Synthesis and Antitubercular and Antibacterial Activities of Triethylammonium 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-D-glucopyranosyl Decyl Phosphate

Article abstract of DOI:10.1134/S1070428018090117

Phosphorylation of 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-D-glucose at the anomeric hydroxy group gave previously unknown triethylammonium 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-D-glucopyranosyl phosphonate, and successive treatment of the latter with decan-1-ol and aqueous iodine afforded triethylammonium 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranosyl phosphate.

Full text of DOI:10.1134/S1070428018090117

ISSN 1070-4280, Russian Journal of Organic Chemistry, 2018, Vol. 54, No. 9, pp. 1333–1336. © Pleiades Publishing, Ltd., 2018.
Original Russian Text © B.F. Garifullin, R.R. Sharipova, A.D. Voloshina, M.A. Kravchenko, V.E. Kataev, 2018, published in Zhurnal Organicheskoi Khimii,
2018, Vol. 54, No. 9, pp. 1321–1324.
Synthesis and Antitubercular and Antibacterial Activities
of Triethylammonium 2-Acetamido-3,4,6-tri-O-acetyl-
2-deoxy-D-glucopyranosyl Decyl Phosphate
B. F. Garifullina, R. R. Sharipovaa, A. D. Voloshinaa, M. A. Kravchenkob, and V. E. Kataeva*
a Arbuzov Institute of Organic and Physical Chemistry, Kazan Scientific Center, Russian Academy of Sciences,
ul. Arbuzova 8, Kazan, 420088 Tatarstan, Russia
*e-mail: kataev@iopc.ru
b Ural Research Institute of Phthisiopulmonology, ul. 22 Parts’’ezda 50, Yekaterinburg, 620039 Russia
Received February 21, 2018
Abstract—Phosphorylation of 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-D-glucose at the anomeric hydroxy
group gave previously unknown triethylammonium 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-D-glucopyranosyl
phosphonate, and successive treatment of the latter with decan-1-ol and aqueous iodine afforded triethyl-
ammonium 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranosyl phosphate.
DOI: 10.1134/S1070428018090117
We have recently reported the synthesis of phos-
phorylated glycolipids based on glucuronic acid, which
showed antitubercular activity against Mycobacterium
tuberculosis H37Rv (MBT) comparable with the
activity of the known antitubercular drug pyrazinamide
(MIC 12.5 μg/mL) [1]. In continuation of our studies
in the field of synthesis and antitubercular activity of
glucosamine derivatives [2, 3], herein we describe
a novel approach to phosphorylated glucosamine-
based glycolipids. This approach utilizes commercially
available glucosamine hydrochloride (1) as starting
compound which was acylated with acetic anhydride
in pyridine to pentaacetyl derivative 2. Regioselective
removal of the acetyl protection from the anomeric
hydroxyl of 2 by the action of methylamine in metha-
nol gave tetraacetyl glucosamine 3. The latter reacted
with 2-chloro-2H,4H-1,3,2-benzodioxaphosphinin-4-
one (5) [4] acording to the procedure described in [1]
to give triethylammonium 2-acetamido-3,4,6-tri-O-
acetyl-2-deoxy-α/β-D-glucopyranosyl phosphonate (6)
in 34% yield (after isolation by chromatography). The
1H NMR spectrum of 6 contained signals typical of
tetraacetyl glucosamine 3, as well as signals of protons
of the triethylammonium cation and PH proton
(δ 6.90 ppm, d, 1JHP = 668 Hz). The 31P NMR spectrum
of 6 showed a singlet at δP 1.93 ppm. The anomeric
proton in the carbohydrate moiety of 6 resonated as
a multiplet in the region δ 5.61–5.69 ppm, indicating
formation of a mixture of anomers. In the next stage,
phosphonate 6 was activated with pivaloyl chloride
and treated with decan-1-ol. Glycolipid 7 thus formed
was oxidized without isolation with aqueous iodine to
produce 23% (after isolation by chromatography) of
target phosphorylated glycolipid 8. The formation of 8
was confirmed primarily by the disappearance of PH
singlet at δP 1.93 ppm from the 31P NMR spectrum and
appearance of a singlet at δP –2.27 ppm, which is
typical of phosphate monoanion (PO4–). In the MALDI
mass spectrum of 8 we observed the molecular ion
peak [M]– with m/z 566.4 (calcd.: m/z 566.2,
C24H41NO12P–). The anomeric proton signal of 8 was
located in the 1H NMR spectrum at δ 5.51 ppm (3JHP
=
7.34, J1,2 = 3.22 Hz), in keeping with α-orientation of
the glycoside bond.
