Anti-HIV benzylisoquinoline alkaloids and flavonoids from the leaves of Nelumbo nucifera, and structure-activity correlations with related alkaloids

Article abstract of DOI:10.1016/j.bmc.2004.10.020

(+)-1(R)-Coclaurine (1) and (-)-1(S)-norcoclaurine (3), together with quercetin 3-O-β-d-glucuronide (4), were isolated from the leaves of Nelumbo nucifera (Nymphaceae), and identified as anti-HIV principles. These compounds can serve as new leads for furt

Full text of DOI:10.1016/j.bmc.2004.10.020

Bioorganic & Medicinal Chemistry 13 (2005) 443–448
Anti-HIV benzylisoquinoline alkaloids and flavonoids from
the leaves of Nelumbo nucifera, and structure–activity
correlations with related alkaloids
Yoshiki Kashiwada,a,* Akihiro Aoshima,a Yasumasa Ikeshiro,a Yuh-Pan Chen,b
Hiroshi Furukawa,c Masataka Itoigawa,d Toshihiro Fujioka,e Kunihide Mihashi,e
L. Mark Cosentino,f Susan L. Morris-Natschkeg and Kuo-Hsiung Leeg,*
aFaculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata 950-2081, Japan
bBrion Research Institute of Taiwan, Taipei, Taiwan 100, Republic of China
cFaculty of Pharmacy, Meijo University, Tempaku-ku, Nagoya 468, Japan
dTokai Gakuen University, Miyoshi, Aichi 470-0207, Japan
eFaculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka 814-0180, Japan
fBBI-Biotech Research Laboratories, Perry Parkway, Gaithersburg, MD 2087, USA
gNatural Products Laboratory, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
Received 22 April 2004; accepted 6 October 2004
Available online 2 November 2004
Abstract—(+)-1(R)-Coclaurine (1) and (ꢀ)-1(S)-norcoclaurine (3), together with quercetin 3-O-b-D-glucuronide (4), were isolated
from the leaves of Nelumbo nucifera (Nymphaceae), and identified as anti-HIV principles. Compounds 1 and 3 demonstrated potent
anti-HIV activity with EC50 values of 0.8 and <0.8lg/mL, respectively, and therapeutic index (TI) values of >125 and >25, respec-
tively. Compound 4 was less potent (EC50 2lg/mL). In a structure–activity relationship study, other benzylisoquinoline, aporphine,
and bisbenzylisoquinoline alkaloids, including liensinine (14), negferine (15), and isoliensinine (16), which were previously isolated
from the leaves and embryo of Nelumbo nucifera, were evaluated for anti-HIV activity. Compounds 14–16 showed potent anti-HIV
activities with EC50 values of <0.8lg/mL and TI values of >9.9, >8.6, and >6.5, respectively. Nuciferine (12), an aporphine alkaloid,
had an EC50 value of 0.8lg/mL and TI of 36. In addition, synthetic coclaurine analogs were also evaluated. Compounds 1, 3, 12,
and 14–16 can serve as new leads for further development of anti-AIDS agents.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
<20lg/mL, TI > 5). Bioassay-guided fractionation and
repeated chromatography of this extract has led to the
isolation and identification of (+)-1(R)-coclaurine (1)2
and (ꢀ)-1(S)-norcoclaurine (3),3 together with quercetin
3-O-b-D-glucuronide (4),4 as anti-HIV principles. We re-
port herein the anti-HIV activity of 1, 3, and 4, as well as
related alkaloids isolated from N. nucifera and synthetic
coclaurine analogs.
Nelumbo nucifera Gaertn. (Nymphaceae) is a perennial
aquatic crop grown and consumed throughout Asia.
All parts of N. nucifera have been used for various
medicinal purposes in oriental medicine. In particular,
the leaves are known for diuretic and astringent proper-
ties, and are used to treat fever, sweating, and strangury
and as a styptic.1 During our continuing search for
plant-derived anti-HIV agents from natural products,
the 95% EtOH extract of the leaves of N. nucifera was
found to display significant anti-HIV activity (EC50
2. Results and discussion
The leaves of N. nucifera were extracted successively
with n-hexane, CHCl3 and 95% EtOH, and anti-HIV
activity was found in the 95% EtOH extract
(EC50 < 20lg/mL). The 95% EtOH extract was further
partitioned between n-BuOH and water yielding an
Keywords: Anti-HIV; Nelumbo nucifera; Benzylisoquinoline alkaloid;
Flavonoid.
