6-OH-Phenanthroquinolizidine Alkaloid and Its Derivatives Exert Potent Anticancer Activity by Delaying S Phase Progression

Article abstract of DOI:10.1021/acs.jmedchem.6b01502

To discover new phenanthroindolizidine and phenanthroquinolizidine alkaloids as potential anticancer drug candidates, non-natural 6-O-desmethylcryptopleurine (2) and its derivatives were prepared. Most of the new compounds exhibited potent antiproliferati

Full text of DOI:10.1021/acs.jmedchem.6b01502

Article
pubs.acs.org/jmc
6‑OH-Phenanthroquinolizidine Alkaloid and Its Derivatives Exert
Potent Anticancer Activity by Delaying S Phase Progression
Yuxiu Liu,†,§ Lihua Qing,‡,§ Chuisong Meng,†,§ Jiajie Shi,‡,§ Yan Yang,‡ Ziwen Wang,† Guifang Han,†
Yi Wang,‡ Jian Ding,‡ Ling-hua Meng,*,‡ and Qingmin Wang*,†
†State Key Laboratory of Elemento-Organic Chemistry, Research Institute of Elemento-Organic Chemistry, Nankai University,
Tianjin 300071, P. R. China
‡Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai 201203, P. R. China
S
* Supporting Information
ABSTRACT: To discover new phenanthroindolizidine and
phenanthroquinolizidine alkaloids as potential anticancer drug
candidates, non-natural 6-O-desmethylcryptopleurine (2) and
its derivatives were prepared. Most of the new compounds
exhibited potent antiproliferative activity against A549 and
BEL-7402 cells, with the lowest IC50 being 3 nM. Optically
pure 2-R was further evaluated against a panel of 30 cancer cell
lines and found to inhibit the proliferation of all tested cell
lines, including three multidrug-resistant cell lines, with an
average IC50 value of 2.1 nM, which is much lower than that of
previously reported phenanthroindolizidine DCB-3503 (1,
IC50: 166.7 nM). A mechanistic evaluation showed that 2-R
potently inhibited cell growth and colony formation, which are
associated with a delay in S phase progression through the inhibition of DNA synthesis. These results along with further study on
the safety profile of these compounds will facilitate the discovery of new phenanthroindolizidine and phenanthroquinolizidine
alkaloids for use as anticancer drug candidates.
zines and have shown more potent anticancer activity9,12 than
INTRODUCTION
■
phenanthroindolizines.
Naturally occurring compounds and their derivatives are
Various biological mechanisms have been proposed to
important constituents for cancer therapy or prevention.
explain the antitumor activities of these alkaloids, but the
Much effort has been drawn to the discovery of new drug
exact target(s) and the mechanisms of action have not yet been
candidates from natural compounds due to their diversified
clearly established. Tylocrebrine was reported to inhibit protein
synthesis in Ehrlich ascites-tumor cells13 and to inhibit DNA
structures and distinct mechanisms of action. Tylophorine
and RNA syntheses in HeLa cells.14 Phenanthroindolizidine
(Figure 1) is a phenanthroindolizidine alkaloid that was first
separated from Tylophora indica in 1935.1 Tylophorine and its
alkaloids pergularinine and tylophorinidine were found to exert
their activities by inhibiting thymidylate synthase15 and
analogues have drawn great interest from academia and
dihydrofolate reductase.16 More recently, Chen et al. discovered
industry due to their unique chemical structures and
that S-(1)-deoxytylophorinidine intercalates nucleic acids.17
remarkable biological activities. In the 1960s, tylocrebrine
entered phase I clinical trials as an anticancer drug candidate,2
Furthermore, a phenanthroindolizidine alkaloid was found to
inhibit protein synthesis in a cell-free translation assay.18 These
but the trial was terminated due to its undesirable central
nervous system side effects.3 Tyloindicine F (NSC-650393)
and tyloindicine G (NSC-650394) were reported to exhibit
alkaloids also displayed potent regulatory activity on cell cycle
progression,5 which might be associated with a downregulation
potent activity in a NCI-60 screen (NCI, DTP), with an
average GI50 value of 0.1 nM.4 A number of analogues were
then discovered that displayed significant activity against a wide
variety of tumor cell lines.5−11 DCB-3503 (NSC-716802, 1), a
synthetic analogue, was also reported to inhibit the proliferation
of multidrug-resistant cells, including KB-V1, KB-MDR, KB-
7D, and KB-HU-R, with an average GI50 value of around 30
nM.5 Phenanthroquinolizidine alkaloids, represented by
cryptopleurine, are structural analogues of phenanthroindoli-
of cyclin D1.6,19 In addition, it was demonstrated that these
alkaloids inhibited the transcriptional activities mediated by key
transcription factors such as activator protein-1 (AP1),5,20
nuclear factor-kappa B (NF-κB),5,6 and hypoxia inducible
factor-1 (HIF1).21 Previous structure−activity relationship
studies indicated that changes in their structures would not
Received: October 30, 2016
© XXXX American Chemical Society
A
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Figure 1. Representative phenanthroindolizidines and phenanthroquinolizidines with anticancer activity.
a
Scheme 1. Synthetic Route for 6-O-Desmethylcryptopleurine 2
a
Reagents and conditions: (a) Ac2O, Et3N, reflux; (b) CH3OH, H2SO4,reflux; (c) MnO2, CF3COOH, rt; (d) Ac2O, 40 °C; (e) H2O, 70 °C; (f)
BnBr, K2CO3, acetone, reflux; (g) LiAlH4, THF, reflux; (h) Br2, CHCl3, rt; (i) K2CO3, DMF, reflux; (j) tetramethylethylenediamine, n-BuLi, THF,
−78 °C; (k) CH3OH, NaBH4, − 40 °C, then rt; (l) H2, Pd/C, THF/CH3OH, rt; (m) Et3SiH, CF3COOH, rt.
only result in changes in the potency of their activities but also
alter the targets of their actions.12,22 Therefore, to find new
potent analogues and explore their mechanisms of action, it is
important to identify new druggable compounds.
It was reported that 6-O-desmethylantofine exhibited more
potent anticancer activity than antofine.9 The group at the 6-
position of the phenanthrene ring might have a non-negligible
role on the activity. We hypothesized that 6-O-desmethyl-
cryptopleurine (2, Figure 1) may also possess better anticancer
activity than cryptopleurine. In this study, we synthesized 6-O-
desmethylcryptopleurine, referring to the structure of 6-O-
desmethylantofine. The esters and ethers of 6-O-desmethyl-
cryptopleurine were also prepared to investigate whether
changes at this position would enhance its activity. To analyze
the structure−activity relationships, we compared the cytotox-
icity of all new synthesized compounds as well as 1 against
A549 and BEL-7402 cells. Furthermore, the most potent
compound was selected to evaluate its anticancer spectrum in
vitro and investigate its anticancer mechanism.
B
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a
Scheme 2. Synthetic Route for Ester Derivatives 11−18
a
Reagents and conditions: (a) for 11: Ac2O, Et3N, CH2Cl2, rt; (b) for 12: pivaloyl chloride, Et3N, cat. DMAP, CH2Cl2, rt; (c) for 13−18: RCO2H,
EDCI, Et3N, cat. DMAP, CH2Cl2, rt
the carbonyl in the amide, affording the C15-oxo compound via
a Parham cyclization process. Because this compound is not
stable, it was not separated but directly reduced by sodium
borohydride to form stable C15-hydroxy compound 9. This
step had high stereoselectivity: the C15-hydroxyl and C14a-
hydrogen were always in the trans position.27 After
deprotection of the benzyl group by palladium-catalyzed
hydrogenation to 10 and two subsequent steps to remove the
hydroxyl at C15, final product 2,3-dimethoxy-6-hydroxy-
phenanthroquinolizidine (2-rac, i.e., 6-O-desmethylcrypto-
pleurine) was obtained, with an ee value of 2-S and 2-R as
high as 98%.
The derivatives of 2-rac were divided into esters and ethers.
The esterification conditions were somewhat different when
different acylation reagents were used. When preparing acetate
11, the reaction used acetic anhydride as the acylation reagent
and triethylamine as the deacid agent; when preparing pivalate
12, pivaloyl chloride was used with triethylamine as the deacid
agent and DMAP was used as the catalyst. The syntheses of
13−18 used the corresponding acids as reactants, using (3-
(dimethylamino)propyl)ethyl carbodiimide hydrochloride
(EDCI) as the dehydrating reagent in the presence of
triethylamine and DMAP (Scheme 2).
RESULTS
■
Chemistry. We previously reported a synthetic method for
2-rac.23 To obtain optically pure compounds of 2 and their
derivatives, we designed another route suitable for providing
chiral 2-S and 2-R24,25 (Scheme 1) according to the synthetic
methods for 6-O-desmethylantofine9 and cryptopleurine.26 At
the beginning, 3,4-dimethoxybenzaldehyde reacted with 4-
hydroxyphenylacetic acid to give diaryl propionic acid through
a Perkin reaction; after esterification, the resulting 3 was
converted to phenanthreneformate 4 through a three-step
sequence in one pot, i.e., a MnO2/CF3COOH-catalyzed
oxidative coupling, an acid-catalyzed rearrangement, and then
a hydrolysis reaction. To eliminate the influence of the phenolic
hydroxyl group on subsequent reactions, we chose to protect
the hydroxyl with a benzyl group and obtained 5. Next, we
reduced the formate to corresponding methanol, which was
then treated with bromine to afford a double brominated
product, 7. Compound 7 was allowed to react with racemic or
optically pure pipecolamide to form key intermediate 8, with
the chirality well maintained (8-S was obtained from L-
pipecolamide, 8-R was from D-pipecolamide, and 8-rac was
from racemic pipecolamide). Compound 8 was treated with n-
butyl lithium at −78 °C, and the generated carbon ion attacked
C
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Scheme 3. Synthetic Route for Ether Derivatives 19−22
a
Scheme 4. Synthetic Route for Amino Acid Derivative 25
a
Reagents and conditions: (a) LiAlH4, THF, ice bath, then rt; (b) EDCI, Et3N, DMAP, CH2Cl2, rt; (c) CF3COOH, CH2Cl2, rt.
Etherification derivatives 19−22 were prepared by reacting 2
with a bromide (bromoacetamide, propargyl bromide,
bromoacetonitrile, or ethyl bromoacetate) via a nucleophilic
substitution reaction in the presence of a base. Using optically
pure raw 2-S and 2-R, optically pure products 21-S and 21-R
were obtained, respectively (Scheme 3).
When an amino acid directly connects to the phenol
hydroxyl, the resulting ester is found to be unstable and easily
undergoes hydrolysis. To introduce an amino acid moiety into
the structure of 2, e.g., compound 25, we first reduced ester 22
to corresponding alcohol 23; then, 23 was allowed to react with
Boc-protected L-alanine to form 24, which was further treated
with trifluoroacetic acid to remove the Boc group, affording 25
(Scheme 4).