The described method of synthesis of glycolipids 8
essentially differs from the known procedures for the
preparation of phosphorylated glucosamine-based
glycolipids containing a phosphate or pyrophosphate
(diphosphate) fragment [5–11]. First, the key glucos-
amine derivative with protected hydroxyl groups in
positions 3, 4, and 6 and free anomeric hydroxyl group
(monosaccharide 3), which was subjected to phos-
phorylation, was readily prepared by regioselective
deprotection of the anomeric hydroxyl group of penta-
1333

GARIFULLIN et al.
1334
Scheme 1.
OH
O
OAc
O
OAc
O
Ac2O
pyridine
MeNH2
MeOH
HO
HO
AcO
AcO
AcO
AcO
OH
OAc
OH
OAc
O
NH2·HCl
NHAc
NHAc
AcO
AcO
Et3N, THF
1
2
3
O
P
O–
O
H
O
COOH
NHAc
POCl3
O
P
Et3NH
6
OH
O
Cl
4
5
OAc
O
OAc
O
CH3(CH2)9OH
PivCl, pyridine
(1) I2, H2O
(2) Na2S2O3
AcO
AcO
AcO
AcO
Et3NH
O–
O
O
P
P
H
OC10H21
O
O
OC10H21
NHAc
NHAc
7
8
acetylglucosamine 2 by the action of methylamine in
methanol. Second, by analogy with [1], we utilized
a different procedure for the synthesis of glycolipids
with a phosphate group from tetraacetylated glucos-
amine 3. Monosaccharide 3 is usually phosphorylated
by reaction with dibenzyl N,N-diisopropylphosphor-
amidite and 1H-tetrazole [9], followed by oxidation
with hydrogen peroxide, debenzylation, and treatment
with tetrabutylammonium hydroxide. The salt thus
obtained is then alkylated with alkyl bromide in the
presence of molecular sieves. In our case, the alkyla-
tion involved not glucosamine phosphate but phospho-
nate which was readily synthesized from tetraacetyl-
glucosamine 3 and phosphorochloridite 5. The synthe-
sis of target compound 8 from salt 6 was carried out in
a one-pot fashion with the oxidation of phosphonate 7
to phosphate with iodine in the final stage.
lower than the activity of the first line antitubercular
drug isoniazid (MIC 0.1 μg/mL) but 4 times higher
than that of the second line antitubercular drug pyra-
zinamide [12] and macrocyclic glucosamine deriva-
tives [3] (MIC 13 μg/mL). We are continuing studies
on the synthesis and biological activity of phos-
phorylated glycolipids based on glucosamine.
EXPERIMENTAL
The 1H, 13C, and 31P NMR spectra were recorded on
a Bruker Avance-400 spectrometer (Germany) at 400
(1H) and 100.6 MHz (13C, 31P) using the solvent
signals as reference (CDCl3). Signals were assigned on
the basis of published data [1, 3, 13]. The mass spectra
(MALDI) were obtained on a Bruker Daltonik
UltraFlex III TOF/TOF instrument operating in the
linear mode (Nd:YAG laser, λ 355 nm); the data were
processed by Bruker Daltonik FlexAnalysis 3.0; a.m.u.
range 200–6000, negative ion detection, metal target,
p-nitroaniline matrix; samples were dissolved in
methanol to a concentration of 10 μg/mL. The optical
rotations were measured on a Perkin Elmer-341
polarimeter at λ 589 nm (temperature 20°C). The
progress of reactions was monitored, and the purity of
the isolated compounds was checked, by thin-layer
chromatography on Sorbfil plates; spots were detected
by treatment with 5% sulfuric acid, followed by
heating to 120°C.
