Allobetulin N-acetylglucosaminide. Synthesis and antimicrobial activity

Article abstract of DOI:10.1134/S1070363217040399

Allobetulin N-acetylglucosaminide has been synthesized and shown to exhibit selective bacteriostatic activity against Staphylococcus aureus ATCC 209p with a minimum inhibitory concentration of 15.5 μg/mL.

Full text of DOI:10.1134/S1070363217040399

ISSN 1070-3632, Russian Journal of General Chemistry, 2017, Vol. 87, No. 4, pp. 890–893. © Pleiades Publishing, Ltd., 2017.
Original Russian Text © I.Yu. Strobykina, B.F. Garifullin, A.S. Strobykina, A.D. Voloshina, R.R. Sharipova, V.E. Kataev, 2017, published in Zhurnal
Obshchei Khimii, 2017, Vol. 87, No. 4, pp. 694–697.
LETTERS
TO THE EDITOR
Allobetulin N-Acetylglucosaminide.
Synthesis and Antimicrobial Activity
I. Yu. Strobykina, B. F. Garifullin, A. S. Strobykina, A. D. Voloshina,
R. R. Sharipova, and V. E. Kataev*
Arbuzov Institute of Organic and Physical Chemistry, Kazan Scientific Center, Russian Academy of Sciences,
ul. Arbuzova 8, Kazan, Tatarstan, 420088 Russia
*e-mail: kataev@iopc.ru
Received January 26, 2017
Abstract—Allobetulin N-acetylglucosaminide has been synthesized and shown to exhibit selective
bacteriostatic activity against Staphylococcus aureus ATCC 209p with a minimum inhibitory concentration of
15.5 μg/mL.
Keywords: triterpenoids, betulin, allobetulin, glucosamine
DOI: 10.1134/S1070363217040399
The natural triterpenoid betulin (1) is known to
undergo Wagner–Meerwein rearrangement to allo-
betulin (2) (Scheme 1) by the action of silica- or
alumina-supported inorganic acids and sulfonic acids,
as well as Lewis acids (see [1] and references therein).
Further modification of allobetulin (2) afforded its
derivatives through the hydroxy group [2], products of
Wagner–Meerwein rearrangement of the A ring [3, 4],
Scheme 1.
.....
H+
+
H
H
OH
OH
HO
1
+
O
H
H
OH
_H+
H
HO
2
890

ALLOBETULIN N-ACETYLGLUCOSAMINIDE
891
Scheme 2.
OH
OH
OAc
NaHCO3
TrocCl
H2O
OH
OH
OAc
O
O
O
Ac2O-Py
Cl_
+
HO
HO
NHTroc
AcO
NH3
NHTroc
OH
OH
OAc
3
4
5
33% HBr, AcOH,
CH2Cl2
29
30
OAc
19
18
H
Br
O
OH
H
28
NHTroc
AcO
ZnCl2,
CH2Cl2
OAc
HO
6
1
29
21
30
19
O
H
18
OAc
17
11
6'
5'
13
25
26
H
AcO
AcO
28
1
9
4'
3'
O
15
1'
7
27
3
5
2'
O
24
23
RHN
7 R = Troc
8 R = H
Zn, AcOH
Ac2O, Et3N
9 R = Ac
4,5-seco derivatives [3], and glycosides where the carbo-
hydrate moiety was represented by glucose, rhamnose,
arabinose, galactose, mannose, and xylose residues [1].
betulin (1) in methylene chloride in the presence of
ZnCl2 (by analogy with [6]) to give glycoside 7. The
formation of 7 was confirmed by the presence of ion
peaks with m/z 926.7 [M + Na]+ and 942.7 [M + K]+
(C45H68Cl3NO11, M 903.4) in its mass spectrum. The
1H NMR spectrum of 7 lacked characteristic signals
assignable to methylidene protons on C29 (δ 4.58 ppm,
d.d and 4.48 ppm, d in the spectrum of 1), whereas the
singlet of the C30H3 protons (δ 1.68 ppm in the
spectrum of 1) appeared in a stronger field. The 19-H
signal of 1 (δ 2.34–2.43 ppm, m) transformed to a
singlet at δ 3.52 ppm in the spectrum of 7. Methylene
protons on C28 in the spectrum of 7 resonated as two
doublets at δ 3.43 (3J = 7.7 Hz) and 3.76 ppm (3J =
We have synthesized previously unknown allobetulin
glycoside 9 (allobetulin N-acetylglucosaminide) with a
3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-D-glucopyranose
residue as glycone (Scheme 2). The synthesis was
carried out for the first time in one preparative step
directly from betulin (1). By analogy with the
procedure described in [5], commercial glycosamine
hydrochloride (3) was converted to 3,4,6-tri-O-acetyl-
2-deoxy-2-[(2,2,2-trichloroethoxy)carbonylamino]-α-
D-glucopyranosyl bromide (6). The latter reacted with
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 87 No. 4 2017

