Synthesis and biological evaluation of tylophorine-derived dibenzoquinolines as orally active agents: Exploration of the role of tylophorine e ring on biological activity

Article abstract of DOI:10.1021/jm300705j

A series of novel tylophorine-derived dibenzoquinolines has been synthesized and their biological activity evaluated. Three assays were conducted: inhibition of cancer cell proliferation, inhibition of TGEV replication for anticoronavirus activity, and su

Full text of DOI:10.1021/jm300705j

Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of Tylophorine-Derived
Dibenzoquinolines as Orally Active Agents: Exploration of the Role of
Tylophorine E Ring on Biological Activity
Yue-Zhi Lee,† Cheng-Wei Yang,†,‡ Hsing-Yu Hsu,† Ya-Qi Qiu,†,‡ Teng-Kuang Yeh,† Hsin-Yu Chang,†
Yu-Sheng Chao,† and Shiow-Ju Lee*,†
†Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, No. 35 Keyan Road, Zhunan Town,
Miaoli County 350, Taiwan
‡Graduate Program of Biotechnology in Medicine, Institute of Molecular Medicine, National Tsing Hua University, Hsinchu, Taiwan
S
* Supporting Information
ABSTRACT: A series of novel tylophorine-derived dibenzoquinolines has been synthesized and their biological activity
evaluated. Three assays were conducted: inhibition of cancer cell proliferation, inhibition of TGEV replication for anticoronavirus
activity, and suppression of nitric oxide production in RAW264.7 cells (a measure of anti-inflammation). The most potent
compound from these assays, dibenzoquinoline 33b, showed improved solubility compared to tylophorine 9a, in vivo efficacies in
a lung A549 xenografted tumor mouse model and a murine paw edema model, good bioavailability, and no significant
neurotoxicity (as tested by a rota-rod test for motor coordination). This is the first study to explore in detail the role of the
tylophorine E ring on biological activity and very strongly suggests that tylophorine-derived dibenzoquinolines merit further
development into orally active agents.
One class of tylophorine derivatives, the tyloindicine I analogue
7-(4-methoxyphenyl)-6-phenyl-2,3,8,8a-tetrahydroindolizin-
5(1H)-one (4) was proposed to operate through an unknown,
novel mechanism of action.9 Another class of tylophorine
derivatives, the phenanthrene-based 9-N replaced compounds,
e.g., 5, was proposed to exert anticancer activity through a
different mode of action by inactivation of Akt and inhibition of
the NF-κB pathway signaling.10 The direct biological targets of
these molecules, however, remain to be elucidated. Further-
more, although they all exert anticancer activity, their
pharmacophores (for the molecular recognition by biological
macromolecules) are likely differentiated, leading to changes in
targeted molecules or mechanism of action.
INTRODUCTION
■
Tylophorine is a phenanthroindolizidine alkaloid found in
various herbs, some of which, e.g., Tylophora indica, Tylophora
atrofolliculata, and Tylophora ovata, are used in the traditional
medicines of several countries.1−3 Although their direct
molecular targets are not known,2,4 they have been found to
impart a multitude of biological activities, including anti-
cancer,5−7 anti-inflammation,5,6 and anticoronavirus.8 Cancer
cell toxicity is accomplished by interfering in the cell cycle and
inhibiting the signaling of the transcriptional factors NF-κB and
AP1.5,7 Their anti-inflammatory activity was found to arise
through enhanced phosphorylation of Akt and down-regulation
of AP1, thereby suppressing nitric oxide production in LPS/
IFNγ stimulated RAW264.7 cells.5 In TGEV (transmissible
gastroenteritis coronavirus) infected ST cells, they inhibit
coronavirus induced apoptosis and viral replication.8
Development of tylophorine-related novel compounds, e.g.,
seco-tylophorine compounds9,11 and phenanthrene-based 9-N
replaced compounds12,13 (Figure 1), into cytotoxic agents for
In addition to the conventional pentacyclic phenanthroindo-
lizidines and phenanthroquinolizidines, the biological activity of
nonpentacyclic tylophorine derivatives has also been explored.
Received: May 21, 2012
© XXXX American Chemical Society
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Figure 1. Tylophorine and derived compounds.
a
Scheme 1. Synthesis of Tylophorine-Derived Dibenzoquinolines with Varied N-Substitutes
a
Reagents and conditions: (a) R-Br, NaH, DMF; (b) (i) NaI, CH3CN, 130 °C, overnight, (ii) Bu3SnH, AIBN, toluene, reflux, 6 h; (c)
NaAl(OCH2CH2OMe)2H2, dioxane, 120 °C, 2 h; (d) LiAlH4, AlCl3, THF, rt, 4 h; (e) LiAlH4, THF, rt, 48 h, quench, MeOH/H2O (100:1), rt; (f)
PPTS, MeOH, 55 °C, 20 h; (g) H2SO4, CH2Cl2, rt, 20 h.
cancer treatment faces formidable challenges. For example, the
solubility of tylophorine in water is very poor. Also, the
stereochemistry of these compounds affects their potency9,14
and thus isolation of enantiopure samples is needed to clarify
structure−activity relationships and further development.
Furthermore, a clinical trial of tylocrebrine in the 1960s failed
due to the central nervous system (CNS) side effects.15
Increasing the polarity of tylophorine and derived compounds
should prevent them from crossing the blood−brain barrier,
thereby lowering CNS toxicity. Thus, the solubility, polarity,
pharmacokinetic properties, oral availability, neurotoxicity, and
synthetic routes toward these compounds must all be
improved.
series of derivatives bearing modifications at the E ring and/or
differing N-substitutions. All derivatives were submitted for a
variety of tests: anticell growth against a panel of cancer cell
lines, antiviral replication in TGEV infected ST cells detected
by inhibition of TGEV N and S protein expression, and
suppression of nitric oxide production in LPS/IFNγ stimulated
RAW264.7 cells. The counterpart C13a atom of the E ring-
uncyclized derivatives, dibenzoquinolines, is not a stereocenter.
Several potent derivatives possessing the same biological
activities as tylophorine in terms of anticancer cell proliferation,
anti-TGEV activity, and anti-inflammation were discovered and
their structure and activity relationships analyzed. Of them,
dibenzoquinoline 33b exhibited improved solubility compared
to tylophorine 9a, potent in vivo efficacy, and good
bioavailability as an orally active agent without neurotoxicity.
Herein, we investigate the role of the tylophorine E ring
(Figure 1) on biological activity through the synthesis of a
B
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a
Scheme 2. Synthesis of Tylophorine-Derived Dibenzoquinolines with Varied Phenanthrene Substitutes
a
Reagents and conditions: (a) Et3N, Ac2O, 90 °C, 15 h; (b) FeSO4·7H2O, NH4OH, 100 °C, 2 h; (c) i-C5H11ONO, H2SO4, NaI, acetone, 0 °C, 6 h;
(d) BH3·THF, THF, 38 °C, 1 h; (e) PCC, CH2Cl2, rt, 2 h; (f) H2SO4, MeOH, 75 °C, 4 h; (g) FeCl3, CH2Cl2, rt, 5 h; (h) LiAlH4, THF, rt, 3 h; (i)
Ph3P=CHCO2Et, toluene, reflux, 5 h; (j) KOH, EtOH/H2O (2:1), reflux, 5 h; (k) (i) (COCl)2, toluene, 70 °C, 14 h, (ii) NaN3, acetone, rt, 2 h, (iii)
I2, 1, 2-dichlorbenzol, reflux, 2.5 h; (l) R-Br, NaH, DMF, 80 °C, 4 h; (m) LiAlH4, AlCl3, THF, rt, 4 h; (n) LiAlH4, THF, rt, 48 h, quench, MeOH/
H2O (100:1), rt.
11. After experimentation, it was found that lithium aluminum
hydride (LiAlH4) alone or in combination with aluminum
chloride (AlCl3) was able to achieve this transformation. The
use of LiAlH4 resulted in generation of products 10 (2−11%
yields) and 11 (5−25% yields), whereas LiAlH4 in combination
with AlCl3 greatly favored product 10 over 11 and thus
generated high yields for 10 (42−95%) (see Supporting
Information Table S1 and Experimental Section).
CHEMISTRY
■
For the preparation of compounds 9−11, the procedure
described in Chuang et al.16 was used for 9 and modified for 10
and 11 (Scheme 1). Increasing the size of the E ring was found
to disfavor ring formation, but the synthesis of 9c was
nevertheless achieved using the same route as for 7-
methoxycryptopleurine 9b, albeit in a lower yield. Reduction
of 8 to give 9 was accomplished using sodium bis(2-
methoxyethoxy) aluminum hydride in dioxane, but these
conditions were not effective in the reduction of 7 to 10 and
The route used to synthesize 22a−22d is depicted in Scheme
2A. Nitrocinnamic acids (14) were synthesized through a
Perkin reaction17 from commercially available phenylacetic
C
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acids (12) and substituted o-nitrobenzaldehydes (13). Reduc-
tion of the nitro group of 14 afforded aminocinnamic acids
(15). Phenanthrene acids (16) were obtained through a
Pschorr cyclization17 from 15. Reduction of 16 with diborane
in THF afforded 22a−22d.18 The route shown in Scheme 2B
was followed as previously described19,20 to achieve the
synthesis of 22e. Oxidation of phenanthrene alcohols (22a−
22e) with pyridinium chlorochromate in CH2Cl2 afforded the
target intermediate phenanthrene aldehydes (23a−23e).21
Subsequently, aldehydes (23) were converted to isoquinoli-
nones (28−33) with a modification of a procedure reported by
Chuang et al.16 (Scheme 2C). Reduction of 27 with LiAlH4 in
THF at room temperature for 48 h afforded dibenzoisoquino-
lines 28−32 in 13−29% and 33 in 14−31% yields, respectively.
This reaction was also greatly improved by reducing 27 with
LiAlH4 and AlCl3 (3:1 ratio) in THF at room temperature for 4
h to afford high yields of dibenzoquinolines 28−32 (40−80%).
The position of hydroxylation was further confirmed by 2D-
NMR analyses (Figure 2).
hydrogen atom projection of the E ring optimized the potency
of these compounds.
Considering the N-alkyl substituent analogues formed by
uncyclizing the tylophorine E ring, the propyl 10b analogue is
the most potent compound when compared to ethyl, butyl, and
pentyl analogues 10a, 10c, and 10d. When compared to the
uncyclized B ring analogues,3 e.g., septicine (2 in Figure 1 and
Table 1), the uncyclized E ring analogues retained most of the
tylophorine activity (Tables 1 and 2), e.g., 2 and 10b compared
to 9a. Of the branch analogues, in general, the branched methyl
group at C1- or C2-position of alkyl substitution slightly or
moderately increased the potency of these compounds. For
example, isobutyl 10f and sec-butyl 10g were more potent than
butyl 10c by ∼2−6-fold (Table 2). However, the introduction
of polar hydroxyl and amine groups to the propyl alcohol 10′m
and propylamine 10′n decreased their potency dramatically by
∼10−30-fold. Further replacement of heterocycle groups for
the N-substituents, e.g., 3-cyclohexene (10h), 2-methyl-[1,3]-
dioxolane (10i), 2-ethyl-[1,3]dioxolane (10j), and 2-propox-
ytetrahydro-2H-pyranyl group (10k) also failed to improve
potency compared to the alkyl-replaced analogues (Table 2).
Further investigation of the methoxy substituents in the
phenanthrene moiety of 10a and 10b was also carried out. Of
these analogues, 29a and 29b, which were demethoxylated at
position C2, were found to be more potent than 10a and 10b.
Thus, the demethoxylation at C2 of phenanthrene moiety
improved the activity of the uncyclized E-ring tylophorine
analogues. However, the demethoxylation at the positions of 6
or 7 to give 30a, 30b, 31, and 32 dramatically weakened the
potency. Methoxylation at C4 did not affect the potency
significantly when comparing the activities of 28a to 10a, 28b
to 10b, 28c to 10c, and 28d to 10e (Table 3). Further
introduction of a hydroxyl group to C14 of the C4
demethoxylated compounds with a N-alkyl group (Table 4)
increased the potency by ∼3−6-fold for 11a compared to 10a,
by ∼9−20-fold for 33b compared to 29a, by ∼4−9-fold for 33c
compared to 29b, and by ∼2-fold for 33d compared to 29c.
However introduction of a hydroxyl group to the same position
C14 of other compounds did not show same tendency for
improving activity, e.g., no significant improvement observed
for 33a compared to 28b; decreased potency occurred to 11b
Figure 2. Key COSY, NOESY, and HMBC correlations of 33a.
