Novel spirobicyclic artemisinin analogues (artemalogues): Synthesis and antitumor activities

Article abstract of DOI:10.1016/j.ejmech.2015.08.035

The sesquiterpene lactone framework of artemisinin was used as a drug repositioning prototype for the development of novel antitumor drugs. Several series of novel artemisinin analogues (artemalogues) were designed and synthesized through 1,3-dipolar cycloaddition of artemisitene with nitrile oxides or nitrones. The isoxazolidine-containing spirobicyclic artemalogue 11b turns out to be the most potent with low micromolar IC₅₀ values against all three tumor cells, which were at least 4-to 14-fold more potent than the parent artemisinin.

Full text of DOI:10.1016/j.ejmech.2015.08.035

European Journal of Medicinal Chemistry 103 (2015) 17e28
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel spirobicyclic artemisinin analogues (artemalogues): Synthesis
and antitumor activities
Gang Liu 1, Shanshan Song b 1, Shiqi Shu , Zehong Miao , Ao Zhang , Chunyong Ding
a
,
,
c
b
a
a, *
a
CAS Key Laboratory of Receptor Research, and Synthetic Organic & Medicinal Chemistry Laboratory, Shanghai Institute of Materia Medica (SIMM), Chinese
Academy of Sciences, Shanghai 201203, China
b
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences, Shanghai 201203, China
Department of Medicinal Chemistry, China Pharmaceutical University, Tongjiaxiang 24, Gulou District, Nanjing 210009, China
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 June 2015
Received in revised form
The sesquiterpene lactone framework of artemisinin was used as a drug repositioning prototype for the
development of novel antitumor drugs. Several series of novel artemisinin analogues (artemalogues)
were designed and synthesized through 1,3-dipolar cycloaddition of artemisitene with nitrile oxides or
nitrones. The isoxazolidine-containing spirobicyclic artemalogue 11b turns out to be the most potent
with low micromolar IC50 values against all three tumor cells, which were at least 4- to 14-fold more
potent than the parent artemisinin.
14 August 2015
Accepted 15 August 2015
Available online 18 August 2015
©
2015 Elsevier Masson SAS. All rights reserved.
Keywords:
Artemisinin
Spirobicyclic artemalogues
1,3-dipolar cycloaddition
Stereoconfiguration
Antiprolifereative effect
1
. Introduction
activity might be similar to that of antimalarial activity [14e16].
Unfortunately, compared to the success in the development of
antimalarial drugs, the majority of reported artemalogues only
showed limited potency in certain cancer cell lines and few was
further profiled in vivo in animal models.
The natural product artemisinin (1) is a unique sesquiterpene
lactone complex bearing a 1,2,4-trioxane structural framework. It
was isolated from the Chinese herb Artemisia annua L. and repre-
sented the prototypic structural template for the development of
therapeutical drugs. Dihydroartemisinin (2), artemether (3), and
artesunate (4) are the well-known analogues of artemisinin pos-
sessing significant efficacy against multidrug resistant malaria and
are prescribedin clinic(Fig.1)[1,2]. Althoughtheexact mechanism is
still unclear, it is believedthat the antimalarial activityof artemisinin
and its analogues is due to the existence of the endoperoxide frag-
ment that upon activation by the cellular Fe (II) species can generate
toxic carbon-centeredfree radicals and eventuallycause the death of
the parasite [3e5]. Meanwhile, artemisinin was also repositioned to
the pursuit of antitumor drugs two decades ago and many diverse
artemisinin analogues (artemalogues) have been reported pos-
sessing potent anticancer activities in a variety of cancer cell lines
Most of the currently reported artemalogues is derived from the
structural modification on the C10-carbonyl function with the aim
to enhance both the antitumor activity and the pharmacokinnetical
properties. Among these compounds, amide 5 [17] and ether 6
[18,19] are the representatives showing relatively high potency
with IC50 values of 460 and 10 nM against the proliferation of hu-
man hepatocellular carcinoma HepG2 and mouse lymphocytic
leukemia L1210 cell lines, respectively. To generate structurally
more diverse artemalogues, we recently conducted an alternative
medicinal chemistry campaign by introducing an isoxazoline or
isoxazolidine functionality to the C9 site thus forming a series of
structurally novel spirobicyclic artemalogues (I, Fig. 1) through 1,3-
dipolar cycloaddition. Herein, we report the synthesis, structural
characterization and antitumor activities of these new analogues.
[6e13]. The mechanism underlying the repositioned antitumor
2. Chemistry
*
Corresponding author.
E-mail address: chding@simm.ac.cn (C. Ding).
These two authors contributed equally to this work.
1
As illustrated in Scheme 1, artemisitene 7 bearing a C9-exocyclic
http://dx.doi.org/10.1016/j.ejmech.2015.08.035
223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
0

18
G. Liu et al. / European Journal of Medicinal Chemistry 103 (2015) 17e28
Fig. 1. Artemisinin, its analogues and the proposed new artemalogues I.
Scheme 1. Synthesis of isoxazolines 9aeg.
vinyl moiety was prepared from artemisinin (1) in two steps with
3% overall yield by following reported selenoxide elimination
methods [20]. Treatment of the aldoxime chlorides 8a-g with Et
generated the corresponding nitrile oxides and then underwent
,3-dipolar addition with artemisitene 7 in CH Cl at room tem-
perature leading to isoxazoline artemalogues 9a-g exclusively in
apparently due to the steric repulsion between the cyclohexyl ring
of artemisinin and the newly formed isoxazoline ring.
Meanwhile, 1,3-dipolar cycloaddition of various cyclic nitrones
with artemisitene 7 was investigated as well. As described in
Scheme 2, substituted 3,4-dihydroisoquinoline 2-oxides 10a-c
were obtained by treating corresponding tetrahydroisoquinolines
5
3
N
1
2
2
6
8e86% overall isolated yields.
The regiochemistry of the 1,3-dipolar cycloaddition was
with oxone and NaHCO
dition with artemisitene
3
in MeCN-THF [21]. Subsequent cycload-
ꢀ
7
in toluene at 60
C provided
confirmed by the appearance of two doublets with respective
chemical shift of 3.99 and 3.69 ppm in the H NMR spectra of
isoxazolidine-containing spirocyclic artemalogues 11a-c as the
major cycloadducts in 41e55% isolated yields, together with the
diastereomers 11a'ec' as the minor products in less than 10% yields.
Similarly, nitrone 10d was obtained from oxidation of pyrrolidine.
Subsequent 1,3-dipolar cycloaddition with 7 yielded artemalogue
11d in 51% yield, however, the corresponding diastereomer 11d'
was not observed. It was found that similar oxidation condition
1
isoxazoline-containing spirobicyclic artemalogue 9a, which were
0
assigned to the proton signals of C4 -methylene on the isoxazoline
ring. Meanwhile, in the HMBC spectrum of 9a (Fig. 2), a typical
long-range correlation between H-8a (
d
H
¼ 2.24 ppm) and C-10
(d
C
¼ 169.08 ppm) was observed, but the long-range correlation
0
between H-8a and C-3 (
d
C
¼ 156.60 ppm) was absent that excludes
(Oxone/EDTA/NaHCO
sponding nitrone 10e. Instead, using NaWO
3
) failed to convert morpholine to corre-
together with H as
the structure of regiomer 9a' (Fig. 2). The newly formed C9 R-
configuration was confirmed by the X-ray crystal of 9a. The excel-
lent regioselectivity of the 1,3-dipolar cycloaddition was likely due
to the electronic effect of the vinyl moiety under the effect of the
ortho-carbonyl moiety, and the exclusive 9R-stereoselectivity was
4
2 2
O
the co-oxidant reagents smoothly delivered 10e in quantitative
yield. Treatment of 10e with artemistene 7 provided cycloadduct
11e in 43% yield, and the diastereomeric isomer 11e' was not
detected again. The relative low yields in all cases were due to the

G. Liu et al. / European Journal of Medicinal Chemistry 103 (2015) 17e28
19
incomplete transformation of artemistene substrate, and the
absence of 11d' and 11e' might be ascribed to the relatively high
diastereoselectivity during the cycloaddition (nearly 7:1 for 11a-c
vs 11a'ec') and the rather low yields of these minor products.