In Vitro Anticancer Activity Screening and Mecha-
nistic Study. New Phenanthroquinolizidine Alkaloids
Possess Potent Activity against the Proliferation of Human
Cancer Cells. To test the anticancer activity of the newly
synthesized phenanthroquinolizidine alkaloids, we first inves-
tigated their antiproliferative activities against human lung
adenocarcinoma A549 cells and human hepatocellular
carcinoma BEL-7402 cells. As shown in Table 1, all of the
compounds exhibited potent antiproliferative activity against
A549 and BEL-7402 cells, with IC50 values in the low
nanomolar range. Although compound 9-rac is structurally
similar to 1, 9-rac (IC50 values of 1.534 and 0.917 μM for A549
and BEL-7402 cells, respectively) is somehow less active than 1
(IC50 values of 0.283 and 0.110 μM), which suggested that
substituents on the phenanthrene ring greatly influence the
activity. When the benzyl group on the 6-hydroxy of
phenanthroquinolizidine is removed, the resulting compound
10-rac displayed a remarkably increased activity (IC50 values of
12 and 8 nM). Compound 2-rac, which was generated by
further removing the 15-hydroxy group of phenanthroquino-
lizidine, exhibited remarkable activity, but it was slightly inferior
to 10-rac (IC50 values of 19 and 16 nM). Because both 2-rac
and 10-rac are poorly soluble and 10-rac is unstable, 2-rac was
chosen as the substrate to make esters or ethers to improve the
solubility. Most of the newly synthesized compounds had better
solubility and were more potent than 1 (Table 1). For esters
11−18, the electrical properties and volume of the acyl moiety
played an important role. An electron-donating group with a
small volume on the carbonyl improved the activity more than
an electron-withdrawing group with a large volume. For
example, acetate 11, cyclopropylcarboxylate 13, and 2-
furancarboxylate 15 were more potent than the corresponding
pivilate 12, cyclohexylcarboxylate 14, and 2-pyridylcarboxylate
17. Compound 18 could be regarded as an exception as it
showed similar activity with a relatively larger volume compared
to that of compound 13. For ethers 19−22 and 25, most of
D
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21-rac was the most potent compound against both A549 and
BEL-7402 cells, with IC50 values of 3 and 6 nM, respectively.
Because compounds 2 and 21 displayed potent antiprolifer-
ative activity in the pilot screening, we conducted chiral
syntheses of 2 and 21 to obtain enantiomerically pure
compounds to investigate the influence of the optical
configuration on their anticancer activity. As shown in Table
2, both 2-R and 21-R were significantly more potent than their
Table 1. IC50 Values of Compounds against the Proliferation
of A549 and BEL-7402 Cells
a
Table 2. IC50 Values of Compounds against A549 and BEL-
a
7402 Cells
a
Values are the mean SD (nM).
S-configuration enantiomers. 2-R displayed the most potent
activity among the compounds tested, with IC50 values of 0.7
nM against A549 cells and 1.7 nM against BEL-7402 cells.
Because 2-R and 21-R displayed potent antiproliferative
activity against lung cancer A549 cells and hepatocellular
carcinoma BEL-7402 cells, we next tested the antiproliferative
activity of the two compounds against normal lung fibroblast
MRC-5 and MRC-9 cells and normal liver LO2 cells. As shown
in Table 3, the IC50 values of 2-R and 21-R obtained against
normal MRC-5 or MRC-9 cells are 3−4-fold higher than those
obtained against A-549 cells, suggesting that 2-R and 21-R
might be more potent inhibitors of tumor cell proliferation.
Similar results were obtained by comparing the IC50 values
against normal liver cells and liver cancer cells. The clinical trial
of tylocrebrine was terminated due to its undesirable central
nervous system side effects. We also evaluated the cytotoxicity
of 2-R and 21-R against primary neurons. The viability of
neurons decreased in a concentration-dependent manner up to
10 nM and remained at a similar level up to 300 nM (Table
S1). Both compounds inhibited cell viability by about 50% at
300 nM, indicating that primary neurons tolerate 2-R and 21-R.
Since 2-R was more potent than 21-R against all tested cell
lines, 2-R was selected for further evaluation and mechanistic
study.
2-R Exerts Potent Antiproliferative Activity against a
Panel of Human Cancer Cell Lines. The antiproliferative
activity of 2-R was further evaluated in a panel of 30 cancer cell
lines including 8 hematoma cell lines, 4 breast cancer cell lines,
3 gastric cancer cell lines, and others. Compound 2-R displayed
potent activity, inhibiting the proliferation of all tested cell lines
a
Values are the mean SD (μM).
them displayed superior activity compared to 1, which may also
be due to the small volume of the substituents. Among them,
E
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a
Table 3. IC50 Values of Compounds against the Indicated Cell Lines
compound
A549
MRC-5
MRC-9
BEL-7402
LO2
2.7
3.9 1.4
2-R
0.7 0.1
0.8 0.1
2.6 0.4
3.2 0.7
2.2 0.7
2.7 0.2
1.7 0.7
2.4 0.1
1
21-R
a
Values are the mean SD (nM).
Figure 2. 2-R potently inhibits cell proliferation. Cells seeded in 96-well plates were treated with 1 (A) or 2-R (B) for 72 h. Cell proliferation of HL-
60, K-562, and SU-DHL-6 cells was measured by an MTT assay, and the rest of the cells were tested by an SRB assay. IC50 values are depicted. Data
shown are the mean SD of three independent experiments.
with an average IC50 value of 2.1 nM, which is much lower than
that of control compound 1 (IC50: 166.7 nM), indicating its
superior activity (Figure 2).
in a dose-dependent manner upon 2-R treatment. The
population of cells in S phase increased from 27.5 1 to
50.3 3.5% in the presence of 10 nM 2-R. Similar results were
obtained in the presence of 1, although a much higher
concentration was required to induce S phase arrest.
To closely investigate the effect of 2-R on S phase
progression, BEL-7402 cells were synchronized in early S
phase by aphidicolin, and cells were then incubated in fresh
media with or without the test compounds. As shown in Figure
5A, cells in the vehicle control group entered G2-metaphase 4 h
after being released from aphidicolin, whereas most cells
remained in S phase at the same time in the presence of 2-R,
indicating that 2-R treatment slowed the progression from S to
G2 phase. Moreover, 2-R delayed the progression of S phase up
to 24 h at 10 nM. Similar results were obtained with 1,
suggesting that both compounds induce S phase arrest by a
similar mechanism.
To detect the activity of 2-R and 1 against cell proliferation
in a real-time manner, an IncuCyte ZOOM system28 was
utilized to monitor cell proliferation continuously. As shown in
Figure 3B, treatment of BEL-7402 cells with different
concentrations of 2-R significantly attenuated cell proliferation,
and 3.3 nM 2-R completely blocked this process up to 10 days.
Similar results were obtained in the presence of 1, but a much
higher concentration was required to display a potency similar
to that of 2-R (Figure 3A), further proving that 2-R is more
potent than 1.
To further evaluate the effect of 2-R on the clonogenicity of
cancer cells, a plate colony formation assay was performed. As
shown in Figure 3C,D, the clonogenic ability of BEL-7402 cells
decreased in a dose-dependent manner in the presence of 2-R,
and almost no colony was observed upon treatment with 100
nM 2-R. Similarly, 1 inhibited colony formation, but it did so
with much lower potency than 2-R.
We also confirmed that 2-R delayed S phase progression in
human hepatoma cancer SMMC-7721 and ZIP-177 cells
(Figure 5B,C).
2-R Significantly Delays S Phase Progression. Because 2-R
significantly inhibited cell proliferation, we further explored its
mechanism of action by investigating its impact on cell cycle
progression. BEL-7402 cells were treated with 2-R for 24 h, and
flow cytometry analysis was performed to detect the cell cycle
distribution. As shown in Figure 4, cells in S phase accumulated
2-R Inhibits DNA Synthesis without Inducing DNA
Damage in BEL-7402 Cells. We next investigated the
mechanism by which 2-R delays S phase progression. Because
DNA replication is an important process during S phase,
interruption of DNA synthesis would result in aberrant S phase
progression. A BrdU incorporation assay was performed to
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Figure 3. 2-R inhibits cell proliferation and colony formation. (A, B) Representative growth curves of BEL-7402 cells in the presence of various
concentrations of 1 (A) or 2-R (B). (C) Representative images of colony formation. (D) Quantitation of colonies formed in the presence of 1 or 2-
R. Data shown are the mean SD of three independent experiments.
Figure 4. 2-R induces S phase accumulation. BEL-7402 cells were treated with 1 (A) or 2-R (B) for 24 h, and the cell cycle distribution was detected
with flow cytometry. **, P < 0.01; *, P < 0.05 compared with the population of cells in S phase in the control group.
detect the effect of 2-R on DNA synthesis. As shown in Figure
6A,B, BrdU-positive cells decreased in a concentration-
dependent manner after BEL-7402 cells were treated with 2-
R or 1, demonstrating the inhibition of DNA synthesis.
DNA single- or double-strand breaks may result in DNA
synthesis inhibition. As DNA single-strand breaks transform
into double-strand breaks as DNA replication forks progress,
we tested the level of phospho-H2AX (γH2AX) as a marker for
DNA damage. As shown in Figure 6C, a robust increase in
γH2AX, phosphorylated CHK2 at Thr68, and phosphorylated
CHK1 at Ser345 was observed after treatment of BEL-7402
cells with CPT (1 μM), which is known to induce DNA
damage by trapping the topoisomerase I−DNA complex.29
However, the level of the tested proteins remained unchanged
upon treatment with 2-R or 1, even at relative high
concentrations, indicating that 2-R and 1 arrest cells in S
phase without inducing DNA damage.
than 20% cells were apoptotic in the presence of 100 nM 2-R
(Figure 7A,B).
Because 2-R failed to significantly induce apoptosis, we
further assessed whether 2-R induces necrosis. A Trypan blue
exclusion assay was performed after BEL-7402 cells were
exposed to 2-R or 1 for 72 h. As shown in Figure 7C, 2-R
slightly induced cell death in a dose-dependent manner, which
is consistent with the results presented in Figure 7B, where 2-R
induced comparable late apoptosis in BEL-7402 cells.
2-R Exhibits Potent Activity against Multidrug-Resistant
Cell Lines. Multidrug resistance is one of the main causes of
cancer treatment failure, and it often happens with drugs that
originate from natural products. As 2-R is modified from
tylophorine, the activity of 2-R against a number of well-
characterized multidrug-resistant cell lines including K562-R,
KB-R, and HL-60-R was studied (Table 4). As shown in Table
5, both 2-R and 1 displayed similar antiproliferative activities
against the resistant cell lines and their parental cell lines with
resistant factor values of about 1, whereas the resistant cell lines
were resistant to ADR, NVT, and VCR, respectively. These
results indicated that multidrug-resistant cell lines are sensitive
to 2-R, demonstrating its potent ability to overcome multidrug
2-R Does Not Significantly Induce Apoptosis or Necrosis in
BEL-7402 Cells. As accumulated S phase arrest may induce
apoptosis, we next examined whether 2-R induces apoptosis by
an Annexin V/PI double staining assay. Annexin V-positive
cells slightly increased in the presence of 2-R and 1, and fewer
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Figure 5. 2-R significantly delays S phase progression. BEL-7402 (A), SMMC-7721 (B), and ZIP-177 (C) cells were synchronized with aphidicolin
in early S phase and then incubated with fresh media with or without test compounds for the indicated amount of time. Cell cycle distribution was
determined with flow cytometry. Data shown are representative from three independent experiments.
resistance. To investigate whether this activity is due to a
reduction in the expression of P-gp, we detected the protein
level of P-gp in K562/ADR cells after treatment with
compound 1 or 2-R for 24 h. Compound 1 or 2-R had little
effect on the expression of P-gp (Figure S1), indicating that
they did not overcome multidrug resistance by downregulating
the expression of P-gp. The exact mechanism deserves further
investigation.
