Alstiphyllanines E-H, picraline and ajmaline-type alkaloids from Alstonia macrophylla inhibiting sodium glucose cotransporter

Article abstract of DOI:10.1016/j.bmc.2010.01.077

Three new picraline-type alkaloids, alstiphyllanines E-G (1-3) and a new ajmaline-type alkaloid, alstiphyllanine H (4) were isolated from the leaves of Alstonia macrophylla together with 16 related alkaloids (5-20). Structures and stereochemistry of 1-4 were fully elucidated and characterized by 2D NMR analysis. Alstiphyllanines E and F (1 and 2) showed moderate Na⁺-glucose cotransporter (SGLT1 and SGLT2) inhibitory activity. A series of a hydroxy substituted derivatives 21-28 at C-17 of the picraline-type alkaloids have been derived as having potent SGLT inhibitory activity. 10-Methoxy-N(1)-methylburnamine-17-O-veratrate (6) exhibited potent inhibitory activity, suggesting that the presence of an ester side chain at C-17 may be important to show SGLT inhibitory activity. Structure activity relationship of alstiphyllanines on inhibitory activity of SGLT was discussed.

Full text of DOI:10.1016/j.bmc.2010.01.077

Bioorganic & Medicinal Chemistry 18 (2010) 2152–2158
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Alstiphyllanines E–H, picraline and ajmaline-type alkaloids
from Alstonia macrophylla inhibiting sodium glucose cotransporter
Hiroko Arai a, Yusuke Hirasawa a, Abdul Rahman b, Idha Kusumawati b, Noor Cholies Zaini b, Seizo Sato c,
Chihiro Aoyama c, Jiro Takeo c, Hiroshi Morita a,
*
a Faculty of Pharmaceutical Sciences, Hoshi University, Shinagawa, Tokyo 142-8501, Japan
b Faculty of Pharmacy, Airlangga University, Jalan Dharmawangsa Dalam, Surabaya 60286, Indonesia
c Central Research Laboratory, Nippon Suisan Kaisha, Ltd, 559-6 Kitano-machi, Hachioji, Tokyo 192-0906, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Three new picraline-type alkaloids, alstiphyllanines E–G (1–3) and a new ajmaline-type alkaloid, alsti-
phyllanine H (4) were isolated from the leaves of Alstonia macrophylla together with 16 related alkaloids
(5–20). Structures and stereochemistry of 1–4 were fully elucidated and characterized by 2D NMR anal-
ysis. Alstiphyllanines E and F (1 and 2) showed moderate Na+-glucose cotransporter (SGLT1 and SGLT2)
inhibitory activity. A series of a hydroxy substituted derivatives 21–28 at C-17 of the picraline-type alka-
loids have been derived as having potent SGLT inhibitory activity. 10-Methoxy-N(1)-methylburnamine-
17-O-veratrate (6) exhibited potent inhibitory activity, suggesting that the presence of an ester side chain
at C-17 may be important to show SGLT inhibitory activity. Structure activity relationship of alstiphylla-
nines on inhibitory activity of SGLT was discussed.
Received 10 December 2009
Revised 29 January 2010
Accepted 30 January 2010
Available online 6 February 2010
Keywords:
Indole alkaloids
Alstonia macrophylla
Alstiphyllanines E–H
SGLT
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
bition of SGLT could decrease glucose re-absorption and that this
could thus result in an increase in urinary sugar excretion, and a
Na+-glucose cotransporter (SGLT) is a membrane protein that
plays an important role in the re-absorption of glucose in the kid-
neys. SGLT is known to have three isoforms (SGLT1, SGLT2, and
SGLT3).1–3 SGLT1 is expressed primarily in the brush border mem-
brane of mature enterocytes in the small intestine, where it ab-
sorbs dietary glucose and galactose from the gut lumen.4 SGLT2
is only expressed in the renal cortex, where it is assumed to be
present in the brush border membrane of the S1 and S2 segments
of the proximal tubule, and to be responsible for the re-absorption
of glucose from the glomerular filtrate.4 It is expected that the inhi-
decrease in blood glucose level. Thus, SGLT inhibitors have thera-
peutic potential for type 2 diabetes.5
Our screening study on SGLT inhibitors in traditional medicine6
discovered that the methanol extract of Alstonia macrophylla shows
moderate SGLT inhibitory activity. The genus Alstonia, which is
widely distributed in tropical regions of Africa and Asia, are well-
known rich sources of unique monoterpene indole alkaloids with
various biological activities such as anticancer, antibacterial, anti-
inflammatory, antitussive, and antimalarial properties.7 Recently,
several new indole alkaloids were isolated from extracts of Alstonia
MeO
MeO
MeO
3'
2'
O
O
4'
23
MeO
HO
CO2Me
1'
O
O
HO
CO2Me
9
6
5'
22
6'
MeO
8
7
10
11
CO2Me
6
5
MeO
MeO
CO2Me
N
5
17
17
16
16
9
O
21
O
2
N
1
8
N
7
10
11
13
N
N
3
12
H
H
2
H
4
1
O
O
N
18
13
21
20
14
20
N
N
H
3
12
15
19
H
H
H
14
19
18
15
1
2
3
4
* Corresponding author. Tel./fax: +81 3 5498 5778.