RESULTS AND DISCUSSION
■
Of the tylophorine cyclized E ring analogues synthesized, the
quinolizidine 9b was the most potent inhibitor of cancer cell
growth (Table 1), exerting greater activity than indolizidine 9a
by ∼2-fold and than 9c by ∼6−8-fold. Similar results were
reported for antofine cyclized E ring analogues. This result is
consistent with that obtained from the corresponding antofine
analogues reported.22 The same trends of potency for anti-
TGEV repliction in ST cells were also found for these
compounds (Table 1). Conceivably, an appropriate size and
Table 1. The Effect of the E Ring Size and seco-Structure of Tylophorine Derivatives on the Anti-TGEV in ST Cells and
Antiproliferative Activities against Carcinoma Cells
a
a
GI50 (μM)
EC50 (μM)
compd ID
NCI-H460
MCF7
SF268
HONE-1
NUGC-3
TGEV
9a
9b
9c
0.23
0.10
0.93
17.0
0.24
0.11
0.96
18.5
0.27
0.14
0.96
24.2
0.27
0.14
1.15
17.3
0.19
0.14
0.62
14.5
0.08
0.03
1.05
15.4
b
2
a
GI50 and EC50 values expressed in μM as the mean values of at least three experiments each in duplicate. Values of SD were less than 30% of GI50
and EC50 values and data not shown. (+)-S-Septicine isolated from Tylophora ovata.3
b
D
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Table 2. Anti-TGEV in ST Cells and Antiproliferative Activity against Carcinoma Cells of Tylophorine-Derived
Dibenzoquinolines with a Variety of N-Substituents
a
a
compd
GI50 (μM)
EC50 (μM)
ID
10a
R
NCI-H460
MCF7
HepG2
HONE-1
NUGC-3
A549
TGEV-IFA
CH2CH3
1.01
0.45
2.05
6.11
0.29
0.87
0.45
2.30
18.52
4.15
7.49
2.89
9.41
11.73
0.03
0.75
0.51
1.97
9.51
0.34
0.91
0.40
2.21
22.45
4.60
9.80
3.35
7.14
15.32
0.03
1.00
0.77
3.04
8.05
0.67
1.81
0.51
3.64
41.04
6.49
13.98
4.15
16.89
36.93
0.18
1.57
1.21
2.75
6.20
0.94
1.72
1.15
2.53
22.06
6.13
9.65
4.52
16.60
21.13
0.02
0.62
0.47
1.93
7.67
0.32
1.04
0.44
1.88
19.09
4.95
8.22
3.40
6.63
10.33
0.02
0.97
0.73
2.22
1.65
0.83
10b
10c
(CH2)2CH3
(CH2)3CH3
4.39
b
10d
10e
(CH2)4CH3
ND
6.53
CH(CH3)2
0.38
1.32
0.53
1.92
31.50
4.82
3.78
3.55
5.47
6.04
0.06
0.60
10f
CH2CH(CH3)2
CH(CH3)CH2CH3
3-cyclohexene
2-methyl-[1,3]dioxolane
2-ethyl-[1,3]dioxolane
(CH2)3OTHP
(CH2)3NHBoc
(CH2)3OH
1.36
10g
0.73
10h
10i
2.99
>50.00
31.89
30.84
1.027
19.28
23.42
10j
10k
10l
10′m
10′n
paclitaxel
(CH2)3NH2
c
b
ND
a
GI50 and EC50 values expressed in μM as the mean values of at least three experiments each in duplicate. Values of SD were less than 30% of GI50
b
c
and EC50 values and data not shown. ND, not determined. Paclitaxel, a positive control.
Table 3. Anti-TGEV in ST Cells and Antiproliferative Activity against Carcinoma Cells of Tylophorine-Derived
Dibenzoquinolines with a Variety of N-Substituents
a
a
substitution
R3
GI50 (μM)
EC50 (μM)
compd ID
R1
R2
R4
R5
NCI-H460 MCF7 HepG2 HONE-1 NUGC-3
A549
TGEV-IFA
28a
28b
28c
28d
29a
29b
29c
30a
30b
31
OCH3
OCH3
OCH3
OCH3
H
OCH3
OCH3
OCH3
OCH3
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
CH2CH3
0.87
0.49
2.68
0.57
0.30
0.26
3.51
3.39
3.82
2.40
3.76
1.18
0.62
3.19
0.72
0.37
0.42
4.73
3.12
3.75
2.83
4.11
2.50
0.86
5.01
1.61
0.66
0.48
4.04
4.01
4.48
3.17
6.29
1.99
0.96
4.06
1.12
0.57
0.77
4.77
4.87
5.56
3.31
4.75
1.70
0.54
2.54
0.64
0.32
0.43
3.99
3.48
4.63
2.94
3.72
1.41
0.79
3.01
0.76
0.40
0.47
2.39
4.38
4.64
2.95
3.67
1.14
0.77
2.92
0.78
0.58
0.68
5.60
14.18
8.91
8.68
2.63
(CH2)2CH3
(CH2)3CH3
CH(CH3)2
CH2CH3
H
H
(CH2)2CH3
(CH2)3CH3
CH2CH3
H
H
OCH3
OCH3
OCH3
H
H
H
H
(CH2)2CH3
(CH2)2CH3
(CH2)2CH3
H
OCH3
H
32
OCH3
OCH3
a
GI50 and EC50 values expressed in μM as the mean values of at least three experiments each in duplicate. Values of SD were less than 30% of GI50
and EC50 values and data not shown.
and 11c compared to 10b and 10e, respectively, by a factor of
∼2−4. Thus, only the introduction of C14 hydroxyl group to
29a (a demethoxylated dibenzoquinoline at C2 and C4)
synergized the effect of demethoxylation at C2 to give the most
potent dibenzoquinoline 33b. Demethoxylation at C2 may
allow the C14 hydroxyl group to participate in an important
bonding interaction in the target binding pocket, giving rise to
the significantly improved potency.
The dibenzoquinolines assayed above for anticancer cell
growth also were tested for anti-TGEV (Tables 2−4) and anti-
inflammatory activity (Table 5) in vitro and found to exert the
same trends of potency in these three different biological
actvities. In addition, the cellular activities which were reported
E
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Table 4. Anti-TGEV in ST Cells and Antiproliferative Activities against Carcinoma Cells of Tylophorine-Derived
Dibenzoquinolines with a Hydroxyl Group at the C14 Position
a
a
compd
GI50 (μM)
HepG2
EC50 (μM)
ID
R
NCI-H460
MCF7
HONE-1
NUGC-3
A549
TGEV-IFA
11a
11b
11c
33a
33b
33c
33d
CH2CH3
0.17
1.08
1.07
0.59
0.02
0.05
1.48
0.16
1.02
1.21
0.59
0.04
0.11
2.33
0.36
1.68
1.86
0.83
0.04
0.11
1.85
0.44
1.74
1.71
0.82
0.06
0.14
3.00
0.15
0.73
0.87
0.49
0.03
0.11
1.86
0.19
1.19
1.23
0.82
0.02
0.05
1.32
0.34
0.67
2.74
0.66
0.04
0.12
2.92
(CH2)2CH3
CH(CH3)
2
(CH2)2CH3
CH2CH3
(CH2)2CH3
(CH2)3CH3
a
GI50 and EC50 values expressed in μM as the mean values of at least three experiments each in duplicate. SD values were less than 30% of GI50 and
EC50 and data not shown.
direct targets of these compounds and their fundamental
mechanisms of action is under investigation in our laboratory.
These compounds also potently inhibited the cytopathic
effect induced by murine hepatitis virus in DBT cells (Figure 4
and Supporting Information Figure S2). Solubility testing
found them to be ∼4−5-fold more soluble in DMSO (Table 6)
and in DMA (data not shown), compared to their respective
related pentacyclic compounds, e.g., 10b vs 9a and 10c vs 9b.
For comparison of 33a to tylophorine 9a, ∼6-fold increase was
found. This improvement gave the guide for the formula choice
of in vivo tests followed.
On the basis of these results, 10b, 29a, and 33b were selected
for in vivo evaluation. Efficacy tests using paw edema and
tumor xenograft murine models, as well as pharmacokinetic
tests, were conducted. For the pharmacokinetic studies,
compounds were administered to rats intravenously and orally
each at 3 mg/kg body weight. Blood samples were taken and
the plasma analyzed. It was found that 9a and 9b exhibited oral
bioavailabilities of 66% and 53%, as described.8 Tylophovatine
C isolated from T. ovata3 was tested and analyzed in parallel as
a reference C14-hydroxytylophorine derivative. Pharmacoki-
netic parameters were also obtained (Table 7). Tylophovatine
C, 10b, 29a, and 33b all exhibited good oral bioavailabilities of
54%, 63%, 105%, and 64%, respectively.
In an A549 xenografted tumor mouse model, 33b
significantly and effectively reduced the tumor volume at the
dose of 10 mg/kg (oral administration) and resulted in a 61%
volume reduction by the end of the test. This is the first report
of an antitumor tylophorine-derived compound, orally active in
vivo. There were no signs of overt toxicity during the course of
the experiment (Figure 5A). In the rat paw edema model,
intraperitoneal administration of 33b resulted in significant
inhibition of acute inflammation by reducing the paw edema
volume greater than 60% at doses of 3 and 5 mg/kg body
weight, respectively (Figure 5B). The neurotoxicity of 33b was
examined by a rota-rod test for motor coordination25 for three
consecutive days. No adverse effect on motor coordination was
observed from the 33b treated groups when compared to the
control vehicle and nontumor-bearing groups. Nontheless, the
group of 33b at the dose 10 mg/kg on the day 3 was found to
have better coordination than those from the nontumor-
Table 5. Anti-inflammatory Activity of Tylophorine Derived
Dibenzoquinolines in Terms of Suppression Nitric Oxide
Production in LPS/IFNγ Stimulated RAW264.7 Cells
Raw 264.7/LPS + IFNγ
a
a
compd ID
EC50 (μM)
CC50 (μM)
9a
0.27
1.42
1.38
0.86
0.38
2.20
2.05
0.07
1.24
>4.0
>4.0
>4.0
>2.0
>4.0
>4.0
0.24
10a
10b
10e
11a
11b
11c
33b
a
EC50 and CC50 values expressed in μM as the mean values of at least
three experiments each in duplicate. SD values were less than 30% of
EC and CC50 and data not shown. EC50 was measured for the
50
effective concentration for 50% inhibition of the compound treatment
on the production of nitric oxide. CC50 was measured for the
concentration of 50% inhibition of the compound treatment on the
cell growth to distinguish the potency and cytotoxicity.
to account for modes of actions exerted by tylophorine in these
three biological systems5,8,23,24 were also examined for these
dibenzoquinolines. These compounds down-regulated the
cyclin A2 expression and caused the accumulation of c-Jun in
carcinoma cells (Figure 3A and Supporting Information S1A),
inhibited iNOS and COX-2 protein expression in LPS/IFNγ
stimulated RAW264.7 cells (Figure 3B and Supporting
Information Figure S1B), and exerted anti-TGEV nucleocapsid
(N) protein expression and TGEV induced apoptosis through
inhibition of the activation of caspase 3 from cleavage of
procaspase 3 in ST cells (Figure 3C,D and Supporting
Information Figure S1C). Thus, these compounds likely retain
the same modes of actions as tylophorine.5,8,23 In the three
biological systems tested above, synthesis of RNA or protein
are largely in demand, e.g., for cancer cell proliferation, viral
replication, or production of proinflammatory factors. Com-
mon properties might be shared by the direct molecular targets
of these compounds in the three biological systems, e.g.,
interfering in RNA or protein synthesis. Elucidation of the
F
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Figure 3. Pharmacological activities of tylophorine and tylophorine-derived dibenzoquinolines in three biological systems. (A) Western analysis for
expression of cyclin A2 and c-Jun in carcinoma cells after compound treatment for 24 h. Tylophorine compounds down-regulated cyclin A2
expression and induced c-Jun protein accumulation to exert their anticancer effect. (B) Western analysis for iNOS, COX-2, and GAPDH protein
expression in LPS/INFγ stimulated RAW264.7 cells after compound treatment for 20 h. Tylophorine compounds inhibited the induction of iNOS
and COX-2 expression in LPS/INFγ stimulated RAW264.7 cells to exert their anti-inflammatory effect. (C) Western analysis for TGEV nucleocapsid
(N) protein, caspase 3, and GAPDH in TGEV infected ST cells at 16 hpi. Tylophorine compounds inhibited TGEV N protein expression and
activation of caspase 3 through cleavage of pro-casapase 3 to exert their anti-TGEV effect. (D) Immunofluorescent assay for TGEV N and spike
protein expression in TGEV infected ST cells at 6 hpi. Phase contrast images were shown for the field of ST cells assayed. (C) and (D) are for anti-
TGEV effect. The concentrations of treatment used for all the above experiments are 2 μM for 9a, 29a, and 11a, 4 μM for 10b and 10e, and 0.5 μM
for 33b. The results shown are representative of at least three independent experiments.
bearing group with a *p value of <0.001 (Figure 5C).
