Novel dihydroisoxazoline-alkyl carbon chain hybrid artemisinin analogues (artemalogs): Synthesis and antitumor activities

Article abstract of DOI:10.1039/c6ra17323c

Two new series of dihydroisoxazoline-alkyl carbon chain hybrid artemisinin analogues (artemalogs) were designed and synthesized though a 1,3-dipolar cycloaddition. Subsequent pharmacological screening led to several compounds having dramatically improved antiproliferative effects against several human tumor cell lines compared to artemisinin and dihydroartemisinin. Mechanistic studies on the most potent artemalogs were investigated.

Full text of DOI:10.1039/c6ra17323c

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ARTICLE
Novel Dihydroisoxazoline-Alkyl Carbon Chain Hybrid Artemisinin
Analogues (Artemalogs): Synthesis and Antitumor Activities
Received 00th January 20xx,
Accepted 00th January 20xx
Gang Liu,†,§ Shanshan Song,ǂ,§ Xiaohua Liu,† Ao Zhang,†,∫ Zehong Miao,ǂ,∫,* and Chunyong Ding†,*
DOI: 10.1039/x0xx00000x
Two new series of dihydroisoxazoline-alkyl carbon chain hybrid artemisinin analogues (artemalogs) were designed and
synthesized though a 1,3-dipolar cycloaddition. Subsequent pharmacological screeneing led to several compounds having
dramatically improved antiproliferative effects against several human tumor cell lines compared to artemisinin and
dihydroartemisinine. Mechanistic studies on the most potent artemalogs were investigated.
www.rsc.org/
activities,22-37 compound
1 bearing a lipophilic carbon chain is
Introduction
one of the most potent artemalogs showing an IC50 value of
0.46 μM against the proliferation of HepG2 cell lines, whereas
the natural artemisinin only showed a much weak cytotoxicity
(IC50 = 97 μM).34 Recently, our group prepared a series of new
artemalogs bearing an isoxazoline/isoxazolidine motif and
tested them for the antiproliferative effects against quamous
carcinoma KB cells, vincristine-resistant KB/VCR cells, and
human lung cancer A549 cells. Among these artemalogs,
The unique molecular frameworks of natural products have
offered medicinal chemists ever-lasting inspiration for the
design and development of biologically useful chemical probes
and therapeutical drugs.1-6 Though a few are used directly as
disease treatment without any chemical modifications, the
majority of natural products needs major cut-offs or adds-on in
order to transfer to clinically useful drugs due to the strict
molecule druggability and safety criteria.1-2 Artemisinin, the
active component of the sweet wormwood plant (Artemisia
annua L.) is a natural sesquiterpene lactone containing a 1,2,4-
trioxane.7-8 The unique structure of artemisinin and its
significant activity against multidrug resistant malaria has built
up a new paradigm of antimalarial drug discovery, and several
of these drugs (dihydroartemisinin, artemether, and
artesunate) have been used for decades as the standard
treatment of P. falciparum.9-10 Meanwhile, artemisinin is also
profiled extensively in many other disease fields, especially
cancer.11-17 Although the excise anticancer mechanism of
artemisinin has been unknown, it is believed that a similar
mechanism as to the antimalarial activity may exist involving
cytotoxic carbon-centered free radicals generated by
interaction of the endoperoxide moiety of artemisinin with
heme iron.18-21 Among the various artemisinin analogues
(artemalogs) that have been reported to show antitumor
spirobicyclic artemalogue
2 was found to be the most potent
with IC50s of 1.47, 3.16 and 5.01 µM, respectively against the
three tumor cells, which were more than four-fold more
potent than that of the parent artemisinin.37
H
H
O
O
H
O
O
O
O
O
H
N
H
H
O
O
O
O
H
OMe
H
N
O
O
O
C16H33
2
O
1
O
KB, IC50 = 1.47
artemisinin
KB/VCR, IC50 = 3.16
A549, IC50 = 5.01
(HepG2, IC50 = 0.46
)
(HepG2, IC50 = 97
)
H
H
O
O
O
O
O
H
O
H
O
O
O
O
X
N
R
N
X
O
O
II
R
†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;
I
(X = O or N, R = long carbon chain)
(proposed dihydroisoxazoline-long carbon chainhybrid artemalogs)
ǂState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica
(SIMM), Chinese Academy of Sciences, Shanghai,201203, China;
∫University of Chinese Academy of Sciences, Beijing 100049, China
§These two authors contributed equally to this work.
Fig. 1 Artemisinin and its analogues.
∗To whom correspondence should be addressed. Zehong Miao,
zhmiao@simm.ac.cn; Tel: 86-21-50806820; Fax: 86-21-50806600; Chunyong
Ding, Fax: +86-50806035; Fax: 86-21-50806600; E-mail: chding@simm.ac.cn
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
As an extension of our interest in the development of
antitumor agents based on the unique endoperoxide motif of
artemisinin, we recently proposed to take advantage of both
structural features of compounds
hrbrid artemalogs
1 and 2 to generate new
and II, which contains both the lipophilic
I
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Journal Name
carbon chain as that in
isoxazoline heterocycle as the linker as that in
1
as the tail terminus and the
(Fig. 1).
Scheme 1 Synthesis of compounds 7a-7f.
2
Herein, we report the synthesis of these two new series of
artemalogs and their cytotoxicity against several tumor cells.
To diversify artemisinin scaffold, we shifted our synthetic effort
to the ring-contracted artemisinin derivatives. The aldehyde
derivative 9, readily accessible starting from artemisinin according
to the literature procedure,40 was treated with hydroxylamine
hydrochloride to give the oxime 10 in 97% yield, which was then
used to generate the nitrile oxide intermediate in the presence of
excess NaClO. Further 1,3-dipolar cycloaddition afforded 11 and 11'
in 44% and 28% yields, respectively. In this case, diastereoisomers
11 and 11' could be separated by silica gel column. The absolute
configuration of the newly formed C-5' center could not be deduced
by NOE and efforts to crystallize these isomers were unsuccessful.
Results and discussion
Chemistry
As shown in Scheme 1, the aldehyde derivative 3 was obtained over
4
steps starting from artemisinin by following
a literature
procedure.38 Treatment of aldehyde
3
with hydroxylamine
hydrochloride gave the oxime 4, which was further oxidized into the
nitrile oxide intermediate by NaClO to undergo the 1,3-dipolar
cycloaddition39 with ethyl acrylate, affording the dihydroisoxazoline
ester product 5 as a mixture of two diastereomers in 76% yield.
Without further isolation, hydrolysis of 5 followed by amide
condensation with alkyl amines of various length provided the
dihydroisoxazoline amides 7a-7e in 63-82% yields. The
dihydroisoxazoline ester 7f could be achieved in 46% yield when
dodecyl acrylate was used as the dipolarophile in the corresponding
1,3-dipolar cycloaddition. It should be noted that neither
dihydroisoxazoline esters 5 and 7f nor dihydroisoxazoline amide 7a-
7e could be separated by TLC or silica gel column, but the NMR
spectra indicated that all of them were diastereomeric mixtures.
The molar ratio of the isomers of amides 7a-7e was determined by
the HPLC data (see SI). To obtain optically pure derivatives, 7d was
separated by preparation chiral-HPLC to provide the (-)-
diastereomer 7d-1 and the (+)-diastereomer 7d-2, respectively
(Scheme 1). Nevertheless, their absolute stereochemistry at C-5'
could not be assigned by 2D NMR, and crystallization of these
isomers were also unsuccessful.
As shown in experiment procedure section, [α] 20
value of
D
compound 11 is -50.0 (c 0.051, MeOH), while the [α]2D0 value of 11' is
+130.0 (c 0.058, MeOH). After hydrolysis of the dihydroisoxazoline
esters 11 and 11', acid 12 was condensed with dodecan-1-amine,
tetradecan-1-amine and hexadecan-1-amine, respectively, to
provide dihydroisoxazoline amides 13-15 in 65-86% yields, whereas
condensation of 12' with tetradecan-1-amine and hexadecan-1-
amine yielded 14'and 15' in 74% yields (Scheme 2).
