Design, synthesis of a novel 4-O-methylsaucerneol analogue LXY7824 as potent HIF-1 inhibitor and anti-cancer agent

Article abstract of DOI:10.1080/10286020.2018.1473386

Hypoxia-inducible factor-1 (HIF-1), an important transcription factor for tumor survival, is an attractive target for anti-cancer treatment. Herein, we present the design and synthesis of LXY7824, a simplified analogue of 4-O-methylsaucerneol. In addition

Full text of DOI:10.1080/10286020.2018.1473386

Journal of Asian Natural Products Research
ISSN: 1028-6020 (Print) 1477-2213 (Online) Journal homepage: http://www.tandfonline.com/loi/ganp20
Design, synthesis of a novel 4-O-methylsaucerneol
analogue LXY7824 as potent HIF-1 inhibitor and
anti-cancer agent
Xiao-Yu Liu, Xiao-Yu Li, Fei-Long Yang, Yan Li, Xiao-Zhen Jiao & Ping Xie
To cite this article: Xiao-Yu Liu, Xiao-Yu Li, Fei-Long Yang, Yan Li, Xiao-Zhen Jiao & Ping
Xie (2018): Design, synthesis of a novel 4-O-methylsaucerneol analogue LXY7824 as potent
HIF-1 inhibitor and anti-cancer agent, Journal of Asian Natural Products Research, DOI:
10.1080/10286020.2018.1473386
To link to this article: https://doi.org/10.1080/10286020.2018.1473386
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Journal of asian natural Products research, 2018
ꢁꢁpꢂ://ꢃꢄꢅ.ꢄꢆg/10.1080/10286020.2018.1473386
ꢀ
Design, synthesis of a novel 4-O-methylsaucerneol analogue
LXY7824 as potent HIF-1 inhibitor and anti-cancer agent
Xiao-Yu Liu, Xiao-Yu Li, Fei-Long Yang, Yan Li, Xiao-Zhen Jiao and Ping Xie
sꢁꢇꢁꢈ Kꢈy lꢇbꢄꢆꢇꢁꢄꢆy ꢄꢉ Bꢅꢄꢇꢊꢁꢅvꢈ sꢋbꢂꢁꢇꢌꢊꢈ ꢇꢌꢃ fꢋꢌꢊꢁꢅꢄꢌ ꢄꢉ nꢇꢁꢋꢆꢇꢍ Mꢈꢃꢅꢊꢅꢌꢈ, Bꢈꢅjꢅꢌg Kꢈy lꢇbꢄꢆꢇꢁꢄꢆy ꢄꢉ
aꢊꢁꢅvꢈ sꢋbꢂꢁꢇꢌꢊꢈ dꢅꢂꢊꢄvꢈꢆy ꢇꢌꢃ dꢆꢋggꢇbꢅꢍꢅꢁy evꢇꢍꢋꢇꢁꢅꢄꢌ, iꢌꢂꢁꢅꢁꢋꢁꢈ ꢄꢉ Mꢇꢁꢈꢆꢅꢇ Mꢈꢃꢅꢊꢇ, cꢀꢅꢌꢈꢂꢈ aꢊꢇꢃꢈmy ꢄꢉ
Mꢈꢃꢅꢊꢇꢍ sꢊꢅꢈꢌꢊꢈꢂ ꢇꢌꢃ Pꢈkꢅꢌg uꢌꢅꢄꢌ Mꢈꢃꢅꢊꢇꢍ cꢄꢍꢍꢈgꢈ, Bꢈꢅjꢅꢌg 100050, cꢀꢅꢌꢇ
ABSTRACT
ARTICLE HISTORY
rꢈꢊꢈꢅvꢈꢃ 1 fꢈbꢆꢋꢇꢆy 2018
aꢊꢊꢈpꢁꢈꢃ 2 Mꢇy 2018
Hypoxia-inducible factor-1 (HIF-1), an important transcription factor
for tumor survival, is an attractive target for anti-cancer treatment.
Herein, we present the design and synthesis of LXY7824, a simplified
analogue of 4-O-methylsaucerneol. In addition, its significant HIF-1
inhibitory activity and potent anti-cancer activity in vivo and in vitro
were also reported.
KEYWORDS
lXY7824; hif-1; ꢇꢌꢇꢍꢄgꢋꢈ;
ꢅꢌꢀꢅbꢅꢁꢅꢄꢌ
1
. Introduction
Cancer has the characteristic features of high morbidity and high mortality and becomes
a serious threat to public health and life. However, the toxicity of the traditional chemo-
therapeutic drugs restricted them to be used. erefore, more and more attention has been
paid to low-toxicity and high-selectivity therapy.
CONTACT Xꢅꢇꢄ-Zꢀꢈꢌ Jꢅꢇꢄ
jꢅꢇꢄxz@ꢅmm.ꢇꢊ.ꢊꢌ
tꢀꢈ ꢂꢋppꢍꢈmꢈꢌꢁꢇꢆy mꢇꢁꢈꢆꢅꢇꢍ ꢉꢄꢆ ꢁꢀꢅꢂ pꢇpꢈꢆ ꢅꢂ ꢇvꢇꢅꢍꢇbꢍꢈ ꢄꢌꢍꢅꢌꢈ ꢇꢁ ꢀꢁꢁpꢂ://ꢃꢄꢅ.ꢄꢆg/10.1080/10286020.2018.1473386.
©
2018 iꢌꢉꢄꢆmꢇ uK lꢅmꢅꢁꢈꢃ, ꢁꢆꢇꢃꢅꢌg ꢇꢂ tꢇyꢍꢄꢆ & fꢆꢇꢌꢊꢅꢂ Gꢆꢄꢋp

