Synthesis and in vitro cytotoxicity of acetylated 3-fluoro, 4-fluoro and 3,4-difluoro analogs of D-glucosamine and D-galactosamine

Article abstract of DOI:10.3762/bjoc.12.75

Background: Derivatives of D-glucosamine and D-galactosamine represent an important family of the cell surface glycan components and their fluorinated analogs found use as metabolic inhibitors of complex glycan biosynthesis, or as probes for the study of protein-carbohydrate interactions. This work is focused on the synthesis of acetylated 3-deoxy-3-fluoro, 4-deoxy-4-fluoro and 3,4-dideoxy-3,4-difluoro analogs of D-glucosamine and D-galactosamine via 1,6-anhydrohexopyranose chemistry. Moreover, the cytotoxicity of the target compounds towards selected cancer cells is determined. Results: Introduction of fluorine at C-3 was achieved by the reaction of 1,6-anhydro-2-azido-2-deoxy-4-O-benzyl-β-D-glucopyranose or its 4-fluoro analog with DAST. The retention of configuration in this reaction is discussed. Fluorine at C-4 was installed by the reaction of 1,6:2,3-dianhydro-β-D-talopyranose with DAST, or by fluoridolysis of 1,6:3,4-dianhydro-2-azido-β-D-galactopyranose with KHF₂. The amino group was introduced and masked as an azide in the synthesis. The 1-O-deacetylated 3-fluoro and 4-fluoro analogs of acetylated D-galactosamine inhibited proliferation of the human prostate cancer cell line PC-3 more than cisplatin and 5-fluorouracil (IC₅₀ 28 ± 3 μM and 54 ± 5 μM, respectively). Conclusion: A complete series of acetylated 3-fluoro, 4-fluoro and 3,4-difluoro analogs of D-glucosamine and D-galactosamine is now accessible by 1,6-anhydrohexopyranose chemistry. Intermediate fluorinated 1,6-anhydro-2-azido-hexopyranoses have potential as synthons in oligosaccharide assembly.

Full text of DOI:10.3762/bjoc.12.75

Synthesis and in vitro cytotoxicity of acetylated 3-fluoro,
4-fluoro and 3,4-difluoro analogs of D-glucosamine and
D-galactosamine
Štěpán Horník1, Lucie Červenková Šťastná1, Petra Cuřínová1, Jan Sýkora1,
Kateřina Káňová2, Roman Hrstka2, Ivana Císařová3, Martin Dračínský4
and Jindřich Karban*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2016, 12, 750–759.
1Institute of Chemical Process Fundamentals of the CAS, Rozvojová
135, 165 02 Praha, Czech Republic, 2Regional Centre for Applied and
Molecular Oncology, Masaryk Memorial Cancer Institute, Žlutý kopec
7, 656 53 Brno, Czech Republic, 3Department of Inorganic Chemistry,
Charles University, Hlavova 2030, 128 43 Praha 2, Czech Republic
and 4Institute of Organic Chemistry and Biochemistry, Flemingovo
nám. 2, 166 10 Praha 6, Czech Republic
doi:10.3762/bjoc.12.75
Received: 08 February 2016
Accepted: 30 March 2016
Published: 20 April 2016
Associate Editor: S. Flitsch
Email:
© 2016 Horník et al; licensee Beilstein-Institut.
License and terms: see end of document.
Jindřich Karban* - karban@icpf.cas.cz
* Corresponding author
Keywords:
amino sugars; cytotoxicity; fluorinated carbohydrates; fluorine;
hexosamines
Abstract
Background: Derivatives of D-glucosamine and D-galactosamine represent an important family of the cell surface glycan compo-
nents and their fluorinated analogs found use as metabolic inhibitors of complex glycan biosynthesis, or as probes for the study of
protein–carbohydrate interactions. This work is focused on the synthesis of acetylated 3-deoxy-3-fluoro, 4-deoxy-4-fluoro and 3,4-
dideoxy-3,4-difluoro analogs of D-glucosamine and D-galactosamine via 1,6-anhydrohexopyranose chemistry. Moreover, the cyto-
toxicity of the target compounds towards selected cancer cells is determined.