To identify whether 2-R affects S phase progression in
multidrug-resistant cells, cells synchronized with aphdicolin,
released, and incubated with the designated compounds for
different lengths of time. As shown in Figure 8A,B, KB parental
cells and multidrug-resistant cells entered G2-metaphase after 4
h in the absence of 1 or 2-R, whereas both KB parental cells
and multidrug-resistant cells remained in S phase up to 12 h
upon treatment with 2-R (10 nM), indicating that 2-R delayed
S phase progression in multidrug-resistant cells as well as in
parental cells. This effect was confirmed in another pair of
multidrug-resistant cells (K562/ADR) and its counterparts
(Figure 8C,D).
study revealed that 2-R significantly inhibited DNA synthesis
without inducing DNA double-strand breaks. However, the
molecular target for its inhibition of DNA synthesis needs to be
clarified to better understand its mechanism of action.
Furthermore, the anticancer activity of 2-R is not due to the
induction of apoptosis or cytotoxicity based on the following
observations: first, 2-R treatment up to 72 h failed to induce
annexin V-positive cells or accumulate cells in sub-G1 phase;
second, 2-R-treated cells remained intact upon 72 h treatment;
and third, 2-R completely blocked cell proliferation up to 10
days without reducing cell number during the incubation.
These results indicate that 2-R may exert its selective cytostatic
activity on rapidly proliferating cells while sparing non-
proliferating cells. Though one important characteristic of
tumor cells is uncontrolled proliferation, the safety of 2-R
should be carefully evaluated for further development. Finally,
2-R displayed favorable activity against MDR cells with an RF
value of about 1 and delayed S phase progression in MDR cell
lines, which makes 2-R an ideal drug candidate to overcome
multiple drug resistance. These results together with further
study on the safety profile and in vivo efficacy of 2-R will
facilitate the discovery of new phenanthroindolizidine and
phenanthroquinolizidine alkaloids for use as anticancer drug
candidates.
DISCUSSION AND CONCLUSIONS
■
6-O-Desmethylcryptopleurine (1) as well as its ester and ether
derivatives were synthesized and found to exert excellent
antiproliferative activities against A549 and BEL-7402 cells, and
compound 2-R was found to possess the most potent activity in
the primary screening. This study revealed that 2-R significantly
inhibits the proliferation of a panel of 30 cancer cell lines
including 3 multidrug-resistant cell lines, with an average IC50
value of 2.1 nM, which is much lower than that of 1 (IC50:
166.7 nM). 2-R displayed a potent inhibitory effect against cell
growth and colony formation. It induced cell cycle arrest in S
phase, which was due to delayed S phase progression. Further
EXPERIMENTAL SECTION
■
Materials and Methods. General. 1H and 13C NMR spectra were
obtained using a Bruker AV400 spectrometer in CDCl3 or DMSO-d6
with tetramethylsilane as the internal standard. HRMS data were
obtained on an FTICR-MS instrument (Ionspec 7.0 T). The melting
points were determined on an X-4 binocular microscope melting point
apparatus and are uncorrected. Reaction progress was monitored by
thin-layer chromatography on silica gel GF-254 with detection by UV.
The enantiomeric excess of 2-rac, 2-S, and 2-R was determined by
H
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Figure 6. 2-R potently inhibits DNA synthesis without inducing DNA damage. BEL-7402 cells were treated with various concentrations of 2-R or 1
for 1 h before cells were labeled with BrdU for 30 min. BrdU-positive cells were stained with an antibody and detected by flow cytometry. (A)
Representative dot plots of BrdU incorporation. (B) Quantitation of relative DNA synthesis in the presence of 1 or 2-R. Data shown are the mean +
SD of three independent experiments. (C) Western blot of phospho-Chk1 Ser345, phospho-Chk2 Thr68, and γH2AX after treatment of BEL-7402
cells with the designated compounds for 1 h.
HPLC on a Shimadzu LC-20AT instrument with a ChiralPak AD-H
column (250 mm, 4.6 mm, 5μ) using a mixture of hexane:isopropa-
nol:triethyl amine (75:25:0.1) as the eluent at a flow rate of 1.0 mL/
min under 254 nm. The purities of compounds 9−22 and 25 were all
determined by HPLC on a Shimadzu LC-20AT instrument with a
Silica column (250 mm, 4.6 mm, 5μ) using a mixture of
dichloromethane:isopropanol (80:20) as the eluent and at a flow
rate of 1.0 mL/min under 254 nm. All compounds used for biological
tests were ≥95% pure.
Synthesis of (E)-2-(4-Acetoxyphenyl)-3-(3,4-dimethoxyphenyl)-
acrylic Acid. A mixture of 3,4-dimethoxybenzaldehyde (19.6 g, 0.1
mol), 4-hydroxyphenylacetic acid (12.2 g, 0.1 mol), acetic anhydride
(40 mL), and triethyl amine (20 mL) was refluxed under mechanical
stirring. When the reaction was complete, water (60 mL) was added to
the reaction mixture, and the reaction was refluxed for 1 h, cooled to
room temperature with stirring for another 1 h, and then filtered. The
cake was washed with water and ethanol and then dried to give (E)-2-
(4-acetoxyphenyl)-3-(3,4-dimethoxyphenyl)acrylic acid (27.4 g, 87%)
as a yellow solid. mp 243−246 °C; 1H NMR (400 MHz, DMSO-d6): δ
12.58 (br, 1H), 7.71 (s, 1H), 7.23 (d, J = 7.6 Hz, 2H), 7.16 (d, J = 7.6
Hz, 2H), 6.85−6.91 (m, 2H), 6.41 (s, 1H), 3.72 (s, 3H), 3.30 (s, 3H),
2.22 (s, 3H); 13C NMR (400 MHz, DMSO-d6): δ 169.1, 168.4, 149.9,
149.6, 147.8, 139.4, 134.3, 130.6, 129.7, 126.7, 125.3, 122.2, 111.8,
111.1, 55.3, 54.5, 20.8; HRMS (ESI): calcd. for C19H16O6 [M − H]−,
341.1031; found, 341.1031.
and then condensed to remove most of the methanol. When water
(100 mL) was added, a large amount of solid precipitated. The solid
was collected by filtration, washed with saturated sodium bicarbonate,
and then dried to give 3 (9.0 g, 90%) as a white solid. mp 127−128
°C; 1H NMR (400 MHz, CDCl3): δ 7.77 (s, 1H), 7.11 (d, J = 8.4 Hz,
2H), 6.83−6.86 (m, 3H), 6.74 (d, J = 8.4 Hz, 1H), 6.49 (d, J = 1.6 Hz,
1H), 3.85 (s, 3H), 3.79 (s, 3H), 3.46 (s, 3H); 13C NMR (400 MHz,
CDCl3): δ 169.3, 155.7, 149.9, 148.1, 140.7, 131.1, 129.6, 128.1, 127.4,
125.6, 115.9, 112.5, 110.5, 55.6, 55.1, 52.4. HRMS (ESI): calcd. for
C18H18NaO5 [M + Na]+, 337.1046; found, 337.1041.
Synthesis of Methyl 6-Hydroxy-2,3-dimethoxyphenanthrene-9-
carboxylate (4). To a solution of 3 (1.20 g, 3.82 mmol) in
trifluoroacetic acid (20 mL) was added manganese dioxide (0.33 g,
3.82 mmol), and the mixture was stirred at room temperature for 6 h.
When the reaction was complete, as indicated by TLC, acetic
anhydride (20 mL) was added, and the mixture was then stirred at 40
°C for 4 h. Water was slowly added (50 mL) at room temperature, and
the mixture was heated to 70 °C and monitored by TLC. When the
reaction was complete, water and dichloromethane were added to the
mixture; the water layer was separated and extracted with dichloro-
methane. The combined organic layer was washed with sodium
bicarbonate, dried over sodium sulfate, filtered, and then concentrated.
The residue was purified on silica gel column using petroleum ether
and ethyl acetate (2:1) as eluent to give 4 (0.87 g, 73%) as a yellow
1
solid. mp 188−190 °C; H NMR (400 MHz, DMSO-d6): δ 9.91 (s,
1H), 8.66 (d, J = 9.2 Hz, 1H), 8.26 (s, 1H), 8.03 (s, 1H), 7.94 (s, 1H),
7.59 (s, 1H), 7.18 (d, J = 8.8 Hz, 1H), 4.04 (s, 3H), 3.93 (s, 3H), 3.92
(s, 3H); 13C NMR (400 MHz, CDCl3): δ 168.2, 154.2, 150.9, 149.8,
131.9, 129.7, 128.5, 126.6, 125.6, 123.6, 123.2, 116.5, 109.2, 106.3,
Synthesis of (E)-Methyl 3-(3,4-Dimethoxyphenyl)-2-(4-
hydroxyphenyl)acrylate (3). A mixture of (E)-2-(4-acetoxyphenyl)-
3-(3,4-dimethoxyphenyl)acrylic acid (10.0 g, 29.3 mmol), methanol
(150 mL), and concentrated sulfuric acid (6.5 g) was refluxed for 4 h
I
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Article
Figure 7. 2-R failed to induce a significant amount of cell apoptosis or necrosis. (A) BEL-7402 cells were treated with various concentrations of 2-R
or 1 for 72 h, and annexin V/PI labeling was performed. Representative dot plots obtained from flow cytometry are shown. (B) Quantitation of early
and late apoptotic cells. (C) BEL-7402 cells were treated with various concentrations of 2-R or 1 for 72 h, and necrotic cells were counted by a
Trypan blue exclusion assay. Data shown are the mean + SD of three independent experiments.
Synthesis of Methyl 6-(Benzyloxy)-2,3-dimethoxyphenanthrene-
9-carboxylate (5). A mixture of 4 (2.5 g, 8.02 mmol), anhydrous
Table 4. Background of Drug-Resistant Cell Lines
resistant
drug
concentration
(μM/L)
potassium carbonate (1.62 g, 12.02 mmol), benzyl bromide (1.65 g,
9.62 mmol), and dried acetone (50 mL) was refluxed for 8 h and then
cooled to room temperature. To the mixture was added dichloro-
methane (100 mL), which was stirred and then filtered. The filtrate
was condensed and then washed with petroleum ether and ethyl
acetate (10:1) to give 5 (2.83 g, 88%) as a white solid. mp 156−157
°C; 1H NMR (400 MHz, CDCl3): δ 8.94 (d, J = 9.2 Hz, 1H), 8.32 (s,
1H), 7.98 (d, J = 2.8 Hz, 1H), 7.78 (s, 1H), 7.55 (d, J = 7.2 Hz, 2H),
740−7.45 (m, 2H), 7.34−7.38 (m, 2H), 7.26 (s, 1H), 5.31 (s, 2H),
cell line
gene alteration
K562/ADR
HL60/MX2
KB/VCR
ADR
NVT
VCR
2
P-gp overexpression
BCRP overexpression
P-gp overexpression
0.2
0.2
103.2, 55.9, 52.2; HRMS (ESI): calcd. for C18H16NaO5 [M + Na]+,
335.0890; found, 335.0889.