*
Corresponding authors. Tel.: +81 25 269 3140; fax: +81 25 268
1230; e-mail: kasiwada@niigata-pharm.ac.jp
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.10.020

444
Y. Kashiwada et al. / Bioorg. Med. Chem. 13 (2005) 443–448
anti-HIV active n-BuOH-soluble fraction. Subsequent
bioassay-guided fractionation and repeated chromato-
graphy of this fraction led to the isolation of two benzyl-
isoquinoline alkaloids (1 and 3) and a flavonoid
glycoside (4) as anti-HIV principles. Compounds 1 and
3 were identified as (+)-1(R)-coclaurine (1)2 and (ꢀ)-
1(S)-norcoclaurine (3),3 respectively, by comparison of
their physical and spectral data with those reported in
the literature. Although the enantiomer of 3, (+)-1(R)–
norcoclaurine, was previously isolated from the embryo
of N. nucifera5 this is the first example of the isolation of
(ꢀ)-1(S)-norcoclaurine (3) from the leaves of N. nuci-
fera. The major component of the leaves of N. nucifera
was identified as the flavonoid quercetin 3-O-b-D-glu-
curonide (4) by comparison of its physical and spectral
data with those reported in the literature.4 In addition,
(ꢀ)-1(R)-N-methylcoclaurine (2),6 quercetin 3-O-b-D-
xylopyranosyl-(1!2)-b-D-galactopyranoside (5),7 rutin
(6),8 isoquecitrin (7),8 and hyperin (8)8 were also isolated
and identified from the 95% EtOH extract (Fig. 1).
with an EC50 value of 4lg/mL and a TI of >25, while
the remaining quercetin glycosides (6–8) showed no
anti-HIV activity. As quercetin itself also did not show
anti-HIV activity (IC50 7.24lg/mL, EC50 no suppres-
sion), the glycosyl moiety bound to the C-3 hydroxyl
group might play an important role in the anti-HIV
activity.
Based on the data of compounds 1–3, benzylisoquino-
line-type [d,l-armepavine (9),9 and lotusine (10)],4 apor-
phine-type [roemerine (11),10 nuciferine (12),10 and
nornuciferine (13)],10 and bisbenzylisoquinoline-type
[liensinine (14),11 neferine (15),12 and isoliensinine
(16)]11 alkaloids, which were previously isolated from
the leaves and embryo of Nelumbo nucifera, were evalu-
ated for anti-HIV activity in a structure–activity rela-
tionship study. Lotusine (10), a quaternary amine
alkaloid, showed low cytotoxicity (IC50 > 100lg/mL),
but also was not a potent anti-HIV agent. Similar to
2, the N-methyl derivative d,l-armepavine (9) showed
good anti-HIV activity (EC50 < 0.8lg/mL), but was also
cytotoxic (IC50 1.77lg/mL). Bisbenzylisoquinoline-type
alkaloids 14–16 showed potent anti-HIV activities with
EC50 values of <0.8lg/mL, and they were less cytotoxic
than 2 or 9, although they had the same partial struc-
tures. In addition, the aporphine-type alkaloids 11–13,
which are N-methyl derivatives similar to 2 and 9, were
also less cytotoxic as compared with 2 and 9. Nuciferine
(12) and nornuciferine (13) demonstrated potent anti-
HIV activity with EC50 values of 0.8 and <0.8lg/mL,
respectively, and TI values of 36.3 and >44, respectively.
Roemerine (11) showed comparable anti-HIV activity
Alkaloids 1 and 3 demonstrated potent anti-HIV activ-
ity with EC50 values of 0.8 and <0.8lg/mL, respectively,
and therapeutic index (TI) values of >125 and >25,
respectively. Although (ꢀ)-1(R)-N-methyl coclaurine
(2)
showed
comparable
anti-HIV
activity
(EC50 < 0.8lg/mL), N-methylation led to increased
cytotoxicity (IC50 1.45lg/mL for 2 compared with
>100 lg/mL for 1). Quercetin glucuronide 4 showed
moderate anti-HIV activity (EC50 2lg/mL) and low
cytotoxicity (IC50 >100lg/mL), resulting in a TI value
of >50. Flavonoid 5 also showed weak anti-HIV activity
R
HOOC
O
O
4
OH
HO
OH
H3CO
HO
O
HO
O
5
6
N
OH
HO
HO
R
H
O
O
R
H
CH3
OH
OH
OH
1
2
HO
HO
O
O
OH
O
HO
O
O
O
R
OH
OH
CH3
OHOH
HO
HO
HO
OH
OH
N
H
O
H
O
OH
7
8
3
HO
OH
HO
HO
HO
O
O
OH
OH
Figure 1. Structures of alkaloids (1–3) and flavonoids (4–8) isolated from the leaves of N. nucifera.

