Synthesis and Antimicrobial Activity of Dihydrobetulin N-Acetylglucosaminides

Article abstract of DOI:10.1007/s10600-017-2210-1

Dihydrobetulin N-acetylglucosaminides were synthesized for the first time. A study of their antimicrobial activity against a standard set of Gram-positive and Gram-negative bacteria and fungi showed highly selective bacteriostatic activity for glycosides 9 and 13 against Staphylococcus aureus ATCC 209p.

Full text of DOI:10.1007/s10600-017-2210-1

DOI 10.1007/s10600-017-2210-1
Chemistry of Natural Compounds, Vol. 53, No. 6, November, 2017
SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF
DIHYDROBETULIN N-ACETYLGLUCOSAMINIDES
I. Yu. Strobykina, B. F. Garifullin, R. R. Sharipova,
A. D. Voloshina, A. S. Strobykina,
*
A. B. Dobrynin, and V. E. Kataev
Dihydrobetulin N-acetylglucosaminides were synthesized for the first time. A study of their antimicrobial
activity against a standard set of Gram-positive and Gram-negative bacteria and fungi showed highly selective
bacteriostatic activity for glycosides 9 and 13 against Staphylococcus aureus ATCC 209p.
Keywords: betulin, dihydrobetulin, D-glucosamine, antimicrobial activity.
Many betulin glycosides have now been synthesized and studied [1–9]. The used glycones include D-glucopyranose
[1–4, 7–9], D-galactopyranose [4, 8, 9], D-mannopyranose [4, 8, 9], D-rhamnopyranose [4, 8], and D-arabinopyranose [4, 5, 8].
Many glycosides exhibited cytostatic activity against various human cancer cell lines [4, 6–8]. Several glycosides were
investigated for hypocholesterolemic [2] and growth-regulating activity [3]. The betulin glycosides were not studied for other
types of biological activity.
In continuation of a series of publications on the synthesis of glucosamine conjugates with natural terpenoids
[
10, 11], we turned our attention to the triterpenoid betulin (1). The double bond of 1 was hydrogenated over Pd by the
literature method [13] to prevent a Wagner–Meerwein rearrangement of 1 to form allobetulin [12] during a ZnCl -activated
2
Koenigs–Knorr reaction of ꢀ-D-glucopyranosyl bromide with glycosyl acceptors [10, 11]. Then, the hydroxyl of dihydrobetulin,
which was obtained in 67% yield, was acylated as before [13]. Next, the C-28 acyl protecting group was selectively removed
from dihydrobetulin diacetate (2) by magnesium methoxide in THF according to the literature [14] to produce in 61% yield
3
ꢁ-O-acetyldihydrobetulin (3).
29
30
20
19 21
H
H
H
18
1
1
OAc
25
26
OH
OH
H
1
H
H
28
i, ii
iii
9
15
3
7
27
HO
5
AcO
AcO
1
2
3
24
23
29
OAc
O
Br
30
26
20
18
H
NHR
AcO
NHTroc
2
5
O
OAc
H
1's
5: R = Troc
6: R = H
4
28
3's
OAc
v
1
9
O
1
5
iv
5
's
27
OAc
AcO
5
6's
vi
OAc
24
23
5 - 7
7: R = Ac
i. H , Pd–C, THF, CH OH; ii. Ac O – Py; iii. Mg(OCH ) , CH OH, THF; iv. ZnCl , CH Cl ; v. Zn, AcOH; vi. Ac O, Et N, CH Cl
2
2
3
2
3 2
3
2
2
2
2
3
2
A. E. Arbuzov Institute of Organic and Physical Chemistry, Kazan Scientific Center, Russian Academy of Sciences,
Arbuzova St., Kazan, 420088, Russian Federation, fax: (843) 273 22 53, e-mail: kataev@iopc.ru. Translated from Khimiya
Prirodnykh Soedinenii, No. 6, November–December, 2017, pp. 936–940. Original article submitted February 7, 2017.
