Synthesis and antibacterial activity of betulin esters

Article abstract of DOI:10.1007/s10600-011-0001-7

Betulin isonicotinate, 6-chloronicotinate, 2-chlorocinnamate, and benzoates were synthesized by the reaction of betulin and the acid chlorides. It was shown that the 3,4-difluorobenzoate exhibited antibacterial activity against Ochrobactrum anthropi, Sten

Full text of DOI:10.1007/s10600-011-0001-7

Chemistry of Natural Compounds, Vol. 47, No. 4, September, 2011 [Russian original No. 4, July-August, 2011]
SYNTHESIS AND ANTIBACTERIAL ACTIVITY
OF BETULIN ESTERS
1
2
1*
K.-T. Chue, M.-S. Chang, and L. N. Ten
UDC 547.597+547.918
Betulin isonicotinate, 6-chloronicotinate, 2-chlorocinnamate, and benzoates were synthesized by the reaction
of betulin and the acid chlorides. It was shown that the 3,4-difluorobenzoate exhibited antibacterial activity
against Ochrobactrum anthropi, Stenotrophomonas maltophilia, and S. nitritireducens whereas the
3,5-dinitrobenzoate was active against the last two microorganisms.
Keywords: betulin, esters, antibacterial activity.
The outer part of the bark of several white birch species is a rich source of pentacyclic lupane triterpenoids [1]. The
most famous of these is betulin [2]. The high content, simple isolation methods, and presence of reactive functional groups
make it an available and convenient starting material for synthesizing new biologically active compounds [3–5]. Compounds
with anti-inflammatory, antioxidant, antibacterial, hepatoprotective, antiviral, antitumor, and immunostimulant properties were
found among synthesized betulin derivatives [6, 7]. The increasing number of publications in this area indicates that active
research is continuing.
Herein we present results from the synthesis of betulin esters and data on their antibacterial activity.
Betulin (1) was obtained by extraction of birch (Betula platyphylla) bark collected in Chungbuk Province (South
13
Korea). The structure of 1 was confirmed by comparison of its PMR and C NMR spectra with those published [8]. Esters
2–8 were prepared by reaction of 1 with the acid chlorides in Py (1:1.2 mole ratio) in the presence of dimethylaminopyridine
(1.2 equivalents). The 28-O-monosubstituted betulin derivatives were isolated in 56–69% yields.
30
O
O
O
O
8'
OCH
20
5'
C
1'
NO
2
R =
C
1'
3'
19 21
3
3
C
C
3'
29
7'
7'
3'
7'
N1'
7'
H
9'
OCH
11
N1'
3'
25
10
Cl
5'
NO
26
5'
17
13
5'
CH OR
2
1
OCH
2
3
28
10'
15
4
3
5
2
H
3
27
7
5
HO
O
O
O
Cl
7'
C
1'
3' F
24
C
1'
3' Cl
C
1'
23
1 - 8
1: R = H
3'
7'
7'
9'
8'
F
Cl
5'
5'
5'
6
7
8
The structures of the esters were elucidated using IR and NMR spectra and comparison of them with those of analogous
13
compounds [5, 9, 10]. C NMR spectra of synthesized 2–8 exhibited resonances for the betulin part of the molecule and the
corresponding number of C resonances for the acyl components (Table 1). A weak-field shift of the C-28 resonances and the
appearance of resonances at 163.1–167.9 ppm indicated that the ester bonds had formed. This was also confirmed by the
–1
appearance of absorption bands at ~1700 cm in the IR spectra of these compounds.
Synthesized 2–8 were checked for antibacterial activity that was assessed using the diameter of the colony growth
inhibition zone around paper disks with the applied compound (values are given below after the name of the bacteria). Betulin
3,4-difluorobenzoate (8) exhibited activity against Ochrobactrum anthropi (12 mm), Stenotrophomonas nitritireducens
(13 mm), and S. maltophilia (11 mm) whereas the 3,5-dinitrobenzoate (5) inhibited growth of the last two test cultures (11 and
10 mm, respectively).