Scheme 2 Synthesis of compounds 13-15 and 14'-15'
.
To improve aqueous solubility, substituted piperazines
bearing carbon chains of various length were employed to
couple with the dihydroisoxazoline carbolic acid 12 leading to
the dihydroisoxazoline amides 18a-18c in 43-64% yields
(Scheme 3a). The dihydroisoxazoline amide 22 bearing an oxa-
alkyl
carbon
chain
could
be
achieved
when
tetraoxatetradecan-1-amine was used (Scheme 3b). To further
diversify artemisinin scaffold as well as investigate the role of
the endoperoxide moiety on antitumor activity, 14 was
.
subjected to FeCl2 4H2O/CH3CN reduction leading to the Ring-
contracted furan acetate product 23 in 82% yield (Scheme
3c).41
2 | J. Name., 2012, 00, 1-3
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Biological activity
In the case of ring-contracted artemalogs 13-15 and 14',15',
In vitro cytotoxic activity. The growth inhibitory effects of all the
synthesized dihydroisoxazoline artemalogs bearing long alkyl chains
were evaluated against three human cancer cell lines, including
squamous carcinoma KB cells, vincristine-resistant KB/VCR cells,
and human lung cancer A549 cells using SRB assays as described in
the in vitro screening protocol (Experimental Section).42 The result
was summarized in Table 1 and Table 2. Both artemisinin and
dihydroartemisinine were chosen as positive controls. As shown in
Table 1, most of the newly synthesized artemalogs (7a−7f) not only
exhibited significantly improved antiproliferative activity against KB
and A549 cells, but also displayed marked growth inhibitory effects
against vincristine-resistant KB/VCR cells, for which artemisinin had
only weak activity with an average IC50 value greater than 20 μM.
Among them, amide analogue 7d with a tetradecyl chain displayed
the most potent antiproliferative activity against all tested cancer
cell lines with IC50 values of 0.68, 0.47 and 0.49 µM, respectively.
Both the (-)-diastereomer 7d-1 and the (+)-diastereomer 7d-2
exhibited comparable antiproliferative potency to that of 7d,
indicating the stereochemistry at C-5' position generally has no
significant impact on the cellular activity. Thus, diastereomeric
mixture 7d could be directly used without separation in the
following pharmacological characterization. Artemalogs with longer
or shorter alkyl chains exhibited less potent activity. Replacement
of the amide bond with an ester bond also led to the decreased
activity against all tested cell lines, especially for A549 cell.
most of them possessed similar potency with submicromolar
IC50 values relative to the aforementioned artemalogs. New
artemalogs bearing a tetradecyl chain also showed the most
potent activity. Generally, the stereochemistry at C-5' position
also has no significant impact on the cellular effect. The (-)-
diastereomer 14 and the (+)-diastereomer 14' possessed
similar potency against KB (0.21 vs 0.24 uM) and KB/VCR (0.58
vs 0.72 μM) cells, but 14 is about one-fold more potent than
14' in terms of A549 cell (0.64 vs 1.21 μM). Similarly, for
artemalogs 15 and 15', there is no significant difference on
their potency against KB (0.55 vs 0.49 μM) and A549 (0.87 vs
1.11 μM) cells, but 15' is one-fold more potent than 15 against
KB/VCR cells (0.86 vs 1.61 μM).
As shown in Table 2, slightly lower activity was observed
when piperazine was inserted into the lipophilic alkyl carbon
chain to give artemalog 18c with IC50 values of 0.88 and 2.42
μM against KB and KB/VCR cells, respectively. Shortening the
length of the alkyl carbon chain led to 18a with dramatically
decreased potency against the two tested cancer cell lines.
Installation of the aqueous hydroxyl group at the end of the
alkyl carbon chain resulted in 18b totally losing the activity.
Artemalog 22 with the tetraoxatetradecanyl chain is
completely inactive, indicating the crucial role of an all carbon
alkyl chain. The ring-contracted furan acetate artemalog 23
lacking the endoperoxide bridge moiety only displayed a
marginal activity (around 15 μM), suggesting the endoperoxide
moiety is another key pharmacophoric function.
(a)
TFA, CH2Cl2
RBr, K2CO3
MeCN, 50o
NH
BocN
NR
HN
NR
BocN
C
Compounds 7d and 14 elicited broad-spectrum in vitro
antitumor effects. To characterize the anticancer activity of the
most potent compounds 7d and 14, they were further tested
against a panel of 7 human cancer cell lines, including
leukemia, breast cancer, ovarian cancer, gastric cancer, colon
cancer, hepatoma, and lung cancer cells. As shown in Table 3,
compounds 7d and 14 exhibited a similar in vitro anticancer
spectrum with IC50 values ranging from 0.15 to 1.01 μM
against almost all of the above human cancer cell lines, except
the leukemia K562 cells in which compounds 7d and 14 were
much less effective with IC50 values of ~10 μM. The results
indicate that the new artemalogs possess a broad anticancer
spectrum.
17a-c
16a-c
H
O
O
O
H
HATU, HOAt, DIPEA
O
O
N
12, CH2Cl2, rt, 4 h
18a, R = C11H23 (54%)
N
O
18b, R = C10H20CH2OH (43%)
18c, R = C14H29 (64%)
NR
(b)
O
O
LAH, THF
NH3/MeOH
rt, 12 h
O
3
O
O
OEt
O
NH2
3
0 oC, 5h
19
20, 95%
H
O
O
O
H
HATU, HOAt, DIPEA
Table 1 Antiproliferative effects of new artemalogs against
O
NH2
O
O
human cancer cell lines
4
12
, CH2Cl2, rt, 4 h
N
O
O
21, 83%
N
H
4
22, 73%
(c)
H
H
O
O
AcO
O
H
O
H
FeCl2.4H2O
MeCN, rt
HN C14H29
H
O
O
O
O
(IC50, µM)
N
N
O
O
HN C14H29
23, 82%
Compound
X
R
14
KB
KB/VCR
A549
Scheme 3 Synthesis of compounds 18a-18c, 22 and 23.
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ARTICLE
Journal Name
Artemisinin
>20
4.79
10.3
5.36
3.65
0.68
0.41
0.63
1.55
3.13
1.30
0.21
0.24
0.55
0.49
>20
>20
10.66
ND
Dihydroarte
misinin
3.09
12.6
5.49
3.82
0.47
0.88
0.84
3.13
6.21
5.27
0.58
0.72
1.61
0.86
0.88
2.42
ND
7a
7b
NH
NH
NH
NH
NH
NH
NH
O
C6H13
C9H19
ND
7c
C12H25
C14H29
C14H29
C14H29
C16H33
C12H25
C12H25
C14H29
C14H29
C16H33
C16H33
ND
7d
0.49
1.52
0.67
3.28
> 20
ND
>20
>20
ND
7d-1
7d-2
7e
15.77
14.94
ND
7f
13
NH
NH
NH
NH
NH
14
0.64
1.21
0.87
1.11
ND - not determined.
14'
15
Compounds 7d and 14 produced direct cytotoxic effects on
multidrug resistant cancer cell lines. Multidrug resistance (MDR),
especially to drugs of natural origin, is an important
impediment to the effective chemotherapy of cancer.43
Therefore, new agents with capacity to circumvent MDR are of
critical importance. To examine the activity of artemalogs 7d
and 14 against MDR, we used three classical MDR cell lines
(K562/A02, KB/VCR, and MCF-7/ADR) that were significantly
resistant to the corresponding drugs.44
15'
ND - not determined.