2
X.ꢀY. LIU ET AL.
In human solid tumors, there are hypoxic regions and the tumor cells within these regions
show resistance to both radiotherapy and chemotherapy [1,2]. Hypoxia-inducible factor-1
(
HIF-1), which regulates cellular oxygen homeostasis, is an important transcription factor
for tumor survival. It is a heterodimeric (α/β) transcriptional factor and its activity relies
on HIF-1α because this protein is degraded in O -dependent manner [3–6]. e levels of
2
HIF-1α are low under normal oxygen conditions (normoxia), but increase in response to
hypoxia in solid tumor. HIF-1 is always over-expressed in various cancers as a result of this
hypoxia and genetic alterations affecting key oncogenes (VEGF, HER2, FRAP, H-RAS, and
c-SRC) and tumor suppressor genes. erefore, HIF-1 is regarded as an attractive target for
anti-cancer treatment [7–10].
Due to the importance of HIF-1 in tumor development and progression, a considerable
amount of effort has been made to identify HIF-1 inhibitors for treatment of cancer [11].
As shown in Figure 1, various HIF-1 inhibitors have been reported, such as Topotecan [12],
PX-478 [13,14], AC1-001 [15,16] and GN6767 [17,18].
In our previous studies, LXY6006, a simplified compound of manassantin A, has shown
potential HIF-1 inhibitory and anti-cancer activity [19]. But in our further research we
find LXY6006 possesses some toxicities such as red eyes of mice, death of mice as admin-
istration up to 60 mg/kg. 4-O-methylsaucerneol (IC 20 nmol), a simplified compound
5
0
of manassantin A from Saururus chinensis, was reported as a sensitive inhibitor of HIF-1
20]. is illustrated that the single side derivative could keep the HIF-1 inhibitory activity.
[
In order to find novel, efficient, and low-toxicity HIF-1 inhibitor, a 4-O-methylsaucerneol
analogue LXY7824, replacing the tetrahydrofuran moiety of 4-O-methylsaucerneol with
a pyrrolidine ring, was designed and synthesized (Figure 2). Such a molecular design not
only improves the chemical accessibility but also achieves structural novelty. e antitumor
activity of LXY7824 was investigated both in vitro and in vivo.
2
. Results and discussion
2
.1. Chemistry
e preparation of target compound LXY7824 was described in Scheme 1.
N
HO
O
NH
OH
2
N
+
Cl
N
N
O
-
O
HCl
O
O
Cl
PX-478
topotecan
COOH
H
H
N
O
O
N
N
MeOOC
O
H
N
H
OH
AC1-001
GN6767
Figure 1. sꢁꢆꢋꢊꢁꢋꢆꢈꢂ ꢄꢉ hif-1 ꢅꢌꢀꢅbꢅꢁꢄꢆꢂ.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
3
Figure 2. tꢀꢈ ꢂꢁꢆꢋꢊꢁꢋꢆꢈꢂ ꢄꢉ mꢇꢌꢇꢂꢂꢇꢌꢁꢅꢌ a, lXY6006, 4-O-mꢈꢁꢀyꢍꢂꢇꢋꢊꢈꢆꢌꢈꢄꢍ ꢇꢌꢃ lXY7824.
Our synthesis commenced with 4-benzyloxy-3-methoxyphenylacetic acid using Evans
oxazolidinone methodology to get compound 3 in high yield [21]. Hydrolysis of Evans
product 3 with LiBH gave 4 with 99% yield. Treatment of 4 with methanesulfonyl chloride
4
and NEt followed by NaN in DMF gave azide product 6. Staudinger reduction of azide 6
3
3
with triphenylphosphine (PPh ) yielded primary amine 7 in 97% yield. Af er hydrolysis of
3
amine 7 with trifluoroacetic acid, an intramolecular amidation produced compound 9 in
great yield. With chiral lactam 9 in hand, we next embarked to introduce aromatic moiety.
Buchwald–Hartwig [22] N-arylation of 9 gave 10 in moderate yield. Af er reduction of
lactam to pyrrolidine with LiAlH , followed by catalytic hydrogenation reduction, we got key
4
intermediate 12 in 71.4% yield for two steps. Treatment of 12 with 13 in acetonitrile (Cs CO
2
3
as a base) gave 14 in 85% yield. At last, compound 14 with 2, 6-di-tert-butyl-4-methylphenol
and diisobutyl aluminum hydride provided the target compound LXY7824.
2
.2. Biological evaluation
2
.2.1. HIF-1 reporter activity assays
LXY7824 was initially evaluated using a T47D human breast tumor cell-based lucif-
erase reporter assay for HIF-1 inhibitory activity. LXY7824 dose-dependently inhib-
ited the HIF activity with the IC values of 22.8 nmol/L, the same level compared with
5
0
4
-O-methylsaucerneol (Table 1).

4
X.ꢀY. LIU ET AL.
Table 1. hif-1-ꢅꢌꢀꢅbꢅꢁꢄꢆy ꢇꢊꢁꢅvꢅꢁy ꢄꢉ lXY7824.
Inhibition (%)
Compound
0.5
5
50
IC50 (nmol/L)
lXY7824
3.1
30.2
60.9
22.8
Scheme 1. syꢌꢁꢀꢈꢂꢅꢂ ꢄꢉ ꢁꢇꢆgꢈꢁ ꢊꢄmpꢄꢋꢌꢃ lXY7824. rꢈꢇgꢈꢌꢁꢂ ꢇꢌꢃ ꢊꢄꢌꢃꢅꢁꢅꢄꢌꢂ: (ꢇ) socꢍ , ꢉꢄꢍꢍꢄwꢈꢃ by n-
2
Bꢋlꢅ, (s)-4-bꢈꢌzyꢍ-2-ꢄxꢇzꢄꢍꢅꢃꢅꢌꢄꢌꢈ, thf; (b) Bꢆch cooꢁBꢋ, nꢇhMds, thf; (ꢊ) lꢅBh , thf/eꢁ o; (ꢃ) Mꢂcꢍ,
2
4
2
neꢁ , ch cꢍ ; (ꢈ) nꢇn , dMf; (ꢉ) PPꢀ , thf, ꢁꢀꢈꢌ h o; (g) cf cooh, ch cꢍ ; (ꢀ) n-(3-dꢅmꢈꢁꢀyꢍꢇmꢅꢌꢄpꢆꢄpyꢍ)-
3
2
2
3
3
2
3
2
2
n’-ꢈꢁꢀyꢍꢊꢇꢆbꢄꢃꢅꢅmꢅꢃꢈ ꢀyꢃꢆꢄꢊꢀꢍꢄꢆꢅꢃꢈ (edci), 1-hyꢃꢆꢄxybꢈꢌzꢄꢁꢆꢅꢇzꢄꢍꢈ (hoBt), n,n-dꢅꢅꢂꢄpꢆꢄpyꢍꢈꢁꢀyꢍꢇmꢅꢌꢈ
diPea), ch cꢍ ; (ꢅ) Pꢃ(oaꢊ) , Xꢇꢌꢁpꢀꢄꢂ, cꢂ co , 1,4-dꢅꢄxꢇꢌꢈ; (j) lꢅaꢍh , thf; (k) Pꢃ-c, ch cꢍ /Mꢈoh; (ꢍ)
(
2
2
2
2
3
4
2
2
cꢂ co , 13, ch cn; (m) 2,6-ꢃꢅ-ꢁꢈꢆꢁ-bꢋꢁyꢍ-4-mꢈꢁꢀyꢍpꢀꢈꢌꢄꢍ, ꢃꢅꢅꢂꢄbꢋꢁyꢍ ꢇꢍꢋmꢅꢌꢋm ꢀyꢃꢆꢅꢃꢈ, eꢁ o.
2
3
3
2
2
.2.2. Western blotting analysis
LXY7824 was next evaluated for the inhibition of the expression of HIF by western blotting
analysis. In Daoy cells, hypoxic exposure (1% O 6 h) leads to the accumulation of HIF-1α pro-
2,
tein. LXY7284 could inhibit the expression of HIF with dose-dependent manner (Figure 3).

