Synthesis of novel natural product-like diaryl acetylenes as hypoxia inducible factor-1 inhibitors and antiproliferative agents

Article abstract of DOI:10.1039/c9ra02525a

The selaginellin derivatives are a type of novel natural pigments with an unusual alkynyl phenol skeleton from the genus Selaginella. Some of these natural compounds were previously reported to show important bioactivities, including anticancer activity, cardiovascular protection and phosphodiesterase-4 inhibition. We designed and synthesized fifteen biphenyl-containing diaryl acetylene derivatives mimicking the skeleton of natural alkynyl phenols. In MTT assay in cancer cells, compounds 1c, 2d, 2g, 2h, 2i and 2j exhibited potent antiproliferative activity. The evaluation of Hypoxia Inducible Factor-1 (HIF-1) pathway inhibitory activity in dual luciferase assay demonstrated that most tested compounds exhibited moderate to good activities. Compounds 1a, 2f and 2h displayed high HIF-1 inhibitory activities and relatively low cytotoxicity, demonstrating great potential as HIF-1 inhibitors. These results afford a new strategy for the discovery of new HIF-1 inhibitors and anti-proliferative agents from natural or synthetic diaryl acetylene derivatives.

Full text of DOI:10.1039/c9ra02525a

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Synthesis of novel natural product-like diaryl
acetylenes as hypoxia inducible factor-1 inhibitors
Cite this: RSC Adv., 2019, 9, 13878
and antiproliferative agents†
ab
a
ab
Guangzhe Li, *ab Xu Wang,a
Shisheng Wang,
Liqiang Liu, Xiuhan Guo,
a
ab
ab
Huijuan Dong, Yueqing Li and Weijie Zhao
The selaginellin derivatives are a type of novel natural pigments with an unusual alkynyl phenol skeleton
from the genus Selaginella. Some of these natural compounds were previously reported to show
important bioactivities, including anticancer activity, cardiovascular protection and phosphodiesterase-4
inhibition. We designed and synthesized fifteen biphenyl-containing diaryl acetylene derivatives
mimicking the skeleton of natural alkynyl phenols. In MTT assay in cancer cells, compounds 1c, 2d, 2g,
2h, 2i and 2j exhibited potent antiproliferative activity. The evaluation of Hypoxia Inducible Factor-1 (HIF-
1
) pathway inhibitory activity in dual luciferase assay demonstrated that most tested compounds
Received 3rd April 2019
Accepted 26th April 2019
exhibited moderate to good activities. Compounds 1a, 2f and 2h displayed high HIF-1 inhibitory activities
and relatively low cytotoxicity, demonstrating great potential as HIF-1 inhibitors. These results afford
a new strategy for the discovery of new HIF-1 inhibitors and anti-proliferative agents from natural or
synthetic diaryl acetylene derivatives.
DOI: 10.1039/c9ra02525a
rsc.li/rsc-advances
The biological activities of these alkynyl phenol compounds
have been demonstrated to involve hypoglycemic, anti-
1
. Introduction
14
2
6
27
Natural products and their synthetic derivatives have served as inammatory,
and anti-oxidant
activity, cardiovascular
and neuroprotective
28
9
a consistent source of valuable new drug leads for centuries, protection,
anticancer effects,
18,29,30
and natural products with bioactive pharmacophores are bio- effects.
Selaginellin and selaginellins A, C, M, N, O, Q have
logically validated starting points for the development of new been reported to be cytotoxic against U251, HeLa, MCF-7 and
1
9–11
drugs. In the last decade, a type of novel natural pigment with BGC-823 tumor cells.
Selaginpulvilins A–L, with an unprec-
an unusual alkynyl phenol skeleton has been successively iso- edented 9,9-diphenyl-1-(phenylethynyl)-9H-uorene skeleton,
lated from the genus Selaginella, mainly from S. tamariscina and exhibit remarkable phosphodiesterase-4 inhibitory activity with
20,22,25
S. pulvinata which are the two qualied species listed in Chinese IC50 of 0.011–1.38 mM.
The previous work in our laboratory
Pharmacopoeia and used as traditional Chinese medicines for on the bioactive constituents from the genus Selaginella
the treatment of dysmenorrhea, abdominal mass and traumatic revealed selaginellins A and B were moderately cytotoxic against
injury. The pharmacological investigations of the genus Selag- H322 cell, and selaginellin A displayed inhibitory activity on
inella have revealed that it has anticancer, antivirus, antioxidant hypoxia inducible factor-1 (HIF-1) transcription in dual lucif-
2
and anti-inammatory activities. The phytochemical investi- erase reporter assay (unpublished).
gations of the genus Selaginella have led to the identication of
more than forty natural alkynyl phenols hitherto, including
3
4–19
14,17
selaginellin, selaginellins A–W,
selariscinins A–D,
and
20–25
selaginpulvilins A–L.
All these natural products possess
unique skeletons characterized by biphenyl-containing diaryl
acetylene, as shown in Fig. 1.
a
Department of Pharmacy, School of Chemical Engineering, Dalian University of
Technology, Dalian 116023, China. E-mail: liguangzhe@dlut.edu.cn
b
State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian
1
16023, China
†
Electronic supplementary information (ESI) available: Spectral data of synthetic
Fig. 1 Selected examples of natural diaryl acetylene compounds from
the genus Selaginella.
compounds. See DOI: 10.1039/c9ra02525a
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HIF-1 is a central regulator involved in detection and adap- starting with halogenated benzoate via the Suzuki coupling and
tion to cellular oxygen stress through regulation of the hypoxic Sonogashira reaction.
transcriptional program in angiogenesis, erythropoiesis, and
31
metabolism. The HIF-1 pathway is involved in many diseases,
including anemia, ischemia, most tumors, and other hypoxic-
ischemic diseases, making HIF-1 an attractive target for thera-
peutic application. A considerable proportion of solid tumors With the aim to synthesize selaginellin N, we rst synthesized
are induced by hypoxia which results in overexpression of the 1a using o-bromobenzoic acid as the starting material via iod-
HIF-1 pathway. Moreover, overexpression of the HIF-1 pathway ization, methyl esterication, regioselective Suzuki coupling
2
. Results and discussion
2.1. Chemistry
32
has been correlated with poor prognosis, invasive tumor reaction and Sonogashira reaction (Scheme 2). Using THF–H O
2
33
growth, and resistance to radiation for tumor patients.
as the solvent in Suzuki reaction, compound 6 was obtained
Therefore, the inhibition of HIF-1 has been identied as an under mild condition with a better yield than reported.
24
34
15
effective therapeutic strategy for various solid tumors.
Compound 1a has a skeleton similar to selariscinin D, which
The unique skeletons associated with important bioactivities exhibited stimulatory effect on glucose uptake in 3T3-L1
of this family of alkynyl phenols have attracted great attention adipocyte cells and potent inhibitory effect against PTP1B.
of scientists from elds of organic synthesis and medicinal
We attempted to synthesize selaginellin N through the
chemistry. Up to now, the reports on the total synthesis of demethylation and dehydration of triphenyl methol interme-
natural alkynyl phenols are rare and all focus on the diary- diate 1f. However, the nucleophilic addition of 1a with 4-
21,23–25
luorene derivatives selaginpulvilins,
whereas the total methoxyphenyl bromide under the presence of n-butyllithium
synthesis of selaginellins and selariscinins has not been re- produced the diphenyl ketone 1c instead of triphenyl methol
ported yet. In our effort to develop synthetic method for sela- (Scheme 3). The steric hindrance of 1c is presumably the
ginellins, we achieved the precursors of selaginellin N (1a–1d) dominated factor to prevent the further reaction to produce
with the skeleton of selariscinin D and selaginellin S. The triphenyl methol. We tried using the more reactive acyl
difficulty in the synthesis of selaginellins with moiety of benzyl
para-quinone methide is considered to result from the large
steric hindrance of polycyclic skeleton. Considering the unique
diaryl acetylene skeleton as a good privileged structure, we
designed and synthesized a series of non-natural diaryl acety-
lene derivatives (2a–2j) analogous to selaginellins but with less
steric hindrance. Herein we report the synthesis of this family of
diaryl acetylene derivatives, their cytotoxic activity against
tumor cells and HIF-1 inhibitory activity.