Y. Kashiwada et al. / Bioorg. Med. Chem. 13 (2005) 443–448
445
(EC50 0.84lg/mL), but had a lower TI value (TI 7.8)
3. Experimental
than 12 and 13. These results suggested that N-methyl
benzylisoquinoline-type alkaloids (such as 9) were more
cytotoxic, whereas oxidative coupling at C-8 (bisbenzyl-
isoquinoline-type, such as 12) or dimerization at C-20
(aporphine-type, such as 14) yielded less cytotoxic
compounds.
3.1. General experimental procedures
Melting points were measured on a Yanako micro
melting point apparatus, and are uncorrected. Optical
rotations were measured on a Perkin–Elmer 241 pola-
rimeter. Mass spectra were determined on a JEOL
1
To investigate the effect of various substitutions in the
benzyl ring, racemic coclaurine analogs were prepared
according to a previously reported procedure13 and evalu-
ated for anti-HIV activity. Compounds with additional
OMe groups (17, 18) and with halogen rather than
OMe (19–21) were inactive. Compounds 26 and 27,
which are N-benzyl-7-benzyl derivatives of the 40-fluoro
and -chloro analogs 20 and 21, showed weak anti-HIV
activity with EC50 values of 5.3 and 3.2lg/mL, but also
had low TI values (Fig. 2).
HX-110 spectrometer. H and 13C NMR spectra were
measured on JEOL JNM-A-400 and JNM-FX200 spec-
trometers with TMS as an internal standard.
3.2. Isolation of alkaloids and flavonoids from the leaves
of Nelumbo nucifera
The dried leaves of N. nucifera (3.0kg) were extracted
successively with n-hexane, CHCl3, and 95% EtOH,
three times at reflux for 2h. The combined extracts were
HO
R1O
H3CO
CH3
CH3
N
N
H
N
H3CO
R2O
CH3
H3CO
HO
CH3
H
9
10
HO
HO
R1
CH2
R2
11
12 CH3 CH3
13
H
CH3
OCH3
OR1
H3CO
N
N
H3C
O
CH3
H
H
OR2
R3O
R1
R2 R3
14 CH3
H
H
15 CH3 CH3
H
16
H
H
CH3
H3CO
H3CO
NH
N
Ph
HO
R1
BnO
R1
R2
R2
R3
R3
R1
R2
R3
17 OCH3 OH
H
23
24
25
26
27
28
18 OCH3 OCH3 OCH3
19
20
21
H
H
H
Br
Br
Cl
Cl
H
H
H
H
22 Cl
Figure 2. Structures for coclaurine related alkaloids of N. nucifera (9–16) and synthesized benzylisoquinoline alkaloids (17–28).

446
Y. Kashiwada et al. / Bioorg. Med. Chem. 13 (2005) 443–448
concentrated under reduced pressure to give hexane
(15g), CHCl3 (90g), and 95% EtOH extracts (460g).
The 95% EtOH extract was further partitioned between
n-BuOH and water yielding n-BuOH-soluble fraction,
which was subsequently subjected to Diaion HP20 chro-
matography. Elution with H2O containing increasing
amounts of MeOH gave five fractions, Frs. I–V. Frac-
tion II consisted mainly of compound 4, which was crys-
tallized from 0.5M HCl to yield 1.0g of pure compound.
The mother liquor was chromatographed over MCI-gel
CHP20P [H2O–MeOH (1:0!0:1)] to give compounds 1
(175mg) and 3 (87mg). Fraction III was further fract-
ionated by MCI-gel CHP20P chromatography with
H2O–MeOH (1:0!0:1) to give fractions III-1–III-3.
