Design, Synthesis, and Structural Optimization of Lycorine-Derived Phenanthridine Derivatives as Wnt/β-Catenin Signaling Pathway Agonists

Article abstract of DOI:10.1021/acs.jnatprod.5b00825

Lycorine is a benzylphenethylamine-type alkaloid member of the Amaryllidaceae family. A lycorine derivative, HLY78, was previously identified as a new Wnt/β-catenin signaling pathway agonist that targets the DAX domain of axin. Herein, the structural opti

Full text of DOI:10.1021/acs.jnatprod.5b00825

Article
pubs.acs.org/jnp
Design, Synthesis, and Structural Optimization of Lycorine-Derived
Phenanthridine Derivatives as Wnt/β-Catenin Signaling Pathway
Agonists
Duo-Zhi Chen,† Chen-Xu Jing,† Jie-Yun Cai,† Ji-Bo Wu,‡ Sheng Wang,‡ Jun-Lin Yin,† Xiao-Nian Li,†
Lin Li,*,‡ and Xiao-Jiang Hao*,†
†State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of
Sciences, Kunming 650201, People’s Republic of China
‡State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai 200000, People’s Republic of China
S
* Supporting Information
ABSTRACT: Lycorine is a benzylphenethylamine-type alkaloid
member of the Amaryllidaceae family. A lycorine derivative,
HLY78, was previously identified as a new Wnt/β-catenin
signaling pathway agonist that targets the DAX domain of axin.
Herein, the structural optimization of HLY78 and analyses of the
structure−activity relationships of lycorine-derived phenanthri-
dine derivatives as agonists of the Wnt/β-catenin signaling
pathway are presented. This research suggests that triazole
groups are important pharmacophores for Wnt activation;
triazole groups at C-8 and C-9 of phenanthridine compounds
markedly enhanced Wnt activation. A C-11−C-12 single bond is
also important for Wnt activation. On the basis of these findings,
two Wnt agonists were designed and synthesized. The results for
these agonists indicated that the combination of a 4-ethyldihydrophenanthridine skeleton and a triazole substituent improves
Wnt activation. These compounds may be useful in further pharmacological or biological studies.
nt/β-catenin is a highly conserved signaling pathway1,2
that is important for development. Aberrant Wnt
mechanisms.15,16 A new small-molecule activator of the Wnt/β-
catenin signaling pathway, 4-ethyl-5-methyl-5,6-dihydro[1,3]-
dioxolo[4,5-j]phenanthridine (HLY78, Figure 1b), which acts
in a Wnt ligand-dependent manner, has also been identified.
Mechanistic studies have indicated that HLY78 targets the
DAX domain of axin and potentiates axin/LRP6 association,
which subsequently promotes LRP6 phosphorylation and Wnt
signaling transduction.17 These findings not only provide new
insights into the regulation of the Wnt/β-catenin signaling
pathway via a Wnt-specific small molecule but also may
facilitate therapeutic applications, such as HSC expansion and
the treatment of osteoporosis.
However, the activity of HLY78 is insufficient for further
pharmacological study. To identify a Wnt agonist, the structure
of HLY78 must be further optimized using rational drug design
approaches. Here, the structural optimization of phenanthridine
analogues based on X-ray diffraction and molecular docking
techniques is described, and an evaluation of derivatives that
exhibit Wnt-activating effects in vitro is presented. The
structure−activity relationships (SARs) of these derivatives
are also analyzed.
W
signaling is involved in many diseases, such as cancer (in
which the pathway is inappropriately activated) and Alzheimer’s
disease and osteoporosis (in which the pathway is attenu-
ated).3−5 In recent years, Wnt antagonists have attracted
attention due to the potential applications in the clinical
treatment of cancer.6−9 Appropriate activation of Wnt signaling
pathways could also be useful in other clinical applications, such
as hematopoietic stem cell (HSC) expansion and the treatment
of osteoporosis.10
Lycorine is a naturally occurring multifunctional benzylphe-
nethylamine alkaloid (Figure 1a) of considerable interest.11−14
Previous studies have identified that lycorine derivatives are
novel HCV (hepatitis C virus) inhibitors that act via novel
Received: September 15, 2015
Figure 1. Structures of lycorine and HLY78.
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b00825
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RESULTS AND DISCUSSION
■
Design and Synthesis of Phenanthridine Derivatives.
The well-characterized X-ray crystal structures of axin protein
(PDB 1WSP)18 and HLY78 were examined to predict the
binding modes; molecular docking of HLY78 into the axin
binding domain, DAX, was analyzed using AutoDock19 to
understand the interaction between HLY78 and axin. A total of
11 conformations of HLY78 docking to the DAX domain were
analyzed, and six of the conformations localized to the same
cavity formed by the juxtaposition of the residues from two
neighboring protomers. The model with the lowest estimated
free energy for binding (6.23 kcal/mol) was selected. The
modeled complex structure of axin-HLY78 revealed that a
group of residues in the cavity contact HLY78 via electrostatic
or hydrophobic interactions. The docking results indicated that
the binding of the phenanthridine compound to the axin
protein likely occurs primarily via two oxygen-bearing
substituents at C-8 and C-9. In addition, due to the steric
hindrance of the active cavity of axin, substituents at C-11 also
affect the binding of the compounds to the protein (Figure 2).
Consequently, the design of novel molecules focused on the
optimization of the two oxygen-bearing substituents at C-8 and
C-9 and the exploration of C-11−C-12 bonds.
Figure 3. Syntheses of phenanthridine-type derivatives. Reagents and
conditions: (a) MeI, DMF, rt, 12 h; (b) t-BuOK, t-BuOH, N2, reflux, 4
h, 70%; (c) 10% Pd/C, CH2Cl2, rt, 24 h, 90%; (d) BBr3, CH2Cl2, −78
°C, 4 h, 70% (for 4), 80% (for 5); (e) RX, NaH, THF, N2, rt, 24 h,
55−87%.
Table 1. Wnt Activating Efficacy of the Phenanthridine
Derivatives
compound
concentration that doubled the activation of Wnt
2
NA
3 (HLY78)
5 μM
10 μM
NA
4
4a
5
5 μM
5 μM
2.5 μM
5 μM
NA
5a
5b
5c
5d
5e
5f
5g
5h
5i
NA
Figure 2. Molecular docking studies of HLY78. The X-ray crystal
structure of HLY78 and the schematic representation of HLY78
docked onto the crystal structure of DAX (PDB 1WSP) are shown.
The two related protomers of DAX are indicated in red and blue.