Therefore, 33b was suggested to be an orally active agent not
only effective against tumors without neurotoxicity but also of
potential utility for treating inflammatory related diseases and
coronaviral infections.
solubility compared to tylophorine 9a, (2) in vivo efficacies
in an A549 xenografted mouse model and a murine paw edema
model, when administrated orally and intraperitoneally
respectively, (3) good bioavailability, and (4) no measurable
neurotoxicity as tested by a rota-rod test for motor
coordination. To the best of our knowledge, dibenzoquinoline
33b is the first example of an orally active tylophorine related
compound. By uncyclizing the tylophorine E ring, a new class
of compounds has been revealed which may prove useful for
improving solubility and potency and diminishing the neuro-
toxicity of tylophorine derived compounds in the future.
CONCLUSION
■
A series of tylophorine derived dibenzoquinolines has been
synthesized and the constituents tested for anticancer, anti-
inflammatory, and anticoronavirus activity. The most potent
compound dibenzoquinoline 33b showed: (1) improved
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Figure 4. Tylophorine 9a and dibenzoquinolines 33b exerted activities
for anti-MHV induced cytopathic effect and antiviral protein
expression in infected DBT cells. (A) Phase contrast images were
shown for cytopathic effect in MHV infected DBT cells at 24 hpi. (B)
Immunofluorescent assay for MHV N protein expression in MHV
infected DBT cells at 24 hpi. Compound concentrations used as
indicated. The results shown are representative of at least three
independent experiments.
Table 6. Solubility of Tylophorine-Related Compounds in
DMSO
solubility compd
mM
mg/mL
9a
10.0
12.3
50.0
50.0
45.0
60.5
3.9
5.0
9b
10a
10b
10c
33b
18.3
19.8
18.4
22.2
EXPERIMENTAL SECTION
■
Chemistry. Reagents and all solvents were analytically pure and
used without further purification. All reactions were carried out in
oven-dried flasks with magnetic stirring. Column chromatography was
done using silica gel (Merck Kieselgel 60, no. 9385, 230−400 mesh
ASTM). Reactions were monitored with thin-layer chromatography
(TLC) using Merck 60 F254 silica gel glass-backed plates and
visualized under UV (254 nm). Melting points were measured with a
Yanaco micromelting point apparatus (MP-500D). Nuclear magnetic
resonance (1H and 13C NMR) spectra were recorded on Varian 300 or
400 or Varian Inova-600 spectrometers in δ (ppm) referenced to
tetramethylsilane. Proton coupling patterns were described as singlet
(s), doublet (d), triplet (t), multiplet (m), and broad (br). Low
resolution mass spectra (LRMS) were given with electric, electrospray,
and atmospheric pressure chemical ionization (EI, ESI, and APCI)
produced by a Finnigan MAT 95XL, Agilent 1100 series, and Agilent
1200 series. High resolution mass spectra (HRMS) were given with
electric ionization (EI) produced by a Finnigan MAT 95XL.
Preparative high pressure liquid chromatography (HPLC) was
performed using a JASCO model PU-2087 HPLC system equipped
with a JASCO model UV-2075 detector and a Thermo hypersil silica
column (5 μm, 10 mm × 250 mm). Purities of the target compounds
were initially confirmed by less than two degrees interval of melting
point, TLC, and HRMS and subsequently determined using a Hitachi
2000 series HPLC system with a reverse phase C18 column (Agilent
ZORBAX Eclipse XDB-C18: 5 μm, 4.6 mm × 150 mm, 0.5 mL/min
flow rate). Mobile phase A was acetonitrile. Mobile phase B was 10
mM NH4OAc aqueous solution containing 0.1% formic acid. The
gradient system started from A/B (10%:90%) at 0 min to A/B
(90%:10%) at 45 min. All compounds tested in the biological assay
H
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Figure 5. In vivo efficacies of 33b. (A(a)) Tumor growth curves in NU/NU mice treated with 33b using a lung A549 xenograft model. (A(b)) Body
weight curves in NU/NU mice treated with 33b. (B) Anti-inflammatory effect in Sprague−Dawley rats treated with 33b and indomethacin using a
murine paw edema model. (C) Neurotoxicity determination by a rota-rod test for motor coordination of NU/NU mice treated with 33b. *p < 0.001;
#p < 0.01; ns, no significance; bar, SD.
showed >95% purity at 254 nm except 87.8% for 10′n, which could
not be purified further (see Supporting Information Table S2).
Solubility Determination in DMSO. Saturation concentrations for
solubility of the test compounds in DMSO were determined by
dissolving compounds in DMSO with assistance of sonication and the
maximal volume before compound precipitation occurred was
measured for calculation of solubility.
General Procedure for the Preparation of 14a−c and 19. 6-
Nitroveratraldehyde (5 g, 23.7 mmol) and homoanisic acid (3.93 g,
23.7 mmol) were dissolved in acetic anhydride (20 mL), and then
triethylamine (3.3 mL) was added slowly. The resultant solution was
stirred at 90 °C for 15 h. At this point, H2O (2 mL) was added and the
reaction was allowed to continue for 15 min. After, K2CO3 (26 g) in
water (240 mL) was added slowly and the mixture was stirred for 60
°C for 1 h. The reaction mixture was cooled to 4 °C and acidified with
12N HCl. The aqueous solution was extracted with CH2Cl2, and the
obtained organic phases were combined, washed with brine, dried, and
then evaporated. Pure 14a (6.86 g) was obtained by further
crystallization from EtOAc.
added to turn the mixture yellow, whereupon it was poured into water
(500 mL) and extracted with CHC13, washed with water, dried, and
evaporated to give the phenanthrene acid 16a (1.07 g).
General Procedure for the Preparation of 22a−d. The hydro-
boration solvent BH3·THF (1 M, 8.75 mL) was added, in three
portions, to a stirred suspension of acid 16a (910 mg, 2.9 mmol) in
THF (29 mL) over 1 h. Upon completion of addition, the reaction
mixture was warmed (38 °C) for another hour, quenched (HOAc),
and evaporated, and the residue was partitioned between 30 mL
portions of CH2Cl2 and 10% NaOH. The organic layer was dried,
filtered, and evaporated to give the alcohol 22a (778 mg).
General Procedure for the Preparation of 20. Compound 19
(4.03 g, 12.8 mmol) was dissolved in 77 mL of MeOH containing 5.2
mL of concentrated H2SO4, and the mixture was refluxed for 4 h. The
solvent was evaporated under reduced pressure, and the residue was
partitioned between portions of CH2Cl2 and saturated NaHCO3
solution. The organic phase was dried, evaporated, and purified by
silica gel column chromatography (EtOAc: hexane = 1:3 v/v) to give
20 (4.06 g).
General Procedure for the Preparation of 15a−c. A solution of
25% NH4OH (40 mL) was degassed with nitrogen for 30 min and
heated to 100 °C before the addition of a FeSO4·7H2O (15 g, 56
mmol) with stirring. A solution of the nitrocinnamic acid 14a (2 g, 5.6
mmol) in 70 mL of 25% NH4OH was added slowly to the reaction
mixture, under nitrogen at 100 °C for 2 h. After reaction was
completed, the reaction mixture was cooled to room temperature to
add decolorizing carbon and then the mixture was filtered. The filtrate
was cooled to 4 °C, acidified to pH 3 with 85% H3PO4, and extracted
with CHC13/2-propanol (3:1 v/v), washed with water, dried, and
evaporated to give the aminocinnamic acid 15a (1.51 g).
General Procedure for the Preparation of 16a−d. The amino-
cinnamic acid 15a (1.51 g, 4.6 mmol) was dissolved in acetone (250
mL) before subsequent slow additions of H2SO4 (0.5 mL, 9.2 mmol)
and isoamyl nitrite (1.23 mL, 9.2 mmol) at 4 °C. In those cases where
a precipitate was still present after 30 min of stirring, water was added
until the solution became homogeneous. After 1 h, sodium iodide (3.6
g, 23.9 mmol) was added in five portions for 5 h. Sodium bisulfite was
General Procedure for the Preparation of 21. Anhydrous FeCl3
(6.12 g, 37.8 mmol) was added to a solution of 20 (3.54 g, 10.8 mmol)
dissolved in dry CH2Cl2 (108 mL) to react at room temperature under
nitrogen for 20 h. The reaction solution was then evaporated under
reduced pressure and the crude product purified by silica gel column
chromatography (EtOAc:hexane = 1:3 v/v) to give 21 (1.88 g).
General Procedure for the Preparation of 22e. A suspension of
LiAlH4 (480 mg, 11.6 mmol) in 20 mL of dry THF was added to a
solution of 21 (1.88 g, 5.8 mmol) dissolved in 30 mL of dry THF. The
reaction mixture was stirred at room temperature under nitrogen for 3
h. The mixture was then quenched by 1.2 mL of H2O, 1.2 mL of 10%
NaOH, and 2.4 mL of H2O, added sequentially. The mixture was
filtered, and the filtrate was evaporated under reduced pressure. The
residue was dissolved in CHC13, and the solution was washed with
brine and dried over MgSO4. The combined organic phase was
evaporated under reduced pressure. The residue was purified by silica
gel column chromatography (EtOAc:hexane = 1:1 v/v) to give alcohol
22e (1.52 g).
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General Procedure for the Preparation of 23a−e. Phenanthrene
alcohol 22a (835 mg, 2.8 mmol) dissolved in 75 mL of dry CH2Cl2
was added to a suspension of pyridinium chlorochromate (903 mg, 4.2
mmol) in 10 mL of dry CH2Cl2 at 0 °C. The reaction mixture was
stirred at room temperature under nitrogen for 2 h. The mixture was
diluted with 80 mL of ether and filtered. The solids were washed with
CHC13, and the combined organic phase was concentrated by
evaporation under reduced pressure. The residue was purified by silica
gel column chromatography (CH2Cl2) to give the aldehyde 23a (726
mg).
General Procedure for the Preparation of 24a−e. A mixture of
23a (0.73 g, 2.5 mmol) and (carboethoxymethylene)-
triphenylphosphorane (1.2 g, 3.4 mmol) in 20 mL of toluene was
refluxed under nitrogen for 4 h. After cooling, the resulting solution
was directly purified by silica gel column chromatography (CH2Cl2) to
afford the ethyl ester 24a (0.89 g).
General Procedure for the Preparation of 25a−e. A solution of 8
mL of 1 N KOH was added to a solution of the ester 24a (0.81 g, 2.2
mmol) in 16 mL of EtOH, and the reaction mixture was heated to
reflux for 3 h. After cooling, the reaction solution was evaporated and
the residue was dissolved in 16 mL water, acidified with 10% HCl, and
extracted with 70 mL of CHCl3 and 60 mL of EtOAc. The combined
extracts were dried with anhydrous MgSO4, filtered, and evaporated
under reduced pressure to give the acrylic acid 25a (0.73 g)
General Procedure for the Preparation of 26a−e. A mixture of
25a (1.26 g, 3.7 mmol) and oxalyl chloride (1.89 g, 14.8 mmol) in 63
mL of toluene was heated for 15 h at 70 °C. After cooling, the
resulting mixture was concentrated under reduced pressure to afford
the acyl chloride. The acyl chloride was added immediately to a
suspension of NaN3 (0.73 g, 11.1 mmol) in 120 mL of dry acetone in
an ice bath. The reaction mixture was stirred for 2 h at room
temperature and filtered. The solvent was evaporated under reduced
pressure, and the residue was purified by silica gel column
chromatography (CHCl3) to afford acryloyl azide. A mixture of
azide and I2 (0.02 g, catalytic amount) in 18 mL of o-dichlorobenzene
was refluxed for 2 h. After cooling, compound 26a was isolated by
filtration and washed with CH2Cl2 to afford pure isoquinolinone 26a
(1.01 g)
General Procedure for the Preparation of 7a−l and 27a−k. A
suspension of NaH (60% dispersion in oil, 119 mg, 2.98 mmol) in 1
mL of DMF cooled in an ice bath was added to a solution of 26a (400
mg, 1.2 mmol) in 15 mL of DMF with stirring at room temperature
under nitrogen for 30 min. After the addition was completed, the
mixture was added slowly to a solution of bromoethane (4.77 mmol)
in 1 mL of DMF. The mixture was stirred at 80 °C for 4 h. The solvent
was evaporated under reduced pressure, and water was then added.
The mixture was extracted with CH2Cl2, dried over MgSO4, and
concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (EtOAc:CH2Cl2 = 1:8 v/v) to afford
the alkyll isoquinolinone of 27a (400 mg).
mixture was filtered, and the filtrate was evaporated under reduced
pressure. The residue was purified by silica gel column chromatog-
raphy (MeOH:EtOAc:CH2Cl2 = 0:10:20, 1:10:20, 2:10:20; all solvent
with 0.001% diethylamine) to afford the 29a and 33b mixtures. The
mixtures were further purified by HPLC (5 μm, 10 mm × 250 mm;
MeOH:EtOAc:CH2Cl2 = 1:10:20, 2:10:20; all solvent with 0.001%
diethylamine) at 1 mL/min flow rate to afford 29a (15 mg) and 33b
(31 mg).
