Design, synthesis and structure-activity relationship optimization of phenanthridine derivatives as new Wnt/β-catenin signalling pathway agonists

Article abstract of DOI:10.1016/j.bioorg.2018.11.020

Phenanthridine derivativeHLY78 has previously been identified as the first Wnt/β-catenin signalling pathway agonist that targets the DAX domain of axin. However, due to the relatively weak activation on the Wnt/β-catenin signalling pathway, HLY78 is insuf

Full text of DOI:10.1016/j.bioorg.2018.11.020

BioorganicChemistry84(2019)285–294
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and structure-activity relationship optimization of
phenanthridine derivatives as new Wnt/β-catenin signalling pathway
agonists
Duo-zhi Chena,1, Bi-juan Yanga,c,1, Xiao-li Heb,1, Shi-rui Fana, Jie-yun Caid, Chen-xu Jinga,
⁎⁎
⁎
Heng Zhangb, Yu Zhanga, Lin Lib, , Xiao-jiang Haoa,
a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China
b State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai,
China
c University of Chinese Academy of Sciences, Shanghai, China
d Yunnan C.T. Quality Inspection & Text Station, Kunming 650106, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Phenanthridine derivativeHLY78 has previously been identified as the first Wnt/β-catenin signalling pathway
agonist that targets the DAX domain of axin. However, due to the relatively weak activation on the Wnt/β-
catenin signalling pathway, HLY78 is insufficient for further pharmacological study. Herein, the structural op-
timization of HLY78 and analyses of the structure-activity relationships (SARs) of HLY78-derived phenan-
thridine derivatives as agonists of the Wnt/β-catenin signalling pathway are presented. In this work, 36 deri-
vatives were designed and synthesized with some derivatives exhibiting stronger Wnt activity than the activity of
HLY78. In particular, one of them, 8-((1,3-dimethy-pyrazol-5-yl)methoxy)-5-ethyl-4-methyl-5,6-dihydro-phe-
nanthridin-9-ol, exhibited strong Wnt active activity and is 10 times more potent than HLY78. The following SAR
analysis suggests that a pyrazole group, especially at the C-8 position, is important for Wnt activation; a methyl
group at the C-4position seems to be more beneficial for Wnt activation than ethyl; and oxidation of the C-6
position reduces the Wnt activation.
Wnt/β-catenin signalling pathway agonist
Phenanthridine derivatives
Structural optimization
Structure-activity relationships (SARs)
1. Introduction
(hepatitis C virus) inhibitors that act via novel mechanisms [15,16]. A
new small-molecule activator of the Wnt/β-catenin signalling pathway,
Wnt/β-catenin is a highly conserved signalling pathway [1,2] that is
important for development. Aberrant Wnt signalling is involved in
many diseases, such as cancer (in which the pathway is inappropriately
activated), Alzheimer’s disease and osteoporosis (in which the pathway
is attenuated) [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 signalling pathways could
also be useful in other clinical applications, such as haematopoietic
stem cell (HSC) expansion, the treatment of osteoporosis and vitiligo
[10].
4-ethyl-5-methyl-5,6-dihydro-[1,3] dioxolo [4,5-j] phenanthridine
(HLY78), 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 signal trans-
duction (Fig. 1) [18]. These findings not only provide new insights into
the regulation of the Wnt/β-catenin signalling pathway via a Wnt-
specific small molecule but also may facilitate therapeutic applications,
such as HSC expansion and the treatment of osteoporosis and vitiligo.
However, the activity of HLY78 is insufficient for further pharma-
cological 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
Lycorine is a naturally occurring multifunctional benzylphenethy-
lamine alkaloid (Fig. 1a) of considerable interest [11–14]. Previous
studies have identified that lycorine derivatives are novel HCV
⁎ Corresponding author.
⁎⁎ Corresponding author.
E-mail addresses: lli@sibs.ac.cn (L. Li), haoxj@mail.kib.ac.cn (X.-j. Hao).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2018.11.020
Received 10 July 2018; Received in revised form 1 November 2018; Accepted 17 November 2018
Availableonline19November2018
0045-2068/©2018PublishedbyElsevierInc.

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BioorganicChemistry84(2019)285–294
Fig. 1. HLY78 and the mode of action on active the Wnt/β-catenin signalling pathway. HLY78 could affect the stability of the LRP6-Axin transcription complex to
regulate the activation of Wnt by targeting axin [18].
diffraction and molecular docking techniques is described, and an
evaluation of derivatives that exhibit Wnt-activating effects in vitro are
presented. The structure-activity relationships (SARs) of these deriva-
tives are also analysed.
at C-6 also affects the binding of the compounds to the protein (Fig. 2).
Consequently, it is necessary to appropriately modify the phenan-
thridine skeleton of HLY78, and eight phenanthridineskeletons (5–12)
that focused on the optimization of the C-4, N-5 and C-6 positions were
designed and synthesized by referencing the results of the docking
analysis (Fig. 3). Another23phenanthridine derivatives bearing dif-
ferent substituents at C-8 or C-9were further designed and synthesized
(Scheme1).
2. Results and discussion
2.1. Design and synthesis of phenanthridine derivatives
2.2. The Wnt activity of phenanthridine derivatives
As shown in Fig. 2, our initial attempts to modify the structure of
HLY78 focused on manipulating the C-4substituents (highlighted in
red) and C-6substituents (highlighted in pink). In addition, we in-
vestigated the effects of modifying the properties of the nitrogen atom
at the 5-position of the HLY78 skeleton (highlighted in blue) and the
benzodioxole group (highlighted in green). Nine phenanthridine deri-
vatives were ultimately synthesized, bearing various combinations of
these modifications, but unfortunately, such modifications did not lead
to active compounds [17].
The effects of all 36 derivatives on the reporter gene of the Wnt/
βcatenin signalling pathway were evaluated in HEK293T cells using
HLY78 as a positive control. As shown in Table 1, compounds 3f and 3g
exhibit much stronger Wnt activity than the activity of HLY78, and 3f in
particular could double the activation of the Wnt signalling pathway at
the low concentration of 0.5 μM, which is 10 times more potent than
HLY78. Furthermore, the Wnt activation effects of compounds 1d, 2d,
2f, 4 and 6fare equal to HLY78. In addition, nine of these compounds
(1, 1e, 2, 2d, 3, 3d, 3e, 4e and 5b) show weaker Wnt activity than that
of HLY78 (Fig. 4). The other remaining phenanthridine derivatives did
not activate the Wnt/β-catenin pathway, even at high concentrations.
In addition, a dose response analysis of compounds 3f and 3g were
The aforementioned failed attempts caused an in-depth structural
analysis of HLY78. Predictionsof the binding modes between HLY78
and the target protein axin were initially conducted. The X-ray crystal
structure of axin (PDB 1WSP) [19] and HLY78 were examined to pre-
dict the binding modes; molecular docking of HLY78 into the axin
binding domain, DAX, was analysed using AutoDock [20]. All the
conformations of HLY78 docking to the DAX domain were analysed,
and six of the conformations were localized to the same cavity formed
by the juxtaposition of residues from two neighbouring protomers. The
modelled complex structure of axin-HLY78 revealed that a group of
residues in the cavity interact with HLY78 via electrostatic or hydro-
phobic interactions. The docking results indicated that the binding of
the phenanthridine compound to axin likely occurs via the substituents
at C-8 and C-9, and due to the steric hindrance in the active cavity of
axin, an ethyl group at the C-4 position seems to adversely affect the
entrance of compounds into the narrow cavity. In addition, amidation
conducted and the EC50 value of 3f and 3g is 0.27
0.11 μM and
0.79
0.21 μM, respectively. To further confirm the activating func-
tion, the effects of compounds 3f and 3g on the expression of the en-
dogenous Wnt target genes (Axin2 and NKD1) were tested. These
compounds could clearly upregulate the expression of endogenous Wnt
target genes (Fig. 5).
2.3. SAR analysis of the Wnt activation effects of phenanthridine
derivatives
The SAR study initially focused on investigating the importance of
Fig. 2. Regions of the phenanthridine skeleton that were targeted for modification. The C-4substituent (highlighted in red); the N-5substituent (highlighted in blue);
the C-6substituent (highlighted in pink); and the benzodioxole group between C-8 and C-9 (highlighted in green).
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Fig. 3. Molecular docking of HLY78 into the axin binding domain, DAX, using AutoDock.
the substituents at the C-4 and N-5 positions. When comparing the
previously reportedC-4 ethyl-substituted compounds, the newly syn-
thesized derivatives bearing a C-4 methyl substituent, such as 3e, 4e,
and 2g, exhibit stronger Wnt activation than that of the equivalent
compounds that bear C-4 ethyl substituents. From these results, it can
be concluded that relative to an ethyl group, a methyl substituent at the
C-4 position would improve the Wnt activation of phenanthridine de-
rivatives. On the other hand, substituents at the N-5 position seem to
have limited effects on the Wnt activities of the compounds.
The nitrogen atoms of alkaloids are known to have profound effects
on their biological activities. The amidated derivatives 5a-d, 6a-h and
8a-bwere therefore prepared to determine how the conversion of the
parent compound’s basic nitrogen into an amide would affect its bio-
logical activity. Nearly all of these derivatives had substantially lower
activity thanthat of the equivalent non-amidated compounds, regard-
less of the other substituents. The basic nitrogen at the N-5 position thus
seems be very important to Wnt activity.
activation activity. The nitrogen heterocyclic substitution, especially
smaller nitrogen heterocycles such as pyrazole or pyridineat the C-8 or
C-9 position should improve the Wnt activity of phenanthridine deri-
vatives. These findings provide constructive guidance for further
structural modifications of lycorine.
