 3.61 (1H, dd, H5α, J5,4 10.8, Jgem 11.2), 3.97 (1H,
ddd, H5'α, J 4.6, J 6.3, Jgem 11.1), 4.18 (1H, dt, H5'β, J5',4 = J 4.7, Jgem 12.2), 4.63 (1H, dt, H3β, J3,2 =
J3,4 7.7, J3,F 49.9), 4.82 (1H, dt, H3α, J3,2 = J3,4 9.3, J3,F 53.3), 5.02-5.18 (4H, m, H2α, H2β, H4α,
H4β), 5.69 (1H, d, H1β, J1,2 6.6), 6.27 (1H, t, H1α, J1,2 = J1,F 3.7); δC (CDCl3,100 MHz): 20.5, 20.6,
20.7 (x 3), 20.8 (CH3), 60.1 (d, C5α, J5,F 6.4), 61.8 (d, C5β, J5,F 5.6), 68.2 (d, C4β, J4,F 21.5), 68.8 (d,
C4α, J4,F 17.5), 69.2 (d, C2β, J2,F 21.5), 69.6 (d, C2α, J2,F 17.5), 88.5 (d, C3α, J3,F 190.0), 89.1 (d,
C3β, J3,F 188.4), 89.6 (d, C1α, J1,F 9.5), 91.6 (d, C1β, J1,F 8.7), 169.7, 169.1 (x2), 169.6, 169.7 (C=O);
δF (CDCl3, 376 MHz) -194.9 (ddt, Fβ, JF,5 4.6, JF,2 = JF,4 12.6, JF,3 50.4), -199.0 (dddt, Fα, JF,1 3.4, JF,5
4.6, JF,2 = JF,4 12.6, JF,3 53.1); m/z (ESI+ve): 301 ([M+Na]+, 100%).
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Methyl 3-deoxy-2,4-di-O-acetyl-3-fluoro-β-D-xylopyranoside, 40
Hydrobromic acid (33% wt. in acetic acid, 24.3 mL, 98.9 mmol) was added dropwise to a solution of
triacetate 39 (5.50 g, 19.8 mmol) in acetic acid and DCM (7:3, 50 mL) and the reaction was stirred at
5 °C for 5 h until TLC (cyclohexane/ethyl acetate, 1:1) showed the disappearance of starting material
(Rf 0.63) and the formation of a major product (Rf 0.77). The reaction mixture was diluted with DCM
(20 mL), washed successively with ice water (50 mL), cold NaHCO3 (sat. aq., 50 mL) and ice water
(50 mL). The solvent was concentrated in vacuo to afford the crude bromide as an orange solid.
Silver carbonate (9.30 g, 33.4 mmol) was added to a solution of the crude bromide in methanol (90
mL) and the reaction mixture was stirred in the dark at rt for 15 h until TLC (cyclohexane/ethyl
acetate, 1:1) showed the formation of major product (Rf 0.47) and no remaining starting material.
After filtration, the solvent was removed in vacuo to afford a residue which was purified by column
chromatography (cyclohexane/ethyl acetate, 8:1 → 5:1) to afford the pure methyl diacetate 40 (3.00 g,
60%) as a white solid.
+
HRMS m/z (ESI+ve): found 273.0747 [M+Na]+; C10H15FNaO6 requires 273.0745; mp. 78 – 80 °C;
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[α]D -71.3 (c 0.85, CHCl3); {lit.49 mp 80 - 81 °C; [α]D -73 (c 1.00, CHCl3)}; νmax (thin film): 1749
(s, C=O); δH (CDCl3, 400 MHz): 2.10 (3H, s, COCH3), 2.12 (3H, s, COCH3), 3.31 (1H, dd, H5, J5,4
8.3, Jgem 12.0), 3.46 (3H, s, OCH3), 4.15 (1H, dt, H5', J5',4 = J5',F 4.9, Jgem 12.0), 4.36 (1H, d, H1, J1,2
6.4), 4.57 (1H, dt, H3, J3,2 = J3,4 7.9, J3,F 50.5), 4.99-5.08 (2H, m, H2, H4); δC (CDCl3, 100 MHz): 20.7
(x2) (COCH3), 56.5 (OCH3), 60.8 (d, C5, J5,F 6.4), 68.9 (d, C4, J4,F 20.7), 70.4 (d, C2, J2,F 20.7), 89.7
(d, C3, J3,F 188.4), 101.0 (d, C1, J1,F 8.0), 169.2 (C=O), 169.7 (C=O); δF (CDCl3, 376 MHz): -194.7
(ddt, JF,5' 5.0, JF,2 = JF,4 12.9, JF,3 50.4); m/z (ESI+ve): 273 ([M+Na]+, 100%).
Methyl 3-deoxy-3-fluoro-β-D-xylopyranoside, 41
Sodium methoxide (177 mg, 3.3 mmol) was added to a solution of diacetate 40 (8.20 g, 32.8 mmol) in
methanol (100 mL). The reaction mixture was stirred at 40 °C for 15 h when TLC analysis
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(cyclohexane/ethyl acetate, 1:1) indicated the disappearance of starting material (Rf 0.47) and the
formation of a product (Rf 0.19). The solvent was concentrated in vacuo and the residue purified by
column chromatography (cyclohexane/ethyl acetate, 1:1 → 0:1) to obtain the title compound 41 (5.27
g, 97%) as a white solid.
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HRMS m/z (ESI+ve): found 189.0527 [M+Na]+; C6H11FNaO4+ requires 189.0534; mp. 100 – 102 °C;
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[α]D -61.8 (c 0.43, MeOH); {lit.49 mp. 102 – 104 °C; [α]D -56.0 (c 1.00, MeOH)}; νmax (thin film):
3395 (br, m, O-H), 1749 (s, C=O); δH (CD3CN, 400 MHz): 3.15 (1H, ddd, H5, J5,F 1.2, J5,4 10.2, Jgem
11.6), 3.35 (1H, dddd, H2, J2,OH 4.2, J2,1 7.5, J2,3 8.7, J2,F 14.2), 3.43 (3H, s, CH3), 3.54 (1H, d, OH, J
4.8), 3.58 (1H, d, OH, J 4.8), 3.72 (1H, ddddd, H4, J4,OH 5.1, J4,5' 5.6, J4,3 8.3, J4,5 10.2, J4,F 13.4), 3.84
(1H, dt, H5', J5',4 = J5',F 5.9, Jgem 11.6), 4.11 (1H, dd, H1, J 1.0 J1,2 7.6), 4.17 (1H, dt, H3, J3,2 = J3,4 8.6,
J3,F 52.8); δC (CD3CN, 100 MHz): 57.3 (CH3), 65.2 (d, C5, J5,F 8.8), 69.0 (d, C4, J4,F 17.5), 72.7 (d,
C2, J2,F 18.3), 97.7 (d, C3, J3,F 182.0), 104.9 (d, C1, J1,F 11.1); δF (CD3CN, 376 MHz): -194.8 (ddt,
JF,5' 5.7, JF,2 = JF,4 13.7, JF,3 52.6); m/z (ESI+ve): 189.1 ([M+Na]+, 100%).
Methyl N-benzyl-3-fluoro-2,4-imino-2,3,4-trideoxy-β-L-ribo-pyranoside, 26
Triflic anhydride (14.0 mL, 125.2 mmol) was added dropwise to a solution of diol 41 (5.20 g, 31.3
mmol) and pyridine (11.0 mL, 187.8 mmol) in DCM (50 mL) at -20 °C. The reaction mixture was
stirred between -20 and -10 °C for 2 h, after which TLC (cyclohexane/ethyl acetate, 1:1) showed the
consumption of starting material (Rf 0.12) and the formation of one major product (Rf 0.65). The
reaction mixture was diluted with DCM (30 mL) and washed with HCl (2 M, aq., 2 x 70 mL). The
organic layer was dried (MgSO4), filtered and the solvent was removed in vacuo to afford the crude
triflate (13.3 g) as an orange solid.
Benzylamine (13.0 mL, 156.5 mmol) was added to a solution of crude triflate (13.3 g) in acetonitrile
(80 mL) and the reaction mixture was stirred at 65 – 70 °C for 2 h until TLC analysis
(cyclohexane/ethyl acetate, 1:1) indicated the consumption of the starting material (Rf 0.65) and the
formation of a single product (Rf 0.67). The solvent was concentrated in vacuo and the residue was
purified by column chromatography (cyclohexane/ethyl acetate, 7:1 → 2:1) to obtain ribopyranoside
26 (7.20 g, 97%) as a light yellow oil.
HRMS m/z (ESI+ve): found 238.1239 [M+H]+; C13H17FNO2+ requires 238.1238; [α]D -13.7 (c 1.13,
20
CHCl3); νmax (thin film): fingerprint region only; δH (CDCl3, 400 MHz): 3.42 (3H, s, OCH3), 3.64 (1H,
ddt, H4, J4,5 = J4,5' 1.7, J4,2 4.5, J4,F 11.0), 3.73 (1H, ddd, H2, J 1.3, J2,4 4.5, J2,F 11.6), 3.75 (1H, br-d,
H5, Jgem 11.0), 4.09 (2H, s, H6), 4.33 (1H, ddd, H5', J 1.3, J 5.1, Jgem 10.9), 4.67 (1H, t, H1, J 1.2),
4.93 (1H, d, H3, J3,F 58.9), 7.15-7.37 (5H, m, ArH); δC (CDCl3, 100 MHz): 51.3 (C6), 55.9 (OCH3),
61.5 (d, C5, J5,F 5.6), 64.5 (d, C4, J4,F 18.3), 67.4 (d, C2, J2,F 18.3), 93.3 (d, C3, J3,F 210.6), 100.2 (d,
C1, J1,F 8.0), 126.7, 128.2, 128.3 (ArCH), 138.9 (ArC); δF (CDCl3, 376 MHz): -206.5 (br-d, JF,3 59.3)
m/z (ESI+ve): 238.2 ([M+H]+, 100%), 260 ([M+Na]+, 2%).