By analyzing the structures of selaginellin and its reported
derivatives, P. F. Tu et al. proposed a potential biosynthetic
35
pathway of selaginellin and its derivatives. We compared the
different skeletons of natural alkynyl phenols as displayed in
Fig. 1 and presumed that the formation of selaginellin N
analogues might undergo the intermediates of selariscinin D
Scheme 2 Synthesis of compound 1a. Reagents and conditions: (a)
ꢀ
Pd(OAc)
2
, NIS, DMF, 100 C, 12 h, 90%; (b) K
2
CO
3
, CH
3
I, acetone,
ꢀ
40
C,
5
h, 100%; (c) Na
2 3
CO ,
4-methoxyphenylboronic acid,
ꢀ
PdCl
2
(PPh
3
)
2 2
, THF/H O ¼ 1 : 1, 60 C, 8 h, 82%; (d) 4-methox-
analogues, selaginellin
S
analogues and selariscinin
B
ꢀ
yphenylacetylene, CuI, PdCl
2 3
(PPh )
2
, PPh
3
, Et
3
N, DMF, 120 C, 12 h,
analogues successively. Based on this presumption, a retro- 48%.
synthetic analysis of selaginellin N was established as shown in
Scheme 1, where selariscinin D analogues were synthesized
Scheme 3 Synthesis of compounds 1b–1e. Reagents and conditions:
ꢀ
(
2
a) NaOH, CH
3
OH/H
2
O ¼ 5 : 1, 80 C, 8 h, 77%; (b) SOCl
2 2 2
, CH Cl , r.t.
ꢀ
h, then n-BuLi, 1-bromo-4-methoxybenzene, THF, ꢁ83 C, 2 h, 38%;
ꢀ
ꢀ
(c) BBr
3
, 0 C, 30 min, 17%; (d) BBr
3
, 0 C, 2 h, 33%. (e) n-BuLi, 1-
ꢀ
Scheme 1 Retrosynthetic analysis of diaryl acetylene compounds.
bromo-4-methoxybenzene, THF, ꢁ83–0 C, 2 h, not detected.
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chloride, which was prepared via the hydrolysis and chlorina-
tion of 1a. As a result, 1c was still yielded as the major product
and no product of triphenyl methol was detected. Although the
efforts to synthesize selaginellin N failed, the diphenyl ketones
1c and 1d are still important products since they bear the same
skeleton with selaginellin S, which showed inhibitory effects
16
against hepatitis B virus gene expression and replication.
During the demethylation of 1a with boron tribromide, an
unexpected isocoumarin 1e was obtained as the main product.
In view of the high steric hindrance in natural selaginellins,
we designed and synthesized a series of selaginellin analogs
(2a–2j) with a different attached position of 4-methoxyl phe-
nylacetylene group (Scheme 4). Using the similar synthetic
approach of 1a, 2a was obtained with a high yield starting with
o-iodobenzoic acid via bromination, methyl esterication,
regioselective Suzuki coupling and Sonogashira reaction.
Compared with the yield of 1a in Sonogashira reaction, the yield
Scheme 5 Synthesis of compounds 2c–2f. Reagents and conditions:
ꢀ
(
a) 4-methoxyphenylmagnesium bromide, THF, ꢁ83 C / r.t. 6 h,
ꢀ
of 2a was greatly increased due to less side reaction between 36%; (b) BBr , 0 C, 30 min, 61%; (c) n-BuLi, 1-bromo-4-methox-
3
36
ꢀ
ꢀ
meta-substituted carboxylic ester and alkynyl.
ybenzene, THF, ꢁ83 C / 0 C, 4 h, 2e: 82%, 2f: 18%.
In the next nucleophilic addition of 2a, using 4-methoxy-
phenylmagnesium bromide as the nucleophilic regent yielded
a diphenyl ketone product 2c, whereas using 4-methoxyphenyl 2.2. Biological results
bromide under the presence of n-butyllithium produced the
2
.2.1. MTT assay for tumor cell growth inhibition. The in
triphenyl methol 2e along with a side product 2f (Scheme 5). As
we expected, the less steric hindrance of 2a than that of 1a
allowed the production of triphenyl methol under the same
conditions.
vitro cytotoxic activity of the synthesized compounds against
MCF-7, HepG2 and L-O2 cells was tested using MTT assay with
cisplatin as a positive control. From the experiment data listed
in Table 1, the tested compounds with different skeletons
exhibited diverse cytotoxic effects. Compounds 1a–1e showed
weak cytotoxic effects with the exception of 1c (IC50 ¼ 7.18 mM
against MCF-7). Among compounds 2a–2j, 2a–2f showed weak
cytotoxic effects with the exception of 2d, while 2g–2j showed
moderate cytotoxic effects. The cytotoxicity of 2d was more
potent than that of 1d, suggesting that the attached position of
phenylacetylene greatly inuenced their cytotoxicity.
Compounds 2g and 2h showed much more cytotoxic effects
As Scheme 6 described, the demethylation of 2e with BBr3
under different conditions gave three different products. By
ꢀ
controlling the reaction temperature at ꢁ83 C and using small
equivalents of BBr
3
, we obtained two demethylation products 2g
ꢀ
and 2h. When the reaction temperature was set at 0 C, the
diaryluorene derivative 2i, the analog of selaginpulvilin K, was
yielded, probably from the Friedel–Cras reaction under the
catalysis of boron tribromide. In order to hydrolyze more methyl
groups, we used excessive equivalents of BBr and slowly raised
3
ꢀ
ꢀ
the reaction temperature from ꢁ83 C to 0 C. As we expected,
the demethylation and dehydration occurred simultaneously to
produce the benzyl para-quinone methide derivative 2j which
resembles selaginellin N in molecular skeleton.
Scheme 4 Synthesis of compounds 2a and 2b. Reagents and condi-
ꢀ
ꢀ
tions: (a) NBS, H
2
SO
4
, 60 C, 2 h, 86%; (b) K
CO
2 3
, CH
3
I, acetone, 40 C,
(PPh
5
h, 100%; (c) Na
2
CO
3
, 4-methoxyphenylboronic acid, PdCl
2
3 2
) ,
ꢀ
THF/H
2
O ¼ 1 : 1, 60 C, 12 h, 80%; (d) 4-methoxyphenylacetylene, CuI, Scheme
6
Synthetic route to compounds 2g–j. Reagents and
ꢀ
ꢀ
ꢀ
PdCl
2
3
(PPh )
2
, PPh
3
ꢀ
, Et
3
N, DMF, 120 C, 12 h, 78%; (e) NaOH, CH
3
OH/ conditions: (a) BBr
3
, ꢁ83 C, 1 h, 2g: 30%, 2h: 50%; (b) BBr
3
, 0 C,
ꢀ
H
2
O ¼ 4 : 1, 100 C, 2 h, 99%.
30 min, 27%; (c) BBr
3
, ꢁ83 / 0 C, 4 h, 48%.