Table 5. IC50 Values of 2-R and 1 against Parental and Drug-Resistant Cell Lines
IC50 (mean SD, nM)
cell line
1
2-R
ADR
NVT
VCR
K562/ADR
K562
137.5 29.4
98.4 16.8
1.4
2.6 0.9
2.2 0.7
1.2
3310.2 85.9
77.7 2.5
42.6
RF
HL60/MX2
HL60
278.3 33.7
212.8 100.1
1.3
1.7 0.2
1.8 0.9
0.9
350.7 87.9
7.2 4.4
48.7
RF
KB/VCR
KB
65.2 15.3
58.2 3.7
1.1
1
0.2
238.7 16
0.2 0.1
1193.5
1.2 0.2
0.8
RF
RF (mean)
1.26
0.96
42.6
48.7
1193.5
J
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Figure 8. 2-R delays S phase progression in multidrug-resistant cells and their parental lines. KB (A), KB/VCR (B), K562 (C), and K562/ADR (D)
cells were synchronized with aphidicolin in early S phase and then incubated with fresh media with or without the tested compounds for different
amounts of time. Cell cycle distribution was determined with flow cytometry.
4.11 (s, 3H), 4.02 (s, 3H), 4.02 (s, 3H); 13C NMR (400 MHz,
CDCl3): δ 168.2, 157.3, 150.9, 149.7, 136.9, 131.7, 129.6, 128.7, 128.5,
128.1, 127.6, 126.7, 125.6, 123.7, 123.3, 116.3, 109.2, 106.1, 103.1,
70.4, 56.1, 55.9, 52.1; HRMS (ESI): calcd. for C25H22NaO5 [M +
Na]+, 425.1359; found, 425.1352.
was filtered. The filtrate was concentrated in vacuo, and the residue
was purified on a silica gel column to give amide 8-rac (3.75 g, 91%) as
a white solid. mp 131−133 °C; H NMR (400 MHz, CDCl3): δ 9.07
1
(d, J = 8.8 Hz, 1H), 7.89 (s, 1H), 7.85 (s, 1H), 7.80 (s, 1H), 7.54−7.53
(m, 2H), 7.42−7.40 (m, 3H), 7.37−7.35 (m, 1H), 5.27 (s, 2H), 4.44
(d, J = 12.0 Hz, 1H), 4.17 (d, J = 12.0 Hz, 1H), 4.08 (s, 6H), 3.54−
3.47 (m, 3H), 3.42−3.37 (m, 1H), 3.30 (d, J = 9.6 Hz, 1H), 2.91 (d, J
= 10.4 Hz, 1H), 2.34−2.32 (m, 1H), 1.88−1.86 (m, 1H), 1.74 (s, 2H),
1.41−1.29 (m, 6H), 1.19−1.16 (m, 3H); 13C NMR (400 MHz,
CDCl3): δ 172.6, 157.4, 149.6, 149.3, 137.2, 130.9, 130.5, 130.3, 128.7,
128.1, 127.6, 126.5, 125.9, 125.3, 123.1, 115.5, 109.7, 105.8, 103.3,
70.4, 57.8, 56.0, 50.5, 42.1, 40.9, 29.7, 25.3, 23.8, 15.0, 13.15. HRMS
(ESI) calcd for C34H40BrN2O [M + H]+, 619.2171, found, 619.2168.
Synthesis of 8-S. Synthesis was performed according to the
procedure for 8-rac using (S)-N,N-diethylpyrrolidine-2-carboxamide
instead of (DL)-N,N-diethylpyrrolidine-2-carboxamide. Yield, 90%.
Synthesis of 8-R. Synthesis was performed according to the
procedure for 8-rac using (R)-N,N-diethylpyrrolidine-2-carboxamide
instead of (DL)-N,N-diethylpyrrolidine-2-carboxamide. Yield, 91%.
Synthesis of 6-Benzyloxy-2,3-dimethoxy-11,12,13,14,14a,15-hex-
ahydro-15-hydroxy-9H-dibenzo[f,h]pyrido[1,2-b]isoquinoline (9-
rac). To a solution of amide 8-rac (10.0 g, 16.18 mmol) and
N,N,N′,N′-tetramethylethylenediamine (4.31 g, 37.21 mmol) in dry
tetrahydrofuran (500 mL) at −78 °C was added a solution of n-BuLi
in hexane (14.83 mL, 2.4 mol/L, 35.60 mmol), and the resulting
mixture was stirred at this temperature for 5 h under Ar in the dark.
Then, methanol (200 mL) and sodium borohydride (0.33 g, 70.65
mmol) were added to the mixture. After stirring at −40 °C for 1 h at
room temperature for 8 h, the reaction mixture was quenched with
water (200 mL) and the product was extracted with methylene
dichloride (380 mL). The combined organic extracts were dried with
anhydrous magnesium sulfate and concentrated in vacuo. The residue
was purified by flash column chromatography on silica gel to give 9-rac
(6.0 g, 80%) as a white solid. Purity 97% (HPLC); dr 15:1 (NMR);
Synthesis of (6-(Benzyloxy)-2,3-dimethoxyphenanthren-9-yl)-
methanol (6). A suspension of ester 5 (2.19 g, 5.45 mmol) in dry
tetrahydrofuran (250 mL) was added in small portions to a suspension
of lithium aluminum hydride (0.62 g, 16.34 mmol) in dry
tetrahydrofuran (150 mL) at ice-bath temperature. After the reaction
mixture had been refluxed under dry nitrogen for 1 h, the mixture was
cooled and acidified with diluted hydrochloric acid to pH 2. Water
(100 mL) was added, and the precipitate was collected by filtration to
1
afford 6 (1.94 g, 95%) as a white solid. mp 184−186 °C; H NMR
(400 MHz, CDCl3): δ 8.10 (d, J = 8.8 Hz, 1H), 7.99 (d, J = 2.8 Hz,
1H), 7.78 (s, 1H), 7.53−7.56 (m, 3H), 7.40−7.45 (m, 2H), 7.30−7.38
(m, 2H), 7.19 (s, 1H), 5.29 (s, 2H), 5.14 (d, J = 6.0 Hz, 2H), 4.09 (s,
3H), 4.01 (s, 3H), 1.76 (t, J = 6.0 Hz, 1H); 13C NMR (400 MHz,
DMSO-d6): δ 156.7, 149.4, 148.7, 137.3, 133.9, 131.0, 128.5, 127.9,
127.9, 126.7, 125.9, 123.8, 123.5, 121.9, 115.7, 108.6, 106.0, 104.0,
69.5, 61.6, 55.8, 55.4; HRMS (ESI): calcd. for C24H22NaO4 [M +
Na]+, 397.1410; found, 397.1402.
Synthesis of 6-(Benzyloxy)-10-bromo-9-(bromomethyl)-2,3-dime-
thoxyphenanthrene (7). A solution of bromine (1.48 g, 9.25 mmol)
in chloroform (20 mL) was added to a solution of alcohol 6 (3.19 g,
8.5 mmol) in chloroform (70 mL) at 0 °C. The mixture was stirred at
room temperature for 10 h and was filtered and washed with
petroleum ether and ethyl acetate (10:1) to afford 7 (3.75 g, 98%) as a
white solid. mp 200−202 °C; 1H NMR (400 MHz, CDCl3): δ 7.99 (d,
J = 8.8 Hz, 1H), 7.77 (s 1H), 7.66 (s, 1H), 7.58 (s, 1H), 7.52 (d, J =
7.2 Hz 2H), 7.43 (t, J = 7.2 Hz 2H), 7.37 (d, J = 7.2 Hz, 1H), 7.30 (d,
J = 8.4 Hz, 1H), 5.23 (s, 2H), 5.14 (s, 2H), 4.03 (s, 6H); 13C NMR
(400 MHz, CDCl3): δ 157.4, 149.92, 149.88, 136.8, 130.7, 129.3,
128.8, 128.2, 127.5, 126.4, 125.7, 125.3, 123.9, 122.8, 116.3, 109.2,
106.2, 103.0, 70.4, 56.0, 55.9, 32.8.
Synthesis of 1-((6-(Benzyloxy)-10-bromo-2,3-dimethoxyphe-
nanthren-9-yl)methyl)-N,N-diethylpiperidine-2-carboxamide (8-
rac). A solution of bromide 7 (3.0 g, 6.67 mmol), (DL)-N,N-
diethylpyrrolidine-2-carboxamide (1.35 g, 7.33 mmol), and potassium
carbonate (4.05 g, 29.3 mmol) in N,N-dimethylformamide (60 mL)
was stirred for 1 h and heated at reflux for 8 h. After cooling,
dichloromethane (60 mL) was added to the reaction mixture, and it
1
mp 223−225 °C; H NMR (400 MHz, CDCl3): main component δ
8.18 (s, 1H), 8.04 (s, 1H), 7.65 (s, 1H), 7.63−7.57 (m, 3H), 7.44 (t, J
= 7.3 Hz,2H), 7.38 (d, J = 7.2 Hz,1H), 7.28 (dd, J = 8.7, 1.5 Hz, 1H),
5.38 (s, 2H), 5.00 (d, J = 9.2 Hz,1H), 4.62 (d, J = 9.2 Hz,1H), 4.04 (s,
3H), 3.93 (s, 3H), 3.25 (d, J = 9.2 Hz, 1H), 2.97 (s, 1H), 2.1 (d, J =
12.0 Hz, 1H), 2.05 (d, J = 12.3 Hz, 1H), 1.83 (d, J = 10.6 Hz,1H),
1.75−1.65 (brs, 2H), 1.22−1.17 (m, 1H), 0.89−0.82 (m, 2H); 13C
K
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NMR (400 MHz, DMSO-d6): δ 156.7, 149.1, 148.2, 137.2, 130.3,
128.5, 128.0, 127.9, 127.6, 126.0, 125.7, 124.4, 123.3, 122.4, 115.7,
105.9, 105.5, 104.2, 69.6, 65.6, 62.0, 55.8, 55.7, 55.4, 26.9, 25.0, 24.0;
HRMS (ESI) calcd for C30H32NO4 [M + H]+, 470.2287; found,
470.2329.
After adding saturated sodium bicarbanate, the two phases were
separated. The water layer was extracted with dichloromethane three
times. The combined organic layer was washed with brine, dried with
anhydrous magnesium sulfate, filtered, and concentrated in vacuo. The
residue was purified by flash column chromatography on silica gel
(dichloromethane:methanol 40:1 as eluent) to give 11 as a light-yellow
Synthesis of 9-(14aS,15S). Synthesis was performed according to
the procedure for 9-rac using 8-S instead of 8-rac. Yield, 82%.
Synthesis of 9-(14aR,15R). Synthesis was performed according to
the procedure for 9-rac using 8-R instead of 8-rac. Yield, 81%.