4. Experimental
4.1. Chemistry
4.1.1. General experimental procedures
Melting points were measured using an X-4 apparatus
(YingyuYuhua Instrument Factory, Gongyi, Henan Province, P. R.
China). ESI and HRMS data were recorded using aFinnigan MAT 90
instrument and a VG Auto Spec-3000 spectrometer, respectively. NMR
experiments were conducted on a Bruker AM-400, DRX-500, or Avance
III 600 spectrometer using residual CDCl3 and DMSO‑d6 or TMS as in-
ternal standards. 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). Pre-coated silica gel 60
GF254 (Merck, Darmstadt, Germany) was used for TLC analyses. Semi-
preparative 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 (r.t.). All regular solvents and reagents were
reagent grade and were purchased from Sigma-Aldrich Chemical Co.
(St. Louis, MO, United States), 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 X-Bridge RP-C18 column (4.6 × 250 mm, 5 μm, 5 mL/
min) with CH3OH-H2O at r.t. All reported yields are for dry compounds
that required no further purification for use in subsequent reactions.
The SAR analysis of the phenanthridine derivatives was continued
by investigating the effects of varying the substituents on the C-8 or C-9
oxygen atoms of the phenanthridine skeleton. Nitrogen heterocyclic,
aromatic, and linear alkyl derivatives were therefore prepared and in-
vestigated. Among these compounds, the ones bearing smaller nitrogen
heterocyclic substituents at C-8 or C-9 (2f and 2g) had a stronger effect
on Wnt than bigger heterocycles (2h), aromatics (5c) and linear alkyl
(1e, 2e and 3e) derivatives. These results indicated that a smaller ni-
trogen heterocyclic group at the C-8 or C-9 position was good for Wnt
activation, and linear alkyl groups are likewise helpful to Wnt activa-
tion, except that the improvements are limited. However, the bigger
nitrogen heterocycles and aromatic groups would diminish the activity
of a compound.
There is a phenomenon that was quite interesting through com-
parison of compounds 3f and 3g. Compound 3fbears a nitrogen het-
erocyclic substituent at the C-8 position, and displays better perfor-
mance than 3g, which bears the same substituent but at the C-9
position. Subsequent docking analysis of 3f and 3gindicated that3fcan
associate with the protein residues in the axin cavity in which HLY78
docks tighter than 3g does (Fig. 6). These result simply that substituents
at the C-8 position of phenanthridine have a greater influence on Wnt
activation.
4.1.2. Synthesis of phenanthridine skeletons
2-Bromo-N-ethyl-5-methoxybenzamide(1b).
Compound1a
(260 mg, 1 mmol) was dissolved in DCM (10 mL), to which DMF
(0.1 mL) and SOCl2 (0.5 mL, 4 mmol) were added. The reaction solution
was stirred for 2 h at 50 °C, and then concentrated to remove DCM. The
residue was then added to a 30% solution of ethylamine in water
(20 mL) at 5 °C and filtered. The cake was purified bycolumn chroma-
tography to give 1b as a pale yellow solid (205 mg, 75% yield). 1H NMR
(400 MHz, CDCl3) δ 7.43 (d, J = 8.8 Hz, 1H), 7.08 (d, J = 3.1 Hz, 1H),
6.81 (dd, J = 8.8, 3.1 Hz, 1H), 3.80 (s, 3H), 3.49 (q, J = 7.0 Hz, 2H),
1.26 (t, J = 7.3 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 167.23 (C),
158.94 (C), 138.53 (C), 134.13 (CH), 117.81 (CH), 114.63 (CH), 109.35
(C), 55.66 (CH3), 35.06 (CH2), 14.69 (CH3); HREIMS m/z 257.0050
[M]+ (calcd for C10H12BrNO2, 257.0051).
3. Conclusions
HLY78 could become a more efficient Wnt/β-catenin signalling
pathway agonist through appropriate modifications. According to the
process of the structure–activity relationship optimization of phenan-
thridine derivatives, it could be concluded that the C-4methyl sub-
stituent and the basic nitrogen at the N-5 position are crucial to Wnt
4-Methyl-N-ethyl-8-methoxyphenanthridin-6(5H)-one (1c).
A
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Scheme 1. Design and syntheses of phenanthridinederivatives.a.(aReagents and conditions: (a) SOCl2, DCM, 50–80 °C, 3 h; (b) RNH2 (30%), 5 °C, 1 h; (c) K2CO3,
norbornene, Pd(OAc)2, TFP, MeCN or DMF, 85–115 °C, 6–20 h; (d) BH3-THF, THF, 60 °C, 2 h; (e). BBr3, CH2Cl2, -78 °C, 4 h, 80%. (f). NaH, alkylating agent, CH2Cl2 or
THF, 60–100 °C, 6–20 h.)
flask was charged under nitrogen with Pd(OAc)2 (3.0 mg, 0.013 mmol),
tri(2-furyl) phosphine (6.2 mg, 0.027 mmol), K2CO3 (72.3 mg,
0.52 mmol), the amide 1b (0.26 mmol), a solution of norbornene
(26.9 mg, 0.286 mmol) in anhydrous solvent (5.8 mL), and 1-iodo-2-
methylbenzene (0.26 mmol). The reaction mixture was heated with
stirring at 85 °C for 6 h and then cooled to rt. After the addition of sa-
turated NH4Cl (30 mL) and extraction with EtOAc (3 × 15 mL), the
combined organic extracts were washed with brine (30 mL) and dried
over Na2SO4. Removal of the solvent under reduced pressure gave the
crude product, which was purified by flash chromatography on silica
gel to furnish 1c as a white wax (55 mg, 80% yield). 1H NMR (400 MHz,
CDCl3) δ 8.15 (d, J = 9.0 Hz, 1H), 8.06 (d, J = 8.2 Hz, 1H), 7.92 (d,
J = 2.8 Hz, 1H), 7.32 (dd, J = 8.9, 2.8 Hz, 1H), 7.28 (d, J = 7.2 Hz,
1H), 7.20 (t, J = 7.7 Hz, 1H), 4.51 (q, J = 6.9 Hz, 2H), 3.96 (s, 3H),
2.70 (s, 3H), 1.37 (t, J = 6.9 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ
163.90 (C), 159.37 (C), 137.15 (C), 133.12 (CH), 127.56 (C), 126.84
(C), 125.82 (C), 123.69 (CH), 122.74 (CH), 122.37 (CH), 121.81 (C),
120.90 (CH), 108.76 (CH), 55.64 (CH3), 42.60 (CH2), 23.87 (CH3),
14.83 (CH3); HREIMS m/z 267.1263 [M]+ (calcd for C17H17NO2,
267.1259).
4-Methyl-N-ethyl-8-methoxy-5,6-dihydrophenanthridine (1d).
A solution of 1c (30 mg, 0.1 mmol) in THF (5 mL) was added to BH3-
THF (0.12 mol) at 60 °C. The reaction was stirred for 2 h and then
quenched using H2O (5 mL). The mixture was then extracted with
EtOAc (20 mL) twice. The organic phase was washed with brine and
concentrated, and the residue was purified by column chromatography
to give 1 as a colourless solid (18 mg, 70% yield). 1H NMR (400 MHz,
CDCl3) δ 7.67 (d, J = 8.5 Hz, 1H), 7.58 (d, J = 7.5 Hz, 1H), 7.13 (d,
J = 6.6 Hz, 1H), 7.08 (t, J = 7.5 Hz, 1H), 6.90 (dd, J = 8.5, 2.6 Hz, 1H),
6.77 (d, J = 2.6 Hz, 1H), 4.10 (s, 2H), 3.86 (s, 3H), 2.70 (q, J = 7.1 Hz,
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Table 1
saturated NaHCO3. The solution was extracted twice with CH2Cl2
(15 mL), and the organic layer was washed with brine, concentrated, and
then purified by column chromatography using chloroform–methanol
(20:1) as the eluent to yield compound1 (19.5 mg, 82% yield). 1H NMR
(400 MHz, CDCl3) δ 7.61 (d, J = 8.3 Hz, 1H), 7.56 (d, J = 7.5 Hz, 1H),
7.12 (d, J = 7.3 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 6.81 (dd, J = 8.3,
2.4 Hz, 1H), 6.71 (d, J = 2.2 Hz, 1H), 4.05 (s, 2H), 2.69 (q, J = 7.1 Hz,
2H), 2.36 (s, 3H), 1.08 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ
155.29 (C), 146.18 (C), 135.33 (C), 133.38 (C), 129.71 (CH), 129.32 (C),
125.99 (C), 124.51 (CH), 123.87 (CH), 120.78 (CH), 114.42 (CH),
113.39 (CH), 50.02 (CH2), 46.08 (CH2), 17.79 (CH3), 13.59 (CH3);
HRESIMS m/z240.1386 [M+H]+ (calcd for C16H18NO, 240.1383).
2-Bromo-N-methyl-5-methoxybenzamide(2b). By referencing the
synthesis method of compound 1b, compound 2b was synthesized
through the amidation of compound 1a in the presence of a 30% so-
lution of methylamine (20 mL) for a yield of 72% (200 mg). m.p. 152 °C.