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Table 1 Anti-proliferative activity of synthesized compounds
Table 2 HIF-1 inhibition in dual luciferase-reporter assay and cell
growth inhibition of synthesized compounds
a
IC50 (mM)
Compounds
Cisplatin
MCF-7
HepG2
L-O2
7.24 ꢃ 0.3
>100
>100
7.18 ꢃ 1.2
51.16 ꢃ 0.6
95.57 ꢃ 0.4
>100
9.66 ꢃ 0.1
>100
3.64 ꢃ 0.1
>100
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
a
b
c
d
e
a
b
c
d
e
f
>100
>100
b
b
—
—
54.75 ꢃ 1.9
55.51 ꢃ 1.9
>100
>100
>100
64.00 ꢃ 1.0
92.51 ꢃ 2.0
>100
>100
>100
>100
>100
a
a
Compounds
HRE IC50 (mM)
GI50 (mM)
6.49 ꢃ 0.1
24.99 ꢃ 0.1
7.83 ꢃ 1.2
>100
>100
>100
1
1
1
1
1
1
2a
2b
2
1
a
b
c
d
e
4.46 ꢃ 0.2
5.41 ꢃ 1.1
20.69 ꢃ 2.5
2.36 ꢃ 0.4
9.52 ꢃ 0.4
13.66 ꢃ 0.4
18.30 ꢃ 1.6
15.57 ꢃ 1.6
39.93 ꢃ 3.1
4.47 ꢃ 0.6
20.81 ꢃ 0.8
1.89 ꢃ 0.4
8.10 ꢃ 1.4
2.52 ꢃ 0.4
5.55 ꢃ 1.3
10.80 ꢃ 1.9
>100
>100
3.75 ꢃ 0.2
44.81 ꢃ 0.2
86.31 ꢃ 0.5
>100
>100
>100
>100
g
h
i
12.98 ꢃ 0.7
11.90 ꢃ 0.5
9.23 ꢃ 0.0
12.10 ꢃ 0.1
15.13 ꢃ 0.6
11.81 ꢃ 0.8
8.96 ꢃ 0.4
11.63 ꢃ 0.6
16.36 ꢃ 1.3
10.58 ꢃ 1.1
10.91 ꢃ 1.0
11.07 ꢃ 0.7
j
a
Data are mean values of three independent experiments. Errors
b
>100
>100
represent standard deviation. The IC50 value was not determined
owing to the nonlinear correlation between inhibition rates and
concentrations.
c
2d
3.24 ꢃ 0.1
2
2
2
2
e
f
g
h
>100
>100
15.48 ꢃ 0.1
10.78 ꢃ 0.3
7.58 ꢃ 0.1
4.47 ꢃ 0.5
than their methylated precursor 2e, suggesting the presence of
phenolic hydroxyl favors the antitumor activity of these 2i
b
2
j
—
compounds. Compounds 2h, 2i and 2j showed similar cytotoxic
effects although they possess different scaffolds. Interestingly,
a
Data are mean values of three independent experiments. Errors
b
compound 2j with a non-natural skeleton showed more cyto- represent standard deviation. The IC50 value was not determined.
toxic effects than its natural analogs selaginellins, enabling 2j to
be a more promising anti-tumor agent due to its advantage of
easier synthesis.
400 MHz NMR Spectrometer instrument (Bruker Avance II).
2.2.2. Cell-based reporter assays for hypoxia-inducible HRMS data were detected with LTQ Orbitrap XL ETD Mass
factor-1 (HIF-1) inhibition. Almost all solid tumors have Spectrometer instrument (Thermo Scientic). The binary
a common feature, that is, hypoxia, resulting in overexpression of solvent system (A/B) of HPLC was as follows: water (A) and
37
the HIF pathway in the tumor microenvironment. Therefore, CH OH (B); gradient condition: from 60% B to 100% B in
3
HIF inhibition has been suggested to be an attractive and 60 min. The absorbance was detected at 254 nm. Reactions were
promising therapeutic strategy for various solid tumors. To date, monitored by the thin layer chromatography (TLC) from Qing-
the mechanism underlying the anticancer effects of alkynyl dao Haiyang Chemical Co. Ltd (200 ꢂ 200 mm, 0.2–0.25 mm).
phenol derivatives has not been investigated. We here explored Chromatograms were visualized by 254 nm UV light.
the effect of synthesized alkynyl phenol derivatives on HIF
3.1.1. 2-Bromo-6-iodobenzoic acid (4). Compound
3
pathway by dual luciferase reporter system. The carborane (6.03 g, 30.16 mmol), N-iodosuccinimide (8.10 g, 36 mmol),
38
derivative 11, an HIF-1 inhibitor reported by Nakamura, was palladium acetate (673.5 mg, 3 mmol) and 50 mL DMF were
used as a reference substance. As the data listed in Table 2, all added to a 100 mL sealed tube. Then the reaction mixture was
ꢀ
tested compounds exhibited moderate to strong HIF inhibitory stirred at 100 C for 12 hours. The mixture was cooled down to
activities except 2j. Compounds 1a, 2f and 2h displayed room temperature and diluted with ethyl acetate. The solution
comparative HIF inhibitory activities and low cytotoxicity was washed with 0.5 M HCl and water. The combined extract
compared with compound 11. Different from the rest synthetic was dried by anhydrous sodium sulfate. Ethyl acetate was
derivatives, compound 2j showed potent anti-proliferative effect, evaporated under vacuum and the residue was puried by silica
but little HIF inhibitory activity was determined, suggesting gel column chromatography to afford compound 4 (8.84 g,
ꢀ
1
a distinct effect resulting from its para-quinone methide moiety. 90%). White solid; mp 153–155 C; H NMR (400 MHz, DMSO-
d
6
) d 7.09 (dd, J ¼ 7.8, 7.9 Hz, 1H), 7.68 (d, J ¼ 7.9 Hz, 1H), 7.87
ꢁ
(d, J ¼ 7.8 Hz, 1H). HRMS (ESI) (m/z): [M ꢁ H] calcd for
3. Experimental section
C H BrIO , 324.8367; found, 324.8369.
7
4
2
3.1. Chemistry
3.1.2. Methyl 2-bromo-6-iodobenzoate (5). Compound 4
All chemicals and solvents were commercially available, at least (7.80 g, 23.93 mmol), potassium carbonate (6.66 g, 48.22 mmol),
1
13
reagent grade. H NMR and C NMR spectra were recorded on 50 mL acetone and excess methyl iodide were added to a 100 mL
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round bottom ask. Then the reaction mixture was stirred at was cooled to room temperature. The solvent was evaporated
ꢀ
40
C for 5 h. Acetone was evaporated under vacuum. The under vacuum. The residue was diluted with ethyl acetate and
residue was dissolved with ethyl acetate and washed with washed with dilute hydrochloric acid and saturated brine. The
saturated brine. The combined extract was dried by anhydrous combined extract was dried by anhydrous sodium sulfate. Ethyl
sodium sulfate. Ethyl acetate was evaporated under vacuum to acetate was evaporated under vacuum. The residue was puried
ꢀ
1
afford compound 5 (8.13 g, 100%). White solid; mp 87–89 C; H by silica gel chromatography to afford compound 1b (538.0 mg,
ꢀ
NMR (400 MHz, CDCl
3
) d 7.77 (d, J ¼ 7.9 Hz, 1H), 7.56 (d, J ¼ 77%). White solid; mp 145–147 C; the purity of the compound
1
8
.1 Hz, 1H), 6.96 (t, J ¼ 8.0 Hz, 1H), 3.99 (s, 3H). HRMS (ESI) (m/ was detected by analytical HPLC to be over 95%. H NMR (400
+
z): [M + H] calcd for C
.1.3. Methyl
8
H
6
BrIO
2
, 340.8669; found, 340.8675.