Subsequent fractionation of Fr. III-1 by Sephadex
LH-20 (50% MeOH–80% MeOH) chromatography gave
two fractions (frs. III-1-1 and III-1-2). Fraction III-1-1
was chromatographed over Toyopearl HW40F [H2O–
MeOH (1:0!0:1)], and then separated by preparative
scale HPLC on Nova Pak HRC18 [MeOH–2% AcOH
(2:3)] to furnish compounds 5 (1.45g) and 6 (60mg).
After crystallization of compound 4 (2.0g) from fraction
III-1-2 by treating with 0.5M HCl, the mother liquor
was further chromatographed on MCI-gel CHP20P
[H2O–MeOH (1:0!0:1)] to give compound 1 (100mg).
Column chromatography of Fraction III-2 over Sepha-
dex LH-20 [H2O–MeOH (1:0!0:1)], followed by crys-
tallization gave compound 4 (3.2g). The mother liquor
was subsequently chromatographed on MCI-gel
CHP20P [H2O–MeOH (1:0!0:1)] to furnish com-
pounds 3 (420mg) and 2 (28mg). Column chromatogra-
phy of Fraction IV over MCI-gel CHP20P [H2O–MeOH
(7:3!0:1)] and subsequent preparative scale HPLC on
Nova Pak HRC18 [MeOH–2% AcOH (2:3)] afforded
compounds 7 (330mg) and 8 (280mg). All compounds
were identified by comparison with literature physical
and spectral data. Complete data are given only for 3,
(+)-1(R)–norcoclaurine, isolated first herein from this
plants source as the R enantiomer.
3.2.5. Quercetin 3-O-b-D-xylopyranosyl-(1!2)-b-D-gal-
16
D
actopyranoside (5). Yellow amorphous powder; ½aꢁ
ꢀ101.5 [c 0.67, MeOH–H2O (1:1)].
3.2.6. Rutin (6). Pale yellow needles, mp 193–194°C (de-
16
comp.), ½aꢁ +12.3 (c 0.7, MeOH).
D
3.2.7. Isoquecitrin (7). Pale yellow needles, mp
16
D
175–177°C, ½aꢁ ꢀ134.6 [c 0.52, MeOH–H2O (1:1)].
3.2.8. Hyperin (8). Pale yellow needles, mp 221–222°C,
16
½aꢁ ꢀ392.9 [c 0.28, MeOH–H2O (1:1)].
D
3.3. General procedure for preparation of coclaurine
analogs
The racemic coclaurine analogs were prepared accord-
ing to the procedure reported previously.13 Thus, a solu-
tion of an appropriate substituted phenylacetic acid
(0.6–1.2mmol) and thionyl chloride (2.8–6.9mmol) in
dry benzene (10mL) was refluxed for 2–4h. After
removing solvent in vacuo, the residue was dissolved
in CH2Cl2 (8mL) and treated with N-benzyl-b-(3-meth-
oxy-4-benzyloxy)-phenylamine (0.4–0.8mmol) in the
presence of triethylamine (3mL) for 1–2h with stirring.
The reaction mixture was diluted with CHCl3 and
washed with H2O, dried over Na2SO4, and concen-
trated. The residue was purified by silica gel chromato-
graphy to give an amide (60–95% yield). A mixture of
amide (0.26–0.73mmol) and phosphorus oxychloride
(2.7–8.7mmol) in dry toluene (10mL) was refluxed for
1–5h. The reaction mixture was concentrated to give
an oily residue, which was washed with petroleum ether.
A solution of the residue in MeOH (10mL) was treated
with NaBH4 (2.6–8.5mmol) for 1h with stirring. After
addition of acetone (1mL), the reaction mixture was di-
luted with 5% NH4OH (30mL), and then extracted with
CHCl3 three times. The combined CHCl3 extract was
washed with H2O, dried over Na2SO4, and concentrated
under reduced pressure. The product was purified by sil-
ica gel chromatography (hexane–EtOAc or benzene–
EtOAc) to give the N-benzyl coclaurine analog benzyl
ether (yield 75–95%), which was deprotected by hydro-
genolysis. A mixture of N-benzyl coclaurine analog ben-
zyl ether (0.2–0.52mmol), Pd–C (23–130mg), and
trifluoroacetic acid (0.3mL) in EtOH (6mL) was stirred
under a hydrogen atmosphere overnight. After removal
of the catalyst by filtration, the filtrate was concentrated
to a syrup. The product was crystallized from acetone
solution (55–96% yield).