1H NMR (400 MHz, CDCl3) δ 7.43 (d, J = 8.8 Hz, 1H), 7.07 (d,
J = 3.1 Hz, 1H), 6.81 (dd, J = 3.1, 8.8 Hz, 1H), 3.79 (s, 3H), 3.00 (s,
3H); 13C NMR (100 MHz, CDCl3) δ 168.0 (C), 158.8 (C), 138.3 (C),
134.1 (CH), 117.8 (CH), 114.6 (CH), 109.3 (C), 55.6 (CH3), 26.7 (CH3);
HREIMS m/z 242.9889 [M]+ (calcd for C9H10BrNO2, 242.9895).
4-Methyl-N-methyl-8-methoxyphenanthridin-6(5H)-one (2c).
By referencing the synthesis method of compound 1c, compound 2b
(0.1 mmol) was coupled to 1-iodo-2-methylbenzene (0.12 mol) to afford
compound 2c (68% yield). 1H NMR (400 MHz, CDCl3) δ 8.13 (d,
J = 9.0 Hz, 1H), 8.01 (d, J = 7.8 Hz, 1H), 7.91 (d, J = 2.8 Hz, 1H), 7.31
(dd, J = 8.9, 2.8 Hz, 1H), 7.28–7.23 (m, 1H), 7.18 (d, J = 7.5 Hz, 1H),
3.95 (s, 3H), 3.81 (s, 3H), 2.66 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
163.99 (C), 159.42 (C), 138.5 (C), 132.77 (CH), 127.54 (C), 126.67 (C),
126.12 (C), 123.74 (CH), 122.31 (CH), 122.9 (CH), 121.38 (C), 120.50
(CH), 108.80 (CH), 55.71 (CH3), 38.51 (CH3), 23.63 (CH3); HREIMS m/
z 253.1102 [M]+ (calcd for C16H15NO2, 253.1103).
Wnt activating efficacy of phenanthridine derivatives.
Compound
Concentration that
Compound
Concentration that
doubled Wnt activation
doubled Wnt activation
HLY78
1
5 μM
4e
5
10 μM
> 20 μM
NAa
10 μM
> 20 μM
5 μM
1c
1d
1e
2
5a
5b
5c
5d
6
10 μM
NA
10 μM
5 μM
> 20 μM
NA
2c
2d
2e
2f
> 20 μM
5 μM
6a
6b
6c
6d
6e
6f
6g
6h
7
> 20 μM
> 20 μM
10 μM
> 20 μM
> 20 μM
5 μM
> 20 μM
5 μM
2g
2h
3
10 μM
NA
10 μM
> 20 μM
10 μM
10 μM
0.5 μM
1 μM
3c
3d
3e
3f
NA
NA
> 20 μM
NA
8
3g
4
8a
> 20 μM
10 μM
2H), 2.37 (s, 3H), 1.10 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3)
δ 159.30 (C), 146.27 (C), 135.05 (C), 133.38 (C), 129.69 (CH), 129.38
(C), 125.87 (C), 124.31 (CH), 123.86 (CH), 120.83 (CH), 112.92 (CH),
111.92 (CH), 55.33 (CH3), 50.25 (CH2), 46.11 (CH2), 17.81 (CH3),
13.64 (CH3); HRESIMS m/z 254.1539 [M+H]+ (calcd for C17H20NO,
254.1539).
N-ethyl-4-methyl-5,6-dihydrophenanthridin-8-ol (1). Compound
1d (0.1 mmol) was dissolved in 10 mL CH2Cl2. The reaction solution was
then cooled to -78 °C and BBr3 (100 μL, 0.2 mmol) was added. The
mixture was then stirred for 4 h after which it was diluted in 10 mL
Fig. 4. The Wnt active activities of phenanthridine derivatives. Compounds 1, 1e, 2, 2d, 3, 3d, 3e, 4e and 5bexhibit weaker activity than that of HLY78; Compounds
1d, 2f, 4 and 6fshow equal activity to HLY78; Compounds 3f and 3g exhibit stronger activity than that of HLY78.
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Fig. 5. Compounds 3g and 3fclearly activated the expression of endogenous Wnt target genes (Axin2 and DKK1), and 3f is more efficient than 3g.
4-Methyl-N-methyl-8-methoxy-5,6-dihydrophenanthridine
(2d). By referencing the synthesis method of compound 1, compound
2d was synthesized through the reduction of compound 2c (0.1 mmol)
in the presence of BH3-THF (0.25 mmol) for a yield of 72%. 1H NMR
(500 MHz, CDCl3) δ 7.70 (d, J = 8.6 Hz, 1H), 7.61 (dd, J = 7.5, 1.5 Hz,
1H), 7.18–7.08 (m, 2H), 6.93 (dd, J = 8.5, 2.7 Hz, 1H), 6.81 (d,
J = 2.6 Hz, 1H), 4.09 (s, 2H), 3.88 (s, 3H), 2.52 (s, 3H), 2.43 (s, 3H);
13C NMR (125 MHz, CDCl3) δ 159.33 (C), 145.89 (C), 134.22 (C),
133.30 (C), 129.53 (CH), 125.47 (C), 125.14 (C), 124.26 (CH), 124.19
(CH), 120.93 (CH), 113.02 (CH), 112.01 (CH), 55.39 (CH2), 55.21
(CH3), 40.37 (CH3), 17.50 (CH3); HRESIMS m/z 240.1385 [M+H]+
(calcd for C16H18NO, 240.1383).
2-Bromo-N-ethyl-4,5-dimethoxybenzamide(3b). By referencing
the synthesis method of compound 1b, compound 3b was synthesized
through the amidation of compound 3a (1 mmol) in the presence of
ethylamine (50 mL) for a yield of 85%. 1H NMR (400 MHz, CDCl3) δ
7.20 (s, 1H), 6.98 (s, 1H), 3.89 (s, 3H), 3.89 (s, 3H), 3.50 (q, J = 7.2 Hz,
2H), 1.27 (t, J = 7.3 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 166.72 (C),
150.69 (C), 148.42 (C), 129.27 (C), 115.67 (CH), 112.90 (CH), 109.70
(C), 56.29 (CH3), 56.15 (CH3), 35.13 (CH2), 14.70 (CH3); HREIMS m/z
Fig. 6. Molecular dockinganalysis of compound 3f. Compound 3fcan bind tightertoaxin than 3g.
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287.0157 [M]+ (calcd for C11H14BrNO3, 287.0157).
(dd, J = 8.8, 2.8 Hz, 1H), 6.80 (t, J = 7.7 Hz, 1H), 4.08 (q, J = 7.0 Hz,
2H), 2.28 (s, 3H), 0.90 (t, J = 7.0 Hz, 3H); 13C NMR (125 MHz, MeOD)
δ 165.68 (C), 158.93 (C), 137.64 (C), 134.09 (CH), 127.98 (C), 127.69
(C), 127.41 (C), 125.34 (CH), 124.43 (CH), 123.50 (C), 123.30 (CH),
122.01 (CH), 112.89 (CH), 43.74 (CH2), 23.95 (CH3), 15.01 (CH3);
HRESIMS m/z254.1176 [M+H]+ (calcd for C16H16NO2, 254.1176).
4-Methyl-N-ethyl-8,9-dimethoxyphenanthridin-6(5H)-one (3c).
By referencing the synthesis method of compound 1c, compound 3b
(0.1 mmol) was coupled to 1-iodo-2-methylbenzene (0.12 mmol)to af-
ford compound 3c (75% yield). 1H NMR (500 MHz, CDCl3) δ 7.97 (d,
J = 7.8 Hz, 1H), 7.87 (s, 1H), 7.52 (s, 1H), 7.26 (dd, J = 5.1, 2.1 Hz,
1H), 7.16 (t, J = 7.6 Hz, 1H), 4.47 (q, J = 7.0 Hz, 2H), 4.05 (s, 3H),
4.01 (s, 3H), 2.68 (s, 3H), 1.34 (t, J = 7.0 Hz, 3H); 13C NMR (125 MHz,
CDCl3) δ 163.64 (C), 153.27 (C), 149.69 (C), 137.81 (C), 133.36 (CH),
128.75 (C), 125.90 (C), 122.49 (CH), 121.53 (C), 120.93 (CH), 119.64
(C), 108.75 (CH), 102.83 (CH), 56.17 (CH3), 56.10 (CH3), 42.45 (CH2),
23.95 (CH3), 14.97 (CH3); HRESIMS m/z298.1441 [M+H]+ (calcd for
N-methyl-8-hydroxy-4-methylphenanthridin-6(5H)-one
(6).
80% yield; 1H NMR (600 MHz, MeOD) δ 8.23 (d, J = 8.9 Hz, 1H), 8.10
(d, J = 7.8 Hz, 1H), 7.72 (d, J = 2.7 Hz, 1H), 7.33 (m, 1H), 7.31 (dd,
J = 8.9, 2.7 Hz, 1H), 7.27–7.23 (m, 1H), 3.78 (s, 3H), 2.65 (s, 3H); 13C
NMR (150 MHz, MeOD) δ 165.68 (C), 158.92 (C), 138.97 (C), 133.71
(CH), 127.93 (C), 127.72 (C), 127.39 (C), 125.34 (CH), 124.52 (CH),
123.20 (CH), 122.96 (C), 121.56 (CH), 112.82 (CH), 39.04 (CH3), 23.66
(CH3); HRESIMS m/z 240.1017 [M+H]+ (calcd for C15H14NO2,
240.1019).