E-mail address: moritah@hoshi.ac.jp (H. Morita).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.077

H. Arai et al. / Bioorg. Med. Chem. 18 (2010) 2152–2158
2153
species collected in Indonesia and Malaysia.8,9 With an aim to iso-
late additional alkaloids against SGLT inhibitory activity, purifica-
tion of extracts of A. macrophylla Wall.ex G. Don (Apocynaceae)
collected in Indonesia led to four new alkaloids alstiphyllanines
E–H (1–4) together with 16 known alkaloids (5–20). Herein we re-
port the isolation and structure elucidation of four new indole
alkaloids, alstiphyllanines E–H (1–4) from A. macrophylla as well
as SGLT inhibitory activity and structure activity relationship
(SAR) study of some picraline-type indole alkaloids.
by HMBC correlations of O-Me to C-30 and C-40. HMBC correlations
for H-5 to C-2, H2-17 to C-7, and H2-6 to C-16 indicated alstiphylla-
nine E possessed picraline-type skeleton. The molecular formulae
of alstiphyllanine E was smaller than that of burnamin-17-O-
30,40,50-trimethoxybenzoate9 by CH2O unit. Compared with 1H
NMR data of burnamin-17-O-30,40,50-trimethoxybenzoate,9 alsti-
phyllanine E was suggested a picraline-type backbone without O-
Me at C-50. The relative stereochemistry of 1 was elucidated by
NOESY correlations as shown in computer-generated 3D drawing
(Fig. 1). The NOESY correlation of H3-18 to H-15 indicated that
the geometry of ethylidene side chain was E. The b-orientation of
C-17 was elucidated by the NOESY correlation of H-14b/H-17a.
2. Results and discussion
Alstiphyllanine F {2, ½a D26
ꢁ32 (c 1.0, MeOH)} was revealed to
ꢀ
2.1. Structures of alstiphyllanines E–H (1–4)
have the molecular formula C32H36N2O9, by HRESITOFMS [m/z
593.2511 (M+H)+,
D
ꢁ1.2 mmu], which was larger than that of
Leaves of A. macrophylla were extracted with MeOH, and the ex-
tract was partitioned between EtOAc and 3% aqueous tartaric acid.
Water-soluble materials, adjusted to pH 9 with satd aq Na2CO3,
were extracted with CHCl3. The CHCl3-soluble materials were sub-
jected to an LH-20 column (CHCl3/MeOH, 1:1) followed by a silica
gel column (CHCl3/MeOH, 1:0–0:1). The eluted fractions were fur-
ther separated by ODS HPLC (MeOH/H2O/TFA) to afford 1 (1.8 mg,
0.00050% dry weight), 2 (1.3 mg, 0.00036%), 3 (10.4 mg, 0.0029%),
and 4 (3.6 mg, 0.0013%), together with 16 known alkaloids,
burnamine-17-O-30,40,50-trimethoxybenzoate10 (5), 10-methoxy-
N(1)-methylburnamine-17-O-veratrate10 (6), alstiphyllanine D9
(7), alstiphyllanine B9 (8), alstiphyllanine C9 (9), picralinal11 (10),
picrinine11 (11), quaternine12 (12), O-deacetylpicraline13 (13), vin-
camedine14 (14), vincamajine15 (15), alstiphyllanine A9 (16),
vincamajine-17-O-veratrate16 (17), vincamajine-17-O-30,40,50-
trimethoxybenzoate16 (18), alstonal17 (19), and alstonerine14 (20).
burnamin-17-O-30,40,50-trimethoxybenzoate by CH2O unit. Com-
pared with 1H NMR data of burnamin-17-O-30,40,50-trim-
ethoxybenzoate, alstiphyllanine F was suggested a picraline-type
backbone with O-Me. The HMBC cross-peak of H3-O-Me (dH 3.27)
to C-10 (dC 156.5) revealed the presence of an indole moiety with
a methoxy group at C-10. HRESITOFMS data [m/z 413.2080
(M+H)+,
D
ꢁ0.4 mmu] of alstiphyllanine G {3, ½a D26
ꢁ42 (c 1.0,
ꢀ
MeOH)} established the molecular formula, C23H28N2O5, which
was larger than that of O-deacetylpicraline13 by C2H4O unit. The
NMR data of 3 were analogous to those of O-deacetylpicraline13 ex-
cept for the following observation: a methoxy signal (dH 3.70) and
an N-methyl signal (dH 2.89) lacking in O-deacetylpicraline ap-
peared for 3. The presence of both methyl groups was verified by
the HMBC correlations of the methoxy protons to C-10 and the
N-methyl protons to C-2 and C-13.