Likewise, the structures of the new isoxazolidine-containing
spirocyclic artemalogues 11a-e and 11a'ec' were deduced on the
basis of their 1D and 2D NMR spectroscopic analysis that were
similar to that of 9a-g. For instance, the regioselectivity of the
0
cycloaddition was evidenced by the existence of C-10 proton sig-
0
nals at the chemical shifts of 2.88 and 3.26 ppm for H-10 a, 3.37 and
0
2
1
.65 ppm for H-10 b, respectively in the NMR spectra of 11a and
0 0
1a'. The (9R,9 R)-configuration for 11a and (9R,9 S)-configuration
for 11a' were ascertained by the correlations between H-8a, H-10
and H-9 in their NOESY spectroscopic analysis. As shown in Fig. 3,
the observed correlations between H-10 b and H-8a at chemical
0
0
0
shifts of 3.37 and 2.13 ppm respectively for compound 11a and 2.65
and 1.92 ppm respectively for compound 11a' indicated the twisted
configuration of the spirobicyclic pyran-2one-isoxazolidine frag-
ment in both isomers. The presence of the correlation between H-
0
0
d
1
0 b and H-9 (
¼ 4.58 ppm) in the NOESY spectra of 11a indicated
0
the R-configuration of the C-9 center, whereas the correlation
between H-10 a and H-9
configuration in the minor diastereomer 11a'. Meanwhile, opposite
Cotton effects in the range of 210e260 nm (correlative to the aro-
matic phenyl moiety) were observed in the CD spectra of the two
0
0
0
(d
¼ 5.03 ppm) suggested the 9 S-
0
diastereomers 11a and 11a' reflecting their opposite C-9 configu-
X-ray of compound 9a
rations. Such assignment was further secured by the X-ray analysis
Fig. 2. HMBC spectra and X-ray crystal structure of 9a.
of 11d (Fig. 3).
Scheme 2. Synthesis of ixoxazolidines 11aee.

20
G. Liu et al. / European Journal of Medicinal Chemistry 103 (2015) 17e28
Fig. 3. (a) NOESY analysis of 11a and 11a'; (b) CD spectra of compounds 11a and 11a'; (c) X-ray structure of 11d.
Since the lactone moiety in the artemisinin skeleton is one of
the major metabolic sites, modifications on this moiety were also
conducted. As shown in Scheme 3, reduction of spirocyclic
4
artemalogues 9a and 9c with NaBH provided the alcoholic in-
termediates 12a and 12b as an inseparable mixture of the two
corresponding diastereomers (~1.7:1) in high yields. Subsequent
methylation with trimethylorthoformate and TsOH in methanol
offered the 10S-configurated diastereomers 13a (47%) and 13b
52%), respectively, as the major products, and the corresponding
0R-configured diastereomers 13a' and 13b' were isolated as the
the signal of H-10 (4.62 ppm), whereas a significant enhance-
ment was observed on the signal of H-10 (4.75 ppm) in 13a'
upon irradiation of the proton signal of H-12 (5.43 ppm). This
result indicated that the H-12 and H-10 are apofacial in 13a
whereas the two similar protons in the diastereomer 13a' are
cofacial. The preference of 10S-configuration (13a) over the 10R-
configuration (13a') is reasonable since the reduction by NaBH
from the -face of the lactone carbonyl group in 9b is favored
than from the -face due to the steric effect of the cyclohexyl
4
a
(
1
b
ring of artemisinin.
minor products. The absolute configurations of these newly
formed C-10 centers in the acetal artemalogues were determined
by the NOE spectra. For example (bottom, Scheme 3), irradiation
of H-12 proton (5.52 ppm) in 13a led to a minor enhancement on
Similarly, isoxazolidine 11b was also reduced by NaBH
4
yielding the intermediate 14 as a 1.7:1 diastereomeric mixture
(Scheme 4). Treating the mixture 14 with trimethylorthoformate
and TsOH in methanol or triethylorthoformate and TsOH in

G. Liu et al. / European Journal of Medicinal Chemistry 103 (2015) 17e28
21
Scheme 3. Synthesis of new acetal artemalogues.
ethanol provided a pair of methyl ethers 15a and 15a' in 53% and
2% isolated yields, respectively or a pair of ethyl ethers 15b and
5b' in 45% and 26% yields, respectively. Fluorination [22] of the
intermediate 14 with DAST in CH Cl led to diastereomers 16 and
6′ in 51% and 34% yields, respectively. The configurations of the
two isomers were deduced from their NOE correlation analysis of
H-12 and H-10, which were similar to that of 13a and 13a'.
Meanwhile, treatment of intermediate 14 with cyanotrichloro-
methane and DBU followed by addition of TMSOTf and 2-
bromoethanol provided bromide 17 in 48% yield, which then
underwent a substitution reaction by morpholine or N-methyl
piperazine to offer artemalogues 18 and 19 in 81% and 86% yields,
respectively (Scheme 4).
The endoperoxide moiety plays a pivotal role on the antima-
larial activity of artemisinin (1) and its analogues 2e4. To deter-
mine whether this is also the case for the antitumor activity of the
new synthesized artemalogues, 1 was subjected to Zn/HOAc
reduction leading to the deoxygenated lactone 20 in 85% yield by
following a literature procedure [23]. Subsequent reaction with
PhSeCl/LDA followed by selenoxide elimination gave rise to pre-
cursor 21 in 50% overall yield. The 1,3-dipolar cycloaddition of 21
with nitrone 10b was conducted by following a similar reaction
condition as that for preparation of 11b and 11b' to afford the
deoxygenated artemalogues 22 and 22′ in 47% and 11% isolated
yields, respectively (Scheme 5). The absolute configurations of the
two diastereomers were deduced similarly by their NMR spec-
troscopic data.
3. Results and discussion
3
1
All the synthesized spirocyclic artemisinin analogues (arte-
malogues) were screened for their anticancer activity against
three human cancer cell lines, including squamous carcinoma KB
cells, vincristine-resistant KB/VCR cells, and human lung cancer
A549 cells. Among the small series of isoxazolinic artemalogues
9a-9g, only para-chlorophenyl analog 9b showed a marginal in-
crease against both KB and KB/VCR cells with IC50 values of around
2
2
1
13.9 mM. All other artemalogues in this subseries displayed similar
low potency against all the tested cancer cells as that of artemi-
sinin (1) (Table 1). In the series of isoxazolidinic artemalogues
11a-c and 11a'ec' that were derived from dihydroisoquinoline N-
oxides, significant discrepancy in the cellular potency was
0
observed. Only the 9 R-configured artemalogues 11a-c showed
significant boost in potency against all the three cells. The non-
substituted aryl analog 11a was more three-four fold more
potent against KB than KB/VCR and A549 cells with IC50 values of
2.91, 14.02, and 9.93 mM, respectively. Higher potency was ach-
ieved when one or two methoxy substituents were introduced
and the corresponding artemalogues 11b and 11c both showed
IC50 values less than 5
m
M in the inhibition of proliferation of all
0
the three cancer cell lines. Surprisingly, all the corresponding 9 S-
configured artemalogues 11a'ec' were completely inactive
(IC50 > 20 mM) in the same cellular test, indicating that the ste-
reochemistry played a pivotal role in the antitumor activity of this
subseries of artemalogues. The tetrahydropyrrole-containing

22
G. Liu et al. / European Journal of Medicinal Chemistry 103 (2015) 17e28
Scheme 4. Synthesis of 10-substituted isoxazolidine artemalogues.
Scheme 5. Synthesis of deoxygenated derivatives of 22 and 22'.
isoxazolidine 11d and the morpholine fused isoxazolidine 11e
were similar spirobicyclic artemalogues. However, only 11d
showed high antiproliferative effects against both KB and KB/VCR
artemalogues (15a, 15b, 16, 18, 19, 15a' and 15b') derived from the
C-10 carbonyl moiety were inactive as well in the three cell lines,
and only compound 19 bearing an N-methyl piperazine moiety
showed rather marginal cellular potency. Compared to the
cells with IC50 values of 2.34 and 3.35 mM, respectively and 11e
was inactive in the cell lines. It was quite disappointingly that in
the subseries of 10S-configurated (13a, 13b) and 10R-configurated
high potency of compound 11b (1e5
deoxygenated spirobicyclic analogues 22 was dramatically
decreased (13e20 M), indicating the crucial role of the endo-
peroxide bridge in the cellular antiproliferative effects. Similar to
11b', the deoxygenated diaseteromer 22′ was also inactive in all
three cell lines.
mM), the potency of the
(
13a', 13b') acetal artemalogues, only the 2,4-dichlorosubstituted
phenyl analog 13b showed moderate cellular potency with IC50
values of 5.29, 5.06 and 14.43 M against KB, KB/VCR and A549
cells, respectively. Similarly, most of the isoxazolidine
m
m

G. Liu et al. / European Journal of Medicinal Chemistry 103 (2015) 17e28
Table 1 (continued )
23
Table 1
Antiproliferative effects of new artemalogues against human cancer cell lines.