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Methyl N-benzyl-3-fluoro-2,4-imino-2,3,4-trideoxy-L-ribonate, 24L
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Ribopyranoside 26 (200 mg, 0.84 mmol) was dissolved in 2 M aq. HCl/1,4-dioxane (5:1, 2 mL). The
reaction mixture was stirred at 40 °C for 21 h. After the consumption of starting material and the
formation of aldehyde was confirmed by mass spectrometry (m/z (ESI+ve): 278 [M+MeOH+Na]+),
solvent was removed in vacuo to give a black solid. A solution of the black solid and potassium
carbonate (349 mg, 2.53 mmol) was stirred at 0 °C under nitrogen atmosphere. Iodine solution (271
mg, 1.09 mmol), which was pre-dissolved in anhydrous methanol (4 mL) by sonication, was added
dropwise into the reaction mixture at 0 °C. Then the mixture was stirred under 5 °C for 2 h until the
completion of reaction was confirmed by mass spectrum. Sodium sulfite (sat. aq., 5 mL) was poured
into the reaction mixture and distilled water (40 mL) was added to dissolve the precipitate. The
aqueous layer was exacted with ethyl acetate (4 x 50 mL), and the combined organic layers were dried
(MgSO4), filtrated and concentrated in vacuo to afford the crude product which was further purified
by column chromatography (cyclohexane/ethyl acetate, 5:1 → 1:1) to yield the title compound 24L as
a brown oil (157 mg, 74%).
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Large scale: Ribopyranoside 26 (1.565 g, 6.6 mmol) was dissolved in 2 M aq. HCl/1,4-dioxane (5:1,
120 mL). The reaction mixture was stirred at 40 °C for 21 h. After the consumption of starting
material and the formation of aldehyde was confirmed by mass spectrometry (m/z (ESI+ve): 278
[M+MeOH+Na]+), solvent was removed in vacuo to give a black solid. A solution of the black solid
and potassium carbonate (2.74 g, 19.8 mmol) was stirred at 0 °C under nitrogen atmosphere in MeOH
(60 mL). Iodine (2.18g, 8.6 mmol), which was pre-dissolved in anhydrous methanol (60 mL) by
sonication, was added dropwise into the reaction mixture at 0 °C. Then the mixture was stirred at 0 °C
for 2 h until the completion of reaction was confirmed by mass spectrum. Sodium sulfite (sat. aq., 30
mL) was poured into the reaction mixture and distilled water (100 mL) was added to dissolve the
precipitate. The aqueous layer was exacted with ethyl acetate (4 x 100 mL), and the combined organic
layers were dried (MgSO4), filtrated and concentrated in vacuo to afford the crude product which was
further purified by column chromatography (cyclohexane/ethyl acetate, 5:1 → 1:1) to yield the title
compound 24L as a brown oil (920 mg, 55%).
+
20
HRMS m/z (ESI+ve): found 276.1014 [M+Na]+; C13H16FNNaO3 requires 276.1006; [α]D -43.8 (c
0.54, CHCl3); νmax (thin film): 3439 (br, w, O-H), 1740 (s, C=O); δH (CDCl3, 400 MHz): 2.35 (1H, s,
OH), 3.17 (1H, dd, H5, J5,4 2.7, Jgem 12.1), 3.37 (1H, ddddd, H4, J 0.6, J4,5' 2.2, J4,5 2.7, J4,3 4.9, J4,F
21.7), 3.44 (1H, dd, H5', J5',4 2.1, Jgem 12.1), 3.70 (3H, s, OCH3), 3.73 (1H, d, H6, Jgem 12.5), 3.78 (1H,
ddd, H2, J 0.6, J2,3 4.8, J2,F 22.2), 3.99 (1H, d, H6', Jgem 12.5), 5.05 (1H, dt, H3, J3,2 =J3,4 4.9, J3,F 55.9),
7.26-7.36 (5H, m, ArH); δC (CDCl3, 100 MHz): 52.1 (OCH3), 60.1 (d, C5, J5,F 4.0), 60.5 (C6), 68.0 (d,
C2, J2,F 21.5), 69.7 (d, C4, J4,F 20.7), 84.4 (d, C3, J3,F 213.8), 128.0, 128.6, 129.3 (ArCH), 135.9
(ArC), 170.2 (d, C1, J 5.6); δF (CDCl3, 376 MHz): -181.5 (dt, JF,2 = JF,4 22.0 JF,3 55.7; m/z (ESI+ve):
254.2 ([M+H]+, 100%), 276.1 ([M+Na]+, 22%).
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Methylamine (0.3 mL, 2.6 mmol, in absolute ethanol) was added to a solution of methyl ester 24L (32
mg, 0.13 mmol) and calcium chloride (14 mg, 0.13 mmol) in anhydrous methanol (1.5 mL). The
reaction mixture was stirred at 40 °C for 2 h until the completion of reaction was confirmed by mass
spectrometry (m/z (ESI+ve): 253 [M+H]+). The reaction mixture was poured onto ethyl acetate (50
mL), dried (MgSO4), filtered and the solvent removed in vacuo to yield the title compound 49 as a
yellow oil which was used without further purification (24 mg, 74%).
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HRMS m/z (ESI+ve): found 275.1166 [M+Na]+; C13H17FN2NaO2 requires 275.1166; [α]D -16.5 (c
1.22, CHCl3); νmax (thin film): 3337 (br, m, O-H, N-H), 1654 (s, C=O); δH (CDCl3, 400 MHz): 2.62
(3H, dt, H7, J 1.2, J7,NH 4.9), 3.36-3.46 (1H, m, H4), 3.40 (1H, dd, H5, J5,4 2.8, Jgem 11.9), 3.53 (1H,
dd, H5', J5,4 3.1, Jgem 12.1), 3.71 (1H, ddt, H2, J 1.6, J2,3 4.9, J2,F 23.2), 3.75 (1H, d, H6, Jgem 12.0),
3.80 (1H, d, H6', Jgem 12.2), 4.81 (1H, dt, H3, J3,2 = J3,4 = 4.6, J3,F 56.2), 6.70 (1H, br-s, NH), 7.26-7.35
(5H, m, ArH); δC (CDCl3, 100 MHz): 25.6 (C7), 60.8 (d, C5, J5,F 4.0), 70.3 (d, C4, J2,F 19.1), 61.6
(C6), 70.5 (d, C2, J4,F 19.1), 85.8 (C3, d, J3,F 216.2), 128.2, 128.8, 129.3 (ArCH), 136.2 (ArC), 170.1
(C1); δF (CDCl3, 376 MHz): -177.1 (dt, JF,2 = JF,4 23.4 JF,3 56.3); m/z (ESI+ve): 253 ([M+H]+, 100%),
275 ([M+Na]+, 25%).
Methyl 3-fluoro-2,4-imino-2,3,4-trideoxy-L-ribonamide, 11
10% Palladium on charcoal (10 % wt., 5 mg) was added to a solution of protected riboamide 49 (24
mg, 0.095 mmol) in 1,4-dioxane/H2O (1:2, 3 mL). The reaction mixture was flushed with argon and
hydrogen gas sequentially. The reaction mixture was stirred vigorously for 3 h at rt under hydrogen
until the completion of reaction was confirmed by mass spectrometry (m/z (ESI+ve): 163 [M+H]+).
After filtration, the solvent was removed in vacuo to afford the title compound 11 as light yellow oil
which was used without further purification (14 mg, 90%).
+
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HRMS m/z (ESI+ve): found 185.0696 [M+Na]+; C6H11FN2NaO2 requires 185.0697; [α]D -61.5 (c
0.69, MeOH); νmax (thin film): 3275 (br, m, O-H, N-H), 1659 (s, C=O); δH (CD3OD, 400 MHz): 2.81
(3H, s, H6), 3.74 (2H, s, H5), 4.28 (1H, br-d, H4, J4,F 16.8), 4.65 (1H, br-d, H2, J2,F 17.3), 5.10 (1H,
br-d, H3, J3,F 55.5); δC (CD3OD, 100 MHz): 26.0 (C6), 63.4 (d, C5, J5,F 4.0), 64.1 (d, C2, J2,F 16.0),
64.3 (d, C4, J4,F 16.0), 90.6 (d, C3, J3,F 214.0), 174.2 (C1); δF (CD3OD, 376 MHz): -177.0 (dt, JF,2
JF,4 19.8 JF,3 54.3); m/z (ESI+ve): 163 ([M+H]+, 100%), 185 ([M+Na]+, 16%).
=
N-Benzyl-3-fluoro-2,4-imino-2,3,4-trideoxy-L-ribonic acid, 35L
Potassium carbonate (29 mg, 0.21 mmol) was added to a solution of methyl ester 24L (40 mg, 0.16
mmol) in 1,4-dioxane/H2O (1:2, 3 mL). The reaction mixture was stirred at 40 °C for 18 h until the
completion of reaction was confirmed by mass spectrometry. HCl (2 M, aq., 0.4 mL) was added to
adjust the mixture to pH 4. The solvent was removed in vacuo to obtain the crude acid which was then
loaded in 1,4-dioxane/H2O (1:2) onto a short column of Dowex® (50W X8, H+) (pre-washed with
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water, 1,4-dioxane and water sequentially until the eluent was neutral). After washing again with
water and 1,4-dioxane and water (1:2), the pure product was released with aqueous ammonia (2 M).
Then solvent was removed in vacuo to yield the title compound 35L as a light yellow glass (22 mg,
43%).