8
0
009-3130/17/5306-1101 ©2017 Springer Science+Business Media New York
1101

C30
C20
C25
C1
O3
C28
C17
C26
C13
C19
C11
C10
C24
C4
C9
C6
C18
C29
C22
O1
C8
C14
C5
C3
C23
C15 C16
C7
C27
O2
Fig. 1. X-ray molecular structure of 3ꢁ-O-acetyldihydrobetulin 3
H atoms and molecule DMSO not shown).
(
An X-ray crystal structure analysis (XSA) of this triterpenoid was performed because its molecular structure had not
been reported (Fig. 1). In the next step, 3 was glycosylated by 3,4,6-tri-O-acetyl-2-deoxy-2-(2ꢂ,2ꢂ,2ꢂ-
trichloroethoxycarbonylamino)-ꢀ-D-glucopyranosyl bromide (4), which was prepared from commercially available glucosamine
hydrochloride as before [15].
The ZnCl -activated Koenigs–Knorr reaction of glycosyl donor 4 and glycosyl acceptor 3 was carried out as before
2
[
[
11]. Glycoside 5 was obtained in 35% yield. Apeak in the electrospray-ionization (ESI) mass spectrum (MS) at m/z 970.5 for
+
M + Na] (C H Cl NO , MM 947.41) indicated that it had formed. The anomeric proton of glycoside 7 resonated in
4
7
72
3
12
the PMR spectrum as a doublet at 4.84 ppm with vicinal SSCC 3.6 Hz. This indicated that the glycoside bond had
the ꢀ-orientation. Next, the 2,2,2-trichloroethoxycarbonyl (Troc) protecting group was removed using Zn dust in glacial
AcOH as before [10]. The resulting amine 6 was acylated without further purification as before [11]. Glycoside 7 was
+
+
obtained in 79% yield. Peaks in the MALDI MS at m/z 838.5 for [M + Na] and 854.4 for [M + K] (C H NO ,
4
6
73
11
MM 815.52) indicated that it had formed. The conversion from 5 to 7 was accompanied by the disappearance in the PMR
spectrum of a multiplet at 4.66–4.76 ppm for the Troc methylene protons, the appearance of a singlet at 1.93 ppm for
the acetamide acyl group, and a low-field shift of the resonance for the amide proton that appeared as a doublet at 5.60 ppm
3
(
J = 9.3 Hz). The anomeric proton of 7 resonated as a doublet at 4.80 ppm with vicinal SSCC 3.7 Hz. This indicated that
the ꢀ-orientation of the glycoside bond was retained.
In the next step, the reaction of 3ꢁ-O-acetyldihydrobetulin (3) with an excess of succinic anhydride in Py produced in
3
8% yield 3ꢁ-O-acetyl-28-O-succinyldihydrobetulin (8), the formation of which was indicated by a peak in the ESI-MS at
+
m/z 609.6 for [M + Na] (C H O , MM 586.42). Then, glycosyl donor 4 underwent a reaction catalyzed by
3
6 58 6
tetrabutylammonium bromide (TBAB) with the carboxylic acid of 8 to give in 38% yield glycoside 9, the formation of which
+
+
was indicated by peaks in the MALDI MS at m/z 1070.8 for [M + Na] and 1086.8 for [M + K] (C H Cl NO ,
5
1
76
3
15
MM 1047.4). The anomeric proton of glycoside 9 resonated in the PMR spectrum as a doublet at 5.76 ppm with vicinal SSCC
.7 Hz, indicating that the resulting glycoside bond had the ꢁ-orientation. This agreed with the literature on the course of
8
the Koenigs–Knorr reaction between glucopyranoside bromide and a carboxyl-containing glycosyl acceptor under
phase-transfer conditions with TBAB catalysis and K CO [16, 17]. Then, the Troc protection on 9 was removed using Zn
2
3
dust in glacial AcOH as before [10]. The resulting amine 10 was acylated without further purification according to
+
the literature [11] for afford in 43% yield glycoside 11. Peaks in the MALDI MS at m/z 938.6 for [M + Na] and 954.6 for
+
[
M + K] (C H NO , MM 915.53) indicated that it had formed. The conversion from 9 to 11 was accompanied by
5
0
77
14
3
3
the disappearance in the PMR spectrum of doublets at 4.67 ppm ( J = 12.1 Hz) and 4.80 ppm ( J = 12.1 Hz) corresponding to
Troc methylene proton resonances and the appearance of a singlet at 1.94 ppm that corresponded to the resonance of
the acetamide acyl protons. Also, a singlet belonging to the amide proton of 11 underwent a low-field shift and was observed
3
as a doublet of doublets at 5.51 ppm ( J = 9.0 Hz). The anomeric proton of 11 resonated as a doublet at 5.72 ppm with vicinal
SSCC 9.0 Hz, indicating that the glycoside bond had the ꢁ-orientation.