Compound Structure
(IC50
KB
, mM)
Compound Structure
(IC50
KB
, mM)
KB/VCR
A549
KB/VCR
A549
1
8
9
R ¼
R ¼
>20
>20
>20
Artemisinin
e
>20
>20
>20
1
17.85
15.43
16.91
13.36
>20
22
NC
9
9
9
9
9
9
9
a
b
c
d
e
f
Ar ¼ Ph
>20
>20
13.95
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
NC
Ar ¼ 4-Cl-Ph
13.89
>20
>20
>20
>20
>20
Ar ¼ 2,6-diCl-Ph
Ar ¼ 4-MeO-Ph
Ar ¼ 3,4-diMeO-Ph
Ar ¼ 3,4,5-triMeO-Ph
g
Ar ¼ 4-CO
2
Me-Ph
22′
>20
>20
NC
Vincristine
e
0.87 (nM) 387.43 (nM) 36.83 (nM)
NC e not calculated.
1
1
1
1
1
1
1a
R ¼ H
2.91
>20
1.47
>20
2.53
>20
14.02
>20
3.16
>20
2.35
>20
9.93
NC
5.01
NC
4.69
>20
1a'
1b
1b'
1c
R ¼ H
0
0
0
0
R ¼ 6 -MeO
4. Conclusion
R ¼ 6 -MeO
R ¼ 6 ,7'-diMeO
In summary, the sesquiterpene lactone framework of artemisi-
nin was used as a drug repositioning prototype for the development
of novel antitumor drugs. Several series of novel artemisinin ana-
logues (artemalogues) were designed and synthesized through 1,3-
dipolar cycloaddition of artemisitene with nitrile oxides or nitro-
nes. Further structural modifications on the lactone fragment or the
endoperoxide bridge were conducted leading to more series of
metabolically more stable artemalogues. All the new synthetic
artemalogues were fully characterized by 1D and 2D NMR spec-
trum and X-ray crystal analysis. Meanwhile, the antiproliferative
effects of these compounds were evaluated against KB, KB/VCR and
A549 cell lines. Generally, the isoxazolidine derivatives exhibited
higher potency than the isoxazoline series. Notably, isoxazolidine-
containing spirobicyclic artemalogue 11b was the most potent with
low micromolar IC50 values against all three cells, which were at
least 4- to 14-fold more potent than the parent artemisinin. The
significant discrepancy of potency between the diastereomeric
isoxazolidine-containing artemalogues as well as the abolished
activity of the deoxygenated derivatives proposed critical impact
1c'
R ¼ 6 ,7'-diMeO
11d
2.34
3.35
10.52
11e
>20
>20
NC
0
factors of the stereochemistry at C-9 position and the endoper-
1
1
1
1
3a
Ar ¼ 4-Cl-Ph
>20
>20
5.29
>20
>20
>20
5.06
>20
NC
NC
14.43
>20
oxide functionality for the antitumor activity. Further structural
modification and antitumor mechanism investigation on the new
potent artemalogues are undergoing.
3a'
3b
3b'
Ar ¼ 4-Cl-Ph
Ar ¼ 2,6-diCl-Ph
Ar ¼ 2,6-diCl-Ph
5. Experimental protocols
5.1. General experimental information
All reactions were performed in glassware containing a Teflon-
coated stir bar. Solvents and chemical reagents were obtained from
commercial sources and used without further purifications. Optical
rotations were measured on Autopol VI, serial number 90079,
manufactured by Rudolph Research Analytical, Hackettstown, NJ.
1
1
1
1
1
1
5a
5a'
5b
5b'
6
R ¼ MeO
R ¼ MeO
R ¼ EtO
R ¼ EtO
R ¼ F
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
NC
1
H and 13C spectra were recorded on Varian Mercury 300 MHz,
Mercury 400 MHz or Mercury 500 MHz and the data were recorded
using CDCl as the solvent. Chemical shifts ( ) are reported in ppm
6′
R ¼ F
NC
3
d

24
G. Liu et al. / European Journal of Medicinal Chemistry 103 (2015) 17e28
downfield from an internal TMS standard. Low-resolution mass
spectra were obtained in the EI mode. Flash column chromatog-
raphy on silica gel (200e300 mesh) was used for the routine pu-
rification of reaction products. The column output was monitored
by TLC on silica gel (100e200 mesh) precoated on glass plates
1.83e1.77 (m, 1H), 1.51e1.28 (m, 7H), 1.12e0.95 (m, 4H); 13C NMR
(126 MHz, CDCl 168.70, 155.87, 150.61, 148.61, 120.88, 120.17,
3
) d
109.93, 108.48, 105.20, 93.63, 83.01, 80.74, 55.50, 55.45, 49.89,
49.87, 49.39, 37.26, 35.39, 32.76, 24.67, 24.23, 23.71, 19.33; EI-MS
þ
20
D
(m/z) 459 (M ); ½aꢂ ¼ þ 102 (c 0.053, CHCl
3
).
(
3
15 ꢁ 50 mm), and spots were visualized by UV light at 254 or
0
65 nM. HMBC and NOE were used in the structural assignment.
5.2.6. (9R)-3 -(3,4,5-trimethoxyphenyl)-spiroisoxazoline
artemalogue (9f)
5.2. Synthesis of spiroisoxazoline artemalogue 9aeg
White solid, (66 mg, 68%); 1H NMR (300 MHz, Chloroform-d)
d
6.86 (s, 2H), 5.99 (s, 1H), 3.91 (d, J ¼ 17.7 Hz, 1H), 3.85 (s, 9H), 3.59
To a solution of hydroximoyl chloride 8aeg (0.21 mmol) and
(d, J ¼ 17.7 Hz, 1H), 2.44e1.97 (m, 5H), 1.83e1.77 (m, 1H), 1.51e1.33
13
artemisitene 7 (0.2 mmol) in CH
2
Cl
2
(3 mL) was added Et
3
N
3
(m, 7H), 1.12e0.98 (m, 4H); C NMR (126 MHz, CDCl ) d 168.56,
ꢀ
(
0.25 mmol) at 0 C. The resulting mixture was stirred at room
155.95, 152.81 (2C), 139.63, 123.51, 105.23, 103.83 (2 CH), 93.64,
83.26, 80.77, 60.48, 55.86, 49.90, 49.38, 37.25, 35.38, 32.76, 24.70,
temperature for 12 h. The solvent was evaporated in vacuo and the
residue was purified via silica column chromatography with pe-
troleum ether/ethyl acetate (3:1) as eluent to provide compounds
þ
20
24.22, 23.65, 19.32; EI-MS (m/z) 489 (M ); ½aꢂ ¼ þ 120 (c 0.049,
D
CHCl
3
).
9
5
d
a-g.