Alstiphyllanine H {4, ½a D26
ꢁ21 (c 1.0, MeOH)} was obtained as a
ꢀ
Alstiphyllanine E {1, ½a D26
ꢁ93 (c 1.0, MeOH)} was revealed to
ꢀ
brown amorphous solid and was revealed to have the molecular
have the molecular formula C30H32N2O7, by HRESITOFMS [m/z
formula C22H26N2O4, by HRESITOFMS [m/z 383.1971 (M+H)+,
D
533.2272 (M+H)+,
D
ꢁ1.6 mmu]. The 1H NMR data (Table 1)
ꢁ2.7 mmu], which was larger than that of vincamajine15 by an
oxygen unit. The 1H NMR data (Table 1) showed the presence of
four aromatic protons, an ethylidene side chain, a methyl ester
function, and an N-methyl group. Partial structures C-9–C-12,
showed the presence of seven aromatic protons, an ethylidene side
chain, a methyl ester function, and two methoxy groups. The
HMBC cross-peak of H2-21 to C-19 indicated the ethylidene side
chain at C-20. The position of each methoxy group was confirmed
Table 1
1H NMR data [dH (J, Hz)] of alstiphyllanines E-H (1–4)
1a
2a
3b
4a
2
3
3.55 (d, 4.8)
4.39 (m)
4.07 (s)
4.00 (s)
3.73 (d, 3.6)
5
5.56 (s)
5.56 (s)
4.72 (d, 2.6)
4.42 (m)
6a
6b
9
10
11
12
14a
14b
15
2.67 (d, 15.4)
3.17 (d, 15.4)
7.56 (d, 7.4)
6.54 (dd, 7.4, 7.2)
6.87 (dd, 7.5, 7.2)
6.73 (d, 7.5)
2.23 (d, 14.8)
2.41 (d, 14.8)
3.40 (s)
2.66 (15.1)
3.25 (m)
7.03 (s)
2.33 (dd, 13.9, 2.6)
3.30 (d, 13.9)
6.93 (d, 2.6)
2.53 (d, 14.4)
2.73 (d, 14.4)
7.20 (d, 7.2)
6.83 (dd, 7.2, 7.2)
7.19 (dd, 7.6, 7.2)
6.77 (d, 7.6)
2.10 (m)
6.60 (d, 8.4)
6.33 (d, 8.4)
2.29 (d, 15.1)
2.37 (d, 15.1)
3.35 (s)
6.73 (dd, 8.5, 2.6)
6.59 (d, 8.5)
1.97 (m)
2.74 (m)
3.36 (s)
3.48 (s)
17a
17b
18
4.08 (d, 11.4)
4.57 (d, 11.4)
1.70 (d, 6.8)
5.74 (q, 6.8)
4.25 (d, 17.1)
4.00 (d, 17.1)
3.75 (s)
4.09 (d, 10.9)
4.94 (d, 10.9)
1.76 (d, 7.2)
5.76 (q, 7.2)
4.02 (m)
4.16 (d, 16.1)
3.80 (s)
3.27 (s)
3.47 (d, 12.3)
3.73 (d, 12.3)
1.56 (dd, 7.1, 2.0)
5.35 (q, 7.1)
3.11 (d, 15.9)
3.66 (d, 15.9)
3.72 (s)
4.16 (s)
1.61 (d, 6.5)
5.55 (q, 6.5)
4.46 (d, 15.1)
4.55 (d, 15.1)
3.73 (s)
19
21a
21b
CO2Me
10-O-Me
30-O-Me
40-O-Me
50-O-Me
N(1)-Me
20
3.70 (s)
3.86 (s)
3.88 (s)
3.89 (s)
3.81 (s)
3.89 (s)
2.89 (s)
2.66 (s)
7.15 (s)
6.94 (d, 8.4)
7.28 (d, 8.4)
6.91 (s)
6.91 (s)
50
60
a
TFA salt in CD3OD.
Free base in CDCl3.
b

2154
H. Arai et al. / Bioorg. Med. Chem. 18 (2010) 2152–2158
MeO
3' 2'
O
23
4'
MeO
1'
O
6'
5'
22
17
16 CO2Me
9
6
8
10
11
5
7
2
1
N4
O
21
13
3
N
H
12
20
14
15
1H-1H COSY
HMBC
19
18
Figure 1. Selected 2D NMR correlations for alstiphyllanine E (1).
Table 2
13C NMR data (dC) of alstiphyllanines E–H (1–4)
C-5–C-6, C-2–C-15, and C-18–C-19 were deduced from a detailed
analysis of 1H-1H COSY spectrum of 4. The HMBC cross-peaks of
H3-18 to C-20 and H-19 to C-15 indicated the presence of an eth-
ylidene side chain at C-20 (Fig. 2). And the presence of an indoline
ring was elucidated by HMBC correlations for H-9 to C-7 and N-Me
to C-2 and C-13. HMBC correlations for H-2, H-5, and H-6a to C-17
and H-6a and H-14a to C-16 indicated alstiphyllanine H possessed
ajmaline-type skeleton. Comparison of 13C chemical shifts of C-3,
C-5, and C-21 (dC 70.5, 77.4, and 67.3, respectively) in 4 with those
(dC 53.2, 61.7, and 55.6, respectively) of vincamedine14 indicated
the presence of an N-oxide functionality at N-4. The relative stereo-
chemistry of 4 was elucidated by NOESY correlations as shown in
computer-generated 3D drawing (Fig. 2). NOESY correlations of
H3-18 to H-21 indicated that the geometry of the ethylidene side
chain was Z. The NOESY correlations of H-3/H-2 and H-14a and
1a
2a
3b
4a
70.9
70.5
77.4
2
3
5
6
7
8
9
10
11
12
13
14
15
16
17
109.5
54.8
106.6
53.3
109.6
49.4
86.9
90.5
89.9
41.6
44.6
44.8
32.1
53.1
53.1
52.7
57.3
132.9
128.4
122.4
129.6
112.1
149.4
20.4
136.0
117.4
156.5
112.9
112.8
143.8
22.5
134.2
113.3
154.5
112.9
109.5
145.4
21.6
130.1
126.4
120.9
129.7
110.7
155.2
22.9
39.9
37.0
33.0
36.3
59.0
58.5
57.5
63.3
67.3
69.6
64.1
74.6
H-14b/H-17 indicated that H-2 was
b-oriented. Oxidation of vincamajine with m-chloroperoxybenzoic
acid (m-CPBA) afforded the N-oxide derivative, whose spectral data
and the [a]D value were identical with those of natural alstiphylla-
nine H. Thus, the structure of asltiphyllanine H was elucidated as
shown in Figure 2.
a
-orientated and H-17 was
18
19
20
21
22
23
CO2Me
13.1
14.7
13.1
12.8
128.2
132.9
42.2
172.8
166.3
52.4
126.0
132.5
47.2
174.8
166.5
53.1
56.2
57.3
61.8
57.3
119.9
137.8
46.7
122.1
129.5
67.3
174.6
171.3
55.8
51.8
52.9
10-O-Me
30-O-Me
56.4
56.4
2.2. SGLT inhibitory activity
40-O-Me
50-O-Me
The in vitro SGLT inhibitory potential of alkaloids 1–20 was as-
N(1)-Me
30.1
35.0
10
20
30
40
50
60
122.5
129.6
149.8
153.1
111.6
125.0
125.8
108.7
154.9
143.9
154.9
108.7
sessed by monitoring inhibition of uptaking of methyl-
a-D-gluco-
pyranoside in cultured cells expressing SGLT1 or SGLT2 at 50
lM
(Table 3). As shown in Table 3, picraline-type alkaloids with vera-
trate or trimethoxybenzoate at C-17 such as compounds 1, 2, and
5–7, showed inhibitory activity against SGLT1 and SGLT2. How-
ever, compounds 8 and 9 which have an N(4)-Me group were found
to have no SGLT inhibitory activity. Any ajmaline and macroline
type alkaloids (4 and 14–20) did not show inhibition on SGLT1
and SGLT2.
a
TFA salt in CD3OD.