C
18H20NO3, 298.1438).
4-Methyl-N-ethyl-8,9-dimethoxy-5,6-dihydrophenanthridine
(3d). By referencing the synthesis method of compound 1, compound
3d was synthesized through the reduction of compound 3c (0.1 mmol)
in the presence of BH3-THF (0.3 mmol) for a yield of 65%. 1H NMR
(500 MHz, CDCl3) δ 7.54 (d, J = 6.9 Hz, 1H), 7.24 (s, 1H), 7.12 (d,
J = 7.4 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 6.73 (s, 1H), 4.06 (s, 2H),
3.96 (s, 3H), 3.93 (s, 3H), 2.70 (q, J = 7.1 Hz, 2H), 2.36 (s, 3H), 1.09 (t,
J = 7.1 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 148.43 (C), 146.36 (C),
140.87 (C), 133.45 (C), 129.81 (CH), 129.38 (C), 125.95 (C), 125.47
(C), 123.75 (CH), 120.70 (CH), 109.64 (CH), 106.54 (CH), 56.12 (CH3),
56.01 (CH3), 49.65 (CH2), 45.96 (CH2), 17.83 (CH3), 13.69 (CH3);
HRESIMS m/z284.1648 [M+H]+ (calcd for C18H22NO2, 284.1645).
2-Bromo-N-methyl-4,5-dimethoxybenzamide(4b). By referen-
cing the synthesis method of compound 1b, compound 4b was syn-
thesized through the amidation of compound 4a (1 mmol) in the pre-
sence of ethylamine (50 mL) for a yield of 85%. 1H NMR (500 MHz,
MeOD) δ 7.13 (s, 1H), 7.00 (s, 1H), 3.84 (s, 3H), 3.82 (s, 3H), 2.88 (s,
3H); 13C NMR (125 MHz, MeOD) δ 171.29 (C), 152.14 (C), 149.82 (C),
131.56 (C), 117.11 (CH), 113.11 (CH), 111.16 (C), 56.75 (CH3), 56.63
(CH3), 26.73 (CH3); HREIMS m/z 273.0000 [M]+ (calcd for
N-ethyl-8,9-dihydroxy-4-methylphenanthridin-6(5H)-one (7).
80% yield; 1H NMR (500 MHz, MeOD) δ 8.00 (dd, J = 6.5, 4.8 Hz, 1H),
7.69 (s, 1H), 7.62 (d, J = 2.9 Hz, 1H), 7.28 (d, J = 6.5 Hz, 1H), 7.18
(td, J = 7.7, 3.7 Hz, 1H), 4.46 (q, J = 6.9 Hz, 2H), 2.67 (s, 3H), 1.28 (t,
J = 7.0 Hz, 3H); 13C NMR (125 MHz, MeOD) δ 165.67 (C), 152.77 (C),
148.00 (C), 138.16 (C), 134.12 (CH), 129.97 (C), 127.37 (C), 124.15
(CH), 123.29 (C), 122.08 (CH), 119.57 (C), 113.47 (CH), 108.12 (CH),
43.47 (CH2), 23.99 (CH3), 15.12 (CH3); HREIMS m/z 269.1050 [M]+
(calcd for C16H15NO3, 269.1052).
N-methyl-8,9-hydroxy-4-methylphenanthridin-6(5H)-one (8).
80% yield; 1H NMR (500 MHz, MeOD) δ 7.83 (dd, J = 37.5, 7.8 Hz,
1H), 7.59 (s, 1H), 7.53 (s, 1H), 7.18 (d, J = 7.3 Hz, 1H), 7.09 (t,
J = 7.7 Hz, 1H), 3.66 (s, 3H), 2.55 (s, 3H); 13C NMR (125 MHz, MeOD)
δ165.70 (C), 152.72 (C), 147.99 (C), 139.56 (C), 133.75 (CH), 129.93
(C), 127.71 (C), 124.26 (CH), 122.77 (C), 121.63 (CH), 119.26 (C),
113.38 (CH), 108.18 (CH), 38.87 (CH3), 23.67 (CH3); HRESIMS m/z
256.0972 [M+H]+ (calcd for C15H14NO3, 256.0968).
C
10H12BrNO3, 273.0001).
4.1.3. Alkylation of compounds 1–8
Synthesis of Compounds 2–8. By referencing the synthesis method
The phenanthridine skeletons1-8 (0.1 mmol) were dissolved in dry
THF (10 mL), and NaH (50 mg, 2 mmol), and 3-(bromomethyl)pyridine,
3-(chloromethyl)-1,5-dimethyl-1H-pyrazole or 5-bromomethyl-1,3-di-
methyl-1H-pyrazole (1 mmol) were added. The mixture was stirred at
rt. for 12 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-EtOAc as the
eluent to afford about 35 mg of each phenanthridine compound.
5-ethyl-4-methyl-8-(prop-2-yn-1-yloxy)-5,6-dihydrophenan-
thridine (1e). 1H NMR (600 MHz, CDCl3) δ 7.60 (d, J = 8.5 Hz, 1H),
7.50 (d, J = 7.6 Hz, 1H), 7.06 (d, J = 7.0 Hz, 1H), 7.00 (t, J = 7.5 Hz,
1H), 6.89 (dd, J = 8.5, 2.6 Hz, 1H), 6.76 (d, J = 2.6 Hz, 1H), 4.67 (d,
J = 2.4 Hz, 2H), 4.02 (s, 2H), 2.62 (q, J = 7.1 Hz, 2H), 2.48 (t,
J = 2.3 Hz, 1H), 2.29 (s, 3H), 1.01 (t, J = 7.1 Hz, 3H); 13C NMR
(150 MHz, CDCl3) δ 157.52 (C), 146.42 (C), 136.44 (C), 133.59 (C),
129.90 (CH), 129.88 (C), 124.49 (C), 124.29 (CH), 123.85 (CH), 120.90
(CH), 113.90 (CH), 112.94 (CH), 76.22 (C), 75.85 (CH), 58.88 (CH2),
50.17 (CH2), 46.09 (CH2), 17.80 (CH3), 13.62 (CH3); HREIMS m/z
277.1463 [M]+ (calcd for C19H19NO, 277.1467).
of compound 1, compounds2-8weresynthesized by the deprotection of
compounds 1c, 2c, 3c, 2d, and 3d (0.1 mmol), respectively, in the
presence of BBr3 (0.3 mmol) for a yield of 65%.
N-methyl-4-methyl-5,6-dihydrophenanthridin-8-ol (2). 1H NMR
(400 MHz, CDCl3) δ 7.59 (d, J = 8.4 Hz, 1H), 7.57–7.52 (m, 1H),
7.14–7.01 (m, 2H), 6.83 (dd, J = 8.4, 2.5 Hz, 1H), 6.73 (d, J = 2.4 Hz,
1H), 4.00 (s, 2H), 2.45 (s, 3H), 2.37 (s, 3H); 13C NMR (100 MHz, CDCl3)
δ 155.76 (C), 145.82 (C), 134.52 (C), 133.37 (C), 129.60 (CH), 129.12
(C), 125.10 (C), 124.55 (CH), 124.31 (CH), 120.96 (CH), 114.66 (CH),
113.75 (CH), 55.23 (CH2), 40.42 (CH3), 17.59 (CH3); HRESIMS m/z
226.1228 [M+H]+ (calcd for C15H16NO, 226.1226).
N-ethyl-4-methyl-5,6-dihydrophenanthridin-8,9-diol (3). 80%
yield; 1H NMR (500 MHz, CDCl3) δ 7.45 (d, J = 7.7 Hz, 1H), 7.24 (s,
1H), 7.11 (d, J = 7.0 Hz, 1H), 7.05 (t, J = 7.6 Hz, 1H), 6.73 (s, 1H),
3.99 (s, 2H), 2.68 (q, J = 7.1 Hz, 2H), 2.34 (s, 3H), 1.07 (t, J = 7.1 Hz,
3H); 13C NMR (125 MHz, CDCl3) δ 143.44 (C), 142.85 (C), 141.90 (C),
133.42 (C), 129.84 (CH), 129.17 (C), 126.59 (C), 126.14 (C), 123.87
(CH), 120.86 (CH), 113.57 (CH), 110.43 (CH), 49.35 (CH2), 45.93
(CH2), 17.78 (CH3), 13.58 (CH3); HRESIMS m/z256.1322 [M+H]+
(calcd for C16H18NO2, 256.1332).
N-methyl-4-methyl-5,6-dihydrophenanthridin-8,9-diol
(4).