Table 2 Cytotoxicities of 18a-c and 22, 23 against human
cancer cell lines
(IC50, µM)
Table 3 Inhibitory effects of 7d and 14 against cell
Compound
proliferation
KB
KB/VCR A549
H
(IC50, µM)
O
Cell lines
O
O
H
7d
12.48
0.52
0.73
0.68
0.23
0.75
0.35
0.15
14
O
15.66
8.07
ND
ND
O
N
N
K562
9.25
0.88
0.80
0.21
0.28
1.01
0.38
0.19
O
N
C11H23
18a
MCF-7
SK-OV-3
KB
> 20
15.83
SGC-7901
SW-620
BEL-7402
SPC-A4
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effect was induced by a cell-cycle arrest. We treated SGC-7901
cells with compound 7d for 24 or 48 h at 0.25 µM, 0.5 µM, 1.0
As shown in Table 4, the IC50 values of compound 7d against µM and 2.0 µM, respectively, and the DNA content of cell
these MDR cell lines were 10.23, 1.71, and 0.47 μM, while to nuclei was detected by flow cytometry. The result showed that
their corresponding sensitive cell lines (K562, MCF-7, and KB), 7d did not induce typical cell cycle arrest in SGC-7901 cells at
its IC50 values were 12.48, 0.52, and 0.68 μM, respectively. The any concentrations (Fig. 2).
results indicated that compound 7d showed approximately
Compound 7d induced apoptosis of SW-620 cells. To determine
equipotent cytotoxicity against each MDR cell lines as whether the growth inhibition induced by 7d was attributed to
compared with their parental cell lines. The corresponding apoptosis. SW-620 cells were treated with vehicle and Taxol
resistance factors are 0.82, 3.29, and 0.69, respectively (Table (TAX) as controls. Meanwhile, similar treatments of SW-620
4). Compared to 7d, Compound 14 displayed similar cells with 7d were conducted at different concentrations (5,
antiproliferative effects against MDR cell lines, with slightly 10, or 20 μM) for 48 h and then stained with FITC-Annexin V
less sensitivity against KB/VCR with higher RF values.
and propidium iodide (PI). The percentages of apoptotic SW-
620 cells were determined by flow cytometry. As shown in Fig.
Table 4 Inhibitory effects of 7d and 14 on proliferation of 3, compound 7d displayed moderate effects to induce
drug-resistant tumor cells
apoptosis of SW-620 cells in a dose-dependent manner, and
resulted in 8.6%, 12.2%, and 49.2% of apoptotic cells (early
and late apoptosis) with 5, 10, and 20 μM, respectively, as
compared to 3.1% in an untreated vehicle control.
7d
14
IC50 (μM)
Cell lines
IC50 (μM)
RF
RF
K562/A02
MCF-7/ADR
KB/VCR
10.23
1.71
0.47
0.82
3.29
0.69
9.81
2.13
0.58
1.06
2.42
2.76
NOTE: Resistance factor (RF) was calculated as the ratio of the
IC50 value of the drug-resistant cells to that of the
corresponding parent cells in Table 3.
Fig. 4 7d induced the cleavage of PARP and Caspase-3 in SW-
620 cells. ADR, adriamycin.
Effects of 7d on cell apoptosis proteins PARP and caspase-
3. To elucidate the potential mechanism of cell apoptosis
induced by the new artemalog 7d, several proteins related to
apoptosis were determined by Western blotting. As shown in
Fig. 4, treatment of SW-620 cells with compound 7d at
moderate concentrations (5−20 μM) slightly triggered PARP
and caspase-3 cleavages from their full-length form to the
cleaved form as indicated by the weak appearance of PARP
fragments and activated caspase-3 in a concentration-
dependent manner.
Fig. 2 7d did not induce typical cell cycle arrest in SGC-7901
cells.
Conclusions
Two new series of dihydroisoxazoline-lipophilic carbon chain
hybrid artemalogs were designed and synthesized though 1,3-
dipolar cycloaddition. Most of these compounds displayed
significantly improved antiproliferative effects against three
human tumor cell lines, compared to artemisinin and
dihydroartemisinine. Among these new artemalogs,
dihydroisoxazoline amide artemalogs 7d and 14 with
tetradecyl chain demonstrated the most potent activity with
submicromolar IC50 values. Further investigations indicated
that artemalogs 7d and 14 not only elicited broad-spectrum in
vitro antitumor effects, but also produced direct cytotoxic
Fig. 3 7d induced apoptosis in SW-620 cells. TAX, taxol.
Cell-cycle analysis. On the basis of the promising
antiproliferative effect, compound 7d was selected for further
mechanistic study to determine whether the growth inhibitory
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effects on multidrug resistant cancer cell lines. Mechanism afford a mixture of diastereoisomers
Journal Name
5
(128 mg, 76%) as a
1
studies showed that 7d did not induce cell cycle arrest colorless oil. H NMR (300 MHz, Chloroform-d) δ 5.33 – 5.32
significantly, but at least partially mediated apoptosis to exert (m, 1H), 5.03 – 4.94 (m, 1H), 4.59 – 4.51 (m, 1H), 4.28 – 4.19
its antiproliferative activity. Further in vivo studies and (m, 1H), 3.52 – 3.32 (m, 2H), 2.78 – 2.49 (m, 3H), 2.36 – 2.26
antitumor mechanism investigation of the new potent (m, 1H), 2.06 – 1.92 (m, 2H), 1.83 – 1.65 (m, 3H), 1.50 – 1.21
artemalogs are undergoing.
(m, 9H), 0.98 – 0.84 (m, 8H).
Preparation of the acid intermediate 6
Experimental Section
To a solution of compound
5 (85 mg, 0.2 mmol) in EtOH (2 mL)
General experimental information
and H2O (2 mL) was added lithium hydroxide monohydrate (10
mg, 0.24 mmol), and the reaction was stirred at rt for 4 h. The
aqueous layer was adjusted to pH 3 by adding hydrochloride
acid (0.5 M) and extracted with CH2Cl2 (3 × 10 mL). The
combined organic layers were washed with water and brine,
dried over anhydrous Na2SO4, filtered, and concentrated under
All reactions were performed in glassware containing a
Tefloncoated 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. 1H and 13C spectra were recorded
on Varian Mercury 300 MHz, 400MHz and 500 MHz and the
data were recorded using CDCl3 as the solvent. Chemical shifts
(δ) are reported in ppm downfield from an internal TMS
standard. Low and high-resolution mass spectra were obtained
in the ESI and EI mode. Flash column chromatography on silica
gel (200-300 mesh) was used for the routine purification of
reaction products. The column output was monitored by TLC
on silica gel (100-200 mesh) precoated on glass plates (15 x 50
mm). HPLC analysis was conducted for all bioassayed
compounds on an Agilent Technologies 1260 series LC system
(Agilent ChemStation Rev.A.10.02; SB-C18, 4.6 mm × 150 mm,
2 μM, MeOH/H2O, rt) with the ultraviolet wavelengths of 214
nm. All the assayed compounds displayed chemical purity
greater than 95%.
reduced pressure to give compound
6 (74 mg, 94%). The crude
product was used to the next step without further purification.
Preparation of 7a-7e
To a solution of compound
6 (0.1 mmol) in CH2Cl2 (2 mL),
o
HATU (0.2 mmol) and HOAT (0.1 mmol) was added at 0 C.
After stirring for 10 min at 0 oC, an appropriate aliphatic amine
(0.1 mmol) and N, N-diisopropylethylamine (0.3 mmol) were
added to this solution. The resulting mixture was stirred at rt
for 4 h and then diluted with CH2Cl2 (15 mL), washed with
saturated NH4Cl solution, water, and brine. The organic layer
was dried over anhydrous Na2SO4, filtered, and concentrated
in vacuo. The residue was purified via silica gel
chromatography with petroleum ether/ethyl acetate (2/1) as
the eluent to afford a mixture of diastereoisomers 7a-7e
.
