Synthesis of 4-ethyl-5-methyl-5,6-dihydrophenanthridine-8,9-diol as the key intermediate of potent agonists of the Wnt signalling pathway

Article abstract of DOI:10.3184/174751915X14285852307852

The synthesis of 4-ethyl-5-methyl-5,6-dihydro-phenanthridine-8,9-diol as the key intermediate of a series of potent Wnt signalling pathway agonists is reported. This synthesis features a consecutive aryl-aryl and N-aryl coupling, leading to 4-ethyl-5-meth

Full text of DOI:10.3184/174751915X14285852307852

JOURNAL OF CHEMICAL RESEARCH 2015
VOL. 39 APRIL, 247–250
RESEARCH PAPER 247
Synthesis of 4-ethyl-5-methyl-5,6-dihydrophenanthridine-8,9-diol as the key
intermediate of potent agonists of the Wnt signalling pathway
Chen-xu Jinga,b, Jie-yun Caic, Yu Zhangb, Duo-zhi Chenb* and Xiao-jiang Haob
aCollege of Traditional Chinese Medicine, Yunnan University of Traditional Chinese Medicine, Kunming 650500, P.R. China
bState Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201,
Yunnan, P.R. China
cYunnan Tobacco Quality Inspection & Supervision Station, Kunmimg 650106, Yunnan, P.R. China
The synthesis of 4-ethyl-5-methyl-5,6-dihydro-phenanthridine-8,9-diol as the key intermediate of a series of potent Wnt signalling
pathway agonists is reported. This synthesis features a consecutive aryl-aryl and N-aryl coupling, leading to 4-ethyl-5-methyl-8,9-
dimethoxyphenanthridin-6-(5H)-one, whose structure was determined by spectroscopic analysis and X-ray crystallography, in a one-pot
sequence with the presence of palladium catalyst and TFP (tri(2-furyl)phosphine) as the ligand.
Keywords: phenanthridin, palladium catalyst, aryl–aryl coupling, N-aryl coupling
The evolutionarily conserved Wnt signalling pathway plays an
important role in the progress of embryonic development and
human diseases.It regulates cell fate specification, proliferation,
differentiationandsurvivalduringembryogenesis.Inappropriate
regulation of the Wnt signalling pathway often links to many
human diseases, such as cancer, Alzheimer’s disease, familial
exudative vitreoretinopathy and bone formation disorders. 1-4
By screening a synthetic chemical library of phenanthridine
derivatives, we previously identified one of the phenanthridine
derivatives, hlyc 60 (Fig. 1), as an activator of the Wnt signalling
pathway. It targets the DIX domain of Axin and potentiates the
Axin–LRP6 association, thus promoting LRP6 phosphorylation
and Wnt signalling transduction. Moreover, we identified that it
may weaken the autoinhibition of Axin. Collectively, our study
not only provides new insights into the regulation of the Wnt/b-
catenin signalling pathway by a Wnt-specific small molecule
but will also facilitate therapeutic applications, such as HSC
expansion. 3
their potent activities in bioassays, including in vivo assays.
According to the structural analysis of hlyc-60, we found that a
key intermediate, 4-ethyl-5-methyl-5,6-dihydrophenanthridine-
8,9-diphenol (1), can simply transformed into hlyc-60 through
a process of alkylation and click reaction. We designed a
synthetic route for the production of this key intermediate 1 in
the preparation of hlyc60.
Results and discussion
According to the retrosynthesis analysis of compound 1, we
concluded that 1 could be prepared from halogenated benzamide
analogues and halogenated ethylbenzene through a consecutive
aryl-aryl and N-aryl coupling process. Commercially
purchased halogenated benzaldehyde can simply transform
into halogenated benzamide by the processes of oxidation and
amidation (Scheme 2).
As shown in Scheme 3, compound 1 was synthesised from
6-bromoveratraldehyde (2) in five steps. First, compound 2 was
oxidised using KMnO4 to afford 3, which was amidated with
methylamine to obtain 4. Compound 4 was then condensed
with iodoarene 5 using palladium catalysis to give 6, followed
by reduction of the C-6 carbonyl to afford 7. Finally, compound
7 was obtained by deprotection of 1 in the presence of boron
tribromide.
Inspired by the above research results, further bioactivity and
structural modification, of these type of compounds should be
employed and it needs larger amounts of the phenanthridine-
type derivatives than could be readily obtained through
semi-synthesis are required to confirm and further explore
The preparation of 4 was the key step of the whole route,
so the reaction conditions were optimised. We studied the
correlations between the yield and the quantity of TFP and from
the results, the highest yield was with 0.1 molar equiv. of TFP
(Fig. 2).
N
N
N
H2N
O
O
N
N
N
H2N
Experimental
N
ESI and high-resolution mass spectra were recorded using a Finnigan
Mat 90 instrument and VG Auto Spec-3000 spectrometer, respectively.
Melting points were measured using X-4 apparatus (Yingyu Yuhua
Instrument Factory, Gongyi, Henan Province, China). NMR
c
hl
60
y
Fig. 1 The structure of hlyc-60.
N
N
N
H2N
H
H
O
O
O
alkylation
N
N
click reaction
O
N
N
H2N
N
c
hl
y
60
1
Scheme 1 The transformation from key intermediate 1 to hlyc60.
* Correspondent. E-mail: chenduozhi@mail.kib.ac.cn