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HRMS m/z (ESI+ve): found 240.1020 [M+H]+; C12H15FNO3+ requires 240.1030; [α]D -13.5 (c 1.07,
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H2O); νmax (thin film): 3070 (br, s, O-H), 1630 (s, C=O); δH (Py-d5, 400 MHz): 3.63 (1H, dq, H4, J4,3 =
J4,5 = J4,5' 4.5, J4,F 22.1), 3.73 (1H, dd, H5, J5,4 4.6, Jgem 11.7), 3.77 (1H, dd, H5', J5',4 4.0, Jgem 11.6),
3.95 (1H, d, H6, Jgem 13.0), 4.15 (1H, dd, H2, J2,3 5.0, J2,F 23.3), 4.35 (1H, d, H6', Jgem 13.0), 5.62 (1H,
dt, H3, J3,2=J3,4 4.9, J3,F 57.0), 7.22-7.64 (5H, m, ArH), 7.87 (2H, br-s, OH); δC (Py-d5, 100 MHz):
61.4 (C6), 62.4 (d, C5, J5,F 3.2), 69.6 (d, C2, J2,F 18.3), 70.7 (d, C4, J4,F 18.3), 88.0 (d, C3, J3,F 210.6),
128.1, 129.0, 130.6 (ArCH), 137.8 (ArC), 174.0 (C1); δF (Py-d5, 376 MHz): -178.5 (dt, JF,2 = JF,4 22.8,
JF,3 56.8); m/z (ESI+ve): 240 ([M+H]+, 100%), 262 ([M+Na]+, 25%).
3-Fluoro-2,4-imino-2,3,4-trideoxy-L-ribonic
acid
[(2R,3S,4S)-3-fluoro-4-
(hydroxymethyl)azetidine-2-carboxylic acid], 10L
10% Palladium on charcoal (10% wt., 5 mg) was added to a solution of N-benzyl protected ribonic
acid 35L (21 mg, 0.09 mmol) in 1,4-dioxane/H2O (2 mL, 1:2). The reaction was flushed with
nitrogen, argon and hydrogen gas sequentially and stirred vigorously for 15 h at rt under hydrogen
until mass spectrometry showed the completion of reaction. After filtration, the solvent was removed
in vacuo to obtain a residue which was purified on a short column of Dowex® (50W X8, H+) (as
illustrated above). The solvent was removed in vacuo to afford the title compound 10L as a light
yellow glass (11 mg, 82%).
-
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HRMS m/z (ESI-ve): found 148.0409 [M-H]-; C5H7FNO3 requires 148.0415; [α]D -30.6 (c 0.50,
H2O); νmax (thin film): 3233 (br, s, O-H), 1630 (s, C=O); δH (D2O, 400 MHz): 3.91 (1H, dd, H5, J5,4
3.7, Jgem 13.2), 3.97 (1H, dd, H5', J5',4 3.9, Jgem 13.2), 4.66 (1H, dq, H4, J4,3 = J4,5 = J4,5' 4.2, J4,F 19.3),
4.92 (1H, dd, H2, J2,3 4.8, J2,F 21.3), 5.31 (1H, dt, H3, J3,2 = J3,4 4.8, J3,F 56.1); δC (D2O, 100 MHz):
58.0 (d, C5, J5,F 4.0), 63.8 (d, C2, J2,F 24.6), 64.9 (d, C4, J4,F 26.2), 87.9 (d, C3, J3,F 210.6), 170.4
(C1); δF (D2O, 376 MHz): -178.3 (dt, JF,2 = JF,4 20.4, JF,3 56.1); m/z (ESI-ve): 148 ([M-H]+, 100%).
N-Benzyl-3-fluoro-2,4-imino-2,3,4-trideoxy-meso-ribitol, 42
Method 1
The bicyclic azetidine 26 (100 mg, 0.42 mmol) was dissolved in 2 M aq. HCl/1,4-Dioxane (5:1, 1
mL). The reaction mixture was stirred at 40 °C for 23 h after which the consumption of starting
material and the formation of aldehyde was confirmed by mass spectrometry (m/z (ESI+ve): 278
[M+MeOH+Na]+). The solvent was removed in vacuo to give a black glass.
Sodium borohydride (196 mg, 2.52 mmol) was added to a solution of the black residue in methanol (4
mL). After stirring at rt for 2 h, mass spectrometry (m/z 248) showed the completion of reaction. The
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solvent was concentrated in vacuo to obtain a residue which was purified on a short column of
Dowex® (50W X8, H+) (pre-washed with water, 1,4-dioxane and water sequentially until the eluent
was neutral) to yield the desired diol 42 as a brown oil (39 mg, 40% over 2 steps).
Method 2
Sodium borohydride (15 mg, 0.39 mmol) was added into a solution of methyl ester 24L (100 mg, 0.39
mmol) in methanol (2 mL) at 0°C. After stirring at rt for 3 h, mass spectrometry (m/z 248) showed the
completion of reaction. The solvent was removed in vacuo to obtain a residue that was purified on a
short column of Dowex® (50W X8, H+) (pre-washed with water, 1,4-dioxane and water sequentially
until the eluent was neutral) to yield the desired diol 42 as a brown oil (79 mg, 90%)
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HRMS m/z (ESI+ve): found 248.1057 [M+Na]+; C12H16FNNaO2 requires 248.1057; [α]D20 0.0 (c 1.0,
CH3OH); νmax (thin film): 3355 (br, s, O-H); δH (CDCl3, 400 MHz): 2.81 (2H, br-s, OH x 2), 3.25 (2H,
ddt, H2(4), J2,1 = J2,1' 3.2, J2,3 5.1, J2,F 22.8), 3.36 (2H, dd, H1(5), J1,2 3.2, Jgem 11.7), 3.52 (2H, dd,
H1'(5'), J1',2 2.8, Jgem 11.9), 3.78 (2H, s, H6), 4.93 (1H, dt, H3, J3,2 = J3,4 4.7, J3,F 56.8), 7.27-7.36 (5H,
m, ArH); δC (CDCl3, 100 MHz): 60.8 (d, C1(5), J 4.8), 61.2 (C6), 70.3 (d, C2(4), J 19.9), 83.9 (d, C3,
J 207.4), 127.9, 128.6, 129.1 (ArCH), 137.0 (ArC); δF (CDCl3, 376 MHz): -184.2 (dt, JF,2 = JF,4 22.9,
JF,3 56.1); m/z (ESI+ve): 248 ([M+Na]+, 100%).
3-Fluoro-2,4-imino-2,3,4-trideoxy-meso-ribitol, 15
10% Palladium on charcoal (10% wt., 5 mg) was added to a solution of N-benzyl protected ribonic
acid 42 (20 mg, 0.09 mmol) in 1,4-dioxane/H2O (1:2). The reaction was flushed with nitrogen, argon
and hydrogen gas sequentially and stirred vigorously for 5 h at rt under hydrogen until mass
spectrometry showed the completion of reaction (m/z (ESI+ve): 136 [M+H]+). After filtration, the
solvent was concentrated in vacuo to obtain a residue which was purified by a short column of
Dowex® (50W X8, H+) (as illustrated above). The solvent was concentrated in vacuo to afford the title
compound 15 as a light yellow oil (12 mg, 100%).
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HRMS m/z (ESI+ve): found 158.0590 [M+Na]+; C5H10FNNaO2 requires 158.0588; [α]D 0.0 (c 1.0,
CH3OH); νmax (thin film): 3330 (br, s, O-H); δH (D2O, 400 MHz): 3.66 (4H, d, H1(5), J1,2 5.7), 3.90
(2H, dq, H2(4), J2,1 = J2,3 = J 5.8, J2,F 21.4), 4.80 (1H, dt, H3, J3,2 = J3,4 5.7, J3,F 56.0); δC (D2O, 100
MHz): 62.3 (d, C2(4), J2,F 20.0), 62.8 (d, C1(5), J1,F 3.8), 89.3 (d, C3, J3,F 209.8); δF (D2O, 376 MHz):
-174.8 (dt, JF,2 = JF,4 21.8, JF,3 56.4); m/z (ESI+ve): 136 ([M+H]+, 100%).
Methyl N-benzyl-3-fluoro-2,4-imino-5-O-mesyl-2,3,4-trideoxy-L-ribonate, 43
Methanesulfonyl chloride (0.05 mL, 0.60 mmol) was added to a solution of methyl ester 24L (100
mg, 0.40 mmol) in pyridine (3 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 1 h when
TLC (cyclohexane/ethyl acetate, 1:1) showed the consumption of starting material (Rf 0.38) and the
formation of product (Rf 0.46). The solvent was removed in vacuo and the residue was purified by
flash column chromatography (cyclohexane/ethyl acetate, 5:1 → 2:1) to give the product 43 as a
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yellow oil (133 g, 100%).
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HRMS m/z (ESI+ve): found 354.0777 [M+Na]+; C14H18FNNaO5S+ requires 354.0782; [α]D -22.1 (c
0.57, CHCl3); νmax (thin film): 1744 (s, C=O); δH (CDCl3, 400 MHz): 2.97 (3H, s, CH3), 3.44 (1H, dq,
H4, J4,3 = J4,5 = J4,5' 4.6, J4,F 20.7), 3.66 (3H, s, CH3), 3.73 (1H, dd, H2, J2,3 5.1, J2,F 21.3), 3.79 (1H, d,
H6, Jgem 12.7), 3.93 (1H, d, H6', Jgem 12.5), 3.97 (1H, dd, H5, J5,4 4.2, Jgem 11.3), 4.10 (1H, dd, H5',
J5',4 4.5, Jgem 11.6), 4.95 (1H, dt, H3, J3,2 = J3,4 5.0, J3,F 55.3), 7.25-7.36 (5H, m, ArH); δC (CDCl3, 100
MHz): 37.6 (SO2CH3), 52.1 (OCH3), 60.5 (C6), 66.4 (d, C4, J4,F 21.5), 67.7 (d, C2, J2,F 21.5), 67.9 (d,
C5, J5,F 4.0), 84.5 (d, C3, J3,F 217.0), 128.0, 128.5, 129.6 (ArCH), 135.3 (ArC), 169.8 (C1, d, J 4.8);
δF (CDCl3, 376 MHz): -179.6 (dt, JF,2 = JF,4 20.9, JF,3 55.3); m/z (ESI+ve): 332 ([M+H]+, 100%).