H
O
H
O
NHR
O
O
H
OH
H
O
i
4
ii
3
O
O
OAc
O
AcO
AcO
OAc
OAc
8
9 - 11
iii
iv
9
: R = Troc
10: R = H
11: R = Ac
i. Succinic anhydride, DMAP, Py; ii. TBAB, K CO , H O, CH Cl ; iii. Zn, AcOH; iv. Ac O, Et N, CH Cl
2
2
3
2
2
2
2
3
2
1
102

Antimicrobial activity of synthesized glycosides 3, 7, 8, 11, and 12 [18] was studied against Gram-positive bacteria
Staphylococcus aureus ATCC 209p and Bacillus cereus ATCC 8035; Gram-negative bacteria Escherichia coli CDCF-50 and
Pseudomonas aeruginosa ATCC 9027; and fungi Aspergillus niger BKMF-1119, Trichophyton mentagrophytes var. Gypseum
1
773, and Candida albicans 855–653.
Glycosides 7 and 11 inhibited the growth of only S. aureus with the activity of 11 (MIC = 15.6 ꢃg/mL) exceeding by
four times and that of glycoside 7 (MIC = 7.8 ꢃg/mL) by eight times that of the antibiotic chloramphenicol (MIC = 62.5 ꢃg/mL).
Betulin derivatives 3 and 8 exhibited antibacterial activity only if they were glycosylated. They themselves and 1,3,4,6-tetra-
O-acetyl-2-deoxy-2-acetamido-D-glucopyranose (12), which was the aglycone of 7 and 11 and was synthesized by analogy
with the literature [18], were inactive.
EXPERIMENTAL
The XSA of 3 was performed at the Spectro-Analytical Joint Use Center of the RFFI (JUC SAC) on a Bruker Smart
°
APEX II CCD diffractometer (ꢄ Mo Kꢀ = 0.71073 A, ꢅ-scanning, 2ꢆ < 52°, R = 0.048). A total of 12,707 reflections were
int
measured, of which 6,356 were independent. The number of observed reflections with I > 2ꢇ(I) was 4,352. Absorption
corrections were made using the SADABS program [19]. The structure was solved by direct methods using the SIR program
[
20] and refined by full-matrix least-squares methods using the SHELXL97 program [21]. Hydroxyl H atoms were found in
an electron-density difference synthesis and refined isotropically. The coordinates of other H atoms were calculated geometrically
and refined using a rider model. All calculations used the WinGX [22] and APEX2 programs [23]. The final agreement
parameters R = 0.1344, wR = 0.3975, GOF = 1.41. The number of refined parameters was 364 with Flack parameter 0.4(4).
2
°
Crystals of 3 (C H O 4C H OS, MM 564.89) were monoclinic at 296K with a = 13.291(8), b = 7.257(5), c = 17.757(11) A ,
32
54
3
2 6
°
3
3
–1
ꢁ
= 104.484(9)°, V = 1658.3(18) A , Z = 2, space group P2 , d
= 1.131 g/cm , ꢃ = 0.132 mm , and F(000) = 624.
1
calcd
A complete dataset for the structure of 3 was deposited in the Cambridge Structural Database (registration No. CCDC 1530699).