4,5-dimethyl-8-(prop-2-yn-1-yloxy)-5,6-dihydrophenan-
80% yield; 1H NMR (500 MHz, CDCl3) δ 7.29 (d, J = 7.9 Hz, 1H), 7.19
(d, J = 3.1 Hz, 2H), 6.92 (d, J = 7.8 Hz, 1H), 6.65 (s, 1H), 3.88 (s, 2H),
2.42 (s, 3H), 2.33 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 143.27 (C),
142.60 (C), 141.80 (C), 137.16 (C), 133.32 (C), 128.32 (CH), 127.90
(C), 126.09 (C), 120.94 (CH), 120.30 (CH), 113.81 (CH), 110.16 (CH),
54.56 (CH2), 42.98 (CH3), 22.76 (CH3); HREIMS m/z 241.1102 [M]+
(calcd for C15H15NO2, 241.1103).
thridine (2e). 1H NMR (500 MHz, CDCl3) δ 7.70 (d, J = 8.6 Hz, 1H),
7.60 (dd, J = 7.5, 1.3 Hz, 1H), 7.14 (dd, J = 7.3, 0.8 Hz, 1H), 7.10 (t,
J = 7.5 Hz, 1H), 6.99 (dd, J = 8.5, 2.7 Hz, 1H), 6.86 (d, J = 2.6 Hz,
1H), 4.74 (d, J = 2.4 Hz, 2H), 4.08 (s, 2H), 2.57 (t, J = 2.4 Hz, 1H),
2.50 (s, 3H), 2.41 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 157.38 (C),
146.09 (C), 134.38 (C), 133.44 (C), 129.85 (CH), 128.94 (C), 126.14
(C), 124.36 (CH), 124.29 (CH), 121.13 (CH), 113.91 (CH), 113.20 (CH),
78.62 (C), 75.67 (CH), 55.90 (CH2), 55.42 (CH2), 40.48 (CH3), 17.60
(CH3); HREIMS m/z 263.1309 [M]+ (calcd for C18H17NO, 263.1310).
8-((1,5-dimethyl-1H-pyrazol-3-yl)methoxy)-4,5-dimethyl-5,6-
N-ethyl-8-hydroxy-4-methylphenanthridin-6(5H)-one (5). 80%
yield; 1H NMR (500 MHz, MeOD) δ 7.82 (d, J = 8.9 Hz, 1H), 7.73 (d,
J = 7.5 Hz, 1H), 7.29 (d, J = 2.7 Hz, 1H), 6.90 (d, J = 7.4 Hz, 1H), 6.85
291

D.-z. Chen et al.
BioorganicChemistry84(2019)285–294
dihydrophenanthridine (2f). 1H NMR (400 MHz, CDCl3) δ 7.66 (d,
J = 8.6 Hz, 1H), 7.57 (d, J = 8.7 Hz, 1H), 7.15–7.04 (m, 2H), 6.99 (dd,
J = 8.5, 2.5 Hz, 1H), 6.87 (d, J = 2.4 Hz, 1H), 6.14 (s, 1H), 5.04 (s, 2H),
4.05 (s, 2H), 3.79 (s, 3H), 2.47 (s, 3H), 2.39 (s, 3H), 2.28 (s, 3H); 13C
NMR (100 MHz, CDCl3) δ 158.65 (C), 146.92 (C), 146.01 (C), 139.69
(C), 134.19 (C), 133.36 (C), 129.57 (CH), 129.15 (C), 125.33 (C),
124.27 (CH), 124.22 (CH), 121.01 (CH), 113.92 (CH), 113.03 (CH),
105.01 (CH), 64.22 (CH2), 55.45 (CH2), 40.45 (CH3), 36.08 (CH3),
17.58 (CH3), 11.27 (CH3); HRESIMS m/z 334.1920 [M+H]+ (calcd for
z364.2022 [M+H]+ (calcd for C22H26N3O2, 364.2020).
5-ethyl-4-methyl-8-(prop-2-yn-1-yloxy)phenanthridin-6(5H)-
one (5a). 1H NMR (600 MHz, CDCl3) δ 8.12 (d, J = 9.0 Hz, 1H), 8.01
(d, J = 7.9 Hz, 1H), 7.96 (d, J = 2.8 Hz, 1H), 7.33 (dd, J = 8.9, 2.8 Hz,
1H), 7.25 (d, J = 6.8 Hz, 1H), 7.16 (t, J = 7.6 Hz, 1H), 4.81 (d,
J = 2.3 Hz, 2H), 4.46 (q, J = 7.0 Hz, 2H), 2.66 (s, 3H), 2.55 (t,
J = 2.3 Hz, 1H), 1.34 (t, J = 7.0 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ
163.89 (C), 157.39 (C), 137.45 (C), 133.54 (CH), 128.44 (C), 126.92
(C), 126.01 (C), 124.04 (CH), 122.96 (CH), 122.80 (CH), 121.79 (C),
121.17 (CH), 110.33 (CH), 78.17 (C), 76.20 (CH), 56.24 (CH2), 42.81
(CH2), 24.05 (CH3), 15.05 (CH3); HRESIMS m/z292.1336 [M+H]+
(calcd for C19H18NO2, 292.1332).
C
21H24N3O, 334.1914).
4,5-dimethyl-9-(pyridin-3-ylmethoxy)-5,6-dihydrophenan-
thridine (2g). 1H NMR (400 MHz, CDCl3) δ 8.62 (d, J = 4.7 Hz, 1H),
7.74 (td, J = 7.7, 1.8 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.57 (dd,
J = 10.1, 4.8 Hz, 2H), 7.23 (dd, J = 11.0, 4.9 Hz, 1H), 7.16–7.04 (m,
2H), 6.98 (dd, J = 8.6, 2.7 Hz, 1H), 6.87 (d, J = 2.6 Hz, 1H), 5.26 (s,
2H), 4.05 (s, 2H), 2.47 (s, 3H), 2.39 (s, 3H); 13C NMR (100 MHz, CDCl3)
δ 158.22 (C), 157.26 (C), 149.25 (CH), 146.02 (C), 136.93 (CH), 134.40
(C), 133.41 (C), 129.73 (CH), 128.99 (C), 125.73 (C), 124.42 (CH),
124.26 (CH), 122.71 (CH), 121.34 (CH), 121.05 (CH), 113.87 (CH),
113.14 (CH), 70.68 (CH2), 55.41 (CH2), 40.46 (CH3), 17.59 (CH3);
HRESIMS m/z 317.1650 [M+H]+ (calcd for C21H21N2O, 317.1648).
9-((5-chlorobenzo[b]thiophen-2-yl)methoxy)-4,5-dimethyl-
8-((1,5-dimethyl-1H-pyrazol-3-yl)methoxy)-5-ethyl-4-methyl-
phenanthridin-6(5H)-one (5b). 1H NMR (400 MHz, CDCl3) δ 8.10 (d,
J = 9.0 Hz, 1H), 8.02 (t, J = 5.0 Hz, 2H), 7.37 (dd, J = 8.9, 2.8 Hz, 1H),
7.24 (d, J = 7.3 Hz, 1H), 7.15 (t, J = 7.6 Hz, 1H), 6.15 (s, 1H), 5.13 (s,
2H), 4.48 (q, J = 6.9 Hz, 2H), 3.77 (s, 3H), 2.67 (s, 3H), 2.25 (s, 3H),
1.35 (t, J = 7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 163.85 (C),
158.49 (C), 146.26 (C), 139.62 (C), 137.13 (C), 133.08 (CH), 127.63
(C), 126.73 (C), 125.75 (C), 123.63 (CH), 122.75 (CH), 122.70 (CH),
121.79 (C), 120.88 (CH), 109.94 (CH), 105.24 (CH), 64.29 (CH2), 42.54
(CH2), 36.06 (CH3), 23.84 (CH3), 14.82 (CH3), 11.19 (CH3); HRESIMS
m/z362.1865 [M+H]+ (calcd for C22H24N3O2, 362.1863).
5,6-dihydrophenanthridine (2h). 1H NMR (400 MHz, CDCl3) δ 7.88
(d, J = 1.8 Hz, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.71 (d, J = 8.5 Hz, 1H),
7.64–7.55 (m, 2H), 7.37 (dd, J = 8.6, 1.6 Hz, 1H), 7.20–7.07 (m, 2H),
7.02 (dd, J = 8.5, 2.3 Hz, 1H), 6.91 (d, J = 2.3 Hz, 1H), 5.29 (s, 2H),
4.09 (s, 2H), 2.51 (s, 3H), 2.41 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
158.33 (C), 146.07 (C), 139.12 (C), 138.79 (C), 134.49 (C), 133.46 (C),
131.37 (C), 130.85 (C), 129.82 (CH), 128.98 (C), 127.24 (CH), 125.92
(C), 125.22 (CH), 124.46 (CH), 124.31 (CH), 123.90 (CH), 121.89 (CH),
121.11 (CH), 113.92 (CH), 113.08 (CH), 64.64 (CH2), 55.46 (CH2),
40.53 (CH3), 17.61 (CH3); HRESIMS m/z 406.1029 [M+H]+ (calcd for
4,5-dimethyl-5,6-dihydro-[1,3]dioxolo[4,5–j]phenanthridine
(4e). 1H NMR (500 MHz, CDCl3) δ 7.49 (dd, J = 7.5, 1.2 Hz, 1H), 7.23
(s, 1H), 7.12 (d, J = 6.5 Hz, 1H), 7.09 (t, J = 7.5 Hz, 1H), 6.72 (s, 1H),
5.98 (s, 2H), 4.00 (s, 2H), 2.48 (s, 3H), 2.39 (s, 3H); 13C NMR (CDCl3,
125 MHz) δ 147.36 (C), 147.15 (C), 145.95 (C), 133.34 (C), 129.81
(CH), 129.20 (C), 126.55 (C), 126.24 (C), 124.21 (CH), 121.12 (CH),
107.19 (CH), 103.76 (CH), 100.92 (CH2), 55.22 (CH2), 40.08 (CH3),
17.54 (CH3); HRESIMS m/z 254.1175 [M+H]+ (calcd for C16H16NO2,
254.1176).