2-Ethyl-6,7,10,11-tetramethoxy-1,2,3,4-tetrahydrodibenzo[f,h]-
isoquinoline (10a). Yield 9% (method e); white crystal; mp 186 °C.
1H NMR (400 MHz, CDCl3): 1.31 (t, J = 7.2 Hz, 3H), 2.81 (quartet, J
= 7.2 Hz, 2H), 2.96 (t, J = 5.6 Hz, 2H), 3.22 (t, J = 5.6 Hz, 2H), 4.02
(s, 2H), 4.04 (s, 3H), 4.05 (s, 3H), 4.12 (s, 6H), 7.15 (s, 1H), 7.29 (s,
1H), 7.82 (s, 1H), 7.83 (s, 1H). 13C NMR (75 MHz, CDCl3): 12.2,
26.7, 49.4, 52.0, 53.4, 55.8, 55.9, 56.9, 102.8, 103.3, 103.4, 103.8, 123.4,
123.5, 124.1, 124.7, 125.5, 125.6, 148.4, 148.5, 148.7. MS (EI) m/z
381 (M+, 100%). HRMS calcd for C23H27NO4 (M+) 381.1940; found
381.1930.
6,7,10,11-Tetramethoxy-2-propyl-1,2,3,4-tetrahydrodibenzo[f,h]-
isoquinoline (10b). Yield 76% (method d), 7% (method e); white
crystal; mp 176−178 °C. 1H NMR (400 MHz, CDCl3): 1.02 (t, J = 7.6
Hz, 3H), 1.74 (sextet, J = 7.6 Hz, 2H), 2.67 (t, J = 7.6 Hz, 2H), 2.92 (t,
J = 6.0 Hz, 2H), 3.18 (t, J = 6.0 Hz, 2H), 3.98 (s, 2H), 4.03 (s, 3H),
4.04 (s, 3H), 4.10 (s, 6H), 7.12 (s, 1H), 7.27 (s, 1H), 7.79 (s, 1H),
7.80 (s, 1H). 13C NMR (100 MHz, CDCl3): 12.0, 20.5, 27.2, 50.1,
54.3, 55.8, 55.9, 56.0, 60.6, 102.9, 103.2, 103.4, 103.8, 123.3, 123.4,
124.3, 125.6, 125.9, 148.3, 148.4, 148.6. MS (EI) m/z 395 (M+, 100%).
HRMS calcd for C24H29NO4 (M+) 395.2097; found 395.2098.
2-Butyl-6,7,10,11-tetramethoxy-1,2,3,4-tetrahydrodibenzo[f,h]-
isoquinoline (10c). Yield 66% (method d); white needle; mp 172−
1
173 °C. H NMR (300 MHz, CDCl3): 1.00 (t, J = 7.5 Hz, 3H), 1.45
(sextet, J = 7.5 Hz, 2H), 1.70 (quintet, J = 7.5 Hz, 2H), 2.70 (t, J = 7.5
Hz, 2H), 2.91 (t, J = 5.7 Hz, 2H), 3.18 (t, J = 5.7 Hz, 2H), 3.97 (s,
2H), 4.03 (s, 3H), 4.04 (s, 3H), 4.10 (s, 6H), 7.12 (s, 1H), 7.26 (s,
1H), 7.79 (s, 1H), 7.80 (s, 1H). 13C NMR (75 MHz, CDCl3): 14.1,
20.8, 27.2, 29.4, 50.1, 54.3, 55.8, 55.9, 58.4, 102.8, 103.2, 103.3, 103.8,
123.3, 123.4, 124.2, 125.6, 125.9, 148.2, 148.3, 148.5, 148.6. MS (EI)
m/z 409 (M+, 100%). HRMS calcd for C25H31NO4 (M+) 409.2253;
found 409.2252.
6,7,10,11-Tetramethoxy-2-pentyl-1,2,3,4-tetrahydrodibenzo[f,h]-
1
isoquinoline (10d). Yield 5% (method e); white needle. H NMR
(300 MHz, CDCl3): 0.95 (t, J = 7.2 Hz, 3H), 1.38−1.43 (m, 4H),
1.70−1.73 (m, 2H), 2.71 (t, J = 7.8 Hz, 2H), 2.94 (t, J = 5.4 Hz, 2H),
3.21 (t, J = 5.4 Hz, 2H), 4.01 (s, 2H), 4.04 (s, 3H), 4.05 (s, 3H), 4.12
(s, 6H), 7.16 (s, 1H), 7.30 (s, 1H), 7.83 (s, 1H), 7.84 (s, 1H). MS
(APCI) m/z 424.2 [M + H]+.
2-Isopropyl-6,7,10,11-tetramethoxy-1,2,3,4-tetrahydrodibenzo-
[f,h]isoquinoline (10e). Yield 59% (method d), 2% (method e); white
needle; mp 185 °C. 1H NMR (300 MHz, CDCl3): 1.26 (d, J = 6.6 Hz,
6H), 2.95 (t, J = 5.7 Hz, 2H), 3.09 (septet, J = 6.6 Hz, 1H), 3.17 (t, J =
5.7 Hz, 2H), 4.03 (s, 3H), 4.04 (s, 3H), 4.08 (s, 2H), 4.10 (s, 6H),
7.13 (s, 1H), 7.27 (s, 1H), 7.80 (s, 1H), 7.81 (s, 1H). 13C NMR (75
MHz, CDCl3): 18.5, 27.8, 45.1, 49.9, 54.1, 55.8, 55.9, 102.9, 103.2,
103.4, 103.8, 123.3, 124.4, 125.7, 126.1, 148.2, 148.3, 148.5. MS (EI)
m/z 395 (M+, 100%). HRMS calcd for C24H29NO4 (M+) 395.2097;
found 395.2103.
General Procedure for the Preparation of 10a−n, 11a−c, 28a−
d, 29a−c, 30a−b, 31, 32, and 33a−d. 1. Reduction by LiAlH4 and
AlCl3 for Method d in Scheme 1 and Method m in Scheme 2. A
solution of AlCl3 (18 mg, 0.13 mmol) in 0.5 mL of dry THF was
added to a stirred suspension of 27a (48 mg, 0.13 mmol) in 2 mL of
dry THF cooled at −15 °C. The mixture was stirred at room
temperature under nitrogen for 15 min, and then LiAlH4 (26 mg, 0.65
mmol) was added at −15 °C. The reaction mixture was warmed up to
room temperature for 4 h and then quenched by 5 mL of MeOH, and
5 mL of 10% NaOH sequentially. The mixture was filtered, and the
filtrate was evaporated under reduced pressure. The residue was
purified by silica gel column chromatography (MeOH:EtOAc:CH2Cl2
= 1:10:20 v/v) to afford the dibenzo[f,h]isoquinolinone of 29a (19
mg).
2. Reduction by LiAlH4 for Method e in Scheme 1 and Method n
in Scheme 2. A solution of LiAlH4 (136 mg, 3.58 mmol) in 5 mL of
dry THF was added to a stirred suspension of 27a (100 mg, 0.28
mmol) in 8 mL of dry THF cooled at −15 °C,. The reaction mixture
was warmed up to room temperature for 2 days, then 5.5 mL of
MeOH/H2O (100:1) was added slowly using a syringe pump. The
2-Isobutyl-6,7,10,11-tetramethoxy-1,2,3,4-tetrahydrodibenzo-
[f,h]isoquinoline (10f). Yield 69% (method d); light-yellow needle;
1
mp 160−161 °C. H NMR (300 MHz, CDCl3): 1.02 (d, J = 6.6 Hz,
6H), 2.04 (septet, J = 6.9 Hz, 1H), 2.47 (d, J = 7.5 Hz, 2H), 2.90 (t, J
= 6.0 Hz, 2H), 3.18 (t, J = 6.0 Hz, 2H), 3.97 (s, 2H), 4.03 (s, 3H), 4.04
(s, 3H), 4.11 (s, 6H), 7.13 (s, 1H), 7.29 (s, 1H), 7.80 (s, 1H), 7.81 (s,
1H). 13C NMR (75 MHz, CDCl3): 21.0, 25.8, 27.1, 50.2, 54.8, 55.8,
55.9, 56.0, 66.8, 102.9, 103.2, 103.4, 103.8, 123.3, 123.4, 124.3, 125.6,
125.8, 126.0, 148.3, 148.4, 148.6. MS (EI) m/z 409 (M+, 100%).
HRMS calcd for C25H31NO4 (M+) 409.2253; found 409.2249.
2-(sec-Butyl)-6,7,10,11-tetramethoxy-1,2,3,4-tetrahydrodibenzo-
[f,h]isoquinoline (10g). Yield 95% (method d); light-yellow crystal;
1
mp 157−158 °C. H NMR (300 MHz, CDCl3): 1.00 (t, J = 7.5 Hz,
3H), 1.19 (d, J = 6.3 Hz, 3H), 1.42−1.55 (m, 1H), 1.76−1.87 (m,
J
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2H), 2.81−2.90 (m, 1H), 2.93−3.01 (m, 1H), 3.15 (t, J = 5.4 Hz, 2H),
4.03 (s, 3H), 4.04 (s, 3H), 4.08 (s, 2H), 4.10 (s, 6H), 7.13 (s, 1H),
7.27 (s, 1H), 7.78 (s, 1H), 7.80 (s, 1H). 13C NMR (75 MHz, CDCl3):
11.5, 13.8, 26.3, 28.0, 29.6, 44.8, 49.8, 55.8, 55.9, 60.4, 102.9, 103.2,
103.3, 103.8, 123.3, 124.4, 125.8, 126.3, 126.5, 148.2, 148.3, 148.5. MS
(EI) m/z 409 (M+, 31%) and 380 (100%). HRMS calcd for
C25H31NO4 (M+) 409.2253; found 409.2257.
2-(6,7,10,11-Tetramethoxy-3,4-dihydrodibenzo[f,h]isoquinolin-
2(1H)-yl)ethanamine (10′n). Yield 71% (method d); colorless rock;
1
mp 161−162 °C. H NMR (300 MHz, CDCl3): 2.80 (t, J = 6.0 Hz,
2H), 2.94 (t, J = 6.0 Hz, 2H), 2.99 (t, J = 6.0 Hz, 2H), 3.19 (t, J = 5.7
Hz, 2H), 4.02 (s, 2H), 4.04 (s, 6H), 4.11 (s, 6H), 7.13 (s, 1H), 7.28 (s,
1H), 7.81 (s, 1H), 7.82 (s, 1H). MS (EI) m/z 396 (M+, 9%) and 349
(100%). HRMS calcd for C23H28N2O4 (M+) 396.2049; found
396.2043.
2-(Cyclohex-2-en-1-yl)-6,7,10,11-tetramethoxy-1,2,3,4-
tetrahydrodibenzo[f,h]isoquinoline (10h). Yield 70% (method d);
2-Ethyl-6,7,10,11-tetramethoxy-1,2,3,4-tetrahydrodibenzo[f,h]-
isoquinolin-4-ol (11a). Yield 25% (method e); white needle; mp 185
°C. 1H NMR (300 MHz, CDCl3): 1.25 (t, J = 7.2 Hz, 3H), 2.26 (d, J =
11.1 Hz, 1H), 2.47−2.58 (m, 1H), 2.68−2.79 (m, 1H), 3.00 (d, J =
15.6 Hz, 1H), 3.30−3.36 (m, 1H), 3.35 (d, J = 11.1 Hz, 1H), 3.84 (s,
3H), 4.07 (s, 3H), 4.09 (s, 3H), 4.12 (s, 3H), 4.91 (s, 1H), 6.25 (s,
1H), 7.40 (s, 1H), 7.55 (s, 1H), 7.79 (s, 1H). 13C NMR (100 MHz,
CDCl3): 11.3, 52.2, 53.3, 55.6, 55.7, 55.8, 57.2, 64.6, 102.5, 102.5,
102.6, 105.1, 122.6, 123.6, 123.9, 125.3, 126.4, 126.6, 147.9, 148.2,
148.4, 148.5. MS (EI) m/z 397 (M+, 47%) and 340 (100%). HRMS
calcd for C23H27NO5 (M+) 397.1889; found 397.1880.
6,7,10,11-Tetramethoxy-2-propyl-1,2,3,4-tetrahydrodibenzo[f,h]-
isoquinolin-4-ol (11b). Yield 14% (method e); white rock; mp 202
°C. 1H NMR (400 MHz, CDCl3): 1.02 (t, J = 7.2 Hz, 3H), 1.70−1.81
(m, 2H), 2.46 (d, J = 11.6 Hz, 1H), 2.52−2.59 (m, 1H), 2.65−2.71
(m, 1H), 3.28 (d, J = 15.2 Hz, 1H), 3.35 (d, J = 11.6 Hz, 1H), 3.80 (d,
J = 15.2 Hz, 1H), 3.95 (s, 3H), 4.08 (s, 3H), 4.11 (s, 3H), 4.13 (s,
3H), 5.03 (s, 1H), 6.67 (s, 1H), 7.60 (s, 1H), 7.68 (s, 1H), 7.76 (s,
1H). 13C NMR (150 MHz, CDCl3): 12.0, 19.7, 54.0, 55.8, 55.9, 56.0,
58.0, 60.4, 64.7, 102.9, 103.0, 104.9, 123.0, 123.8, 124.2, 125.3, 126.7,
127.3, 148.3, 148.5, 148.7, 148.8. MS (EI) m/z 411 (M+, 50%) and
340 (100%). HRMS calcd for C24H29NO5 (M+) 411.2046; found
411.2049.