1) Reaction and Separation Materials Research Center, Korea Institute of Energy Research, 102, Gajeong-ro, Yuseong-
gu, Daejeon, 305-343, Republic of Korea, fax: (8242) 860 31 35, e-mail: lten@kier.re.kr; l_ten@yahoo.com; 2) Biotechnology
& Natural Products Lab. Co., Ltd., Suwon, 443-702, Republic of Korea. Translated from Khimiya Prirodnykh Soedinenii,
No. 4, July–August, 2011, pp. 516–519. Original article submitted December 11, 2010.
©
0009-3130/11/4704-0583 2011 Springer Science+Business Media, Inc.
583

13
TABLE 1. C NMR Spectra of Betulin (1) and Esters 2–8 (ꢀ, ppm, J/Hz)
1
2
3
4
5
6
7
8
C atom
1
2
3
4
5
6
7
8
38.89
27.58
79.18
39.06
55.47
18.49
34.41
41.10
50.58
37.34
21.01
25.39
37.49
42.91
27.23
29.36
47.98
48.94
48.00
150.67
29.93
34.16
28.18
15.56
16.31
16.17
14.95
60.75
109.90
19.21
39.07
27.61
79.15
38.92
55.51
18.47
34.40
41.11
50.58
37.36
20.99
25.41
37.87
42.97
27.37
30.36
46.96
49.05
48.03
150.29
29.88
34.99
28.18
15.57
16.30
16.26
15.02
63.76
110.17
19.35
125.69
117.74
153.15
142.44
153.15
117.74
166.72
56.46
61.13
56.46
39.07
27.60
79.12
38.92
55.50
18.48
34.40
41.10
50.57
37.36
20.98
25.40
37.92
42.96
27.30
30.08
46.90
49.06
47.95
150.09
29.76
34.84
28.19
15.58
16.31
16.27
15.02
64.41
110.27
19.35
39.07
27.60
79.14
38.92
55.50
18.48
34.39
41.10
50.56
37.36
20.98
25.38
37.92
42.96
27.29
30.09
46.90
49.06
47.95
150.07
29.75
34.84
28.19
15.58
16.31
16.25
15.02
64.31
110.28
19.35
39.07
27.58
79.14
38.91
55.49
18.48
34.40
41.11
50.54
37.36
20.98
25.38
37.99
42.99
27.30
30.13
46.95
49.09
47.95
149.89
29.74
34.87
28.19
15.58
16.32
16.26
15.04
65.78
110.43
19.34
134.31
129.58
148.90
122.56
148.90
129.58
163.09
39.08
27.62
79.18
38.93
55.52
18.51
34.41
41.11
50.60
37.37
21.01
25.43
37.84
42.95
27.34
30.10
46.80
49.06
47.96
150.36
29.86
34.87
28.21
15.58
16.32
16.27
15.01
63.29
110.11
19.38
135.16
132.93
127.84
131.22
127.27
130.38
140.64
121.09
167.14
39.07
27.61
79.16
38.92
55.51
18.49
34.40
41.11
50.58
37.36
20.99
25.41
37.90
42.97
27.32
30.14
46.90
49.07
47.95
150.16
29.80
34.88
28.19
15.58
16.31
16.26
15.03
64.21
110.23
19.37
131.66
130.50
133.15
137.73
128.85
130.75
165.29
39.05
27.59
79.14
38.90
55.49
18.47
34.38
41.09
50.56
37.34
20.98
25.39
37.88
42.95
27.29
30.10
46.87
49.04
47.94
150.15
29.77
34.85
28.18
15.56
16.30
16.25
15.00
64.05
110.20
19.34
126.72
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1ꢁ
2ꢁ
3ꢁ
4ꢁ
5ꢁ
150.80
137.82
123.06
137.82
150.80
165.64
155.85
151.35
139.75
125.49
124.40
164.96
119.07 (d, J = 15.1)a
150.28 (dd, J = 196.4, 15.5)a
153.72 (dd, J = 211.5, 9.6)a
117.55 (d, J = 14.6)a
127.67
ꢁ
ꢁ
6
7
167.87
8ꢁ
9ꢁ
10ꢁ
______
a
Splitting constants (Hz) of C resonances by F atoms are given in parentheses.