0
5
.2.7. (9R)-3 -(4-methoxycarbonylphenyl)-spiroisoxazoline
0
.2.1. (9R)-3 -phenyl-spiroisoxazoline artemalogue (9a)
White solid, (67 mg, 84%); H NMR (500 MHz, Chloroform-d)
7.71e7.69 (m, 2H), 7.46e7.38 (m, 3H), 6.03 (s, 1H), 3.99 (d,
artemalogue (9g)
1
White solid, (65 mg, 71%); 1H NMR (400 MHz, Chloroform-d)
d
8.07 (d, J ¼ 6.0 Hz, 2H), 7.75 (d, J ¼ 6.0 Hz, 2H), 6.03 (s, 1H),
J ¼ 17.9 Hz, 1H), 3.69 (d, J ¼ 17.9 Hz, 1H), 2.49e2.43 (m, 1H),
4.01e3.95 (m, 4H), 3.69 (d, J ¼ 17.9 Hz, 1H), 2.48e2.02 (m, 5H),
13
2
2
.40e2.35 (m, 1H), 2.24 (dd, J ¼ 13.4, 4.3 Hz, 1H), 2.15e2.11 (m, 1H),
.07e2.01 (m, 1H), 1.87e1.83 (m, 1H), 1.54e1.39 (m, 7H), 1.16e1.07
1.87e1.83 (m, 1H), 1.50e1.31 (m, 7H), 1.13e1.02 (m, 4H); C NMR
(101 MHz, CDCl 168.84, 166.39, 155.99, 132.73, 131.59, 129.90 (2
3
) d
13
(
m, 1H), 1.04 (d, J ¼ 6.0 Hz, 3H); C NMR (101 MHz, CDCl
3
)
d
169.08,
CH), 126.84 (2 CH), 105.76, 94.21, 84.12, 81.15, 52.33, 50.32, 49.39,
37.69, 35.83, 33.18, 25.11, 24.68, 24.11, 19.78; EI-MS (m/z) 457 (M );
½aꢂ ¼ 92 (c 0.049, CHCl
þ
1
8
2
56.60, 130.45, 128.70 (2 CH), 128.58, 126.95 (2 CH), 105.69, 94.15,
2
0
3.63, 81.19, 50.32, 50.30, 49.72, 37.71, 35.85, 33.21, 25.13, 24.69,
4.17, 19.81; EI-MS (m/z) 399 (M ); ½aꢂ ¼ þ 100 (c 0.056, CHCl
3
).
D
þ
20
D
3
).
5
.2.8. Preparation of intermediates 10aed
To a solution of secondary amine (2.0 mmol) in a mixture of
MeCN-THF (3.5 mL, 4:1) was added EDTA aqueous solution (2.8 mL,
0.01 M) and NaHCO
2.1 mmol) was added portionwise over 2 h under vigorous stirring
to maintain the temperature at 5 C. The resulting mixture was
stirred for another 20 min at 5 C, and then diluted with ethyl ac-
0
5.2.2. (9R)-3 -(4-chlorophenyl)-spiroisoxazoline artemalogue (9b)
1
White solid, (74 mg, 86%); H NMR (300 MHz, Chloroform-d)
7.58 (d, J ¼ 8.1 Hz, 2H), 7.33 (d, J ¼ 8.1 Hz, 2H), 5.98 (s, 1H), 3.90 (d,
ꢀ
d
3
(0.84 g, 10.0 mmol) at 5 C. Oxone (1.29 g,
J ¼ 17.1 Hz, 1H), 3.61 (d, J ¼ 17.1 Hz, 1H), 2.46e1.97 (m, 5H),
13
ꢀ
1.82e1.77 (m, 1H), 1.54e1.22 (m, 7H), 1.12e0.94 (m, 4H); C NMR
ꢀ
(
151 MHz, CDCl 168.94, 155.73, 136.42, 129.00 (2 CH), 128.19 (2
3
)
d
CH), 127.14, 105.74, 94.19, 83.90, 81.17, 50.32, 50.30, 49.51, 37.71,
etate (10 mL). The two phases were separated, and the aqueous
þ
3
5.84, 33.19, 25.13, 24.69, 24.14, 19.80; EI-MS (m/z) 433 (M );
layer was extracted with ethyl acetate (3 ꢁ 10 mL). The combined
2
0
½aꢂ ¼ þ 118 (c 0.053, CHCl
3
).
2 4
organic layer was washed with brine, dried over Na SO and
D
concentrated under reduced pressure. The crude product was
directly used in the next reaction without purification.
0
5
.2.3. (9R)-3 -(2,6-dichlorophenyl)-spiroisoxazoline artemalogue (9c)
1
White solid, (75 mg, 80%); H NMR (300 MHz, Chloroform-d)
d
7.35e7.24 (m, 3H), 5.98 (s, 1H), 3.83 (d, J ¼ 18.3 Hz, 1H), 3.65 (d,
5.2.9. Preparation of intermediate 10e
J ¼ 18.3 Hz, 1H), 2.41e2.22 (m, 3H), 2.08e1.97 (m, 2H), 1.84e1.79
To a solution of morpholine (2 mmol) in MeOH (3 mL) was
13
(
m, 1H), 1.50e1.29 (m, 7H), 1.13e0.99 (m, 4H); C NMR (126 MHz,
added Na
2
WO
4
(0.1 mmol). And then H
2
O
2
(6 mmol) was added
ꢀ
CDCl 166.37, 152.11, 133.20 (2C), 129.22, 126.07 (2 CH), 125.48,
3
)
d
slowly at 0 C. The reaction mixture was stirred at room tempera-
1
2
03.59, 92.13, 81.97, 79.14, 49.52, 48.28, 48.23, 35.66, 33.79, 31.16,
2
ture for 1.5 h, and diluted with ethyl acetate (10 mL) and H O
(5 mL). The two phases were separated and the aqueous layer was
＋
3.01, 22.62, 22.17, 17.77; ESI-MS (m/z) 490.0 [M þ Na] ;
2
0
½aꢂ ¼ þ136 (c 0.050, CHCl
3
).
extracted with ethyl acetate (3 ꢁ 10 mL). The combined organic
D
2 4
layer was washed with brine, dried over Na SO and concentrated
0
5.2.4. (9R)-3 -(3-methoxyphenyl)-spiroisoxazoline artemalogue (9d)
under reduced pressure. The crude product was directly used in the
following reaction without purification.
1
White solid, (67 mg, 78%); H NMR (300 MHz, Chloroform-d)
d
7.58 (d, J ¼ 8.1 Hz, 2H), 6.87 (d, J ¼ 8.1 Hz, 2H), 5.97 (s, 1H), 3.91 (d,
J ¼ 17.7 Hz, 1H), 3.81 (s, 3H), 3.61 (d, J ¼ 17.7 Hz, 1H), 2.45e2.29 (m,
5.3. Synthesis of spiroisoxazolidine artemalogue 11aee, 11a'ec'
2
H), 2.21e2.15 (m, 1H), 2.10e1.97 (m, 2H),1.82e1.77 (m, 1H),
13
1
d
9
2
0
.54e1.28 (m, 7H), 1.12e0.97 (m, 4H); C NMR (151 MHz, CDCl
169.24, 161.29, 156.14, 128.54 (2 CH), 121.13, 114.10 (2 CH), 105.68,
4.13, 83.35, 81.22, 55.38, 50.36, 50.31, 49.97, 37.74, 35.88, 33.25,
3
)
A mixture of artemisitene 7 (0.2 mmol) and nitrone (0.24 mmol)
ꢀ
in dry toluene (2 mL) were stirred at 60 C for 12 h. The solvent was
evaporated in vacuo, and the resulting crude residue was purified
via silica column chromatography to provide the desired products.
þ
20
D
5.15, 24.72, 24.21, 19.82; EI-MS (m/z) 429 (M ); ½aꢂ ¼ þ 123 (c
.054, CHCl
3
).
0
5
.3.1. (9R,9 R)-tetrahydroisoquinoline-fused spiroisoxazolidine
0
5
.2.5. (9R)-3 -(3,4-dimethoxyphenyl)-spiroisoxazoline artemalogue
artemalogue (11a)
(9e)
White solid, (35 mg, 41%); 1H NMR (500 MHz, Chloroform-d)
White solid, (67 mg, 73%); 1H NMR (300 MHz, Chloroform-d)
7.37 (s, 1H), 7.01 (d, J ¼ 8.4 Hz, 1H), 6.80 (d, J ¼ 8.4 Hz, 1H), 5.98 (s,
H), 3.95e3.88 (m, 7H), 3.61 (d, J ¼ 17.6 Hz, 1H), 2.45e1.97 (m, 5H),
d
7.19e7.08 (m, 4H), 5.97 (s, 1H), 4.58 (dd, J ¼ 11.5, 6.1 Hz, 1H),
d
1
3.60e3.55 (m, 1H), 3.42e3.36 (m, 2H), 310e3.03 (m, 1H), 2.91e2.84
(m, 2H), 2.43e2.37 (m, 2H), 2.13 (dd, J ¼ 13.0, 4.5 Hz, 1H), 2.10e1.99

G. Liu et al. / European Journal of Medicinal Chemistry 103 (2015) 17e28
25
(
1
1
6
2
0
m, 2H), 1.84e1.79 (m, 1H), 1.53e1.42 (m, 6H), 1.31e1.22 (m, 1H),
.14e0.98 (m, 4H); 13C NMR (101 MHz, CDCl
170.94, 133.30,
33.27, 127.88, 126.88, 126.45, 125.73, 105.08, 93.28, 82.43, 80.95,
35.83, 33.44, 28.72, 25.11, 24.97, 24.70, 19.79; ESI-MS (m/z) 488.2
＋
20
3
)
d
[M þ H] ; ½aꢂ ¼ þ72 (c 0.048, CHCl
D
3
).