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Methyl N-benzyl-5-azido-3-fluoro-2,4-imino-2,3,4,5-tetradeoxy-L-ribonate, 27
Sodium azide (13 mg, 0.20 mmol) was added to a solution of mesylate 43 (50 mg, 0.15 mmol) in
DMF (2 mL) and the reaction mixture was stirred at 60 °C for 26 h. After this time TLC analysis
(cyclohexane/ethyl acetate, 1:1) indicated the disappearance of the starting material (Rf 0.48) and the
formation of a single product (Rf 0.78). The reaction mixture was diluted with ethyl acetate (20 mL)
and washed with 1:1 H2O/brine (sat., aq.) (3 x 20 mL). The organic layer was dried (MgSO4), filtered
and the solvent was removed in vacuo to obtain a residue that was purified by flash column
chromatography (cyclohexane/ethyl acetate, 7:1 → 5:1) to yield the title compound 27 as a light
yellow oil (32 mg, 80%)
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HRMS m/z (ESI+ve): found 301.1072 [M+Na]+; C13H15FN4NaO2 requires 301.1071; [α]D -61.7 (c
1.62, CHCl3); νmax (thin film): 2102 (s, N3), 1743 (s, C=O); δH (CDCl3, 400 MHz): 3.05 (1H, dd, H5,
J5,4 4.4, Jgem 13.2), 3.10 (1H, dd, H5', J5',4 4.4, Jgem 13.2), 3.33 (1H, ddq, H4, J 0.7, J4,3 = J4,5 = J4,5' 4.7,
J4,F 21.2), 3.70 (3H, s, CH3), 3.70 (1H, ddd, H2, J 0.7, J2,3 5.0, J2,F 21.5), 3.75 (1H, d, H6, Jgem 12.5),
4.00 (1H, d, H6', Jgem 12.7), 4.96 (1H, dt, H3, J3,2 = J3,4 5.0, J3,F 55.7), 7.30-7.35 (5H, m, ArH); δC
(CDCl3, 100 MHz): 51.5 (d, C5, J5,F 4.0), 52.1 (CH3), 60.9 (C6), 67.4 (d, C4, J4,F 20.7), 68.0 (d, C2,
J2,F 21.5), 85.3 (d, C3, J3,F 215.4), 127.9, 128.5, 129.7 (ArCH), 135.6 (ArC), 170.0 (d, C1, J1,F 5.6); δF
(CDCl3, 376 MHz): -179.7 (dt, JF,2 = JF,4 21.5, JF,3 55.7); m/z (ESI+ve): 301 ([M+Na]+, 100%).
Methyl N-benzyl-5-O-(tert-butyldimethylsilyl)-3-fluoro-2,4-imino-2,3,4-trideoxy-L-ribonate, 50
tert-Butyldimethylsilyl chloride (111 mg, 0.74 mmol) was added to a solution of methyl ester 24L
(157 mg, 0.62 mmol) in DMF (10 mL) and the reaction mixture was stirred at rt for 2 h. After this
time TLC analysis (cyclohexane/ethyl acetate, 1:1) indicated the disappearance of starting material (Rf
0.50) and the formation of a major product (Rf 0.87). The reaction mixture was diluted with ethyl
acetate (20 mL) and washed with 1:1 H2O/brine (sat., aq.) (2 x 20 mL). The organic layer was dried
(MgSO4), filtered and the solvent was removed in vacuo to yield the title compound 50 as a brown oil
(218 mg, 95%).
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HRMS m/z (ESI+ve): found 390.1878 [M+Na]+; C19H30FNNaO3Si+ requires 390.1871; [α]D -16.4 (c
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0.90, CHCl3); νmax (thin film): 1745 (s, C=O); δH (CDCl3, 400 MHz): 0.00, 0.01 (2 x 3H, 2 x s, CH3),
0.87 (9H, s, C(CH3)3), 3.27 (1H, dq, H4, J4,3 = J4,5 = J4,5' 5.4, J4,F 21.5), 3.47 (1H, ddd, H5, J 1.0, J5,4
5.4, Jgem 10.8), 3.51 (1H, dd, H5', J5',4 5.7, Jgem 10.8), 3.64 (1H, dd, H2, J2,3 5.1, J2,F 21.5), 3.65 (3H, s,
OCH3), 3.82 (1H, d, H6, Jgem 12.9), 3.91 (1H, d, H6', Jgem 12.9), 4.85 (1H, dt, H3, J3,2 = J3,4 5.1, J3,F
56.2), 7.25-7.35 (5H, m, ArH); δC (CDCl3, 100 MHz): -5.5, -5.4 (CH3Si), 18.2 (C(CH3)2), 25.8
(C(CH3)2), 51.9 (OCH3), 60.9 (C6), 63.7 (d, C5, J5,F 4.0), 67.8 (d, C2, J2,F 21.5), 69.3 (d, C4, J4,F
19.1), 86.2 (d, C3, J3,F 214.6), 127.6, 128.3, 129.7 (ArCH), 136.0 (ArC), 170.6 (d, C1, J1,F 4.8); δF
(CDCl3, 376 MHz): -179.0 (dt, JF,2 = JF,4 21.8, JF,3 56.1); m/z (ESI+ve): 368 ([M+H]+, 100%), 390
([M+Na]+, 30%).
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Methyl N-benzyl-5-O-(tert-butyldimethylsilyl)-3-fluoro-2,4-imino-2,3,4-trideoxy-L-ribonamide,
52
Methylamine (0.30 mL, 2.6 mmol, in absolute ethanol) was added to a solution of methyl ester 50 (42
mg, 0.11 mmol) and calcium chloride (12.0 mg, 0.11 mmol) in anhydrous methanol (1.0 mL). The
reaction mixture was stirred at 45 °C for 2 h when the completion of reaction was confirmed by mass
spectrometry (m/z (ESI+ve): 367 [M+H]+). The pH of the reaction mixture was adjusted to pH 5 using
NH4Cl (sat. aq.)/H2O (1:3, 2 mL) and the mixture was extracted with ethyl acetate (3 x 15 mL). The
organic layer was dried (MgSO4), filtered and solvent was removed in vacuo to yield the amide 52 as
a yellow oil (41 mg, 100%).
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HRMS m/z (ESI+ve): found 389.2037 [M+Na]+; C19H31FN2NaO2Si+ requires 389.2031; [α]D -4.4 (c
0.82, CHCl3); νmax (thin film): 1681 (s, C=O); δH (CDCl3, 400 MHz): 0.01 (6H, s, CH3), 0.88 (9H, s,
C(CH3)3), 2.62 (3H, d, NCH3, JCH3,NH 4.9), 3.32-3.42 (1H, m, H4), 3.38 (1H, dd, H5, J 3.3, Jgem 11.1),
3.53 (1H, dd, H5', J5',4 3.5, Jgem 10.9), 3.68 (1H, dd, H2, J2,3 4.5, J2,F 24.1), 3.73 (1H, d, H6, Jgem 12.5),
3.80 (1H, d, H6', Jgem 12.2), 4.73 (1H, dt, H3, J3,2 = J3,4 4.4, J3,F 56.2), 6.88 (1H, q, NH, JNH,CH3 5.0),
7.25-7.35 (5H, m, ArH); δC (CDCl3, 100 MHz): -5.6, -5.5 (CH3Si), 18.2 (C(CH3)2), 25.4 (NCH3), 25.7
(C(CH3)2), 61.7 (C6), 62.2 (d, C5, J5,F 4.8), 70.4 (d, C4, J4,F 19.9), 70.5 (d, C2, J2,F 20.7), 86.2 (d, C3,
J3,F 216.2), 127.9, 128.6, 129.2 (ArCH), 136.6 (ArC), 170.4 (d, C1, J1,F 5.6); δF (CDCl3, 376 MHz): -
176.3 (dt, JF,2 = JF,4 24.3, JF,3 56.4); m/z (ESI+ve): 367 ([M+H]+, 100%), 389 ([M+Na]+, 30%).
Methyl
N-(N-benzyl-8-fluoro-7,9-imino-8,7,9-trideoxy-10-O-(tert-butyldimethylsilyl)-L-
ribonamido)-3-fluoro-2,4-imino-2,3,4-trideoxy-5-O-(tert-butyldimethylsilyl)-L-ribonamide, 54
Potassium carbonate (19 mg, 0.14 mmol) was added to a solution of methyl ester 50 (40 mg, 0.11
mmol) in 1,4-dioxane/H2O (2 mL, 1:1). The reaction mixture was stirred at 40 °C for 26 h until mass
spectrometry indicated completion of the hydrolysis (m/z (ESI-ve): 352 [M-H]-) and the solvent was
removed in vacuo.
10% Palladium on charcoal (10% wt., 5 mg) was added to a solution of 52 (40 mg, 0.11 mmol) in 1,4-
dioxane/H2O (3 mL, 1:2). The reaction was flushed with argon and hydrogen gas sequentially and
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then stirred vigorously for 5 h at rt under hydrogen until mass spectrometry showed the completion of
reaction (m/z (ESI+ve): 277 [M+H]+). After filtration, the solvent was removed in vacuo to afford a
residue that was used without further purification.
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N,N,N’,N’-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU, 50 mg, 0.13
mmol) was added to a solution of the crude acid (57 mg) and amine (30 mg) in anhydrous DMF (1.5
mL). After stirring for 20 min, triethylamine (0.02 mL) was added to the reaction mixture which was
stirred rt for a further 20 h until TLC analysis (cyclohexane/ethyl acetate, 1:1) showed the
consumption of the starting materials and formation of one major product (Rf 0.75). The reaction
mixture was diluted with ethyl acetate (20 mL) and washed with half saturated brine (20 mL). The
organic layer was dried (MgSO4), filtered and the solvent removed in vacuo to give a residue that was
purified by flash column chromatography (cyclohexane/ethyl acetate, 1:1) to give the pure peptide 54
as a yellow oil (30 mg, 45%).