MHz, acetone-d
6
) d 7.55 (dd, J ¼ 7.7, 1.3 Hz, 1H), 7.52–7.45 (m,
0
0
3
3-bromo-4 -methoxy-[1,1 -biphenyl]-2- 3H), 7.45–7.41 (m, 2H), 7.38 (dd, J ¼ 7.6, 1.3 Hz, 1H), 7.01–6.94
1
3
carboxylate (6). Compound 5 (8.13 g, 23.93 mmol), p-methox- (m, 4H), 3.83 (s, 6H); C NMR (101 MHz, acetone-d ) d 169.79,
6
yphenylboronic acid (4.36 g, 28.72 mmol), 50 mL THF and 161.07, 160.45, 140.02, 137.66, 133.87, 133.11, 130.90, 130.45,
5
0 mL water were added to a 250 mL three-necked ask and 130.36, 129.97, 121.94, 115.67, 115.05, 114.66, 93.48, 86.65,
ꢁ
stirred at room temperature. Bis-triphenylphosphine palladium 55.71, 55.56. HRMS (ESI) (m/z): [M ꢁ H] calcd for C23
H
18 4
O ,
dichloride (842.3 mg, 1.2 mmol) was added under nitrogen 357.1132; found, 357.1138.
ꢀ
0
0
atmosphere. When the temperature reached 60 C, sodium
3.1.6. (4 -Methoxy-3-((4-methoxyphenyl)ethynyl)-[1,1 -biph
carbonate (5.07 g, 47.86 mmol) was added. Then the reaction enyl]-2-yl)(4-methoxyphenyl)methanone (1c). Compound 1b
mixture was reuxed for 12 hours. The mixture was cooled to (365.4 mg, 1.02 mmol) was dissolved in 3 mL dichloromethane.
room temperature and diluted with ethyl acetate. The solution Then the reaction mixture was stirred in an ice water bath and
was washed with saturated brine. The combined extract was 0.2 mL thionyl chloride was added, followed by a drop of DMF.
dried by anhydrous sodium sulfate. Ethyl acetate was evapo- Aer being stirred for 2 hours, the solvent was evaporated under
rated under vacuum. The residue was puried by silica gel vacuum. The residue was dissolved in 2 mL THF under nitrogen
column chromatography to afford compound 6 (6.28 g, 82%). atmosphere for use. Parabromoanisole (308 mL, 2.4 mmol) and
ꢀ
1
White solid; mp 89–91 C; H NMR (400 MHz, acetone-d
6
) d 7.64 3 mL anhydrous THF were added to a 50 mL dry round bottom
(
dd, J ¼ 7.0, 1.9 Hz, 1H), 7.47–7.42 (t, 1H), 7.41 (d, J ¼ 6.0 Hz, ask under nitrogen atmosphere. Then the reaction mixture
ꢀ
1
3
3
H), 7.33 (d, J ¼ 8.7 Hz, 2H), 7.00 (d, J ¼ 8.7 Hz, 2H), 3.83 (s, 3H), was stirred at ꢁ83 C and 1.7 mL nBuLi in n-hexane solution (1.6
+ –1
.69 (s, 3H). HRMS (ESI) (m/z): [M + H] calcd for C H BrO , M L ) was added. Aer being stirred for 30 min, the prepared
1
5
13
3
21.0121; found, 321.0126.
acid chloride was added to the reaction mixture and stirred for
0
3.1.4. Methyl
4 -methoxy-3-((4-methoxyphenyl)ethynyl)- 2 h. The reaction mixture was quenched by adding saturated
0
[1,1 -biphenyl]-2-carboxylate (1a). Compound 6 (5.08 g, 15.88 ammonium chloride solution. The reaction mixture was diluted
mmol), triphenylphosphine (416.5 mg, 1.59 mmol), cuprous with ethyl acetate and washed with saturated brine. The
iodide (151.2 mg, 0.79 mmol) and 30 mL DMF were added to combined extract was dried by anhydrous sodium sulfate. Ethyl
a 250 mL three-necked ask and stirred at room temperature. acetate was evaporated under vacuum. The residue was puried
Bis-triphenylphosphine palladium dichloride (557.3 mg, 0.79 by silica gel chromatography to afford compound 1c (170.3 mg,
ꢀ
mmol) and 4-ethynylanisole (8.24 mL, 63.52 mmol) were added 38%). White solid; mp 48–49 C; the purity of the compound
1
under nitrogen atmosphere. Then 30 mL triethylamine was was detected by analytical HPLC to be over 95%. H NMR (400
ꢀ
added and reuxed at 120 C for 12 hours. The reaction mixture MHz, DMSO-d
6
) d 7.65 (d, J ¼ 8.9 Hz, 2H), 7.61–7.56 (m, 2H),
was cooled to room temperature and diluted with ethyl acetate. 7.46 (dd, J ¼ 6.1, 2.9 Hz, 1H), 7.20 (d, J ¼ 8.7 Hz, 2H), 7.03–6.97
The solution was washed with saturated ammonium chloride (m, 4H), 6.89–6.83 (m, 4H), 3.80 (s, 3H), 3.74 (s, 3H), 3.70 (s, 3H);
1
3
solution. The combined extract was dried by anhydrous sodium
6
C NMR (101 MHz, DMSO-d ) d 195.25, 163.39, 159.65, 158.73,
sulfate. Ethyl acetate was evaporated under vacuum. The 141.09, 139.22, 132.50, 131.49, 131.41, 129.99, 129.92, 129.80,
residue was puried by silica gel column chromatography to 129.33, 120.45, 114.30, 114.12, 113.78, 113.59, 93.82, 86.17,
ꢀ
+
afford compound 1a (3.0 g, 48%). White solid; mp 98–99 C; the 55.55, 55.25, 55.02. HRMS (ESI) (m/z): [M + H] calcd for
purity of the compound was detected by analytical HPLC to be C H O , 449.1747; found, 449.1744.
3
0
24 4
1
0
0
over 95%. H NMR (400 MHz, CDCl
1
8
3
) d 7.51 (dd, J ¼ 7.7, 1.1 Hz,
3.1.7. (4 -Hydroxy-3-((4-hydroxyphenyl)ethynyl)-[1,1 -biphe
H), 7.44 (d, J ¼ 8.8 Hz, 2H), 7.40 (d, J ¼ 7.7 Hz, 1H), 7.33 (d, J ¼ nyl]-2-yl)(4-methoxyphenyl)methanone (1d). Compound 1c
.7 Hz, 2H), 7.32–7.30 (m, 1H), 6.94 (d, J ¼ 8.7 Hz, 2H), 6.87 (d, J (150.8 mg, 0.34 mmol) was dissolved in 2 mL dry dichloro-
1
3
¼
8.8 Hz, 2H), 3.84 (s, 3H), 3.83 (s, 3H), 3.74 (s, 3H); C NMR methane in a 10 mL chicken heart ask. Then the reaction
(
101 MHz, DMSO-d
6
) d 168.57, 159.88, 159.11, 138.87, 135.18, mixture was stirred in an ice water bath and boron tribromide
1
1
33.03, 131.29, 129.99, 129.58, 129.17, 120.65, 114.56, 114.16, (320 mL, 3.38 mmol) was added. Aer being stirred for 30 min,
13.66, 92.89, 85.36, 55.34, 55.18, 52.21. HRMS (ESI) (m/z): [M + 3 mL diethyl ether was added. The reaction mixture was stirred
+
H] calcd for C H O , 373.1434; found, 373.1439.