10-(5'-(Hexylcarbamoyl)-4',5'-dihydroisoxazol-3'-
1
Preparation of intermediate 4
yl)methyldeoxoartemisinin (7a). Colorless oil (36 mg, 76%); H
NMR (400 MHz, Chloroform-d) δ 6.76 – 6.71 (m, 1H), 5.32 and
5.29 (s, s, 1H, H-12), 4.97 – 4.91 (m, 1H), 4.59 – 4.55 (m, 1H),
3.53 – 3.15 (m, 4H), 2.77 – 2.61 (m, 2H), 2.47 – 2.42 (m, 1H),
2.34 – 2.22 (m, 1H), 2.05 – 1.92 (m, 2H), 1.81 – 1.66 (m, 4H),
1.50 – 1.25 (m, 16H), 0.97 – 0.88 (m, 8H); 13C NMR (126 MHz,
Chloroform-d) δ 170.53 and 170.37 (1C), 158.40 and 158.37
(1C), 102.34 and 102.31 (1C), 89.36 and 89.24 (1C), 80.49,
77.73 and 77.56 (1C), 69.96 and 69.37 (1C), 51.38, 43.18,
41.39, 40.67, 38.66, 37.03 and 36.99 (1C), 36.11, 33.80, 30.97
and 30.91 (1C), 29.74 and 29.69 (1C), 28.86, 28.45 and 28.34
(1C), 26.02 and 25.99 (1C), 25.43 and 25.38 (1C), 24.32 and
24.29 (1C), 22.03, 19.53, 13.53 and 13.50 (1C), 11.94 and 11.91
(1C); MS (ESI) 501.2 [M + Na] ＋ . HRMS (ESI) calcd for
The aldehyde
procedure.39
3
was prepared according to the literature
To a mixture of compound
3
(310 mg, 1 mmol) and NaHCO3
(420 mg, 5 mmol) in MeOH (5 mL) was added hydroxylamine
hydrochloride (345 mg, 5 mmol). The resulting mixture was
stirred at rt for 1 h, and concentrated in vacuo. The residue
was diluted with water (20 mL) and washed with CH2Cl2 (3×10
mL). The combined organic layers were washed with water
and brine, dried over anhydrous Na2SO4, filtered, and
concentrated under reduced pressure to give compound
4
(286 mg, 88%) as a colorless oil. The crude product was used to
the next step without further purification.
C26H43N2O6, 479.3116; found, 479.3118. [α]20 = +54 (c 0.049,
D
Preparation of 10-(5'-(methoxycarbonyl)-4',5'-dihydroisoxazol-3'-
yl)methyldeoxoartemisinin (5)
MeOH).
10-(5'-(Nonylcarbamoyl)-4',5'-dihydroisoxazol-3'-
To a solution of compound
4 (130 mg, 0.4 mmol) and ethyl
yl)methyldeoxoartemisinin (7b). Colorless oil (33 mg, 63%); 1H
NMR (400 MHz, Chloroform-d) δ 6.79 – 6.73 (m, 1H), 5.32 and
5.30 (s, s, 1H, H-12), 4.98 – 4.91 (m, 1H), 4.60 – 4.54 (m, 1H),
3.46 – 3.17 (m, 4H), 2.78 – 2.60 (m, 2H), 2.48 – 2.42 (m, 1H),
2.35 – 2.27 (m, 1H), 2.06 – 1.92 (m, 2H), 1.82 – 1.65 (m, 3H),
1.52 – 1.21 (m, 23H), 0.98 – 0.86 (m, 8H); 13C NMR (126 MHz,
Chloroform-d) δ 170.52 and 170.36 (1C), 158.36 and 158.34
(1C), 102.32 and 102.28 (1C), 89.34 and 89.23 (1C), 80.46,
acrylate (44 mg, 0.44 mmol) in dry CH2Cl2 (10 mL) was added a
solution of sodium hypochlorite (2.24 mL, 13 % active
chlorine), then triethylamine (61 mg, 0.6 mmol) was added
dropwise at rt. The mixture was stirred at rt for 6 h and then
washed with water (2 x 10 mL). The organic layer was washed
with brine, dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by silica gel
chromatography using petroleum ether/ethyl acetate (5/1) to
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77.71 and 77.55 (1C), 69.90 and 69.36 (1C), 51.36, 43.16, 2.05 – 1.92 (m, 2H), 1.81 – 1.65 (m, 3H), 1.52 – 1.25 (m, 33H),
41.36, 40.66, 38.65, 37.01 and 36.97 (1C), 36.10 and 36.08 1.03 – 0.83 (m, 8H). 13C NMR (126 MHz, CDCl3) δ 171.87,
(1C), 33.79, 31.36 and 31.33 (1C), 29.72 and 29.68 (1C), 29.18 – 159.70, 103.66, 90.71, 81.82, 78.91, 70.68, 52.72, 44.52, 42.00,
28.33 (5C), 26.35 and 26.32 (1C), 25.42 and 25.37 (1C), 24.30 39.99, 38.32, 37.43, 35.13, 32.75, 31.03, 30.70 – 29.97 (9C),
and 24.27 (1C), 22.15, 19.51, 13.60, 11.91 and 11.89 (1C); MS 29.80, 27.68, 26.71, 25.62, 23.52, 20.85, 14.95, 13.25. [α]2D0 =
(ESI) 543.3 [M + Na]＋ . HRMS (ESI) calcd for C29H49N2O6, +100 (c 0.051, MeOH).
521.3585; found, 521.3597. [α]2D0 = +72 (c 0.050, MeOH).
10-(5'-(Hexadecylcarbamoyl)-4',5'-dihydroisoxazol-3'-
yl)methyldeoxoartemisinin (7e). colorless oil (48 mg, 77%); 1H
10-(5'-(Dodecylcarbamoyl)-4',5'-dihydroisoxazol-3'-
1
yl)methyldeoxoartemisinin (7c). Colorless oil (40 mg, 72%); H NMR (400 MHz, Chloroform-d) δ 6.76 – 6.70 (m, 1H), 5.32 and
NMR (400 MHz, Chloroform-d) δ 6.75 – 6.72 (m, 1H), 5.32 and 5.29 (s, s, 1H, H-12), 4.97 – 4.91 (m, 1H), 4.60 – 4.55 (m, 1H),
5.29 (s, s, 1H, H-12), 4.97 – 4.90 (m, 1H), 4.59 – 4.54 (m, 1H), 3.49 – 3.16 (m, 4H), 2.73 – 2.62 (m, 2H), 2.48 – 2.42 (m, 1H),
3.50 – 3.14 (m, 4H), 2.77 – 2.59 (m, 2H), 2.48 – 2.42 (m, 1H), 2.34 – 2.22 (m, 1H), 2.05 – 1.92 (m, 2H), 1.81 – 1.65 (m, 3H),
2.34 – 2.26 (m, 1H), 2.04 – 1.92 (m, 2H), 1.81 – 1.65 (m, 3H), 1.49 – 1.25 (m, 37H), 0.97 – 0.84 (m, 8H); 13C NMR (126 MHz,
1.50 – 1.21 (m, 29H), 0.97 – 0.88 (m, 8H); 13C NMR (126 MHz, Chloroform-d) δ 170.54 and 170.38 (1C), 158.40 and 158.37
Chloroform-d) δ 170.52 and 170.35 (1C), 158.37 and 158.35 (1C), 102.34 and 102.31 (1C), 89.37 and 89.25 (1C), 80.50,
(1C), 102.32 and 102.29 (1C), 89.36 and 89.25 (1C), 80.48, 77.73 and 77.57 (1C), 69.93 and 69.37 (1C), 51.39, 43.19, 41.39,
77.72 and 77.56 (1C), 69.91 and 69.35 (1C), 51.37, 43.17, 40.67, 38.67, 37.03 and 36.99 (1C), 36.10, 33.80, 31.43, 29.70,
41.38, 40.67, 38.66, 37.03 and 36.98 (1C), 36.11 and 36.09 29.20 – 28.35 (12C), 26.37 and 26.35 (1C), 25.44 and 25.39
(1C), 33.80, 31.41, 29.74 and 29.69 (1C), 29.17 – 28.35 (8C), (1C), 24.29, 22.20, 19.53, 13.63, 11.94; MS (ESI) 641.4 [M + Na]
26.37 and 26.34 (1C), 25.43 and 25.38 (1C), 24.31 and 24.28 ＋. HRMS (ESI) calcd for C36H63N2O6, 619.4681; found, 619.4695.