The antimicrobial activity of glycolipid 8 against
gram positive and gram negative bacteria and fungi, as
well as its antitubercular activity, was studied. Com-
pound 8 inhibited the growth of S. aureus at a mini-
mum inhibitory concentration (MIC) of 62.5 μg/mL,
which is comparable to the activity of Chloramphe-
nicol used as control. However, compound 8 turned
out to be inactive against other microorganisms. It
should be noted that no data on antibacterial activity of
glucosamine derivatives were reported previously.
Glycolipid 8 inhibited the growth of MBT at a MIC of
3.1 μg/mL; i.e., its antitubercular activity was much
RUSSIAN JOURNAL OF ORGANIC CHEMISTRY Vol. 54 No. 9 2018

SYNTHESIS AND ANTITUBERCULAR AND ANTIBACTERIAL ACTIVITIES
1335
Glucosamine hydrochloride was commercial prod-
uct (Acros Organics). Compounds 2, 3, and 5 were
synthesized as described in [14–16].
1.32 t (9H, CH3CH2N, J = 7.3 Hz); 1.94 s, 1.98 s,
1.99 s, and 2.05 s (3H each, CH3CO); 3.06 q (6H,
CH3CH2N, J = 7.31 Hz), 3.83–3.94 m (2H, CH2O),
4.05–4.10 m (1H, 2-H), 4.18-4.25 m (2H, 6-H), 4.30–
4.40 m (1H, 5-H), 5.16 t (1H, 4-H, J = 9.80 Hz), 5.30 t
(1H, 3-H, J = 10.04 Hz), 5.51 d.d (1H, 1-H, J = 7.34,
3.22 Hz), 7.06 d [1H, NHC(O)CH3, J = 9.35 Hz],
Triethylammonium 2-acetamido-3,4,6-tri-O-
acetyl-2-deoxy-α/β-D-glucopyranosyl phosphonate
(6). Compound 5, 0.42 g (2 mmol), was added with
stirring to a solution of 0.67 g (2 mmol) of tetraacetyl-
glucosamine 3 and 1.87 mL (18.5 mmol) of triethyl-
amine in 10 mL of THF. The mixture was stirred for
3 h, 1 mL of water was added, and the mixture was
stirred for 1 h more. The mixture was then evaporated
to dryness, and the product was isolated by chromatog-
raphy on silica gel using methylene chloride–methanol
(40:1 to 5:1 with addition of 1% of triethylamine) as
eluent. Yield 0.33 g (34%), colorless oily material,
13
11.98 br.s (1H, HNEt3). C NMR spectrum, δC, ppm:
8.71 s (3C, CH3CH2N), 14.14 s [CH3(CH2)9], 20.68 s
(CH3CO), 20.78 s (CH3CO), 22.71 s (CH3CO), 22.99 s
(CH3CONH), 25.88 s [CH3CH2(CH2)8], 27.41 s
(CH3CH2CH2), 29.36 s (CH3CH2CH2CH2)), 29.47 s
[CH3(CH2)3CH2], 29.62 s [CH3(CH2)4CH2], 29.68 s
[CH2(CH2)3O], 30.88 s (CH2CH2CH2O), 31.93 s
(CH2CH2O), 45.71 s (3C, CH2N), 52.15 s (C2), 61.96 s
(C6), 66.16 s (CH2O), 68.36 s (C4), 68.65 s (C5),
71.53 s (C3), 94.16 d (C1, JCP = 5.42 Hz), 169.48 s
(CH3CO), 170.84 s (CH3CO), 171.02 s (CH3CO),
182.24 s (CH3CO). 31P NMR spectrum (100.6 MHz,
CDCl3): δP –2.27 ppm (PO4–). Mass spectrum:
m/z 566.4 (Irel 100%) [M – Et3N]–. C24H41NO12P.
Calculated: M – Et3N 566.2. Found, %: C 54.09;
H 8.78; N 4.21; O 28.37; P 4.55. C30H57N2O12P. Cal-
culated, %: C 53.88; H 8.59; N 4.19; O 28.71; P 4.63.