892
STROBYKINA et al.
7.7 Hz). These data are consistent with the allobetulin
structure of 7 [1, 4]. The anomeric proton signal of
glycoside 7 was a doublet at δ 5.03 ppm with a vicinal
coupling constant of 3.6 Hz, which indicated α-
orientation of the glycosidic bond.
ZnCl2 was added, and a solution of 0.3 g (0.68 mmol)
of betulin (1) in 20 mL of chloroform was then added
with stirring under argon. The mixture was stirred for
9 h at 20°C and diluted with chloroform, and the
precipitate was filtered off and washed with a 5%
solution of NaHCO3. The filtrate was dried over
MgSO4, the solvent was distilled off under reduced
pressure, and the residue was subjected to dry column
chromatography using hexane–ethyl acetate (20 : 1 to
1:1) as eluent. Yield 0.14 g (23%), white amorphous
powder, mp 110–115°C, [α]D20 = 73.3° (c = 1.67,
The 2,2,2-trichloroethoxycarbonyl (Troc) protecting
group was then removed by treatment with zinc dust in
glacial acetic acid according to [6]. Unstable amine 8
thus formed was acylated as described in [7] to obtain
N-acetylglucosaminide 9 which showed ion peaks with
m/z 794.7 [M + Na]+ and 810.7 [M + K]+ in the mass
spectrum (C44H69NO10, M 771.5). The transformation
of 7 to 9 was accompanied by disappearance from the
1H NMR spectrum of the multiplet signal corres-
ponding to the methylene protons of the Troc group,
appearance of a singlet at δ 1.93 ppm due to acetyl
protons of the acetamide fragment, and low-field shift
of the NH proton signal which was observed as a
doublet at δ 5.55 ppm (3J 9.4 Hz). The anomeric
proton of 9 resonated as a doublet at δ 4.99 ppm with a
vicinal coupling constant of 3.9 Hz, corresponding to
α-glycosidic bond.
1
CH2Cl2). H NMR spectrum (CDCl3), δ, ppm: 0.80–
1.78 m (23H, oleanane skeleton), 0.70 d (1H, 5-H, J =
9.6 Hz); 0.78 s, 0.85 s, 0.86 s, 0.90 s, 0.92 s, 0.98 s,
and 0.99 s (3H each, C23H3, C24H3, C25H3, C26H3,
C27H3, C29H3, C30H3); 2.01 s, 2.03 s, and 2.08 s [3H
each, CH3C(O)]; 3.20 d.d (1H, 3-H, J = 11.7, 4.4 Hz),
3.43 d (1H, 28-H, J = 7.7 Hz), 3.52 s (1H, 19-H), 3.76
d (1H, 28-H, J = 7.7 Hz), 4.01–4.07 t.d (1H, 2′-H, J =
10.2, 3.7 Hz), 4.08–4.15 m (2H, 5′-H, 6′-H), 4.22 d.d
(1H, 6′-H, J = 12.3, 4.5 Hz), 4.65–4.77 m (2H,
OCH2CCl3), 5.03 d (1H, 1′-H, J = 3.6 Hz), 5.05–5.13
13
m (2H, 4′-H, NH), 5.18 t (1H, 3′-H, J = 10.2 Hz). C
Glycoside 9 was tested for antibacterial and anti-
fungal activities against gram-positive (Staphylococcus
aureus ATCC 209p, Bacillus cereus ATCC 8035) and
gram-negative bacteria (Escherichia coli CDCF-50,
Pseudomonas aeruginosa ATCC 9027) and fungi
(Aspergillus niger BKMF-1119, Trichophyton menta-
grophytes var. Gypseum 1773, Candida albicans 855-
653). Compound 9 showed selective bacteriostatic
effect against S. aureus with a minimum inhibitory con-
centration (MIC) of 15.5 μg/mL and thus turned out to
be superior to the antibiotic chloramphenicol (MIC
62.5 μg/mL). However, glycoside 9 was inactive
against the other bacterial and fungal strains up to a
concentration of 500 μg/mL and higher. It should be
noted that our result is the first mention of anti-
microbial activity of allobetulin derivatives.
NMR spectrum (CDCl3), δC, ppm: 13.4, 15.7, 16.4,
16.5, 18.2, 20.6, 20.7, 21.0, 22.9, 24.5, 26.2, 26.39,
26.42, 28.6, 28.8, 32.7, 33.9, 34.1, 36.2, 36.7, 37.1,
38.5, 38.6, 40.6, 40.7, 41.5, 46.8, 51.1, 54.2, 55.9,
62.0, 68.32, 68.35, 70.9, 71.2, 74.5, 85.5, 87.9, 94.5,
95.4, 154.2, 169.4, 170.6 (2C), 170.9. Mass spectrum
(ESI), m/z: 926.7 [M + Na]+, 942.7 [M + K]+. Found,
%: C 59.66; H 7.62. C45H68Cl3NO11. Calculated, %: C
59.70; H 7.57.
3-O-(3,4,6-Tri-O-acetyl-2-amino-2-deoxy-α-D-
glucopyranosyl)allobetulin (8). Activated zinc dust,
0.5 g (7.6 mmol), was added under argon to a solution
of 0.1 g (0.1 mmol) of glycoside 7 in 4 mL of glacial
acetic acid. The mixture was stirred for 3 h at 20°C and
concentrated under reduced pressure, the residue was
diluted with 10 mL of methylene chloride, and the
solution was washed with a 5% solution of NaHCO3
and with water, dried over MgSO4, and concentrated
under reduced pressure. Yield 0.08 g (93%). The
product was used in the next step without additional
purification.
Betulin (1) was kindly provided by N.I. Medvedeva
(Ufa Institute of Chemistry, Russian Academy of
Sciences). 3,4,6-Tri-O-acetyl-2-deoxy-2-[(2,2,2-trichloro-
ethoxy)carbonylamino]-α-D-glucopyranosyl bromide
(6) was synthesized according to [5]. Glucosamine
hydrochloride was commercial product (Acros Organics,
Belgium).
3-O-(3,4,6-Tri-O-acetyl-2-acetamido-2-deoxy-α-
D-glucopyranosyl)allobetulin (9). Amine 8, 1 mmol,
was dissolved in 5 mL of methylene chloride, 3 mmol
of triethylamine and 10 mmol of acetic anhydride were
added under argon, and the mixture was stirred for 2 h
at 20°C. It was then diluted with 10 mL of methylene
3-O-[3,4,6-Tri-O-acetyl-2-deoxy-2-(2,2,2-trichloro-
ethoxycarbonylamino)-α-D-glucopyranosyl]allobetulin
(7). Bromide 6, 0.92 g (1.69 mmol), was dissolved in
10 mL of methylene chloride, 0.23 g (1.69 mmol) of
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 87 No. 4 2017