(1C), 22.18, 19.52, 13.62, 11.93 and 11.90 (1C); MS (ESI) 585.3 [α]2D0 = +67 (c 0.046, MeOH).
[M + Na]＋. HRMS (ESI) calcd for C32H55N2O6, 563.4055; found,
563.4046. [α]2D0 = +64 (c 0.057, MeOH).
Preparation of 7f
10-(5'-(Tetradecylcarbamoyl)-4',5'-dihydroisoxazol-3'-
To a solution of compound
4 (65 mg, 0.2 mmol) and dodecyl
yl)methyldeoxoartemisinin (7d). Colorless oil (48 mg, 82%); 1H
NMR (300 MHz, Chloroform-d) δ 6.77 – 6.71 (m, 1H), 5.32 and
5.29 (s, s, 1H, H-12), 4.97 – 4.90 (m, 1H), 4.61 – 4.53 (m, 1H),
3.54 – 3.14 (m, 4H), 2.78 – 2.57 (m, 2H), 2.48 – 2.41 (m, 1H),
2.36 – 2.25 (m, 1H), 2.07 – 1.92 (m, 2H), 1.82 – 1.64 (m, 3H),
1.52 – 1.13 (m, 33H), 1.00 – 0.83 (m, 8H); 13C NMR (126 MHz,
Chloroform-d) δ 171.04 and 170.88 (1C), 158.90 and 158.55
(1C), 102.86 and 102.82 (1C), 89.87 and 89.75 (1C), 81.00,
78.23 and 78.07 (1C), 70.45 and 69.89 (1C), 51.89, 43.69, 41.89,
41.18, 39.18, 37.54 and 37.50 (1C), 36.60, 34.30, 31.93, 30.24
and 30.20 (1C), 29.70 – 28.85 (10C), 26.88 and 26.85 (1C),
25.94 and 25.89 (1C), 24.79, 22.70, 20.03, 14.13, 12.44 and
12.42 (1C); MS (ESI) 613.4 [M + Na]＋. HRMS (ESI) calcd for
acrylate (53 mg, 0.22 mmol) in dry CH2Cl2 (5 mL) was added a
solution of sodium hypochlorite (1.12 mL, 13 % active
chlorine), then triethylamine (31 mg, 0.3 mmol) was added
dropwise. The mixture was stirred at rt overnight and then
washed with water (2 x 10 mL). The organic layer was washed
with brine, dried over anhydrous Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by silica
column chromatography using petroleum ether/ethyl acetate
(5/1) to afford a mixture of diastereoisomers
a colorless oil.
7 (52 mg, 46%) as
10-(5'-Dodecyloxycarbonyl)-4',5'-dihydroisoxazol-3'-
yl)methyldeoxoartemisinin (7f). 1H NMR (400 MHz,
Chloroform-d) δ 5.32 (s, 1H), 5.01 – 4.93 (m, 1H), 4.57 – 4.52
(m, 1H), 4.18 – 4.13 (m, 2H), 3.50 – 3.30 (m, 2H), 2.76 – 2.49
(m, 3H), 2.35 – 2.27 (m, 1H), 2.04 – 1.92 (m, 2H), 1.82 – 1.58
(m, 5H), 1.38 – 1.25 (m, 27H), 0.97 – 0.86 (m, 8H); 13C NMR
(126 MHz, Chloroform-d) δ 170.66, 157.56 and 156.30 (1C),
102.87 and 102.81 (1C), 89.82 and 89.79 (1C), 81.04 and 81.01
(1C), 77.31 and 77.20, (1C), 71.06 and 70.38 (1C), 65.87, 51.95
and 51.91 (1C), 43.77 and 43.71 (1C), 41.18, 40.49, 37.54 and
37.49 (1C), 36.60, 34.31, 31.92, 30.30 and 30.19 (1C), 29.66 –
28.48 (8C), 25.89, 25.78, 24.80, 22.69, 20.05 and 19.96 (1C),
14.13, 12.49 and 12.40 (1C); MS (ESI) 586.4 [M + Na]＋. HRMS
C34H59N2O6, 591.4368; found, 591.4354. [α]20 = +75 (c 0.044,
D
MeOH).
(-)-10-(5'-(Tetradecylcarbamoyl)-4',5'-dihydroisoxazol-3'-
1
yl)methyldeoxoartemisinin (7d-1). Colorless oil. H NMR (400
MHz, CDCl3) δ 6.75 – 6.74 (m, 1H), 5.31 (s, 1H), 4.95 (dd, J =
11.6, 6.0 Hz, 1H), 4.60 – 4.56 (m, 1H), 3.49 (dd, J = 18.0, 11.6
Hz, 1H), 3.40 – 3.18 (m, 3H), 2.73 – 2.61 (m, 2H), 2.48 (dd, J =
11.2, 3.2 Hz, 1H), 2.36 – 2.28 (m, 1H), 2.05 – 1.94 (m, 2H), 1.83
– 1.67 (m, 2H), 1.51 – 1.27 (m, 33H), 0.99 – 0.86 (m, 8H). 13C
NMR (126 MHz, CDCl3) δ 171.72, 159.75, 103.66, 90.60, 81.83,
79.08, 71.29, 52.72, 44.52, 42.75, 40.02, 38.38, 37.46, 35.15,
32.77, 31.09, 30.69 – 29.83 (9C), 29.70, 27.72, 26.79, 25.66,
23.53, 20.87, 14.96, 13.25. [α]2D0 = -8 (c 0.049, MeOH).
(ESI) calcd for C32H54NO7, 564.3895; found, 564.3886. [α]20 =
D
+59 (c 0.051, MeOH).
(+)-10-(5'-(Tetradecylcarbamoyl)-4',5'-dihydroisoxazol-3'-
1
yl)methyldeoxoartemisinin (7d-2). Colorless oil. H NMR (500
Preparation of intermediate 10
The ring-contracted artemisinin aldehyde
according to the literature procedure.40
9 was prepared
MHz, CDCl3) δ 6.76 – 6.74 (m, 1H), 5.32 (s, 1H), 4.95 (dd, J =
11.5, 6.0 Hz, 1H), 4.57 (ddd, J = 11.5, 6.5, 3.0 Hz, 1H), 3.49 (dd,
J = 17.5, 12.0 Hz, 1H), 3.36 – 3.26 (m, 2H), 3.15 (td, J = 13.0, 7.0
Hz, 1H), 2.73 (dd, J = 15.0, 11.5 Hz, 1H), 2.66 – 2.57 (m, 1H),
2.44 (dd, J = 15.0, 3.0 Hz, 1H), 2.30 (tt, J = 14.5, 7.5 Hz, 1H),
To a mixture of compound
9 (282 mg, 1 mmol) and NaHCO3
(420 mg, 5 mmol) in MeOH (5 mL) was added hydroxylamine
hydrochloride (345 mg, 5 mmol). The resulting mixture was
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stirred at rt for 1 h, concentrated in vacuo. The residue was chromatography with petroleum ether/ethyl acetate (2/1) as
diluted with water (20 mL) and washed with CH2Cl2 (3 × 10 mL). the eluent to afford 13-15 and 14'-15'
The combined organic layer were washed with water and (-)-9-(5'-(Dodecylcarbamoyl)-4',5'-dihydroisoxazol-3'-yl)
.
1
brine, dried over anhydrous Na2SO4, filtered, and concentrated ring-contracted artemalog (13). Colorless oil (46 mg, 86%); H
under reduced pressure to give compound 10 (286 mg, 97%) NMR (400 MHz, Chloroform-d) δ 6.83 – 6.79 (m, 1H), 5.69 (s,
as colorless oil. The crude product was used to the next step 1H), 4.93 (dd, J = 11.5, 3.9 Hz, 1H), 3.58 (dd, J = 18.3, 11.5 Hz,
without further purification.