Results: Introduction of fluorine at C-3 was achieved by the reaction of 1,6-anhydro-2-azido-2-deoxy-4-O-benzyl-β-D-glucopyra-
nose or its 4-fluoro analog with DAST. The retention of configuration in this reaction is discussed. Fluorine at C-4 was installed by
the reaction of 1,6:2,3-dianhydro-β-D-talopyranose with DAST, or by fluoridolysis of 1,6:3,4-dianhydro-2-azido-β-D-galactopyra-
nose with KHF2. The amino group was introduced and masked as an azide in the synthesis. The 1-O-deacetylated 3-fluoro and
4-fluoro analogs of acetylated D-galactosamine inhibited proliferation of the human prostate cancer cell line PC-3 more than
cisplatin and 5-fluorouracil (IC50 28 ± 3 μM and 54 ± 5 μM, respectively).
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Conclusion: A complete series of acetylated 3-fluoro, 4-fluoro and 3,4-difluoro analogs of D-glucosamine and D-galactosamine is
now accessible by 1,6-anhydrohexopyranose chemistry. Intermediate fluorinated 1,6-anhydro-2-azido-hexopyranoses have poten-
tial as synthons in oligosaccharide assembly.
Introduction
Derivatives of D-glucosamine (GlcN) and D-galactosamine galactosamine analog 4 [19] (Figure 1), inhibited the
(GalN) are essential amino sugar components of glycans in growth of murine L1210 leukemia cells in micromolar range
glycoproteins, glycolipids and proteoglycans. As such, they (IC50 27–35 μM). Compound 5 also inhibited the proliferation
participate in functions performed by cell-surface glycans in- of the human pancreatic cancer cell line KP1-NL (IC50 30 μM)
cluding cell adhesion and signaling [1]. Unnatural analogs of [20] and 4-fluoro-D-glucosamine analogs 1 and 2 were re-
these amino sugars prepared by a selective replacement of a ported to inhibit the proliferation of the human prostate cancer
hydroxy group by fluorine have proved valuable tools to perturb cell line PC-3 (IC50 61 µm for 2) [2].
glycan and glycosaminoglycan biosynthesis [2,3], to inhibit
amino sugars processing enzymes [4,5], to probe interactions of The remarkable ability of acetylated fluoro analogs of GlcNAc
amino sugars with their target enzymes and lectins [6,7], or to and GalNAc to perturb the (glycosamino)glycan biosynthesis
increase the hydrolytic stability of the glycosidic bond in amino and their antiproliferative properties aroused our interest in
sugar glycosides [8,9]. For example, acetylated 4-fluoro analogs developing a methodology for the preparation of a complete
of D-glucosamines 1–3, D-galactosamine 4, and 6-fluoro-D- series of acetylated 3-fluoro, 4-fluoro-, and 3,4-difluoro analogs
galactosamine 9 (Figure 1) acted as metabolic inhibitors of the 1, 4–8 (Figure 1) including previously unknown members of
biosynthesis of cell-surface O- and N-linked glycans (including this class of hexosamine mimics: acetylated 3-fluoro-D-GalNAc
their lactosamine and sialyl LewisX terminal epitopes) 6, 3,4-difluoro-D-GlcNAc 7 and 3,4-difluoro-D-GalNAc 8, as
[2,3,10,11], and glycosaminoglycans [3]. The resulting disrup- well as 3-fluoro-D-GlcNAc 5, in which case the reported syn-
tion of protein–(glycosamino)glycan interactions had important thesis was troublesome and low-yielding [20]. To carry out the
biomedical consequences such as reduced selectin-mediated synthesis, flexible synthetic methods for the stereoselective
tumor cell adhesion [12,13], suppressed selectin-mediated introduction of fluorine at one or more designated positions of
leukocyte migration [11,14,15], reduced angiogenesis [3], or the hexosamine skeleton are necessary. The elaborated chem-
inhibition of tumor growth by decreased galectin-mediated anti- istry of 1,6-anhydrohexopyranose derivatives is suitable for this
tumor T cell apoptosis [16].
purpose [21-23]. Building on previous results from our [24] and
other groups [25-28], we designed an approach based on stereo-
selective introduction of an azide as a masked amine group at
C-2, and fluorine at C-3 and C-4 by nucleophilic displacement.