These microorganisms are Gram-negative pathogenic bacteria. S. maltophilia causes absess–septic infections [11]. S.
nitritireducens can cause various types of biological damage [12]. O. anthropi causes sepsis in people with weakened immunity
[13]. Compounds that exhibit activity against Bacillus subtilis, Escherichia coli, Enterrococcus faecalis, Micrococcus luteus,
Staphylococcus aureus, S. epidermidis [14, 15], and Klebsiella pneumoniae [16] are known among betulin derivatives. This
list of bacteria sensitive to betulin compounds can be extended to the three microorganisms noted above as a result of the
present work.
EXPERIMENTAL
13
C NMR and PMR spectra were recorded in CDCl on a Varian Unity Inova 600 spectrometer at operating frequencies
3
125 and 600 MHz, respectively, using TMS as an internal standard. IR spectra were recorded on an FT-IR spectrometer
584

(JASCO FT-IR 4100) using attenuated total internal reflectance (ATIR). Elemental analysis was performed on an EA1110-
FISONS instrument (ThermoQuest Italia SPA, CE Instruments). Melting points were determined on a Fisher-Jones instrument
(Fisher Scientific Co.). All acid chlorides were purchased from Sigma-Aldrich.
Preparative reversed-phase HPLC was carried out on an OptimaPaK C column (250 ꢂ 21.1 mm, 10 ꢃm particle
18
size) with isocratic elution by CH CN:H O (86:14, vol%) at flow rate 7 mL/min. Absorbance of effluents was estimated at
3
2
212 nm using a UV detector (UV730D, Young Lin Instrument, Korea).
Isolation of Betulin from Birch Bark. Dried and ground bark from B. platyphylla (265.0 g) was soaked in EtOAc
and refluxed for 2 h. The yield of dry extract after double extraction was 53.1 g (20.0%). The resulting extract was recrystallized
twice from CHCl :EtOH (82:18, vol%). Small impurities of polar compounds were removed over a column of Al O to afford
3
2 3
pure 1 (99.0%, 32.5 g, HPLC analysis).
General Method for Synthesizing 2–8. A solution of 1 (2.0 g, 4.5 mmol), acid chloride (5.4 mmol), and
dimethylaminopyridine (0.64 g, 5.4 mmol) in anhydrous Py (35 mL) was stirred at 40°C under a N atmosphere for 24 h. The
2
mixture was diluted with EtOAc, washed several times with HCl solution (5%) and then H O until neutral, and dried over
2
anhydrous Na SO . The solvent was vacuum distilled. The solid was purified by preparative HPLC to afford highly pure
2
4
esters 2–8 in 56–69% yields.
Antibacterial activity was determined using paper disks as described previously [17]. Disks of diameter 8 mm
(Advantec Toyo Roshi Kaisha, Ltd., Japan) were treated with a solution (60 ꢃL) of the compound in DMSO (1 mg/mL) and
placed on the surface of inoculated agar in Petri dishes. All tests were performed in triplicate. Pure solvent was used as a
T
negative control; gentamicin, as a positive control. The test cultures Ochrobactrum anthropi KCTC 22833 and
T
Stenotrophomonas maltophilia KCTC 1773 were obtained from the Korean Collection for Type Culture (KCTC);
T
Stenotrophomonas nitritireducens KACC 10891 , from the Korean Agricultural Culture Collection (KACC).
Betulin 28-O-3,4,5-trimethoxybenzoate (2), C H O , 61% yield, white crystalline compound, 97.9% pure (HPLC
40 60
6
–1
analysis), mp 223–224°C. IR spectrum (ATIR, ꢄ, cm ): 3410 (br), 2939, 2866, 1701, 1640, 1588, 1462, 1415, 1334, 1227,
1125, 1009, 874, 768, 732.