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HRMS m/z (ESI+ve): found 634.3288 [M+Na]+; C30H51F2N3NaO4Si2+ requires 634.3278; [α]D -44.0
(c 0.93, CHCl3); νmax (thin film): 1682 (s, C=O); δH (CDCl3, 400 MHz): 0.01, 0.03, 0.06, 0.07 (12H, 4
x s, CH3), 0.85 (9H, s, C(CH3)3), 0.88 (9H, s, C(CH3)3), 2.74 (3H, d, NCH3, JCH3,NH 4.9), 3.36 (1H, dq,
H9, J9,8 = J9,10 = J9,10' 5.0, J9,F 21.9), 3.56 (1H, dd, H7, J7,8 4.9, J7,F 20.3), 3.50-3.64 (1H, m, H4), 3.57-
3.63 (1H, m, H5), 3.70 (2H, br-s, -CH2Ar), 3.74 (2H, br-s, H10), 4.19 (1H, d, H5', Jgem 12.2), 4.31
(1H, br-d, H2, J2,F 23.7), 4.79 (1H, dt, H8, J8,7 = J8,9 4.8, J8,F 56.1), 5.08 (1H, br-d, H3, J3,F 55.3), 7.26-
7.35 (5H, m, ArH), 7.43 (1H, br-s, NH); δC (CDCl3, 100 MHz): -5.7, -5.4 (x 2) (CH3Si), 18.2
(C(CH3)2), 25.7 (NHCH3), 25.8 (C(CH3)3), 62.5 (C5, -CH2Ar), 64.1 (C10), 66.0 (C7), 67.0 (C2), 68.6
(d, C4, J4,F 25.4), 70.9 (d, C9, J9,F 18.3), 85.5 (d, C3, J3,F 194.7), 86.1 (d, C8, J8,F 218.6), 128.3, 128.6,
128.9 (ArCH), 136.2 (ArC), 167.7, 167.8 (C1, C6); δF (CDCl3, 376 MHz): -177.7 (dt, JF,7 = JF,9 21.2,
JF,8 56.1), -184.4 (dt, JF,2 = JF,4 22.9, JF,3 55.3); m/z (ESI+ve): 612 ([M+H]+, 100%), 634 ([M+Na]+,
30%).
N-Benzyl-3R,4R-difluoro-L-proline methyl ester, 28
A solution of azetidine methyl ester 24L (289 mg, 0.79 mmol) in DCM (2 mL) was added dropwise to
a solution of XtalFluor-M (289 mg, 1.19 mmol) and TEA3HF (0.26 mL, 1.19 mmol) in DCM (2
mL) at -78 °C. After stirring for 1 h, the mixture was stirred at rt for 18 h until TLC
(cyclohexane/ethyl acetate, 2:1) showed the disappearance of starting material (Rf 0.16) and the
formation of major product (Rf 0.56). The mixture was diluted with half saturated NaHCO3 (10 mL)
and stirred for a further 20 min, before extraction with DCM (3 x 20 mL). The combined organic
layers were dried (MgSO4), filtered and concentrated under reduced pressure to obtain a crude residue
which was purified by flash chromatography (cyclohexane/ethyl acetate, 7:1 → 6:1) to yield the
desired product 28 as a yellow oil (169 mg, 84%).
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HRMS m/z (ESI+ve): found 278.0958 [M+Na]+; C13H15F2NNaO2 requires 278.0963; [α]D -64.2 (c
0.31, CHCl3); νmax (thin film): 1746 (s, C=O); δH (CDCl3, 400 MHz): 2.85 (1H, dddd, H5, J5,3F 2.0, J5,4
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5.0, Jgem 11.7, J5,4F 31.9), 3.26 (1H, ddt, H5', J5',3 = J5',4 1.4, Jgem 11.6, J5',4F 20.9), 3.51 (1H, dd, H2, J2,3
3.7, J2,3F 26.9), 3.66 (1H, d, H6, Jgem 13.1), 3.76 (3H, s, OCH3), 4.05 (1H, d, H6', Jgem 13.2), 5.05 (1H,
dddt, H4, J4,3 = J4,5' 1.3, J4,5 4.9, J4,3F 14.9, J4,4F 50.7), 5.27 (1H, dddt, H3, J3,4 = J3, 5' 1.3, J3,2 3.7, J3,4F
16.6, J3,3F 50.1), 7.25-7.36 (5H, m, ArH); δC (CDCl3,100 MHz): 52.4 (OCH3), 56.2 (dd, C5, J5,3F 2.4,
J5,4F 23.1), 57.5 (C6), 69.8 (dd, C2, J2,3F 0.8, J2,4F 26.2), 97.9 (dd, C4, J4,3F 29.0, J4,4F 182.4), 97.9 (dd,
C3, J3,4F 32.7, J3,3F 186.4), 127.5, 128.4, 128.9 (ArCH), 136.7 (ArC), 170.4 (d, C1, J1,F 8.8); δF
(CDCl3, 376 MHz): -184.6 (ddddd, 3F, J3F,5 2.3, J3F,4F 8.0, J3F,4 14.9, J3F,2 26.3, J3F,3 50.4), -186.6
(ddddd, 4F, J4F,3F 7.9, J4F,3 16.4, J4F,5' 20.9, J4F,5 32.0, J4F,4 50.7); m/z (ESI+ve): 256 ([M+H]+, 100%).
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N-Benzyl-2R,3R-difluoro-2,4-imino-1,2,3,4-tetradeoxyl-D-arabinofuranose, 47
Lithium aluminum hydride (1 M in THF) (0.24 mL, 0.24 mmol) was added dropwise to a solution of
difluoromethyl ester 28 (30 mg, 0.120 mmol) in anhydrous THF at -78 °C. The reaction mixture was
stirred for 1 h until TLC analysis (cyclohexane/ethyl acetate, 2:1) showed the consumption of the
starting material (Rf 0.67) and formation of a major product (Rf 0.37). The reaction was quenched
with NH4Cl (sat. aq.) to pH 6 and extracted with ethyl acetate (2 x 15 mL). The organic layers were
dried (MgSO4), filtered and concentrated in vacuo to yield pure alcohol 47 as a clear oil which was
used without further purification (18 mg, 66%).
20
HRMS m/z (ESI+ve): found 250.1008 [M+Na]+; C12H15F2NNaO+ requires 250.1014; [α]D -70.6 (c
0.90, MeOH); νmax (thin film): 3348 (br, w, O-H); δH (CD3OD, 400 MHz): 2.72 (1H, dddd, H1, J1,3F
3.0, J1,2 4.6, Jgem 12.1, J1,2F 36.7), 2.74-2.85 (1H, m, H4), 3.09 (1H, ddq, H1', J = J = J 1.0, Jgem 12.1,
J1',2F 20.5), 3.48 (1H, d, H6, Jgem 13.2), 3.64 (1H, ddt, H5, J = J 0.7, J5,4 6.1, Jgem 11.5), 3.67 (1H, ddt,
H5', J = J 1.1, J5',4 4.9, Jgem 11.6), 4.05 (1H, d, H6', Jgem 13.1), 4.91-5.11 (2H, m, H3, H2), 7.23-7.36
(5H, m, ArH); δC (CD3OD,100 MHz): 58.3 (dd, C1, J1,3F 2.4, J1,2F 22.3), 59.5 (C6), 61.6 (d, C5, J5,4F
6.4), 71.3 (dd, C4, J 1.6, J4,3F 22.3), 95.3 (dd, C2, J2,3F 29.4, J2,2F 179.6), 98.6 (dd, C3, J3,2F 28.6, J3,3F
177.2), 128.4, 129.4, 130.1 (ArCH), 139.2 (ArC); δF (CD3OD, 376 MHz): -187.7 (ddddd, 3F, J3F,1 3.4,
J3F,2F 6.9, J3F,2 13.7, J3F,4 28.6, J3F,3 50.1), -188.1 (ddddd, 2F, J2F,3F 6.9, J2F,3 17.2, J2F,1' 20.6, J2F,1 36.6,
J2F,2 50.4); m/z (ESI+ve): 228 ([M+H]+, 100%).
2R,3R-Difluoro-2,4-imino-1,2,3,4-tetradeoxy-D-arabinofuranose, 16
10% Palladium on charcoal 47 (10% wt., 5 mg) was added to a solution of N-benzyl-difluoro alcohol
(15 mg, 0.066 mmol) in 1,4-dioxane/H2O (1:1, 2 mL). The reaction was flushed with argon and
hydrogen gas sequentially and stirred vigorously for 2 h at rt under hydrogen when mass spectrometry
showed the completion of reaction (m/z (ESI+ve): 138 [M+H]+). After filtration, the solvent was
removed in vacuo to afford the title compound 16 as a clear oil (7 mg, 82%).