[α]D20 = 56.7 (c = 0.8, CHCl3). H NMR spectrum, δ,
1
ppm: 1.36 t (9H, CH3CH2N, J = 7.30 Hz); 1.97 s (3H),
2.01 s (6H), and 2.07 s (3H) (CH3CO); 3.08 q (6H,
CH3CH2N, J = 7.30 Hz), 4.09–4.27 m (3H, 2-H, 6-H),
4.36–4.47 m (1H, 5-H), 5.17 t (1H, 4-H, J = 9.62 Hz),
5.30 t (1H, 3-H, J = 9.92 Hz), 5.61–5.69 m (1H, 1-H),
6.90 d (1H, PH, J = 668.04 Hz), 6.90–7.05 m [1H,
NHC(O)CH3], 11.73 br.s (1H, HNEt3). 13C NMR spec-
trum, δC, ppm: 8.67 s (3C, CH3CH2N), 20.72 br.s (2C,
CH3CO), 20.83 s (CH3CO), 23.05 s (CH3CONH),
45.88 s (3C, CH3CH2N), 52.09 s (C2), 61.93 s (C6),
68.38 s (C4), 68.99 s (C3), 71.21 s (C5), 93.05 d (C1,
JCP = 3.91 Hz); 169.55 s, 170.15 s, 171.19 s, 171.27 s
(CH3CO). 31P NMR spectrum: δP 1.93 ppm (PH).
Found, %: C 47.05; H 7.49; N 5.53; O 33.95; P 5.98.
C20H37N2O11P. Calculated, %: C 46.87; H 7.28; N 5.47;
O 34.34; P 6.04.
The antimicrobial activity of glycolipid 8 was
evaluated by the serial dilution method on liquid
nutrient media according to the procedures described
in [17, 18]; the minimum inhibitory concentrations
were determined. The test cultures were gram positive
bacteria S. aureus ATCC 209p and B. cereus ATCC
8035, gram negative bacteria E. coli CDC F-50 and
P. aeruginosa ATCC 9027, and fungi A. niger BKMF-
1119, T. mentagrophytes var. gypseum 1773, and
C. albicans 855–653.
Triethylammonium 2-acetamido-3,4,6-triacetyl-
2-deoxy-α-D-glucopyranosyl decyl phosphate (8).
A solution of 0.37 g (0.72 mmol) of phosphonate 6
in 10 mL of pyridine was cooled to –20°C, 0.2 g
(1.26 mmol) of decan-1-ol was added with stirring, and
a solution of 0.3 mL (2.5 mmol) of pivaloyl chloride in
5 mL of pyridine was then added. The mixture was
stirred for 1 h, 1 mL of water and 0.18 g (0.71 mmol)
of iodine were added, the mixture was stirred for 2 h,
and a 1 M solution of Na2S2O3 was added dropwise
until the iodine color disappeared. The light yellow
mixture was evaporated to dryness, and the product
was isolated from the residue by chromatography on
silica gel using methylene chloride–methanol (40:1 to
5 :1 with addition of 1 vol % of triethylamine) as
eluent. Yield 0.11 g (23%), colorless oily material,
[α]D20 = 26.6 (c = 0.642, MeOH). 1H NMR spectrum, δ,
ppm: 0.86 t [3H, CH3(CH2)9, J = 7.0 Hz], 1.18 s (4H,
CH3CH2CH2), 1.21–1.29 m [12H, [(CH2)6CH2O],
The antitubercular activity of glycolipid 8 against
Mycobacterium tuberculosis H37Rv (MBT) laboratory
strain was evaluated by the vertical diffusion method
on Novaya solid nutrient medium. The medium was
placed in 5-mL test tubes which were inoculated with
0.1 mL of a suspension of mycobacteria diluted to
a turbidity of 10 GKI units; the test tubes were in-
cubated for 24 h to grow MBT. The test tubes were
set vertically, and 0.3 mL of a solution of 8 in DMSO
with a concentration of 12.5, 6.2, 3.1, 1.5, 0.7, 0.35, or
0.1 μg/mL was added dropwise to each test tube. The
test tubes were then placed in a thermostat and
incubated for 10 days at 37°C under sterile conditions.