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
5
Figure 3. lXY7824 ꢅꢌꢀꢅbꢅꢁꢂ hif-1 ꢈxpꢆꢈꢂꢂꢅꢄꢌ. aꢉꢁꢈꢆ ꢀypꢄxꢅꢊ ꢈxpꢄꢂꢋꢆꢈ (1% o , 6 ꢀ) ꢅꢌ ꢁꢀꢈ pꢆꢈꢂꢈꢌꢊꢈ ꢄꢉ ꢁꢈꢂꢁ
2
ꢊꢄmpꢄꢋꢌꢃ, ꢍꢈvꢈꢍꢂ ꢄꢉ hif-1 pꢆꢄꢁꢈꢅꢌ ꢅꢌ dꢇꢄy ꢊꢈꢍꢍꢂ wꢈꢆꢈ ꢃꢈꢁꢈꢆmꢅꢌꢈꢃ by wꢈꢂꢁꢈꢆꢌ bꢍꢄꢁ.
Table 2. tꢀꢈ ꢅꢌꢀꢅbꢅꢁꢅꢄꢌ ꢄꢉ lXY7824, Gdc0449, iꢆꢈꢂꢂꢇ ꢄꢌ ꢁꢀꢈ pꢆꢄꢍꢅꢉꢈꢆꢇꢁꢅꢄꢌ ꢄꢉ vꢇꢆꢅꢄꢋꢂ ꢊꢇꢌꢊꢈꢆ ꢊꢈꢍꢍꢂ.
Comp.
IC50 (μmol/L)
U-87MG
Hs683
U251
T98G
Daoy
SH-SY5Y
SK-N-SH
SVGP12
lXY7824
Gdc0449
iꢆꢈꢂꢂꢇ
16.37
11.46
38.71
34.59
245.00
18.53
29.98
>500
29.08
48.43
304.84
13.76
12.25
>500
24.6
55.97
243.37
53.92
41.00
303.04
19.63
307.31
474.43
44.67
2
.2.3. In vitro cytotoxicity evaluation of LXY7824
In order to determinate the anti-cancer activity of LXY7824, it was tested the inhibi-
tion activity against eight tumor cell lines and a human normal brain cell SVGP12 using
3
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method with Iressa
and GDC0449 as the positive control. e eight tumor cell lines included human glioma
cells U-87 MG, Hs683, U251, T98G, medulloblastoma cell line Daoy, human neuroblas-
toma cell lines SH-SY5Y and SK-N-SH. e results indicated that U-87 MG and Daoy were
more sensitivity, and the IC values were 16.37 and 12.25 μmol/L respectively, suggesting
5
0
LXY7824 was sensitive to brain tumor (Table 2, Figure 4).
2

.2.4. In vivo studies
e results of the cytotoxicity evaluation of LXY7824 showed that it was sensitive to brain
tumor. erefore, in vivo studies of LXY7824 were further evaluated using Daoy xenograf s
model. e results showed that tumor growth was significantly suppressed in mice admin-
istrated with LXY7824 (100 mg/kg), with relative tumor volume (RTV, T/C) to 40.57% at
the end of the treatment, and inhibition of tumor weight was 55.14% (Table 3, Figure 5).
3
. Conclusions
Starting from 4-benzyloxy-3-methoxyphenylacetic acid, we get a simplified product LXY7824
of 4-O-methylsaucerneol in 13 steps with 18.3% overall yield. In this study, we found that
LXY7284 could inhibit the expression of HIF with dose-dependent manner. Moreover,
LXY7824 is sensitive to brain tumor and significantly inhibited growth of brain tumor
in nude mice. It can be concluded that chemical modification of 4-O-methylsaucerneol
certainly holds great promise, and that further exploration in this field may lead to potent
anticancer agents.