Free base in CDCl3.
b
To discuss SAR of picraline-type alkaloids showing SGLT inhib-
itory activity, we prepared eight picraline-type derivatives 21–28
from 6 and 7 by use of acyl anhydride, m-CPBA, and boron tribro-
mide, respectively (Table 4). As shown in Table 4, the presence of
an N(1)-Me group promoted SGLT1 inhibitory activity when com-
pared to those of 1, 2 and 5. Compound 22 which was converted
a methoxy group at C-10 of 7 into a hydroxyl showed less activity
against SGLT1, whereas N(4)-oxide derivatives 23 and 24 with a
HO
CO2Me
5
9
6
8
10
11
7
2
O
21
1
N
13
N
3
12
18
14
20
19
15
1H-1H COSY
HMBC
Figure 2. Selected 2D NMR correlations for alstiphyllanine H (4).

H. Arai et al. / Bioorg. Med. Chem. 18 (2010) 2152–2158
2155
Table 3
Structures and SGLT inhibitory activity of alkaloids 1–20
R1
R2
R3
R4
Inhibition %a
SGLT2
SGLT1
Picraline-type alkaloids
1
2
3
5
6
7
8
9
10
11
12
13
CH2O-Bz(OMe)2
CH2O-Bz(OMe)3
CH2OH
CH2O-Bz(OMe)3
CH2O-Bz(OMe)2
CH2O-Bz(OMe)3
CH2O-Bz(OMe)2
CH2O-Bz(OMe)3
CHO
H
H
H
Me
H
Me
Me
Me
Me
H
H
H
H
H
H
H
H
H
H
H
OMe
H
60.3
65.2
14.0
53.0
95.8
89.9
-10.3
-8.2
16.5
9.6
85.9
103.8
31.6
87.3
102.6
101.4
-0.2
OMe
OMe
H
OMe
OMe
OMe
OMe
H
19Z
N(4)-Me
N(4)-Me
-6.1
36.3
27.3
30.0
-0.2
H
H
H
OMe
H
H
H
H
11.7
10.1
CH2OH
Ajmaline-type alkaloids
4
Me
Me
Me
Me
Me
Me
OH
OAc
OH
OAc
OBz(OMe)2
OBz(OMe)3
N(4)-oxide
N(4)-oxide
4.3
23.1
47.4
30.6
38.5
44.0
47.6
14
15
16
17
18
22.0
11.5
5.3
26.0
7.2
Macroline-type alkaloids
19
20
15.8
20.7
26.8
27.7
R1 CO2Me
R2 CO2Me
N
H
H
16
H
H
R2
O
10
O
O
N
NH
N
N4
O
N
R4
H
H
N
H
N
R1
O
H
H
H
H
R3
19
O
ajmaline type
(4 and 14-18)
macroline type
macroline type
picraline type
(1-3 and 5-13)
19
20
a
Inhibition (%) at 50 lM.
Table 4
Structures and SGLT inhibitory activity of picraline-type derivatives 1, 2, 5–7, and 21–28
R1
R2
R3
Inhibition %a (IC50
l
M)
SGLT1
SGLT2
1
2
5
6
7
21
22
23
24
25
26
27
28
CH2O-Bz(OMe)2
CH2O-Bz(OMe)3
CH2O-Bz(OMe)3
CH2O-Bz(OMe)2
CH2O-Bz(OMe)3
CH2O-Bz
CH2O-Bz(OH)3
CH2O-Bz(OMe)2
CH2O-Bz(OMe)3
CH2O-cinnamoyl
CH2O-Ac
H
OMe
H
H
H
H
60.3 (44)
65.2 (39)
53.0 (50)
95.8 (4)
85.9 (40)
103.8 (40)
87.3 (35)
102.6 (0.5)
101.4 (2)
100.1 (1)
95.6 (7)
64.9 (35)
91.4 (11)
102.8 (1)
39.9 (78)
86.9 (12)
25.7 (>100)
19Z
OMe
OMe
OMe
OH
OMe
OMe
OMe
OMe
OMe
OMe
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
89.9 (5)
85.2 (17)
46.9 (50)
94.6 (5)
93.8 (4)
96.3 (5)
5.4 (>100)
27.1 (97)
7.6 (>100)
N(4)-oxide
N(4)-oxide
CH2OCOCH2CH3
CH2O-Bn
R1 CO2Me
16
R2
10
N4
O
N
R3
H
picraline type (1, 2, 5-7, and 21-28)
a
Inhibition (%) at 50 lM.
methoxy group at C-10 showed less activity against SGLT2. Ali-
phatic esters at C-17 such as 26 and 27 showed less activity against
both SGLT1 and SGLT2, and the presence of an aromatic long side
chain at C-17 such as cinnamoyl derivative 25 potentiated the
inhibitory activity against SGLT1 and SGLT2. On the other hand,
the benzyl ether derivative 28 at C-17 did not show inhibitory
activity.