for 20 min and saturated sodium bicarbonate solution was
slowly added. Then the reaction mixture was diluted with ethyl
2
4
20 4
0
0
3
.1.5. 4 -Methoxy-3-((4-methoxyphenyl)ethynyl)-[1,1 -biphe
nyl]-2-carboxylic acid (1b). Compound 1a (722.0 mg, 1.94 acetate and washed with saturated brine. The combined extract
mmol), 50 mL methanol and 10 mL 60% sodium hydroxide was dried by anhydrous sodium sulfate. Ethyl acetate was
solution were added to a 100 mL round bottom ask. Then the evaporated under vacuum. The residue was puried by silica gel
ꢀ
reaction mixture was reuxed at 80 C for 8 hours. The mixture chromatography to afford compound 1d (24.5 mg, 17%). White
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ꢀ
+
solid; mp 91–93 C; the purity of the compound was detected by (s, 3H). HRMS (ESI) (m/z): [M + H] calcd for C
8
H
6
BrIO
2
,
1
analytical HPLC to be over 95%. H NMR (400 MHz, acetone-d ) 340.8669; found, 340.8675.
d 8.73 (s, 1H), 8.39 (s, 1H), 7.70 (d, J ¼ 8.8 Hz, 2H), 7.57–7.51 (m,
6
0
0
3.1.11. Methyl
4-bromo-4 -methoxy-[1,1 -biphenyl]-2-
2
H), 7.42 (dd, J ¼ 5.9, 3.0 Hz, 1H), 7.18 (d, J ¼ 8.5 Hz, 2H), 7.01 carboxylate (10). Compound 9 (9.99 g, 29.4 mmol), p-methox-
(
2
d, J ¼ 8.5 Hz, 2H), 6.93 (d, J ¼ 8.8 Hz, 2H), 6.75 (d, J ¼ 8.5 Hz, yphenylboronic acid (5.36 g, 35.28 mmol), 50 mL THF and
1
3
H), 6.73 (d, J ¼ 8.5 Hz, 2H), 3.82 (s, 3H); C NMR (101 MHz, 50 mL water were successively added to a 250 mL three-necked
) d 195.31, 163.70, 158.01, 157.02, 141.74, 140.12, ask and stirred at room temperature. Bis-triphenylphosphine
32.86, 131.63, 131.06, 130.77, 130.21, 129.81, 129.55, 128.90, palladium dichloride (1.03 g, 1.47 mmol) was then added
21.49, 115.47, 115.06, 113.75, 113.53, 94.09, 85.79, 55.02. under nitrogen atmosphere. When the temperature reached
acetone-d
6
1
1
ꢁ
ꢀ
HRMS (ESI) (m/z): [M ꢁ H] calcd for C H O , 449.1289; 60 C, sodium carbonate (6.23 g, 58.8 mmol) was added. Then
2
8
20 4
found, 419.1293.
the reaction mixture was reuxed for 12 hours. The mixture was
3.1.8. 3,8-Bis(4-hydroxyphenyl)-1H-isochromen-1-one (1e). cooled to room temperature and diluted with ethyl acetate. The
Compound 1a (186.5 mg, 0.50 mmol) was dissolved in 2 mL dry solution was washed with saturated brine. The combined
dichloromethane in a 10 mL chicken heart ask. Then the extract was dried by anhydrous sodium sulfate. Ethyl acetate
reaction mixture was stirred in an ice water bath and boron was evaporated under vacuum. The residue was puried by
tribromide (470 mL, 4.97 mmol) was added. Aer being stirred silica gel chromatography to afford compound 10 (7.53 g, 80%).
ꢀ
1
6
for 2 hours, 3 mL diethyl ether was added. The reaction mixture White solid; mp 76–77 C; H NMR (400 MHz, acetone-d ) d 7.86
was stirred for 20 min and saturated sodium bicarbonate (d, J ¼ 2.2 Hz, 1H), 7.75 (dd, J ¼ 8.3, 2.2 Hz, 1H), 7.38 (d, J ¼
solution was slowly added. The reaction mixture was diluted 8.3 Hz, 1H), 7.28–7.21 (m, 2H), 7.02–6.95 (m, 2H), 3.84 (s, 3H),
+
with ethyl acetate and washed with saturated brine. The 3.66 (s, 3H). HRMS (ESI) (m/z): [M + H] calcd for C H BrO ,
1
5
13
3
combined extract was dried by anhydrous sodium sulfate. Ethyl 321.0121; found, 321.0126.
0
acetate was evaporated under vacuum. The residue was puried
3.1.12. Methyl 4 -methoxy-4-((4-methoxyphenyl)ethynyl)-
0
by silica gel chromatography to afford compound 1e (54.9 mg, [1,1 -biphenyl]-2-carboxylate (2a). Compound 10 (7.53 g, 23.535
3%). Light yellow solid; mp 297–299 C; the purity of the mmol), triphenylphosphine (617.3 mg, 2.35 mmol), cuprous
compound was detected by analytical HPLC to be over 95%. H iodide (224.1 mg, 1.18 mmol) and 40 mL DMF were added to
ꢀ
3
1
NMR (400 MHz, acetone-d ) d 8.87 (s, 1H), 8.36 (s, 1H), 7.80 (d, J a 250 mL three-necked ask and stirred at room temperature.
6
¼
8.7 Hz, 2H), 7.74 (t, J ¼ 7.7 Hz, 1H), 7.58 (d, J ¼ 7.8 Hz, 1H), Bis-triphenylphosphine palladium dichloride (826.0 mg, 1.18
7
.29 (d, J ¼ 7.5 Hz, 1H), 7.21 (d, J ¼ 8.4 Hz, 2H), 7.16 (s, 1H), 6.98 mmol) and 4-ethynylanisole (9.5 mL, 73.25 mmol) were added
13
(d, J ¼ 8.7 Hz, 2H), 6.87 (d, J ¼ 8.4 Hz, 2H); C NMR (101 MHz, under nitrogen atmosphere. Then 40 mL triethylamine was
ꢀ
acetone-d
6
) d 160.51, 160.13, 157.68, 154.57, 146.79, 140.77, added and reuxed at 120 C for 8 hours. The reaction mixture
1
1
C
34.62, 133.82, 131.82, 130.85, 127.65, 126.18, 124.49, 117.92, was cooled to room temperature and diluted with ethyl acetate.
ꢁ
16.66, 115.25, 100.76. HRMS (ESI) (m/z): [M ꢁ H] calcd for The solution was washed with saturated ammonium chloride
21
H
14
O
4
, 329.0819; found, 329.0829.
solution. The combined extract was dried by anhydrous sodium
sulfate. Ethyl acetate was evaporated under vacuum. The
3.1.9. 5-Bromo-2-iodobenzoic acid (8). Compound
7
(
8.44 g, 34.03 mmol) and 60 mL H SO were added to a 250 mL residue was puried by silica gel chromatography to afford
2 4
ꢀ
round bottom ask. NBS (4.82 g, 38.06 mmol) was then added at compound 2a (6.84 g, 78%). White solid; mp 138–139 C; the
6
mixture was cooled to room temperature and dropped into ice over 95%. H NMR (400 MHz, acetone-d
ꢀ
0 C and the mixture was stirred for 2 hours. Then the reaction purity of the compound was detected by analytical HPLC to be
1
6
) d 7.83 (d, J ¼ 1.8 Hz,
water. The mixture was diluted with ethyl acetate and washed 1H), 7.69 (dd, J ¼ 8.0, 1.8 Hz, 1H), 7.57–7.50 (m, 2H), 7.46 (d, J ¼
with saturated brine. The combined extract was dried by 8.0 Hz, 1H), 7.32–7.26 (m, 2H), 7.03–6.96 (m, 4H), 3.86 (s, 3H),
1
3
6
anhydrous sodium sulfate. Ethyl acetate was evaporated under 3.85 (s, 3H), 3.67 (s, 3H); C NMR (101 MHz, DMSO-d )
vacuum. The residue was puried by silica gel chromatography d 168.05, 159.75, 158.97, 140.26, 133.48, 133.09, 131.73, 131.70,
to afford compound 8 (9.54 g, 86%). White solid; mp 159– 131.26, 130.81, 129.31, 121.33, 114.45, 113.86, 90.81, 86.79,
ꢀ
1
+
1
(
61 C; H NMR (400 MHz, CDCl ) d 8.14 (d, J ¼ 2.4 Hz, 1H), 7.90 55.30, 55.14, 52.14. HRMS (ESI) (m/z): [M + H] calcd for
3
d, J ¼ 8.4 Hz, 1H), 7.33 (dd, J ¼ 8.4, 2.4 Hz, 1H). HRMS (ESI) (m/
C
24
H
20
O
4
, 373.1434; found, 373.1442.