C
24H21ClNOS, 406.1027).
Methyl 4′-(((5-ethyl-4-methyl-6-oxo-5,6-dihydrophenanthridin-
8-yl)oxy)methyl)-[1,1′-biphenyl]-2-carboxylate (5c). 1H NMR
(500 MHz, CDCl3) δ 8.17 (d, J = 9.0 Hz, 1H), 8.07 (d, J = 7.7 Hz, 1H),
8.04 (d, J = 2.8 Hz, 1H), 7.84 (dd, J = 7.8, 1.0 Hz, 1H), 7.52 (d,
J = 8.2 Hz, 3H), 7.44–7.38 (m, 3H), 7.36 (d, J = 8.1 Hz, 2H), 7.28 (d,
J = 3.4 Hz, 1H), 7.20 (t, J = 7.6 Hz, 1H), 5.27 (s, 2H), 4.51 (q,
J = 7.0 Hz, 2H), 3.65 (s, 3H), 2.70 (s, 3H), 1.37 (t, J = 7.0 Hz, 3H); 13C
NMR (125 MHz, CDCl3) δ 169.04 (C), 163.90 (C), 158.57 (C), 142.15 (C),
141.23 (C), 137.24 (C), 135.46 (C), 133.24 (CH), 131.38 (CH), 130.80
(CH), 130.75 (C), 129.90 (CH), 128.64 (CH), 128.53 (CH), 128.47 (CH),
127.86 (C), 127.44 (CH), 127.30 (CH), 126.98 (C), 126.88 (C), 123.84
(CH), 122.90 (CH), 122.81 (CH), 121.83 (C), 120.98 (CH), 109.99 (CH),
70.10 (CH2), 52.04 (CH3), 42.65 (CH2), 23.91 (CH3), 14.87 (CH3);
HRESIMS m/z478.2023 [M+H]+ (calcd for C31H28NO4, 478.2013).
8-((5-(1H-pyrazol-1-yl)pyridin-2-yl)methoxy)-5-ethyl-4-methyl-
phenanthridin-6(5H)-one (5d). 1H NMR (400 MHz, CDCl3) δ 8.55 (d,
J = 25.7 Hz, 2H), 8.17 (d, J = 8.7 Hz, 1H), 8.06 (d, J = 7.9 Hz, 1H),
8.04–8.00 (m, 2H), 7.95 (d, J = 8.5 Hz, 1H), 7.75 (s, 1H), 7.39 (d,
J = 8.9 Hz, 1H), 7.28 (dd, J = 10.2, 4.4 Hz, 1H), 7.20 (t, J = 7.3 Hz,
1H), 6.48 (d, J = 1.7 Hz, 1H), 5.24 (s, 2H), 4.51 (q, J = 6.8 Hz, 2H),
2.70 (s, 3H), 1.37 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ
163.81 (C), 158.08 (C), 151.48 (C), 147.46 (CH), 142.20 (CH), 138.50
(CH), 137.27 (C), 133.39 (CH), 129.78 (C), 128.20 (C), 127.17 (CH),
126.87 (C), 125.94 (C), 124.00 (CH), 122.87 (CH), 122.82 (CH), 121.71
(C), 121.03 (CH), 112.32 (CH), 109.90 (CH), 107.93 (CH), 67.44 (CH2),
42.68 (CH2), 23.91 (CH3), 14.85 (CH3); HRESIMS m/z411.1818 [M
+H]+ (calcd for C25H23N4O2, 411.1816).
5-ethyl-4-methyl-8,9-bis(prop-2-yn-1-yloxy)-5,6-dihy-
drophenanthridine (3e). 1H NMR (500 MHz, CDCl3) δ 7.53 (d,
J = 7.0 Hz, 1H), 7.44 (s, 1H), 7.14 (dd, J = 7.4, 0.7 Hz, 1H), 7.08 (t,
J = 7.5 Hz, 1H), 6.90 (s, 1H), 4.82 (d, J = 2.4 Hz, 2H), 4.80 (d,
J = 2.4 Hz, 2H), 4.06 (s, 2H), 2.70 (q, J = 7.1 Hz, 2H), 2.54 (q,
J = 2.3 Hz, 2H), 2.36 (s, 3H), 1.08 (t, J = 7.1 Hz, 3H); 13C NMR
(125 MHz, CDCl3) δ 147.32 (C), 146.92 (C), 146.47 (C), 133.46 (C),
130.13 (CH), 129.07 (C), 127.46 (C), 126.96 (C), 123.82 (CH), 120.98
(CH), 113.09 (CH), 110.64 (CH), 78.68 (C), 78.54 (C), 75.98 (CH),
75.95 (CH), 57.35 (CH2), 57.06 (CH2), 49.58 (CH2), 46.00 (CH2), 17.80
(CH3), 13.65 (CH3); HRESIMS m/z332.1648 [M+H]+ (calcd for
C
22H22NO2, 332.1645).
8-((1,3-dimethyl-1H-pyrazol-5-yl)methoxy)-5-ethyl-4-methyl-
5,6-dihydrophenanthridin-9-ol (3f). 1H NMR (400 MHz, CDCl3) δ
7.52 (d, J = 7.6 Hz, 1H), 7.35 (s, 1H), 7.14 (d, J = 7.2 Hz, 1H), 7.07 (t,
J = 7.5 Hz, 1H), 6.81 (s, 1H), 6.13 (s, 1H), 5.06 (s, 2H), 4.05 (s, 2H),
3.84 (s, 3H), 2.70 (q, J = 7.1 Hz, 2H), 2.36 (s, 3H), 2.27 (s, 3H), 1.10 (t,
J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 147.51 (C), 146.43 (C),
145.27 (C), 144.90 (C), 137.33 (C), 133.40 (C), 130.10 (CH), 129.02
(C), 127.30 (C), 125.38 (C), 123.91 (CH), 121.17 (CH), 110.41 (CH),
110.22 (CH), 107.19 (CH), 61.42 (CH2), 49.62 (CH2), 45.93 (CH2),
36.33 (CH3), 17.78 (CH3), 13.69 (CH3), 13.40 (CH3); HRESIMS m/z
364.2022 [M+H]+ (calcd for C22H26N3O2, 364.2020).
9-((1,3-dimethyl-1H-pyrazol-5-yl)methoxy)-5-ethyl-4-methyl-
5,6-dihydrophenanthridin-8-ol (3g). 1H NMR (500 MHz, CDCl3) δ
7.49 (d, J = 7.0 Hz, 1H), 7.31 (s, 1H), 7.13 (d, J = 6.8 Hz, 1H), 7.07 (t,
J = 7.5 Hz, 1H), 6.81 (s, 1H), 6.14 (s, 1H), 5.10 (s, 2H), 4.02 (s, 2H),
3.84 (s, 3H), 2.69 (q, J = 7.1 Hz, 2H), 2.36 (s, 3H), 2.27 (s, 3H), 1.08 (t,
J = 7.1 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 147.54 (C), 146.42 (C),
145.68 (C), 144.83 (C), 137.46 (C), 133.62 (C), 129.88 (CH), 129.25
(C), 127.95 (C), 125.30 (C), 123.77 (CH), 120.54 (CH), 113.42 (CH),
107.45 (CH), 107.17 (CH), 61.63 (CH2), 49.38 (CH2), 45.92 (CH2),
36.35 (CH3), 17.81 (CH3), 13.63 (CH3), 13.41 (CH3); HRESIMS m/
4,5-dimethyl-8-(prop-2-yn-1-yloxy)phenanthridin-6(5H)-one
(6a). 1H NMR (500 MHz, CDCl3) δ 8.17 (dd, J = 8.9, 4.0 Hz, 1H), 8.03
(d, J = 7.8 Hz, 1H), 8.00 (d, J = 2.9 Hz, 1H), 7.38 (dd, J = 8.9, 2.8 Hz,
1H), 7.28 (d, J = 7.3 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 4.85 (d,
J = 2.4 Hz, 2H), 3.82 (s, 3H), 2.67 (s, 3H), 2.56 (t, J = 2.3 Hz, 1H); 13
C
NMR (125 MHz, CDCl3) δ 163.84 (C), 157.29 (C), 138.66 (C), 133.01
(CH), 128.27 (C), 126.62 (C), 126.18 (C), 123.92 (CH), 122.99 (CH),
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BioorganicChemistry84(2019)285–294
122.62 (CH), 121.24 (C), 120.60 (CH), 110.25 (CH), 77.99 (C), 76.01
(CH), 56.15 (CH2), 38.49 (CH3), 23.62 (CH3); HRESIMS m/z 278.1177
[M+H]+ (calcd for C18H16NO2, 278.1176).
13C NMR (125 MHz, CDCl3) δ 163.91 (C), 158.17 (C), 138.72 (C),
138.58 (C), 132.94 (CH), 130.86 (C), 130.82 (C), 128.05 (C), 127.72
(CH), 126.64 (C), 126.18 (C), 125.32 (C), 125.21 (CH), 123.98 (CH),
123.84 (CH), 123.00 (CH), 122.81 (CH), 121.89 (CH), 121.27 (C),
120.56 (CH), 109.80 (CH), 64.71 (CH2), 38.53 (CH3), 23.61 (CH3);
HREIMS m/z 419.0750 [M]+ (calcd for C24H18ClNO2S, 419.0747).