1H), 3.36 (dd, J = 18.3, 3.9 Hz, 1H), 3.31 – 3.13 (m, 2H), 2.36 –
2.22 (m, 2H), 2.12 – 1.95 (m, 2H), 1.84 (s, 3H), 1.75 – 1.70 (m,
1H), 1.55 – 1.25 (m, 27H), 0.99 – 0.86 (m, 8H); 13C NMR (126
MHz, Chloroform-d) δ 170.56, 165.20, 103.38, 97.13, 85.51,
83.97, 77.43, 53.26, 48.05, 41.62, 38.73, 36.57, 36.06, 31.95,
31.40, 29.20 – 28.78 (7C), 27.97, 26.35, 25.90, 24.75, 23.73,
22.18, 19.24, 13.62; MS (ESI) 557.4 [M + Na]＋. HRMS (ESI)
Preparation of 11 and 11'
To a solution of compound 10 (118 mg, 0.4 mmol) and ethyl
acrylate (44 mg, 0.44 mmol ) in dry CH2Cl2 (10 mL) was added a
solution of sodium hypochlorite (2.24 mL, 13 % active chlorine)
and then triethylamine (61 mg, 0.6 mmol) was added dropwise
at rt. The mixture was stirred at rt overnight and then washed
with water (2 x 10 mL). The organic layer was washed with
brine, dried over anhydrous Na2SO4, filtered, and concentrated
in vacuo. The residue was purified by silica gel chromatography
using petroleum ether/ethyl acetate (5/1) to afford 11 (70 mg,
44%) and 11' (44 mg, 28%) as a colorless oil.
calcd for C30H51N2O6, 535.3742; found, 535.3743. [α]20 = -33 (c
D
0.057, MeOH).
(-)-9-(5'-(Tetradecylcarbamoyl)-4',5'-dihydroisoxazol-3'-yl)
1
ring-contracted artemalog (14). Colorless oil (45 mg, 81%); H
NMR (400 MHz, Chloroform-d) δ 6.84 – 6.81 (m, 1H), 5.69 (s,
1H), 4.93 (dd, J = 11.6, 4.0 Hz, 1H), 3.58 (dd, J = 18.4, 11.6 Hz,
1H), 3.36 (dd, J = 18.4, 4.0 Hz, 1H), 3.30 – 3.14 (m, 2H), 2.36 –
2.23 (m, 2H), 2.12 – 1.95 (m, 2H), 1.84 (s, 3H), 1.75 – 1.68 (m,
1H), 1.56 – 1.25 (m, 31H), 1.02 – 0.86 (m, 8H); 13C NMR (126
MHz, Chloroform-d) δ 170.57, 165.21, 103.39, 97.13, 85.52,
83.97, 77.43, 53.25, 48.06, 41.62, 38.73, 36.57, 36.07, 31.95,
31.42, 29.18 – 28.78 (9C), 27.97, 26.35, 25.90, 24.75, 23.73,
22.19, 19.24, 13.62; MS (ESI) 585.4 [M + Na]＋. HRMS (ESI)
(-)-9-(5'-(Methoxycarbonyl)-4',5'-dihydroisoxazol-3'-yl)
1H NMR (300 MHz,
ring-contracted artemalog (11)
.
Chloroform-d) δ 5.71 (s, 1H), 4.97 (dd, J = 11.6, 5.6 Hz, 1H),
4.23 (q, J = 7.1 Hz, 2H), 3.58 (dd, J = 18.0, 11.6 Hz, 1H), 3.29
(dd, J = 18.0, 5.6 Hz, 1H), 2.37 – 2.29 (m, 2H), 2.11 – 1.95 (m,
3H), 1.85 (s, 3H), 1.57 – 1.47 (m, 5H), 1.41 – 1.22 (m, 5H), 1.03
– 0.98 (m, 5H). [α]2D0 = -50 (c 0.051, MeOH).
calcd for C32H55N2O6, 563.4055; found, 563.4070. [α]20 = -33 (c
D
(+)-9-(5'-(Methoxycarbonyl)-4',5'-dihydroisoxazol-3'-yl)
0.044, MeOH).
ring-contracted artemalog (11')
.
H
NMR (300 MHz,
(+)-9-(5'-(Tetradecylcarbamoyl)-4',5'-dihydroisoxazol-3'-yl)
ring-contracted artemalog (14'). Colorless oil (42 mg, 74%); 1H
NMR (400 MHz, Chloroform-d) δ 6.76 – 6.73 (m, 1H), 5.71 (s,
1H), 4.93 (dd, J = 11.2, 4.8 Hz, 1H), 3.48 (dd, J = 17.6, 4.8 Hz,
1H), 3.34 – 3.14 (m, 3H), 2.36 – 2.20 (m, 2H), 2.11 – 1.96 (m,
2H), 1.78 – 1.72 (m, 4H), 1.60 – 1.25 (m, 31H), 1.10 – 0.86 (m,
8H); 13C NMR (126 MHz, Chloroform-d) δ 171.12, 163.60,
103.35, 96.84, 85.69, 84.16, 77.67, 52.53, 48.08, 41.41, 38.72,
36.58, 36.27, 31.91, 31.43, 29.20 – 28.80 (9C), 27.21, 26.34,
26.19, 24.71, 23.83, 22.20, 19.30, 13.63; MS (ESI) 585.4 [M +
Na]＋ . HRMS (ESI) calcd for C32H55N2O6, 563.4055; found,
563.4054. [α]2D0 = +82 (c 0.053, MeOH).
Chloroform-d) δ 5.63 (s, 1H), 4.89 (dd, J = 11.6, 6.4 Hz, 1H),
4.16 (q, J = 7.2 Hz, 2H), 3.50 (dd, J = 17.9, 6.4 Hz, 1H), 3.21 (dd,
J = 17.9, 11.4 Hz, 1H), 2.30 – 2.21 (m, 2H), 2.04 – 1.89 (m, 3H),
1.76 (s, 3H), 1.54 – 1.40 (m, 5H), 1.36 – 1.17 (m, 5H), 1.00 –
0.86 (m, 5H). [α]2D0 = +130 (c 0.058, MeOH).
Preparation of the acid 12 and 12'
To a solution of compound 11 or 11' (79 mg, 0.2 mmol) in
EtOH (2 mL) and H2O (2 mL) was added lithium hydroxide
monohydrate (10 mg, 0.24 mmol). The reaction was stirred at
rt for 4 h, and then adjusted to pH 3 by adding hydrochloride
acid (0.5 M) and extracted with CH2Cl2 (3 × 10 mL). The
combined organic layers were washed with water and brine,
dried over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure. The crude product 12 (68 mg, 93%) or 12'
(64 mg, 88%) was used to the next step without further
purification.