6
X.ꢀY. LIU ET AL.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
7
Figure 4. tꢀꢈ ꢃꢄꢂꢈ-ꢈffꢈꢊꢁ ꢆꢈꢍꢇꢁꢅꢄꢌꢂꢀꢅp ꢄꢉ lXY7824 ꢄꢌ pꢆꢄꢍꢅꢉꢈꢆꢇꢁꢅꢄꢌ ꢅꢌ dꢇꢄy ꢊꢈꢍꢍ ꢍꢅꢌꢈ.
Figure 5. tꢀꢈ ꢅꢌꢀꢅbꢅꢁꢅꢄꢌ ꢈffꢈꢊꢁ ꢄꢉ lXY7824 ꢄꢌ ꢁꢀꢈ gꢆꢄwꢁꢀ ꢅꢌ mꢈꢃꢋꢍꢍꢄbꢍꢇꢂꢁꢄmꢇ dꢇꢄy xꢈꢌꢄgꢆꢇꢉꢁ. aꢌꢅmꢇꢍꢂ
3
wꢈꢆꢈ ꢆꢇꢌꢃꢄmꢍy ꢃꢅvꢅꢃꢈꢃ ꢅꢌꢁꢄ ꢁwꢄ gꢆꢄꢋpꢂ, wꢀꢈꢌ ꢁꢋmꢄꢆꢂ gꢆꢈw ꢁꢄ ꢇꢌ ꢇvꢈꢆꢇgꢈ vꢄꢍꢋmꢈ ꢄꢉ 200 mm . oꢌꢈ gꢆꢄꢋp
ꢆꢈꢊꢈꢅvꢈꢃ p.ꢄ (ꢄꢆꢇꢍ gꢇvꢇgꢈ) ꢄꢉ cꢆꢈmꢄpꢀꢄꢆ el/ꢈꢁꢀꢇꢌꢄꢍ/wꢇꢁꢈꢆ ꢇꢌꢃ ꢂꢈꢆvꢈꢃ ꢇꢂ ꢇ vꢈꢀꢅꢊꢍꢈ ꢊꢄꢌꢁꢆꢄꢍ; ꢁꢀꢈ ꢄꢁꢀꢈꢆ gꢆꢄꢋp
ꢆꢈꢊꢈꢅvꢈꢃ ꢇꢌ ꢄꢆꢇꢍ ꢃꢄꢂꢈ ꢄꢉ 100 mg/kg lXY7824 ꢈvꢈꢆy ꢃꢇy ꢉꢄꢆ 10 ꢃꢇyꢂ. Mꢅꢊꢈ wꢈꢆꢈ ꢈꢋꢁꢀꢇꢌꢅzꢈꢃ ꢇꢁ ꢁꢀꢈ ꢈꢌꢃ ꢄꢉ
ꢁꢀꢈ ꢁꢆꢈꢇꢁmꢈꢌꢁ pꢈꢆꢅꢄꢃ. tꢋmꢄꢆꢂ wꢈꢆꢈ ꢆꢈmꢄvꢈꢃ ꢇꢌꢃ wꢈꢅgꢀꢈꢃ. tꢋmꢄꢆ gꢆꢄwꢁꢀ ꢊꢋꢆvꢈꢂ wꢈꢆꢈ pꢍꢄꢁꢁꢈꢃ ꢉꢄꢆ dꢇꢄy
xꢈꢌꢄgꢆꢇꢉꢁꢂ wꢅꢁꢀ ꢇꢃmꢅꢌꢅꢂꢁꢆꢇꢁꢅꢄꢌ ꢄꢉ lXY7824 100 mg/kg ꢇꢁ gꢅvꢈꢌ ꢁꢅmꢈ-pꢄꢅꢌꢁꢂ. Mꢈꢇꢌ rtV ꢇꢁ gꢅvꢈꢌ-pꢄꢅꢌꢁꢂ
ꢉꢄꢆ dꢇꢄy xꢈꢌꢄgꢆꢇꢉꢁ.
4
. Experimental
4
.1. General experimental procedures
1H NMR and 13C NMR were recorded on Mercury 400, Bruker AV500, AV600, JEOL
ECZ400S spectrometer. Coupling constants are given in Hz and chemical shif s are expressed
as δ values in ppm. e following multiplicity abbreviations are used: (s) singlet, (d) doublet,
(
t) triplet, (q) quartet, (m) multiplet. ESI-HRMS data were measured on ermo Exactive
Orbitrap plus spectrometer. All the chemicals were purchased from commercial sources:
Sigma-Aldrich Chemical Co., Arcos Chemical Co., or J&K Chemical Co. with the purity
of more than 95%. Solvents were dried according to standard procedures when needed.
Flash column chromatography was performed on Biotage Isolera one or carried out on
silica gel (200–300 mesh).