9-(2-(1,3-dioxolan-4-yl)ethoxy)-4,5-dimethylphenanthridin-
6(5H)-one (6h). 1H NMR (600 MHz, CDCl3) δ 8.14 (d, J = 8.9 Hz, 1H),
8.03 (d, J = 7.8 Hz, 1H), 7.92 (d, J = 2.4 Hz, 1H), 7.32 (dd, J = 8.9,
2.6 Hz, 1H), 7.27 (s, 1H), 7.20 (t, J = 7.6 Hz, 1H), 5.14 (t, J = 4.6 Hz,
1H), 4.30 (t, J = 6.5 Hz, 2H), 4.07–3.98 (m, 2H), 3.95–3.86 (m, 2H),
3.82 (s, 3H), 2.67 (s, 3H), 2.23 (q, J = 6.4 Hz, 2H); 13C NMR (150 MHz,
CDCl3) δ 163.96 (C), 158.66 (C), 138.51 (C), 132.72 (CH), 127.51 (C),
126.64 (C), 126.10 (C), 123.67 (CH), 122.89 (CH), 122.48 (CH), 121.38
(C), 120.46 (CH), 109.66 (CH), 101.99 (CH), 64.97 (CH2), 64.01 (CH2),
38.49 (CH3), 33.66 (CH2), 29.69 (CH2), 23.59 (CH3); HRESIMS m/z
340.1548 [M+H]+ (calcd for C20H22NO4, 340.1543).
8-((1,5-dimethyl-1H-pyrazol-3-yl)methoxy)-4,5-dimethylphe-
nanthridin-6(5H)-one (6b). 1H NMR (500 MHz, CDCl3) δ 8.11 (d,
J = 9.0 Hz, 1H), 8.02 (d, J = 2.8 Hz, 1H), 7.99 (d, J = 7.9 Hz, 1H), 7.38
(dd, J = 8.9, 2.9 Hz, 1H), 7.23 (d, J = 6.8 Hz, 1H), 7.17 (t, J = 7.6 Hz,
1H), 6.15 (s, 1H), 5.13 (s, 2H), 3.80 (s, 3H), 3.77 (s, 3H), 2.64 (s, 3H),
2.26 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 163.85 (C), 158.47 (C),
146.19 (C), 139.54 (C), 138.42 (C), 132.64 (CH), 127.51 (C), 126.50
(C), 125.97 (C), 123.59 (CH), 122.80 (CH), 122.59 (CH), 121.29 (C),
120.39 (CH), 109.92 (CH), 105.19 (CH), 64.29 (CH2), 38.36 (CH3),
36.03 (CH3), 23.51 (CH3), 11.15 (CH3); HRESIMS m/z 370.1532
[M + Na]+ (calcd for C21H21N3O2, 370.1526).
4,5-dimethyl-9-(pyridin-3-ylmethoxy)phenanthridin-6(5H)-one
(6c). 1H NMR (500 MHz, CDCl3) δ 8.62 (d, J = 4.7 Hz, 1H), 8.13 (d,
J = 9.0 Hz, 1H), 8.00 (dd, J = 9.4, 5.4 Hz, 2H), 7.71 (td, J = 7.7,
1.7 Hz, 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.40 (dd, J = 8.9, 2.9 Hz, 1H),
7.23 (dd, J = 10.1, 4.8 Hz, 2H), 7.17 (t, J = 7.6 Hz, 1H), 5.32 (s, 2H),
3.78 (s, 3H), 2.63 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 163.73 (C),
158.12 (C), 156.46 (C), 149.37 (CH), 138.48 (C), 136.79 (CH), 132.80
(CH), 127.82 (C), 126.64 (C), 126.03 (C), 123.80 (CH), 122.86 (CH),
122.75 (CH), 122.11 (CH), 121.49 (CH), 121.19 (C), 120.45 (CH),
110.55 (CH), 70.82 (CH2), 38.38 (CH3), 23.52 (CH3); HREIMS m/z
330.1379 [M]+ (calcd for C21H18N2O2, 330.1368).
4,5-dimethyl-[1,3]dioxolo[4,5–j]phenanthridin-6(5H)-one
(8a). 1H NMR (500 MHz, CDCl3) δ 7.90 (d, J = 7.9 Hz, 1H), 7.85 (s,
1H), 7.56 (s, 1H), 7.27 (dd, J = 7.4, 0.7 Hz, 1H), 7.18 (t, J = 7.7 Hz,
1H), 6.10 (s, 2H), 3.79 (s, 3H), 2.65 (s, 3H); 13C NMR (125 MHz, CDCl3)
δ 163.50 (C), 152.23 (C), 148.25 (C), 139.05 (C), 133.15 (CH), 130.85
(C), 126.13 (C), 122.74 (CH), 121.19 (C), 121.10 (C), 120.77 (CH),
106.64 (CH), 101.93 (CH2), 100.74 (CH), 38.36 (CH3), 23.64 (CH3);
HRESIMS m/z 268.0969 [M+H]+ (calcd for C16H14NO3, 268.0968).
Methyl 4′-(((4,5-dimethyl-6-oxo-5,6-dihydrophenanthridin-8-
yl)oxy)methyl)-[1,1′- biphenyl]-2-carboxylate (6d). 1H NMR
(500 MHz, CDCl3) δ 8.18 (d, J = 9.0 Hz, 1H), 8.08–8.01 (m, 2H), 7.84
(dd, J = 7.7, 1.0 Hz, 1H), 7.53 (d, J = 7.8 Hz, 3H), 7.44–7.40 (m, 2H),
7.39–7.34 (m, 3H), 7.28 (d, J = 7.4 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H),
5.27 (s, 2H), 3.83 (s, 3H), 3.65 (s, 3H), 2.67 (s, 3H); 13C NMR
(125 MHz, CDCl3) δ 169.05 (C), 163.98 (C), 158.60 (C), 142.14 (C),
141.23 (C), 138.57 (C), 135.43 (C), 132.86 (CH), 131.38 (CH), 130.80
(CH), 130.75 (C), 129.90 (CH), 128.64 (2CH), 127.80 (C), 127.45
(2CH), 127.31 (CH), 126.68 (C), 126.17 (C), 123.86 (CH), 122.99 (CH),
122.81 (CH), 121.38 (C), 120.54 (CH), 110.02 (CH), 70.13 (CH2), 52.05
(CH3), 38.53 (CH3), 23.63 (CH3); HRESIMS m/z 464.1865 [M+H]+
(calcd for C30H26NO4, 464.1856).
4.2. Bioactivity assays
4.2.1. Cell culture
HEK293T cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% (v/v) heat-inactivated FBS in
a humidified 5% CO2/95% air (v/v) atmosphere at 37 °C.
4.2.2. 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 (GFP) in the resultant cell lysates was first
determined in a Wallac multi-counter 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 intensity using the same counter. The luminescence in-
tensities were normalized against the fluorescence intensities [20].
8-((3,5-dimethoxybenzyl)oxy)-4,5-dimethylphenanthridin-
6(5H)-one (6e). 1H NMR (500 MHz, CDCl3) δ 8.16 (d, J = 9.0 Hz, 1H),
8.03 (d, J = 7.9 Hz, 1H), 8.00 (d, J = 2.8 Hz, 1H), 7.40 (dd, J = 8.9,
2.8 Hz, 1H), 7.28 (s, 1H), 7.20 (t, J = 7.6 Hz, 1H), 6.64 (d, J = 2.2 Hz,
2H), 6.43 (t, J = 2.2 Hz, 1H), 5.16 (s, 2H), 3.82 (s, 3H), 3.81 (s, 6H),
2.67 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 163.96 (C), 161.04 (2C),
158.49 (C), 138.79 (C), 138.58 (C), 132.85 (CH), 127.79 (C), 126.69
(C), 126.16 (C), 123.83 (CH), 122.97 (CH), 122.66 (CH), 121.37 (C),
120.53 (CH), 110.12 (CH), 105.40 (2CH), 100.14 (CH), 70.27 (CH2),
55.42 (2CH3), 38.52 (CH3), 23.62 (CH3); HRESIMS m/z 390.1701 [M
+H]+ (calcd for C24H24NO4, 390.1700).
9-((1,3-dimethyl-1H-pyrazol-5-yl)methoxy)-4,5-dimethylphe-
nanthridin-6(5H)-one (6f). 1H NMR (500 MHz, CDCl3) δ 8.03 (d,
J = 9.0 Hz, 1H), 7.90 (t, J = 5.0 Hz, 2H), 7.23 (dd, J = 8.9, 2.7 Hz, 1H),
7.20–7.15 (m, 1H), 7.10 (t, J = 7.6 Hz, 1H), 6.08 (s, 1H), 5.04 (s, 2H),
3.78 (s, 3H), 3.72 (s, 3H), 2.56 (s, 3H), 2.17 (s, 3H); 13C NMR
(125 MHz, CDCl3) δ 163.73 (C), 157.71 (C), 147.33 (C), 138.51 (C),
137.39 (C), 132.98 (CH), 128.11 (C), 126.53 (C), 126.11 (C), 123.95
(CH), 122.95 (CH), 122.58 (CH), 121.10 (C), 120.52 (CH), 109.65 (CH),
106.95 (CH), 60.65 (CH2), 38.44 (CH3), 36.43 (CH3), 23.58 (CH3),
13.40 (CH3); HREIMS m/z 347.1639 [M]+ (calcd for C21H21N3O2,
347.1634).