In this work, three new picraline-type alkaloids, alstiphylla-
nines E–G (1–3) and a new ajmaline-type alkaloid, alstiphyllanine
H (4) were isolated from the leaves of A. macrophylla, and their

2156
H. Arai et al. / Bioorg. Med. Chem. 18 (2010) 2152–2158
structures were fully elucidated by 2D NMR analysis. SAR study of
these alkaloids and synthetic analogue against STLT1 and SGLT2
suggested that the presence of picraline-type alkaloid with an ester
side chain at C-17 may be important to show inhibitory activity.
(M+H)+; HRESITOFMS m/z 593.2511 [(M+H)+,
D +1.2 mmu, calcd
for C32H37N2O9, 593.2499].
3.3.3. Alstiphyllanine G (3)
Brown amorphous solid; ½a D26
ꢀ
ꢁ42 (c 1.0, MeOH); IR (film) mmax
1500), 240 (3500),
3420 and 1720 cmꢁ1; UV (MeOH) kmax 306 (
e
3. Experimental section
3.1. General methods
and 204 (12,000) nm; 1H and 13C NMR data (Tables 1 and 2); ESIMS
m/z 413 (M+H)+; HRESITOFMS m/z 413.2080 [(M+H)+,
D +0.4 mmu,
calcd for C23H28N2O5, 413.2076].
1H and 2D NMR spectra were recorded on a Bruker AV 400 spec-
trometer and chemical shifts were reported using residual CD3OD
(dH 3.31 and dC 49.0) as internal standards. Standard pulse se-
quences were employed for the 2D NMR experiments. 1H–1H COSY,
HOHAHA, and NOESY spectra were measured with spectral widths
of both dimensions of 4800 Hz, and 32 scans with two dummy
scans were accumulated into 1 K data points for each of 256 t1
increments. NOESY spectra in the phase sensitive mode were mea-
sured with a mixing time of 800 ms. For HMQC spectra in the phase
sensitive mode and HMBC spectra, a total of 256 increments of 1 K
data points were collected. For HMBC spectra with Z-axis PFG, a
50 ms delay time was used for long-range C–H coupling. Zero-fill-
ing to 1 K for F1 and multiplication with squared cosine-bell win-
dows shifted in both dimensions were performed prior to 2D
Fourier transformation.
3.3.4. Alstiphyllanine H (4)
Brown amorphous solid; ½a D26
ꢀ
ꢁ21 (c 1.0, MeOH); IR (film) mmax
1400) and 204
3420 and 1740 cmꢁ1; UV (MeOH) kmax 291 (
e
(10,000) nm; 1H and 13C NMR data (Tables 1 and 2); ESIMS m/z
383 (M+H)+; HRESITOFMS m/z 383.1944 [(M+H)+,
D
ꢁ2.7 mmu,
calcd for C22H26N2O4, 383.1971].
3.3.5. Conversion of vincamajine (15) to alstiphyllanine H (4)
m-Chloroperoxybenzoic acid (0.9 mg) was added to a stirred
solution of vincamajine (15, 0.9 mg) in CH2Cl2 (0.2 mL) at room
temperature. The mixture was stirred at 0 °C for 10 min, and
washed with 20% Na2SO2 (5 mL) and H2O (5 mL), and concentrated
to give a pale yellow solid. The residue was subjected to a silica gel
column (CHCl3/MeOH, 10:1) to give the N-oxide derivative
(1.5 mg), whose spectral data and [a] value were identical with
D
those of alstiphyllanine H (4).
3.2. Material
3.3.6. Conversion of 6 to 3
The leaves of A. macrophylla were collected at Purwodadi Botan-
ical Garden, Indonesia in 2006. The botanical identification was
made by Ms. Sri Wuryanti, Purwodadi Botanical Garden, Indonesia.
A voucher specimen has been deposited in the herbarium at Pur-
wodadi Botanical Garden, Pasuruan, Indonesia.
A mixture of 39.6 mg of alkaloid 6 and 20 mL of 5% NaOMe were
heated for 30 min under stirring. The solution was diluted with
water and extracted with CHCl3. The extract was treated with 3%
tartaric acid (pH 2) and then partitioned with EtOAc. The aqueous
layer was treated with saturated Na2CO3 aq to pH 9 and extracted
with CHCl3 to give 3 (27.1 mg, 95.8%).
3.3. Extraction and isolation
3.3.7. Conversion of 3 to Its benzoate derivative (21)
The leaves of A. macrophylla (363.5 g) were extracted with MeOH.
The MeOH extract (43.8 g) was treated with 3% tartaric acid (pH 2)
and then partitioned with EtOAc. The aqueous layer was treated
with satd aq Na2CO3 aq to pH 9 and extracted with CHCl3 to give
alkaloidal fraction (2.06 g). The alkaloidal fraction was purified by
LH-20 column (CHCl3/MeOH, 1:0) and SiO2 column (CHCl3/MeOH,
1:0?0:1) and the fraction eluted by MeOH was purified by ODS
HPLC (CH3CN/H2O/CF3CO2H, 45:55:0.1; flow rate, 2 mL/min; UV
detection at 254 nm) to afford alstiphyllanines E (1, 1.8 mg,
0.00050% yield), F (2, 1.3 mg, 0.00036%), G (3, 10.4 mg, 0.0029%),
and H (4, 3.6 mg, 0.0013%), together with known alkaloids, burn-
amine-17-O-30,40,50-trimethoxybenzoate10 (5), 10-methoxy-N(1)-
methylburnamine-17-O-veratrate10 (6), alstiphyllanine D9 (7), alsti-
phyllanine B9 (8), alstiphyllanine C9 (9), picralina11 (10), picrinine11
(11), quaternine12 (12), O-deacetylpicraline13 (13), vincamedine14
(14), vincamajine15 (15), alstiphyllanine A9 (16), vincamajine-17-
O-veratrate16 (17), vincamajine-17-O-30,40,50-trimethoxybenzo-
ate16 (18), alstonal17 (19), alstonerine14 (20).