0
2.61, 50.12, 50.04, 49.30, 49.23, 37.37, 35.44, 33.06, 28.76, 24.66,
5.3.7. (9R,9 R)-tetrahydropyrrole-fused spiroisoxazolidine
artemalogue (11d)
＋
20
D
4.40, 23.49, 19.48; ESI-MS (m/z) 450.0 [M þ Na] ; ½aꢂ ¼ þ 114 (c
.047, CHCl
3
).
White solid, (37 mg, 51%); 1H NMR (300 MHz, Chloroform-d)
d
5.92 (s, 1H), 3.72e3.66 (m, 1H), 3.58e3.50 (m, 1H), 3.13e2.95 (m,
0
5
.3.2. (9R,9 S)-tetrahydroisoquinoline-fused spiroisoxazolidine
2H), 2.69e2.63 (m, 1H), 2.41e1.89 (m, 8H), 1.80e1.73 (m, 2H),
13
artemalogue (11a')
1.50e1.23 (m, 7H), 1.16e0.98 (m, 4H); C NMR (126 MHz, CDCl
170.26,105.41, 93.83, 81.43, 81.39, 66.41, 56.64, 51.04, 50.61, 49.06,
37.87, 35.86, 33.50, 30.11, 25.16, 24.81, 24.13, 23.82, 19.90; EI-MS (m/
3
)
White solid, (5 mg, 6%); 1H NMR (500 MHz, Chloroform-d)
d
d
7.20e7.11 (m, 4H), 5.93 (s, 1H), 5.03 (dd, J ¼ 11.4, 6.2 Hz, 1H),
.48e3.44 (m, 1H), 3.26 (dd, J ¼ 13.8, 6.2 Hz, 1H), 3.16e3.10 (m, 1H),
.03e2.98 (m, 1H), 2.81e2.76 (m, 1H), 2.65 (dd, J ¼ 13.9, 11.3 Hz,
H), 2.41e2.33 (m, 2H), 2.10e2.06 (m, 1H), 2.02e1.97 (m, 1H), 1.92
þ
20
D
3
3
1
(
1
z) 365 (M ); ½aꢂ ¼ þ 76 (c 0.045, CHCl
3
).
0
5.3.8. (9R,9 R)-morpholine-fused spiroisoxazolidine artemalogue
(11e)
dd, J ¼ 13.4, 4.1 Hz, 1H), 1.84e1.79 (m, 1H), 1.53e1.28 (m, 7H),
13
white solid, (33 mg, 43%); 1H NMR (300 MHz, Chloroform-d)
5.91 (s, 1H), 4.09e3.34 (m, 6H), 3.17e3.12 (m, 1H), 2.93e2.73 (m,
.10e1.05 (m, 1H), 1.01 (d, J ¼ 6.3 Hz, 3H); C NMR (126 MHz,
CDCl
3
)
d
170.57,133.96, 132.71, 127.62, 127.26, 126.25, 125.91,104.97,
d
9
3
3.14, 82.32, 80.52, 61.86, 52.61, 50.49, 49.88, 49.86, 37.30, 35.42,
2H), 2.40e2.19 (m, 2H), 2.06e1.92 (m, 3H), 1.77e1.72 (m, 1H),
1.47e1.14 (m, 7H), 1.10e0.94 (m, 4H); C NMR (101 MHz, CDCl )
3
þ
13
3.01, 28.67, 24.66, 24.54, 24.27, 19.38; EI-MS (m/z) 427 (M );
2
0
½
aꢂ ¼ þ 83 (c 0.051, CHCl
3
).
d
5
170.54, 105.05, 93.38, 81.55, 80.92, 64.25, 63.84, 58.62, 50.36,
0.09, 49.48, 44.44, 37.30, 35.39, 33.01, 24.66, 24.35, 23.44, 19.43;
D
0
0
þ
20
5.3.3. (9R,9 R)-6 -methoxytetrahydroisoquinoline-fused
EI-MS (m/z) 381 (M ); ½aꢂ ¼ þ 97 (c 0.052, CHCl
3
).
D
spiroisoxazolidine artemalogue (11b)
White solid, (50 mg, 55%); 1H NMR (300 MHz, Chloroform-d)
6.99 (d, J ¼ 8.4 Hz, 1H), 6.75 (d, J ¼ 8.4 Hz, 1H), 6.66 (s, 1H), 5.97 (s,
5.4. General procedure of the synthesis of 13aeb, 13a'eb', 15aeb,
15a'eb'
d
1
H), 4.58e4.52 (m, 1H), 3.78 (s, 3H), 3.61e3.52 (m, 1H), 3.41e3.32
(
2
(
1
5
2
m, 2H), 3.10e2.99 (m, 1H), 2.89e2.81 (m, 2H), 2.46e2.37 (m, 2H),
.17e2.00 (m, 3H), 1.84e1.79 (m, 1H), 1.55e1.23 (m, 7H), 1.19e0.96
m, 4H); 13C NMR (126 MHz, CDCl
171.31, 158.41, 134.99, 128.28,
25.59, 112.81, 112.77, 105.53, 93.74, 83.04, 81.39, 62.67, 55.26,
0.57, 50.53, 49.67, 49.62, 37.82, 35.90, 33.49, 29.34, 25.11, 24.85,
The solution of 9b, 9c or 11b (0.2 mmol) in MeOH (3 mL) was
ꢀ
cooled to 0 C, and then NaBH
4
(0.3 mmol) was added slowly. The
ꢀ
3
)
d
reaction mixture was stirred at 0 C for 2 h. The solution was
quenched with H O (5 mL), and extracted with CH Cl
(2 ꢁ 10 mL).
The combined organic layer was washed with brine, dried over
Na SO and concentrated under reduced pressure. The crude res-
2
2
2
þ
20
3.96, 19.92; EI-MS (m/z) 457 (M ); ½aꢂ ¼ þ 106 (c 0.050, CHCl
3
).
2
4
D
idue 12a, 12b or 14 was directly used in the next reaction without
purification.
0 0
.3.4. (9R,9 S)-6 -methoxytetrahydroisoquinoline-fused
5
spiroisoxazolidine artemalogue (11b')
To a solution of 12a, 12b or 14 (0.2 mmol) in MeOH (3 mL) was
added trimethylorthoformate or triethylorthoformate (2.0 mmol)
1
White solid, (7 mg, 8%); H NMR (300 MHz, Chloroform-d)
d
7.01
(
4
3
1
d
d, J ¼ 8.4 Hz, 1H), 6.72 (d, J ¼ 8.4 Hz, 1H), 6.61 (s, 1H), 5.88 (s, 1H),
and methanesulfonic (0.24 mmol). The resulting mixture was stir-
ꢀ
.95e4.89 (m, 1H), 3.75 (s, 3H), 3.41e3.36 (m, 1H), 3.21e2.94 (m,
H), 2.74e2.52 (m, 2H), 2.36e2.27 (m, 2H), 2.05e1.70 (m, 4H),
red at 50 C for 3 h. The reaction solution was quenched with H
2
O
(10 mL) and diluted with CH
separated and the aqueous layer was extracted with CH
(2 ꢁ 10 mL). The combined organic layer was washed with brine,
dried over Na SO and concentrated under reduced pressure. The
2
Cl
2
(10 mL). The two phases were
.45e1.23 (m, 7H), 1.09e0.95 (m, 4H); 13C NMR (126 MHz, CDCl
)
Cl
2
3
2
170.97, 158.27, 134.40, 128.68, 126.39, 112.77, 112.70, 105.41, 93.58,
2.89, 80.96, 61.88, 55.28, 52.99, 50.82, 50.36, 50.31, 37.74, 35.86,
3.44, 29.27, 25.10, 24.97, 24.71, 19.82; ESI-MS (m/z) 480.0
8
3
2
4
crude residue was purified via silica column chromatography with
petroleum ether/ethyl acetate (4:1) to provide 13a and 13a', 13b
and 13b', 15a and 15a' or 15b and 15b'.