4.2.3. 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 fol-
lowing primer pairs were used for the target genes: Axin2: 5′-AGGCT
AGCTGAGGTGT-3′ and 5′-AGGCTTG-GATTGGAGAA-3′; NKD1: 5′-GTC
AACCACTCCCCAACATC-3′ and 5′-AATGGTGGTAG-CAGCCAGAC-3′;
GAPDH: 5′-AGGTCGGAGTCAACGGATTTG-3′ and 5′-TGTAAACCA-
TGTAGTTGAGGTCA-3′.
8-((5-chlorobenzo[b]thiophen-2-yl)methoxy)-4,5-dimethyl-
phenanthridin-6(5H)-one (6g). 1H NMR (500 MHz, CDCl3) δ 8.19 (d,
J = 9.0 Hz, 1H), 8.10 (d, J = 2.8 Hz, 1H), 8.05 (d, J = 7.6 Hz, 1H), 7.88
(d, J = 2.0 Hz, 1H), 7.79 (d, J = 8.6 Hz, 1H), 7.64 (s, 1H), 7.41 (dd,
J = 8.9, 2.9 Hz, 1H), 7.36 (dd, J = 8.6, 1.9 Hz, 1H), 7.29 (d, J = 7.8 Hz,
1H), 7.22 (t, J = 7.6 Hz, 1H), 5.42 (s, 2H), 3.84 (s, 3H), 2.68 (s, 3H);
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4.3. Molecular docking protocol
[6] T. Ji, Y. Guo, K. Kim, P. McQueen, S. Ghaffar, A. Christ, C. Lin, R. Eskander, X. ZiB,
H. Hoang, Neuropilin-2 expression is inhibited by secreted Wnt antagonists and its
down-regulation is associated with reduced tumor growth and metastasis in os-
teosarcoma, Mol. Cancer. 14 (2015).
Compounds 12 and HLY78 were docked with EPAC2 using the X-ray
structure of axin (PDB code: 1WSR) and AutoDock 4.2. The H2O mo-
lecules 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
programmefor interactive growth and subsequent scoring.
[7] A.L. Silva, S.N. Dawson, M.J. Arends, K. Guttula, N. Hall, E.A. Cameron,
T.H.M. Huang, J.D. Brenton, S. Tavare, M. BienzA, E.K. Ibrahim, Boosting Wnt
activity during colorectal cancer progression through selective hypermethylation of
Wnt signaling antagonists, Bmc Cancer 14 (2014).
[8] S.A. Von Schulz-Hausmann, L.C. Schmeel, F.C. Schmeel, I.G.H. Schmidt-Wolf,
Targeting the Wnt/beta-catenin pathway in renal cell carcinoma, Anticancer Res.
34 (2014) 4101–4108.
[9] R.M. Kypta, J. Waxman, Wnt/beta-catenin signalling in prostate cancer, J. Nat. Rev.
Urol. 9 (2012) 418–428.
[10] B. Cosimelli, S. Laneri, C. Ostacolo, A. Sacchi, E. Severi, E. Porcu, E. Rampazzo,
E. Moro, G. Basso, G. Viola, Synthesis and biological evaluation of imidazo 1,2-a
pyrimidines and imidazo 1,2-a pyridines as new inhibitors of the Wnt/beta-catenin
signaling, Euro. J. Med. Chem. 83 (2014) 45–56.
Author contributions
[11] B. Gabrielsen, T.P. Monath, J.W. Huggins, D.F. Kefauver, G.R. Pettit, G. Groszek,
M. Hollingshead, J.J. Kirsi, W.M. Shannon, E.M. Schubert, J. Dare, B. Ugarkar,
M.A. UsseryM, J. Phelan, Antiviral (RNA) activity of selected amar-
yllidaceaeisoquinoline constituents and synthesis of related substances, J. Nat.
Prod. 55 (1992) 1569–1581.
D-Z Chen and B-J Yang designed and performed the chemical
syntheses under the guidance of X-J Hao; J-Y Caiand J-J Guo purified
all the compounds; X-LHe, HZhangand Y Zhang evaluated the Wnt
activities and other bioactivities of all compoundsunder the guidance of
L Lin.; D-Z Chen and X-L He wrote the manuscript with advice from all
of the authors; X.H. guided all aspects of this study.
[12] M. Ieven, A.J. Vlietinck, D.A. Vandenberghe, J. Totte, R. Dommisse, E. Esmans,
F. Alderweireldt, Plant antiviral agents .3. isolation of alkaloids from clivia-miniata
regel (amaryllidaceae), J. Nat. Prod. 45 (1982) 564–573.
[13] G. Van Goietsenoven, A. Andolfi, B. Lallemand, A. Cimmino, D. Lamoral-Theys,
T. Gras, A. Abou-Donia, J. Dubois, F. Lefranc, V. Mathieu, A. Kornienko, R. Kiss,
A. Evidente, Amaryllidaceae alkaloids belonging to different structural subgroups
display activity against apoptosis-resistant cancer cells, J. Nat. Prod. 73 (2010)
1223–1227.
Acknowledgements
Funding: This work was supported by the National Natural Science
Foundation of China [grant numbers 81773610 and 21432010],
Central Asian Drug Discovery and Development Centre of Chinese
Academy of Sciences [grant number CAM201702], Youth Innovation
Promotion Association of CAS [grant number 2018429], and Yunnan
Applied Basic Research Projects [grant number 2016FB015].
[14] D. Lamoral-Theys, A. Andolfi, G. Van Goietsenoven, A. Cimmino, B. Le Calve,
N. Wauthoz, V. Megalizzi, T. Gras, C. Bruyere, J. Dubois, V. Mathieu, A. Kornienko,
R. Kiss, A. Evidente, Lycorine, the main phenanthridine amaryllidaceae alkaloid,
exhibits significant antitumor activity in cancer cells that display resistance to
proapoptotic stimuli: an investigation of structure-activity relationship and me-
chanistic insight, J. Med. Chem. 52 (2009) 6244–6256.
[15] D.Z. Chen, J.Y. Cai, J.L. Yin, J.D. Jiang, C.X. Jing, Y.P. Zhu, J.J. Cheng, Y.T. Di,
Y. Zhang, M.M. Cao, S.L. Li, Z.G. PengX, J. Hao, Lycorine-derived phenanthridine
downregulators of host Hsc70 as potential hepatitis C virus inhibitors, Future Med.
Chem. 7 (2015) 561–570.
Appendix A. Supplementary material
[16] D.Z. Chen, J.D. Jiang, K.Q. Zhang, H.P. He, Y.T. Di, Y. Zhang, J.Y. Cai, L. Wang,
S.L. Li, P. Yi, Z.G. Peng, X.J. Hao, Evaluation of anti-HCV activity and SAR study of
(+)-lycoricidine through targeting of host heat-stress cognate 70 (Hsc70), Bioorg.
Med. Chem. Lett. 23 (2013) 2679–2682.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2018.11.020.
[17] D.Z. Chen, C.X. Jing, J.Y. Cai, J.B. Wu, S. Wang, J.L. Yin, X.N. Li, L. LiX, J. Hao,
Design, synthesis, and structural optimization of lycorine-derived phenanthridine
derivatives as Wnt/beta-catenin signaling pathway agonists, J. Nat. Prod. 79 (2016)
180–188.
References
[1] H. Clevers, R. Nusse, Wnt/beta-catenin signaling and disease, Cell 149 (2012)
1192–1205.
[18] S. Wang, J. Yin, D. Chen, F. Nie, X. Song, C. Fei, H. Miao, C. Jing, W. Ma, L. Wang,
S. Xie, C. Li, R. Zeng, W. Pan, X. HaoL, Li, Small-molecule modulation of Wnt
signaling via modulating the Axin-LRP5/6 interaction, Nature Chem. Biol. 9 (2013)
579–582.
[2] N. Maruotti, A. Corrado, A. Neve, F.P. Cantatore, Systemic effects of Wnt signaling,
J. Cell. Physiol. 228 (2013) 1428–1432.
[3] V. Dormoy, D. Jacqmin, H. Lang, T. Massfelder, From development to cancer: les-
sons from the kidney to uncover new therapeutic targets, Anticancer Res. 32 (2012)
3609–3617.
[19] T. Schwarz-Romond, M. Fiedler, N. Shibata, P.J.G. Butler, A. Kikuchi, Y. Higuchi,
M. Bienz, The DIX domain of Dishevelled confers Wnt signaling by dynamic poly-
merization, Nat. Struct. Mol. Biol. 14 (2007) 484–492.
[4] Z.F. Zimmerman, R.T. MoonA, J. Chien, Targeting Wnt pathways in disease, Cold
Spring Harbor Perspect. Biol. 4 (2012).
[20] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. GoodsellA,
J. Olson, AutoDock4 and AutoDockTools4: automated docking with selective re-
ceptor flexibility, J. Comput. Chem. 30 (2009) 2785–2791.
[5] J.B. Regard, Z. Zhong, B.O. Williams, Y. Yang, Wnt signaling in bone development
and disease: making stronger bone with Wnts, Cold Spring Harbor Perspect. Biol. 4
(2012).
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