Journal of Natural Products
Article
mp 185 °C; [α]23 +3.4 (c 2.8, CH Cl ); R 0.13 (CH Cl −MeOH−
powder (0.008 g, 19% yield): R 0.23 (CH Cl −MeOH, 96%:4%); H
1
D
2
2
f
2
2
f
2
2
1
NH OH, 95:4:1); H NMR (CDCl , 400 MHz) δ 4.66 (1H, s), 4.56
NMR (CDCl , 500 MHz) 6.88 (1H, d, J = 8.6 Hz), 5.91 (1H, d, J =
4
3
3
(
1H, s), 4.22 (1H, d, J = 12 Hz), 3.83 (1H, d, J = 12 Hz), 2.42 (1H, td,
9.6 Hz), 4.86−4.72 (2H, m), 4.68 (1H, s), 4.58 (1H, s), 4.23−4.10
J = 11, 6 Hz), 2.26 (1H, m, H-3α), 2.04 (3H, s, OCOCH ), 2.00−1.86
(2H, m), 4.02 (1H, m), 3.80 (1H, d, J = 10.8 Hz), 3.67−3.50 (2H, m),
3
(
2H, m), 1.81 (1H, ddd, J = 10, 4, 2 Hz) 1.74 (1H, dd, J = 12, 8 Hz),
.70−0.60 (22H, m, CH , CH ring protons), 1.65, 1.00, 0.95, 0.89,
3.50−3.30 (9H, m), 3.40, 3.32 (s, OCH ), 3.14 (1H, s), 3.01(s, 2H),
3
1
0
1
3
1
2.51−2.31 (5H, m), 2.28−2.22 (6H, nm), 2.25 (s, N(CH ) , 2.13−
2
3 2
13
.77, 0.67 (18H, s, CH ); C NMR (CDCl , 100 MHz) δ 171.8,
0.74 (CH, CH , and CH betulin ring and peptide protons, m);
3
3
2 3
+
50.3, 110.0, 63.0, 59.8, 56.1, 50.6, 49.0, 47.9, 46.5, 42.9, 41.0, 39.6,
7.8, 37.5, 34.7, 34.4, 30.0, 29.8, 28.5, 27.2, 25.4, 21.3, 20.9, 19.3, 18.9,
6.2, 15.6, 15.0; (+)-HRAPCIMS m/z 484.4150 (calcd for
(+)-HRAPCIMS m/z 1022.8243 [M + H] (calcd for C H N O ,
61 108 5 7
1022 0.8250).
3,28-Di-O-acetyl-30-bromobetulin (12). The bromination of
13
C H NO , 484.4155).
diacetoxy betulin was carried out according to the literature.
3
2
3
54
2
1
0
-β-Amino-N-(Boc-Dap)-28-O-acetylbetulin (10). Boc-Dap
Diacetoxybetulin (0.5 g, 0.8 mmol) was dissolved in dry CCl (10
4
(
0.03 g, 0.10 mmol) was dissolved in dry CH Cl (l mL) and added
mL), NBS (0.3 g, 1.7 mmol) was added, and the reaction mixture was
heated at reflux for 1.5 h. The precipitated solid was collected and
crystallized from ethanol to give a colorless solid: 0.35 g (61%); mp
2
2
to a solution of 9b (0.05 g, 0.01 mmol) in dry CH Cl (3 mL), the
2
2
solution was cooled to 0 °C (ice bath), and TEA (80 μL, 3.5 equiv)
followed by DEPC (0.025 mL, 0.16 mmol, 1.6 equiv) were then
added. The solution was stirred for 18 h with warming to rt over time
and concentrated to an amber oil. The crude product was separated by
column chromatography on silica gel eluting with hexanes−EtOAc
1
13
189−190 °C; R 0.43 (hexanes−EtOAc, 8:2); H and C NMR
f
spectroscopic data in accord with published data.
3,28-Di-O-acetyl-30-azidobetulin (13). To a solution of
bromobetulin 12 (1.25 g, 2.0 mmol) in anhydrous DMF (28 mL)
(
(
(
3:7) to yield a colorless oil, which crystallized from CHCl −hexanes
was added NaN (0.60 g, 9.2 mmol, 4.5 equiv), the reaction mixture
3
3
22
0.042 g, 56% yield): mp 105 °C; [α] −7.1 (c 1.3, CHCl ); R 0.3
hexanes−EtOAc, 2:8); H NMR (CDCl , 400 MHz) δ 5.83 (0.5H,
was heated to 90 °C for 30 min and cooled, and EtOAc (40 mL) was
added followed by water (40 mL). The organic layer was extracted and
D
3
f
1
3
bs, NH), 5.51 (0.5H, bs, NH), 4.65(1H, s), 4.56 (1H, s), 4.22 (1H, d, J
washed with brine (2 × 40 mL), dried (MgSO ), and concentrated to
4
=
11 Hz), 3.98−3.74 (3H, m), 3.62−3.48 (2H, m), 3.41 (3H, s,
3
an off-white residue, and crystallization from EtOAc gave azide 13 as a
1
4
OCH ), 3.20 (1H, m), 2.41 (1H, m), 2.27 (1H, m), 2.04 (2H, s,
OCOCH ), 1.99−0.70 (34 H, m), 1.65, 0.99, 0.94, 0.83, 0.78, 0.72 (21
H, 6s, CH ); C NMR (CDCl , 100 MHz) (two conformers
colorless solid (0.82 g, 70%): mp 185 °C [lit. 192−193 °C]; R 0.5
f
1 13
(EtOAc−hexanes, 2:8); H and C NMR spectroscopic data
consistent with published data; (+)-HRAPCIMS m/z 540.4052 [M
+ H − N ] (calcd for C H NO , 540.4053).