1
light-yellow crystal; mp 128−129 °C. H NMR (400 MHz, CDCl3):
1.62−1.71 (m, 1H), 1.75−1.77 (m, 1H), 1.92−1.94 (m, 1H), 2.02−
2.03 (m, 1H), 2.04−2.08 (m, 2H), 2.91−2.96 (m, 1H), 3.04−3.09 (m,
1H), 3.17 (t, J = 5.2 Hz, 2H), 3.63 (br s, 1H), 4.03 (s, 3H), 4.04 (s,
3H), 4.10 (s, 6H), 4.16 (d, J = 6.0 Hz, 2H), 5.86 (d, J = 10.4 Hz, 1H),
5.95 (d, J = 10.4 Hz, 1H), 7.12 (s, 1H), 7.27 (s, 1H), 7.80 (s, 1H), 7.81
(s, 1H). 13C NMR (100 MHz, CDCl3): 21.7, 23.2, 25.4, 28.0, 45.2,
49.9, 55.8, 56.0, 60.0, 103.0, 103.2, 103.4, 103.9, 123.3, 123.4, 124.4,
125.8, 126.2, 126.3, 129.2, 130.6, 148.3, 148.4, 148.6. MS (EI) m/z
433 (M+, 100%). HRMS calcd for C27H31NO4 (M+) 433.2253; found
433.2259.
2-((1,3-Dioxolan-2-yl)methyl)-6,7,10,11-tetramethoxy-1,2,3,4-
tetrahydrodibenzo[f,h]isoquinoline (10i). Yield 42% (method d);
white needle; mp 182−183 °C. 1H NMR (300 MHz, CDCl3): 2.96 (d,
J = 4.2 Hz, 2H), 3.07 (t, J = 5.1 Hz, 2H), 3.19 (t, J = 5.1 Hz, 2H), 3.91
(AA′BB′, 2H), 4.02 (s, 3H), 4.03 (s, 3H), 4.04 (AA′BB′, 2H), 4.10 (s,
6H), 4.12 (s, 2H), 5.21 (t, J = 4.5 Hz, 1H), 7.12 (s, 1H), 7.26 (s, 1H),
7.79 (s, 1H), 7.80 (s, 1H). 13C NMR (75 MHz, CDCl3): 26.9, 51.0,
54.7, 55.8, 55.9, 60.9, 64.9, 102.9, 103.2, 103.3, 103.8, 123.3, 123.4,
124.2, 125.5, 125.7, 148.3, 148.4, 148.6. MS (EI) m/z 439 (M+, 13%)
and 366 (100%). HRMS calcd for C25H29NO6 (M+) 439.1995; found
439.1987.
2-(2-(1,3-Dioxolan-2-yl)ethyl)-6,7,10,11-tetramethoxy-1,2,3,4-
2-Isopropyl-6,7,10,11-tetramethoxy-1,2,3,4-tetrahydrodibenzo-
[f,h]isoquinolin-4-ol (11c). Yield 5% (method e); yellow crystal; mp
170−172 °C. 1H NMR (300 MHz, CDCl3): 1.24−1.28 (m, 6H), 2.68
(d, J = 11.7 Hz, 1H), 3.11−3.17 (m, 1H), 3.31 (d, J = 11.7 Hz, 1H),
3.89 (d, J = 15.3 Hz, 1H), 4.04 (s, 3H), 4.05 (s, 3H), 4.07 (s, 3H), 4.08
(s, 3H), 4.23 (d, J = 15.3 Hz, 1H), 5.14 (s, 1H), 7.12 (s, 1H), 7.70 (s,
1H), 7.79 (s, 1H), 7.81 (s, 1H). 13C NMR (150 MHz, CDCl3): 17.4,
19.5, 50.3, 52.8, 53.9, 55.9, 56.0, 56.1, 64.6, 103.1, 103.2, 103.3, 104.6,
123.6, 123.9, 124.4, 125.4, 127.4, 128.2, 148.6, 148.7, 149.0, 149.1. MS
(ESI) m/z 412 (M + H)+.
2-Ethyl-6,7,8,10,11-pentamethoxy-1,2,3,4-tetrahydrodibenzo-
[f,h]isoquinoline (28a). Yield = 29% (method n); yellow rock; mp
199−200 °C. 1H NMR (400 MHz, CDCl3): 1.31 (t, J = 7.2 Hz, 3H),
2.79 (q, J = 7.2 Hz, 2H), 2.93 (t, J = 5.6 Hz, 2H), 3.19 (t, J = 5.6 Hz,
2H), 3.98 (s, 3H), 4.01 (s, 2H), 4.02 (s, 3H), 4.04 (s, 3H), 4.05 (s,
3H), 4.09 (s, 3H), 7.16 (s, 1H), 7.18 (s, 1H), 9.20 (s, 1H). 13C NMR
(150 MHz, CDCl3): 10.1, 22.7, 46.9, 49.4, 50.6, 55.7, 55.8, 56.0, 60.6,
61.4, 100.3, 101.7, 108.3, 118.6, 123.1, 123.4, 124.1, 127.0, 143.1,
148.5, 148.8, 151.8, 152.2. MS (EI) m/z 411 (M+, 100%). HRMS
calcd for C24H29NO5 (M+) 411.2046; found 411.2050.
tetrahydrodibenzo[f,h]isoquinoline (10j). Yield 45% (method d);
1
white crystal; mp 186−187 °C. H NMR (300 MHz, CDCl3): 2.10
(td, J = 7.5, 4.5 Hz, 2H), 2.87 (t, J = 7.5 Hz, 2H), 2.94 (t, J = 5.7 Hz,
2H), 3.18 (t, J = 5.7 Hz, 2H), 3.88 (AA′BB′, 2H), 3.99 (s, 2H), 4.02
(AA′BB′, 2H), 4.03 (s, 6H), 4.10 (s, 6H), 5.04 (t, J = 4.8 Hz, 1H),
7.10 (s, 1H), 7.26 (s, 1H), 7.79 (s, 1H), 7.80 (s, 1H). 13C NMR (75
MHz, CDCl3): 27.2, 31.7, 50.1, 53.3, 54.2, 55.8, 55.9, 56.0, 64.9, 102.8,
102.9, 103.2, 103.3, 103.4, 103.8, 123.3, 123.4, 124.2, 125.4, 125.5,
125.8, 148.3, 148.4, 148.6. MS (EI) m/z 453 (M+, 51%) and 324
(100%). HRMS calcd for C26H31NO6 (M+) 453.2151; found
453.2141.
6,7,10,11-Tetramethoxy-2-(3-((tetrahydro-2H-pyran-2-yl)oxy)-
propyl)-1,2,3,4-tetrahydrodibenzo[f,h]isoquinoline (10k). Yield 7%
1
(method e); yellow crystal. H NMR (400 MHz, CDCl3): 1.46−1.55
(m, 4H), 1.65−1.70 (m, 1H), 1.75−1.81 (m, 1H), 1.97 (quintet, J =
7.2 Hz, 2H), 2.73−2.77 (m, 2H), 2.87 (t, J = 5.6 Hz, 2H), 3.12 (t, J =
5.6 Hz, 2H), 3.41−3.52 (m, 2H), 3.81−3.86 (m, 2H), 3.93 (s, 2H),
3.96 (s, 3H), 3.97 (s, 3H), 4.00 (s, 6H), 4.53−4.55 (m, 1H), 7.06 (s,
1H), 7.20 (s, 1H), 7.73 (s, 1H), 7.74 (s, 1H). MS (EI) m/z 495 (M+,
9%) and 410 (100%). HRMS calcd for C29H37NO6 (M+) 495.2621;
found 495.2627.
6,7,8,10,11-Pentamethoxy-2-propyl-1,2,3,4-tetrahydrodibenzo-
[f,h]isoquinoline (28b). Yield 17% (method n); light-yellow crystal;
1
mp 124−125 °C. H NMR (300 MHz, CDCl3): 1.03 (t, J = 7.5 Hz,
tert-Butyl (2-(6,7,10,11-Tetramemthoxy-3,4-dihydrodibenzo[f,h]-
isoquinolin-2(1H)-yl)ethyl) carbamate (10l). Yield 47% (method d);
yellow crystal; mp 123−124 °C. 1H NMR (300 MHz, CDCl3): 1.43 (s,
9H), 2.84 (t, J = 5.7 Hz, 2H), 2.94 (t, J = 5.7 Hz, 2H), 3.19 (t, J = 5.4
Hz, 2H), 3.44 (quartet, J = 5.7 Hz, 2H), 4.00 (s, 2H), 4.04 (s, 6H),
4.11 (s, 6H), 5.20 (br s, 1H), 7.10 (s, 1H), 7.28 (s, 1H), 7.80 (s, 1H),
7.81 (s, 1H). 13C NMR (75 MHz, CDCl3): 27.2, 28.4, 49.9, 54.0, 54.8,
55.8, 55.9, 56.0, 57.1, 102.8, 103.3, 103.4, 103.8, 123.4, 123.5, 124.1,
125.3, 125.5, 125.9, 148.4, 148.6, 148.7. MS (ESI) m/z 497 (M + H)+.
3-(6,7,10,11-Tetramethoxy-3,4-dihydrodibenzo[f,h]isoquinolin-
2(1H)-yl)propan-1-ol (10′m). Yield 93% (method d); white needle.
1H NMR (300 MHz, CDCl3): 1.95 (quintet, J = 5.6 Hz, 2H), 2.99−
3H), 1.74 (sextet, J = 7.5 Hz, 2H), 2.68 (t, J = 7.5 Hz, 2H), 2.92 (t, J =
6.0 Hz, 2H), 3.18 (t, J = 6.0 Hz, 2H), 3.98 (s, 3H), 4.00 (s, 2H), 4.02
(s, 3H), 4.04 (s, 3H), 4.05 (s, 3H), 4.09 (s, 3H), 7.16 (s, 1H), 7.18 (s,
1H), 9.19 (s, 1H). 13C NMR (150 MHz, CDCl3): 12.0, 20.2, 27.1,
49.8, 54.0, 55.7, 55.8, 60.0, 60.5, 61.3, 100.4, 102.2, 108.1, 117.8, 123.5,
124.8, 125.6, 126.6, 128.4, 142.1, 148.0, 151.6, 151.7. MS (EI) m/z
425 (M+, 100%). HRMS calcd for C25H31NO5 (M+) 425.2202; found
425.2195.
2-Butyl-6,7,8,10,11-pentamethoxy-1,2,3,4-tetrahydrodibenzo-
[f,h]isoquinoline (28c). Yield 16% (method n); light-yellow crystal;
1
mp 120−123 °C. H NMR (400 MHz, CDCl3): 1.00 (t, J = 7.6 Hz,
3.07 (m, 4H), 3.23 (t, J = 5.6 Hz, 2H), 3.90 (t, J = 5.6 Hz, 2H), 4.04
(s, 3H), 4.06 (s, 3H), 4.11 (s, 2H), 4.12 (s, 6H), 7.13 (s, 1H), 7.28 (s,
1H), 7.83 (s, 1H), 7.84 (s, 1H). MS (EI) m/z 411 (M+, 97%) and 349
(100%). HRMS calcd for C24H29NO5 (M+) 411.2046; found
411.2043.
3H), 1.45 (sextet, J = 7.6 Hz, 2H), 1.70 (quintet, J = 7.6 Hz, 2H), 2.71
(t, J = 7.6 Hz, 2H), 2.92 (t, J = 5.6 Hz, 2H), 3.18 (t, J = 5.6 Hz, 2H),
3.98 (s, 3H), 4.00 (s, 2H), 4.02 (s, 3H), 4.05 (s, 3H), 4.06 (s, 3H),
4.09 (s, 3H), 7.16 (s, 1H), 7.18 (s, 1H), 9.19 (s, 1H). MS (ESI) m/z
440 (M + H)+.