248 JOURNAL OF CHEMICAL RESEARCH 2015
O
X
O
O
H
H
O
O
O
X
N
H
+
N
N
O
O
1
H
O
O
C O
X
Scheme 2 The retrosynthesis analysis of hlyc60.
r
r
B
r
O
O
B
O
O
B
O
O
c
b
,
a
H
N
H
C O
H
COO
2
3
4
O
I
d
5
6
O
O
O
e
N
N
O
O
7
f
H
O
N
H
O
1
Scheme 3 Total synthesis of compound 1. Reagents and conditions: (a) NaHCO3, KMnO4, H2O, 90 °C, 3 h, 85%; (b) SOCl2, DMF, THF, 50 °C, 2 h; (c) CH3NH2
(30%), 5 °C, 1 h, 75%; (d) K2CO3, norbornene, Pd(OAc)2, TFP, MeCN, 85°C, 6 h, 75%; (e) LAH, THF, –78 °C, 2 h, 60%; g. BBr3, CH2Cl2, –78 °C, 4 h, 80%.
Fig. 2 Correlations between yields and the dosage of TFP.

JOURNAL OF CHEMICAL RESEARCH 2015 249
experiments were carried out on a Bruker AM-400 spectrometer or
a DRX-500 spectrometer or an Avance III 600 spectrometer with
the solvents CDCl3 and DMSO-d6, and Me4Si as internal standard.
Column chromatography was performed on Silica gels (60–80 mesh,
200–300 mesh, 300–400 mesh, Qingdao Haiyang Chemical Co. Ltd,
Qingdao, China). Pre-coated silica gel 60 F254 (Merck, Darmstadt,
Germany) was used for TLC. Semipreparative HPLC was performed
on a Hypersil Gold RP-C18 column (i.d. 10 × 250 mm; Thermo
Fisher Scientific Inc., Waltham, Massachusetts, USA) developed with
CH3CN–H2O at r.t. All regular solvents and reagents were reagent
grade and purchased from Aldrich-Sigma Chemical Co., Acros
Organics and J&K Scientific. The purities of all compounds were
more than 95% as determined by HPLC. All yields reported are for
dry compounds that require no further purification for use in other
reactions.
2-Bromo-4,5-dimethoxybenzoic acid (3):
A
solution of
Figure 3 X-ray crystal structure of compound 7 (CCDC 948062), crystal
data: C18H21NO2, M = 283.36, monoclinic, a = 20.412(7) Å, b = 9.126(3)
Å, c = 8.164(3) Å, α = 90.00°, β = 95.825(5)°, γ = 90.00°, V = 1513.0(9)
Å3, T = 100(2) K, space group P21/c, Z = 4, μ(MoKα) = 0.081 mm-1, 12550
reflections measured, 3649 independent reflections (Rint = 0.0612). The
final R1 values were 0.0789 (I > 2σ(I)). The final wR(F2) values were 0.1941
(I > 2σ(I)). The final R1 values were 0.1238 (all data). The final wR(F2) values
were 0.2230 (all data). The goodness of fit on F2 was 1.089.
6-bromoveratraldehyde (2, 250 mg, 1 mmol), NaHCO3 (200 mg), and
KMnO4 (500 mg) in H2O (20 mL) was heated to 90 °C with stirring
for 3 h, then extracted with CH2Cl2 (20 mL) twice. The organic phase
was washed with saturated NH4Cl and brine, dried over MgSO4,
filtered and concentrated. The residue was then purified by column
chromatography to give 3 as a pale yellow solid (220 mg, 85% yield).
m.p. 183–185 °C, (lit. 6 184–185 °C); IR (KBr) nmax 2960, 1702, 1598,
1568, 1514, 1436, 1263, 663, 511 cm-1; 1H NMR (500 MHz, CDCl3 ) δ