Resulting 3-fluoro, 4-fluoro, and 3,4-difluoro analogs of
2-azido-1,6-anhydrohexopyranoses were then converted into the
target fluoro analogs of D-glucosamine and D-galactosamine
(Scheme 1). Dual protection of the anomeric and primary
hydroxy groups in the form of the 1,6-anhydro bridge reduced
the number of protecting groups, and the rigid bicyclic skeleton
of 1,6-anhydrohexopyranoses enabled a high degree of regio-
and stereocontrol necessary for the introduction of heteroatomic
substituents at C-2, C-3, and C-4. The synthesis of the analogs
6–8 has not yet been reported to our knowledge, while the syn-
thesis of 1, 4 and 5 represents an alternative to the published
procedures [2,20,29]. Herein we also report on the cytotoxicity
of prepared fluoro analogs in the human ovarian cancer A2780
Figure 1: Examples of deoxofluorinated hexosamines.
Some acetylated fluoro hexosamines also displayed antiprolifer-
ative properties in vitro [17]. For example, 3- and 4-fluoro-D-
glucosamine analogs 5 [18] and 1 [19], and 4-fluoro-D-
Scheme 1: Retrosynthetic plan.
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Beilstein J. Org. Chem. 2016, 12, 750–759.
and prostate cancer PC-3 cell lines. Preliminary results for the derivative 18. Lithium azide was found superior to sodium
synthesis of compounds 5 and 6 were communicated earlier in a azide in conversion of the triflate to the azide 18, and the yield
letter [30].
of 18 increased from 48% reported earlier [46] to 82%.
Results and Discussion
Synthesis
The synthesis of mono- and difluoro analogs of 2-azido-2-
deoxy-1,6-anhydrohexopyranoses, which are key intermediates,
The synthesis commenced with the introduction of an azide at is shown in Scheme 3. We first explored the reactions of azido
the 2α-position of the 1,6-anhydropyranose skeleton by nucleo- alcohols 11, 12 and 15 (Scheme 3) with diethylaminosulfur
philic cleavage of a 2β,3β-epoxide or the displacement of the trifluoride (DAST) to achieve the introduction of a nucleophilic
C-2β triflate ester using mainly a combination of published pro- fluorine atom. Reaction of 11 with DAST using a minor modifi-
cedures (Scheme 2). 2-Azido alcohols 11, 12 and 15 were ob- cation of the reported procedure [28] provided the D-gluco-
tained from 1,6:2,3-dianhydro-4-O-benzyl-β-D-mannopyranose configured 3-fluoro-derivative 19 (Scheme 3) with clean reten-
(10, one of the Černý epoxides available from D-glucal [31-33] tion of configuration. To prepare the D-galacto-configured
or levoglucosan [34]). Regioselective azidolysis [21,35,36] of analog of 19, deoxofluorination of 15 by reaction with DAST in
10 yielded 2-azido alcohol 11 which was converted to 12 by benzene at 75 °C (conditions used for fluorination of 11), or in
benzoylation at O-3 and debenzylation at O-4. To prevent azide dichloromethane at rt was attempted. Disappointingly, only
reduction, oxidative debenzylation [24] was applied instead of unreacted 15 was recovered. A very slow formation of several
the more common hydrogenation. Debenzylation of 10 gave unidentified fluorine-containing compounds (19F NMR) was
dianhydro derivative 13 [34,37] (available also directly from observed on prolonged reaction times. Oxidative debenzylation
D-glucal [31,33] or from levoglucosan [38]) which was con- of 19 gave fluorhydrin 20 which was subjected to epimeriza-
verted to 14 by Latrell–Dax inversion at C-4 [39]. O-Benzyla- tion at C-4 by Latrell–Dax inversion to furnish 2-azido-3-fluoro
tion [40] of 14 followed by azidolysis [41] furnished 15. compound 21 with the desired D-galacto configuration
2-Azido-3,4-epoxide 18 was prepared from readily available (Scheme 3). The geminal fluorine–carbon coupling value
[42] 2,3-isopropylidene-D-mannosan (16) in five steps 2JC4,F = 17.7 Hz in 21 is characteristic for a gauche
(Scheme 2). Tosylation of 16 [43], followed by hydrolysis of relationship between the C3–F and C4–O bonds whereas
the benzylidene acetal [44] and oxirane ring closure [45] at C-4 2JC4,F = 31.5 Hz in 20 is consistent with an antiperiplanar
delivered 1,6:3,4-dianhydro derivative 17. Formation of the arrangement. The inversion at C-4 is also manifested by an
triflate ester and azidolysis furnished 1,6:3,4-dianhydro-2-azido increase in the 3JH4,H3/5 values (1.7 → 4.5 Hz) together with an
increase of the 3JF,H4 coupling (13.9 → 26.4 Hz).