ꢁ
0
0
z): [M ꢁ H] calcd for C
7
H
4
BrIO
2
, 324.8367; found, 324.8369.
3.1.13. 4 -Methoxy-4-((4-methoxyphenyl)ethynyl)-[1,1 -biph
3
.1.10. Methyl 5-bromo-2-iodobenzoate (9). Compound 8 enyl]-2-carboxylic acid (2b). Compound 2a (372.1 mg, 1 mmol),
(9.54 g, 29.4 mmol), potassium carbonate (8.13 g, 58.8 mmol), sodium hydroxide (1.0 g), 20 mL methanol and 5 mL water were
5
0 mL acetone and excess methyl iodide were successively added to a 100 mL round bottom ask. Then the reaction
ꢀ
added to a 100 mL round bottom ask. Then the reaction mixture was reuxed at 100 C for 2 hours. The mixture was
mixture was stirred at 40 C for 5 h. Acetone was evaporated cooled to room temperature. The solvent was evaporated under
ꢀ
under vacuum. The residue was diluted with ethyl acetate and vacuum. The residue was diluted with ethyl acetate and washed
washed with saturated brine. The combined extract was dried by with dilute hydrochloric acid and saturated brine. The
anhydrous sodium sulfate. Ethyl acetate was evaporated under combined extract was dried by anhydrous sodium sulfate. Ethyl
vacuum to afford compound 9 (9.99 g, 100%). White solid; mp acetate was evaporated under vacuum. The residue was puried
ꢀ
1
4
1
8–49 C; H NMR (400 MHz, acetone-d
6
) d 7.98 (d, J ¼ 8.4 Hz, by silica gel chromatography to afford compound 2b (354.5 mg,
ꢀ
H), 7.91 (d, J ¼ 2.4 Hz, 1H), 7.48 (dd, J ¼ 8.4, 2.4 Hz, 1H), 3.92 99%). White solid; mp 191–193 C; the purity of the compound
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1
0
was detected by analytical HPLC to be over 95%. H NMR (400 methoxy-4-((4-methoxyphenyl)ethynyl)-[1,1 -biphenyl]-2-yl)-1-(4-
MHz, DMSO-d ) d 12.98 (s, 1H), 7.79 (d, J ¼ 1.7 Hz, 1H), 7.66 (dd, methoxyphenyl)pentan-1-ol (2f). Parabromoanisole (2.03 g,
6
J ¼ 8.0, 1.8 Hz, 1H), 7.56–7.51 (m, 2H), 7.41 (d, J ¼ 8.0 Hz, 1H), 10.84 mmol) and 5 mL anhydrous THF were added to a 100 mL
7
.33–7.27 (m, 2H), 7.03–6.96 (m, 4H), 3.81 (s, 3H), 3.80 (s, 3H); dry round bottom ask under nitrogen atmosphere. Then the
1
3
ꢀ
C NMR (101 MHz, DMSO-d
6
) d 169.65, 160.20, 159.38, 140.51, reaction mixture was stirred at ꢁ83 C and 7.45 mL n-BuLi in n-
ꢁ1
1
1
33.55, 133.38, 133.31, 132.63, 131.97, 131.24, 129.92, 121.66, hexane solution (1.6 M L ) was added. Aer being stirred for 1
14.92, 114.44, 114.21, 91.06, 87.49, 55.77, 55.61. HRMS (ESI) hour, 15 mL solution of compound 2a (2.02 g, 5.42 mmol) in
ꢁ
(m/z): [M ꢁ H] calcd for C23
18 4
H O , 357.1132; found, 357.1139. THF was added to the reaction mixture. The temperature was
0
0
ꢀ
naturally raised to 0 C. The reaction mixture was stirred for 4
3
.1.14. (4 -Methoxy-4-((4-methoxyphenyl)ethynyl)-[1,1 -
biphenyl]-2-yl)(4-methoxyphenyl)methanone (2c). Compound hours and quenched by adding saturated ammonium chloride
a (744.3 mg, 2 mmol) and 5 mL of anhydrous THF were added solution. The reaction mixture was diluted with ethyl acetate
2
to a 50 mL dry round bottom ask under nitrogen atmosphere. and washed with saturated brine. The combined extract was
ꢀ
Then the reaction mixture was stirred at ꢁ83 C and 2.5 mL of p- dried by anhydrous sodium sulfate. Ethyl acetate was evapo-
ꢁ
1
methoxyphenylmagnesium bromide (1.0 M L in THF) was rated under vacuum. The residue was puried by silica gel
added. The temperature was naturally raised to room temper- chromatography to afford compound 2e (2.47 g, 82%) and
ature. Aer being stirred for 6 hours, the reaction mixture was compound 2f (493.9 mg, 18%). The purity of the two
quenched by adding saturated ammonium chloride solution. compounds was detected by analytical HPLC to be over 95%.
ꢀ
1
The reaction mixture was diluted with ethyl acetate and washed Compound 2e: pink solid; mp 179–181 C; H NMR (400 MHz,
with saturated brine. The combined extract was dried by DMSO-d ) d 8.09 (d, J ¼ 1.5 Hz, 1H), 7.51 (d, J ¼ 8.6 Hz, 2H), 7.34
6
anhydrous sodium sulfate. Ethyl acetate was evaporated under (dd, J ¼ 7.7, 1.4 Hz, 1H), 6.98 (d, J ¼ 8.6 Hz, 2H), 6.85–6.89 (m,
vacuum. The residue was puried by silica gel chromatography 3H), 6.68 (d, J ¼ 8.7 Hz, 2H), 6.61 (d, J ¼ 8.4 Hz, 2H), 6.44 (d, J ¼
to afford compound 2c (319.2 mg, 36%). White solid; mp 153– 8.2 Hz, 2H), 4.79 (s, 1H), 3.81 (s, 3H), 3.72 (s, 6H), 1.99–1.84 (m,
ꢀ
1
55 C; the purity of the compound was detected by analytical 2H), 1.30–1.11 (m, 3H), 1.03–0.88 (m, 1H), 0.79 (t, J ¼ 7.2 Hz,
1
13
6
6
HPLC to be over 95%. H NMR (400 MHz, DMSO-d ) d 7.72 (dd, J 3H); C NMR (101 MHz, DMSO-d ) d 159.51, 157.76, 157.67,
¼
8.0, 1.8 Hz, 1H), 7.64–7.59 (m, 2H), 7.52 (m, J ¼ 4.8, 3.9 Hz, 145.84, 140.71, 140.39, 134.13, 132.95, 132.33, 129.64, 128.64,
4
6
1
1
1
H), 7.23–7.16 (m, 2H), 7.01–6.97 (m, 2H), 6.97–6.93 (m, 2H), 127.71, 120.74, 114.42, 114.37, 112.71, 112.17, 89.16, 88.54,
1
3
.88–6.82 (m, 2H); C NMR (101 MHz, DMSO-d ) d 195.37, 75.70, 55.28, 55.05, 54.98, 38.47, 25.73, 22.60, 14.11. ESI MS (m/
6
+
63.36, 159.70, 158.83, 139.10, 139.06, 133.05, 132.40, 131.97, z): [M ꢁ OH] calcd for C H O , 489.24; found, 489.36.