1
3
PMR and C NMR spectra were recorded on Avance-400 and Avance-600 spectrometers (Bruker, Germany) at
1
13
operating frequencies 400 and 600 MHz for H and 100 MHz for C using the solvent [CD(H)Cl ] for calibration. Resonances
3
in the spectra were attributed according to the literature [13, 15, 24]. MALDI mass spectra were obtained on an UltraFlex III
TOF/TOF time-of-flight mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) in linear mode. The Nd YAG laser
had ꢄ = 355 nm. Data were processed using the FlexAnalysis 3.0 program (Bruker Daltonik GmbH, Bremen, Germany).
Measurements were made in positive-ion mode in the range m/z 200–6000. The matrix was 2,5-dihydroxybenzoic acid (DHB)
–
3
and p-nitroaniline (p-NA). Samples were dissolved in CH Cl at a concentration of 10 mg/mL. A solution of the matrix in
2
2
MeCN at a concentration of 10 mg/mL was prepared. Samples were deposited by the dried-drop method. The matrix solution
was applied using a 0.5-ꢃL pipette onto an Anchor Chip target (Bruker Daltonik GmbH, Bremen, Germany). Analyte solution
(
0.5 ꢃL) was deposited on the target after the solvent evaporated. Electrospray ionization (ESI) mass spectra were obtained on
an AmazonX mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany). ESI-MS were recorded in positive-ion mode in
the range m/z 100–2800 at capillary potential 4500 V. The drying gas (N ) was at 250°C with flow rate 8 L/min. The eluent
2
–
6
was MeOH–H O (70:30) at flow rate 0.2 mL/min. Samples were diluted with MeOH to a concentration of 10 g/L.
2
The injected sample volume was 20 ꢃL. Data were processed using the DataAnalysis 4.0 program (Bruker Daltonik GmbH,
Bremen, Germany). The course of reactions and purity of compounds were monitored by TLC on Sorbfil plates (OOO Imid,
Krasnodar, Russia). Compounds were detected by treating plates with H SO solution (5%) followed by heating to 120°C.
2
4
Specific rotation was measured on a Model 341 polarimeter (PerkinElmer, USA) in a thermostatted cell at 20°C using ꢄ = 589 nm.
Melting points were determined on a Boetius apparatus.
Betulin (1) was supplied by Cand. N. I. Medvedeva (UIC, RAS). 3ꢁ-O-Acetyl-28-O-acetyldihydrobetulin (2),
3
ꢁ-O-acetyldihydrobetulin (3), and 3,4,6-tri-O-acetyl-2-deoxy-2-(2ꢂ,2ꢂ,2ꢂ-trichloroethoxycarbonylamino)-ꢀ-D-glucopyranosyl
bromide (4) were prepared in analogy with the literature [13–15]. Constants and spectral characteristics of 2, 3, and 4 agreed
with the literature [13, 15]. Commercial glucosamine hydrochloride and ZnCl (Acros, Belgium) were used.
2
3
-O-Acetyl-28-O-[3ꢂ,4ꢂ,6ꢂ-tri-O-acetyl-2ꢂ-deoxy-2ꢂ-(2ꢈ,2ꢈ,2ꢈ-trichloroethoxycarbonylamino)-ꢀ-D-gluco-
pyranosyl]-dihydrobetulin (5). A solution of 3 (0.15 g, 0.31 mmol) and glycoside donor 4 (0.15 g, 0.37 mmol) in anhydrous
CH Cl (5 mL) under Ar was treated with ZnCl (0.05 g, 0.37 mmol). The reaction mixture was stirred in the dark for 28 h at
2
2
2
2
0°C; diluted with CH Cl ; washed successively with NaHCO solution (5%), H O, saturated NaCl solution, and water; dried
2 2 3 2
over CaCl ; and concentrated at reduced pressure. Flash chromatography of the residue over a dry column (silica gel,
2
1
103

2
0
hexane–EtOAc, from 20:1 to 5:1) produced 5 (0.10 g, 35%) as a white amorphous powder, [ꢀ] +73.9ꢉ (ñ 1.2, CH Cl ).