PMR spectrum (600 MHz, CDCl , ꢀ, ppm, J/Hz): 0.76, 0.84, 0.97, 1.01, 1.07 (15H, 5s, CH -23, 24, 25, 26, 27), 1.71
3
3
(3H, s, CH -30), 2.54 (1H, td, J = 11.4, 5.4, H-19), 3.19 (1H, dd, J = 11.4, 4.8, H-3), 3.90 (6H, s, CH -8ꢁ, 10ꢁ), 3.91 (3H, s,
3
3
CH -9ꢁ), 4.09 and 4.53 (2H, both d, J = 10.8, H-28), 4.61 and 4.72 (2H, both br.s, H-29), 7.31 (2H, s, H-2ꢁ, 6ꢁ) (for all
3
compounds, only characteristic proton resonances are given).
Betulin 28-O-isonicotinate (3), C H NO , 69% yield, white crystalline compound, 98.3% pure (HPLC analysis),
36 53
–1
3
mp 221–222°C. IR spectrum (ATIR, ꢄ, cm ): 3420 (br), 2936, 2868, 1715, 1639, 1599, 1560, 1455, 1406, 1377, 1347, 1325,
1287, 1255, 1120, 1066, 1046, 1013, 970, 915, 883, 858, 849, 841, 831, 822, 810, 760, 708, 675.
PMR spectrum (600 MHz, CDCl , ꢀ, ppm, J/Hz): 0.77, 0.84, 0.97, 1.01, 1.06 (15H, 5s, CH -23, 24, 25, 26, 27), 1.71
3
3
(3H, s, CH -30), 2.52 (1H, td, J = 11.4, 5.4, H-19), 3.19 (1H, dd, J = 11.4, 4.8, H-3), 4.13 and 4.57 (2H, both d, J = 10.8, H-28),
3
4.62 and 4.73 (2H, both br.s, H-29), 7.85 (2H, d, J = 4.8, H-3ꢁ, 5ꢁ), 8.79 (2H, d, J = 4.8, H-2ꢁ, 6ꢁ)
Betulin 28-O-6-chloronicotinate (4), C H ClNO , 56% yield, white crystalline compound, 98.5% pure (HPLC
36 52
–1
3
analysis), mp 193–194°C. IR spectrum (ATIR, ꢄ, cm ): 3430 (br), 2936, 2868, 1720, 1638, 1586, 1455, 1389, 1367, 1288,
1269, 1122, 1105, 1045, 1019, 971, 879, 766.
PMR spectrum (600 MHz, CDCl , ꢀ, ppm, J/Hz): 0.77, 0.84, 0.97, 1.01, 1.06 (15H, 5s, CH -23, 24, 25, 26, 27), 1.71
3
3
(3H, s, CH -30), 3.18 (1H, dd, J = 11.4, 4.8, H-3), 4.13 and 4.57 (2H, both d, J = 10.8, H-28), 4.62 and 4.72 (2H, both br.s,
3
H-29), 7.43 (1H, m, H-5ꢁ), 8.25 (1H, m, H-4ꢁ), 9.01 (1H, m, H-2ꢁ).
Betulin 28-O-3,5-dinitrobenzoate (5), C H N O , 65% yield, white crystalline compound, 98.2% pure (HPLC
37 52
–1
2 7
analysis), mp 239–240°C. IR spectrum (ATIR, ꢄ, cm ): 3420 (br), 2932, 1717, 1640, 1542, 1458, 1386, 1340, 1279, 1177,
1074, 1044, 984, 922, 884, 773 721.
PMR spectrum (600 MHz, CDCl , ꢀ, ppm, J/Hz): 0.77, 0.84, 0.97, 1.02, 1.08 (15H, 5s, CH -23, 24, 25, 26, 27), 1.71
3
3
(3H, s, CH -30), 2.52 (1H, td, J = 10.2, 5.4, H-19), 3.19 (1H, dd, J = 11.4, 4.8, H-3), 4.24 and 4.68 (2H, both d, J = 10.8, H-28),
3
4.63 and 4.74 (2H, both br.s, H-29), 9.15 (2H, s, H-2ꢁ, 6ꢁ), 9.23 (1H, s, H-4ꢁ).