3.2.1. (+)-1(R)-Coclaurine (1). Colorless needles; mp
222–224°C; [a]D +4.8 (c 0.48, MeOH).
3.2.2. (ꢀ)-1(R)-N-Methylcoclaurine (2). Colorless plates;
16
mp 135–137°C; ½aꢁ ꢀ115 (c 0.6, MeOH).
D
3.2.3. (ꢀ)-1(S)-Norcoclaurine (3). Colorless prisms; mp
16
D
1
239–242°C (decomp.); ½aꢁ ꢀ39.6 (c 0.63, MeOH); H
NMR (acetone–d6+D2O, 400MHz) d 2.93, 3.06 (each
1H, dt, J = 6, 17.5Hz, H2–4), 3.18 (1H, dd, J = 8,
14.5Hz, H-70), 3.39 (1H, dd, J = 6, 14.5Hz, H-70),
3.40, 3.56 (each 1H, dt, J = 6, 13Hz, H2–3), 4.70 (1H,
dd, J = 6, 8Hz, H-1), 6.70, 6.72 (each 1H, s, H-5, 8),
6.83, 7.21 (each 2H, d,J = 8.5Hz, H-20, 30); 13C NMR
(acetone–d6+D2O, 100MHz) d 39.6 (C-70), 40.4 (C-3),
57.0 (C-1), 114.0 (C-5), 115.9 (C-8), 116.4 (C-30,50),
123.4 (C-5a), 123.7 (C-8a). 126.7 (C-10), 131.5 (C-
20,60), 144.8 (C-6), 145.7 (C-7), 157.3 (C-40).
3.3.1. 1-(30-Methoxy-40-hydroxybenzyl)-6-methoxy-7-hydr-
oxy-1,2,3,4-tetrahydroisoquinoline (17). White pow-
der, mp 183–185°C; 1H NMR (400MHz, CD3OD):
2.93–3.08 (3H, m, H2-4 and H-3), 3.25 (1H, dd,
J = 8.5, 14.5Hz, H-70), 3.36 (1H, dd, J = 5.5, 14.5Hz,
H-70), 3.46 (1H, m, H-30), 3.82, 3.84 (each 3H, s,
OCH3), 4.63 (1H, dd, J = 5.5, 8.5Hz, H-1), 6.67 (1H,
s, H-5), 6.77 (1H, s, H-8), 6.74 (1H, dd, J = 1.7,
7.8Hz, H-60), 6.80 (1H, d, J = 7.8Hz, H-50), 6.83 (1H,
d, J = 1.7Hz, H-20); FABMS m/z 316 (M+H)+; HR-
FABMS (positive) m/z 316.1547 (calcd for C18H22O4N,
316.1549).
3.2.4. Quercetin 3-O-b-D-glucuronide (miquelianin) (4).
16
D
Pale yellow needles; mp 190–192°C; ½aꢁ ꢀ24.2 (c 0.66,
MeOH).

Y. Kashiwada et al. / Bioorg. Med. Chem. 13 (2005) 443–448
447
3.3.2.
1-(30,40,50-Trimethoxy)-6-methoxy-7-hydroxy-
1,2,3,4-tetrahydroisoquinoline (18). Colorless needles,
3.3.8. N-Benzyl-1-(40-chlorobenzyl)-6-methoxy-7-benzyl-
oxy-1,2,3,4-tetrahydroisoquinoline (27). A white amor-
phous powder, 1H NMR (200MHz, CDCl3): 2.4–3.4
(4H in total, m, H2-3,4), 2.72, 2.90 (1H, dd, J = 6.5,
14Hz, H2-70), 3.68 (1H, t, J = 6.5Hz, H-1), 3.73 (2H,
s, N-benzyl CH2), 3.86 (3H, s, OCH3), 4.91 (2H, s, ben-
zyl CH2), 6.19 (1H, s, H-5), 6.61 (1H, s, H-8), 6.91 (2H,
d, J = 8.5Hz, H-30,50), 7.24(1H, d, J = 8.5Hz, H-20,60),
7.15–7.40 (10H in total, m, aromatic H); FABMS m/z
484 (M+H)+.