8
X.ꢀY. LIU ET AL.
4
.2. (S)-4-Benzyl-3-(2-(4-(benzyloxy)-3-methoxyphenyl)acetyl)oxazolidin-2-one
(
2)
To a solution of (S)-4-benzyl-2-oxazolidinone (6.43 g, 36.4 mmol) in 100-ml THF, n-BuLi
(
2.5 M, 14.5 ml, 36.4 mmol) was added at −78 °C, and then stirred for 30 min. Af er that,
acyl chloride of 1 (10.5 g, 36.4 mmol) which was prepared from 1 with SOCl was added
2
to the reaction mixture and allowed to stir for 3 h at room temperature. en the reaction
was quenched by sat. NH Cl aq solution, diluted with ethyl acetate (300 ml), and washed
4
with brine, dried, and evaporated to afford the crude product, which was purified by col-
umn chromatography on silica gel (EtOAc/petroleum ether, 1:5) to give 2 (15.7 g, 99%) as
a white solid;
2
0
1
[
ꢀ] + 60.5 (c 0.5, CHCl ); R 0.28 (3:1 petroleum ether–ethyl acetate); H NMR (400 MHz,
D
3 f
CDCl ) δ 6.83–7.45 (m, 13H), 5.16 (s, 2H), 4.66–4.69 (m, 1H), 4.26–4.29 (m, 1H), 3.89–4.22
3
1
3
(
m, 3H), 3.89 (s, 3H), 3.25 (d, J = 12.8 Hz, 1H), 2.73–2.78 (m, 1H); C NMR (100 MHz,
CDCl ) δ 171.3, 153.3, 149.5, 147.3, 137.1, 135.0, 129.4, 128.9, 128.5, 127.7, 127.3, 127.2,
3
+
1
(
26.4, 121.9, 113.8, 113.4, 70.9, 66.0, 55.9, 55.2, 40.9, 37.6; HRESIMS: m/z 432.1796 [M + H]
calcd for C H O N, 432.1806).
26 26 5
4
.3. tert-Butyl (S)-4-((S)-4-benzyl-2-oxooxazolidin-3-yl)-3-(4-(benzyloxy)-3-
methoxyphenyl)-4-oxobutanoate (3)
A solution of NaHMDS in THF (2.0 M, 3 ml, 6 mmol) was added to a solution of 2 (2.23 g,
5
mmol) in 50-ml THF at −78 °C. Af er 1.5 h, tert-Butyl bromoacetate (0.9 ml, 6 mmol)
was added and the reaction was stirred for 2 h. e reaction was quenched by sat. NH Cl aq
4
solution, diluted with ethyl acetate (200 ml), and washed with brine, dried, and evaporated
to afford the crude product, which was purified by column chromatography on silica gel
(
(
EtOAc/petroleum ether, 1:15) to give 3 (2.3 g, 85%) as a white solid.
20
1
[
ꢀ] + 104.5 (c 1.0, CHCl ); R 0.48 (3:1 petroleum ether–ethyl acetate); H NMR
D
3 f
400 MHz, CDCl ) δ 6.79–7.43 (m, 13H), 5.42 (dd, J = 4.4, 11.2 Hz, 1H), 5.12 (s, 2H),
3
4
2
1
8
.55–4.60 (m, 1H), 4.02–4.11 (m, 2H), 3.88 (s, 3H), 3.24–3.39 (m, 2H), 2.78–2.81 (m, 1H),
1
3
.60 (dd, J = 4.4, 16.8 Hz, 1H), 1.43 (s, 9H); C NMR (100 MHz, CDCl ) δ 173.5, 171.0,
3
52.8, 149.6, 147.7, 137.1, 135.7, 129.9, 129.5, 128.9, 128.6, 127.8, 127.3, 120.7, 113.9, 112.2,
+
0.9, 71.0, 65.7, 56.0, 55.8, 44.2, 40.2, 37.6, 28.1; HRESIMS: m/z 568.2296 [M + Na] (calcd
for C H O NNa, 568.2306).
3
2
35
7
4
.4. tert-Butyl (S)-4-((S)-4-benzyl-2-oxooxazolidin-3-yl)-3-(4-(benzyloxy)-3-
methoxyphenyl)-4-oxobutanoate (4)
To a stirred solution of 3 (2.4 g, 4.4 mmol) in 5-ml THF and 50-ml Et O was added LiBH
2
4
(
4 M, 2.42 ml, 9.7 mmol) at 0 °C. e mixture was allowed to warm to room temperature and
stirred for 2 h. e reaction was quenched by addition of 10-ml 1 N NaOH and extracted
with 100-ml Et O. Organic layers were washed with H O (20 ml), followed by brine (20 ml),
2
2
and then dried (MgSO ). e solution was concentrated in vacuo to give the crude product,
4
which was purified by column chromatography on silica gel (EtOAc/ petroleum ether, 1:2)
to give 4 (1.6 g, 99%) as an oil.

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
9
2
0
1
[
ꢀ]D + 11.3 (c 0.3, CHCl ); R 0.14 (3:1 petroleum ether–ethyl acetate); H NMR (400 MHz,
3
f
CDCl ) δ 6.69–7.43 (m, 8H), 5.13 (s, 2H), 3.88 (s, 3H), 3.72 (m, 2H), 3.21–3.25 (m, 1H), 2.67
3
1
3
(
dd, J = 7.2, 15.2 Hz, 1H), 2.52 (dd, J = 7.2, 15.2 Hz, 1H), 1.34 (s, 9H); C NMR (150 MHz,
CDCl ) δ 171.8, 149.7, 147.1, 137.2, 134.2, 128.5, 127.8, 127.2, 119.6, 114.2, 111.7, 80.6,
3
+
7
7.2, 77.0, 76.8, 71.0, 67.0, 56.0, 44.3, 38.8, 27.9; HRESIMS: m/z 395.1817 [M + Na] (calcd
for C H O Na, 395.1829).
2
2
28
5
4
.5. tert-Butyl (S)-3-(4-(benzyloxy)-3-methoxyphenyl)-4-((methylsulfonyl)oxy)
butanoate (5)
To a stirred solution of 4 (1.64 g, 4.41 mmol) in 40-ml CH Cl , NEt (0.9 ml, 6.62 mmol)
2
2
3
was added dropwise at 0 °C, followed by MsCl (0.4 ml, 4.85 mmol). e reaction mixture
allowed to warm to 25 °C. Af er stirring at 25 °C for 1 h, the reaction was diluted by 50-ml
CH Cl . e organic layers were washed with 20-ml H O, followed by 20-ml brine, then
2
2
2
dried, and evaporated to afford the crude product to give 5 (2 g, 99%) as an oil.
4
.6. tert-Butyl (S)-4-azido-3-(4-(benzyloxy)-3-methoxyphenyl)butanoate (6)
To a solution of 5 (0.6 g, 1.33 mmol) in DMF (5 ml), NaN (0.26 g, 4 mmol) was added. e
3
reaction was stirred at 65 °C for 5 h. And then, H O (50 ml) and EtOAc (50 ml) were added.
2

e organic layers were washed with brine (50 ml) and then dried (MgSO ). e mixture
4
was filtered, and the filtrate was concentrated under reduced pressure to give the crude
product, which was purified by column chromatography on silica gel (EtOAc/petroleum
ether, 1:20) to give 6 (0.45 g, 85%) as an oil.
2
0
1
[
ꢀ] -6.5 (c 0.3, CHCl ); R 0.25 (10:1 petroleum ether–ethyl acetate); H NMR (400 MHz,
D
3 f
CDCl ) δ 6.69–7.43 (m, 8H), 5.13 (s, 2H), 3.89 (s, 3H), 3.41–3.53 (m, 2H), 3.25–3.29 (m,
3
1
3
1
H), 2.66 (dd, J = 6.8, 15.2 Hz, 1H), 2.51 (dd, J = 6.8, 15.2 Hz, 1H), 1.33 (s, 9H); C NMR
(
150 MHz, CDCl ) δ 170.9, 149.6, 147.3, 137.1, 133.8, 128.5, 127.8, 127.2, 119.5, 114.1,
3
+
1
11.53, 80.8, 71.0, 56.5, 56.0, 41.8, 39.3, 27.9; HRESIMS: m/z 420.1879 [M + Na] (calcd
for C H O N Na, 420.1894).
2
2
27
4
3
4
.7. tert-Butyl (S)-4-amino-3-(4-(benzyloxy)-3-methoxyphenyl)butanoate (7)
Compound 6 (1 g, 2.5 mmol) was dissolved in THF (25 ml) and PPh (860 mg, 3.3 mmol)
3
was added at 0 °C. e solution was stirred at 25 °C for 24 h, and then water (10 ml) was
added. e mixture was concentrated af er 6 h and extracted with EtOAc (100 ml). e
combined organics were washed with brine, dried over Na SO , filtered, and concentrated.
2
4