30-Aminobetulin (14). To a cooled (ice bath) solution of azide
3
13
14
3
3
1
0
c
+
observed) δ 173.7, 173.2, 171.8, 154.7, 154.5, 150.2, 110.0, 84.1,
2
34 54
4
8
4
2
1
2.3, 79.9, 79.3, 62.9, 60.9, 60.5, 59.0, 56.5, 56.2, 56.1, 50.4, 48.9, 47.8,
7.1, 46.6, 46.4, 44.5, 43.9, 42.8, 40.9, 39.2, 37.9, 37.7, 37.1, 34.7, 34.2,
9.8, 29.7, 28.8, 27.2, 26.3, 25.7, 25.5, 25.3, 24.9, 24.7, 24.3, 21.2, 20.8,
13 (0.l g, 0.18 mmol) in dry THF (2.5 mL) was added LiAlH (l M in
4
THF, 0.75 mL, 0.75 mmol, 4 equiv). The reaction mixture was stirred
with warming to rt for 2 h and cooled, and water (0.03 mL), 15%
NaOH (0.03 mL), and water (0.09 mL) were added successively. The
resulting mixture was stirred for 20 min before extraction with ether
(25 mL). Anhydrous sodium sulfate was added, and the mixture was
allowed to stand overnight, filtered, and concentrated to yield 14 as an
off-white solid (68 mg, 85% yield), which was recrystallized from
9.2, 18.7, 16.6, 16.1, 14.8, 14.6, 14.3; (+)-HRAPCIMS m/z 753.5768
+
[
M + H] (calcd for C H N O , 753.5782).
46
77
2
6
3
β-Amino-N-(Dap-Dil-Val-Dov)-28-O-acetylbetulin (11a).
Amide 10 (0.05 g, 0.067 mmol) was dissolved in anhydrous CH Cl2
2
(
1 mL) and cooled in an ice bath. TFA (0.1 mL, 0.149 g. 1.3 mmol, 19
equiv) was added, and the solution stirred for 3 h. Excess TFA was
removed under reduced pressure for 2 h to yield a foamy solid. The
TFA salt (0.43 g, 0.07 mmol) was stirred together with Dov-Val-Dil-
2
2
CHCl −CH OH: mp 210−215 °C; [α] −2.1 (c 0.52, EtOH); R
3
3
D
f
1
0.1 (CH Cl −CH OH, 9:1); H NMR (CDCl , 400 MHz, sparingly
2
2
3
3
11
TFA (0.038 g, 0.07 mmol) in anhydrous CH Cl (2 mL) under N
soluble) δ 4.83 (2H, s), 3.77 (1H, d, J = 11 Hz), 3.69 (1H, m), 3.30
(1H, d, J = 11 Hz), 3.25 (1H, d, J = 7.4 Hz), 3.21−3.14 (1H, m),
2.32−2.21 (1H, m), 2.13−2.00 (2H, m), 1.95−1.81 (4H, m), 1.73−
2
2
2
at 0 °C. TEA (0.05 mL, 0.36 mmol, 15 equiv) and DEPC (0.011 mL,
.7l mmol, 10 equiv) were then added. The reaction mixture was
0
stirred for 18 h at rt, then concentrated and separated using column
chromatography eluting with CH Cl −MeOH (97:3) to give 11a as a
colorless frothy solid (0.052 g, 74% yield). Further purification on
Sephadex LH-20 eluting with methanol gave a colorless crystalline
solid: mp 135 °C; R 0.3 (CH Cl −MeOH, 95:5); H NMR (CDCl ,
5
1.01 (21 H, CH , CH ring protons), 1.00, 0.96, 0.95, 0.80, 0.74 (15H,
2
13
5s, CH ), 0.66 (1H, d, J = 9 Hz); C NMR (CD OD, 100 MHz) δ
2
2
3
3
156.7, 106.6, 79.6, 62.8, 60.1, 56.7, 51.5, 50.6, 48.9, 43.8, 42.1, 39.9,
38.6, 38.3, 35.5, 35.0, 30.9, 30.4, 30.1, 28.6, 28.2, 28.0, 27.8, 22.1, 19.4,
1
+
16.7, 16.6, 16.2, 15.2; (+)-HRAPCIMS m/z 458.3998 [M + H] (calcd
f
2
2
3
00 MHz) (two conformers were present and some signals were
for C H NO , 458.3998).