＋
20
D
[M þ Na] ; ½aꢂ ¼ þ 82 (c 0.050, CHCl
3
).
0
0
0
5
.3.5. (9R,9 R)-6 ,7 -dimethoxytetrahydroisoquinoline-fused
0
spiroisoxazolidine artemalogue (11c)
5.4.1. (9R,10S)-10-methoxy-3 -(4-chlorophenyl)-spiroisoxazoline
White solid, (45 mg, 46%); 1H NMR (300 MHz, Chloroform-d)
artemalogue (13a)
d
6
2
6.58 (s, 1H), 6.51 (s, 1H), 5.95 (s, 1H), 4.50e4.44 (m, 1H), 3.82 (s,
H), 3.58e3.50 (m, 1H), 3.36e3.27 (m, 2H), 2.99e2.70 (m, 3H),
.41e2.35 (m, 2H), 2.12e1.97 (m, 3H), 1.82e1.77 (m, 1H), 1.51e1.23
White solid, (42 mg, 47%); 1H NMR (400 MHz, Chloroform-d)
d
7.63 (d, J ¼ 8.2 Hz, 2H), 7.36 (d, J ¼ 8.2 Hz, 2H), 5.52 (s, 1H), 4.62 (s,
1H), 3.71e3.52 (m, 5H), 2.39e2.32 (m, 1H), 2.21e1.89 (m, 4H),
1.69e1.62 (m, 1H), 1.53e1.25 (m, 7H), 1.08e0.92 (m, 4H); C NMR
13
13
(
m, 7H), 1.12e0.98 (m, 4H); C NMR (126 MHz, CDCl
3
)
d
171.30,
1
8
3
47.94, 147.61, 125.84, 125.22, 110.87, 109.80, 105.51, 93.66, 82.92,
3
(126 MHz, CDCl ) d 157.18, 136.13, 128.91 (2 CH), 128.04 (2 CH),
1.46, 62.73, 55.98, 55.83, 50.63, 50.49, 49.83, 49.72, 37.81, 35.90,
3.51, 28.83, 25.11, 24.83, 23.91, 19.92; ESI-MS (m/z) 488.2
127.80, 104.41, 101.72, 87.59, 84.38, 82.58, 56.57, 52.60, 49.34, 46.46,
37.38, 36.40, 33.96, 25.82, 24.72, 24.27, 20.22; ESI-MS (m/z) 472.1
＋
20
D
＋
20
D
[M þ H] ; ½aꢂ ¼ þ87 (c 0.052, CHCl
3
).
[M þ Na] ; ½aꢂ ¼ þ132 (c 0.051, CHCl
3
).
0
0
0
0
5
.3.6. (9R,9 S)-6 ,7 -dimethoxytetrahydroisoquinoline-fused
5.4.2. (9R,10R)-10-methoxy-3 -(4-chlorophenyl)-spiroisoxazoline
artemalogue (13a')
spiroisoxazolidine artemalogue (11c')
1
White solid, (25 mg, 28%); 1H NMR (400 MHz, Chloroform-d)
White solid, (6 mg, 6%); H NMR (300 MHz, Chloroform-d)
d
6.56
(
4
1
d
s, 2H), 5.89 (s, 1H), 4.93e4.87 (m, 1H), 3.82 (s, 6H), 3.38e2.98 (m,
d
7.64 (d, J ¼ 8.4 Hz, 2H), 7.34 (d, J ¼ 8.4 Hz, 2H), 5.44 (s, 1H), 4.75 (s,
H), 2.67e2.54 (m, 2H), 2.41e2,28 (m, 2H), 2.06e1.75 (m, 4H),
1H), 3.93 (d, J ¼ 18.0 Hz, 1H), 3.51 (s, 3H), 3.35 (d, J ¼ 18.0 Hz, 1H),
13
.45e1.23 (m, 7H), 1.04e0.95 (m, 4H); C NMR (126 MHz, CDCl
171.05, 147.78, 147.73, 126.01, 125.05, 110.65, 110.07, 105.41, 93.53,
2.85, 81.01, 62.09, 55.99, 55.86, 53.09, 50.97, 50.40, 50.30, 37.74,
3
)
2.37e2.29 (m, 2H), 2.09e1.89 (m, 3H), 1.76e1.67 (m, 1H), 1.61e1.29
1
3
(m, 7H), 1.09e0.93 (m, 4H); C NMR (101 MHz, CDCl
135.77, 128.77 (2 CH), 128.10 (2 CH), 104.55, 98.89, 91.24, 85.64,
3
) d 157.67,
8

26
G. Liu et al. / European Journal of Medicinal Chemistry 103 (2015) 17e28
0
0
8
2
2.32, 57.61, 51.74, 50.23, 40.85, 37.50, 36.27, 33.65, 25.74, 24.35,
5.4.8. (9R,9 R,10R)-10-ethoxy-6 -methoxytetrahydroisoquinoline-
fused spiroisoxazolidine artemalogue (15b')
þ
20
3.61, 20.20; EI-MS (m/z) 449 (M ); ½aꢂ ¼ þ28 (c 0.047, CHCl
3
).
D
White solid, (25 mg, 26%); 1H NMR (400 MHz, Chloroform-d)
0
d
1
7.06 (d, J ¼ 8.4 Hz, 1H), 6.72 (d, J ¼ 8.4 Hz, 1H), 6.61 (s, 1H), 5.37 (s,
5
.4.3. (9R,10S)-10-methoxy-3 -(2,6-dichlorophenyl)-
H), 4.73 (s, 1H), 4.55e4.52 (m, 1H), 4.00e3.96 (m, 1H), 3.77 (s, 3H),
spiroisoxazoline artemalogue (13b)
White solid, (50 mg, 52%); 1H NMR (300 MHz, Chloroform-d)
3.57e3.49 (m, 1H), 3.41e3.35 (m, 1H), 3.20e3.09 (m, 2H), 3.01e2.92
m, 1H), 2.83e2.75 (m, 2H), 2.37e2.32 (m, 2H), 2.06e1.89 (m, 3H),
.72e1.69 (m, 1H), 1.50e1.29 (m, 7H), 1.15 (t, J ¼ 7.1 Hz, 3H),
(
1
d
2
(
7.34e7.27 (m, 3H), 5.53 (s, 1H), 4.88 (s, 1H), 3.61e3.57 (m, 5H),
.39e2.18 (m, 2H), 2.15e2.02 (m, 2H), 1.94e1.88 (m, 1H), 1.73e1.68
m, 1H), 1.54e1.25 (m, 7H), 1.02e0.97 (m, 4H); C NMR (126 MHz,
CDCl 155.46, 134.52 (2C), 130.51, 128.17, 127.56 (2 CH), 103.87,
01.31, 87.11, 83.99, 82.03, 56.31, 52.14, 48.96, 48.66, 36.92, 35.93,
13
13
1.05e0.96 (m, 4H); C NMR (101 MHz, CDCl
28.14, 112.15, 112.00, 103.89, 96.00, 90.88, 84.07, 82.33, 65.33,
1.58, 54.80, 51.59, 48.86, 48.74, 39.02, 37.13, 35.91, 33.41, 29.27,
3
) d 157.53, 134.31,
1
6
3
) d
1
3
þ
20
D
＋
25.26, 23.99, 22.88, 19.87, 14.51; EI-MS (m/z) 487 (M ); ½aꢂ ¼ þ10
3.52, 25.28, 24.34, 23.79, 19.78; ESI-MS (m/z) 506.1 [M þ Na] ;
2
0
(c 0.050, CHCl
3
).
½aꢂ ¼ þ124 (c 0.051, CHCl
3
).