Synthesis of 2,3-Dimethoxy-11,12,13,14,14a,15-hexahydro-15-
hydroxy-9H-dibenzo[f,h]pyrido[1,2-b]isoquinolin-6-ol (10-rac). To
a solution of 9-rac (0.9 g, 1.92 mmol) in tetrahydrofuran (500 mL)
and methanol (100 mL) was added Pd/C (10%, 0.09 g), and the
resulting mixture was stirred at room temperature under a H2
atomosphere for 2 days. After being diluted with dichloromethane
(50 mL), the mixture was filtered. The filtrate was condensed to give
10-rac (0.69 g, 95%) as a white solid. Purity 96% (HPLC); mp 234 °C
(dec.); 1H NMR (400 MHz, DMSO-d6): δ 9.75 (s, 1H), 7.96 (s, 1H),
7.91 (s, 1H), 7.61 (s, 1H), 7.55 (d, J = 8.8 Hz, 1H), 7.03 (d, J = 8.8
Hz, 1H), 4.86 (d, J = 9.2 Hz, 1H), 4.58 (d, J = 9.2 Hz, 1H), 4.05 (d, J
= 16.0 Hz, 1H), 4.01 (s, 3H), 3.92 (s, 3H), 3.24 (d, J = 16.0 Hz, 1H),
3.00 (d, J = 10.4 Hz, 1H), 2.84 (s, 1H), 2.13−1.98 (m, 3H), 1.82 (d, J
= 11.6 Hz, 1H), 1.61−1.57 (m, 2H), 1.36−1.27 (m, 1H); 13C NMR
(400 MHz, DMSO-d6): δ 156.4, 149.4, 148.5, 131.3, 127.3, 126.5,
126.4, 125.1, 123.5, 121.8, 116.8, 107.0, 106.0, 104.2, 67.5, 66.1, 62.7,
56.4, 56.3, 55.94, 27.5, 25.6, 24.6. HRMS (ESI) calcd for C23H26NO4
[M + H]+, 380.1862; found, 380.1862.
1
solid (0.3 g, 67%). mp 176 °C; purity 95% (HPLC); H NMR (400
MHz, CDCl3): δ 8.13 (s, 1H), 7.78 (s, 2H), 7.27 (s, 1H), 7.14 (s, 1H),
4.43 (d, J = 15.4 Hz, 1H), 4.07 (s, 3H), 4.02 (s, 3H), 3.55 (d, J = 15.4
Hz, 1H), 3.29 (d, J = 9.9 Hz, 1H), 2.98−2.82 (m, 2H), 2.39 (s, 3H),
2.32−2.27 (m, 2H), 1.97 (d, J = 9.9 Hz, 1H), 1.88−1.78 (m, 3H),
1.59−1.50 (m, 1H), 1.41 (s, 1H); 13C NMR (400 MHz, CDCl3): δ
169.9, 149.5, 148.5, 148.4, 129.7, 126.7, 126.5, 126.1, 124.8, 123.9,
123.5, 120.0, 114.4, 103.7, 57.3, 56.0, 55.9, 55.5, 34.2, 33.2, 25.5, 24.1,
22.1, 21.3; HRMS (ESI) calcd for C25H28NO4 [M + H]+, 406.2018;
found, 406.2010.
Synthesis of 2,3-Dimethoxy-11,12,13,14,14a,15-hexahydro-9H-
dibenzo[f,h]pyrido [1,2-b]isoquinolin-6-yl Pivalate (12). To a
solution of 2-rac (0.5 g, 1.38 mmol) in dichloromethane (50 mL)
were succesively added triethyl amine (0.28 g, 2.75 mmol), DMAP
(0.025 g, 0.21 mmol), and pivaloyl chloride. The mixture was stirred at
room temperature for 4 h and was then quenched with suturated
ammonium chloride. The organic layler was separated, and the water
layer was extracted with dichloromethane three times. The combined
organic layer was washed with brine, dried with anhydrous magnesium
sulfate, filtered, and concentrated in vacuo. The residue was purified by
flash column chromatography on silica gel (dichloromethane:methanol
40:1 as eluent) to give 12 as a light-yellow solid (0.47 g, 77%). mp
92−94 °C; purity 95% (HPLC); 1H NMR (400 MHz, CDCl3): δ 8.14
(d, J = 2.1 Hz, 1H), 7.87−7.85 (m, 2H), 7.27−7.23 (m, 2H), 4.46 (d, J
= 15.5 Hz, 1H), 4.12 (s, 3H), 4.07 (s, 3H), 3.67 (d, J = 15.5 Hz, 1H),
3.30 (d, J = 11.2 Hz, 1H), 3.13 (dd, J = 3.1 Hz, J = 16.5 Hz, 1H),
2.96−2.89 (m, 1H), 2.46−2.41 (m, 1H), 2.33 (td, J = 3.6 Hz, J = 11.2
Hz, 1H), 2.06 (dd, J = 2.0 Hz, J = 11.2 Hz, 1H), 1.89 (d, J = 11.8 Hz,
1H), 1.81−1.77 (m, 2H), 1.61−1.50 (m, 2H), 1.45 (s, 9H); 13C NMR
(400 MHz, CDCl3): δ 177.5, 149.6, 148.9, 148.6, 129.8, 126.7, 126.5,
126.2, 125.1, 123.9, 123.6, 120.0, 114.3, 103.9, 103.8, 57.4, 56.1, 55.9,
55.8, 39.2, 34.5, 33.4, 27.3 (3C), 27.2, 25.7, 24.2; HRMS (ESI) calcd
for C28H34NO4 [M + H]+, 448.2488; found, 406.2483.
Synthesis of 10-(14aS,15S). Synthesis was performed according to
the procedure for 10-rac using 9-(14aS,15S) instead of 9-rac. Yield,
96%.
Synthesis of 10-(14aR,15R). Synthesis was performed according to
the procedure for 10-rac using 9-(14aR,15R) instead of 9-rac. Yield,
95%.
Synthesis of 2,3-Dimethoxy-11,12,13,14,14a,15-hexahydro-9H-
dibenzo[f,h]pyrido[1,2-b]isoquinolin-6-ol (2-rac). To a solution of
10-rac (0.69 g, 1.84 mmol) in trifluoroacetic acid (20 mL) was added
triethylsilane (0.48 g, 4.21 mmol), and the resulting mixture was
stirred at room temperature for 2 h in the dark. Dichloromethane (20
mL) was added, and the mixture was made basic with a 10% aqueous
solution of sodium carbonate and then filtered. The filtrate was
concentrated and washed with cold methanol to give 2-rac (0.65 g,
Synthesis of 2,3-Dimethoxy-11,12,13,14,14a,15-hexahydro-9H-
dibenzo[f,h]pyrido[1,2-b]isoquinolin-6-yl Cyclopropanecarboxylate
(13). To a solution of 2-rac (0.4 g, 1.1 mmol) in dichloromethane (50
mL) were succesively added cyclopropanecarboxylic acid (0.12 g, 1.38
mmol), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydro-
chloride (0.43 g, 2.2 mmol), triethyl amine (0.22 g, 2.2 mmol), and
DMAP (0.27 g, 2.2 mmol). The mixture was stirred at room
temperature overnight in the dark and was then quenched with
suturated ammonium chloride. The organic layler was separated, and
the water layer was extracted with dichloromethane three times. The
combined organic layer was washed with brine, dried with anhydrous
magnesium sulfate, filtered, and concentrated in vacuo. The residue
was purified by flash column chromatography on silica gel
(dichloromethane:methanol 80:1 as eluent) to give 13 (0.35 g, 8.03
mmol, yield 73%) as a light-yellow solid. mp 224−226 °C; purity 99%
1
96%) as a yellow solid. Purity 97% (HPLC); mp 175 °C (dec.); H
NMR (400 MHz, DMSO-d6): δ 9.78 (s, 1H, OH), 7.98 (d, J = 1.8 Hz,
1H, 5-H), 7.92 (s, 1H, 4-H), 7.68 (d, J = 8.9 Hz, 1H, 8-H), 7.18 (s,
1H, 1-H), 7.11 (dd, J = 8.8, 1.9 Hz, 1H, 7-H), 4.38 (d, J = 15.6 Hz, 1H,
9-Ha), 4.00 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.51 (d, J = 14.5 Hz,
1H, 9-Hb), 3.22 (d, J = 9.9 Hz, 1H, 11-Ha), 3.00 (d, J = 14.7 Hz, 1H,
15-Ha), 2.70 (dd, J = 15.7, 10.9 Hz, 1H, 15-Hb), 2.43−2.15 (m, 2H,
11-Hb, 14a-H), 1.91 (d, J = 10.9 Hz, 1H, 13-Ha), 1.81−1.65 (m, 2H,
12-Hb, 14-Ha), 1.60 (d, J = 12.1 Hz, 1H, 12-Ha), 1.46−1.27 (m, 2H,
13-Hb, 14-Hb); 13C NMR (400 MHz, DMSO-d6): δ 155.5 (C-6),
149.1 (C-2 or C-3), 148.2 (C-3 or C-2), 130.0, 125.4, 125.2, 123.9 (C-
8), 123.0, 122.7, 121.6, 116.3 (C-7), 106.6 (C-5), 104.0 (C-1 or C-4),
103.9 (C-4 or C-1), 57.2 (C-14a), 55.5 (OCH3), 55.4 (OCH3), 55.2
(C-11), 54.9 (C-9), 33.3 (C-15), 32.6 (C-13), 25.0 (C-12), 23.5 (C-
14); HRMS (ESI) calcd for C23H26NO3 [M + H]+, 364.1913; found,
364.1916.
Synthesis of 2-S. Synthesis was performed according to the
procedure for 2-rac using 10-(14aS,15S) instead of 10-rac. Yield, 94%.
[α]2D0 = 114°, (c = 1.0, DMSO); purity 98% (HPLC); 98% ee [tR
(minor) = 12.43 min, tR (major) = 18.68 min].
Synthesis of 2-R. Synthesis was performed according to the
procedure for 2-rac using 10-(14aR,15R) instead of 10-rac. Yield,
94%. [α]2D0 = −112°, (c = 1.0, DMSO); purity 98% (HPLC); 99% ee
[tR (major) = 12.36 min, tR (minor) = 18.68 min].
1
(HPLC); H NMR (400 MHz, CDCl3): δ 8.18 (d, J = 2.2 Hz, 1H),
7.86−7.84 (m, 2H), 7.29 (dd, J = 8.8 Hz, J = 2.2 Hz, 1H), 7.24 (s,
1H), 4.45 (d, J = 15.5 Hz, 1H), 4.10 (s, 3H), 4.06 (s, 3H), 3.65 (d, J =
15.0 Hz, 1H), 3.29 (d, J = 11.0 Hz, 1H), 3.10 (dd, J = 16.4 Hz, J = 3.2
Hz, 1H), 2.94−2.87 (m, 1H), 2.43−2.38 (m, 1H), 2.35−2.29 (m, 1H),
2.05 (d, J = 12.4 Hz, 1H), 1.98−1.93 (m, 1H), 1.92−1.88 (m, 1H),
1.81−1.77 (m, 1H), 1.57−1.46 (m, 2H), 1.27−1.24 (m, 2H), 1.11−
1.06 (m, 2H); 13C NMR (400 MHz, CDCl3): δ 173.9, 149.6, 148.6,
148.6, 129.8, 126.8, 126.6, 126.3, 125.3, 123.9, 123.6, 120.1, 114.4,
103.9, 103.8, 57.5, 56.2, 56.0, 56.0 (2C), 34.7, 33.6, 25.8, 24.3, 13.2, 9.4
(2C); HRMS (ESI) calcd for C27H30NO4 [M + H]+, 432.2175; found,
432.2169.