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HRMS m/z (ESI+ve): found 138.0725 [M+H]+; C5H10F2NO+ requires 138.0725; [α]D +4.3 (c 0.37,
MeOH); νmax (thin film): 3321 (br, m, O-H); δH (D2O, 400 MHz) free base: 3.18 (1H, dddd, H1, J1,3F
2.2, J1,2 3.9, Jgem 13.9, J1,2F 36.7), 3.24 (1H, dd, H1', Jgem 13.9, J1',2F 23.3), 3.33 (1H, ddt, H4, J4,3 3.5,
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J4,5 = J4,5' 6.5, J4,3F 27.5), 3.69 (1H, dd, H5, J5,4 6.5, Jgem 11.6), 3.73 (1H, ddt, H5', J5',3 = J5',F 1.0, J5',4
6.0, Jgem 11.7), 5.04 (1H, dddt, H3, J3,2 = J3,5' 1.0, J3,4 3.5, J3,2F 16.4, J3,3F 50.1), 5.27 (1H, dddt, H2, J2,1'
= J2,3 1.0, J2,1 4.1, J2,3F 12.5, J2,2F 50.0); δC (D2O, 100 MHz): 50.5 (dd, C1, J1,3F 2.4, J1,2F 23.4), 61.3 (d,
C5, J5,F 7.6), 64.9 (d, C4, J4,3F 23.8), 96.8 (dd, C2, J2,3F 30.5, J2,2F 174.5), 97.8 (dd, C3, J3,2F 29.5, J3,3F
177.3); δF (D2O, 376 MHz): -188.5 (ddddd, 3F, J3F,1 2.3, J3F,2F 6.9, J3F,2 12.6, J3F,4 27.5, J3F,3 50.3), -
188.9 (ddddd, 2F, J3F,2F 6.9, J2F,3 16.0, J2F,1' 22.7, J2F,1 35.2, J2F,2 50.4); m/z (ESI+ve): 138 ([M+H]+,
100%).
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N-Benzyl-3R,4R-difluoro-L-proline, 48
Sodium hydroxide (1 M, aq., 0.24 mL, 0.24 mmol) was added to a solution of difluoromethyl ester 28
(28 mg, 0.11 mmol) in 1,4-dioxane/H2O (1:1, 2 mL). After stirring at 40 °C for 4 h, mass
spectrometry showed the formation of product and disappearance of starting of material. Then solvent
was removed in vacuo to afford a residue which was loaded onto a Serdolit® CG 400 resin column
(the resin was pre-stirred in sodium hydroxide (1 M, aq.) for 15 min, and then flushed with H2O until
the eluent was neutral). After washing with 1,4-dioxane and H2O, acetic acid (2 M, aq.) was used to
release the product. The solvent was removed in vacuo to give the pure product 48 as light yellow
glass (17 mg, 64%).
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HRMS m/z (ESI+ve): found 264.0805 [M+Na]+; C12H13F2NNaO2 requires 264.0807; [α]D -11.7 (c
0.16, MeOH), νmax (thin film): 3030 (OH, br), 1677 (C=O, s); δH (D2O, 400 MHz): 3.96 (1H, ddt, H5,
J5,4 = J5,3F 3.1, Jgem 14.0, J5,4F 40.0), 4.21 (1H, dd, H5', Jgem 13.9, J5',4F 17.3), 4.56 (1H, d, H6, Jgem
12.9), 4.70 (1H, d, H6', Jgem 12.9), 4.73 (1H, d, H2, J2,F 26.6), 5.48-5.63 (2H, m, H3, H4), 7.49-7.59
(5H, m, ArH); δC (D2O,100 MHz): 58.3 (d, C5, J5,4F 21.0), 60.7 (C6), 72.0 (d, C2, J2,3F 23.8), 91.8 (dd,
C3/C4, J 33.4, J 186.0), 95.0 (dd, C3/C4, J 31.5, J 179.3), 129.4, 129.7 (ArCH), 131.1 (ArC), 131.5
(ArCH), 167.5 (d, C1, J1,F 9.5); δF (D2O, 376 MHz): -182.0 (ddddd, 3F, J3F,5 2.9, J3F,4F 6.3, J3F,4 14.3,
J3F,2 26.9, J3F,3 48.2), -193.2 (ddddd, 4F, J4F,3F 8.0, J4F,3 14.9, J4F,5' 17.2, J4F,5 39.7, J4F,4 47.4); m/z
(ESI+ve): 242 ([M+H]+, 100%).
3R,4R-Difluoro-L-proline, 12
10% Palladium on charcoal (10% wt., 5 mg) was added to a solution of N-benzyl-difluoro proline 48
(17 mg, 0.07 mmol) in 1,4-dioxane/H2O (1:1, 2 mL). The reaction was flushed with argon and
hydrogen gas sequentially and stirred vigorously for 4 h at rt under hydrogen until mass spectrometry
showed the completion of reaction (m/z (ESI-ve): 150 [M-H]-). After filtration, the solvent was
removed in vacuo to afford the title compound 12 as a light yellow glass (10 mg, 86%).
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HRMS m/z (ESI-ve): found 150.0367 [M-H]-; C5H6F2NO2 requires 150.0372; [α]D -4.6 (c 0.19,
MeOH); νmax (thin film): 3023 (br, s, O-H), 1636 (s, C=O); δH (D2O, 400 MHz): 3.82 (1H, ddt, H5, J5,4
= J 3.1, Jgem 14.5, J5,4F 39.1), 3.96 (1H, dd, H5', Jgem 14.2, J5',4F 21.8), 4.61 (1H, d, H2, J2,F 24.9), 5.42-
5.54 (1H, m, H4), 5.58 (1H, dd, H3, J3,4 6.3, J4,F 47.9); δC (D2O,100 MHz): 50.3 (d, C5, J5,4F 22.9),
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(d, C1, J1,F 12.4); δF (D2O, 376 MHz, 1H-decoupled): -185.8 (d, J 15.6), -190.5 (d, J 15.6); m/z (ESI-
ve): 150 ([M-H]-, 100%).
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1,2,4,6-Tetra-O-acetyl-3-deoxy-3-fluoro-D-glucopyranoside, 29
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Method 1
A solution of 21 (6.88 g, 26.24 mmol) in 1:1 water/trifluoroacetic acid (20.5 mL) was stirred at rt for
18 h after which TLC analysis (ethyl acetate) showed complete conversion of the starting material (Rf
0.97) to a single major product (Rf 0.06). The mixture was concentrated in vacuo and the crude 3-
deoxy-3-fluoro-glucose was used without any further purification.
Acetic acid anhydride (95.9 mL, 931.5 mmol) was added to a solution of the crude glucopyranose in
pyridine (95.9 mL) the reaction mixture was stirred at rt. After 4 h TLC analysis (cyclohexane/ethyl
acetate, 1:1) showed complete conversion of the starting material (baseline) to two major products (Rf
0.67 and 0.57) and a minor product (Rf 0.16). The mixture was concentrated in vacuo and the
resulting oil was partitioned between ethyl acetate (130 mL) and 1:1, NaHCO3 (sat., aq.)/brine (130
mL). The aqueous layer was extracted with ethyl acetate (2 x 130 mL). The combined organic layers
were dried (MgSO4), filtered, concentrated in vacuo and the residue was purified by flash
chromatography (cyclohexane/ethyl acetate, 77:23) to give the title compound 29 as a white solid
(8.53 g, 93%).
Method 2
A solution of 21 (15.28 g, 58.3 mmol) in a mixture of 1,4-dioxane (75 mL) and water 30 mL was
stirred with Dowex® (50W X8, H+) at 65 °C for 18 h after which TLC analysis (ethyl acetate) showed
complete conversion of the starting material (Rf 0.93) to a single major product (Rf 0.07). The mixture
was concentrated in vacuo and the crude 3-deoxy-3-fluoro-glucose was used without any further
purification.
Acetic anhydride (75 mL, 931.5 mmol) was added to a solution of the crude glucopyranose (10.6 g) in
pyridine (75 mL) and the reaction mixture was stirred at rt. After 18 h TLC analysis
(cyclohexane/ethyl acetate, 1:1) showed complete conversion of the starting material (Rf 0.00) to a
single major product (Rf 0.89). The mixture was concentrated in vacuo and the residue was purified
by flash chromatography (10% → 25% ethyl acetate in cyclohexane) to give the title compound 29 as
a white solid as a 1:1 mixture of anomers (19.75 g, 97%).
HRMS m/z (ESI+ve): found 373.0910 [M+Na]+; C14H19FNaO9 requires 373.0905; mp. 86 - 88 °C;
+
vmax (thin film): 1744 (s, C=O); δH (CDCl3, 400 MHz): 2.05 (3H, s, CH3), 2.06 (3H, s, CH3), 2.07 (3H,
s, CH3), 2.08 (x2) (2 x 3H, 2 x s, CH3), 2.09 (3H, s, CH3), 2.10 (3H, s, CH3), 2.14 (3H, s, CH3), 3.72
(1H, dddd, H5β, J5,F 1.3, J5,6 2.2, J5,6' 4.5, J5,4 10.1), 3.98 – 4.04 (1H, m, H5α), 4.08 (1H, dt, H6α, J6,5
= J6,F 2.1, Jgem 12.5), 4.09 (1H, dt, H6β, J6,5 = J6,F 1.9, Jgem 12.6), 4.22 (1H, dd, H6'α, J6',5 4.3, Jgem
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12.6), 4.24 (1H, dd, H6'β, J6',5 4.5, Jgem 12.6), 4.59 (1H, dt, H3β, J3,2 = J3,4 9.1, J3,F 49.5), 4.80 (1H, dt,
H3α, J3,2 = J3,4 9.4, J3,F 53.3), 5.14 (1H, ddd, H2α, J2,1 3.8, J2,3 9.7, J2,F 12.1), 5.15 - 5.18 (3H, m, H2β,
H4α, H4β), 5.63 (1H, d, H1β, J1,2 8.3), 6.31 (1H, d, H1α, J1,2 3.8); δC (CDCl3, 100 MHz): 20.4 (CH3),
20.5 (x2) (CH3), 20.6 (CH3), 20.7 (CH3), 61.2 (C6α, C6β), 67.6 (d, C4α/C4β, J4,F 19.0), 67.6 (d,
C4α/C4β, J4,F 18.3), 69.5 (d, C2α, J2,F 17.5), 69.6 (d, C5α, J5,F 8.0), 70.2 (d, C2β, J2,F 19.1), 71.8 (d,
C5β, J5,F 7.2), 90.4 (d, C1α, J1,F 9.5), 89.5 (d, C3α, J3,F 189.9), 91.1 (d, C1β, J1,F 11.1), 91.5 (d, C3β,
J3,F 191.5), 168.5, 168.9 (x2), 169.1, 169.5, 170.5, 170.6 (C=O); ); δF (CDCl3, 376 MHz): -200.0 (ddt,
J 3.4, J = J 12.6, JF,3 52.7), -196.0 (dt, JF,2 = JF,4 12.6, JF,3 51.5; m/z (ESI+ve): 373 ([M+Na]+, 100%).