e residue was purified by flash chromatography (EtOAc/ petroleum ether, 1:1 to DCM/
MeOH, 20:1) to afford 7 (0.9 g, 96.8%) as an oil.
2
0
1
[
ꢀ] + 13.8 (c 0.3, CHCl ); R 0. 5 (10:1 DCM–MeOH); H NMR (400 MHz, CDCl ) δ
D
3
f
3
6
.67–7.44 (m, 8H), 5.13 (s, 2H), 3.88 (s, 3H), 3.02–3.05 (m, 1H), 2.80–2.93 (m, 2H), 2.59
1
3
(
dd, J = 7.2, 14.8 Hz, 1H), 2.51 (dd, J = 7.2, 14.8 Hz, 1H), 1.31 (s, 9H); C NMR (150 MHz,
CDCl ) δ 171.6, 149.7, 147.0, 137.3, 135.2, 128.6, 128.5, 127.8, 127.3, 119.8, 114.3, 111.7,
3
+
8
0.4, 77.2, 77.0, 76.8, 71.1, 56.0, 47.7, 45.7, 40.2, 28.0; HRESIMS: m/z 372.2169 [M + H]
(
calcd for C H O N, 372.2169).
22 30 4

10
X.ꢀY. LIU ET AL.
4
.8. (S)-4-Amino-3-(4-(benzyloxy)-3-methoxyphenyl)butanoic acid (8)
A solution of CF COOH (0.5 ml, 6.7 mmol) in CH Cl (3 ml) was added to a solution of 7
3
2
2
(
215 mg, 0.58 mmol) in CH Cl (5 ml) at 0 °C. e reaction was stirred at room temperature
2 2
for 2 h. e reaction was concentrated under reduced pressure to give 8 (180 mg).
.9. (S)-4-(4-(Benzyloxy)-3-methoxyphenyl)pyrrolidin-2-one (9)
4
Without further purification, 8 (180 mg, 0.57 mmol), EDCI (142 mg, 0.74 mmol), and
HOBT (154 mg, 1.14 mmol) were dissolved in CH Cl (30 ml), and then DIPEA (0.25 ml,
2
2
1
.43 mmol) was added to the mixture at 0 °C. e reaction was stirred for 16 h. Water (15 ml)
was added to the reaction and extracted with CH Cl (30 ml). e combined organics were
2
2
washed with 5% NaHCO brine, dried over Na SO , filtered, and concentrated. e residue
3,
2
4
was purified by flash chromatography (DCM/MeOH, 50:1) to afford 9 (140 mg, 82.5%) as
a white solid.
2
0
1
[
ꢀ] + 28.1 (c 0.5, CHCl ); R 0.25 (2 ml: 4 drops DCM–MeOH); H NMR (400 MHz,
D
3 f
CDCl ) δ 6.71–7.36 (m, 8H), 6.42 (s, 1H), 5.13 (s, 2H), 3.89 (s, 3H), 3.72–3.76 (m, 1H),
3
1
3
3
.60–3.64 (m, 1H), 3.36–3.40 (m, 1H), 2.67–2.73 (m, 1H), 2.44–2.50 (m, 1H); C NMR
(
125 MHz, CDCl ) δ 177.8, 149.8, 147.2, 137.1, 135.2, 128.6, 127.9, 127.2, 118.7, 114.2,
3
+
1
2
10.6, 71.1, 56.1, 49.7, 40.1, 38.1; HRESIMS: m/z 298.1440 [M + H] (calcd for C H O N,
1
8
20
3
98.1438).
4
2
.10. (S)-4-(4-(Benzyloxy)-3-methoxyphenyl)-1-(3,4-dimethoxyphenyl)pyrrolidin-
-one (10)
Compound 9 (500 mg, 1.68 mmol), 4-bromoveratrole (493 mg, 2.27 mmol), Pd(OAc)
2
(
20 mg, 5% mmol), Xantphos (75 mg, 7.5% mol), and Cs CO (766 mg, 2.35 mmol) were
2 3
dissolved in 1,4-dioxane (15 ml). e mixture was stirred at 100 °C for 25 h under N . EtOAc
2
(
50 ml) was added, the organic layers were washed with H O (50 ml), brine (50 ml), and
2
then dried (Na SO ). e mixture was filtered, and the filtrate was concentrated in vacuo
2
4
to give the crude product, which was purified by column chromatography on silica gel
(EtOAc/petroleum ether, 1:1) to give 10 (0.45 g, 62%) as an oil.
2
0
1
[
ꢀ] -10.4 (c 0.4, CHCl ); R 0.13 (1:1 petroleum ether–ethyl acetate); H NMR (400 MHz,
D
3 f
CDCl ) δ 7.30–7.51 (m, 6H), 6.77–6.87 (m, 5H), 5.15 (s, 2H), 4.11–4.14 (m, 1H), 3.89 (s,
3
6
1
1
H), 3.87 (s, 3H), 3.81–3.85 (m, 1H), 3.63–3.65 (m, 1H), 2.95–2.97 (m, 1H), 2.78–2.80 (m,
1
3
H); C NMR (125 MHz, CDCl ) δ 172.7, 149.9, 148.9, 147.3, 146.2, 137.0, 134.7, 132.8,
3
28.6, 127.9, 127.2, 118.7, 111.7, 110.9, 110.5, 105.1, 71.1, 56.4, 56.1, 56.0, 56.0, 40.4, 36.9;
+
HRESIMS: m/z 434.1954 [M + H] (calcd for C H O N, 434.1962).
2
6
28
5
4
.11. (S)-3-(4-(Benzyloxy)-3-methoxyphenyl)-1-(3,4-dimethoxyphenyl)pyrrolidine
(
11)
To a solution of LiAlH (18 mg, 0.46 mmol) in 5-ml THF, 10 (80 mg, 0.18 mmol) in THF
4
was added at 0 °C, and then stirred for 3 h at 60 °C. Af er cooling the mixture to 0 °C, the
reaction was quenched by sat. Na SO aq solution, diluted with dichloromethane (100 ml),
2
4
and washed with brine, dried, and evaporated to afford the crude product, which was