An attempt to introduce fluorine at C-4 on reaction of 12 with
DAST furnished orthoester 25 instead of the desired 4-fluoro
derivative. The structure of orthoester 25 was confirmed by
single crystal X-ray analysis. The formation of 25 resulted from
an intramolecular displacement of the unstable alkoxysulfur
intermediate 23 with the oxygen of the benzoyl group and a
subsequent reaction of the salt 24 with methanol upon
quenching. A similar participation of a vicinal O-acetyl
protecting group on reaction with DAST leading to the forma-
tion of an orthoacetate was reported [47]. Reaction of fluoro-
hydrin 20 with DAST proceeded with inversion at C-4 giving
3,4-difluoro analog 22. The D-galacto configuration of 22 was
manifested by the values of the vicinal coupling constants
3JH2,H3 = 1.6 Hz and 3JH3,H4 = 4.5 Hz, and the large value of
2JC5,F4 = 27.2 Hz confirmed the equatorial position of fluorine
at C-4.
Scheme 2: Preparation of starting 2-azido compounds. Reagents and
conditions: (a) NaN3, NH4Cl, MeOC2H4OH, 79%; (b) i) BzCl, py;
ii) NaBrO3, Na2S2O4, AcOEt, H2O, 47% over 2 steps; (c) Pd/C, H2,
EtOH, 96%; (d) Tf2O, then (Bu)4NNO2, 69%; (e) (i) NaH, BnBr, THF,
79%; (ii) LiN3, NH4Cl, MeOC2H4OH, 100 °C, 81%; (f) (i) TsCl, py,
Cleavage of the oxirane ring in 18 on reaction with KHF2 in
ethylene glycol [48,49] furnished the 4-fluoro derivative 26
(ii) AcOH, H2O; (iii) IRA 410 (OH−), MeOH, 78% over 3 steps;
(g) (i) Tf2O, py, CH2Cl2; (ii) LiN3, DMF, 82% over 2 steps.
(Scheme 3). The moderate yield of 26 (40–60%) can be attri-
buted to the formation of the 4-O-(2-hydroxyethyl) derivative
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Beilstein J. Org. Chem. 2016, 12, 750–759.
Scheme 3: Preparation of mono and difluoro analogs of 2-azido-2-deoxy-1,6-anhydro-β-D-gluco- and galactopyranoses. Reagents and conditions:
(a) DAST, benzene, 75–81 °C, 88%; (b) NaBrO3, Na2S2O4, EtOAc, H2O, 75%; (c) Tf2O, py, CH2Cl2; then (Bu)4NNO2, DMF, 67%; (d) DAST,
benzene, 75–81 °C; or DAST, CH2Cl2, −25 °C to rt; (e) DAST, CH2Cl2, −25 °C to rt, 78%; (f) DAST, CH2Cl2, −25 °C to rt, 65%; (g) KHF2, C2H4(OH)2,
175 °C, 40–60% (26); (h) DAST, benzene, 75–81 °C, 46% (28), 12% (29); (i) DAST, CH2Cl2, −50 °C to rt, 52 %, (j) LiN3, NH4Cl, MeOC2H4OH,
100 °C, 74% (31).
27 arising from solvent participation, and to a partial decompo- a side product. Products 28 and 29 can be separated by careful
sition (indicated by TLC) at high temperatures necessary for the chromatography and their structures were verified by single
reaction to proceed. The low values of the vicinal coupling con- crystal X-ray analysis.