Betulin 28-O-2-chlorocinnamate (6), C H ClO , 59% yield, white crystalline compound, 98.2% pure (HPLC
39 55
–1
3
analysis), mp 200–201°C. IR spectrum (ATIR, ꢄ, cm ): 3410 (br), 2941, 2868, 1703, 1638, 1454, 1385, 1323, 1267, 1185,
1107, 1087, 1039, 1017, 977, 887, 766.
PMR spectrum (600 MHz, CDCl , ꢀ, ppm, J/Hz): 0.76, 0.83, 0.97, 0.99, 1.06 (15H, 5s, CH -23, 24, 25, 26, 27), 1.70
3
3
(3H, s, CH -30), 2.50 (1H, td, J = 10.8, 5.4, H-19), 3.19 (1H, dd, J = 11.4, 4.8, H-3), 4.00 and 4.42 (2H, both d, J = 10.8, H-28),
3
585

4.60 and 4.71 (2H, both br.s, H-29), 6.45 (1H, d, J = 16.2, H-8ꢁ), 7.29 (2H, m, H-4ꢁ, 5ꢁ), 7.42 (1H, d, J = 7.8, H-3ꢁ), 7.63 (1H,
d, J = 7.2, H-6ꢁ), 8.10 (1H, d, J = 16.2, H-7ꢁ).
Betulin 28-O-3,4-dichlorobenzoate (7), C H Cl O , 63% yield, white crystalline compound, 98.4% pure (HPLC
37 52
–1
2 3
analysis), mp 240–241°C. IR spectrum (ATIR, ꢄ, cm ): 3420 (br), 2930, 2867, 1704, 1644, 1456, 1387, 1284, 1272, 1237,
1147, 1113, 1045, 1031, 981, 884, 759.
PMR spectrum (600 MHz, CDCl , ꢀ, ppm, J/Hz): 0.76, 0.83, 0.97, 1.00, 1.06 (15H, 5s, CH -23, 24, 25, 26, 27), 1.70
3
3
(3H, s, CH -30), 2.51 (1H, td, J = 10.8, 6.0, H-19), 3.19 (1H, dd, J = 11.4, 4.8, H-3), 4.10 and 4.53 (2H, both d, J = 10.8, H-28),
3
4.63 and 4.72 (2H, both br.s, H-29), 7.53 (1H, d, J = 8.4, H-5ꢁ), 7.86 (1H, dd, J = 8.4, 1.8, H-6ꢁ), 8.10 (1H, d, J = 1.8, H-2ꢁ).
Betulin 28-O-3,4-difluorobenzoate (8), C H F O , 60% yield, white crystalline compound, 98.5% pure (HPLC
37 52 2
–1
3
analysis), mp 200–201°C. IR spectrum (ATIR, ꢄ, cm ): 3410 (br), 2935, 2867, 1702, 1649, 1519, 1436, 1315, 1197, 1115,
1092, 1047, 973, 887, 833, 777, 766.
PMR spectrum (600 MHz, CDCl , ꢀ, ppm, J/Hz): 0.76, 0.83, 0.97, 1.00, 1.06 (15H, 5s, CH -23, 24, 25, 26, 27), 1.70
3
3
(3H, s, CH -30), 2.51 (1H, td, J = 10.8, 5.4, H-19), 3.19 (1H, dd, J = 11.4, 4.8, H-3), 4.09 and 4.52 (2H, both d, J = 10.8, H-28),
3
4.61 and 4.72 (2H, both br.s, H-29), 7.23 (1H, m, H-5ꢁ), 7.84 (2H, m, H-2ꢁ, 6ꢁ).
ACKNOWLEDGMENT
The work was supported financially by the Korea Forest Service [Forest Science & Technology Projects
(S120808L1201106)].
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