(-)-9-(5'-(Hexadecylcarbamoyl)-4',5'-dihydroisoxazol-3'-yl)
ring-contracted artemalog (15). Colorless oil (38 mg, 65%); 1H
NMR (400 MHz, Chloroform-d) δ 6.83 – 6.80 (m, 1H), 5.69 (s,
1H), 4.93 (dd, J = 11.6, 4.0 Hz, 1H), 3.58 (dd, J = 18.4, 11.6 Hz,
1H), 3.36 (dd, J = 18.4, 4.0 Hz, 1H), 3.31 – 3.13 (m, 2H), 2.36 –
2.20 (m, 2H), 2.11 – 1.96 (m, 2H), 1.84 (s, 3H), 1.75 – 1.70 (m,
1H), 1.55 – 1.25 (m, 35H), 1.02 – 0.86 (m, 8H); 13C NMR (126
MHz, Chloroform-d) δ 170.56, 165.20, 103.37, 97.13, 85.51,
Preparation of 13-15 and 14'-15'
To a solution of compound 12 or 12' (0.1 mmol) in CH2Cl2 (2 83.97, 77.43, 53.26, 48.05, 41.62, 38.73, 36.57, 36.06, 31.95,
mL), HATU (0.2 mmol) and HOAT (0.1 mmol) was added at 0 31.42, 29.19 – 28.79 (11C), 27.96, 26.35, 25.90, 24.75, 23.73,
o
oC. After stirring for 10 min at 0 C, an appropriate aliphatic 22.19, 19.24, 13.62; MS (ESI) 591.3 [M + H]＋. HRMS (ESI) calcd
amine (0.1 mmol) and N,N-diisopropylethylamine (0.3 mmol) for C34H59N2O6, 591.4368; found, 591.4366. [α]20 = -24 (c 0.043,
D
were added to this solution. The resulting mixture was stirred MeOH).
at rt for 4 h, and then diluted with CH2Cl2 (15 mL), washed with
(+)-9-(5'-(Hexadecylcarbamoyl)-4',5'-dihydroisoxazol-3'-yl)
saturated NH4Cl solution, water, and brine. The organic layer ring-contracted artemalog (15'). Colorless oil (44 mg, 74%); 1H
was dried over anhydrous Na2SO4, filtered, and concentrated NMR (400 MHz, Chloroform-d) δ 6.79 – 6.75 (m, 1H), 5.73 (s,
in vacuo. The residue was purified via silica gel 1H), 4.94 (dd, J = 11.6, 4.8 Hz, 1H), 3.48 (dd, J = 18.0, 4.8 Hz,
8 | J. Name., 2012, 00, 1-3
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1H), 3.36 – 3.16 (m, 3H), 2.37 – 2.25 (m, 2H), 2.12 – 1.97 (m, (m, 3H), 2.41 – 2.27 (m, 5H), 2.13 – 2.06 (m, 1H), 2.01 – 1.97
2H), 1.80 – 1.70 (m, 4H), 1.58 – 1.27 (m, 35H), 1.12 – 0.88 (m, (m, 1H), 1.88 – 1.84 (m, 4H), 1.63 – 1.40 (m, 9H), 1.37 – 1.11
8H); 13C NMR (126 MHz, Chloroform-d) δ 170.12, 163.67, (m, 17H), 0.99 – 0.86 (m, 4H); 13C NMR (126 MHz, Chloroform-
103.33, 96.83, 85.68, 84.15, 77.67, 52.52, 48.07, 41.41, 38.71, d) δ 165.82, 165.42, 103.82, 97.58, 86.32, 84.73, 76.69, 63.00,
36.57, 36.26, 31.91, 31.43, 29.20 – 28.80 (11C), 27.20, 26.34, 58.53, 53.78, 53.32, 52.70, 48.70, 45.80, 42.50, 39.26, 37.14,
26.18, 24.71, 23.83, 22.19, 19.30, 13.63; MS (ESI) 591.3 [M + 36.68, 32.79, 32.50, 29.69 – 29.40 (3C), 28.80, 27.44, 26.64,
H] ＋ . HRMS (ESI) calcd for C34H59N2O6, 591.4368; found, 25.73, 25.32, 24.27, 19.80; MS (ESI) 606.4 [M + H]＋. HRMS (ESI)
591.4381. [α]2D0 = +74 (c 0.051, MeOH).
calcd for C33H56N3O7, 606.4113; found, 606.4095. [α]20 = -82 (c
D
0.049, MeOH).
Preparation of intermediates 16a-16c
(-)-9-((5'-(4-Tetradecylpiperazine-1-carbonyl)-4',5'-
dihydroisoxazol-3'-yl) ring-contracted artemalog (18c).
Colorless oil (40 mg, 64%); 1H NMR (400 MHz, Chloroform-d) δ
5.74 (s, 1H), 5.18 (dd, J = 11.2, 7.2 Hz, 1H), 3.87 – 3.73 (m, 3H),
3.58 – 3.51 (m, 2H), 3.39 (dd, J = 18.0, 11.2 Hz, 1H), 2.53 – 2.48
(m, 3H), 2.39 – 2.27 (m, 5H), 2.11 – 2.06 (m, 1H), 2.01 – 1.96
(m, 1H), 1.88 – 1.84 (m, 4H), 1.67 – 1.47 (m, 7H), 1.42 – 1.11
(m, 23H), 0.99 – 0.86 (m, 7H); 13C NMR (126 MHz, Chloroform-
d) δ 165.37, 165.49, 103.37, 97.14, 85.87, 84.29, 76.24, 58.06,
53.34, 52.82, 52.20, 48.26, 45.27, 41.96, 38.81, 36.69, 36.24,
32.05, 31.48, 29.24 – 28.91 (7C), 28.36, 27.01, 26.19, 24.89,
23.83, 22.25, 19.35, 13.69; MS (ESI) 632.4 [M + H]＋. HRMS (ESI)
To a solution of tert-Butyl 1-piperazinecarboxylate (3 mmol)
and K2CO3 (6 mmol) in MeCN (10 mL) was added the
appropriate alkyl bromide (3.15 mmol), the resulting mixture
was stirred at rt for 2 h. The residue was diluted with water (20
mL) and washed with CH2Cl2 (3 × 10 mL). The combined
organic layer were washed with water and brine, dried over
anhydrous Na2SO4, filtered, and concentrated under reduced
pressure. The crude product was used to the next step without
further purification.
Preparation of intermediates 17a-17c
calcd for C36H62N3O6, 632.4633; found, 632.4647. [α]20 = -80 (c
D
To a solution of 16a, 16b or 16c (1 mmol) in CH2Cl2 (10 mL) was
0.053, MeOH).
added trifluoroacetic acid (30 mmol). The reaction was stirred
at rt for 4 h and then concentrated in vacuo. The residue was
used to the next step without purification.
Preparation of 3,6,9,12-tetraoxatetradecan-1-amide (20)
A solution of compound 19 (528 mg, 2 mmol) and NH3.MeOH
(5 mL) was stirred at rt for 12 h, the reaction was evaporated
under reduced pressure to afford the crude product 20 (445
mg, 95%) as a colorless oil.
Preparation of 18a-18c
To a solution of compound 12 (0.1 mmol) in CH2Cl2 (2 mL),
o
HATU (0.2 mmol) and HOAT (0.1 mmol) was added at 0 C.
o
After stirring for 10 min at 0 C, 17a
,
17b or 17c (0.1 mmol)
Preparation of 3,6,9,12-tetraoxatetradecan-1-amine (21)
and N, N-diisopropylethylamine (0.3 mmol) were added to this
solution. The resulting mixture was stirred at rt for 4 h and
then diluted with CH2Cl2 (15 mL), washed with saturated NH4Cl
solution, water, and brine. The organic layer was dried over
anhydrous Na2SO4, filtered, and concentrated in vacuo. The
residue was purified via silica gel chromatography with
To a stirred solution of compound 20 (235 mg, 1 mmol) in
o
anhydrous THF (5 mL) at 0 C under nitrogen atmosphere was
carefully added LiAlH4 (152 mg, 4 mmol). The resulting mixture
was stirred at rt for 5 h. The reaction was then slowly
o
quenched at 0 C with water and filtered over a pad of celite.
The filtrate was washed with CH2Cl2 (3 × 10 mL). The combined
organic layer were washed with water and brine, dried over
anhydrous Na2SO4, filtered, and concentrated under reduced
pressure to give reduced compound 21 (184 mg, 83%) as a
colorless oil.
MeOH/CH2Cl2 (40/1) as the eluent to afford 18a
(-)-9-((5'-(4-Undecylpiperazine-1-carbonyl)-4',5'-
dihydroisoxazol-3'-yl) ring-contracted artemalog (18a)
, 18b or 18c.
.