7.54 (s, 1H), 7.52 (s,1H), 3.87 (s, 3H), 3.81 (s, 3H);13C NMR (125 MHz,
CDCl3) δ 169.87, 153.3, 152.14, 121.85, 116.73, 115.31, 111.64, 56.81,
56.64; ESI+MS m/z 261 [M + H]+; HREIMS m/z 259.9690 [M]+ (calcd
for C9H9BrO4, 259.9684).
1542, 1509, 1451, 1260, 643, 576 cm-1;1H NMR (400 MHz, CDCl3) δ
7.57 (d, J = 7.1 Hz, 1H), 7.53 (s, 1H), 7.49 (s, 1H), 7.18 (d, J = 6.9 Hz,
1H), 6.75 (m,1H), 4.06 (s, 2H), 4.02 (s, 3H), 3.98 (s, 3H), 2.96 (q, J =
7.4 Hz, 2H), 2.41 (s, 3H), 1.23 (t, J = 7.5 Hz, 3H);13C NMR (100 MHz,
CDCl3) δ 153.4 (C), 146.5 (C), 140.5 (C), 138.8 (C), 132.9 (C), 131.3
(CH), 130.0 (C), 129.0 (C), 123.2 (CH), 120.5 (CH), 108.9 (CH), 103.1
(CH), 52.3 (CH2), 56.8 (CH3), 56.4 (CH3), 39.9 (CH3), 28.5 (CH2), 15.8
(CH3). HREIMS m/z 283.1567 [M]+ (calcd for C18H21NO2, 283.1572).
4-Ethyl-5-methyl-5,6-dihydrophenanthridine-8,9-diol (1): Com-
pound 7 (29 mg, 0.1 mmol) was dissolved in 5 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 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 give 1 as a pale
yellow powder (20 mg, 80% yield) m.p. 139–141 °C; IR (KBr) nmax
3384, 2965, 1613, 1513, 1451, 1411, 1377, 1304, 1248, 1122, 929, 867,
2-Bromo-4,5-dimethoxy-N-methyl-benzamide (4): Compound
3
(260 mg, 1mmol) was dissolved in THF (10 mL), to which DMF (0.1
mL) and SOCl2 (0.5 mL, 4mmol) were added. The reaction solution
was stirred for 2 h at 50 °C and then concentrated to remove THF.
The residue was then added to a 30% solution of methylamine in
water (20 mL) at 5°C and filtered. The cake was purified by column
chromatography to give 4 as a pale yellow solid (205 mg, 75%
yield). m.p. 119–121 °C; IR (KBr) nmax 3422, 2961, 1597, 1551, 1505,
1
1456, 1259, 1159, 1014, 865 cm-1; H NMR (400 MHz, CDCl3) δ 7.46
(s, 1H), 7.33 (s, 1H), 6.63 (s, 1H), 3.87 (s, 3H), 3.85 (s, 3H), 2.79 (s,
3H); 13C NMR (100 MHz, CDCl3) δ 169.31, 152.32, 151.16, 131.95,
116.69, 116.31, 112.86, 56.81, 56.78, 26.33; ESI+MS m/z 274 [M+H]+;
HREIMS m/z 273.0004 [M]+ (calcd for C10H12BrNO3, 273.0001).
4-Ethyl-5-methyl-8,9-dimethoxyphenanthridin-6(5H)-one (6):
A
flask was charged under nitrogen with Pd(OAc)2 (3.0 mg, 0.013 mmol),
tri-2-furylphosphine (6.2 mg, 0.027 mmol), K2CO3 (72.3 mg, 0.52
mmol), the amide 4 (0.26 mmol), a solution of norbornene (26.9 mg,
0.286 mmol) in anhydrous solvent (5.8 mL), and 1-ethyl-2-iodobenzene
(5, 0.26 mmol). The reaction mixture was heated with stirring at 85
°C for 6 h and then cooled to room temperature. After the addition of
saturated 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 6 as white wax (58 mg, 75% yield). IR (KBr) nmax 3446,
2923, 1642, 1550, 1512, 1464, 1360, 1029, 691, 563, cm-1; 1H NMR