NA
NA
NA
NA
Two initial compounds (4, 5) were generated from lycorine
using a semisynthetic process; these compounds were further
modified to study the effects of structural modifications on Wnt
signaling compared to HLY78. Exhaustive methylation of
lycorine followed by Hoffman degradation afforded intermedi-
ate 2 in 70% yield. Hydrogenation of 2 using 10% Pd/C
afforded HLY78 (3). Subsequently, compounds 2 and 3 were
treated with BBr3 at −78 °C to obtain compounds 4 and 5,
respectively, which were alkylated using different alkylating
agents to afford 14 phenanthridine derivatives (Figure 3).15
Activation of the Wnt/β-Catenin Signaling Pathway
by Phenanthridine Derivatives. The effects of all derivatives
on the reporter gene of the Wnt/β-catenin signaling pathway
were evaluated in HEK293T cells. As shown in Table 1,
compounds 4, 5, 5a, 5b, and 5c activated Wnt signaling, and 5b
was most effective. A concentration of 5b of 2.5 μM doubled
the activity of the Wnt signaling pathway. The remaining
phenanthridine derivatives did not activate the Wnt/β-catenin
pathway, even at high concentrations. To further confirm the
activating function, the effects of compounds 5h and 5i on the
expression of endogenous Wnt target genes (Axin2 and NKD1)
were tested. These compounds did not upregulate the
expression of endogenous Wnt target genes (Figure 4).
SAR Analyses of Phenanthridine Derivatives Based on
Wnt Activation in Vitro. The bioassays of the phenanthridine
derivatives indicated that the C-11−C-12 bond is important for
activating the Wnt signaling pathway. A Δ11,12 double bond
altered the degree of conjugation within the molecular skeleton
and blocked free rotation of the bond, thus increasing steric
hindrance and decreasing the activity of the compounds, as
shown in Figure 5. Thus, a C-11−C-12 single bond is crucial
for the ability of the phenanthridine derivatives to activate Wnt.
Because compound 5 exhibited acceptable Wnt signaling
activation, further structural optimization was focused on the C-
8 and C-9 hydroxy groups of compound 5. However,
compounds 5d−5i exhibited nearly complete loss of Wnt
activation. The above results and docking analysis of HLY78
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biotinylated derivative (HLY179) that exhibited potent Wnt
activation was designed. Specifically, this compound increased
the activation of the Wnt signaling pathway by 2-fold at a
concentration of 0.325 μM.17 Inspired by this result, the
structural characteristics of HLY179 were analyzed and
compared with the precursor, compound 5c. HLY179 features
additional C-8 and C-9 triazole substituents as well as biotin
side chains (Figure 6).
Figure 4. Effects of compounds 5h and 5i on the expression of
endogenous Wnt target genes (Axin2 and NKD1). Neither 5h nor 5i
clearly upregulated the expression of endogenous Wnt target genes.
Figure 6. Comparison of HLY179 and the precursor compound 5c.
HLY179 features additional C-8 and C-9 triazole substituents as well
as biotin side chains (orange).
To confirm the effects of the triazole and biotin moieties, a
series of “model molecules” were designed and synthesized to
simulate different substructures of HLY179 (Figure 7a). Of
these molecules, compounds 8 and 9 were used to study the
effects of the triazole groups, whereas compounds 10, 11, and
biotin were used to explore the effects of the biotin chain in
HLY179. The abilities of these compounds to activate Wnt
signaling were next examined (Figure 7b). Compounds 8 and 9
were clearly Wnt agonists, implying that the triazole moiety
markedly enhances the activation of Wnt signaling. In contrast,
compounds 10, 11, and biotin did not exhibit any activity,
indicating that biotin itself does not activate Wnt signaling.
However, upon the introduction to the phenanthridine
skeleton, biotin improved the activation of Wnt signaling. In
addition, a comparison of 5c with 6 and 7 indicated that the
basic phenanthridine framework was crucial for Wnt agonist
activity.
Figure 5. Effects of the C-11−C-12 bond on Wnt activation. A double
bond between these two carbons markedly reduced the ability of these
phenanthridine derivatives to activate Wnt.
suggested that the cavity or the binding domain of axin may
interact with the C-8 and C-9 substituents via electrostatic or
hydrophobic interactions to facilitate specific binding and allow
only suitable groups to enter this cavity and associate with the
protein residues.
Research on “Model Molecules”. Interestingly, in a
previous study of the activating mechanism of HLY78, a
Design and Synthesis of Potential Wnt Agonists. The
SAR studies prompted us to maintain a C-11−C-12 single bond
and to introduce triazole disubstituents at the free C-8 and C-9
hydroxy groups to optimize the activity of these compounds.
To further explore compounds with these features, compounds
12 and 14, which feature a C-11−C-12 single bond and C-8
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Figure 7. Study of model molecules. (a) Design and syntheses of model molecules. (b) All model molecules except compounds 8 and 9 lacked the
ability to activate Wnt signaling.
and C-9 triazole disubstituents (Figure 8a), were designed and
synthesized. As expected, compounds 12 and 14 effectively
activated Wnt signaling in a reporter gene assay, and 12
exhibited the highest Wnt agonist activity among the synthetic
phenanthridine derivatives. Compound 12 doubled the
activation of the Wnt signaling pathway at a low concentration
of 1.25 μM (Figure 8b). Further evaluation also confirmed the
ability of these compounds to activate the expression of
endogenous Wnt target genes (Figure 8c). Subsequent docking
analysis of 12 indicated that it can associate with the protein
residues, in which HLY78 docks, in the axin cavity (Figure 9).
This result indicates that the combination of a 4-ethyl-
dihydrophenanthridine skeleton and a triazole substituent
enhances Wnt activation. Although HLY179 exhibits greater
activity than compounds 12 and 14, the structure makes it
more of a molecular probe than a lead candidate, and 12 and 14
are more suitable for further pharmacological or biological
studies.
The aforementioned SARs of lycorine-derived HLY78
indicated that the C-11−C-12 single bond is crucial for the
activation of Wnt signaling. Moreover, the triazole group
appears to serve as a pharmacophore for Wnt activation.
Substituting the free C-8 and C-9 hydroxy groups with triazole
groups markedly enhanced the activation of Wnt signaling. In
addition, biotin itself did not activate Wnt signaling, but when
biotin was introduced into the structure of phenanthridine, the
abilities of these two derivatives to activate Wnt signaling were
improved. Guided by these findings, two good Wnt agonists, 12
and 14, were designed and synthesized. The activities of these
two compounds indicate that the combination of a 4-
ethyldihydrophenanthridine skeleton and a triazole substituent
optimizes the activation of Wnt. This study not only confirms
the predictions obtained from SAR data but also provides
suitable lead compounds for use in pharmacological or
biological studies.
D
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Figure 8. Discovery of the new and effective Wnt agonists compounds 12 and 14. (a) Syntheses of 12 and 14. (b) Both 12 and 14 effectively
activated Wnt and doubled the activation of the Wnt signaling pathway at concentrations of 1.25 and 2.5 μM, respectively. Compound 12 was the
most active Wnt agonist among the examined phenanthridine derivatives. (c) Compounds 12 and 14 clearly activated the expression of endogenous
Wnt target genes (Axin2 and NKD1).