1
mp 144–147° C; H NMR (200MHz, CD3OD): 2.91–
3.18 (3H, m, H2-4 and H-3), 3.26 (1H, m, H-30), 3.32–
3.48 (2H,m, H2-7), 3.75, 3.84 (each 3H, s, OCH3), 3.81
(6H, s, OCH3), 4.70 (1H, t, J = 7.0Hz, H-1), 6.61 (2H,
s, H-20,60), 6.69 (1H, s, H-5), 6.78 (1H, s, H-8); FABMS
m/z 360 (M+H)+; HR-FABMS (positive) m/z 360.1813
(calcd for C20H26O5N, 360.1811).
3.3.3. 1-(40-Bromobenzyl)-6-methoxy-7-hydroxy-1,2,3,4-
tetrahydroisoquinoline (19). Colorless needles, mp 215–
3.4. Anti-HIV assay
1
217°C; H NMR (200MHz, CD3OD): 2.99–3.15 (2H,
The T cell line, H9, was maintained in continuous cul-
ture with complete medium (RPMI 1640 with 10% fetal
calf serum [FCS] supplemented with L-glutamine) at 5%
CO2 and 37°C. Aliquots of this cell line were only used
in experiments when in log-phase of growth. Test sam-
ples were first dissolved in dimethyl sulfoxide (DMSO).
The following are the final drug concentrations routinely
used for screening: 100, 20, 4, and 0.8lg/mL. For active
agents, additional dilutions are prepared for subsequent
testing so that an accurate EC50 values (see definition
below) could be achieved. As the test samples were being
prepared, an aliquot of H9 cells was infected with HIV-1
(IIIB isolate), while another aliquot was mock-infected
with complete medium. The mock-infected sample was
used for toxicity determinations (IC50, see definition be-
low). The stock virus used for these studies typically had
a TCID50 value of 104 infectious units (IU)/ml. The
appropriate amount of virus for multiplicity of infection
(m.o.i.) between 0.1 and 0.01IU/cell was added to the
first aliquot of cells. The other aliquot of cells received
m, H2-4), 3.08 (1H, dd, J = 8.5, 14.5Hz, H-70), 3.22
(1H, dd, J = 5.5, 14.5Hz, H-70), 3.43–3.66 (2H, m,
H2-3), 3.85 (3H, s, OCH3), 4.71 (1H, dd, J = 5.5,
8.5Hz, H-1), 6.62 (1H, s, H-5), 6.79 (1H, s, H-8), 7.38
(2H, d, J = 8.5Hz, H-30,50), 7.39 (2H, d, J = 8.5Hz,
H-20,60); FABMS m/z 348 (M+H)+, 346; HR-FABMS
(positive) m/z 348.0596 (calcd for C17H19O2NBr,
348.0600).
3.3.4. 1-(40-Fluorobenzyl)-6-methoxy-7-hydroxy-1,2,3,4-
tetrahydroisoquinoline (20). White powder, mp 197–
1
199°C; H NMR (200MHz, CD3OD): 3.02–3.20 (4H,
m, H2-4 and 70), 3.30–3.66 (2H, m, H2-3), 3.85 (3H, s,
OCH3), 4.66 (1H, t, J = 7.5Hz, H-1), 6.58 (1H, s, H-
5), 6.79 (1H, s, H-8), 7.11 (2H, t, J = 8.5Hz, H-30,50),
7.33 (2H, dd, J = 5.5, 8.5Hz, H-20,60); FABMS m/z
288 (M+H)+; HR-FABMS (positive) m/z 288.1398
(calcd for C17H19O2NF, 288.1400).
3.3.5. 1-(40-Chlorobenzyl)-6-methoxy-7-hydroxy-1,2,3,4-
tetrahydroisoquinoline (21). Colorless granules, mp 206–
1
208°C; H NMR (200MHz, CD3OD): 3.03–3.15 (4H,
m, H2-4 and 70), 3.37–3.56 (2H, m, H2-3), 3.84 (3H, s,
OCH3), 4.70 (1H, t, J = 7.5Hz, H-1), 6.61 (1H, s, H-
5), 6.78 (1H, s, H-8), 7.36 (4H, br s, aromatic H);
FABMS m/z 304 (M+H)+; HR-FABMS (positive) m/z
304.1107 (calcd for C17H18O2NCl, 304.1104).