D
0
5.5. Preparation of 16 and 16
0
5
.4.4. (9R,10R)-10-methoxy-3 -(2,6-dichlorophenyl)-
spiroisoxazoline artemalogue (13b')
Compound 14 (0.1 mmol) was dissolved in dry CH
2
Cl
2
(2 mL)
White solid, (26 mg, 27%); 1H NMR (300 MHz, Chloroform-d)
ꢀ
and cooled to 0 C. DAST (0.15 mmol) was added quickly under N
2
.
d
1
7.33e7.23 (m, 3H), 5.43 (s, 1H), 4.79 (s, 1H), 3.82 (d, J ¼ 18.2 Hz,
ꢀ
After 10 min, the reaction was stirred at 40 C for 3 h. After removal
of the solvent, the residue was purified via silica column chroma-
tography with PE/EA (10:1) to give a pair of isomers.
H), 3.58e3.46 (m, 4H), 2.37e2.31 (m, 2H), 2.08e1.85 (m, 3H),
13
1.76e1.70 (m, 1H), 1.56e1.23 (m, 7H), 1.11e0.94 (m, 4H); C NMR
(
126 MHz, CDCl 155.51,134.66 (2C),130.21,128.55,127.56 (2 CH),
3
) d
1
3
03.99, 98.01, 90.75, 85.43, 81.91, 57.03, 51.26, 49.71, 43.09, 37.04,
0 0
.5.1. (9R,9 R,10R)-10-fluoro-6 -methoxytetrahydroisoquinoline-
5
5.79, 33.18, 25.19, 23.88, 23.08, 19.76; ESI-MS (m/z) 506.1
fused spiroisoxazolidine artemalogue (16)
＋
20
D
[M þ Na] ; ½aꢂ ¼ þ19 (c 0.048, CHCl
3
).
White solid, (47 mg, 51%); 1H NMR (400 MHz, Chloroform-d)
d
7.04 (d, J ¼ 8.7 Hz, 1H), 6.76 (d, J ¼ 8.5 Hz, 1H), 6.62 (s, 1H), 5.58 (s,
1H), 5.04 (d, J ¼ 53.3 Hz, 1H), 4.58e4.56 (m, 1H), 3.78 (s, 3H),
.38e3.23 (m, 3H), 3.07e3.03 (m, 1H), 2.70e2.56 (m, 2H),
2.40e2.30 (m, 2H), 2.08e1.88 (m, 3H), 1.78e1.65 (m, 2H), 1.53e1.25
0
0
5.4.5. (9R,9 R,10S)-10-methoxy-6 -methoxytetrahydroisoquinoline-
3
fused spiroisoxazolidine artemalogue (15a)
White solid, (50 mg, 53%); 1H NMR (400 MHz, Chloroform-d)
7.09 (d, J ¼ 8.4 Hz, 1H), 6.78 (dd, J ¼ 8.4, 2.7 Hz, 1H), 6.64 (d,
13
(
3
m, 6H), 1.03e0.93 (m, 4H); C NMR (126 MHz, CDCl ) d 157.66,
d
1
34.39,127.76,126.58,112.73,112.47,110.57 (d, J ¼ 231.8 Hz),104.08,
J ¼ 2.7 Hz, 1H), 5.45 (s, 1H), 4.61e4.59 (m, 1H), 4.27 (s, 1H), 3.80 (s,
8
4
8.08, 81.67, 79.68 (d, J ¼ 20.2 Hz), 61.09, 54.81, 52.08, 48.96, 48.05,
7.13, 37.12, 35.79, 33.64, 25.08, 24.94, 24.19, 23.91,19.79; EI-MS (m/
3
2
H), 3.41e3.37 (m, 4H), 3.25e3.23 (m, 1H), 3.05e3.02 (m, 1H),
.73e2.62 (m, 2H), 2.41e2.24 (m, 2H), 2.08e1.91 (m, 4H), 1.68e1.28
þ
20
13
z) 461 (M ); ½aꢂ ¼ þ92 (c 0.050, CHCl3).
(
m, 8H), 1.00e0.89 (m, 4H); C NMR (126 MHz, CDCl
3
)
d
157.98,
D
134.85, 128.36, 127.56, 112.84, 105.42, 104.16, 87.38, 82.79, 80.96,
0
0
5
.5.2. (9R,9 R,10S)-10-fluoro-6 -methoxytetrahydroisoquinoline-
61.46, 56.32, 55.25, 52.79, 49.49, 49.16, 48.64, 37.49, 36.48, 34.16,
þ
20
D
fused spiroisoxazolidine artemalogue (16
0
)
2
5.82, 25.59, 24.75, 24.37, 20.34; EI-MS (m/z) 473 (M ); ½aꢂ ¼ þ84
1
White solid, (31 mg, 34%); H NMR (400 MHz, Chloroform-d)
7.06 (d, J ¼ 8.4 Hz, 1H), 6.74 (dd, J ¼ 8.5, 2.7 Hz, 1H), 6.62 (d,
(
c 0.047, CHCl
3
).
d
J ¼ 2.7 Hz, 1H), 5.55 (d, J ¼ 52.9 Hz, 1H), 5.47 (s, 1H), 4.58e4.55 (m,
0
0
5.4.6. (9R,9 R,10R)-10-methoxy-6 -methoxytetrahydroisoquinoline-
1
H), 3.77 (s, 3H), 3.27e3.15 (m, 3H), 2.99e2.91 (m, 1H), 2.79e2.68
fused spiroisoxazolidine artemalogue (15a')
(
m, 2H), 2.42e2.34 (m, 2H), 2.09e1.91 (m, 3H), 1.75e1.71 (m, 1H),
White solid, (30 mg, 32%); 1H NMR (300 MHz, Chloroform-d)
7.03 (d, J ¼ 8.4 Hz, 1H), 6.69 (d, J ¼ 8.4 Hz, 1H), 6.59 (s, 1H), 5.36
13
1
.53e1.42 (m, 4H), 1.36e1.26 (m, 3H), 1.08e0.97 (m, 4H); C NMR
126 MHz, CDCl 157.66, 134.29, 128.08, 126.54, 112.22, 112.19,
04.25, 102.90 (d, J ¼ 219.2 Hz), 91.14 (d, J ¼ 6.3 Hz), 83.04 (d,
J ¼ 21.4 Hz), 81.76, 61.54, 54.80, 51.30, 48.74, 48.69, 38.63, 37.06,
5.71, 33.32, 28.29, 25.00, 23.89, 22.88, 19.77; EI-MS (m/z) 461
d
(
3
) d
(
s, 1H), 4.64 (s, 1H), 4.54e4.47 (m, 1H), 3.75 (s, 3H), 3.50 (s, 3H),
1
3
2
.33e2.89 (m, 4H), 2.78e2.67 (m, 2H), 2.41e2.30 (m, 2H),
.06e1.87 (m, 3H), 1.73e1.25 (m, 8H), 1.08e0.94 (m, 4H); 13C NMR
3
(
126 MHz, CDCl
3
)
d
157.93, 134.81, 128.59, 127.43, 112.57, 112.38,
þ
20
(
M ); ½aꢂ ¼ þ11 (c 0.046, CHCl
3
).
D
104.38, 97.88, 91.39, 84.45, 82.77, 62.04, 57.61, 55.24, 52.03, 49.24,
4
9.18, 39.33, 37.60, 36.35, 33.88, 29.23, 25.69, 24.45, 23.27, 20.34;
5.6. General procedure of the synthesis of 18, 19
þ
20
EI-MS (m/z) 473 (M ); ½aꢂ ¼ þ8 (c 0.051, CHCl
3
).
D
To a solution of 14 (0.15 mmol) in CH
2 2
Cl (2 mL) were added DBU
0
0
5
.4.7. (9R,9 R,10S)-10-ethoxy-6 -methoxytetrahydroisoquinoline-
(0.03 mmol) and CCl CN (0.75 mmol) under N
3
2
at room temperature.