ALLOBETULIN N-ACETYLGLUCOSAMINIDE
893
chloride, washed with a saturated solution of NaHCO3,
1 M aqueous HCl, and water, dried over Na2SO4, and
concentrated under reduced pressure. The residue was
purified by chromatography using hexane–ethyl
acetate (5:1 to 1:1) as eluent. Yield 0.06 g (71%),
The antibacterial and antifungal activities of com-
pound 9 were assessed by the serial dilution method
using liquid nutrition media according to the
procedures described in [8, 9]; minimum inhibitory
concentration (MIC) ensuring 99% growth inhibition
of test cultures was determined.
white amorphous powder, mp 126–128°C, [α]D20
=
83.5° (c = 1.0, CH2Cl2). 1H NMR spectrum (CDCl3), δ,
ppm: 0.80–1.80 m (24H, oleanane skeleton), 0.70 d
(1H, 5-H, J = 9.8 Hz); 0.78 s, 0.84 s, 0.86 s, 0.90 s,
0.92 s, 0.97 s, and 0.99 s (3H each, C23H3, C24H3,
C25H3, C26H3, C27H3, C29H3, C30H3); 1.93 s, 2.01 s,
2.02 s, and 2.07 s [3H each, CH3C(O)]; 3.19 d.d (1H,
3-H, J = 11.8, 4.4 Hz), 3.43 d (1H, 28-H, J = 7.8 Hz),
3.51 s (1H, 19-H), 3.76 d (1H, 28-H, J = 7.8 Hz), 4.06–
4.12 m (2H, 5′-H, 6′-H), 4.20 d. d (1H, 6′-H, J = 12.8,
5.1 Hz), 4.27–4.34 m (1H, 2′-H), 4.99 d (1H, 1′-H, J =
3.9 Hz), 5.10 t (1H, 4′-H, J = 9.4 Hz), 5.14 t (1H, 3′-H,
J = 9.9 Hz). 5.55 d (1H, NH, J = 9.4 Hz). 13C NMR
spectrum (CDCl3), δC, ppm: 13.4, 15.7, 16.5 (2C),
18.2, 20.6, 20.70, 20.72, 21.0, 22.7, 23.2, 24.5, 26.2,
26.37, 26.41, 28.5, 28.8, 32.7, 33.9, 34.1, 36.2, 36.7,
37.1, 38.4, 38.6, 40.6, 40.7, 41.4, 46.8, 51.0, 52.2,
55.8, 62.1, 68.2, 68.3, 71.2, 71.3, 85.0, 87.9, 94.4,
169.3, 169.8, 170.6, 171.4. Mass spectrum (MALDI),
m/z: 794.7 [M + Na]+, 810.7 [M + K]+. Found, %: C
68.38; H 9.09. C44H69NO10. Calculated, %: C 68.45; N
9.01.
ACKNOWLEDGMENTS
This study was performed under financial support
by the Russian Science Foundation (project no. 14-50-
00014).
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