X-Bridge RP-C18 column (4.6 × 250 mm, 5 μm, 5 mL/min) with
CH3OH−H2O at rt. All reported yields are for dry compounds that
required no further purification for use in other reactions.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
measured using an X-4 apparatus (Yingyu Yuhua Instrument Factory,
Gongyi, Henan Province, P. R. China). ESI and HRMS data were
recorded using a Finnigan MAT 90 instrument and a VG Auto Spec-
3000 spectrometer, respectively. NMR experiments were conducted
on Bruker AM-400, DRX-500, or Avance III 600 spectrometers using
residual CDCl3 and DMSO-d6 or TMS as internal standard. Column
chromatography was performed on silica gel (60−80 mesh, 200−300
mesh, 300−400 mesh, Qingdao Haiyang Chemical Co. Ltd., Qingdao,
P. R. China). Precoated silica gel 60 GF254 (Merck, Darmstadt,
Germany) was used for TLC analyses. Semipreparative HPLC analyses
were performed on a Hypersil Gold RP-C18 column (i.d. 10 × 250
mm, 5 μm, 5 mL/min) developed with CH3CN−H2O at room
temperature (rt). All regular solvents and reagents were reagent grade
and were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO,
USA), Acros Organics (Geel, Belgium), and J&K Scientific (Beijing, P.
R. China). The purities of all compounds used in biological assays
exceeded 95%, as determined by HPLC. HPLC was performed on an
Syntheses of Compounds 4 and 5. 5-Methyl-4-vinyl-5,6-
dihydro[1,3]dioxolo[4,5-j]phenanthridine (2).17 A solution of
lycorine (300 mg, 1 mmol) in DMF (10 mL) was poured into a
round-bottomed flask, followed by the addition of CH3I (400 μL, 2
mmol). The resulting mixture was stirred at rt for 12 h, after which the
mixture was evaporated to remove the DMF. The vessel was charged
with t-BuOK (1.1 g, 10 mmol) and t-BuOH (10 mL), heated to 90 °C,
and stirred for 4 h. After cooling the mixture to rt, the reaction was
quenched with 50 mL of saturated NH4Cl and extracted twice with
EtO2 (20 mL). The organic phase was washed with saturated NH4Cl
and brine, dried over MgSO4, filtered, and concentrated. The residue
was purified by column chromatography and eluted with petroleum
ether−EtOAc (5:1) to yield compound 2 (240 mg, 70% yield). Pale
1
yellow crystals (from CHCl3): mp 168−170 °C; H NMR (CDCl3,
400 MHz) δ 7.58 (1H, d, J = 7.7 Hz), 7.47 (1H, d, J = 7.7 Hz), 7.26
(2H, dt, J = 10.7, 7.1 Hz), 7.16 (1H, d, J = 7.7 Hz, 1), 6.72 (1H, s),
5.99 (2H, s), 5.75 (1H, d, J = 17.8 Hz), 5.32 (1H, d, J = 11.1 Hz), 4.03
E
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4-Ethyl-5-methyl-5,6-dihydrophenanthridine-8,9-diphenol (5).17
1
Pale yellow, amorphous powder (from CHCl3); H NMR (CDCl3,
400 MHz) δ 7.46 (1H, d, J = 7.1 Hz), 7.27 (1H, d, J = 2.7 Hz), 7.17
(1H, dt, J = 15.0, 7.5 Hz), 7.00 (1H, s), 6.75 (1H, s), 3.96 (2H, s),
2.82−2.76 (2H, m), 2.31 (3H, s), 1.28 (3H, dd, J = 14.7, 7.1 Hz); 13C
NMR (CDCl3, 100 MHz) δ 143.83 (C), 143.05 (C), 141.22 (C),
139.43 (C), 128.85 (C), 127.66 (CH), 126.32 (C), 125.18 (C), 124.67
(CH), 120.74 (CH), 113.82 (CH), 110.44 (CH), 53.67 (CH2), 40.21
(CH3), 23.26 (CH2), 14.85 (CH3); HREIMS m/z 255.1250 (calcd for
C16H17NO2, 255.1259).
Alkylation of Compounds 4 and 5. Syntheses of Compounds
4a and 5a−5i. The lycorine derivatives (0.1 mmol) were dissolved in
dry THF (10 mL), and NaH (50 mg, 2 mmol) and the appropriate
haloalkane (1 mmol) were added. The mixture was stirred at rt for 24
h and quenched with H2O (50 mL) in an ice bath. The solution was
evaporated to remove the THF and extracted with CH2Cl2 (2 × 30
mL). The organic layer was washed with saturated NaHCO3 and brine,
dried over MgSO4, filtered, and concentrated. The residue was purified
by column chromatography using petroleum ether−EtOAc as the
eluent to afford compounds 4a and 5a−5i.
8,9-Diethoxy-5-methyl-4-vinyl-5,6-dihydrophenanthridine (4a).
Yield: 20.1 mg, 65%. Colorless, amorphous powder (from CHCl3):
1H NMR (CDCl3, 400 MHz) δ 7.96 (d, J = 7.8 Hz, 1H), 7.84 (s, 1H),
7.51 (s, 1H), 7.49 (d, J = 7.5 Hz, 1H), 7.18 (m, 1H), 7.13 (dd, J =
17.3, 10.9 Hz, 1H), 5.71 (d, J = 17.3 Hz, 1H), 5.58 (d, J = 17.9 Hz,
1H), 4.28 (s, 2H), 3.81 (q, J = 7.4 Hz, 2H), 3.76 (q, J = 7.1 Hz, 2H),
2.85 (s, 3H), 1.49−1.43 (m, 6H); 13C NMR (CDCl3, 100 MHz) δ
162.4 (C), 151.4 (C), 150.3 (C), 137.6 (CH), 130.9 (CH), 127.3 (C),
126.1 (C), 121.9 (CH), 121.3 (CH), 120.5 (C), 118.9 (C), 117.2,
(CH), 114.8 (CH2), 103.4 (CH), 59.4 (CH2), 55.3 (CH2), 54.6
(CH2), 38.9 (CH3), 16.5 (CH3), 16.4 (CH3); HREIMS m/z 309.1735
[M]+ (calcd for C20H23NO2, 309.1729).
8,9-Diethoxy-4-ethyl-5-methyl-5,6-dihydrophenanthridine (5a).
Yield: 24.9 mg, 80%. Colorless, amorphous powder (from CHCl3):
1H NMR (CDCl3, 600 MHz) δ 7.57 (1H, d, J = 7.8 Hz), 7.47 (1H, t, J
= 7.8 Hz), 7.41 (1H, s), 7.33 (1H, d, J = 7.9 Hz), 6.83 (1H, s), 4.13
(2H, s), 3.46 (2H, q, J = 7.1 Hz), 3.39 (2H, q, J = 7.1 Hz), 2.58 (3H,
s), 2.35−2.31 (2H, m), 1.53−1.43 (6H, m), 1.29 (3H, t, J = 7.3 Hz);
13C NMR (CDCl3, 150 MHz) δ 157.32 (C), 148.43 (C), 141.41 (C),
139.75 (C), 133.36 (C), 132.11 (CH), 130.83 (C), 129.14 (C), 124.10
(CH), 121.12 (CH), 107.66 (CH), 106.27 (CH), 58.39 (CH2), 58.26
(CH2), 54.03 (CH2), 40.31 (CH3), 28.55 (CH2), 16.84 (CH3), 16.62
(CH3), 15.81 (CH3); HREIMS m/z 311.1874 (calcd for C20H25NO2,
311.1885).