To a solution of 3 (3.2 mg) in CH2Cl2 (0.1 mL) was added benzoic
anhydride (4.5 mg) and DMAP (3.2 mg), and the solution was stir-
red at room temperature. The mixture was diluted with CHCl3 and
washed with water, satd aq NaHCO3, and water. The organic phase
was dried over MgSO4 and concentrated in vacuo, and then puri-
fied by an ODS HPLC (MeOH/H2O/formic acid; flow rate, 2 mL/
min; UV detection at 254 nm) to obtain 21 (2.4 mg, 60.0%): ½a D27
ꢀ
ꢁ38 (c 0.1, MeOH); IR (film) 1740 and 1710 cmꢁ1
;
1H NMR
(CD3OD) d 7.61 (dd, 7.6, 7.6, H-20, 60), 7.55 (dd, 7.6, 7.6, H-40),
7.39 (d, 7.6, H-30, 50), 7.05 (d, 2.5, H-9), 6.57 (d, 8.6, H-12), 6.42
(dd, 2.5, 8.6, H-11), 5.66 (q, 6.9, H-19), 5.49 (br s, H-5), 4.78 (d,
10.9, H-17), 4.24 (d, 10.9, H-17) 4.12 (m, H-21), 3.70 (s, –OMe),
3.64 (br s, H-3, 15), 3.41 (s, –OMe), 3.35 (d, 15.0, H-6), 2.94 (s, –
NMe), 2.53 (d, 15.0, H-6), 2.29 (d, 15.1, H-14), 2.19 (d, 15.1, H-
14), 1.70 (d, 6.92, H-18); HRESIMS m/z 517.2323 [calcd for
C30H33N2O6 (M+H)+, 517.2339].
3.3.8. Conversion of 7 to its hydroxy derivative (22)
A solution of boron tribromide in CH2Cl2 (1.0 M, 8.1
added dropwise to stirred solution of (1.1 mg) in CH2Cl2
(50 L), stirring being continued for 15 min at 0 °C. The reaction
lL) was
3.3.1. Alstiphyllanine E (1)
Brown amorphous solid; ½a D26
ꢀ
ꢁ93 (c 1.0, MeOH); IR (film) mmax
7
3390, 1740, and 1680 cmꢁ1; UV (MeOH) kmax 291 (
e 4700), 264
l
(6200), and 204 (27,000) nm; 1H and 13C NMR data (Tables 1 and
mixture was quenched with water and diluted with EtOAc. The or-
ganic layer was successively washed with water and brine, dried
with MgSO4, and concentrated in vacuo. The residue was
chromatographed on an ODS HPLC (MeOH/H2O/formic acid,
55:45:0.1; flow rate, 2 mL/min; UV detection at 254 nm) to give
2); ESIMS m/z 533 (M+H)+; HRESITOFMS m/z 533.2272 [(M+H)+,
D
ꢁ1.6 mmu, calcd for C30H33N2O7, 533.2288].
compound 22 (0.3 mg, 30.3 %): ½a D27
ꢀ
ꢁ78 (c 0.1, MeOH); IR (film)
3.3.2. Alstiphyllanine F (2)
Brown amorphous solid; ½a D26
ꢀ
ꢁ32 (c 1.0, MeOH); IR (film) mmax
6800) and 204
3420, 1740, and 1710 cmꢁ1 1H NMR (CD3OD) d 7.00 (d, 8.4, H-
;
3420, 1740, and 1680 cmꢁ1; UV (MeOH) kmax 245 (
e
9), 6.87 (s, H-20, 60), 6.58 (d, 2.5, H-12), 6.51 (dd, 8.4, 2.5, H-11),
5.74 (q, 7.7, H-19), 5.41 (br s, H-5), 4.53 (d, 11.4, H-17), 4.25 (d,
(28,000) nm; 1H and 13C NMR data (Tables 1 and 2); ESIMS m/z 593

H. Arai et al. / Bioorg. Med. Chem. 18 (2010) 2152–2158
2157
11.4, H-17), 4.19 (br s, H-3), 3.72 (br s, H-15), 3.99 (m, H-21), 3.70
(s, –OMe), 3.30 (m, H-6), 2.95 (s, –NMe), 2.60 (d, 15.9, H-6), 2.34 (d,
16.1, H-14), 2.28 (d, 16.1, H-14), 1.72 (d, 7.7, H-18); HRESIMS m/z
551.2052 [calcd for C29H31N2O9(M+H)+, 551.2030].
14.6, H-21), 3.72 (s, –OMe), 3.70 (s, –OMe), 3.44 (br s, H-3), 3.36
(m, H-21), 3.35 (m, H-15), 3.30 (m, H-6), 2.89 (s, –NMe), 2.36
(dd, 14.4, 2.8, H-6), 2.09 (d, 15.4, H-14), 2.00 (d, 15.4, H-14), 1.64
(d, 7.3, H-18), 1.54 (s, –COCH3); HRESIMS m/z 455.2161 [calcd for
C25H31N2O6(M+H)+, 455.2182].