K
dx.doi.org/10.1021/jm300705j | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2 - I s o p r o p y l - 6 , 7 , 8 , 1 0 , 1 1 - p e n t a m e t h o x y - 1 , 2 , 3 , 4 -
2H), 3.21 (t, J = 5.7 Hz, 2H), 3.89 (s, 3H), 3.96 (s, 2H), 3.98 (s, 3H),
4.02 (s, 3H), 7.21 (d, J = 2.9 Hz, 1H), 7.24 (dd, J = 10.4, 2.9 Hz, 1H),
7.27 (d, J = 9.0 Hz, 1H), 7.75 (d, J = 9.0 Hz, 1H), 9.62 (d, J = 10.4 Hz,
1H). 13C NMR (150 MHz, CDCl3): 12.1, 20.5, 27.6, 50.2, 54.2, 55.3,
56.3, 59.7, 60.7, 104.2, 111.7, 113.8, 119.5, 122.8, 124.0, 125.9, 126.6,
128.8, 129.8, 132.9, 146.3, 151.0, 157.9. MS (EI) m/z 365 (M+, 86%)
and 336 (100%). HRMS calcd for C23H27NO3 (M+) 365.1991; found
365.1987.
tetrahydrodibenzo[f,h]isoquinoline (28d). Yield 13% (method n);
1
light-yellow crystal; mp 195−198 °C. H NMR (300 MHz, CDCl3):
1.12 (d, J = 6.3 Hz, 6H), 2.97 (t, J = 5.4 Hz, 2H), 3.11 (septet, J = 6.3
Hz, 1H), 3.18 (t, J = 5.4 Hz, 2H), 3.98 (s, 3H), 4.02 (s, 3H), 4.04 (s,
3H), 4.06 (s, 3H), 4.09 (s, 3H), 4.11 (s, 2H), 7.16 (s, 1H), 7.18 (s,
1H), 9.19 (s, 1H). MS (ESI) m/z 426 (M + H)+.
2-Ethyl-7,10,11-trimethoxy-1,2,3,4-tetrahydrodibenzo[f,h]-
isoquinoline (29a). Yield 40% (method m), 16% (method n); yellow
crystal; mp 160−161 °C. 1H NMR (300 MHz, CDCl3): 1.32 (t, J = 7.2
Hz, 3H), 2.80 (q, J = 7.2 Hz, 2H), 2.94 (t, J = 5.7 Hz, 2H), 3.25 (t, J =
5.7 Hz, 2H), 4.00 (s, 2H), 4.01 (s, 3H), 4.05 (s, 3H), 4.10 (s, 3H),
7.14 (s, 1H), 7.21 (dd, J = 9.0, 2.6 Hz, 1H), 7.89 (d, J = 2.6 Hz, 1H),
7.90 (s, 1H), 7.92 (d, J = 9.0 Hz, 1H). 13C NMR (150 MHz, CDCl3):
12.0, 20.4, 50.0, 54.0, 55.5, 55.9, 56.0, 60.3, 102.9, 104.0, 104.6, 114.8,
123.3, 125.1, 125.3, 125.5, 126.5, 127.4, 130.2, 148.2, 149.4, 157.6. MS
(EI) m/z 351 (M+, 100%). HRMS calcd for C22H25NO3 (M+)
351.1834; found 351.1837.
6,7,8,10,11-Pentamethoxy-2-propyl-1,2,3,4-tetrahydrodibenzo-
[f,h]isoquinolin-4-ol (33a). Yield 20% (method n), light-yellow
1
crystal; mp 213−214 °C. H NMR (600 MHz, CDCl3): 1.01 (t, J =
7.8 Hz, 3H), 1.72 (sextet, J = 7.8 Hz, 2H), 2.54 (dd, J = 11.1, 2.1 Hz,
1H), 2.59 (ddt, J = 15.3, 12, 7.8 Hz, 1H), 2.68 (ddt, J = 15.3, 12, 7.8
Hz, 1H), 3.35 (d, J = 11.1 Hz, 1H), 3.47 (d, J = 15.3 Hz, 1H), 3.97 (s,
3H), 4.00 (s, 3H), 4.05 (s, 3H), 4.06 (s, 3H), 4.09 (s, 3H), 4.11 (d, J =
15.3 Hz, 1H), 5.09 (s, 1H), 6.96 (s, 1H), 7.64 (s, 1H), 9.16 (s, 1H).
13C NMR (150 MHz, CDCl3): 11.9, 20.0, 54.4, 55.6, 55.7, 55.8, 57.9,
60.1, 60.5, 61.3, 64.9, 101.4, 102.5, 107.8, 118.2, 123.9, 124.4, 127.7,
128.1, 128.3, 142.2, 147.9, 148.5, 151.4, 151.9. MS (EI) m/z 441 (M+,
61%) and 370 (100%). HRMS calcd for C25H31NO6 (M+) 441.2151;
found 441.2149.
2-Ethyl-7,10,11-trimethoxy-1,2,3,4-tetrahydrodibenzo[f,h]-
isoquinolin-4-ol (33b). Yield 31% (method n); yellow needle; mp
207−208 °C. 1H NMR (300 MHz, CDCl3): 1.28 (t, J = 7.2 Hz, 3H),
2.38 (dd, J = 11.4, 2.4 Hz, 1H), 2.60 (ddq, J = 14.7, 12.3, 7.2 Hz, 1H),
2.78 (ddq, J = 14.7, 12.3, 7.2 Hz, 1H), 3.18 (d, J = 14.7 Hz, 1H), 3.36
(d, J = 11.4 Hz, 1H), 3.62 (d, J = 14.7 Hz, 1H), 3.91 (s, 3H), 4.03 (s,
3H), 4.10 (s, 3H), 5.05 (s, 1H), 6.53 (s, 1H), 7.24 (dd, J = 9.3, 2.7 Hz,
1H), 7.64 (s, 1H), 7.72 (d, J = 2.7 Hz, 1H), 8.34 (d, J = 9.3 Hz, 1H).
13C NMR (150 MHz, CDCl3): 12.1, 52.1, 53.7, 55.5, 55.8, 55.9, 57.3,
7,10,11-Trimethoxy-2-propyl-1,2,3,4-tetrahydrodibenzo[f,h]-
isoquinoline (29b). Yield 13% (method n); yellow needle; mp 159−
1
160 °C. H NMR (400 MHz, CDCl3): 1.03 (t, J = 7.6 Hz, 3H), 1.75
(sextet, J = 7.6 Hz, 2H), 2.67 (t, J = 7.6 Hz, 2H), 2.92 (t, J = 6.0 Hz,
2H), 3.24 (t, J = 6.0 Hz, 2H), 3.98 (s, 2H), 4.02 (s, 3H), 4.05 (s, 3H),
4.11 (s, 3H), 7.15 (s, 1H), 7.21 (dd, J = 9.2, 2.4 Hz, 1H), 7.89 (d, J =
2.4 Hz, 1H), 7.91 (s, 1H), 7.93 (d, J = 9.2 Hz, 1H). 13C NMR (150
MHz, CDCl3): 12.0, 20.4, 26.9, 50.2, 53.9, 55.5, 55.8, 55.9, 60.5, 103.7,
103.8, 104.6, 114.8, 123.3, 123.9, 124.0, 125.0, 126.0, 126.8, 130.1,
148.3, 149.3, 157.4. MS (EI) m/z 365 (M+, 100%). HRMS calcd for
C23H27NO3 (M+) 365.1991; found 365.1995.
2-Butyl-7,10,11-trimethoxy-1,2,3,4-tetrahydrodibenzo[f,h]-
isoquinoline (29c). Yield 15% (method n);yellow crystal; mp 130−
64.3, 103.0, 103.6, 104.5, 115.1, 124.1, 124.5, 124.7, 126.0, 126.2,
128.2, 130.6, 148.7, 149.2, 157.6. MS (EI) m/z 367 (M+, 42%) and
310 (100%). HRMS calcd for C22H25NO4 (M+) 367.1784; found
367.1780.
1
131 °C. H NMR (300 MHz, CDCl3): 1.00 (t, J = 7.5 Hz, 3H), 1.45
(sextet, J = 7.5 Hz, 2H), 1.71 (quintet, J = 7.5 Hz, 2H), 2.71 (t, J = 7.5
Hz, 2H), 2.92 (t, J = 5.7 Hz, 2H), 3.23 (t, J = 5.7 Hz, 2H), 3.98 (s,
2H), 4.02 (s, 3H), 4.05 (s, 3H), 4.11 (s, 3H), 7.12 (s, 1H), 7.21 (dd, J
= 9.0, 2.6 Hz, 1H), 7.88 (d, J = 2.6 Hz, 1H), 7.90 (s, 1H), 7.92 (d, J =
9.0 Hz, 1H). MS (EI) m/z 379 (M+, 88%) and 336 (100%). HRMS
calcd for C24H29NO3 (M+) 379.2147; found 379.2136.
7,10,11-Trimethoxy-2-propyl-1,2,3,4-tetrahydrodibenzo[f,h]-
isoquinolin-4-ol (33c). Yield 16% (method n); yellow needle; mp
209−210 °C. 1H NMR (600 MHz, CDCl3): 1.01 (t, J = 7.2 Hz, 3H),
1.70−1.75 (m, 2H), 2.46 (d, J = 11.4 Hz, 1H), 2.51−2.56 (m, 1H),
2.63−2.68 (m, 1H), 3.29 (d, J = 14.4 Hz, 1H), 3.34 (d, J = 11.4 Hz,
1H), 3.81 (d, J = 14.4 Hz, 1H), 3.96 (s, 3H), 4.03 (s, 3H), 4.10 (s,
3H), 5.08 (s, 1H), 6.71 (s, 1H), 7.25 (dd, J = 9.0, 3.0 Hz, 1H), 7.72 (s,
1H), 7.78 (d, J = 1.8 Hz, 1H), 8.32 (d, J = 8.4 Hz, 1H). 13C NMR (150
MHz, CDCl3): 12.0, 19.7, 53.9, 55.5, 55.7, 55.8, 57.8, 60.4, 64.4, 102.9,
103.4, 104.3, 114.9, 124.0, 124.3, 124.7, 125.9, 126.3, 128.0, 130.5,
148.6, 149.0, 157.5. LRMS (EI+) m/z (rel intensity) 381 (M+, 42%)
and 310 (100%). HRMS calcd for C23H27NO4 (M+) 381.1940; found,
381.1936.
2-Ethyl-6,7,11-trimethoxy-1,2,3,4-tetrahydrodibenzo[f,h]-
isoquinoline (30a). Yield 65% (method m); yellow crystal; mp 111−
1
112 °C. H NMR (300 MHz, CDCl3): 1.31 (t, J = 7.2 Hz, 3H), 2.79
(q, J = 7.2 Hz, 2H), 2.95 (t, J = 5.7 Hz, 2H), 3.23 (t, J = 5.7 Hz, 2H),
3.97 (s, 3H), 4.01 (s, 2H), 4.04 (s, 3H), 4.10 (s, 3H), 7.21 (dd, J = 8.9,
2.6 Hz, 1H), 7.24 (d, J = 2.6 Hz, 1H), 7.30 (s, 1H), 7.93 (s, 1H), 8.46
(d, J = 8.9 Hz, 1H). 13C NMR (150 MHz, CDCl3): 12.4, 27.3, 49.7,
52.3, 53.6, 55.4, 55.8, 55.9, 103.2, 103.8, 103.9, 115.0, 123.2, 124.1,
125.0, 125.5, 128.2, 130.6, 148.5, 148.7, 157.6. MS (EI) m/z 351 (M+,
100%). HRMS calcd for C22H25NO3 (M+) 351.1834; found 351.1836.
6,7,11-Trimethoxy-2-propyl-1,2,3,4-tetrahydrodibenzo[f,h]-
isoquinoline (30b). Yield 80% (method m); yellow crystal; mp 114−
2-Butyl-7,10,11-trimethoxy-1,2,3,4-tetrahydrodibenzo[f,h]-
isoquinolin-4-ol (33d). Yield 14% (method n); light-yellow crystal;
1
1
mp 165−166 °C. H NMR (600 MHz, CDCl3): 1.01 (t, J = 7.2 Hz,
115 °C. H NMR (300 MHz, CDCl3): 1.02 (t, J = 7.5 Hz, 3H), 1.75
3H), 1.41−1.45 (m, 2H), 1.66−1.70 (m, 2H), 2.42 (d, J = 11.4 Hz,
1H), 2.50−2.55 (m, 1H), 2.64−2.69 (m, 1H), 3.22 (d, J = 15.6 Hz,
1H), 3.35 (d, J = 12.0 Hz, 1H), 3.67 (d, J = 14.4 Hz, 1H), 3.94 (s, 3H),
4.02 (s, 3H), 4.10 (s, 3H), 5.05 (s, 1H), 6.61 (s, 1H), 7.24 (dd, J = 9.0,
3.0 Hz, 1H), 7.68 (s, 1H), 7.75 (d, J = 1.8 Hz, 1H), 8.33 (d, J = 9.6 Hz,
1H). 13C NMR (150 MHz, CDCl3): 14.1, 20.8, 28.5, 53.9, 55.5, 55.7,
55.8, 57.8, 58.3, 64.3, 102.8, 103.3, 104.3, 114.9, 123.9, 124.2, 124.8,
125.9, 126.4, 127.9, 130.5, 148.5, 148.9, 157.5. LRMS (EI+) m/z (rel
intensity) 395 (M+, 10%) and 310 (100%). HRMS calcd for
C24H29NO4 (M+) 395.2097; found, 395.2098.