4-Ethyl-8,9-dimethoxy-5-methyl-5,6-dihydrophenanthridine
(5b). Yield: 23.9 mg, 87%. Colorless, amorphous powder (from
CHCl3): 1H NMR (CDCl3, 400 MHz) δ 7.57 (1H, d, J = 7.1 Hz), 7.53
(1H, s), 7.49 (1H, s), 7.18 (1H, d, J = 6.9 Hz), 6.75 (1H, m), 4.06
(2H, s), 4.02 (3H, s), 3.98 (3H, s), 2.96 (2H, q, J = 7.4 Hz), 2.41 (3H,
s), 1.23 (3H, t, J = 7.5 Hz); 13C NMR (CDCl3, 100 MHz) δ 153.44
(C), 146.52 (C), 140.53 (C), 138.81 (C), 132.96 (C), 131.73 (CH),
130.04 (C), 129.01 (C), 123.23 (CH), 120.56 (CH), 108.92 (CH),
103.18 (CH), 52.32 (CH2), 56.83 (CH3), 56.41 (CH3), 39.95 (CH3),
28.58 (CH2), 15.80 (CH3); HREIMS m/z 283.1567 (calcd for
C18H21NO2, 283.1572).
Figure 9. Docking analysis on compound 12. The results indicated
that 12 effectively associates with the axin cavity in which HLY78
docks.
(2H, s), 2.51 (3H, s); 13C NMR (CDCl3, 100 MHz) δ 147.43 (C),
145.16 (C), 133.43 (CH), 133.28 (C), 129.21 (C), 126.43 (C), 125.82
(C), 124.96 (CH), 124.35 (CH), 122.74 (CH), 114.32 (CH2), 107.17
(CH), 103.61 (CH), 100.93 (CH2), 54.86 (CH2), 41.55 (CH);
HREIMS m/z 265.1107 (calcd for C17H15NO2, 265.1103).
4-Ethyl-5-methyl-5,6-dihydro-[1,3]dioxolo[4,5-j]phenanthridine
(3).17 A solution of 2 (27 mg, 0.1 mmol) and 10% Pd/C (30 mg) in
CH2Cl2 (5 mL) was stirred under an atmosphere of H2 for 24 h. The
organic layer was filtered and concentrated. The residue was purified
by column chromatography and was eluted with petroleum ether−
EtOAc (15:1) to afford compound 3 (22 mg, 90% yield). Pale yellow
crystals (from CHCl3): mp 159−160 °C; 1H NMR (CDCl3, 400
MHz) δ 7.51 (1H, t, J = 8.4 Hz), 7.28 (2H, d, J = 6.4 Hz), 7.20 (2H, t,
J = 7.9 Hz), 6.75 (1H, s), 6.01 (1H, s), 4.01 (2H, s, 2.83 (2H, q, J = 7.5
Hz), 2.50 (3H, s), 1.32 (3H, dd, J = 15.7, 8.2 Hz); 13C NMR (CDCl3,
100 MHz) δ 147.3 (C), 147.12 (C), 145.43 (C), 139.45 (C), 129.33
(C), 127.71 (CH), 126.65 (C), 126.32 (C), 124.64 (CH), 121.02
(CH), 107.26 (CH), 103.76 (CH), 100.95 (CH2), 55.22 (CH2), 41.02
(CH), 23.13 (CH2), 14.85 (CH3); HREIMS m/z 267.1261 (calcd for
C17H17NO2, 267.1259).
Syntheses of Compounds 4 and 5.17 Compound 2 or 3 (0.2
mmol) was dissolved in 10 mL of CH2Cl2. The solution was cooled to
−78 °C, and BBr3 (200 μL, 0.4 mmol) was added. The mixture was
stirred for 10 h and diluted in 50 mL of saturated NaHCO3, followed
by extraction with CH2Cl2 (2 × 20 mL). The organic layer was washed
with brine and concentrated. The residue was purified by column
chromatography using CHCl3−MeOH (4, 25:1; 5, 20:1) as the eluent
to afford 4 (35.7 mg, 70% yield) and 5 (40.8 mg, 80% yield),
respectively.
4-Ethyl-5-methyl-8,9-bis(prop-2-yn-1-yloxy)-5,6-dihydro-
phenanthridine (5c). Yield: 22.3 mg, 70%. Pale yellow, amorphous
powder (from CHCl3): 1H NMR (CDCl3, 400 MHz) δ 7.55 (1H, dd,
J = 7.0, 2.1 Hz), 7.47 (1H, s), 7.19−7.14 (2H, m), 6.92 (1H, s), 4.85−
4.82 (4H, m), 4.03 (2H, s), 2.85−2.77 (2H, m), 2.59−2.54 (2H, m),
2.51 (3H, s), 1.30 (3H, q, J = 7.5 Hz); 13C NMR (CDCl3, 100 MHz) δ
147.43 (C), 146.82 (C), 145.61 (C), 139.58 (C), 128.97 (C), 127.94
(CH), 126.82 (C), 126.34 (C), 124.61 (CH), 121.03 (CH), 113.15
(CH), 110.51 (CH), 78.68 (CH), 78.44 (CH), 75.91 (C), 75.89 (C),
55.22 (CH2), 56.9 3 (CH2), 54.81 (CH2), 41.36 (CH3), 23.13 (CH2),
14.87 (CH3); HREIMS m/z 331.1579 (calcd for C22H21NO2,
331.1572).
5-Methyl-4-vinyl-5,6-dihydrophenanthridine-8,9-diol (4).17 Pale
1
yellow, amorphous powder (from CHCl3): H NMR (CDCl3, 800
MHz) δ 7.55 (1H, d, J = 7.7 Hz, H-1), 7.44 (1H, d, J = 7.7 Hz, H-3),
7.21 (2H, m, H-10, H-11), 7.13 (1H, t, J = 7.7 Hz, H-2), 6.73 (1H, s,
H-7), 5.72 (1H, d, J = 17.7 Hz, H-12), 5.30 (1H, d, J = 10.9 Hz, H-12),
3.99 (2H, s, H-6), 2.49 (3H, s, NMe); 13C NMR (CDCl3, 200 MHz) δ
145.0 (C, C-9), 143.4 (C, C-8),142.7 (C, C-4a), 133.2 (CH, C-11),
133.0 (C, C-6a), 128.7 (C, C-10a),125.5 (C, C-4), 124.8 (C, C-10b),
124.6 (CH, C-1), 124.1 (CH, C-3), 122.4 (CH, C-2), 114.2 (CH2, C-
12), 113.4 (CH, C-7), 110.1 (CH, C-10), 54.0 (CH2, C-6),41.5 (CH3,
NMe); HREIMS m/z 253.1109 (calcd for C16H17NO2, 253.1103).