Colorless oil (32 mg, 54%); 1H NMR (400 MHz, Chloroform-d) δ
5.73 (s, 1H), 5.16 (dd, J = 11.3, 7.3 Hz, 1H), 3.85 – 3.72 (m, 3H),
3.59 – 3.47 (m, 2H), 3.37 (dd, J = 18.0, 11.3 Hz, 1H), 2.58 – 2.45
(m, 3H), 2.40 – 2.26 (m, 5H), 2.10 – 2.05 (m, 1H), 2.01 – 1.94
Preparation of 22
(m, 1H), 1.87 – 1.81 (m, 4H), 1.62 – 1.36 (m, 5H), 1.29 – 1.09 To a solution of compound 12 (37 mg, 0.1 mmol) in CH2Cl2 (2
(m, 19H), 0.99 – 0.85 (m, 7H); 13C NMR (126 MHz, Chloroform- mL), HATU (76 mg, 0.2 mmol) and HOAT (14 mg, 0.1 mmol)
o
d) δ 165.32, 164.92, 103.31, 97.09, 85.82, 84.24, 76.19, 58.01, was added at 0 C. After stirring for 10 min at 0 oC, compound
53.29, 52.77, 52.15, 48.21, 45.27, 41.96, 38.77, 36.64, 36.18, 21 (22 mg, 0.1 mmol) and N, N-Diisopropylethylamine (0.3
32.00, 31.40, 29.20 – 28.83 (3C), 28.31, 26.98, 26.13, 24.83, mmol) were added to this solution. The resulting mixture was
23.78, 22.18, 19.30, 13.62; MS (ESI) 590.4 [M + H]＋. HRMS (ESI) stirred at rt for 4 h and then diluted with CH2Cl2 (15 mL),
calcd for C33H56N3O6, 590.4164; found, 590.4178. [α]20 = -108 washed with saturated NH4Cl solution, water, and brine. The
D
(c 0.052, MeOH).
organic layer was dried over anhydrous Na2SO4, filtered, and
(-)-9-((5'-(4-(11-Hydroxyundecyl)piperazine-1-carbonyl)-
concentrated in vacuo. The residue was purified via silica gel
4',5'-dihydroisoxazol-3'-yl) ring-contracted artemalog (18b). chromatography with MeOH/CH2Cl2 (30/1) as the eluent to
Colorless oil (26 mg, 43%); 1H NMR (400 MHz, Chloroform-d) δ afford 22 (42 mg, 73%) as a colorless oil.
5.74 (s, 1H), 5.17 (dd, J = 11.2, 7.2 Hz, 1H), 3.86 – 3.73 (m, 3H),
(-)-9-((5'-((3,6,9,12-Tetraoxatetradecylcarbamoyl)-4',5'-
3.65 – 3.51 (m, 4H), 3.38 (dd, J = 18.0, 11.2 Hz, 1H), 2.52 – 2.49 dihydroisoxazol-3'-yl) ring-contracted artemalog (22). 1H NMR
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
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Journal Name
(400 MHz, Chloroform-d) δ 7.27 – 7.25 (m, 1H), 5.69 (s, 1H), Sigma, MO) assay and the Cell counting kit-8 (CCK-8) (Dojindo
4.94 (dd, J = 11.6, 4.0 Hz, 1H), 3.68 – 3.33 (m, 20H), 2.36 – 2.26 Laboratories, Japan) assay, respectively.
(m, 2H), 2.13 – 1.96 (m, 2H), 1.84 (s, 3H), 1.78 – 1.73 (m, 1H),
1.56 – 1.33 (m, 6H), 1.26 – 1.20 (m, 5H), 0.99 – 0.94 (m, 4H);
13C NMR (126 MHz, Chloroform-d) δ 170.73, 164.87, 103.34,
Cell cycle assays
SGC-7901 were seeded into 6-well plates, cultured overnight
97.10, 85.53, 83.98, 77.44, 70.11, 70.06, 69.98, 69.83, 69.27,
and treated with different concentrations of 7d for 24 h and 48
69.00, 66.10, 53.26, 48.03, 41.51, 38.43, 36.55, 36.05, 32.02,
h. Cells were then harvested and washed with PBS, fixed with
27.94, 25.76, 24.73, 23.72, 19.24, 14.62; MS (ESI) 571.2 [M +
H]＋. HRMS (ESI) calcd for C28H46N2NaO10, 593.3045; found,
pre-cooled 70% ethanol at 4°C. Staining went along in PBS
593.3057. [α]2D0 = -62 (c 0.050, MeOH).
containing 40 g/ml RNase A and 10 g/ml propidium iodide (PI)
in the dark for 30 min. For each sample, at least 1×104 cells
were collected with a FACS Calibur (BD Biosciences, Franklin
Preparation of 23
Lakes, NJ) and analyzed using the CELLQUEST software (BD
To a solution of compound 14 (56mg, 0.1 mmol) in MeCN (2
.
mL) was added FeCl2 4H2O (38 mg, 0.2 mmol) at rt. After 10
Biosciences, Franklin Lakes, NJ).
min, the reaction mixture was filtered over Celite and the
Annexin V-FITC apoptosis assays
filtrate concentrated under reduced pressure. Purification by
silica gel chromatography using petroleum ether/ethyl acetate
(3/1) as the eluent gave compound 23 (46 mg, 82%) as a and treated with different agents. Then, cells were harvested,
colorless oil. washed and stained by using a AnnexinV-FITC apoptosis
SW-620 were seeded into 6-well plates, cultured overnight
Ring-contracted furan acetate artemalog (23). 1H NMR (400 detection kit (KeyGEN BioTECH, Nanjing, China). Fluorescence
MHz, Chloroform-d) δ 6.78 – 6.75 (m, 1H), 6.28 (s, 1H), 4.94 – of the cells was determined immediately by flow cytometry
4.89 (m, 1H), 4.13 – 4.09 (m, 1H), 3.88 – 3.82 (m, 1H), 3.42 – (BD Biosciences, Franklin Lakes, NJ).
3.37 (m, 2H), 3.31 – 3.16 (m, 2H), 2.28 – 2.20 (m, 1H), 2.13 (s,
3H), 2.03 – 1.99 (m, 1H), 1.84 – 1.74 (m, 5H), 1.58 – 1.52 (m,
Western blotting
2H), 1.49 – 1.33 (m, 3H), 1.30 – 1.11 (m, 23H), 1.01 – 0.86 (m,
7H); 13C NMR (101 MHz, Chloroform-d) δ 170.84, 169.82,
SW-620 cells were treated with 7d for 48 h. Western blotting
was used to detect the cleavage of PARP and caspase-3.
163.95, 96.85, 88.90, 85.86, 77.91, 67.71, 52.62, 50.91 , 41.73,
39.20, 34.38, 31.93, 30.32, 29.70, 29.66, 29.61, 29.53, 29.37,
29.29, 27.56, 26.86, 26.59, 26.24, 22.70, 21.50, 20.05, 14.14;
MS (ESI) 585.4 [M + Na]＋. HRMS (ESI) calcd for C32H54N2NaO6,
585.3874; found, 585.3881. [α]2D0 = -72 (c 0.054, MeOH).
Acknowledgements
This work was supported by grants from the Pujiang Talent
Project from Shanghai Commission of Science and Technology
(14PJ1410700), and Chinese NSF (81402790, 81125021,
81373277, 81430080), and National Program on Key Basic
Research Project of China (2015CB910603-004).
Biological assays
Cell culture
Human cancer KB, K562, MCF-7, SW-620, A549 and SPC-A4 cell
lines were purchased from the the American Type Culture
Collection (ATCC; Manassas, VA). The K562/A02 subline was
from the Institute of Hematology, Chinese Academy of Medical
Sciences (Tianjin, China). Both KB/VCR and MCF-7/ADR
sublines were from the Sun Yat-Sen University of Medical
Sciences (Guangzhou, China). SK-OV-3 cells were from the
Japanese Foundation of Cancer Research (Tokyo, Japan). SGC-
7901 and BEL-7402 cell lines were kept in the Shanghai
institute of Materia Medica of the Chinese Academy of
Sciences (Shanghai, China). Cells were periodically
authenticated with morphologic inspection and tested for
mycoplasma contamination. The cell lines were cultured
according to the suppliers’ instructions.
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