stant 3JH3,H4 = 2.0 Hz and the large value of the geminal cou-
pling constant 2JC3,F = 29.6 Hz evidenced a trans-diaxial rela- Since fluorination of 12 with DAST failed to introduce fluorine
tionship between the C-3 and C-4 substituents in 26. The reac- at C-4β due to formation of orthoester 25, the reaction of
tion of fluorohydrin 26 with DAST in benzene under heating 1,6:2,3-dianhydro-β-D-talopyranose 14 with DAST was utilized
afforded, as the main product, 3,4-difluoro-D-gluco analog 28 to give 1,6:2,3-dianhydro-4-deoxy-4-fluoro-β-D-talopyranose
(46%), and the rearranged 2,6-anhydro compound 29 (12%) as (30) with retention of the configuration at C-4 [39]. Azidolysis
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Beilstein J. Org. Chem. 2016, 12, 750–759.
of the oxirane ring in the reaction with lithium azide furnished before [52]. The main product 28 and rearranged difluoride 29
2-azido derivative 31. Although nucleophilic cleavage of a can arise from the same intermediate species 39 (Scheme 4B,
three-membered ring annulated to the 1,6-anhydrohexo- pathways a and b, respectively). Compound 19 can, in principle,
pyranose skeleton usually occurs solely in trans-diaxial fashion be also formed from 11 through azide participation (not shown).
[50], formation of the trans-diequatorial side-product 32 in In such a case the reaction is unexpectedly sensitive to minor
ca. 8% was observed in NMR spectra. The desired D-galacto- steric alterations of the substrate because the C-4 epimer 15
configured 2-azido derivative 31 was separated from 32 by (Scheme 3) did not react. An internal fluorine attack from the
crystallization in 74% yield. The D-galacto configuration of 31 β-face of the tetrahydropyran ring through a concerted
is evidenced by the vicinal coupling values 3JH4,H3/5 = 4.5 Hz (Scheme 4C) or contact ion-pair (Scheme 4D) SNi mechanism
and the long range coupling between equatorial protons H-1, cannot be ruled out [55,56] because the bulky Et2NSF2O sub-
H-3, and H-5 (4JH3,H1/5 = 1.4 Hz).
stituent at C-3 might force the substrate to adopt boat B3,O
conformation [57] bringing the C-2, C-3 and C-4 substituents
The reaction of the D-gluco-configured 3-hydroxy derivatives into a trans-equatorial arrangement unfavorable for anchimeric
11, and 26 with DAST, and the previously reported reactions of assistance.
D-gluco-configured 3-hydroxy derivatives 33 [26], 34 [27], and
35 [25] (Scheme 4) with DAST are an important means of ob- With the D-gluco- and D-galacto-configured deoxofluoro deriv-
taining 3-deoxy-3-fluoro derivatives of D-gluco-configured atives of 2-azido-1,6-anhydrohexopyranoses in hand, we exam-
aldohexopyranoses which are difficult to prepare otherwise. ined their conversion to the target acetylated hexosamine
These reactions characteristically proceed with a clean reten- analogs. Initially, compound 19 was hydrogenated (Pd/C) and
tion of configuration which can be explained by an anchimeric acetylated to afford acetamide 40 in modest yield (37%).
assistance of the trans-diaxially positioned (with respect to Sulfuric acid-catalyzed [35] cleavage of the internal acetal with
C3–OH) polar groups at C-2 or C-4, or by an internal fluorine acetic anhydride gave a mixture containing 1,2-oxazoline 41 as
attack as in SNi substitution. A simple SN2 displacement the main product (Scheme 5). The structure of oxazoline 41 was
leading to configurational inversion is probably suppressed by confirmed by single crystal X-ray diffraction analysis which
the steric effects of the axially positioned groups at C-2 and
C-4, and repulsive effects of their aligned dipoles [51]. Com-
pounds 11, and 33–35, possess a trans-diaxially positioned
benzyloxy group at C-4 capable of participation through an
oxiranium intermediate species (Scheme 4A) [52-54]. The for-
mation of the rearranged difluoride 29 from alcohol 26
(Scheme 3) suggests an anchimeric assistance of the vicinal C-2
Scheme 5: Formation of oxazoline 41 from 19.
azido group. Although rare, azide participation was postulated
Scheme 4: Suggested mechanisms for deoxofluorination at C-3 of 1,6-anhydro-β-D-glucohexopyranose derivatives. A) participation of the O-benzyl
group; B) participation of the azide group; C) internal fluorine attack, concerted mechanism; D) internal fluorine attack, contact ion pair mechanism.