8,9-Bis[(1,3-dimethyl-1H-pyrazol-5-yl)methoxy]-4-ethyl-5-meth-
yl-5,6- dihydrophenanthridine (5d). Yield: 26.8 mg, 57%. Pale yellow
powder (from CHCl3): 1H NMR (CDCl3, 400 MHz) δ 7.50 (1H, dd,
F
DOI: 10.1021/acs.jnatprod.5b00825
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
J = 7.3, 1.6 Hz), 7.36 (1H, s), 7.23−7.12 (2H, m), 6.85 (1H, s), 6.05
(2H, s), 5.04 (2H, s), 5.02 (2H, s), 4.00 (2H, s), 3.82 (3H, s), 3.81
(3H, s), 2.80 (2H, q, J = 7.5 Hz), 2.47 (3H, s), 2.24 (3H, s), 2.24 (3H,
s), 1.30 (3H, t, J = 7.5 Hz); 13C NMR (CDCl3, 100 MHz) δ 148.40
(C), 147.79 (C), 147.21 (C), 147.18 (C), 145.64 (C), 139.67 (C),
137.99 (C), 137.80 (C), 128.70 (C), 128.08 (CH), 127.26 (C), 126.79
(C), 124.67 (CH), 120.97 (CH), 114.06 (CH), 111.55 (CH), 106.82
(CH), 106.81 (CH), 62.51 (CH2), 62.07 (CH2), 54.76 (CH2), 41.31
(2CH3), 36.32 (CH3), 23.15 (CH2), 14.83 (CH3), 13.37 (2CH3);
HREIMS m/z 417.2635 (calcd for C28H33N5O2, 471.2634).
8,9-Bis[(3,5-dimethoxybenzyl)oxy]-4-ethyl-5-methyl-5,6-dihydro-
phenanthridine (5e). Yield: 33.3 mg, 60%. Colorless, amorphous
powder (from CHCl3): 1H NMR (CDCl3, 400 MHz,) δ 7.49 (1H, dd,
J = 7.1, 1.9 Hz), 7.37 (1H, s), 7.22−7.10 (2H, m), 6.83 (1H, s), 6.68
(2H, d, J = 2.2 Hz), 6.66 (2H, d, J = 2.2 Hz), 6.41 (2H, d, J = 1.9 Hz),
5.17 (2H, s), 5.14 (2H, s), 3.99 (2H, s), 3.77 (12H, overlap), 2.80
(2H, q, J = 7.5 Hz), 2.47 (3H, s), 1.31 (1H, t, J = 7.5 Hz); 13C NMR
(CDCl3, 101 MHz,) δ 160.91 (4C), 148.88 (C), 148.25 (C), 145.61
(C), 139.80 (C), 139.61 (C), 139.54 (C), 129.09 (C), 127.70 (CH),
126.31 (C), 125.82 (C), 124.57 (CH), 120.93 (CH), 113.07 (CH),
110.41 (CH),104.99 (2CH), 104.92 (2CH), 99.87 (CH), 99.79 (CH),
71.62 (CH2), 71.13 (CH2), 55.27 (4CH3), 54.85 (CH2), 41.30 (CH3),
23.16 (CH2), 14.85 (CH3); HRESIMS m/z 556.2696 (calcd for
C34H38NO6, 556.2699).
8,9-Bis[(5-chlorobenzothiophen-2-yl)methoxy]-4-ethyl-5-methyl-
5,6- dihydrophenanthridine (5i). Yield: 36.9 mg, 60%. Colorless,
1
amorphous powder (from CHCl3): H NMR (CDCl3, 500 MHz) δ
7.94 (1H, d, J = 1.9 Hz), 7.90 (1H, d, J = 3.1 Hz), 7.75 (2H, dd, J =
8.6, 4.6 Hz), 7.52−7.46 (3H, m), 7.43 (1H, s), 7.33−7.29 (2H, m),
7.24−7.13 (2H, m), 6.91 (1H, s), 5.36 (1H, s), 5.34 (2H, s), 4.01 (2H,
s), 2.80 (2H, q, J = 7.5 Hz), 2.48 (2H, s), 1.30 (3H, t, J = 7.5 Hz); 13C
NMR (CDCl3, 126 MHz) δ 148.89 (C), 148.27 (C), 145.70 (C),
139.66 (C), 139.12 (C), 139.02 (C), 138.76 (C), 138.74 (C), 131.75
(C), 131.47 (C), 130.69 (C), 128.91 (C), 127.99 (CH), 127.12 (CH),
127.08 (C), 127.02 (CH), 126.58 (C), 125.06 (CH), 125.04 (CH),
124.70 (CH), 123.82 (CH), 123.79 (CH), 121.97 (CH), 121.85
(CH), 121.05 (CH), 113.92 (CH), 111.38 (CH), 66.74 (CH2), 66.31
(CH2), 54.87 (CH2), 41.39 (CH3), 23.21 (CH2), 14.89 (CH3);
HREIMS m/z 615.0863 [M]+ (calcd for C34H27Cl2NO2S2, 615.0860).
Syntheses of “Model Molecules”. Syntheses of Compounds 6
and 7.14 Pyrocatechol or resorcinol (0.1 mmol) was dissolved in dry
acetone (3 mL), and K2CO3 (0.4 mmol) and propargyl bromide (20
μL, 0.25 mmol) were added. The mixture was stirred at 60 °C for 24 h
and quenched with H2O (50 mL) in an ice bath. The solution was
evaporated to remove acetone and extracted with CH2Cl2 (2 × 30
mL). The organic layer was washed with saturated NaHCO3 and brine,
dried over MgSO4, filtered, and concentrated. The residue was purified
by column chromatography with CHCl3−MeOH (100:1) to afford
compounds 6 and 7, respectively.