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
11
purified by column chromatography on silica gel (EtOAc/petroleum ether, 1:5) to give 11
65 mg, 84%) as a white solid.
(
2
0
1
[
ꢀ] -17.8 (c 0.15, CHCl ); R 0.5 (2:1 petroleum ether–ethyl acetate); H NMR (400 MHz,
D
3 f
CDCl ) δ 7.30–7.45 (m, 5H), 6.75–6.85 (m, 4H), 6.08–6.21 (m, 2H), 5.14 (s, 2H), 3.88 (s,
3
6
2
1
4
H), 3.82 (s, 3H), 3.64–3.68 (m, 1H), 3.40–3.49 (m, 3H), 3.28–3.32 (m, 1H), 2.36 (m, 1H),
1
3
.05–2.14 (m, 1H); C NMR (125 MHz, CDCl ) δ 150.1, 149.7, 146.9, 143.4, 140.3, 137.2,
3
28.5, 127.8, 127.2, 119.0, 114.1, 113.8, 111.0, 102.5, 97.1, 71.1, 56.9, 56.1, 55.8, 55.4, 48.2,
+
3.9, 33.5; HRESIMS: m/z 420.2163 [M + H] (calcd for C H O N, 420.2169).
2
6
30
4
4
.12. (S)-4-(1-(3,4-Dimethoxyphenyl)pyrrolidin-3-yl)-2-methoxyphenol (12)
Compound 11 (60 mg, 0.14 mmol) was dissolved in DCM/MeOH (1 ml/4 ml). Pd-C (80 mg)
was added and the solution was stirred at 25 °C for 4 h under hydrogen. e mixture was
filtered and concentrated. e residue was purified by flash chromatography (EtOAc/petro-
leum ether, 1:5) to afford 12 (40 mg, 85%) as an oil.
1
R 0.4 (2:1 petroleum ether–ethyl acetate); H NMR (400 MHz, CDCl ) δ 6.79–6.89 (m,
f
3
4
3
1
2
H), 6.09–6.21 (m, 2H), 3.88 (s, 6H), 3.82 (s, 3H), 3.64–3.68 (m, 1H), 3.40–3.49 (m, 3H),
1
3
.27–3.31 (m, 1H), 2.37 (m, 1H), 2.08–2.14 (m, 1H); C NMR (125 MHz, CDCl ) δ 150.1,
3
46.9, 144.4, 119.8, 114.3, 113.9, 119.6, 102.5, 97.1, 57.0, 56.6, 55.9, 55.8, 48.2, 44.1, 33. 6,
+
9.7; HRESIMS: m/z 330.1689 [M + H] (calcd for C H O N, 330.1700).
1
9
24
4
4
.13. (R)-1-(3,4-Dimethoxyphenyl)-2-(4-((S)-1-(3,4-dimethoxyphenyl)pyrrolidin-3-
yl)-2-methoxyphenoxy)propan-1-one (14)
Compound 12 (30 mg, 0.09 mmol) and Cs CO (36 mg, 0.11 mmol) were mixed in acetoni-
2
3
trile (3 ml). 13 (40 mg, 0.11 mmol) in 1-ml acetonitrile was added to the mixture at 0 °C.

e reaction was stirred at room temperature for 3 h. e reaction mixture was filtered and
concentrated. e residue was purified by flash chromatography (EtOAc/petroleum ether,
:2) to afford 14 (40 mg, 85%) as a solid.
1
2
0
1
[
ꢀ] -33 (c 0.13, CHCl ); R 0.4 (1:1 petroleum ether–ethyl acetate); H NMR (400 MHz,
D
3 f
CDCl ) δ 7.83 (d, J = 8.0 Hz, 1H), 7.68 (s, 1H), 6.89–6.90 (m, 5H), 6.19 (s, 1H), 6.08 (d,
J = 8.0 Hz, 1H), 5.40–5.43 (m, 1H), 3.81–3.94 (m, 15H), 3.62 (t, J = 8.0 Hz, 1H), 3.37–3.45
3
(
m, 3H), 3.25 (t, J = 8.0 Hz, 1H), 2.34–2.35 (m, 1H), 2.02–2.12 (m, 1H), 1.72 (d, J = 8.0 Hz,
1
3
3
1
5
H); C NMR (125 MHz, CDCl ) δ 197.7, 153.6, 150.1, 149.8, 149.0, 145.6, 143.4, 140.3,
3
36.8, 127.3, 123.6, 119.2, 115.7, 113.8, 111.4, 111.2, 110.1, 102.4, 97.0, 78.1, 57.0, 56.1, 56.0,
6.0, 55.8, 55.4, 48.2, 43.9, 33.5, 29.7, 19.4; HRESIMS: m/z 522.2472 [M + H] (calcd for
+
C H O N, 522.2486).
3
0
36
7
4
.14. (1R,2R)-1-(3,4-Dimethoxyphenyl)-2-(4-((S)-1-(3,4-dimethoxyphenyl)
pyrrolidin-3-yl)-2-methoxyphenoxy)propan-1-ol (LXY7824)
To a stirred solution of 2,6-di-tert-butyl-4-methylphenol (3.23 g, 14.66 mmol) in 40-ml tol-
uene, was added DIBALH (1.2 M, 12.2 ml, 14.66 mmol) at 0 °C. e mixture was stirred at
this temperature for 1.5 h. And then compound 14 (430 mg, 0.8 mmol) was added dropwise
at −78 °C and stirred for 3 h. e reaction was quenched by addition of 1 N HCl (25 ml)
and extracted with 100-ml EtOAc. Organic layers were washed with H O (20 ml), followed
2