D
2
2
1
Í NMR spectrum (400 MHz, CDCl , ꢊ, ppm, J/Hz): 0.80–1.95 (26Í, m, CH, CH ), 0.77 (3Í, d, J = 6.7, Í -29 or Í -30),
3
2
3
3
0
.83–0.86 (12H, m, 4 ÑÍ ), 0.96, 1.01 (3H each, s, ÑH ), 2.01, 2.03, 2.04, 2.10 [3Í each, s, 4 ÑÍ Ñ(Î)], 3.05 (1H, d, J = 9.5,
3 3 3
Í -28), 3.89–3.96 (2Í, d, Í-5ꢂs, Í -28), 4.01–4.09 (2Í, m, Í-6ꢂs, 2ꢂs), 4.32 (1Í, dd, J = 12.4, 3.9, Í-6ꢂs), 4.47 (1Í, dd
À
B
J = 10.5, 5.6, Í-3), 4.66–4.76 (2Í, m, ÑÍ ÑÑl ), 4.84 (1Í, d, J = 3.6, Í-1ꢂs), 5.07–5.15 (2Í, m, NH, Í-4ꢂs), 5.20 (1Í, t,
2
3
1
3
J = 10.1, Í-3ꢂs). C NMR spectrum (100 MHz, CDCl , ꢊ, ppm): 14.7, 14.9, 16.1, 16.5, 18.1, 20.5, 20.6, 20.7, 20.8, 21.2, 21.3,
3
2
6
1.9, 22.9, 23.6, 26.7, 27.0, 27.9, 29.4, 30.2, 34.2, 37.0, 37.1, 37.8, 38.3, 40.9, 42.8, 44.7, 47.1, 48.1, 49.9, 54.3, 55.3, 61.8,
+
7.6, 67.9, 68.1, 71.0, 74.6, 80.8, 80.9, 95.4, 97.9, 154.1, 169.3, 170.6, 170.9 (2 Ñ). Mass spectrum (ESI) m/z: 970.5 [M + Na]
+
(
calcd [M + Na] 970.4). C H Cl NO .
4
7
72
3
12
3
ꢁ-O-Acetyl-28-(3ꢂ,4ꢂ,6ꢂ-tri-O-acetyl-2ꢂ-deoxy-2ꢂ-amino-ꢀ-D-glucopyranosyl)-dihydrobetulin (6). A solution of
5
(0.1 g, 0.1 mmol) in glacial AcOH (4 mL) under Ar was treated with activated Zn dust (0.5 g, 7.6 mmol), stirred for 3 h at
2
0°C, and concentrated at reduced pressure. The residue was diluted with CH Cl (10 mL); washed with NaHCO solution
2
2
3
(
5%) and H O; dried over MgSO ; and concentrated at reduced pressure to afford 6 (0.08 g, 94%), which was used without
2 4
further purification.