8,9-Bis[(1,5-dimethyl-1H-pyrazol-3-yl)methoxy]-4-ethyl-5-meth-
1
1,3-Bis(prop-2-yn-1-yloxy)benzene (6). Yield: 16.7 mg, 90%. H
yl-5,6- dihydrophenanthridine (5f). Yield: 28.3 mg, 60%. Pale yellow,
1
NMR (CDCl3, 500 MHz) δ 7.24−7.20 (1H, m), 6.64−6.62 (3H, m),
6.39 (1H, t, J = 2.9 Hz), 4.67 (4H, s), 2.56−2.55 (2H, m); 13C NMR
(CDCl3, 125 MHz) δ 158.69 (2C), 129.96 (CH), 107.84 (2CH),
102.39 (CH), 78.49 (2C),75.73 (2CH), 55.82 (2CH2); ESIMS m/z
187 [M + H]+.
amorphous powder (from CHCl3): H NMR (CDCl3, 400 MHz) δ
7.51 (1H, dd, J = 6.5, 2.7 Hz), 7.47 (1H, s), 7.18−7.11 (2H, m), 6.89
(1H, s), 6.16 (1H, s), 6.15 (1H, s), 5.18 (2H, s), 5.14 (2H, s), 3.96
(2H, s), 3.77 (6H, s), 2.79 (2H, q, J = 7.5 Hz), 2.45 (3H, s), 2.26 (3H,
s), 2.24 (3H, s), 1.29 (3H, t, J = 7.5 Hz); 13C NMR (CDCl3, 101
MHz) δ 148.91 (C), 148.14 (C), 147.57 (C), 147.40 (C), 145.54 (C),
139.47 (C), 139.44 (C), 139.38 (C), 129.33 (C), 127.41 (CH), 125.92
(C), 125.39 (C), 124.52 (CH), 121.09 (CH), 113.09 (CH), 110.67
(CH), 104.97 (CH), 104.95 (CH), 65.87 (CH2), 65.58 (CH2), 54.82
(CH2), 41.32 (CH3), 35.96 (2CH3), 23.11 (CH2), 14.86 (CH3), 11.26
(2CH3); HREIMS m/z 471.2621 [M]+ (calcd for C28H33N5O2,
471.2634).
1
1,2-Bis(prop-2-yn-1-yloxy)benzene (7). Yield: 17.7 mg, 95%. H
NMR (CDCl3, 500 MHz) δ 7.06−6.95 (4H, overlap), 4.72 (4H, s),
2.52−2.51 (2H, m); 13C NMR (CDCl3, 125 MHz) δ 159.21 (2C),
126.38 (2CH), 111.26 (2CH), 79.04 (2C), 76.21 (2CH), 56.13
(2CH2); ESIMS m/z 187 [M + H]+.
Syntheses of Compounds 8 and 9. Compound 6 or 7 (66.0 mg,
0.2 mmol) was added to compound 13 (22 μL, 0.25 mmol) in H2O/t-
BuOH (2 mL, 1:1), followed by the addition of CuSO4 (3.0 mg) and a
sodium ascorbate solution (50 μL, 1 M solution). The mixture was
stirred for 15 h at rt and concentrated in vacuo; the resultant residue
was purified by column chromatography with CHCl3−MeOH (50:1)
to yield compounds 8 and 9, respectively.
8,9-Bis{[5-(1H-pyrazol-1-yl)pyridin-2-yl]methoxy}-4-ethyl-5-meth-
yl-5,6- dihydrophenanthridine (5g). Yield: 37.0 mg, 65%. Pale yellow
1
powder (from CHCl3): H NMR (CDCl3, 400 MHz) δ 8.54 (2H, s),
8.47 (2H, s, 2H), 8.02−7.96 (2H, m), 7.92 (2H, s), 7.72 (2H, s), 7.48
(1H, d, J = 6.7 Hz), 7.38 (1H, s), 7.15 (2H, m), 6.85 (1H, s), 6.45
(2H, s), 5.20 (2H, s), 5.18 (2H, s), 3.98 (2H, s), 2.78 (2H, d, J = 7.6
Hz), 2.45 (3H, s), 1.28 (3H, t, J = 7.5 Hz); 13C NMR (CDCl3, 100
MHz) δ 148.96 (C), 148.38 (C), 147.76 (CH), 147.68 (CH), 146.08
(C), 142.59 (CH), 142.55 (CH), 140.09 (C), 138.82 (CH), 138.76
(CH), 130.78 (C), 130.59 (C), 129.61 (C), 129.20 (C), 128.46 (CH),
127.52 (2CH), 127.43 (C), 127.00 (C), 125.10 (CH), 121.38 (CH),
114.10 (CH), 112.70 (CH), 112.68 (CH), 111.46 (CH), 108.33
(CH), 108.30 (CH), 69.68 (CH2), 69.20 (CH2), 55.24 (CH2), 41.76
(CH3), 23.59 (CH2), 15.28 (CH3); HREIMS m/z 568.2510 [M]+
(calcd for C34H31N7O2, 568.2503).
8,9-Bis{[methyl-(1,1′-biphenyl)-2-carboxylate]methoxy}-4-ethyl-
5-methyl-5,6-dihydrophenanthridine (5h). Yield: 49.2 mg, 70%.
Colorless, amorphous powder (from CHCl3): 1H NMR (CDCl3/
methanol-d4, 400 MHz) δ 7.85−7.78 (m, 2H), 7.58−7.29 (m, 16H),
7.21−7.10 (m, 2H), 6.87 (s, 1H), 5.28 (s, 2H), 5.26 (s, 2H), 3.99 (s,
2H), 3.61 (s, 3H), 3.55 (s, 3H), 2.79 (q, J = 7.5 Hz, 2H), 2.47 (s, 3H),
1.30 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ 169.38 (C),
169.30 (C), 149.04 (C), 148.32 (C), 145.48 (C), 142.09 (C), 142.06
(C), 140.91 (C), 140.88 (C), 139.51 (C), 136.35 (C), 136.12
(C),131.37 (2CH), 131.34 (2CH), 130.74 (CH), 130.71 (CH),
129.79 (CH), 129.75 (CH), 129.04 (2C), 128.47 (2CH), 127.79
(2CH), 127.25 (2CH), 127.22 (2CH), 127.15 (CH), 126.33 (C),
125.89 (C), 124.69 (CH), 120.95 (CH), 113.44 (CH), 110.93 (CH),
71.71 (CH2), 71.22 (CH2), 54.82 (CH2), 51.96 (CH3), 51.91 (CH3),
41.26 (CH3), 23.10 (CH2), 14.82 (CH3); HRESIMS m/z 704.3021
[M + H]+ (calcd for C46H42NO6, 704.3012).
2,2′-{[(1,3-Phenylenebis(oxy))bis(methylene)]bis(1H-1,2,3-tria-
1
zole-4,1-diyl)}bis(ethan-1-amine) (8). Yield: 17.9 mg, 50%. H NMR
(methanol-d4, 400 MHz) δ 7.41 (2H, s), 7.21−7.17 (1H, m), 6.46−
6.40 (3H, m), 5.07 (4H, s), 4.29−4.21 (4H, m), 3.21−3.14 (4H, m);
13C NMR (CDCl3, 125 MHz) δ 157.13 (2C), 143.68 (2C), 128.67
(CH), 126.64 (2CH),109.34 (2CH), 103.45 (CH), 62.23 (2CH2),
57.44 (2CH2), 54.39 (2CH2); ESIMS m/z 359 [M + H]+.