Synthesis of 2,3-Dimethoxy-11,12,13,14,14a,15-hexahydro-9H-
dibenzo[f,h]pyrido[1,2-b]isoquinolin-6-yl Acetate (11). To a solution
of 2-rac (0.4 g, 1.1 mmol) in dichloromethane (50 mL) were added
acetic anhydride (0.22 g, 2.2 mmol) and triethyl amine (0.17 g, 1.65
mmol), and then the mixture was stirred at room temperature for 3 h.
Synthesis of 2,3-Dimethoxy-11,12,13,14,14a,15-hexahydro-9H-
dibenzo[f,h]pyrido[1,2-b]isoquinolin-6-yl Cyclohexanecarboxylate
(14). Synthesis was performed according to the procedure for 13
using cyclohexanecarboxylic acid. Obtained 0.41 g (yield 79%) as a
L
DOI: 10.1021/acs.jmedchem.6b01502
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
light yellow solid. mp 199−200 °C; purity 98% (HPLC); H NMR
(400 MHz, CDCl3): δ 8.14 (s, 1H), 7.84−7.82 (m, 2H), 7.26−7.25
(m, 1H), 7.24 (s, 1H), 4.49 (d, J = 15.7 Hz, 1H), 4.10 (s, 3H), 4.06 (s,
3H), 3.71 (d, J = 14.6 Hz, 1H), 3.33 (d, J = 11.2 Hz, 1H), 3.13 (d, J =
16.0 Hz, 1H), 3.00−2.94 (m, 1H), 2.70−2.62 (m, 1H), 2.49 (s, 1H),
2.40−2.34 (m, 1H), 2.18 (s, 1H), 2.15 (s, 1H), 1.92−1.86 (m, 6H),
1.75−1.60 (m, 4H), 1.52−1.31 (m, 4H); 13C NMR (400 MHz,
CDCl3): δ 174.9, 149.6, 148.7, 148.6, 129.8, 126.6, 126.4, 126.1, 123.8,
123.6, 120.1, 114.3, 103.8, 103.7, 57.5, 56.0, 55.9, 55.8, 43.4, 34.3, 33.3,
29.08, 29.06, 25.8, 25.6, 25.5, 24.0; HRMS (ESI) calcd for C30H36NO4
[M + H]+, 474.2644; found, 474.2625.
Hz, 1H), 1.80−1.75 (m, 2H), 1.55−1.42 (m, 2H); 13C NMR (400
MHz, CDCl3): δ 164.2, 158.7, 149.4, 148.3, 148.0, 140.7, 131.1, 129.7,
129.1, 127.2, 127.1, 126.9, 126.1, 125.2, 124.1, 123.3, 122.3, 119.2,
114.6, 103.7, 103.5, 57.3, 56.3, 55.9, 53.5, 34.9, 33.8, 26.0, 24.4; HRMS
(ESI) calcd for C30H28N3O4S [M + H]+, 526.1801; found, 526.1788.
Synthesis of 2-((2,3-Dimethoxy-11,12,13,14,14a,15-hexahydro-
9H-dibenzo[f,h]pyrido[1,2-b]isoquinolin-6-yl)oxy)acetamide (19).
To a solution of 2-rac (0.5 g, 1.38 mmol) in DMF (9 mL) was
added cesium carbonate (0.54 g, 1.66 mmol). After stirring at room
temperature for 0.5 h, a solution of ethyl α-bromoacetamide (0.23 g,
1.65 mmol) in DMF (6 mL) was added dropwise under an ice bath.
The reaction mixture was then stirred at room temperature and
monitored by TLC. After the reaction was complete, ethyl acetate and
water were added. The organic layler was separated, and the water
layer was extracted with ethyl acetate three times. The combined
organic layer was washed with brine, dried with anhydrous magnesium
sulfate, filtered, and concentrated in vacuo. The residue was purified by
flash column chromatography on silica gel (ethyl acetate as eluent) to
give 19 as a brown solid (0.40 g, 69%). mp 205−207 °C; purity 96%
Synthesis of 2,3-Dimethoxy-11,12,13,14,14a,15-hexahydro-9H-
dibenzo[f,h]pyrido[1,2-b]isoquinolin-6-yl Furan-2-carboxylate (15).
Synthesis was performed according to the procedure for 13 using 2-
furoic acid. Yield, 82%. mp 239−242 °C; purity 97% (HPLC); H
1
NMR (400 MHz, CDCl3): δ 8.28 (s, 1H), 7.84 (d, J = 8.7 Hz, 1H),
7.79 (s, 1H), 7.69 (s, 1H), 7.45 (s, 1H), 7.38 (d, J = 8.3 Hz, 1H), 7.17
(s, 1H), 6.61 (s, 1H), 4.38 (d, J = 15.2 Hz, 1H), 4.06 (s, 3H), 4.04 (s,
3H), 3.55 (d, J = 15.2 Hz, 1H), 3.24 (d, J = 12.0 Hz, 1H), 2.99 (d, J =
15.7 Hz, 1H), 2.85−2.79 (m, 1H), 2.28−2.23 (m, 1H), 1.99 (d, J =
10.6 Hz, 1H), 1.87 (d, J = 11.0 Hz, 1H), 1.79 (s, 2H), 1.54−1.40 (m,
2H); 13C NMR (400 MHz, CDCl3): δ 156.3, 148.4, 147.4, 146.8,
146.2, 143.1, 128.7, 125.9, 125.8, 125.2, 124.3, 123.0, 122.4, 118.7,
118.5, 113.5, 111.2, 102.7, 102.6, 56.3, 55.2, 55.0, 54.9, 54.0, 33.8, 32.7,
24.9, 23.3; HRMS (ESI) calcd for C28H28NO5 [M + H]+, 458.1967;
found, 458.1963.
1
(HPLC); H NMR (400 MHz, DMSO-d6): δ 8.11 (s, 1H), 8.04 (s,
1H), 7.80 (d, J = 8.8 Hz, 1H), 7.69 (s, 1H), 7.53 (s, 1H), 7.28−7.24
(m, 2H), 4.68 (s, 2H), 4.31 (d, J = 16.0 Hz, 1H), 4.02 (s, 3H), 3.93 (s,
3H), 3.40 (d, J = 16.0 Hz, 1H), 3.19−3.05 (m, 2H), 2.73−2.66 (m,
1H), 2.21−2.10 (m, 2H), 1.93 (d, J = 9.2 Hz, 1H), 1.78 (d, J = 8.8 Hz,
1H), 1.71 (d, J = 11.6 Hz, 1H), 1.63−1.54 (m, 1H), 1.39−1.32 (m,
2H); 13C NMR (400 MHz, DMSO-d6): δ 170.2, 155.6, 149.3, 148.2,
129.5, 125.7, 125.3, 124.7, 124.1, 123.2, 122.8, 115.8, 105.9, 104.4,
103.9, 67.1, 57.3, 55.7, 55.5, 55.4, 55.3, 33.9, 33.1, 25.5, 24.0; HRMS
(ESI) calcd for C25H29N2O4 [M + H]+, 421.2083; found, 421.2127.
Synthesis of 2,3-Dimethoxy-6-(prop-2-yn-1-yloxy)-
11,12,13,14,14a,15-hexahydro-9H-dibenzo[f,h]pyrido[1,2-b]-
isoquinoline. (20). Synthesis was performed according to the
procedure for 19 using propargyl bromide instead of α-bromoaceta-
mide. Obtained 0.47 g (yield 85%) as a brown solid. mp 202−204 °C;
purity 96% (HPLC); 1H NMR (400 MHz, CDCl3): δ 8.03 (d, J = 2.5
Hz, 1H), 7.90 (s, 1H), 7.80 (d, J = 9.0 Hz, 1H), 7.26−7.22 (m, 2H),
4.89 (s, 2H), 4.42 (d, J = 15.5 Hz, 1H), 4.10 (s, 3H), 4.06 (s, 3H), 3.62
(d, J = 15.4 Hz, 1H), 3.26 (d, J = 11.3 Hz, 1H), 3.09 (dd, J = 16.3, J =
3.1 Hz, 1H), 2.90−2.86 (m, 1H), 2.60 (s, 1H), 2.40−2.34 (m, 1H),
2.29 (td, J = 11.2, J = 3.9 Hz, 1H), 2.03 (d, J = 13.1 Hz, 1H), 1.89 (d, J
= 12.4 Hz, 1H), 1.79−1.72 (m, 2H), 1.57−1.42 (m, 2H); 13C NMR
(400 MHz, DMSO-d6): δ 155.2, 149.3, 148.3, 129.6, 125.8, 125.4,
124.8, 124.1, 123.3, 122.8, 115.6, 106.5, 104.6, 104.0, 79.4, 78.2, 57.2,
55.9, 55.5, 55.4, 35.8, 34.0, 33.2, 30.7, 25.5, 24.0; HRMS (ESI) calcd
for C26H28NO3 [M + H]+, 402.2024; found, 402.2604.
Synthesis of 2-((2,3-Dimethoxy-11,12,13,14,14a,15-hexahydro-
9H-dibenzo[f,h]pyrido[1,2-b]isoquinolin-6-yl)oxy)acetonitrile (21-
rac). Synthesis was performed according to the procedure for 19
using α-bromoacetonitrile instead of α-bromoacetamide. Obtained
0.45 g (yield 87%) as a brown solid. mp 199−201 °C; purity 95%
(HPLC); 1H NMR (400 MHz, CDCl3): δ 7.94 (s, 1H), 7.79−7.75 (m,
2H), 7.20−7.16 (m, 2H), 4.92 (s, 2H), 4.39 (d, J = 16.0 Hz, 1H), 4.08
(s, 3H), 4.04 (s, 3H), 3.57 (d, J = 16.0 Hz, 1H), 3.27 (d, J = 8.0 Hz,
1H), 3.00 (d, J = 16.0 Hz, 1H), 2.87−2.81 (m, 1H), 2.35−2.26 (m,
2H), 2.04−1.99 (m, 1H), 1.88 (d, J = 16.0 Hz, 1H), 1.80−1.75 (m,
2H), 1.58−1.52 (m, 1H), 1.44−1.41 (m, 1H); 13C NMR (400 MHz,
DMSO-d6): δ 162.3, 154.2, 149.4, 148.4, 129.5, 125.8, 125.3, 124.4,
123.9, 122.8, 116.8, 115.3, 106.7, 104.7, 103.9, 57.0, 55.9, 55.4, 53.9,
35.7, 33.8, 33.0, 30.7, 25.4, 23.9; HRMS (ESI) calcd for C25H27N2O3
[M + H]+, 403.1977; found, 403.2016.