ꢁ-O-Acetyl-28-(3ꢂ,4ꢂ,6ꢂ-tri-O-acetyl-2ꢂ-deoxy-2ꢂ-acetamido-ꢀ-D-glucopyranosyl)-dihydrobetulin (7). Asolution
of 6 (1 mmol) in CH Cl (5 mL) under Ar was treated with Et N (3 mmol) andAc O (10 mmol); stirred for 2 h at 20°C; diluted
3
2
2
3
2
with CH Cl (10 mL); washed successively with saturated NaHCO solution, HCl solution (1 M), and H O; dried over Na SO ;
2
2
3
2
2
4
and concentrated at reduced pressure. Glycoside 7 was isolated by chromatography (silica gel, hexane–EtOAc, from 5:1 to
2
0
1
1
:1) as a white amorphous powder. Yield 0.064 g (79%), [ꢀ] +44.9ꢉ (ñ 1.07, CH Cl ). Í NMR spectrum (400 MHz,
D 2 2
CDCl , ꢊ, ppm, J/Hz): 0.88–2.08 (25Í, m, CH, CH ), 0.76 (3H, d, J = 6.8, H -29 or H -30), 0.82–0.87 (12H, m, 4 CH ), 0.97,
3
2
3
3
3
1
.01 (3Í each, s, ÑÍ ), 1.93, 2.02, 2.03, 2.04, 2.10 [3Í each, s, 5 CH C(O)], 3.02 (1Í, d, J = 9.4, H -28), 3.86–3.93 (2Í, m,
3 3 À
H-5ꢂs, H -28), 4.04 (1Í, dd, J = 12.4, 2.0, Í-6ꢂs), 4.26–4.35 (2Í, m, Í-2ꢂs, 6ꢂs), 4.47 (1Í, dd, J = 10.5, 5.5, Í-3), 4.80 (1Í,
B
1
3
d, J = 3.7, Í-1ꢂs), 5.09–5.20 (2Í, m, Í-3ꢂs, 4ꢂs), 5.60 (1Í, d, J = 9.3, NH). C NMR spectrum (100 MHz, CDCl , ꢊ, ppm):
3
1
3
1
8
4.6, 14.9, 16.0, 16.1, 16.5, 18.1, 20.6, 20.7 (2 Ñ), 20.8, 21.3, 21.7, 22.9, 23.1, 23.7, 26.8, 27.0, 27.9, 29.4, 30.2, 34.3, 34.8,
7.0, 37.1, 37.8, 38.4, 40.9, 42.8, 44.7, 47.2, 48.1, 49.9, 52.3, 55.3, 61.9, 67.3, 67.9, 68.0, 71.4, 80.9, 97.9, 169.2, 169.8, 170.7,
71.0, 171.4. Mass spectrum (MALDI): m/z 838.5 [M + Na] (calcd [M + Na] 838.5), m/z 854.4 [M + K] (calcd [M + K]
+
+
+
+
54.5). C H NO .
4
6
73
11
3
ꢁ-O-Acetyl-28-O-succinyldihydrobetulin (8). A solution of 3 (0.64 g, 1.3 mmol), succinic anhydride (0.39 g,
3
.9 mmol), and DMAP (0.48 g, 3.9 mmol) in anhydrous Py (10 mL) was refluxed for 16 h, cooled, acidified with HCl solution
(
(
10%), poured into ice water, and extracted with CHCl . The organic layer was washed successively with H O, HCl solution
3
2
5%), saturated NaCl solution, and H O; dried over MgSO ; and concentrated at reduced pressure. Recrystallization from
2
4
2
0
1
MeOH produced 8 as a white amorphous powder. Yield 0.33 g (43%), [ꢀ] –10.5ꢉ (ñ 1.33, CH Cl ). Í NMR spectrum
D
2
2
(
400 MHz, CDCl , ꢊ, ppm, J/Hz): 0.80–1.90 (27Í, m, CH, CH ), 0.77 (3Í, d, J = 6.7, Í -29 or Í -30), 0.83 (3Í, d, J = 6.7,
3 2 3 3
Í -29 or Í -30), 0.84, 0.85, 0.86, 0.96, 1.04 (3Í each, s, 5 ÑÍ ), 2.04 [3Í, s, ÑÍ Ñ(Î)], 2.60–2.72 (4Í, m, ÎÑÑÍ ÑÍ ÑÎ),
3
3
3
3
2
2
1
3
3
.86 (1Í, d, J = 10.9, Í -28), 4.30 (1Í, d, J = 10.9, Í -28), 4.44–4.51 (1Í, dd, J = 10.4, 6.1, Í-3). C NMR spectrum
À B
(
100 MHz, CDCl , ꢊ, ppm): 14.6, 14.9, 16.0, 16.1, 16.5, 18.2, 20.8, 21.3, 21.6, 22.9, 23.7, 26.8, 26.9, 27.9, 28.9, 29.1, 29.4,
3
2
9.8, 31.6, 34.2, 37.0, 37.2, 37.8, 38.4, 40.9, 42.9, 44.5, 46.6, 48.2, 50.0, 55.3, 63.3, 81.0, 171.1, 172.4, 177.4. Mass spectrum
+
+
(
ESI), m/z 609.6 [M + Na] (calcd [M + Na] 609.4). C H O .