2,2′-{[(1,2-Phenylenebis(oxy))bis(methylene)]bis(1H-1,2,3-tria-
zole-4,1-diyl)}bis(ethan-1-amine) (9).17 Yield: 16.1 mg, 45%. 1H
NMR (CDCl3, 500 MHz) δ 7.41 (2H, s), 7.13−6.99 (4H, overlap),
5.11 (4H, s), 4.29−4.22 (4H, m), 3.21−3.14 (4H, m); 13C NMR
(CDCl3, 125 MHz) δ 159.21 (2C), 144.38 (2C), 127.71 (2CH),
126.92 (2CH), 112.06 (2CH), 61.18 (2CH2), 58.68 (2CH2), 55.19
(2CH2); ESIMS, m/z 359 [M + H]+.
2-Azidoethylamine (13).17 2-Bromoethylamine hydrobromide
(500 mg, 2.44 mmol) and sodium azide (475.9 mg, 7.32 mmol)
were dissolved in H2O (2 mL); the solution was heated to 75 °C,
stirred for 21 h, and cooled to 0 °C. To this mixture were added KOH
(800 mg) and Et2O (2 mL), and the solution was extracted with Et2O
(2 × 10 mL) and concentrated in vacuo. The resultant residue was
purified by column chromatography with CHCl3−MeOH (20:1) to
afford compound 13 (171 mg, 82% yield). Colorless liquid (from ethyl
ether): 1H NMR (CDCl3, 400 MHz) δ 3.30 (2H, t, J = 5.7 Hz,
CH2NH2), 2.80−2.84 (2H, m, CH2N3), 1.27 (2H, s, NH2); 13C NMR
(CDCl3, 100 MHz) δ 54.6 (CH2N3), 41.2 (CH2NH2); ESIMS m/z 87
[M + H]+.
G
DOI: 10.1021/acs.jnatprod.5b00825
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Journal of Natural Products
Article
2′,5′-Dioxopyrrolidin-1-yl-5-[(3″aS,4″S,6″aR)-2″-oxohexahydro-
1H- thieno(3″,4″-d)imidazol-4″-yl]pentanoate (10).17 A solution of
D-biotin (24 mg, 0.1 mmol) in DMF (10 mL) was added to pyridine
(2 mL) and dicyclohexylcarbodiimide (DCC) (41 mg, 0.2 m). The
mixture was stirred for 0.5 h and charged with N-hydroxysuccinimide
(13.8 mg, 0.12 mmol). The resultant solution was stirred for 24 h at rt
and concentrated in vacuo. The residue was recrystallized from
propanol to give 10 (22 mg, 65% yield). Colorless solid (from
1.92 (3H, s), 1.32 (2H, t, J = 7.3 Hz), 1.24 (3H, t, J = 7.6 Hz); 13C
NMR (125 MHz, CDCl3) δ 171.20 (2C), 148.84 (C), 148.29 (C),
145.72 (C), 144.07 (C), 143.81 (C), 139.73 (C), 128.63 (C), 128.23
(CH), 127.74 (C), 127.27 (C), 124.74 (CH), 124.20 (CH), 124.14
(CH), 121.07 (CH), 115.00 (CH), 112.55 (CH), 64.72 (CH2), 64.10
(CH2), 54.78 (CH2), 49.81 (CH2), 49.79 (CH2), 41.42 (CH3), 39.74
(CH2), 39.68 (CH2), 23.21 (CH2), 23.03 (2CH3), 14.87 (CH3);
HREIMS m/z 587.2960 [M]+ (calcd for C30H37N9O4, 587.2969).
Bioactivity Assays. Cell Culture. HEK293T cells were cultured in
Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v)
heat-inactivated fetal bovine serum in a humidified 5% CO2/95% air
(v/v) atmosphere at 37 °C.
Reporter Gene Assay. HEK293T cells were transfected using
Lipofectamine Plus (Invitrogen) according to the manufacturer’s
instructions. For reporter gene assays, HEK293T cells were seeded in
24-well plates. Each well was transfected with 250 ng of plasmids in
total, including 20 ng of TOPFlash and 25 ng of EGFP-C1. The LacZ
plasmid was added to equalize the total amount of plasmid in the wells
to 250 ng. Eighteen hours after transfection, the cells were treated with
Wnt3a-conditioned medium (Wnt3a CM) or control medium (Ctr
CM) for an additional 6 h and were lysed using a Boehringer
Mannheim luciferase assay kit (200 μL/well) for luciferase assays. The
fluorescence intensity emitted by green fluorescent protein in the
resultant cell lysates was first determined in a Wallac multicounter
capable of counting fluorescence and luminescence. Next, the
luciferase substrate was added to the cell lysates, and the luciferase
activities were determined by measuring the luminescence intensities
using the same counter. The luminescence intensities were normalized
against the fluorescence intensities.20
Target Gene Assay. Cells were treated with the synthesized
compounds as indicated and control or Wnt3a CM for 6 h. Total RNA
was extracted with TRIzol. Additionally, purified RNA was reverse
transcribed using oligo(dT) priming and the Superscript III First-
Strand Synthesis System (Invitrogen) according to the manufacturer’s
instructions. The gene transcripts were quantified by quantitative real-
time PCR using a Quantitative SYBR green PCR kit (Takara SYBR
premix Ex Taq) and ABI Quant Studio 6. Gene expression was
normalized by GAPDH. The following primer pairs were used for the
target genes: Axin2: 5′-AGGCTAGCTGAGGTGT-3′ and 5′-
AGGCTTGGATTGGAGAA-3′; NKD1: 5′-GTCAACCACTCCC-
CAACATC-3′ and 5′-AATGGTGGTAGCAGCCAGAC-3′;
GAPDH: 5′-AGGTCGGAGTCAACGGATTTG-3′ and 5′-
TGTAAACCATGTAGTTGAGGTCA-3′.
1
CHCl3): mp 204−206 °C; H NMR (DMSO-d6, 400 MHz) δ 6.42
(1H, s, NH), 6.36 (1H, s, NH), 4.27−4.32 (1H, m, H-6″a), 4.11−4.16
(1H, m, H-3″a), 3.06−3.12 (1H, m, SCH), 2.78−2.85 (5H, m,
CH2CH2 (succinyl), SCH2), 2.66 (2H, t, J = 7.3 Hz, H-2), 2.57 (1H, d,
J = 11.4 Hz, SCH2), 1.58−1.68 (3H, m, H-3, H-5), 1.35−1.54 (3H, m,
H-4, H-5); 13C NMR (DMSO-d6, 100 MHz) δ 170.34 (N(CO)2),
168.91 (CO2), 162.73 ((HN)2CO), 61.05 (C-3″a), 59.23 (C-6″a),
55.27 (SCH), 39.94 (SCH2), 30.08 (C-2), 27.85 (C-4), 27.65 (C-5),
25.44 (CH2CH2 (succinyl)), 24.37 (C-3); ESIMS (m/z) 342[M +
H]+.