3.3.9. Conversion of 6 to Its N(4)-oxide derivative (23)
To a solution of 6 (2.8 mg) in CHCl3 (0.3 mL) was added m-CPBA
3.3.13. Conversion of 3 to its propionate derivative (27)
(1.0 mg) in CHCl3 (300
lL) and the mixture was kept at 4 °C for
To a solution of 3 (1.6 mg) in CH2Cl2 (0.05 mL) was added pro-
10 min. After evaporation, the residue was applied to a silica gel
pionic anhydride (3 lL), and DMAP (1.2 mg) in CH2Cl2 (50 lL) and
column (CHCl3/MeOH, 9:1) to give 23 (1.0 mg, 34.8 %): ½a D27
ꢀ
ꢁ14
the solution was stirred at room temperature. The mixture was di-
luted with CHCl3 and washed with water, satd aq NaHCO3, and
water. The organic phase was dried over MgSO4 and concentrated
in vacuo and then purified by an ODS HPLC (MeOH/H2O/formic
acid; flow rate, 2 mL/min; UV detection at 254 nm) to obtain 27
(c 0.5, MeOH); IR (film) 1740 and 1710 cmꢁ1 1H NMR (CD3OD) d
;
7.24 (dd, 8.5, 2.0, H-50), 7.11 (d, 2.0, H-20), 7.03 (d, 2.6, H-9), 6.93
(d, 8.5, H-50), 6.56 (d, 8.6, H-12), 6.40 (dd, 8.6, 2.6, H-11), 5.69 (q,
6.7, H-19), 5.05 (br s, H-5), 4.81 (d, 11.1, H-17), 4.34 (d, 16.4, H-
21) 4.16 (d, 16.4, H-21), 4.03 (d, 3.2, H-3), 3.89 (s, –OMe), 3.88 (s,
–OMe), 3.74 (s, –OMe), 3.60 (br s, H-15), 3.34 (s, –OMe), 3.30 (m,
H-6), 2.95 (s, –NMe), 2.50 (m, H-6), 2.47 (m, H-14), 2.25 (d, 15.8,
H-14), 1.72 (dd, 6.7, 2.3, H-18); HRESIMS m/z 593.2522 [calcd for
C32H37N2O9(M+H)+, 593.2499].
(0.2 mg, 60.0%). ½a D27
ꢀ
ꢁ143 (c 0.1, MeOH); IR (film) 1740 cmꢁ1
;
1H NMR (CD3OD) d 7.02 (d, 2.6, H-2), 6.80 (dd, 8.6, 2.6, H-11),
6.70 (d, 8.6, H-12), 5.74 (q, 6.5, H-19), 5.55 (br s, H-5) 4.52 (d,
11.2, H-17), 4.21 (m, H-21), 3.98 (m, H-21), 3.93, (d, 11.2, H-17),
3.75 (m, H-3), 3.74 (s, –OMe), 3.73 (s, –OMe), 3.64 (br s, H-15),
3.23 (d, 15.5, H-6), 2.95 (s, –NMe), 2.61 (d, 15.5, H-6), 2.32 (d,
14.6, H-14), 2.21 (d, 14.6, H-14), 1.84 (m, H-10), 1.72 (d, 6.5, H-
18), 0.85 (t, 7.5, H-20); HRESIMS m/z 469.2352 [calcd for
C26H33N2O6(M+H)+, 469.2339].
3.3.10. Conversion of 7 to its N(4)-oxide derivative (24)
To a solution of 7 (1.0 mg) in CHCl3 was added m-CPBA (1.6 mg)
in CHCl3 (300 lL) and the mixture was kept at 4 °C for 10 min.
After evaporation, the residue was applied to a silica gel column
(CHCl3/MeOH, 9:1) to give 24 (1.0 mg, 34.8 %): ½a D27
ꢀ
ꢁ24 (c 0.5,
3.3.14. Conversion of 3 to its benzyl ether derivative (28)
MeOH)); IR (film) 1730 and 1720 cmꢁ1
;
1H NMR (CDCl3) d 7.08
To a solution of 3 (2.7 mg) in dry CH2Cl2 (53
lL) were added tri-
(d, 2.6, H-12), 6.89 (s, H-20, 50), 6.52 (d, 8.6, H-9), 6.52 (d, 8.6, H-
9), 6.52 (d, 8.6, H-12), 6.36 (dd, 8.6, 2.6, H-11), 5.64 (q, 7.12, H-
19), 5.21 (br s, 3.54, H-5), 4.80 (d, 10.9, H-17), 4.44 (d, 16.7, H-
21) 4.32 (d, 16.7, H-21), 4.19, (d, 2.4, H-3), 4.06 (d, 10.9, H-17),
3.91 (s, –OMe), 3.88 (s, –OMe), 3.72 (s, –OMe), 3.45 (br s, H-15),
3.34 (s, –OMe), 3.30 (m, H-6), 3.00 (s, –NMe), 2.58 (dd, 15.6, 3.5,
H-6), 2.54 (d, 15.7, H-14), 2.20 (d, 15.7, H-14), 1.69 (dd, 7.0, 2.0,
H-18); HRESIMS m/z 623.2624 [calcd for C33H39N2O10(M+H)+,
623.2605].
ethylamine (1.27 L), benzyl bromide (0.93 l
l
L), and DMAP
(0.4 mg). The reaction mixture was heated for 3 h, then cooled to
room temperature and diluted with CHCl3. The organic phase
was washed twice with an aqueous solution of NaHCO3 and once
with water. The organic phase was dried Na2SO4 and concentrated
in vacuo. The residue was chromatographed on an ODS HPLC
(MeOH/H2O/formic acid, 61:39:0.1; flow rate, 2 mL/min; UV detec-
tion at 254 nm) to give 28 (0.6 mg, 18.2%): ½a D27
ꢁ4.6 (c 0.5, MeOH);
ꢀ
IR (film) 1730 cmꢁ1 1H NMR (CD3OD) d 7.64 (d, 7.8, H-30, 70), 7.57
;
(m, H-40, 50, 60), 6.85 (m, H-9, 12), 6.78 (d, 9.5, H-11), 5.65 (m, H-5),
5.62 (m, H-19), 4.61 (s, H-10), 4.45 (d, 16.1, H-21), 4.41 (s, H-3), 3.97
(d, 16.1, H-21), 3.77, (m, H-15) , 3.74 (s, –OMe), 3.72 (s, –OMe), 3.66
(d, 17.5, H-17), 3.61 (d, 17.5, H-17), 3.30 (m, H-6), 3.03 (s, –NMe),
2.54 (dd, 16.6, 3.7, H-6), 2.38 (m, H-14), 1.65 (d, 5.5, H-18); HRE-
SIMS 503.2535 [calcd for C30H35N2O5(M+H)+, 503.2546].