12
X.ꢀY. LIU ET AL.
by brine (20 ml), and then dried (Na SO ). e solution was concentrated in vacuo to give
2
4
the crude product, which was purified by column chromatography on silica gel (EtOAc/
petroleum ether, 1:2) to give 4 (380 mg, 88%) as a white solid.
2
0
1
[
ꢀ] -62.5 (c 0.4, CHCl ); R 0.25 (1:1 petroleum ether–ethyl acetate); H NMR (400 MHz,
D
3 f
CDCl ) δ 6.83–6.98 (m, 9H), 6.22 (s, 1H), 6.11 (d, J = 8.4 Hz, 1H), 4.64 (d, J = 8.4 Hz,
3
1
H), 3.90–3.93 (m, 12H), 3.83 (s, 3H), 3.68 (t, J = 8.4 Hz, 1H), 3.41–3.49 (m, 3H), 3.35 (t,
1
3
J = 8.0 Hz, 1H), 2.36–2.41 (m, 1H), 2.12–2.15 (m, 1H), 1.17 (d, J = 6.4 Hz, 3H); C NMR
(
125 MHz, CDCl ) δ 150.9, 150.2, 149.0, 148.9, 146.5, 143.4, 140.5, 138.0, 132.6, 120.0, 119.5,
3
1
1
19.1, 114.0, 111.1, 110.9, 110.1, 102.6, 97.2, 84.2, 78.4, 57.0, 55.9, 55.8, 55.5, 48.2, 44.1, 33.5,
+
7.1; HRESIMS: m/z 524.2634 [M + H] (calcd for C H O N, 524.2643).
3
0
38
7
4
.15. HIF-1 reporter activity assays
e T47D cells (American Type Culture Collection) were incubated in RPIM 1640 Medium

(
Gibco), supplemented with 10% (v/v) fetal bovine serum (PAA), 100 U/ml penicillin G
sodium, and 100 μg/ml streptomycin in a humidified atmosphere (5% CO and 95% air)
2
4
at 37 °C. Exponentially grown T47D cells were plated at 3 × 10 cells into 96-well plates.
Af er 24 h, T47D cells were co-transfected with the pGL2-TK-3HRE luc reporter [23] and
the internal control construct pRL-CMV(Promega) by lipofectamine 2000 (Invitrogen)
following manufacturer’s instructions, 0.2 μg pGL2-TK-3HRE luc reporter and 0.01 μg
pRL-CMV used per well. Af er 24 h, test compounds were added in a volume of 100-μl
RPIM 1640 medium per well, and the incubation continued for another 30 min at 37 °C.

e cells were exposed to hypoxic (1% O /5% CO /94% N ) or normoxic (5% CO /95% air)
2 2 2 2
conditions at 37 °C for 20 h. A dual luciferase assay system (Promega, Madison, USA) was
employed to determine luciferase activities following manufacturer’s instructions. Firefly
luciferase activity was normalized with that of the Renilla luciferase [19,24].
4
.16. Western blotting analysis
Exponentially growing cells were treated with chemicals before exposed to normoxia (21%
O ), 1,10-phenanthroline (10 mM), or hypoxia (1% O ) at 37 °C. Nuclear extracts were pre-
2
2
pared using the Nuclear and Cytoplasmic Extraction Reagents (CWBio Co., Beijing, China).
Cells were lysed using RIPA buffer containing 50 mM Tris-HCl, pH7.4, 150 mM NaCl, 1%
Triton X-100, 0.5% deoxycholate, 0.1% SDS, and protease inhibitor cocktails (Amresco,
Washington, USA). Equal amounts of proteins were resolved by SDS-PAGE gels and trans-
ferred to PVDF membranes (Millipore, Burlington, USA). Membranes were blocked with
5
% non-fat dry milk followed by incubation with primary antibodies and HRP conjugated
secondary antibodies for protein visualization. e HIF-1 antibody was purchased from
CST (Shanghai) Biological Reagents Co., Ltd., China and Santa Cruz (USA), respectively.
4
.17. In vitro cytotoxicity evaluation of LXY7824
Cell lines were seeded in a 96-well plate. Af er incubation for 24 h, cells were treated with
varied concentrations of unnatural ginsenosides or dimethyl sulfoxide as vehicle. Af er
incubation for 96 h, 50-μl MTT stock solution (2 mg/ml, Sigma Chemical) was added to
each well, and the plates were incubated for an additional 4 h at 37 °C. e solution was

JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH
13
then removed from each well, and 150-μl dimethyl sulfoxide was added. Following gentle
agitation, the absorbance was measured using an ELISA reader (Bio-Rad, USA) at 570 nm.

ree parallel samples were measured each time. Absorbance values were normalized to
the values obtained for vehicle-treated cells to determine the percentage of surviving cells.
e median inhibitory concentration (IC ) was assessed from the dose response curve.

5
0
4
.18. In vivo studies
xenograf s. All animal studies were performed in compliance with the policies of the Institute
of Materia Medica Animal Care and Use Committee. Six-week-old female BALB/c/nu nude
mice were used in the present study. ey were purchased from Vital River Laboratory
Animal Technology Co., Ltd. (Beijing, China) and housed in the controlled environment
at 25 °C on a 12-h light/dark cycle (5 mice per group). When tumors grew to an average
3
volume of 200 mm , tumor-bearing mice were randomly separated into two groups of six
animals. One group received p.o (oral gavage) of Cremophor EL/ethanol/waterand served
as a vehicle control; the other group received an oral dose of 100 mg/kg LXY7824 every
day for 10 days. Mice were euthanized at the end of the treatment period. Tumors were
removed and weighed.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding

is work was financially supported by CAMS Innovation Fund for Medical Sciences [grant number
CIFMS, 2016-I2M-3-009].
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