N-(2′-Azidoethyl)-5-[(3″aS,4″S,6″aR)-2″-oxohexahydro-1H-
thieno(3″,4″-d)imidazol-4″-yl]pentanamide (11).17 Compounds 10
(34 mg, 0.1 mmol) and 13 (17 mg 0.2 mmol) were dissolved in DMF
(2 mL), and Et3N (12 mg, 1.2 mmol) was added. The solution was
stirred for 24 h at rt and concentrated in vacuo. The resultant residue
was purified by column chromatography using CHCl3−MeOH (20:1)
to yield 11 (23 mg, 70% yield). Colorless waxy substance (from
1
CHCl3): H NMR (DMSO-d6, 400 MHz) δ 8.03 (1H, t, J = 5.3 Hz,
CONH), 6.42 (1H, s, NH), 6.35 (1H, s, NH), 4.26−4.32 (1H, m, H-
6″a), 4.08−4.14 (1H, m, H-3″a), 3.31 (2H, d, J = 7.6 Hz, CH2N3),
3.19−3.24 (2H, m, CH2CH2N3), 3.05−3.11 (1H, m, SCH), 2.80 (1H,
dd, J = 12.4, 5.1 Hz, SCH2), 2.56 (1H, d, J = 12.9 Hz, SCH2), 2.06
(2H, t, J = 7.3 Hz, H-2), 1.55−1.65 (1H, m, H-5), 1.39−1.55 (3H, m,
H-3, H-5), 1.20−1.38 (2H, m, H-4); 13C NMR (DMSO-d6, 100 MHz)
δ 172.43 (CONH), 162.74 ((HN)2CO), 61.06 (C-3″a), 59.23 (C-
6″a), 55.45 (SCH), 50.01 (CH2N3), 39.97 (SCH2), 38.10
(CH2CH2N3), 35.13 (C-2), 28.22 (C-4), 28.07 (C-5), 25.26 (C-3);
HRESIMS m/z 313.1442 [M + H]+ (calcd for C12H21N6O2S,
313.1446).
2,2′-{[((4-Ethyl-5-methyl-5,6-dihydrophenanthridine-8,9-diyl)bis-
(oxy))bis(methylene)]bis(1H-1,2,3-triazole-4,1-diyl)}bis(ethan-1-
amine) (12).17 Compound 5c (66.0 mg, 0.2 mmol) was added to
compound 13 (22 μL, 0.25 mmol) in H2O/t-BuOH (2 mL, 1:1),
followed by the addition of CuSO4 (3.0 mg) and a sodium ascorbate
solution (50 μL, 1 M solution). The resultant solution was stirred for
20 h at 60 °C and concentrated in vacuo; the resultant residue was
purified by column chromatography with CHCl3−MeOH (9:1) to
Molecular Docking Protocol. Compounds 12 and HLY78 were
docked with EPAC2 using the X-ray structure of axin (PDB code:
1WSR) and AutoDock 4.2. The H2O molecules and ligand within the
crystal structure were removed, and polar hydrogen moieties were
added using AutoDockTools. In the structures of the analogues, all
bonds were rotatable except the aromatic, amide, cyano, and double
bonds; the protein was treated as a rigid structure. The docking runs
were performed using the standard parameters of the program for
interactive growth and subsequent scoring.
1
afford 12 (60 mg, 55% yield). Waxy substance (from CH3OH): H
NMR (methanol-d4, 400 MHz) δ 8.07 (1H, s), 8.05 (1H, s), 7.55 (1H,
dd, J = 7.3, 1.6 Hz, 1), 7.48 (1H, s), 7.18−7.04 (2H, m), 7.00 (1H, s),
5.22 (2H, s), 5.18 (2H, s), 4.45 (4H, dd, J = 11.7, 5.9 Hz), 3.93 (2H,
s), 3.12 (4H, br s), 2.74 (2H, q, J = 7.5 Hz), 2.39 (3H, s), 1.25 (3H, t, J
= 7.5 Hz); 13C NMR (methanol-d4, 101 MHz) δ 149.89 (C), 149.17
(C), 146.73 (C), 145.14 (C), 144.92 (C), 140.63 (C), 130.18 (C),
129.13 (CH), 128.10 (C), 127.65 (C), 126.14 (CH), 126.10 (CH),
125.96 (CH), 122.39 (CH), 114.87 (CH), 112.14 (CH), 64.21 (CH2),
63.73 (CH2), 55.58 (2CH2), 53.29 (2CH2), 42.34 (CH2), 41.70
(CH3), 24.31 (CH2), 15.51 (CH3); HREIMS m/z 503.2759 (calcd for
C26H33N9O2, 503.2757).
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b00825.
N,N′-{[(((4-Ethyl-5-methyl-5,6-dihydrophenanthridine-8,9-diyl)-
bis(oxy))bis(methylene))bis(1H-1,2,3-triazole-4,1-diyl)]bis(ethane-
2,1-diyl)}diacetamide (14). Compound 12 (50 mg 0.1 mmol) was
added to a solution of pyridine (3 mL) and Ac2O (16 mg, 0.16 mmol).
The resultant mixture was stirred at rt for 1 h under N2 and poured
into ice-cold H2O (50 mL) with vigorous stirring. The mixture was
extracted with EtOAc (2 × 30 mL), washed with saturated NaHCO3
and brine, concentrated, and separated by silica gel column
chromatography to afford compound 14 with a yield of 32%. Waxy
NMR spectra of the synthetic compounds (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
*Tel/Fax: 86-21-54921188. E-mail: lli@sibs.ac.cn. (L. Li).
*Tel/Fax: 86-871-65223070. E-mail: haoxj@mail.kib.ac.cn. (X.-
H. Hao).
1
substance (from CHCl3): H NMR (500 MHz, CDCl3) δ 7.57 (1H,
s), 7.54 (1H, s), 7.48−7.44 (1H, m), 7.39 (1H, s), 7.16−7.07 (2H, m),
6.86 (1H, s), 5.10 (2H, s), 5.08 (2H, s), 4.42 (4H, dd, J = 10.8, 7.0
Hz), 3.95 (2H, s), 3.70 (4H, d, J = 4.1 Hz), 2.41 (3H, s), 1.93 (3H, s),
Notes
The authors declare no competing financial interest.
H
DOI: 10.1021/acs.jnatprod.5b00825
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
ACKNOWLEDGMENTS
■
We thank Dr. X. He (Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences) for conducting the
Wnt target gene assays throughout this work and Dr. W. Wang
(Kunming Institute of Botany, Chinese Academy of Sciences, P.
R. China) for assisting with the purification of the compounds
throughout this work. This research was funded by the National
Natural Science Foundation of People’s Republic of China
(81402828 and 21432010), the “Xi Bu Zhi Guang” Foundation
of the Chinese Academy of Sciences, and the Foundation of
State Key Laboratory of Phytochemistry and Plant Resources in
West China (P2015-ZZ14).
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DOI: 10.1021/acs.jnatprod.5b00825
J. Nat. Prod. XXXX, XXX, XXX−XXX