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Beilstein J. Org. Chem. 2016, 12, 750–759.
also confirmed the retention of configuration during the TESOTf as a catalyst for acetolysis gave better results than
preceding fluorine introduction.
sulfuric acid in terms of product purity. Hydrogenolysis of 42
on palladium in ethanol/HCl followed by acetylation of the
To prevent oxazoline formation, the order of reactions was amino group furnished the target acetylated 3-fluoro-D-GlcNAc
reversed, and triethylsilyl triflate (TESOTf)-catalyzed [58] 5 as a chromatographically separable mixture of anomers
acetolysis of the 1,6-anhydro bridge in 19 gave 42 (Table 1) as (Table 1). Addition of HCl was found necessary to effect a
a mixture of anomers from which the α-anomer crystallized. clean hydrogenolytic removal of the O-benzyl group in 42. The
Table 1: Acetolysis of the fluorine-containing intermediates and hydrogenation.
entry
1
starting compound
product of acetolysis
yield (%)a
96
target compound
yield (%)a
82b
42
43
5
6
19
21
2
83
78
74
86
1
3
26
16
n.a.
4
5
6
94
78
44d
70
4
7
8
46
47
31
28
22
c
86
48
aIsolated yield; bH2, Pd/C, EtOH, AcCl, then Ac2O, py was used for hydrogenation of 42; c47 was not isolated as pure compound, see text; doverall
from 28; n.a. – not available.
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Beilstein J. Org. Chem. 2016, 12, 750–759.
D-gluco configuration of 42 and 5 was reflected in the the value of 3JH2,H3 (1.6 → 11.1 Hz), a decrease in the value of
large values of 3JH3,H2/4 (7.6–10.2 Hz), and the chair inversion 2JC5,F4 (27.2 → 18.3 Hz), and an increase of 3JH3,F4 coupling
accompanying the cleavage of the 1,6-anhydro bridge (4.3 → 26.0 Hz) between 22 and 8.
(1C4 → 4C1) in a decrease of the geminal fluorine–carbon cou-
pling value 2JC4,F (31.5 Hz in 40 → 18.4 Hz in 5 (α-anomer)) To study the influence of 1-O-deacetylation on the cytotoxicity,
[59]. 1H and 13C NMR spectra of 5 reported in [20] are compa- the monofluorinated analogs 1, and 4–6 were subjected to
rable with our data for the α-anomer (5α). A discrepancy in the anomeric deacetylation (Scheme 6). Compound 5 provided 1-O-
values of specific optical rotation
(reported +10°, +81° deacetylated product 49 by treatment with BnNH2 in THF.
obtained by us) can be explained assuming that the value for the Since acetylated 4-fluoro-D-GlcNAc 1 under these conditions
β-anomer (5β) was reported in [20]. Our synthesis of 5 is more did not react cleanly, we used piperidine-promoted [60] de-
practical (55% from 10 in 3 steps) than the earlier preparations acetylation to prepare 2 in 74% yield. Similarly, acetylated
which were reported troublesome owing to low yields (28% and 4-fluoro-D-GalNAc 4 gave 50 in 60% yield. The attempted
10%, respectively) of the key fluorination step [18,20].