(sextet, J = 7.5 Hz, 2H), 2.68 (t, J = 7.5 Hz, 2H), 2.94 (t, J = 6.0 Hz,
2H), 3.22 (t, J = 6.0 Hz, 2H), 3.97 (s, 3H), 4.00 (s, 2H), 4.04 (s, 3H),
4.10 (s, 3H), 7.21 (dd, J = 8.7, 2.7 Hz, 1H), 7.24 (d, J = 2.7 Hz, 1H),
7.30 (s, 1H), 7.93 (s, 1H), 8.46 (d, J = 8.7 Hz, 1H). 13C NMR (150
MHz, CDCl3): 11.9, 20.0, 26.5, 49.6, 53.4, 55.5, 55.8, 55.9, 59.9, 103.1,
103.5, 103.8, 115.3, 123.2, 124.1, 124.2, 124.8, 127.8, 130.3, 148.6,
148.8, 157.7. MS (EI) m/z 365 (M+, 90%) and 336 (100%). HRMS
calcd for C23H27NO3 (M+) 365.1991; found 365.1987.
6,7,10-Trimethoxy-2-propyl-1,2,3,4-tetrahydrodibenzo[f,h]-
isoquinoline (31). Yield 60% (method m); yellow needle; mp 159−
1
Biology. Statistical Analysis. InStat version 3.06 software for
Windows (GraphPad, San Diego, CA) was used to evaluate the
statistical significance between two groups by 2-tailed unpaired
Student’s t test. Throughout the text, figures, and legends, the
following terminology is used to denote statistical significance: *p <
160 °C. H NMR (400 MHz, CDCl3): 1.02 (t, J = 7.6 Hz, 3H), 1.74
(sextet, J = 7.6 Hz, 2H), 2.65 (t, J = 7.6 Hz, 2H), 2.93 (t, J = 6.0 Hz,
2H), 3.19 (t, J = 5.6 Hz, 2H), 4.02 (s, 3H), 4.05 (s, 5H), 4.11 (s, 3H),
7.20 (dd, J = 8.8, 2.4 Hz, 1H), 7.30 (s, 1H), 7.81 (d, J = 8.8 Hz, 1H),
7.90 (d, J = 2.4 Hz, 1H), 7.91 (s, 1H). MS (EI) m/z 365 (M+, 100%).
HRMS calcd for C23H27NO3 (M+) 365.1991; found 365.1998.
7,8,11-Trimethoxy-2-propyl-1,2,3,4-tetrahydrodibenzo[f,h]-
isoquinoline (32). Yield 51% (method m); yellow crystal; mp 88−89
#
0.001; p < 0.01; ns, no significance.
Western Analysis. These assays were performed as described.5,23,26
Cyclin A2, c-Jun, iNOS, COX-2, and GAPDH proteins were analyzed
by immunoblotting with anti-Cyclin A2, anti-c-Jun (Santa Cruz
Biotechnology, Santa Cruz, CA), anti-iNOS (BIOMOL Research
1
°C. H NMR (300 MHz, CDCl3): 1.01 (t, J = 7.5 Hz, 3H), 1.74
(sextet, J = 7.5 Hz, 2H), 2.66 (t, J = 7.8 Hz, 2H), 2.90 (t, J = 5.7 Hz,
L
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Laboratories, Plymouth Meeting, PA), anti-COX-2 (Upstate Bio-
technology), and anti-GAPDH (Cell Signaling, Beverly, MA)
antibodies, respectively.
according to committee-approved procedures. Male rats (330−380 g,
9−10 weeks old) were quarantined for 1 week before use. The animals
were surgically implanted with a jugular vein cannula 1 day before
treatment and were fasted before treatment. Compounds 10b, 29a,
and 33b at 3 mg/kg as well as tylophovatine C at 1.5 mg/kg were
given to rats (n = 3) by intravenous or oral administration as prepared
in a mixture of DMA/PEG400 (30/70, v/v) for 10b, 29a, and 33b and
of DMA/PEG400 (50/50, v/v) for tylophovatine C. The volume of
the dosing solution given was adjusted according to the body weight
recorded before the drug was administered. At 0 (immediately before
dosing), 2, 5 (intravenous only), 15, and 30 min and 1, 2, 4, 6, 8, 12,
and 24 h after compound administration, a blood sample (150 μL) was
taken from each animal via the jugular vein cannula and stored in ice
(0−4 °C). The processing of the plasma and subsequent analysis by
high performance liquid chromatography-tandem mass spectrometry
(HPLC-MS) were as described.31 The plasma concentration data were
analyzed by a standard noncompartmental method with the Kinetica
software (InnaPhase, Philadelphia, PA, USA).
In Vivo Anti-inflammatory Activity Measured by Carrageenan-
Induced Hind-Paw Edema Test in Rats. Female Sprague−Dawley rats
(193−214 g, 8−9 week old, n = 5) were used. To produce
inflammation, 100 μL of 1% carrageenan solution in normal saline
was injected into the left hind paw subplantar tissue. One hour before
carrageenan challenge, the sample preparations (3 or 5 mg/kg) were
injected intraperitoneally to the divided groups, with DMSO (100 μL)
injected into the vehicle control group. The tests were performed as
described.28 The positive control indomethacin was purchased from
Sigma-Aldrich (cat. I7378) with purity of ≥99% (TLC). When
inhibition is or greater than 30%, the treatment is considered with a
significant efficacy.
In Vivo Antitumor Activity. Four week-old male nude mice (Nu-
Foxn1nu, BioLASCO Taiwan Co., Ltd.) were used. Nude mice were
subcutaneously injected with 10 million A549 cells (in 200 μL of
RPMI-1640 medium) into the right flank. When the average size of
tumors reached ∼60 mm3, mice were randomly divided into three
groups (n = 8 for treatments of vehicle and 33b at the dose of 10 mg/
kg; n = 7 for treatment of 33b at the dose of 5 mg/kg) and treated
with vehicle control (25% DMA + 75% PEG400), 33b (5 mg/kg), and
33b (10 mg/kg) via oral gavage. Each group was administered once
daily, on day 1−10, day 15−19, and day 22−26, for 20 times in total.
The tumor volume was calculated using the equation V (mm3) = a ×
b2/2, where a is the largest diameter and b is the smallest diameter.32
Tumor growth inhibitions (TGI) were determined for antitumor
effects which are expressed as % (Tday60 − Tday1)/Cday60 − Cday1) (T/C,
treated versus control), dividing the tumor volumes from treatment
groups with the control groups and multiplied by 100%. The effective
criteria for the T/C (%) according to the National Cancer Institute
standard is <42%.33 The reduction in tumor volume for treated versus
vehicle treatment should be greater than 58% to be considered with a
significant efficacy. No overt signs of any adverse were observed from
the test mice during experimental period.
Rota-Rod Test for Motor Coordination. Neurotoxicity was
determined by a rota-rod test for motor coordination. Male nude
mice were used in antitumor test first as described above and
subsequently for the rota-rod test with modification.25 The mice tested
included four groups. Group I: tumor-bearing mice received vehicle
(25% DMA + 75% PEG400, n = 8). Group II: tumor-bearing mice
received 33b at dose of 5 mg/kg for 20 administrations (n = 7). Group
III: tumor-bearing mice received 33b at dose of 10 mg/kg for 20
administrations (n = 8). Group IV: the same age of normal nude mice
(n = 5) without tumor or any treatment as another control group in
addition to group I. On the day of 58−61 after the compound or
vehicle administrations, all the mice were placed onto individual
sections of the apparatus for training (day 58) and test (day 59, 60,
and 61) with the rod setting in motion at a increscent speed from 2 to
30 rpm in 300 s. Performance was measured as the time that elapsed
between the animal being placed on the rod and falling off the rotating
rod with 300 s as the cutoff.
In Vitro Carcinoma Cell Growth Inhibitory Assay. The previously
described assay procedure was used.23,27 The human lung carcinoma
cell line A549 cells (ATCC CCL-185) were grown in normal RPMI
1640 culture medium (GIBCO-Life Technologies, Inc., Gaithersburg,
MD) supplemented with 2 g/L of NaHCO3, 2 mM L-glutamine, and
10% fetal bovine serum (FBS, Hyclone Laboratory Inc., Greeley, Co).
NUGC-3 gastric carcinoma, HONE-1 nasopharyngeal carcinoma,
MCF-7 breast carcinoma, NCI-H460 lung carcinoma, SF-268
glioblastoma, HepG2 hepato carcinoma, and A549 lung carcinoma
cells were seeded at 4500, 6000, 6500, 2500, 7500, 10000, and 5000
cells/well, respectively, in 96-well plates. The positive control
paclitaxel was purchased from Calbiochem-EMD Millipore (cat.
580555) with purity of ≥97% by HPLC.
In Vitro Anti-inflammatory Effect by Determining Nitric Oxide
Production. The previously described assay procedure was em-
ployed.5,28 RAW264.7 cells were seeded (65000 cells/well) and
cultured in 96-well plates. After 24 h incubation, the medium was
replaced with complete medium containing IFNγ (20 ng/mL)/LPS (5
μg/mL), and the test compounds were added at various concen-
trations as indicated for 18 h before measurement of nitric oxide
production by the Nitrate/Nitrite assay kit as described.
Immunofluorescent Assay (IFA). The IFA assay was performed and
the images were captured as described.26 Briefly, ST cells in 96-well
plates, with or without a 2 h pretreatment with test compounds, were
infected with TGEV at a multiplicity of infection (MOI) of 10 for IFA.
The IFA was performed at 6 h postinfection (hpi) with antibodies
against the spike (S) and nucleocapsid (N) proteins of TGEV. The
cells were treated with 10 different concentrations of test compounds.
The results of these assays were used to obtain the dose−response
curves from which 50% maximal effective concentration (EC50) values
were determined.
Cytopathic Effect Induced by Murine Hepatitis Virus in DBT Cells.
The murine astrocytoma cell line DBT (a kind gift from Dr. Lai,
Michael M. C.; Academia Sinica, Taiwan, R.O.C.)29 and embryonic
murine hepatocyte cell line BNL CL.2 (BCRC no. 60180) was
maintained as monolayer culture in Dulbecco's Modified Eagles
Medium (DMEM, Hyclone Laboratory Inc., Greeley, Co) supple-
mented with 10% heat-inactivated FBS. The murine hepatitis virus
(MHV), JHM strain (a gift from Dr. Jan, Jia-Tsrong; Academia Sinica,
Taiwan, R.O.C.), was propagated in BNL CL.2 cells30 and harvested
and the viral titer was measured. The DBT culture condition and the
MOI of MHV-JHM used were optimized and determined in order to
induce evident cytopathic effects at 24 hpi. DBT cells (2.8 × 105 cells/
well) were seeded onto the 24-well plates the day before infection with
MHV-JHM. Prior to the addition of test compounds, the culture
medium was changed to DMEM containing 2% FBS for MHV-JHM
infection. The test compounds were pretreated for 1 h prior to MHV-
JHM infection at MOI of 0.005, and the cytopathic effects in DBT
cells were observed at 24 hpi and recorded using a charge-coupled
device linked to a Nikon Eclipse TE2000 microscopy. Subsequently,
these MHV-infected DBT cells were subject to immunofluorescent
assay as described above with a monoclonal antibody against MHV N
protein generated in our laboratory (data not shown and will publish
elsewhere).
Animal Study Protocols. Animal study protocols [(NHRI-IACUC-
096006-A, 04/20/2007−04/19/2010), (NHRI-IACUC-096036-A,
11/01/2007−10/31/2010), (NHRI-IACUC-099025-A, 04/20/
2010−04/19/2013), (NHRI-IACUC-099047-A, 10/31/2010−10/
31/2013), (NHRI-IACUC-100112-A, 01/01/2012−12/31/2015),
and (NHRI-IACUC-099065-A, 08-10-2010−08-09-2013)] were re-
viewed and approved for the in vivo experiments herein by the
Institutional Animal Care and Use Committee of National Health
Research Institutes, Taiwan.
Pharmacokinetic Analysis. The Sprague−Dawley rats for the
pharmacokinetic study were obtained from BioLASCO Taiwan Co.
(Ilan, Taiwan) and housed in the animal facility at the National Health
Research Institutes, Taiwan. The animal studies were performed
M
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Activity Relations of Phenanthroindolizidines and Phenanthroquino-
lizidines. Biochem. Biophys. Res. Commun. 2007, 354, 942−948.
(7) Gao, W.; Lam, W.; Zhong, S.; Kaczmarek, C.; Baker, D. C.;
Cheng, Y. C. Novel Mode of Action of Tylophorine Analogs as
Antitumor Compounds. Cancer Res. 2004, 64, 678−688.