36 58 6
3
ꢁ-O-Acetyl-28-O-{succinyl-[3ꢂ,4ꢂ,6ꢂ-tri-O-acetyl-2ꢂ-deoxy-2ꢂ-(2ꢈ,2ꢈ,2ꢈ-trichloroethylcarbonylamino)-ꢁ-D-
glucopyranosyl]}-dihydrobetulin (9). A solution of 4 (0.2 g, 0.37 mmol) in anhydrous CH Cl (4 mL) under Ar was treated
2
2
with K CO (0.063 g, 0.46 mmol), H O (5 mL), and TBAB (0.025 g, 0.08 mmol); stirred vigorously, treated dropwise with a
2
3
2
solution of 8 (0.18 g, 0.31 mmol) in CH Cl (6 mL), stirred for 10 h at 20°C, and diluted with CH Cl (10 mL). The aqueous
2
2
2
2
layer was separated and extracted with CH Cl . The combined organic layers were washed with H O and dried over CaCl .
2
2
2
2
The solvent was distilled at reduced pressure. Flash chromatography of the residue over a dry column (silica gel,
2
0
hexane–EtOAc, from 10:1 to 2:1) produced 9 as a white amorphous powder. Yield 0.10 g (38%), [ꢀ] +0.8ꢉꢋ(ñ 1.33, CHCl3).
D
1
Í NMR spectrum (400 MHz, CDCl , ꢊ, ppm, J/Hz): 0.83–2.0 (26Í, m, CH, CH ), 0.76 (3H, d, J = 6.8, H -29 or H -30), 0.83
3
2
3
3
(
3Í, d, J = 6.8, H -29 or H -30), 0.84, 0.85, 0.86, 0.95, 1.04 (3Í each, s, 5 ÑÍ ), 2.04 [9H, br.s, 3 CH C(O)], 2.09 [3H, s,
3 3 3 3
CH C(O)], 2.58–2.73 (4H, m, OÑCH CH CO), 3.79–3.86 (2H, m, H-5ꢂs, H -28), 3.88–3.98 (1H, m, H-2ꢂs), 4.08–4.14 (1H,
3
2
2
À
dd, J = 12.5, 1.4, Í-6ꢂs), 4.25–4.33 (2H, m, H-6ꢂs, H -28), 4.47 (1Í, dd, J = 10.4, 6.0, Í-3), 4.67 (1H, d, J = 12.1, CH CCl ),
B
À
3
4
.80 (1H, d, J = 12.1, CH CCl ), 5.09–5.17 (2H, m, Í-4ꢂs, NH), 5.24 (1H, t, J = 9.9, H-3ꢂs), 5.76 (1Í, d, J = 8.7, H-1ꢂs).
C NMR spectrum (100 MHz, CDCl , ꢊ, ppm): 14.6, 14.8, 16.1 (2Ñ), 16.5, 18.2, 20.5, 20.6, 20.7, 20.8, 21.2, 21.6, 22.9, 23.7,
B 3
1
3
3
1
104

2
6
6.8, 26.9, 27.9, 28.8, 29.1, 29.4, 29.8, 34.2, 34.6, 37.0, 37.2, 37.8, 38.4, 40.9, 42.9, 44.5, 46.5, 48.1, 49.9, 55.3, 61.6, 63.2,
7.9, 72.0, 72.9, 74.6, 80.9, 81.0, 92.5, 95.4, 154.1, 169.3, 170.5, 170.7 (2 Ñ), 171.0, 172.1. Mass spectrum (MALDI):
+
+
+
+
m/z 1070.8 [M + Na] (calcd [M + Na] 1070.4), m/z 1086.8 [M + K] (calcd [M + K] 1086.4). C H Cl NO .
5
1
76
3
15
3
ꢁ-O-Acetyl-28-O-[succinyl-(3ꢂ,4ꢂ,6ꢂ-tri-O-acetyl-2ꢂ-deoxy-2ꢂ-amino-ꢁ-D-glucopyranosyl)]-dihydrobetulin (10).