anomeric deacetylation of 3-fluoro-D-GalNAc 6 by treatment
with piperidine followed by chromatography gave a fraction
For the remaining fluoro derivatives of 1,6-anhydro-2-azido- containing an inseparable side-product in addition to the ex-
hexopyranoses, TESOTf-catalyzed acetolysis and subsequent pected deacetylated product 51. The side-product showed no
hydrogenation (Pd/C in a mixture of ethanol and acetic an- fluorine resonance in 19F NMR and its molecular formula
hydride) was applied to obtain the target acetylated fluoro C17H28N2O7 assigned by LC–HRMS corresponded to a formal
analogs (Table 1). Compound 21 was acetolyzed to 43 which displacement of fluorine by piperidine, leading probably to
was obtained as a mixture of anomers and characterized by compound 53. When pure 51 (prepared by another method, see
NMR and finally hydrogenated to furnish acetylated 3-fluoro- below) was reacted with excess piperidine, high resolution
GalNAc 6 isolated as a separable mixture of anomers. The ESIMS analysis of the reaction detected transient formation of
D-galacto configuration of 6 is manifested by the lower 3JH3,H4 an adduct ion corresponding to a supposed intermediate enal 52
coupling value (3.4 Hz, α-anomer) in comparison with that of (Scheme 6), while the adduct ion corresponding to 53 was the
its C-4 epimer 5 (8.6 Hz, α-anomer). Acetolysis of the internal final product (see Supporting Information File 1). Presumably,
acetal in 26 proceeded more slowly and beside the desired prod- piperidine as a relatively strong base effected dehydrofluorina-
uct 44 (78% yield), 3-O-acetyl derivative 45, in which the 1,6- tion of 51 to enal 52 which then added piperidine to give 53 as a
anhydro bridge remained intact, was also isolated. Hydrogena- byproduct (Scheme 6). To avoid the action of basic amines, a
tion of 44 furnished the known acetylated 4-fluoro-D-GlcNAc 1 silica gel mediated anomeric deacetylation, recommended for
[2]. The large values of the vicinal coupling constants 3JH3,H2/4 2-aminosugars [61], was tried. The reaction proceeded
and 3JH4,H5 (8.9–11.1 Hz) confirmed the D-gluco configuration extremely slowly with our substrate 6 and the product
of 1 and the chair inversion when going from 26 to 1, which 51 was obtained in only 40% yield after chromatography and
was also manifested by a decrease in the geminal coupling con- recrystallization.
stant 2JC3,F (29.6 → 18.9 Hz, α-anomer). Acetolysis and subse-
quent hydrogenation of 31 provided the known peracetylated
4-deoxy-4-fluoro-D-galactosamine 4 [29]. The chair inversion
of the tetrahydropyran ring associated with the cleavage of the
1,6-anhydro bridge is indicated by the increase in the coupling
value 3JH2,H3 (1.4 → 11.6 Hz) and decrease in the coupling
value 2JC5,F (27.4 → 18.4 Hz) when going from 31 to 4
(α-anomer). Acetolysis of 28 afforded diacetate 47 containing
chromatographically inseparable impurities (NMR). They were
removed in the next hydrogenation step to yield the peracety-
lated 3,4-difluoro analog of D-glucosamine 7 isolated as an
α-anomer in 44% yield from 28. The D-gluco configuration of 7
is reflected in the large values of the vicinal coupling constants
3JH2,H3 = 10.5 Hz, 3JH3,H4 = 8.2 Hz, and 3JH4,H5 = 9.8 Hz.
Acetolysis of 22 gave diacetate 48 as a separable mixture of
anomers. Hydrogenation of the α-anomer furnished the
peracetylated 3,4-difluoro analog of D-galactosamine 8. The
D-galacto configuration of 8 was confirmed by an increase in
Scheme 6: 1-O-Deacetylation of monofluorinated hexosamines.
Reagents and conditions: (a) BnNH2, THF, 62%; (b) C5H10NH, THF,
74%; (c) C5H10NH, THF, 60%; (d) silica gel, MeOH, 30 days, 40%.
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Cytotoxicity
PC-3 cell line, and human ovarian cancer A2780 cell line using
Some acetylated fluorinated hexosamines (HexN), including the MTT assay, and the obtained IC50 values were compared
peracetates of the α-methyl glycoside of 3-fluoro-D-ManNAc with those obtained for cisplatin and 5-fluorouracil.