(8) Yang, C. W.; Lee, Y. Z.; Kang, I. J.; Barnard, D. L.; Jan, J. T.; Lin,
D.; Huang, C. W.; Yeh, T. K.; Chao, Y. S.; Lee, S. J. Identification of
Phenanthroindolizines and Phenanthroquinolizidines as Novel Potent
Anti-Coronaviral Agents for Porcine Enteropathogenic Coronavirus
Transmissible Gastroenteritis Virus and Human Severe Acute
Respiratory Syndrome Coronavirus. Antiviral Res. 2010, 88, 160−168.
(9) Kimball, F. S.; Turunen, B. J.; Ellis, K. C.; Himes, R. H.; Georg,
G. I. Enantiospecific Synthesis and Cytotoxicity of 7-(4-Methox-
yphenyl)-6-phenyl-2,3,8,8a-tetrahydroindolizin-5(1H)-One Enan-
tiomers. Bioorg. Med. Chem. 2008, 16, 4367−4377.
(10) Lin, J. C.; Yang, S. C.; Hong, T. M.; Yu, S. L.; Shi, Q.; Wei, L.;
Chen, H. Y.; Yang, P. C.; Lee, K. H. Phenanthrene-Based Tylophorine-
1 (PBT-1) Inhibits Lung Cancer Cell Growth through the Akt and
NF-κB Pathways. J. Med. Chem. 2009, 52, 1903−1911.
(11) Kimball, F. S.; Himes, R. H.; Georg, G. I. Synthesis and
Evaluation of Heteroaromatic 6,7-Diaryl-2,3,8,8a-tetrahydroindolizin-
5(1H)-Ones for Cytotoxicity against the HCT-116 Colon Cancer Cell
Line. Bioorg. Med. Chem. Lett. 2008, 18, 3248−3250.
(12) Wei, L.; Brossi, A.; Kendall, R.; Bastow, K. F.; Morris-Natschke,
S. L.; Shi, Q.; Lee, K. H. Antitumor Agents 251: Synthesis, Cytotoxic
Evaluation, and Structure−Activity Relationship Studies of Phenan-
threne-Based Tylophorine Derivatives (PBTs) as a New Class of
Antitumor Agents. Bioorg. Med. Chem. 2006, 14, 6560−6569.
(13) Wei, L.; Shi, Q.; Bastow, K. F.; Brossi, A.; Morris-Natschke, S.
L.; Nakagawa-Goto, K.; Wu, T. S.; Pan, S. L.; Teng, C. M.; Lee, K. H.
Antitumor Agents 253. Design, Synthesis, and Antitumor Evaluation of
Novel 9-Substituted Phenanthrene-Based Tylophorine Derivatives as
Potential Anticancer Agents. J. Med. Chem. 2007, 50, 3674−3680.
(14) Fu, Y.; Lee, S. K.; Min, H. Y.; Lee, T.; Lee, J.; Cheng, M.; Kim,
S. Synthesis and Structure−Activity Studies of Antofine Analogues as
Potential Anticancer Agents. Bioorg. Med. Chem. Lett. 2007, 17, 97−
100.
(15) Suffness, M.; Douros, J. Anticancer Agents Based on Natural
Product Models; Academic Press: New York, 1980; pp 465−487.
(16) Chuang, T. H.; Lee, S. J.; Yang, C. W.; Wu, P. L. Expedient
Synthesis and Structure−Activity Relationships of Phenanthroindoli-
zidine and Phenanthroquinolizidine Alkaloids. Org. Biomol. Chem.
2006, 4, 860−867.
(17) Duclos, R. I., Jr.; Tung, J. S.; Rapoport, H. A High-Yield
Modification of the Pschorr Phenanthrene Synthesis. J. Org. Chem.
1984, 49, 5243.
(18) Buckley, T. F., III; Rapoport, H. A-Amino Acids as Chiral
Educts for Asymmetric Products, Chirally Specific Syntheses of
Tylophorine and Cryptopleurine. J. Org. Chem. 1983, 48, 4222−4232.
(19) Su, C. R.; Damu, A. G.; Chiang, P. C.; Bastow, K. F.; Morris-
Natschke, S. L.; Lee, K. H.; Wu, T. S. Total Synthesis of
Phenanthroindolizidine Alkaloids ( )-Antofine, ( )-Deoxypergular-
inine, and Their Dehydro Congeners and Evaluation of Their
Cytotoxic Activity. Bioorg. Med. Chem. 2008, 16, 6233−6241.
(20) Wang, K.; Lu, M.; Yu, A.; Zhu, X.; Wang, Q. Iron(III) Chloride
Catalyzed Oxidative Coupling of Aromatic Nuclei. J. Org. Chem. 2009,
74, 935−938.
(21) Bremmer, M. L.; Khatri, N. A.; Weinreb, S. M. Quinolizidine
Alkaloid Synthesis Via the Intramolecular Imino Diels−Alder
Reaction. epi-Lupinine and Cryptopleurine. J. Org. Chem. 1983, 48,
3661−3666.
(22) Yang, X.; Shi, Q.; Bastow, K. F.; Lee, K. H. Antitumor Agents.
274. A New Synthetic Strategy for E-Ring SAR Study of Antofine and
Cryptopleurine Analogues. Org. Lett. 2010, 12, 1416−1419.
(23) Wu, C. M.; Yang, C. W.; Lee, Y. Z.; Chuang, T. H.; Wu, P. L.;
Chao, Y. S.; Lee, S. J. Tylophorine Arrests Carcinoma Cells at G1
Phase by Downregulating Cyclin A2 Expression. Biochem. Biophys. Res.
Commun. 2009, 386, 140−145.
ASSOCIATED CONTENT
* Supporting Information
One-dimensional, two-dimensional NMR data of 33a and
experimental details for intermediates 14a−14c, 15a−15c,
16a−16d, 22a−22d, 23a−23e, 24a−24e, 25a−25e, 26a−26e,
19, 20, 21, 22e, 7a−7l, and 27a−27k This material is available
free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: 886-37-246166 ext 35715. Fax: 886-37-586456. E-
mail: slee@nhri.org.tw.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Health Research
Institutes, Taiwan, R.O.C., and by the National Science Council
of Taiwan, R.O.C. (99-320-B-400-010-MY3). Cheng-Wei Yang
and Ya-Qi Qiu currently are Ph.D. students in the Graduate
Program of Biotechnology in Medicine sponsored by the
National Tsing Hua University and the National Health
Research Institutes. The authors acknowledge Dr. Iou-Jiun
Kang for support with a part of H NMR, 13C NMR, and LC/
1
1
MS measurements and Dr. Yu-Chen Chou a part of the H
NMR and LC/MS measurements. We are also grateful to Dr.
Jinq-Chyi Lee for proof reading the manuscript.
ABBREVIATIONS USED
■
A549, human lung adenocarcinoma epithelial cells; AP1,
activator protein 1; CC50, concentration of 50% cellular
cytotoxicity; DBT, murine astrocytoma cells; GAPDH,
glyceraldehyde 3-phosphate dehydrogenase; GI50, concentra-
tion required for 50% inhibition of cell growth; HepG2, human
liver hepatocellular carcinoma cells; HONE-1, human naso-
pharyngeal cells; LPS, lipopolysaccharides; MCF-7, human
breast carcinoma cells; MHV, murine hepatitis virus; N,
nucleocapsid; NCI-H460, human nonsmall cell lung cancer
cells; NF-κB, nuclear factor κ-light chain-enhancer of activated
B cells; NUGC-3, human gastric carcinoma cells; RAW264.7,
murine macrophage; S, spike; SD, standard deviation; SF-268,
human glioblastoma cells; ST, swine testicular epithelial cells;
TGEV, transmissible gastroenteritis coronavirus
REFERENCES
■
(1) Huang, X.; Gao, S.; Fan, L.; Yu, S.; Liang, X. Cytotoxic Alkaloids
from the Roots of Tylophora atrofolliculata. Planta Med. 2004, 70,
441−445.
(2) Chemler, S. R. Phenanthroindolizidines and Phenanthroquino-
lizidines: Promising Alkaloids for Anti-Cancer Therapy. Curr. Bioactive
Compd. 2009, 5, 2−19.
(3) Lee, Y. Z.; Huang, C. W.; Yang, C. W.; Hsu, H. Y.; Kang, I. J.;
Chao, Y. S.; Chen, I. S.; Chang, H. Y.; Lee, S. J. Isolation and
Biological Activities of Phenanthroindolizidine and Septicine Alkaloids
from the Formosan Tylophora Ovata. Planta Med. 2011, 77, 1932−
1938.
(4) Li, Z.; Jin, Z.; Huang, R. Isolation, Total Synthesis and Biological
Activity of Phenanthroindolizidine and Phenanthroquinolizidine
Alkaloids. Synthesis 2001, 16, 2365−2378.
(5) Yang, C. W.; Chen, W. L.; Wu, P. L.; Tseng, H. Y.; Lee, S. J. Anti-
Inflammatory Mechanisms of Phenanthroindolizidine Alkaloids. Mol.
Pharmacol. 2006, 69, 749−758.
(6) Yang, C. W.; Chuang, T. H.; Wu, P. L.; Huang, W. H.; Lee, S. J.
Anti-Inflammatory Effects of 7-Methoxycryptopleurine and Structure−
N
dx.doi.org/10.1021/jm300705j | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(24) Lee, S. J.; Wu, C. M.; Yang, C. W. Tylophorine Elevates the
Amounts of c-Jun Protein in Carcinoma Cells. ASCB 47th Annual
Meeting, Washington, DC, December 1−5, 2007.
(25) Jones, B. J.; Roberts, D. J. The Quantiative Measurement of
Motor Inco-Ordination in Naive Mice Using an Acelerating Rotarod. J.
Pharm. Pharmacol. 1968, 20, 302−304.
(26) Yang, C. W.; Yang, Y. N.; Liang, P. H.; Chen, C. M.; Chen, W.
L.; Chang, H. Y.; Chao, Y. S.; Lee, S. J. Novel Small-Molecule
Inhibitors of Transmissible Gastroenteritis Virus. Antimicrob. Agents
Chemother. 2007, 51, 3924−3931.
(27) Chang, H. S.; Lin, Y. J.; Lee, S. J.; Yang, C. W.; Lin, W. Y.; Tsai,
I. L.; Chen, I. S. Cytotoxic Alkyl Benzoquinones and Alkyl Phenols
from Ardisia Virens. Phytochemistry 2009, 70, 2064−2071.
(28) Tseng, H. Y.; Wu, S. H.; Huang, W. H.; Wang, S. F.; Yang, Y.
N.; Mahindroo, N.; Hsu, T.; Jiaang, W. T.; Lee, S. J. Benzothiazolium
Compounds: Novel Classes of Inhibitors That Suppress the Nitric
Oxide Production in RAW264.7 Cells Stimulated by LPS/IFNγ.
Bioorg. Med. Chem. Lett. 2005, 15, 2027−2032.
(29) Koetters, P. J.; Hassanieh, L.; Stohlman, S. A.; Gallagher, T.; Lai,
M. M. Mouse Hepatitis Virus Strain JHM Infects a Human
Hepatocellular Carcinoma Cell Line. Virology 1999, 264, 398−409.
(30) O’Keefe, B. R.; Giomarelli, B.; Barnard, D. L.; Shenoy, S. R.;
Chan, P. K.; McMahon, J. B.; Palmer, K. E.; Barnett, B. W.; Meyerholz,
D. K.; Wohlford-Lenane, C. L.; McCray, P. B., Jr. Broad-Spectrum in
Vitro Activity and in Vivo Efficacy of the Antiviral Protein Griffithsin
against Emerging Viruses of the Family Coronaviridae. J. Virol. 2010,
84, 2511−2521.
(31) Yao, H. T.; Wu, Y. S.; Chang, Y. W.; Hsieh, H. P.; Chen, W. C.;
Lan, S. J.; Chen, C. T.; Chao, Y. S.; Chang, L.; Sun, H. Y.; Yeh, T. K.
Biotransformation of 6-Methoxy-3-(3′,4′,5′-trimethoxy-benzoyl)-1H-
Indole (BPR0L075), a Novel Antimicrotubule Agent, by Mouse, Rat,
Dog, and Human Liver Microsomes. Drug Metab. Dispos. 2007, 35,
1042−1049.
(32) Yang, R.; Rescorla, F. J.; Reilly, C. R.; Faught, P. R.; Sanghvi, N.
T.; Lumeng, L.; Franklin, T. D., Jr.; Grosfeld, J. L. A Reproducible Rat
Liver Cancer Model for Experimental Therapy: Introducing a
Technique of Intrahepatic Tumor Implantation. J. Surg. Res. 1992,
52, 193−198.
(33) Bissery, M. C.; Chabot, G. G. History and New Development of
Screening and Evaluation Methods of Anticancer Drugs Used in Vivo
and in Vitro. Bull. Cancer 1991, 78, 587−602.
O
dx.doi.org/10.1021/jm300705j | J. Med. Chem. XXXX, XXX, XXX−XXX