The growth of MBT was evaluated according to
a standard procedure, according to which an inhibition
zone of larger than 10 mm indicates tuberculostatic
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GARIFULLIN et al.
1336
8. Riley, J.G., Xu, C., and Brockhausen, I., Carbohydr.
Res., 2010, vol. 345, p. 586.
9. Dumbre, S., Derouaux, A., Lescrinier, E., Piette, A.,
Joris, B., Terrak, M., and Herdewijn, P., J. Am. Chem.
Soc., 2012, vol. 134, p. 9343.
10. Vinnikova, A.N., Torgov, V.I., Utkina, N.S., Vese-
lovsky, V.V., Druzhinina, T.N., Wang, S., Brock-
hausen, I., and Danilov, L.L., Russ. J. Bioorg. Chem.,
2015, vol. 41, p. 105.
activity. The MBT growth inhibition zone (mm) is
proportional to the tuberculostatic activity. An inhibi-
tion zone of 100 mm and larger is regarded as the
complete inhibition. The antitubercular drug isoniazid
(MIC 0.1 μg/mL) was used as control.
The antitubercular activity of glycolipid 8 was
studied under financial support by the Russian Science
Foundation (project no. 14-50-00014).
11. Chen, C., Liu, B., Xu, Y., Utkina, N., Zhou, D., Dani-
lov, L., Torgov, V., Veselovsky, V., and Feng, L.,
Carbohydr. Res., 2016, vol. 430, p. 36.
REFERENCES
1. Izmest’ev, E.S., Andreeva, O.V., Sharipova, R.R., Krav-
chenko, M.A., Garifullin, B.F., Strobykina, I.Yu., Kata-
ev, V.E., and Mironov, V.F., Russ. J. Org. Chem., 2017,
vol. 53, p. 51.
2. Garifullin, B.F., Strobykina, I.Yu., Sharipova, R.R., Krav-
chenko, M.A., and Kataev, V.E., Macroheterocycles,
2016, vol. 9, p. 320.
3. Garifullin, B.F., Strobykina, I.Yu., Sharipova, R.R., Krav-
chenko, M.A., Andreeva, O.V., Bazanova, O.B., and
Kataev, V.E., Carbohydr. Res., 2016, vol. 431, p. 15.
12. Donald, P.R., Tuberculosis, 2010, vol. 90, p. 279.
13. Coxon, B., Carbohydr. Res., 2005, vol. 340, p. 1714.
14. Cao, Z., Qu, Y., Zhou, J., Liu, W., and Yao, G.,
J. Carbohydr. Chem., 2015, vol. 34, p. 28.
15. Gorityala, B.K., Lu, Z., Leow, M.L., Ma, J., and
Liu, X.-W., J. Am. Chem. Soc., 2012, vol. 134,
p. 15229.
16. Donahue, M.G. and Johnston, J.N., Bioorg. Med. Chem.
Lett., 2006, vol. 16, p. 5602.
17. National Committee for Clinical Laboratory Standards.
Methods for dilution antimicrobial susceptibility. Tests
for bacteria that grow aerobically – Sixth edition:
Approved standard. M7-A5, NCCLS, Wayne, PA, USA.
2000.
4. Young, R.W., J. Am. Chem. Soc., 1952, vol. 74, p. 1672.
5. Beletskaya, I.P., Karlstedt, N.B., Nifant’ev, E.E., Khoda-
rev, D.V., Kukhareva, T.S., Nikolaev, A.V., and
Ross, A.J., Russ. J. Org. Chem., 2006, vol. 42, p. 1780.
6. Montoya-Peleaz, P.J., Riley, J.G., Szarek, W.A., Val-
vano, M.A., Schutzbach, J.S., and Brockhausen, I.,
Bioorg. Med. Chem. Lett., 2005, vol. 15, p. 1205.
18. National Committee for Clinical Laboratory Standards.
Reference method for broth dilution antifungal suscep-
tibility testing of conidium-forming filamentous fungi:
Proposed standard. M38-P, NCCLS, Wayne, PA, USA.
1998.
7. Brockhausen, I., Larsson, E.A., and Hindsgaul, O.,
Bioorg. Med. Chem. Lett., 2008, vol. 18, p. 807.
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