Synthesis of 2,3-Dimethoxy-11,12,13,14,14a,15-hexahydro-9H-
dibenzo[f,h]pyrido[1,2-b]isoquinolin-6-yl Thiophene-2-carboxylate
(16). Synthesis was performed according to the procedure for 13
using thiophene-2-carboxylic acid. Obtained 0.45 g (yield 87%) as a
1
light yellow solid. mp 247−249 °C; purity 98% (HPLC); H NMR
(400 MHz, CDCl3): δ 8.28 (s, 1H), 8.04 (s, 1H), 7.86−7.81 (m, 2H),
7.69 (s, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.19 (s, 2H), 4.41 (d, J = 15.2
Hz, 1H), 4.07 (s, 3H), 4.05 (s, 3H), 3.58 (d, J = 15.1 Hz, 1H), 3.25 (d,
J = 15.1 Hz, 1H), 3.03 (d, J = 15.8 Hz, 1H), 2.88−2.81 (s, 1H), 2.32−
2.25 (s, 2H), 2.01 (d, J = 9.8 Hz, 1H), 1.87 (d, J = 11.8 Hz, 1H), 1.80
(s, 2H), 1.56−1.41 (m, 2H); 13C NMR (400 MHz, CDCl3): δ 161.0,
149.6, 148.6, 148.3, 134.8, 133.6, 133.0, 129.8, 128.1, 127.0, 126.9,
126.3, 125.5, 124.0, 123.6, 120.0, 114.6, 103.9, 103.8, 57.5, 56.3, 56.1,
56.02, 55.97, 34.9, 33.8, 25.9, 24.8, 24.3; HRMS (ESI) calcd for
C28H28NO4S [M + H]+, 474.1739; found, 474.1732.
Synthesis of 2,3-Dimethoxy-11,12,13,14,14a,15-hexahydro-9H-
dibenzo[f,h]pyrido[1,2-b]isoquinolin-6-yl Nicotinate (17). Synthesis
was performed according to the procedure for 13 using nicotinic acid.
Obtained 0.51 g (yield 80%) as a light yellow solid. mp 183 °C (dec.);
purity 96% (HPLC); H NMR (400 MHz, CDCl3): δ 1H NMR (400
1
MHz, CDCl3): δ 9.49 (s, 1H), 8.89 (dd, J = 4.8 Hz, J = 1.4 Hz, 1H),
8.55−8.52 (m, 1H), 8.32 (d, J = 2.0 Hz, 1H), 7.92 (d, J = 9.0 Hz, 1H),
7.86 (s, 1H), 7.52−7.49 (m, 1H), 7.41 (dd, J = 8.0 Hz, J = 2.0 Hz,
1H), 4.48 (d, J = 15.4 Hz, 1H), 4.09 (s, 3H), 4.07 (s, 3H), 3.68 (d, J =
15.4 Hz, 1H), 3.31 (d, J = 11.1 Hz, 1H), 3.14 (dd, J = 16.8 Hz, J = 3.2
Hz, 1H), 3.10−2.90 (m, 1H), 2.44 (t, J = 10.6 Hz, 1H), 2.34 (td, J =
10.8 Hz, J = 3.9 Hz, 1H), 2.10−2.06 (m, 1H), 1.91 (d, J = 12.8 Hz,
1H), 1.84−1.79 (m, 2H), 1.62−1.53 (m, 1H), 1.51−1.42 (m, 1H); 13C
NMR (400 MHz, CDCl3): δ 164.3, 154.1, 151.5, 149.7, 148.7, 148.3,
137.7, 129.9, 127.0, 126.9, 126.3, 125.7, 125.0, 124.1, 123.5, 119.8,
114.5, 106.5, 103.8, 103.8, 57.5, 56.1, 56.0, 56.0, 55.9, 34.6, 33.5, 25.7,
24.2; HRMS (ESI) calcd for C29H29N2O4 [M + H]+, 469.2127; found,
469.2120.
Synthesis of 2,3-Dimethoxy-11,12,13,14,14a,15-hexahydro-9H-
dibenzo[f,h]pyrido[1,2-b]isoquinolin-6-yl Benzo[d][1,2,3]-
thiadiazole-7-carboxylate (18). Synthesis was performed according
to the procedure for 13 using benzo[d][1,2,3]thiadiazole-7-carboxylic
acid. Obtained 0.48 g (yield 83%) as a yellow-green solid. mp 159−
161 °C; purity 98% (HPLC); 1H NMR (400 MHz, CDCl3): δ 8.77 (d,
J = 8.0 Hz, 1H), 8.49 (d, J = 8.0 Hz, 1H), 8.29 (s, 1H), 7.80 (d, J = 9.2
Hz, 1H), 7.67 (s, 2H), 7.36 (d, J = 8.8 Hz, 1H), 7.11 (s, 1H), 4.31 (d, J
= 15.6 Hz, 1H), 4.05 (s, 3H), 4.01 (s, 3H), 3.48 (d, J = 15.6 Hz, 1H),
3.24 (d, J = 10.8 Hz, 1H), 2.96 (d, J = 16.0 Hz, 1H), 2.83−2.75 (m,
1H), 2.29−2.23 (m, 2H), 2.01 (d, J = 11.6 Hz, 1H), 1.90 (d, J = 11.6
Synthesis of 21-(S). Synthesis was performed according to the
procedure for 21-rac using 2-S instead of 2-rac. Purity 95% (HPLC).
Synthesis of 21-(R). Synthesis was performed according to the
procedure for 21-rac using 2-R instead of 2-rac. Purity 95% (HPLC).
Syntheisis of Ethyl 2-((2,3-Dimethoxy-11,12,13,14,14a,15-hexa-
hydro-9H-dibenzo[f,h]pyrido[1,2-b]isoquinolin-6-yl)oxy)acetate
(22). Synthesis was performed according to the procedure for 19 using
ethyl α-bromoacetate instead of α-bromoacetamide. Obtained 0.50 g
(yield 81%) as a brown solid. mp 84−86 °C; purity 96% (HPLC); 1H
NMR (400 MHz, CDCl3): δ 7.96 (d, J = 2.5 Hz, 1H), 7.87 (s, 1H),
M
DOI: 10.1021/acs.jmedchem.6b01502
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Journal of Medicinal Chemistry
Article
7.77 (d, J = 9.0 Hz, 1H), 7.23 (s, 1H), 7.19 (dd, J = 9.0, J = 2.5 Hz,
1H), 4.81 (s, 2H), 4.53 (d, J = 15.4 Hz, 1H), 4.32 (q, J = 7.1 Hz, 2H),
4.10 (s, 3H), 4.06 (s, 3H), 3.61 (d, J = 15.4 Hz, 1H), 3.27 (d, J = 10.8
Hz, 1H), 3.06 (d, J = 8.4 Hz, 1H), 2.89 (t, J = 10.8 Hz, 1H), 2.38 (s,
1H), 2.30 (t, J = 10.4 Hz, 1H), 2.06 (d, J = 12.8 Hz, 1H), 1.92 (s, 1H),
1.84 (s, 2H), 1.66 (d, J = 12.4 Hz, 1H), 1.48 (s, 1H), 1.32 (t, J = 7.1
Hz, 3H); 13C NMR (400 MHz, CDCl3): δ 169.1, 155.6, 149.5, 148.4,
130.1, 126.5, 125.4, 125.1, 124.3, 124.3, 123.4, 114.5, 106.7, 103.8,
103.8, 65.9, 61.5, 57.5, 56.2, 56.0, 56.0, 34.7, 33.7, 25.9, 24.3, 14.3;
HRMS (ESI) calcd for C27H32NO5 [M + H]+, 450.2236; found,
450.2275.
Synthesis of 2-((2,3-Dimethoxy-11,12,13,14,14a,15-hexahydro-
9H-dibenzo[f,h]pyrido[1,2-b]isoquinolin-6-yl)oxy)ethanol (23).
Under an ice bath, LiAlH4 (0.06 g, 1.34 mmol) was added to a
solution of 22 (0.3 g, 0.67 mmol) in tetrahydrofuran (20 mL) in
portions. After the mixture was stirred at room temperature for 8 h,
water was slowly added to quench the reaction, and the mixture was
extracted with dichloromethane three times. The combined organic
layer was washed with brine, dried with anhydrous magnesium sulfate,
filtered, and concentrated in vacuo. The residue was purified by flash
column chromatography on silica gel (dichloromethane:methanol 30:1
as eluent) to give 23 as a yellow-green solid (0.23 g, 84%). mp 223−
225 °C; 1H NMR (400 MHz, DMSO-d6): δ 8.07−8.04 (m, 2H), 7.74
(d, J = 9.2 Hz, 1H), 7.22−7.18 (m, 2H), 4.98 (s, 1H), 4.30−4.22 (m,
3H), 4.01 (s, 3H), 3.91 (s, 3H), 3.84−3.82 (m, 2H), 3.37 (d, J = 16.0
Hz, 1H), 3.12 (d, J = 10.8 Hz, 1H), 2.96 (d, J = 14.4 Hz, 1H), 2.68−
2.62 (m, 1H), 2.19−2.08 (m, 1H), 1.87 (d, J = 10.0 Hz, 1H), 1.76−
1.67 (m, 2H), 1.61−1.56 (m, 1H), 1.38−1.31 (m, 2H); 13C NMR
(400 MHz, DMSO-d6): δ 156.6, 149.2, 148.2, 129.7, 125.7, 125.1,
124.9, 124.2, 124.0, 122.8, 115.5, 105.6, 104.7, 103.8, 69.8, 59.8, 57.1,
55.8, 55.3, 33.8, 33.0, 30.4, 29.1, 25.4, 23.9.
MX2 (Topo II deficient), lung adenocarcinoma cell line A549, breast
carcinoma cell lines T-47D, MCF7, MDA-MB-231, and MDA-MB-
157, oral epidermoid carcinoma cell line KB, gastric carcinoma cell line
KATO III, colorectal adenocarcinoma cell line HCT 116,
hepatocellular carcinoma cell lines Hep3B and HepG2, cervical
carcinoma cell line HeLa, prostate cancer cell line PC-3, ovarian cancer
cell line SKOV-3, pharyngeal carcinoma cell line Detroit 562, B cell
lymphoma cell line SU-DHL-6, pancreas adenocarcinoma cell line
BxPC-3, and lung fibroblast cell lines MRC-5 and MRC-9 were
purchased from American Type Culture Collection. Human
rhabdomyosarcoma cell line Rh-30 was a gift from the St. Jude
Children’s Research Hospital. Human hepatocellular carcinoma cell
lines SMMC-7721 and Zip-177 were gifts from the Second Military
Medical School (Shanghai, China). Stomach cancer cell line MKN-45
was obtained from the Japanese Foundation of Cancer Research.
Adriamycin-selected MDR cell subline K562/ADR was purchased
from Nanjing KeyGEN BioTECH Co., Ltd. MDR cell subline KB/
VCR was obtained from Zhongshan University of Medical Sciences.