Table 1. Anti-HIV activities for benzylisoquinoline and aporphine
alkaloids and flavonoids from Nelumbo nucifera and synthetic cocla-
urine analogse
Compound
IC50 (lg/mL)a
EC50 (lg/mL)b,c
TId
>125
>1.8
1
>100
1.45
20
0.8
<0.8
<0.8
2.0
2
1-(30,40-Dichlorobenzyl)-6-methoxy-7-hydroxy-
1,2,3,4-tetrahydroisoquinoline (22). Colorless needles,
3
>25
>50
>25
3.3.6.
4
>100
>100
>100
1.77
>100
7.8
1
5
4.0
35.8
mp 189–191°C; H NMR (200MHz, CD3OD): 3.03–
3.15 (2H, m, H2-4), 3.29–3.323.30 (2H, m, H2-70),
3.43–3.66 (2H, m, H2-3), 3.85 (3H, s, OCH3), 4.71
(1H, dd, J = 5.5, 8.5Hz, H-1), 6.60 (1H, s, H-5), 6.80
(1H, s, H-8), 7.28 (1H, dd, J = 2, 8Hz, H-20), 7.37 (1H,
d, J = 8Hz, H-50), 7.55 (1H, d, J = 2Hz, H-20); FABMS
m/z 338 (M+H)+; HR-FABMS (positive) m/z 338.0708
(calcd for C17H18O2NCl2, 338.0714).
6
>2.8
9
<0.8
20.7
>2.2
>4.8
9.3
10
11
12
13
14
15
16
26
27
AZT
0.84
0.8
29.0
35.2
7.97
6.87
5.17
7.5
36.3
< 0.8
<0.8
<0.8
<0.8
5.3
>44
>9.9
>8.6
>6.5
1.4
3.3.7. N-Benzyl-1-(40-fluorobenzyl)-6-methoxy-7-benzyl-
oxy-1,2,3,4-tetrahydroisoquinoline (26). A white amor-
phous powder, 1H NMR (200MHz, CDCl3): 2.4–3.4
(6H in total, m, H2–3,4,70), 3.68(1H, t, J = 7Hz, H-1),
3.74 (2H, s, N-benzyl CH2), 3.86 (3H, s, OCH3), 4.91
(2H, s, benzyl CH2), 6.20 (1H, s, H-5), 6.61 (1H, s, H-
8), 6.91 (1H, dd, J = 3, 8.5Hz, H-20,60), 7.21 (2H, t,
J = 8.5Hz, H-30,50), 7.16–7.36 (10H in total, m, aro-
matic H); FABMS m/z 468 (M+H)+.
11.9
1871
3.2
0.045 0.056d
3.7
41,667
a The agent concentration that inhibited H9 cell growth by 50%.
b The agent concentration that inhibited viral replication in H9 cell by
50%.
c In vitro therapeutic index (TI) ratio: IC50/EC50
.
d This EC50 value represents the mean and standard deviation of 65
experimentally determined EC50 values for AZT.14
e Compounds 7, 8, 17–25, and 28 showed no suppression.

448
Y. Kashiwada et al. / Bioorg. Med. Chem. 13 (2005) 443–448
only culture medium and was then incubated under
identical conditions to the HIV-infected cells. After a
4h incubation at 37°C and 5% CO2, both cell popula-
tions were washed three times with fresh medium and
then added to the appropriate wells of a 24 well-plate
containing various concentrations of the test drug or
culture medium (positive infected control/negative-con-
trol drug). In addition, AZT was also assayed during
each experiment as a positive-control drug. The plates
were incubated at 37°C and 5% CO2 for 4days. Cell-free
supernatants were collected on day 4 and tested by an
in-house p24 antigen ELISA assay. P24 antigen is a core
protein of HIV and, therefore, is an indirect measure of
virus present in the supernatants. Toxicity was deter-
mined by performing cell counts by a coulter counter
on the mock-infected cells, which had either received
culture medium (no toxicity) or test sample or AZT. If
a test sample had suppressive capability and was not
toxic, its effects are reported in the following terms:
IC50, the concentration of test sample that was toxic
to 50% of the mock-infected cells; EC50, the concentra-
tion of the test sample that was able to suppress HIV
replication by 50%; and therapeutic index (TI), the ratio
of IC50 to EC50 (Table 1).
National Institute of Allergies and Infectious Diseases
(K.H.L.).
References and notes
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Acknowledgements
This investigation was supported by the Promotion and
Mutual Aid Corporation for Private Schools of
Japan (Y.K.), as well as Grant AI-33066 from the