δ 8.21 (d, J = 7.6 Hz, 1H), 7.71 (s, 1H), 7.60 (s, 1H), 7.32 (d, J = 7.4
Hz, 1H), 7.29–7.25 (m, 1H), 3.92 (s, 3H), 3.89 (s, 3H), 3.76 (s, 3H),
3.01–3.03 (m, 2H), 1.25 (t, J = 8.1 Hz, 3H); 13C NMR: δ 166.1, 139.4,
134.5, 132.5, 132.1, 131.3, 128.7, 127.5, 125.1, 129.6, 122.2, 121.1, 121.0,
58.3, 58.1, 38.6, 28.1, 15.5; HREIMS m/z 297.1361 [M]+ (calcd for
C18H19NO3, 297.1365).
1
804, 755 cm-1; H NMR (400 MHz, CDCl3) δ 7.46 (d, J = 7.1 Hz, 1H),
7.27 (d, J = 2.7 Hz, 1H), 7.17 (dt, J = 15.0, 7.5 Hz, 1H), 7.00 (s, 1H), 6.75
(s, 1H), 3.96 (s, 2H), 2.82–2.76 (m, 2H), 2.31 (s, 3H), 1.28 (dd, J = 14.7,
13
7.1 Hz, 3H)； C NMR (100 MHz, CDCl3) δ 143.8 (C), 143.0 (C), 141.2
(C), 139.4 (C), 128.8 (C), 127.6 (CH), 126.3(C), 125.1 (C), 124.6 (CH),
120.7 (CH), 113.8 (CH), 110.4 (CH), 53.6 (CH2), 40.2 (CH3), 23.2
(CH2), 14.8 (CH3)。HREIMS m/z 255.1250 [M]+ (calcd for C16H17NO2,
255.1259).
Conclusions
To enable the production of phenanthridine-type derivatives in
sufficient quantities to enable their use in further bioassays, a
totally synthetic route to the key intermediate 8 was developed.
This new route hinges on an efficient palladium-catalysed
coupling reaction involving sequential C–N and C–C bond
formation with C–H activation. In conclusion, an efficient
and convenient synthesis of the key intermediate of potent
phenanthridins Wnt agonists has been reported. This route
featured a one-step palladium catalysed preparation of 4-ethyl-
5-methyl-8,9-dimethoxyphenanthridin-6(5H)-one (6). The
relatively simple reaction procedure, utilisation of cheap and
readily available reagents, and ideal yields (23%) of products
are the main advantages of the present approach.
4-Ethyl-5-methyl-8,9-dimethoxy-5,6-dihydrophenanthridine
(7):
A solution of 6 (30 mg, 0.1 mmol) in THF (5 mL) was added to LAH
(20 mg) at –78 °C. The reaction was stirred for 2 h and then quenched
using H2O (5 mL). The mixture was then extracted with Et2O (20 mL)
twice. The organic phase was washed with brine and concentrated,
and the residue was purified by column chromatography to give 7 as
a colourless solid (21 mg, 75% yield), its X-ray crystal structure was
determined (Fig. 3), m.p. 72–74 °C; IR (KBr) nmax 2912, 1653, 1638,

250 JOURNAL OF CHEMICAL RESEARCH 2015
References
This research was supported financially by the National
Natural Science Foundation of China (81402828 and 21432010)
and “Xi Bu Zhi Guang” Foundation of The Chinese Academy
of Sciences.
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Received 27 February 2015; accepted 8 April 2015
Paper 1503221 doi: 10.3184/174751915X14285852307852
Published online: 13 April 2015
6
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