KATO III cells were maintained in IMDM with 20% fetal bovine
serum (FBS, Life Technologies). U-118MG, MCF7, HuH-7, SK-HEP-
1, and SKOV-3 cells were maintained in DMEM with 10% FBS. PC-3
and A549 cells were maintained in F-12 medium with 10% FBS. MDA-
MB-231 and MDA-MB-157 cells were maintained in L15 medium
with 10% FBS. HCT 116 cells were maintained in 5A medium with
10% FBS. Hep3B, HepG2, Detroit 562, MRC-5, and MRC-9 cells
were maintained in EMEM with 10% FBS. KB and KB/VCR cells
were maintained in MEM with 10% FBS. The remaining cells were
maintained in RPMI 1640 medium with 10% FBS. IMDM, EMDM, F-
12, L15, and 5A media were purchased from Life Technologies
Corporation. MEM, DMEM, and RPMI 1640 media were purchased
from Corning Corporation. MDA-MB-157 and MDA-MB-231 cells
were cultured in a humidified atmosphere of air at 37 °C; other cells
were incubated in a humidified atmosphere of 95% air plus 5% CO2 at
37 °C.
SRB and MTT Assays. The antiproliferative activity of compounds
was assessed using an MTT assay with suspended cells (i.e., HL-60, K-
562, SU-DHL-6, K562/ADR, and HL-60/MX2 cells) or by an SRB
assay with the other adherent cells as previously described.9 Briefly,
cells were seeded in 96-well plates and treated in triplicate with
compounds for 72 h. For the MTT assay, 20 μL of MTT solution (5
mg/mL) was added to each well. The plates were further incubated at
37 °C for 4 h, allowing MTT to change into dark-blue formazan
crystals, followed by the addition of 100 μL of water-based solubilizing
solution (10% SDS, 5% isobutyl alcohol, 10 mM HCl) into each well.
For the SRB assay, cells were fixed with 10% ice-cold trichloroacetic
acid and stained with 0.4% SRB in 1% acetic acid. The absorbance of
each well was measured at 570 nm for the MTT assay or at 540 nm for
the SRB assay with a VERSMax plate reader (Molecualr Devices,
Sunnyvale, CA) The antiproliferative activity was expressed as IC50
values, determined by the four-parameter logistic method. Mean IC50
values were obtained from at least three independent experiments.
Cell Proliferation Assay. BEL-7402 cells were seeded in 12-well
plates at a density of 1.5 × 104 cells per well and treated in triplicate
with a gradient of concentrations of compounds. Cells were then
incubated in a IncuCyte ZOOM live cell imaging system (Essen
BioScience) for 10 days. This system takes phase-contrast images in
real time and automatically calculates the relative phase-contrast area
at each time point. The data were retrieved, and proliferation curves
were plotted.
Synthesis of (2S)-2-((2,3-Dimethoxy-11,12,13,14,14a,15-hexahy-
dro-9H-dibenzo[f,h]pyrido[1,2-b]isoquinolin-6-yl)oxy)ethyl 2-Ami-
nopropanoate (25). To a solution of 23 (0.39 g, 0.96 mmol) in
dichloromethane (50 mL) were succesively added N-Boc-alanine (0.2
g, 1.1 mmol), 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide
hydrochloride (0.37 g, 1.92 mmol), triethyl amine (0.19 g, 1.92
mmol), and DMAP (0.23 g, 1.92 mmol). The mixture was stirred at
room temperature for 8 h and was then quenched with suturated
ammonium chloride. The organic layler was separated, and the water
layer was extracted with dichloromethane three times. The combined
organic layer was washed with brine, dried with anhydrous magnesium
sulfate, filtered, and concentrated in vacuo. The residue was purified by
flash column chromatography on silica gel (dichloromethane:methanol
40:1 as eluent) to give 24 as a light-yellow solid. 24 was dissolved in
dichloromethane (30 mL), and trifluoroacetic acid (5 mL) was added.
The mixture was stirred at room temperature for 2 h, and saturated
sodium bicarbonate was then added. The organic layler was separated,
dried with anhydrous magnesium sulfate, filtered, and concentrated in
vacuo to give 25 as a yellow-green solid (0.33 g, 72%). mp 204−206
1
°C; purity 95% in dr 1:3 (HPLC); H NMR (400 MHz, CDCl3): δ
7.93 (d, J = 2.0 Hz, 1H), 7.90 (s, 1H), 7.79 (d, J = 8.8 Hz, 1H), 7.25
(s, 1H), 7.19 (dd, J = 8.8 Hz, J = 2.0 Hz, 1H), 4.58 (s, 1H), 4.47 (d, J =
15.6 Hz, 1H), 4.41 (s, 1H), 4.12 (s, 3H), 4.06 (s, 3H), 3.70−3.60 (m,
2H), 3.31 (d, J = 11.2 Hz, 1H), 3.14−3.09 (m, 1H), 2.97−2.90 (m,
1H), 2.47 (s, 1H), 2.38−2.31 (m, 1H), 2.07−2.05 (m, 1H), 1.92−1.72
(m, 5H), 1.63−1.53 (m, 1H), 1.48−1.43 (m, 1H), 1.36 (d, J = 6.8 Hz,
3H); 13C NMR (400 MHz, CDCl3): δ 176.7, 156.3, 149.5, 148.5,
130.2, 126.4, 125.1, 124.7, 124.2, 123.9, 123.4, 115.2, 105.8, 103.8,
66.1, 63.3, 57.6, 56.1, 56.0, 50.1, 34.5, 33.5, 29.7, 25.7, 24.2, 20.8;
HRMS (ESI) calcd for C28H35N2O5 [M + H]+, 479.2546; found,
479.2532.
Cell Lines and Cell Culture. Human gastric adenocarcinoma cell
line SGC-7901, hepatocellular carcinoma cell lines BEL-7402, BEL-
7404, HuH-7, SK-HEP-1, premyelocytic leukemia cell line HL-60,
chronic myelogenous leukemia cell line K-562, renal carcinoma cell
line 786-O, colon adenocarcinoma cell line COLO 205, and
hepatocyte cell line LO2 were obtained from the cell bank at
Shanghai Institute’s Cell Resource Center, Chinese Academy of
Sciences. Glioblastoma cell line U-118MG, MDR cell subline HL-60/
Colony Formation Assay. BEL-7402 cells were seeded in 12-well
plates at a density of 200 cells per well. Cells were treated in triplicate
with compounds for 24 h, and then compounds were removed from
the medium by washing twice with PBS. Cells were further incubated
in fresh medium for 12 days. Colonies were visualized by staining with
SRB, and the number of colonies formed on each plate was quantified
by counting stained colonies with a diameter ≥ 1 mm.
Cell Cycle Analysis. Treated cells were collected and fixed in 70%
ethanol at −20 °C for at least for 1 h. The fixed cells were stained with
50 μg/mL propidium iodide and 10 μg/mL RNase A for 30 min in the
dark at room temperature. DNA content was measured with a FACS
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DOI: 10.1021/acs.jmedchem.6b01502
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Calibur flow cytometer (BD Biosciences), and cell cycle distribution
was analyzed using ModFit LT software or FlowJo software. For each
sample, ≥1.0 × 104 cells were analyzed. For cell synchronization,
exponentially growing cells were incubated with 0.2 μg/mL aphidicolin
(Santa Cruz) for 16 h.
ORCID
Qingmin Wang: 0000-0002-6062-3766
Author Contributions
§Y.L., L.Q., C.M., and J.S. contributed equally to this work.
Western Blot Analysis. BEL-7402 cells (1.5 × 105) were seeded
in 12-well plates and exposed to compounds at the indicated
concentrations for various amounts of time. Cells were harvested
and subjected standard western blot analysis, using antibodies against
γH2AX, p-Chk1 Ser345, or p-Chk2 Thr68 (all from Cell Signaling
Technology) or β-actin (Sigma-Aldrich). Relative gray intensity of
bands was analyzed with ImageQuant TL software (GE Healthcare
Life Sciences).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to the National Natural Science Foundation of
China (21421062, 21372131, 21672117 and 81321092), the
Specialized Research Fund for the Doctoral Program of Higher
Education (20130031110017) and the Tianjin Natural Science
Foundation (16JCZDJC32400) for generous financial support
for our programs. We would like to Thank Huan Wang from
Shanghai Institute of Materia Medica for the help in the
cytotoxic assay of rat neurons.
Apoptosis Assay. Cells undergoing apoptosis were detected using
an annexin V−FITC apoptosis detection kit (KeyGEN BioTECH)
according to the manufacturer’s instructions. Briefly, BEL-7402 cells (3
× 105) were seeded in 6-well plates and exposed to compounds at the
indicated concentrations for 72 h. Cells were harvested and washed
with PBS, resuspended in 100 μL of cold binding buffer with 5 μL of
annexin V−FITC and 5 μL of PI, and incubated for 15 min in the dark
at room temperature. Annexin V and PI staining were measured with a
FACS Calibur flow cytometer (BD Biosciences) and analyzed with
FlowJo software.
ABBREVIATION USED
■
ADR, adriamycin; BCRP, breast cancer resistance protein;
BrdU, 5-bromodeoxyuridine; CHK1, checkpoint kinase 1;
CHK2, checkpoint kinase 2; CPT, camptothecin; DMSO,
dimethyl sulfoxide; DMAP, 4-dimethylaminopyridine; EDCI,
(3-(dimethylamino)propyl)ethyl carbodiimide hydrochloride;
IC50, 50% inhibitory concentration; MDR, multidrug resistance;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium;
NVT, novantrone; PBS, phosphate buffered saline; P-gp, P-
glycoprotein; SRB, sulforhodamine B; VCR, vincristine
BrdU Labeling. BEL-7402 cells (3 × 105) were seeded in 6-well
plates and exposed to compounds at the indicated concentrations for 1
h. Cells were labeled with 10 μM BrdU for 30 min before harvesting.
Cells were trypsinized, washed with PBS, and fixed in 70% ethanol at
−20 °C for at least 1 h. After fixation, cells were washed with PBS and
incubated in 2 N HCl for 20 min. Cells were neutralized with 0.1 M
borate buffer for 2 min and incubated with anti-BrdU-FITC (BD
Biosciences) and PI for 30 min in the dark at room temperature.
Labeled cells were analyzed by flow cytometry and analyzed with
FlowJo software.
Trypan Blue Staining. BEL-7402 cells (5 × 104) were seeded in
12-well plates and exposed to compounds at the indicated
concentrations for 72 h. Cells were trypsinized and stained with
0.4% Trypan blue solution (Life Technology) at a 9:1 dilution for 3
min and were then loaded into a hemocytometer to count the
unstained (viable) and stained (necrotic) cells separately.
Statistical Analysis. All experiments were repeated independently
three times. Data are presented as the mean SD. Significance was
analyzed with Student’s t test. Results were considered statistically
significant at p < 0.05 (notation: *, p < 0.05; **, p < 0.01).
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b01502.
1H and 13C NMR spectra of 2-rac; DEPT, CH-COSY,
and HH-COSY of 2-R; 1H NMR, 13C NMR, HRMS, and
HPLC spectra of compounds 1 and 7−25; procedure for
primary neuronal cell culture and treatment; effects of 2-
R and 21-R on the viability of rat primary neurons
(Table S1); and effects of compounds 1 and 2-R on the
expression of P-gp (Figure S1) (PDF)
Molecular formula strings and associated biological data
(CSV)
AUTHOR INFORMATION
■
Corresponding Authors
*(L.-h.M.) Phone: 86-21-50806722. Fax: 86-21-50806722. E-
mail: lhmeng@simm.ac.cn.
*(Q.W.) Phone: 86-22-23503952. Fax: 86-22-23503952. E-
mail: wangqm@nankai.edu.cn.
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