3.3.11. Conversion of 3 to its cinnamoyl derivative (25)
Compound
DMAP (5.7 mg), were combined with CH2Cl2 (100
hexylcarbodiimide (DCC) (23.5 mg) in CH2Cl2 (50
3
(13.9 mg), hydrocinnamic acid (5.8 mg), and
L). 1,3-Dicyclo-
L) was added
l
l
dropwise over 10 min at 0 °C. The solution was warmed to room
temperature and stirred overnight. The reaction mixture was par-
titioned with CHCl3 and 1 N aq HCl, 10 % aq NaHCO3, and water.
The combined organic extract was dried (Na2SO4) and concen-
trated in vacuo and then purified by an ODS HPLC (MeOH/H2O/for-
mic acid, 60:40:0.1; flow rate, 2 mL/min; UV detection at 254 nm)
3.3.15. Uptake of Methyl-
a-D-glucopyranoside in cultured cells
expressing SGLT1 or SGLT218
COS-1 cells were cultured at 37 °C in Dulbecco’s modified Ea-
gle’s/Ham’s F-12 medium (1:1) supplemented with 10% fetal calf
serum. For the uptake assay, the cells were plated at 1 ꢂ 105
to obtain compound 25 (0.7 mg, 3.8%): ½a D27
ꢁ49 (c 0.5, MeOH); IR
ꢀ
(film) 1740 and 1710 cmꢁ1 1H NMR (CD3OD) d 7.52 (m, H-40, 80),
;
7.41 (m, H-50, 60, 70), 7.24 (d, 16.1, H-20), 7.08 (s, H-9), 6.58 (s, H-
11, 12), 5.97 (d, 16.1, H-10), 5.52 (q, 7.4, H-19), 4.98 (m, H-5),
4.65 (d, 10.9, H-17), 4.08 (d, 10.9, H-17), 3.83 (m, H-21), 3.79 (m,
H-3), 3.71 (s, –OMe), 3.68 (m, H-21), 3.48 (s, –OMe), 3.48 (m, H-
15), 3.30 (m, H-6), 2.90 (s, –NMe), 2.39 (d, 14.6, H-6), 2.12 (d,
14.3, H-14), 2.04 (d, 14.3, H-14), 1.65 (d, 7.4, H-18); HRESIMS m/
z 543.2490 [calcd for C32H35N2O6(M+H)+, 543.2495].
cells/24-well plate (Asahi Techno Glass, Tokyo, Japan), and 1 lg
of each transporter plasmid was transfected into subconfluent cul-
tures of COS-1 cells using Lipofectamine 2000 (Invitrogen). The
cells were used 2–3 days after transfection. They were incubated
in a pretreatment buffer [140 mM NaCl, 2 mM KCl, 1 mM CaCl2,
1 mM MgCl2, and 10 mM Hepes/Tris (pH 7.5)] with a test sample
at 37 °C for 30 min. An uptake solution containing 80 mM
methyl-
a
-
D
-glucopyranoside
and
4
l
Ci/mL
methyl
a-D-
[U-14C]glucopyranoside was then added into each well and the
mixture was incubated at 37 °C for 30 min. Following incubation,
the plates were washed three times with cold stop buffer
[140 mM choline chloride, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2,
3.3.12. Conversion of 3 to its acetylate derivative (26)
Compound 3 (1.0 mg), acetic anhydride (7.5
lL), triethylamine
(2.5 L), and DMAP (0.5 mg) in CH2Cl2 (50 L) was stirred at room
l
l
temperature for 1.5 h. The reaction mixture was partitioned with
CHCl3 and 10 % aq NaHCO3. The combined organic extract was con-
centrated in vacuo and then purified by a silica gel column (CHCl3/
and 10 mM Hepes/Tris (pH 7.5)] containing 300 lM phlorizin.
The cells were then solubilized with 0.1 M NaOH, and their radio-
activity was measured with a liquid scintillation counter (3100TR,
Perkin–Elmer). Phlorizine was used as a standard drug for this bio-
assay and its IC50 values were 0.2 and 0.1 mM against SGLT1 and
SGLT2, respectively.
MeOH, 1:0–0:1) to obtain compound 26 (0.8 mg, 73.4%). ½a D27
ꢁ32
ꢀ
(c 0.5, MeOH); IR (film) 1740 cmꢁ1; 1H NMR (CD3OD) d 7.02 (d, 2.6,
H-12), 6.73 (dd, 8.6, 2.6, H-11), 6.61 (d, 8.6, H-9), 5.51 (q, 7.3, H-19),
4.94 (m, H-5) 4.53 (d, 11.0, H-17), 3.86, (d, 11.0, H-17), 3.78 (d,

2158
H. Arai et al. / Bioorg. Med. Chem. 18 (2010) 2152–2158
G. C.; Steele, J. C. P.; Houghton, P. J. Planta Med. 1999, 65, 690–694; (d) Husain,
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This work was supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science,
and Technology of Japan and a grant from The Open Research
Center Project in Hoshi University.
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