3
4
34 4
ꢀ
1
31.31, 130.38, 130.28, 129.68, 129.32, 121.13, 114.43, 113.99, Compound 2f: white solid; mp 113–114 C; H NMR (400 MHz,
13.95, 90.74, 87.15, 55.56, 55.29, 55.06. HRMS (ESI) (m/z): [M + DMSO-d ) d 7.48–7.41 (m, 3H), 7.17 (d, J ¼ 1.7 Hz, 1H), 7.03 (d, J
6
+
H] calcd for C30
H
24
O
4
, 449.1747; found, 449.1741.
¼ 7.8 Hz, 1H), 6.95–6.99 (m, 6H), 6.77 (d, J ¼ 8.8 Hz, 4H), 6.64 (d,
J ¼ 8.7 Hz, 2H), 6.55 (d, J ¼ 8.7 Hz, 2H), 5.76 (s, 1H), 3.78 (s, 3H),
0
0
3
.1.15. (4 -Hydroxy-4-((4-hydroxyphenyl)ethynyl)-[1,1 -
1
3
biphenyl]-2-yl)(4-methoxyphenyl)methanone (2d). Compound 3.72 (s, 6H), 3.67 (s, 3H); C NMR (101 MHz, DMSO-d )
6
2c (319.2 mg, 0.71 mmol) was dissolved in 5 mL dry dichloro- d 159.54, 157.69, 157.31, 146.30, 142.35, 139.86, 134.99, 133.21,
methane added in a 50 mL round bottom ask. Then the 132.94, 131.57, 130.02, 129.47, 128.87, 120.18, 114.36, 114.04,
reaction mixture was stirred in an ice water bath and boron 112.66, 111.94, 89.35, 88.05, 80.72, 55.26, 54.99, 54.90. HRMS
+
tribromide (340 mL, 3.6 mmol) was added. Aer being stirred for (ESI) (m/z): [M ꢁ OH] calcd for C H O , 539.2222; found,
3
7
32 5
3
0 min, 10 mL diethyl ether was added. The reaction mixture 539.2209.
0
was stirred for 20 min and saturated sodium bicarbonate
3.1.17. 4-((2-(Hydroxybis(4-methoxyphenyl)methyl)-4 -
solution was slowly added. Then the reaction mixture was methoxy-[1,1 -biphenyl]-4-yl)ethynyl)phenol (2g); 2 -(hydrox-
diluted with ethyl acetate and washed with saturated brine. The ybis(4-methoxyphenyl)methyl)-4 -((4-hydroxyphenyl)ethynyl)-
combined extract was dried by anhydrous sodium sulfate. Ethyl [1,1 -biphenyl]-4-ol (2h). Compound 2e (139.1 mg, 0.25 mmol)
0
0
0
0
acetate was evaporated under vacuum. The residue was puried was dissolved in 2 mL dry dichloromethane in a 10 mL chicken
ꢀ
by silica gel chromatography to afford compound 2d (180.7 mg, heart ask. Then the reaction mixture was stirred at ꢁ83 C and
ꢀ
6
1%). White solid; mp 229–231 C; the purity of the compound boron tribromide (140 mL, 1.4 8 mmol) was added. Aer being
1
was detected by analytical HPLC to be over 95%. H NMR (400 stirred for 1 hour, 5 mL diethyl ether was added. The reaction
MHz, DMSO-d
) d 9.97 (s, 1H), 9.54 (s, 1H), 7.68 (dd, J ¼ 8.0, mixture was stirred for 20 min and saturated sodium bicar-
.7 Hz, 1H), 7.58 (d, J ¼ 8.8 Hz, 2H), 7.49 (d, J ¼ 8.1 Hz, 1H), 7.46 bonate solution was slowly added. Then the reaction mixture
d, J ¼ 1.6 Hz, 1H), 7.39 (d, J ¼ 8.6 Hz, 2H), 7.08 (d, J ¼ 8.5 Hz, was diluted with ethyl acetate and washed with saturated brine.
6
1
(
2
8
1
1
1
H), 6.93 (d, J ¼ 8.9 Hz, 2H), 6.80 (d, J ¼ 8.6 Hz, 2H), 6.65 (d, J ¼ The combined extract was dried by anhydrous sodium sulfate.
1
3
.5 Hz, 2H), 3.79 (s, 3H); C NMR (101 MHz, DMSO-d ) d 195.60, Ethyl acetate was evaporated under vacuum. The residue was
6
63.25, 158.23, 157.08, 139.30, 138.94, 133.13, 132.31, 131.88, puried by silica gel chromatography to afford compound 2g
30.34, 130.03, 129.75, 129.70, 129.35, 121.12, 115.77, 115.34, (40.6 mg, 30%) and compound 2h (66.0 mg, 50%). The purity of
13.89, 112.24, 91.16, 86.60, 55.51. HRMS (ESI) (m/z): [M + H]
, 421.1434; found, 421.1432.
.1.16. (4 -Methoxy-4-((4-methoxyphenyl)ethynyl)-[1,1 -
biphenyl]-2-yl)bis(4-methoxyphenyl)methanol
+
the two compounds was detected by analytical HPLC to be over
95%. Compound 2g: pink solid; mp 199–201 C; H NMR (400
ꢀ
1
calcd for C28
H
0
20
O
4
0
3
MHz, acetone-d ) d 8.79 (s, 1H), 7.44 (dd, J ¼ 7.8, 1.5 Hz, 1H),
6
0
(2e);
1-(4 -
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1
3
7
4
6
1
1
8
.34 (d, J ¼ 8.5 Hz, 2H), 7.09–7.11 (m, 2H), 7.06 (d, J ¼ 8.8 Hz, 2.0 Hz, 1H), 3.75 (s, 3H); C NMR (101 MHz, DMSO-d
H), 6.82–6.86 (m, 6H), 6.68–6.74 (m, 4H), 3.87 (s, 1H), 3.79 (s, 160.71, 159.69, 158.27, 156.77, 142.07, 139.27, 138.91, 138.67,
6
) d 185.55,
1
3
H), 3.76 (s, 3H); C NMR (101 MHz, acetone-d ) d 158.67, 134.37, 133.29, 133.09, 132.20, 130.86, 130.66, 129.96, 129.59,
58.65, 157.93, 146.40, 141.46, 139.97, 134.23, 133.13, 133.02, 128.95, 128.06, 127.90, 121.07, 115.78, 115.01, 113.62, 112.16,
6
ꢁ
32.11, 130.40, 129.59, 129.10, 121.43, 115.60, 113.74, 112.80, 91.18, 86.62, 55.36. HRMS (ESI) (m/z): [M ꢁ H] calcd for
ꢁ
9.68, 87.29, 82.03, 54.58. HRMS (ESI) (m/z): [M ꢁ H] calcd for
34 24 4
C H O , 495.1602; found, 495.1618.