[18], 3-fluoro-D-GlcNAc 5 [18], 4-fluoro-D-GlcNAc 1 [19],
4-fluoro-D-GalNAc 4 [19], 4,4-difluoro-D-xylo-HexNAc [19], All of the tested compounds induced only moderate to weak
and 4,6-difluoro-D-GalNAc [19] were reported to exhibit anti- inhibition of A2780 cell proliferation (IC50 values ranging from
proliferative properties against L1210 leukemia cells in micro- 78 μM to 327 μM, Table 2) in comparison to cisplatin
molar concentrations (IC50 24–43 μM). It was found that (IC50 12.9 μM). Anomeric deacetylation resulted in a higher ac-
O-deacetylated amino sugars were often inactive due to low tivity in the case of 3-fluoro-D-GlcNAc (49, IC50 84 μM,
lipophilicity and poor cellular uptake [19]. Compound 5 was Table 2, entry 6), and especially 4-fluoro-D-GlcNAc (2,
also cytotoxic to the human pancreatic cancer cell line KP1-NL IC50 78 μM, ca. 4-fold higher activity than that of the peracety-
(IC50 = 30 μM) [20], and 1 and its 1-O-deacetylated derivative lated 1, Table 2, entries 1 and 2), while 1-O-deacetylated 50 had
2 inhibited cell proliferation of the human prostate cancer cell a lower activity (IC50 142 μM, Table 2, entry 4) then the parent
line PC-3 (IC50 61 µM for 2) [2]. Interestingly, while all peracetate 4 (IC50 78 μM, Table 2, entry 2). All of the tested
4-fluoro analogs 1–3 (Figure 1) reduced the expression of fluorinated D-glucosamine analogs also induced only weak
highly branched N-glycans in PC-3 cells, the 6-O-deacetylated inhibition on PC-3 cells (IC50 ranging from 134 μM to 337 μM,
analog 3 showed only negligible cytotoxicity [2] implying that Table 2, entries 1, 2, 5, 6, and 9), and 1-O-deacetylation seemed
the inhibition of proliferation and perturbation of N-glycan bio- to have a negative effect here (Table 2, entries 2 and 6). On the
synthesis occur by different mechanisms.
other hand, the 1-O-deacetylated form of 3-fluoro-D-galac-
tosamine 51 (IC50 28 μM, Table 2, entry 8) and 4-fluoro-D-
Increased cytotoxicity as a result of 1-O-deacylation was noted galactosamine 50 (IC50 54 μM, Table 2, entry 4) reduced
for a variety of acylated (nonfluorinated) D-mannosamine and proliferation of PC-3 cells more than cisplatin (IC50 182 μM,
D-glucosamine derivatives [62,63]. Acylated hexosamine deriv- Table 2, entry 12) and 5-fluorouracil (IC50 69 μM, Table 2,
atives were subsequently studied as possible templates for the entry 13). The effect of anomeric deacetylation was
development of anticancer therapeutics [64,65]. While the particularly pronounced in 3-fluoro-D-galactosamine 51 owing
ability of hexosamine derivatives and analogs to inhibit cell to inactivity of the corresponding peracetate 6. Finally,
growth creates an avenue for their use in the development of difluoro analogs 7 and 8, and oxazoline 41 exhibited
anticancer drugs, it also limits their utility as agents to modify weak (compound 7) to none (compounds 8 and 41) activity
the cellular glycome [62]. The cytotoxic activity of peracety- in the PC-cell line (Table 2, entries 9–11). Taken together,
lated monofluoro analogs 1, and 4–6, their 1-O-deacetylated de- our results seem to corroborate in part the observations by
rivatives 2, and 49–51, difluoro analogs 7 and 8, and oxazoline Yarema at al. that the liberation of the anomeric hydroxy
41 was therefore tested for 24 h on the human prostate cancer group in acylated hexosamines resulted in an enhancement
Table 2: IC50 mean values (μM) of selected fluorinated analogs after 24 h treatment using PC-3 and A2780 cancer cell lines.
entry
compd. no.
abbreviationa
PC-3
A2780
1
2
1
4F-Ac3GlcNAc
4F-Ac2GlcNAc-1-OH
4F-Ac3GalNAc
235 ± 26
337 ± 36
101 ± 15
54 ± 5
327 ± 48
78 ± 9
2
3
4
78 ± 9
4
50
4F-Ac2GalNAc-1-OH
3F-Ac3GlcNAc
142 ± 28
218 ± 29
84 ± 6
5
5
134 ± 17
308 ± 38
>500
6
49
3F-Ac2GlcNAc-1-OH
3F-Ac3GalNAc
7
6
249 ± 36
137 ± 24
183 ± 26
250 ± 41
108 ± 14
12.9 ± 1.5
52 ± 7
8
51
3F-Ac2GalNAc-1-OH
3F,4F-Ac2GlcNAc
3F,4F-Ac2GalNAc
3F-Ac2Glc-1,2-oxazoline
28 ± 3
9
7
199 ± 34
>500
10
11
12
13
8
41
>1000
cisplatin
5-fluorouracil
182 ± 13
69 ± 6
aThese abbreviations are provided for quick orientation, anomeric deacetylation is indicated by '-1-OH'.
757

Beilstein J. Org. Chem. 2016, 12, 750–759.
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