20:1 ratio: major β-anomer data below:
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δH (CDCl3, 400 MHz): 2.08 (3H, s, CH3), 2.09 (3H, s, CH3), 2.10 (3H, s, CH3), 2.11 (3H, s, CH3),
3.73 (1H, dddd, H5, J5,6 1.3, J 2.3, J5,6' 4.6, J 10.1), 4.11 (1H, dt, H6, J6,5 1.8, Jgem 12.4), 4.26 (1H, dd,
H6', J6',5 4.7, Jgem 12.5), 4.59 (1H, dt, H3, J 9.1, J3,F 51.8), 5.20 - 5.28 (2H, m, H2, H4), 5.64 (1H, d,
H1, J1,2 8.3); δC (CDCl3, 100 MHz): 20.6 (CH3), 20.6 (CH3), 20.7 (CH3), 20.8 (CH3), 61.3 (C6), 67.6
(d, C2, J2,F 19.1), 70.2 (d, C4, J4,F 19.1), 71.8 (d, C5, J5,F 8.1), 91.1 (d, C1, J1,F 11.1), 91.6 (d, C3, J3,F
191.2), 169.0, 169.0, 169.1, 170.6 (4 x C=O).
Methyl 2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-β-D-glucopyranoside, 30
HBr (33% in acetic acid, 38.7 mL, 213.7 mmol) was added dropwise to a solution of 29 (4.86 g, 13.87
mmol) in DCM (0.13 mL) at 0 °C. The solution was stirred for 5 h at 0 °C after which TLC analysis
(cyclohexane/ethyl acetate, 1:1) showed complete conversion of the starting material (Rf 0.52) to a
single major product (Rf 0.77). The reaction mixture was diluted with DCM (90 mL) and poured onto
iced water (90 mL). The organic layer was washed with ice cold NaHCO3 (sat., aq., 90 mL) and the
aqueous layer was extracted with DCM (90 mL). The combined organic layers were dried (MgSO4),
filtered and concentrated in vacuo at 20 °C to give the crude bromide which was used without further
purification.
Silver carbonate (6.50 g, 23.59 mmol) was added to a solution of crude bromide in methanol (62.3
mL) and the mixture was stirred at rt, in the dark for 18 h after which TLC analysis
(cyclohexane/ethyl acetate, 1:1) showed complete conversion of the starting material (Rf 0.77) to a
single major product (Rf 0.48). The reaction mixture was filtered through celite, concentrated in vacuo
and the residue was purified by flash chromatography (cyclohexane/ethyl acetate, 77:23) to give the
title compound 30 as a white solid (3.91 g, 87%).
+
HRMS m/z (ESI+ve): found: 345.0967 [M+Na]+; C13H19FNaO8 requires 345.0956; mp. 87 - 90 °C;
[α]D20 -26.7 (c 0.81, CHCl3); vmax (thin film): 1744 (s, C=O); δH (CDCl3, 400 MHz): 2.09 (3H, s, CH3),
2.10 (3H, s, CH3), 2.12 (3H, CH3), 3.50 (3H, s, OMe), 3.60 (1H, a-ddq, H5, J5,6 = J = J 1.3, J5,6' 4.7,
J5,4 10.0), 4.16 (1H, ddd, H6, J6,5 1.4, J6,F 2.5, Jgem 12.3), 4.26 (1H, dd, H6', J6',5 4.8, Jgem 12.3), 4.35
(1H, dd, H1, J 0.7, J1,2 8.1), 4.54 (1H, dt, H3, J3,2 = J3,4 9.1, J3,F 52.2), 5.10 (1H, ddd, H2, J2,1 8.0, J2,3
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9.2, J2,F 13.3), 5.21 (1H, ddd, H4, J4,5 9.1, J4,3 10.0, J4,F 12.5); δC (CDCl3, 100 MHz): 20.6 (CH3), 20.7
(CH3), 20.8 (CH3), 57.0 (OCH3), 61.7 (C6), 68.3 (d, C4, J4,F 19.1), 70.9 (d, C5, J5,F 8.1), 71.2 (d, C2,
J2,F 19.1), 91.7 (d, C3, J3,F 190.3), 101.1 (d, C1, J1,F 11.1), 169.1, 170.7 (3 x C=O); δF (CDCl3, 376
MHz): -195.9 (dt, JF,2 =JF,4 12.8, JF,3 52.2); m/z (ESI+ve): 345 ([M+Na]+, 100%).
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Methyl 3-deoxy-3-fluoro-β-D-glucopyranoside, 31
Sodium methoxide (0.65 g, 12.0 mmol) was added to a solution of 30 (6.45 g, 20.03 mmol) in
methanol (71 mL) at rt. After 5 h, TLC analysis (ethyl acetate) showed complete conversion of the
starting material (Rf 0.90) to a single major product (Rf 0.33). The mixture was concentrated in vacuo
and the residue was purified by flash chromatography (ethyl acetate/methanol, 17:3) to give the title
compound 31 as a colourless oil (3.77 g, 96%) which crystallised on standing.
HRMS m/z (ESI+ve): found: 219.0632 [M+Na]+; C7H13FNaO5+ requires 219.0639; mp. 120 - 124 °C;
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[α]D -34.7 (c 0.73, CH3OH); {Lit.50 m.p. 129.5-130 °C; [α]D -33.5 (c 1.2, H2O)}; vmax (thin film):
3361 (br, m, OH); δH (CD3OD, 400 MHz): 3.27 (1H, a-ddq, H5, J5,6' = J = J 1.2, J5,6 5.4, J5,4 9.9), 3.39
(1H, ddd, H2, J2,1 7.8, J2,3 9.1, J2,F 14.2), 3.54 (3H, s, OCH3), 3.57 (1H, ddd, H4, J4,3 8.7, J4,5 9.9, J4,F
14.2), 3.70 (1H, dd, H6, J6,5 5.4, Jgem 12.0), 3.87 (1H, ddd, H6', J6',5 1.3, J6',F 2.3, Jgem 12.0), 4.20 (1H,
dd, H1, J 0.7, J1,2 7.8), 4.24 (1H, dt, H3, J3,2 =J3,4 8.8, J3,F 52.8); δC (CD3OD, 100 MHz): 57.5 (OMe),
62.2 (C6), 69.7 (d, C4, J4,F 18.1), 73.6 (d, C2, J2,F 18.1), 76.7 (d, C5, J5,F 9.1), 98.5 (d, C3, J3,F 182.2),
104.6 (d, C1 J1,F 12.1); δF (CDCl3, 376 MHz) -196.1 (dt, JF,2 = JF,4 14.3, JF,3 52.6); m/z (ESI+ve): 219
([M+Na]+, 100%).
Methyl 6-O-tert-butyldimethysilyl-3-deoxy-3-fluoro-β-D-glucopyranoside, 32
Imidazole (5.42 g, 79.6 mmol) and tert-butyldimethylsilyl chloride (5.20 g, 34.5 mmol) were added to
a solution of 31 (5.20 g, 26.5 mmol) in anhydrous DMF (10.0 mL) at -20 °C. The reaction mixture
was stirred for 2.5 h at -20 °C after which the mixture was allowed to warm to rt. After 3 h TLC
analysis (cyclohexane/ethyl acetate, 1:1) showed complete conversion of the starting material (Rf
0.03) to a single major product (Rf 0.58). The reaction mixture was diluted with ethyl acetate (100
mL) and washed with 1:1 water/brine (3 x 60 mL). The combined aqueous layers were extracted with
ethyl acetate (3 x 40 mL). The combined organic layers were dried (MgSO4), filtered, concentrated in
vacuo and the residue was purified by flash chromatography (cyclohexane/ethyl acetate, 9:1 → 4:1 →
13:7) to give the title compound 32 as a white solid (7.42 g, 98%).
HRMS m/z (ESI+ve): found 333.1504; [M+Na]+; C13H27FNaO5Si+ requires 333.1504; mp. 80-84 °C;
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[α]D -40.0 (c 1.27, CHCl3); vmax (thin film): 3267 (br, m, OH); δH (CDCl3, 400 MHz): 0.10 (6H, 2 x
s, CH3Si), 0.90 (9H, s, (CH3)CSi), 2.66 (1H, br-s, 2-OH), 3.35 (1H, dddd, H5, J 1.0, J5,6' 4.8, J5,6 5.8, J
9.5), 3.39 (1H, br-s, 4-OH), 3.54 (3H, s, OMe), 3.58 (1H, ddd, H2, J2,1 7.8, J2,3 9.1, J2,F 13.6), 3.78 -
3.86 (1H, m, H4), 3.86 (1H, ddd, H6, J 0.8, J6,5 5.8, Jgem 10.6), 3.96 (1H, ddd, H6', J 0.8, J6',5 4.8, Jgem
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10.6), 4.21 (1H, dd, H1, J 0.8, J1,2 7.8), 4.42 (1H, dt, H3, J3,4 = J3,2 8.8, J3,F, 52.8); δC (CDCl3, 100
MHz): -5.5 (2 x CH3Si), 18.2 ((CH3)CSi), 25.8 ((CH3)CSi), 57.3 (OMe), 64.2 (C6), 71.3 (d, C4, J4,F
17.6), 72.3 (d, C2, J2,F 17.6), 73.1 (d, C5, J5,F 8.0), 96.4 (d, C3, J3,F 183.0), 102.8 (d, C1, J1,F 12.0); δF
(CDCl3, 376 MHz): -196.4 (dt, JF,2 =JF,4 13.7, JF,3 52.7); m/z (ESI+ve): 310 ([M+Na]+,100%).