36 30 5
C H O , 541.2020; found, 541.2011. Compound 2h: pink
ꢀ
1
solid; mp 119–121 C; H NMR (400 MHz, acetone-d
6
) d 8.76 (s,
H), 8.37 (s, 1H), 7.43 (d, J ¼ 8.3 Hz, 1H), 7.33 (d, J ¼ 8.5 Hz, 2H),
.05–7.11 (m, 6H), 6.83–6.86 (m, 6H), 6.64 (q, J ¼ 8.7 Hz, 4H),
3.2. Biological assays
1
7
3
3
.2.1. MTT assay for cell growth inhibition. The inhibition
1
3
of compounds 1a–1e and 2a–2j against MCF-7, HepG2, HeLa
and L-O2 cells were evaluated using a standard MTT-based
colorimetric assay. Three thousand corresponding cells per
.78 (s, 6H), 3.75 (s, 1H); C NMR (101 MHz, acetone-d )
6
d 158.70, 157.90, 156.46, 146.40, 141.52, 140.03, 133.17, 133.01,
1
1
32.87, 132.13, 130.50, 129.59, 129.09, 121.35, 115.60, 114.43,
13.77, 112.82, 89.65, 87.32, 82.17, 54.59. HRMS (ESI) (m/z): [M
ꢀ
well were seeded into 96-well plates and incubated at 37 C, 5%
ꢁ
CO2 for 24 h. Then 100 mL drug-containing medium with
a series of concentration were dispensed into wells to maintain
the nal concentration as 100, 50, 25, 12.5, 6.25 and 3.125 mM,
respectively. Each concentration was in triplicate. Aer 48 hours
of incubation, cell survival was determined by the addition of 20
mL MTT (Sigma-Aldrich, St. Louis, USA) work solution (5 mg
ꢁ
H] calcd for C35
H
28
O
5
, 527.1864; found, 527.1866.
3.1.18. 7-((4-Hydroxyphenyl)ethynyl)-9,9-bis(4-methoxy-
phenyl)-9H-uoren-2-ol (2i). Compound 2e (155.7 mg, 0.28
mmol) was dissolved in 2 mL dry dichloromethane in a 10 mL
chicken heart ask. Then the reaction mixture was stirred in an
ice water bath and boron tribromide (110 mL, 1.16 mmol) was
added. Aer being stirred for 30 min, 5 mL diethyl ether was
added. The reaction mixture was stirred for 20 min and satu-
rated sodium bicarbonate solution was slowly added. Then the
reaction mixture was diluted with ethyl acetate and washed with
saturated brine. The combined extract was dried by anhydrous
sodium sulfate. Ethyl acetate was evaporated under vacuum.
The residue was puried by silica gel chromatography to afford
ꢁ1
mL MTT dissolved in phosphate buffer solution). Aer post-
incubation at 37 C for 4 hours, the medium was discarded
ꢀ
followed by adding 150 mL DMSO (Sigma-Aldrich, St. Louis,
USA). The plates were then vortexed for 10 min for complete
dissolution. The optical absorbance was measured at 570 nm.
The data represented the mean of three independent experi-
ments in triplicate and were expressed as mean ꢃ SD. The IC50
value was dened as the concentration at which 50% of the cells
could survive.
3.2.2. Cell-based reporter assays for hypoxia inducible
factor-1 (HIF-1) inhibition. HeLa cells expressing HRE-
dependent rey luciferase reporter construct (HRE-Luc) and
constitutively expressing CMV-driven Renilla luciferase reporter
with SureFECT transfection reagent were established with
Cignal™ Lenti Reporter (SABiosciences, Frederick, MD)
according to the manufacturer's instructions. The consensus
ꢀ
compound 2i (38.8 mg, 27%). White solid; mp 125–127 C; the
purity of the compound was detected by analytical HPLC to be
over 95%. H NMR (400 MHz, acetone-d
1
6
) d 8.75 (s, 1H), 8.53 (s,
1
8
6
1
1
1
H), 7.71–7.75 (m, 2H), 7.46 (d, J ¼ 8.1 Hz, 2H), 7.37 (d, J ¼
.5 Hz, 2H), 7.12 (d, J ¼ 8.8 Hz, 4H), 6.91–6.80 (m, 8H), 3.74 (s,
1
3
6
H); C NMR (101 MHz, acetone-d ) d 158.61, 157.97, 157.77,
54.32, 151.69, 140.25, 137.69, 132.94, 130.90, 130.68, 129.01,
28.52, 121.54, 121.04, 119.21, 115.58, 115.05, 114.12, 113.57,
13.02, 89.44, 88.09, 63.93, 54.56. HRMS (ESI) (m/z): [M ꢁ H]
ꢁ
0
0
sequence of HRE was 5 -TACGTGCT-3 from the erythropoietin
gene. Cells stably expressing the HRE-reporter gene were
selected with puromycin. The cells were incubated for 12 hours
with or without drugs under the normoxic or hypoxic condition
calcd for C35
H
26
O
4
, 509.1758; found, 509.1753.
0
0
3
.1.19. 4-((4 -Hydroxy-4-((4-hydroxyphenyl)ethynyl)-[1,1 -
biphenyl]-2-yl)(4-methoxyphenyl)methylene)cyclohexa-2,5-dien-
1-one (2j). Compound 2e (2.23 g, 4.0 mmol) was dissolved in
(
1% O ). Aer incubation, the luciferase assay was performed
2
40 mL dry dichloromethane in a 250 mL round bottom ask.
ꢀ
using a Luciferase Assay System (Promega, Madison, WI)
according to the manufacturer's instructions. The drug
concentration required to inhibit the relative light units by 50%
IC50) was determined from semi-logarithmic dose–response
plots, and the results represent means ꢃ SD of triplicate
Then the reaction mixture was stirred at ꢁ83 C and boron
tribromide (7.6 mL, 80.39 mmol) was added. The temperature
ꢀ
was slowly raised to 0 C. Aer being stirred for 4 hours, 30 mL
diethyl ether was added. The reaction mixture was stirred for
(
20 min and saturated sodium bicarbonate solution was slowly
samples.
added. Then the reaction mixture was diluted with ethyl acetate
and washed with saturated brine. The combined extract was
dried by anhydrous sodium sulfate. Ethyl acetate was evapo-
rated under vacuum. The residue was puried by silica gel
4
. Conclusion
chromatography to afford compound 2j (943.4 mg, 48%). Red In conclusion, we designed and synthesized a class of natural
ꢀ
solid; mp 191–193 C; the purity of the compound was detected product-like biphenyl-containing diaryl acetylenes mimicking
1
by analytical HPLC to be over 95%. H NMR (400 MHz, DMSO- natural alkynyl phenols from the genus Selaginella. In MTT
d
6
) d 10.01 (s, 1H), 9.49 (s, 1H), 7.66 (dd, J ¼ 8.0, 1.6 Hz, 1H), 7.43 assay in cancer cells, compounds 1c, 2d, 2g, 2h, 2i and 2j
(
1
d, J ¼ 8.0 Hz, 1H), 7.41–7.33 (m, 4H), 7.22 (dd, J ¼ 9.9, 2.6 Hz, exhibited potent cytotoxic activity. The evaluation of HIF-1
H), 6.85–6.94 (m, 6H), 6.81 (d, J ¼ 8.6 Hz, 2H), 6.59 (d, J ¼ inhibitory activity demonstrated that all tested compounds
.5 Hz, 2H), 6.33 (dd, J ¼ 10.0, 1.9 Hz, 1H), 6.29 (dd, J ¼ 9.9, exhibited moderate to good activities except 2j. Compounds 1a,
8
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2
f and 2h displayed high HIF-1 inhibitory activities and rela- 14 P. Nguyen, B. Zhao, M. Y. Ali, J. Choi, D. Rhyu, B. Min and
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Conflicts of interest
There are no conicts to declare.
1
1
2
2
2
Acknowledgements
This research was supported by the National Natural Science
Foundation of China (Grant No. 21606036) and the Funda-
mental Research Funds for the Central Universities
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