30
52
2
doubled) δ 6.90 (1H, d, J = 9.0 Hz), 5.92 (1H, dd, J = 9.5, 4.5 Hz),
.87 (1H, m), 4.79 (1H, dd, J = 9.2, 6.6 Hz), 4.68 (1H, m), 4.59 (1H,
m), 4.27−4.09 (3H, m), 4.02 (1H, d, J = 6.8 Hz), 3.96 (1H, m), 3.88−
.74 (2H, m), 3.71−3.50 (2H, m), 3.49−3.36 (7H, m), 3.41 (s,
OCH ), 3.33 (4H, m), 3.32 (s, OCH ), 3.15 (1H, s), 3.02 (3H, s),
30-N-(Boc-Dap)-betulin (15a). To a cooled (ice bath) solution of
4
amine 14 (0.15 g, 0.33 mmol) in dry CH Cl (10 mL) was added a
2
2
1
0
solution of Boc-Dap (0.09 g, 0.30 mmol) in CH Cl (3 mL). TEA
2
2
3
(0.24 mL, 1.71 mmol) and DEPC (0.033 mL, 2.13 mmol) were added,
and the reaction mixture was stirred for 20 h before terminating with
removal of the solvent under low pressure overnight. Separation by
flash silica gel chromatograpy (eluent: CH Cl −CH OH, 98:2) gave
3
3
2
.51−2.37 (4H, m), 2.25 (9H, s), 2.07 (3H, s, OCOCH ), 2.15−0.72
3
13
(
CH, CH , and CH protons, betulin ring, and peptide protons); C
2 3
2
2
3
NMR (CDCl , 100 MHz) δ 173.6, 173.56, 171.9, 171.8, 170.2, 150.32,
amide 15a as a frothy solid (85 mg, 50% yield): mp 129−131 °C; R
3
f
1
1
5
4
3
2
1
10.0, 88.11, 86.3, 82.3, 78.2, 76.6, 62.9, 61.8, 60.4, 59.3, 58.1, 56.6,
6.2, 53.9, 51.1, 50.4, 48.9, 47.9, 47.8, 46.9, 46.8, 46.4, 45.5, 45.0, 43.8,
3.0, 42.9, 42.8, 41.6, 41.2, 41.0, 40.9, 40.7, 39.3, 38.0, 37.9, 37.7, 37.3,
7.2, 36.8, 36.4, 33.8, 31.9, 31.1, 29.9, 29.7, 28.9, 28.6, 27.8, 27.2, 26.6,
6.5, 26.0, 25.9, 25.5, 25.3, 25.1, 24.7, 23.7, 21.2, 20.9, 20.3, 19.9, 19.8,
0.2 (CH Cl −CH OH, 96:4); H NMR (CDCl , 400 MHz) δ 6.56
2
2
3
3
(0.5H, bs), 5.84 (0.5H, bs), 4.81 (1H, s), 4.72 (1H, s), 3.93−3.64 (5H,
m), 3.59−3.31 (1H, m), 3.40 (3H, s, OCH ), 3.29−3.08 (3H, m),
3
2.45−2.19 (2H, m), 2.12−1.97 (1H, m), 1.96−0.67 (CH, CH ring
2
protons and Boc-Dap protons), 0.97, 0.93, 0.78, 0.73 (15H, 4s, CH ),
3
9.2, 18.7, 17.9, 16.7, 16.6, 16.2, 15.9, 14.9, 14.1, 13.7, 11.0;
0.65 (1H, m); 13C NMR (CDCl , 100 MHz) δ (two conformers
3
+
(
+)-HRAPCIMS m/z 1064.8360 [M + H] (calcd for C H N O ,
present) 174.2, 173.7, 154.9, 154.6, 151.7, 107.9, 84.2, 82.4, 80.9, 79.5,
79.0, 60.9, 60.3, 59.4, 58.8, 55.4, 50.5, 49.5, 47.9, 47.2, 46.7, 44.7, 44.2,
42.8, 41.1, 39.0, 38.9, 37.4, 37.3, 34.4, 34.0, 29.4, 28.7 (m), 28.2, 27.5,
27.2, 26.2, 25.6, 24.8, 24.4, 21.1, 18.4, 16.3, 16.1, 15.6, 14.9;
63
110
5
8
1
064.8350).
3
β-Amino-N-(Dap-Dil-Val-Dov)-betulin (11b). A solution of
1
1a (0.042 g, 0.04 mmol) in THF−MeOH (0.8 mL:0.6 mL) was
+
treated with 2 N NaOH (0.3 mL) as described in the procedure for
the synthesis of 7b. Following separation of the reaction mixture by
chromatography on a silica gel column (gradient elution CH Cl −
(+)-HRAPCIMS m/z 727.5631 [M + H] (calcd for C H N O ,
4
4
75
2
6
727.5625).
30-N-(Boc-Dap)-3,28-di-O-acetoxybetulin (15b). Alcohol 15a
(0.08 g, 0.11 mmol) was taken up in dry pyridine (2 mL). Acetic
2
2
MeOH, 97:3 → 94:4) and drying, 11b was obtained as an amorphous
E
J. Nat. Prod. XXXX, XXX, XXX−XXX