ꢀ
fused spiroisoxazolidine artemalogue (15b)
The mixture was stirred for 30 min and then cooled to 0 C. TMSOTf
(0.3 mmol) and 2-bromoethanol (1.5 mmol) were added into the
reaction mixture, and the resulting solution was warmed to room
temperature and stirred for 40 min. The reaction was quenched with
White solid, (44 mg, 45%); 1H NMR (400 MHz, Chloroform-d)
d
7.06 (d, J ¼ 8.5 Hz, 1H), 6.75 (dd, J ¼ 8.5, 2.7 Hz, 1H), 6.60 (d,
J ¼ 2.7 Hz, 1H), 5.43 (s, 1H), 4.57e4.54 (m, 1H), 4.31 (s, 1H), 3.77 (s,
3
2
H), 3.73e3.65 (m, 1H), 3.49e3.16 (m, 4H), 3.06e3.01 (m, 1H),
.69e2.64 (m, 2H), 2.38e2.19 (m, 2H), 2.06e1.85 (m, 4H), 1.66e1.61
NaHCO
3
aqueous (10 mL) and extracted with CH
2 2
Cl
(2 ꢁ 10 mL). The
combined organic layer was washed with brine, dried over Na
2
SO
4
(
0
1
6
2
m, 1H), 1.52e1.41 (m, 4H), 1.32e1.23 (m, 2H), 1.12 (t, J ¼ 7.1 Hz, 3H),
.99e0.93 (m, 4H); 13C NMR (101 MHz, CDCl
157.50, 134.46,
27.90, 127.19, 112.44, 103.62, 102.52, 87.10, 82.39, 80.34, 63.45,
0.96, 54.80, 52.32, 49.02, 48.86, 48.31, 37.08, 36.06, 33.74, 25.39,
and concentrated under reduced pressure. The crude residue was
purified via silica column chromatography with petroleum ether/
ethyl acetate (5:1) to provide intermediate 17 in 48% yield.
To a solution of compound 17 (0.1 mmol) in MeCN (2 mL) were
3
) d
þ
20
4.89, 24.45, 23.93, 19.88, 14.39; EI-MS (m/z) 487 (M ); ½aꢂ ¼ þ93
added K
2
CO
3
(0.5 mmol) and the appropriate secondary amine
D
ꢀ
(
c 0.053, CHCl
3
).
(2 mmol). The reaction mixture was heated to 50 C and stirred for

G. Liu et al. / European Journal of Medicinal Chemistry 103 (2015) 17e28
27
0
0
3
h. The solution was quenched with H
CH Cl
(2 ꢁ 10 mL). The combined organic layer was washed with
brine, dried over Na SO and concentrated under reduced pressure.
The crude residue was purified via silica column chromatography
2
O (5 mL) and extracted with
5.7.3. (8R,9 S)-6 -methoxytetrahydroisoquinoline-fused
0
2
2
spiroisoxazolidine deoxyartemalogue (22 )
White solid, (10 mg, 11%); 1H NMR (500 MHz, Chloroform-d)
2
4
d
7.00 (d, J ¼ 8.5 Hz, 1H), 6.77 (dd, J ¼ 8.5, 2.7 Hz, 1H), 6.64 (d,
with CH
2
Cl
2
/MeOH (20:1) to provide the desired products.
J ¼ 2.7 Hz, 1H), 5.73 (s, 1H), 4.88 (dd, J ¼ 10.9, 6.3 Hz, 1H), 3.79 (s,
3
H), 3.42e3.39 (m,1H), 3.07e3.01 (m, 2H), 2.91 (dd, J ¼ 13.8, 6.4 Hz,
0
0
5.6.1. (9R,9 R,10S)-10-(2-(4-methylpiperazin-1-yl)ethoxy)-6 -
1H), 2.77e2.74 (m, 1H), 2.61 (dd, J ¼ 13.9, 11.0 Hz, 1H), 2.37e2.33
(m, 1H), 2.13 (dd, J ¼ 13.0, 4.2 Hz, 1H), 1.91e1.88 (m, 1H), 1.83e1.79
(m, 1H), 1.76e1.72 (m, 1H), 1.60e1.54 (m, 4H), 1.31e1.17 (m, 4H),
methoxytetrahydroisoquinoline-fused spiroisoxazolidine
artemalogue (18)
White solid, (46 mg, 81%); 1H NMR (300 MHz, Chloroform-d)
7.04 (d, J ¼ 8.4 Hz, 1H), 6.73 (dd, J ¼ 8.4, 2.6 Hz, 1H), 6.58 (d,
1.13e1.05 (m, 1H), 0.93 (d, J ¼ 6.3 Hz, 3H); C NMR (101 MHz,
13
d
3
CDCl ) d 170.26, 158.16, 134.64, 128.49, 126.53, 112.82, 112.73,
J ¼ 2.6 Hz, 1H), 5.60 (s, 1H), 4.54e4.50 (m, 1H), 4.31 (s, 1H),
110.06, 99.41, 83.52, 82.64, 61.68, 55.27, 50.64, 49.97, 49.49, 44.73,
35.60, 33.74, 33.29, 29.15, 25.62, 24.20, 22.04, 18.60; EI-MS (m/z)
441 (M ); ½aꢂ ¼ ꢃ62 (c 0.054, CHCl
3
.76e3.66 (m, 7H), 3.51e3.18 (m, 4H), 3.01e2.15 (m, 11H),
.06e1.86 (m, 4H), 1.63e1.58 (m, 1H), 1.51e1.24 (m, 7H), 0.98e0.90
þ
20
D
2
3
).
(
m, 4H); 13C NMR (126 MHz, CDCl
3
)
d
157.48, 134.30, 127.88, 127.05,
1
5
2
0
12.38, 103.53, 103.40, 87.14, 82.32, 80.44, 66.41, 65.14, 60.94, 57.52,
5
.8. Proliferation inhibition assays
4.72, 53.33, 52.33, 48.93, 48.80, 48.07, 37.15, 36.00, 33.77, 25.29,
þ
20
D
5.09, 24.33, 23.96, 19.82; EI-MS (m/z) 572 (M ); ½aꢂ ¼ þ110 (c
Sulforhodamine B (SRB) assays were done to measure the IC50
3
.048, CHCl ).
values of different agents in the cells exposed to their gradient
concentrations for 72 h. For this purpose, KB, KB/VCR, A549 cells
were seeded into 96-well plates, cultured overnight, and treated
with agents for the indicated time. The absorbance at 560 nm was
measured with spectra-MAX190 (Molecular Devices). The per-
centage of cell proliferation inhibition was calculated as: prolifer-
ation inhibition (%) [1 ꢃ (A560treated/A560control)] ꢁ 100%. The
averaged IC50 values were determined with the Logit method from
three independent tests.
0 0
.6.2. (9R,9 R,10S)-10-(2-morpholinoethoxy)-6 -
5
methoxytetrahydroisoquinoline-fused spiroisoxazolidine
artemalogue (19)
White solid, (50 mg, 86%); 1H NMR (300 MHz, Chloroform-d)
d
5
7.06 (d, J ¼ 8.4 Hz, 1H), 6.76e6.68 (d, J ¼ 8.4 Hz, 1H), 6.61 (s, 1H),
.59 (s, 1H), 4.56e4.53 (m, 1H), 4.34 (s, 1H), 3.78 (s, 3H), 3.52e2.98
(
m, 6H), 2.65e1.87 (m, 20H), 1.65e1.26 (m, 8H), 1.01e0.95 (m, 4H);
1
3
C NMR (126 MHz, CDCl
03.51, 103.43, 87.15, 82.33, 80.47, 65.52, 60.99, 56.97, 54.73, 54.37,
2.42, 52.32, 48.97, 48.82, 48.06, 45.22, 37.05, 36.01, 33.76, 25.30,
3
) d 157.47, 134.33, 127.87, 127.09, 112.37,
1
Acknowledgment
5
2
0
þ
20
D
5.17, 24.30, 23.95, 19.76; EI-MS (m/z) 585 (M ); ½aꢂ ¼ þ111 (c
This work was supported by grants from the Pujiang Talent
Project from Shanghai Commission of Science and Technology
.048, CHCl
.7. Preparation of compounds 22 and 22
Intermediate 21 was prepared according to the same procedure
3
).
(14PJ1410700), and Chinese NSF (81402790, 81125021, 81373277,
0
5
8
1430080), and National Program on Key Basic Research Project of
China (2015CB910603-004).
for the preparation of artemistene 7 [20].
Appendix A. Supplementary data
A mixture of compound 21 (0.2 mmol) and nitrone 10b
ꢀ
(
0.24 mmol) in dry toluene (2 mL) was stirred at 60 C for 12 h. The
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.08.035.
solvent was evaporated in vacuo and the residue was purified via
silica column chromatography with petroleum ether/ethyl acetate
(
2:1) to give 22 and 22′ as a pair of isomers.
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