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Methyl
N-benzyl-6-O-tert-butyldimethylsilyl-3-fluoro-2,4-imino-2,3,4-trideoxy-β-D-
talopyranoside, 23
Triflic anhydride (16.1 mL, 95.7 mmol) was added to a solution of pyridine (15.5 ml, 191 mmol) and
diol 32 (7.42 g, 23.9 mmol) in DCM (100 mL) at -30 °C. The reaction mixture was stirred at -30 to -
10 °C for 2 h after which time TLC analysis (cyclohexane/ethyl acetate, 3:1) showed almost complete
conversion of the starting material (Rf 0.17) to a single major product (Rf 0.67). A further portion of
pyridine (3.86 mL, 47.8 mmol) and triflic anhydride (4.0 mL, 23.9 mmol) was added and the reaction
stirred for a further 1 h at -30 °C after which the reaction mixture was diluted with DCM (80 mL),
washed with 2 M HCl (100 mL) and the aqueous layer was extracted with DCM (2 x 50 mL). The
combined organic layers were dried (MgSO4), filtered and concentrated in vacuo to give the crude
ditriflate 22 (13.73 g, quant.)
[Partial data: δH (CDCl3, 400 MHz): 0.10 (6H, 2 x s, CH3Si), 0.92 (9H, s, (CH3)CSi), 3.55 - 3.58 (1H,
m, H5), 3.59 (3H, s, OMe), 3.89 (1H, dd, H6, J6,5 3.3, Jgem 12.1), 3.98 (1H, dd, H6', J6',5 2.1, Jgem 12.0),
4.49 (1H, d, H1, J1,2 8.1), 4.69 (1H, ddd, H2, J2,1 7.8, J2,3 9.1, J2,F 12.9), 4.83 (1H, dt, H3, J3,4 = J3,2 8.9,
J3,F 51.6), 5.16 (1H, ddd, H4, J4,3 9.0, J4,5 9.6, J4,F 12.1); δC (CDCl3, 100 MHz): -5.6 (CH3Si), -5.5
(CH3Si), 18.3 ((CH3)3CSi), 25.7 ((CH3)3CSi), 57.7 (OMe), 60.3 (C6), 72.7 (d, C5, J5,F 6.0), 78.1 (d,
C4, J4,F 18.1), 81.8 (d, C2, J2,F 18.1), 89.6 (d, C3, J3,F 196.2), 99.8 (d, C1, J1,F 9.1)]
Benzylamine (13.0 mL, 120 mmol) and N,N-diisopropylethylamine (10.2 mL, 59.8 mmol) where
added to a solution of the crude ditriflate (13.73 g) in acetonitrile (140 mL) and the solution was
heated to 65 °C. After 18 h TLC analysis (cyclohexane/ethyl acetate, 3:1) showed complete
conversion of the starting material (Rf 0.72) to a single major product (Rf 0.80). The reaction mixture
was concentrated and the residue was purified by flash chromatography (1% → 5% → 10%, ethyl
acetate in cyclohexane) to give the title compound 23 as a light yellow oil (7.6 g, 84%).
HRMS m/z (ESI+ve): found 404.2019 [M+Na]+; C20H32FNNaO3Si+ requires 404.2028; [α]D -47.1 (c
20
1.15, CHCl3); vmax (thin film): fingerprint region only; δH (CDCl3, 400 MHz): -0.05 (3H, s, CH3Si), -
0.01 (3H, s, CH3Si), 0.82 (9H, s, (CH3)3CSi), 3.47 (3H, s, OMe), 3.51 (1H, ddd, H2, J2,1 1.3, J2,4 6.0,
J2,F 13.2), 3.68 (1H, ddd, H4, J4,5 1.2, J4,2 6.0, J4,F 13.3), 3.78 (1H, ddd, H6, J6,F 1.7, J6,5 5.4, Jgem 9.5),
3.84 (1H, dd, H6', J6',5 8.2, Jgem 9.2), 3.94 (1H, ddt, H5, J5,4 = J5,F 1.2, J5,6 5.4, J5,6' 8.2), 4.26 (2H, br-s,
CH2Ph), 4.81 (1H, t, H1, J1,2 = J1,F 1.2), 5.07 (1H, d, H3, J3,F 58.4), 7.19 - 7.24 (1H, m, ArH), 7.27 -
7.32 (2H, m, ArH), 7.39 - 7.43 (2H, m, ArH); δC (CDCl3, 100 MHz): -5.5 (2 x CH3Si), 18.2
((CH3)CSi), 25.8 ((CH3)CSi), 56.0 (CH2Ph), 56.1 (OMe), 63.1 (C6), 65.1 (d, C4, J4,F 18.3), 67.7 (d,
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128.3 (ArCH), 139.0 (ArC); δF (CDCl3, 376 MHz): -197.0 (dt, JF,2 = JF,4 13.2, JF,3 58.4); m/z (ESI+ve):
382 ([M+H]+, 100%).
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5,6-Di-O-acetyl-N-benzyl-3-fluoro-2,4-imino-2,3,4-trideoxy-D-talose acetyl methyl acetal, 33
Boron trifluoride diethyl etherate (4.5 mL, 36.7 mmol) was added dropwise to a solution of 23 (3.11
g, 8.2 mmol) in acetic anhydride (30 mL) at -30 °C before warming to rt. After 18 h mass
spectrometry (ESI+ve) showed the formation of the desired product 412 [M+H+] and no remaining
starting material. The mixture was concentrated under reduced pressure, and the residue was
dissolved in ethyl acetate (40 mL) and washed with NaHCO3 (sat. aq., 3 x 20 mL). The organic
fraction was dried (MgSO4), filtered and concentrated under reduced pressure. Purification by flash
column chromatography (10% → 30% ethyl acetate in cyclohexane) gave the title mixed acetal 33
(3.37 g, quant.) as a pale yellow oil, in a 1:1 ratio of diastereoisomers.
+
HRMS m/z (ESI+ve): found 412.1775 [M+H]+; C20H27FNO7 requires 412.1766; vmax (thin film):
1738 (s, C=O); δH (CDCl3, 400 MHz): 1.93 (3H, s, CH3), 1.94 (3H, s, CH3), 1.97 (3H, s, CH3), 2.07
(3H, s, CH3), 2.08 (3H, s, CH3), 2.18 (3H, s, CH3), 3.36 (1H, dt, H2, J2,1 = J2,3 5.1, J2,F 22.5), 3.37 (1H,
dt, H2, J2,1 = J2,3 4.7, J2,F 22.5), 3.39 (3H, s, OCH3), 3.41 - 3.52 (2H, m, H4), 3.43 (3H, s, OCH3), 3.77
(1H, d, CH2Ph, Jgem 13.2), 3.80 (1H, d, CH2Ph, Jgem 13.0), 3.86 (1H, d, CH2Ph, Jgem 13.7), 3.87 (1H, d,
CH2Ph, Jgem 13.2), 4.05 (1H, dd, H6, J6,5 6.2, Jgem 12.1), 4.11 (1H, dd, H6, J6,5 6.2, Jgem 12.1), 4.29
(1H, dd, H6', J6',5 3.7, Jgem 12.0), 4.35 (1H, dd, H6', J6',5 3.8, Jgem 12.1), 4.85 (1H, dt, H3, J3,2 = J3,4 4.8,
J3,F 55.9), 4.89 (1H, dt, H3, J3,2 = J3,4 4.7, J3,F 55.7), 4.89 - 4.94 (1H, m, H5), 5.00 (1H, ddd, H5, J5,6'
3.8, J5,6 6.0, J5,4 7.2), 5.46 (1H, d, H1, J1,2 4.9), 5.66 (1H, d, H1, J1,2 5.1), 7.25 - 7.39 (10H, m, ArH);
δC (CDCl3, 100 MHz): 20.7 (3 x CH3), 20.8 (CH3), 20.9 (CH3), 21.0 (CH3), 57.2 (OMe), 57.7 (OMe),
60.9 (CH2Ph), 61.6 (CH2Ph), 62.2 (2 x C6), 67.7 (d, C4, J4,F 20.7), 68.2 (d, C4, J4,F 20.7), 69.2 (d, C2,
J2,F 20.7), 69.9 (d, C2, J2,F 19.9), 71.4 (d, C5, J5,F 25.4), 71.5 (d, C5, J5,F 25.4), 83.5 (d, C3, J3,F 211.6),
83.5 (d, C3, J3,F 211.4), 97.4 (d, C1, J1,F 19.9), 97.5 (d, C1, J1,F 19.9), 127.6, 127.7, 128.5 (x 2), 128.9,
129.3 (ArCH), 136.6, 137.2 (ArC), 170.0, 170.1, 170.5, 170.6 (x 2), 170.7 (C=O); δF (CDCl3, 376
MHz): -179.8 (dt, JF,4 = JF,5 22.9, JF,3 56.1), -180.0 (dt, JF,4 = JF,5 22.9, JF,3 56.1); m/z (ESI+ve): 412
([M+H]+, 100%), 434 ([M+Na]+, 40%).
N-Benzyl-4-fluoro-3,5-imino-3,4,5-trideoxy-D-altritol, 34
DIBALH (1.5 M in toluene, 6.1 mL, 9.2 mmol) was added dropwise to a solution of 33 (471 mg, 1.1
mmol) in DCM (5 mL) at -78 °C. After 1.5 h mass spectrometry (ESI+ve) showed the formation of
the desired product 254 [M+H+] or gemdiol 272 [M+H+] and no remaining starting material. The
reaction mixture was diluted with ethyl acetate (20 mL) and stirred with sodium potassium tartrate
solution (25 mL, sat. aq.) until two layers were formed. The layers were separated and the aqueous
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