A solution of 9 (0.1 g, 0.1 mmol) in glacial AcOH (4 mL) under Ar was treated with activated Zn dust (0.5 g, 7.6 mmol), stirred
for 3 h at 20°C, and concentrated at reduced pressure. The residue was diluted with CH Cl (10 mL), washed with NaHCO
2
2
3
solution (5%) and H O, dried over MgSO , and concentrated at reduced pressure to afford 10 (0.04 g, 42%) that was used
2
4
without further purification.
3
ꢁ-O-Acetyl-28-O-[succinyl-(3ꢂ,4ꢂ,6ꢂ-tri-O-acetyl-2ꢂ-deoxy-2ꢂ-acetamido-ꢁ-D-glucopyranosyl)]-dihydrobetulin
(
11). A solution of 10 (1 mmol) in CH Cl (5 mL) under Ar was treated with Et N (3 mmol) and Ac O (10 mmol); stirred for
2
2
3
2
2
h at 20°C; diluted with CH Cl (10 mL); washed successively with saturated NaHCO solution, HCl solution (1 M), and
2
2
3
H O; dried over Na SO , and concentrated at reduced pressure. Glycoside 11 was isolated by chromatography (silica gel,
2
2
4
2
0
1
hexane–EtOAc, from 5:1 to 1:1) as a white amorphous powder. Yield 0.016 g (43%), [ꢀ] +2.8ꢉ (ñ 0.65, CH Cl ). Í NMR
D
2
2
spectrum (600 MHz, CDCl , ꢊ, ppm, J/Hz): 0.83–1.90 (26 Í, m, dihydrobetulin core), 0.76 (3H, d, J = 7.0, H -29 or H -30),
3
3
3
0
.83 (3H, d, J = 7.0, H -29 or H -30), 0.84, 0.85, 0.86, 0.94, 1.02 (3Í each, s, 5 ÑÍ ), 1.94, 2.03, 2.037, 2.041, 2.09 [3Í each,
3
3
3
s, 5 ÑÍ Ñ(Î)], 2.58–2.72 (4H, m, COCH CH CO), 3.77–3.81 (1Í, m, Í-5ꢂs), 3.83 (1Í, d, J = 11.0, H -28), 4.11 (1Í, dd,
3
2
2
À
J = 12.5, 2.0, Í-6ꢂs), 4.25–4.34 (3Í, m, Í-2ꢂs, 6ꢂs, H -28), 4.47 (1Í, dd, J = 11.0, 5.5, Í-3), 5.11–5.19 (2Í, m, Í-3ꢂs, 4ꢂs),
B
1
3
5
1
3
1
.51 (1Í, d, J = 9.0, NH), 5.72 (1Í, d, J = 9.0, Í-1ꢂs). C NMR spectrum (100 MHz, CDCl , ꢊ, ppm): 14.6, 14.8, 16.0, 16.1,
6.5, 18.2, 20.5, 20.6, 20.7, 20.8, 21.3, 21.6, 22.9, 23.2, 23.7, 26.8, 26.9, 27.9, 28.7, 29.1, 29.4, 29.7, 34.2, 34.6, 37.0, 37.2,
7.8, 38.4, 40.9, 42.9, 44.6, 46.6, 48.2, 50.0, 53.2, 55.3, 61.6, 63.2, 67.7, 72.5, 73.0, 80.9, 92.8, 169.2, 170.2, 170.6, 171.0,
3
+
+
+
71.08, 171.10, 172.3. Mass spectrum (MALDI): m/z 938.6 [M + Na] (calcd [M + Na] 938.5), m/z 954.6 [M + K]
+
(
calcd [M + K] 954.5). C H NO .
5
0
77
14
ACKNOWLEDGMENT
The Russian Science Foundation (Grant No. 14-50-00014) provided financial support for the study of antimicrobial
activity of the synthesized compounds by serial dilutions in liquid growth media by the literature method [25, 26].
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