Synthesis and anticancer activity of novel deoxoartemisinin-glycolipid hybrids

Article abstract of DOI:10.1248/cpb.c14-00036

The practical synthesis and anticancer activity of novel deoxoartemisinin-glycolipid hybrids, which incorporate two drugs into a single molecule and can impact multiple targets simultaneously are presented. These hybrids exhibited potent in vitro anticanc

Full text of DOI:10.1248/cpb.c14-00036

446
Regular Article
Chem. Pharm. Bull. 62(5) 446–453 (2014)
Vol. 62, No. 5
Synthesis and Anticancer Activity of Novel Deoxoartemisinin–Glycolipid
Hybrids
Dongguk Min,a,# Minkyu Kim,a,# Jérémy Ricci,a Sora Jung,a Kirim Kim,b Won-Yoon Chung,b
,a
Kwang-Kyun Park,b and Mankil Jung*
b
a Department of Chemistry, Yonsei University; Seoul 120–749, Korea: and Department of Oral Biology, College of
Dentistry, Yonsei University; Seoul 120–752, Korea.
Received January 13, 2014; accepted February 28, 2014; advance publication released online March 7, 2014
The practical synthesis and anticancer activity of novel deoxoartemisinin–glycolipid hybrids, which
incorporate two drugs into a single molecule and can impact multiple targets simultaneously are presented.
These hybrids exhibited potent in vitro anticancer activity against several human cancer cell lines. The de-
oxoartemisinin–glycolipid hybrids generally demonstrated better anticancer activity than either artemisinin
or daumone alone and cisplatin.
Key words anticancer activity; oral squamous carcinoma; deoxoartemisinin; glycolipid hybrid
Daumone (1) (Fig. 1), a life expanding glycolipid isolated
from Caenorhabditis elegans, was identified for the first time
by our laboratory as a dauer-inducing pheromone, and its total
synthesis was presented.1) While some previous studies have
presented glycolipid derivatives with anticancer activity, the
relatively weak activity for this compound class has generally
discouraged its use for anticancer chemotherapeutic agents.2–6)
However, glycosphingolipids found in a specific class of in-
vertebrate that do not have sialic acids suppressed melanoma
cell proliferation.7) Furthermore, glycolipids isolated from Fig. 1. Structure of Daumone (1) and Artemisinin (2)
spinach have been shown to inhibit DNA polymerase activity
and mitochondrial pol replication.8) Recently, we reported the artemisinin–glycolipid hybrids show strong antiangiogenic
synthesis of novel glycolipid derivatives through stereospecific activity,22) with twice the potency of the known antiangiogenic
α-glycosylation to afford di- and tri-rhamnoside daumone de- agents fumagillin and thalidomide.22)
rivatives.9) Most of the derivatives possessed potent anticancer
These promising results encouraged us to further prepare
activity against human cancer cell lines with an effective a series of 12β(C–C)-type deoxoartemisinin–glycolipid hy-
concentration in the nanomolar range, comparable to doxoru- brids and to test them as anticancer agents. Although gefitinib
bicin.9)
has been reported to be cytotoxic against an oral cancer cell
Artemisinin (2) (Fig. 1), also known as qinghaosu, a natu- line,34) no other anti-oral cancer agent has been reported in
ral tetracyclic sesquiterpene trioxane isolated from Artemisia the literature to date. In this study we report the practical
annua L.,10) and its derivatives have been used clinically to synthesis and exceptional in vitro anti-oral and anti-lung can-
treat drug-resistant malaria.11–15) In addition to their well- cer activities of novel non-acetal deoxoartemisinin–glycolipid
known antimalarial activity, artemisinin and its derivatives hybrids.
also possess potent antiangiogenic activity,16–23) and several
research groups have demonstrated potential antitumor prop-
Results and Discussion
As outlined in Chart 1, artemisinin derivative 4 was ob-
erties for this compound class.20,24–31) We have shown that
non-acetal 12β(C–C)-type amide derivatives of deoxoartemi- tained through a 4 reaction sequence from either artemisinin35)
sinin exhibit higher in vitro anticancer activity22) along with (2) or artemisinic acid36) (3) according to previously reported
20 times greater acid stability than acetal (C–O)-type deriva- procedures. Direct oxidation of the terminal double bond of
tives of artemisinin.32)
allyl deoxoartemisinin 4 using KMnO4 and NaIO4 provided
One approach that has been developed to improve anti- carboxymethyldeoxoartemisinin (5).22) Esterification of acid
cancer activity is the use of hybrid drugs, which incorporate 5 with methanol in the presence of H2SO4 afforded ester 6
two drugs into a single molecule33) and can impact multiple in 97% yield. β-isomer 6 (12S) in natural configuration was
targets simultaneously. Since cancer is a complex disease, it exclusively obtained (J12,11=4.7 Hz).
is unlikely that a single functional targeted drug will be effec-
In order to examine the role of the hybrid glycolipid link-
tive for treating this most advanced disease. Combined drugs age, different glycolipids were synthesized (Chart 2). Briefly,
that impact multiple targets simultaneously are better at con- in the course of the stereospecific total synthesis of daumone
trolling complex disease systems, are less prone to drug resis- (1) via eight steps from L-rhamnose,1) intermediate 7a had
tance.33) We recently reported that non-acetal 12β(C–C)-type been prepared. Debenzoylation of compound 7a with NaOMe
provided dihydroxy 7b1) in 91% yield. Protection of the alco-
hol moiety of 4-(hydroxymethyl)benzoic acid (8a) using tert-
The authors declare no conflict of interest.
butyl dimethylsilyl chloride and imidazole as nucleophilic cat-
#These authors contributed equally to this work.
© 2014 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: mjung@yonsei.ac.kr

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447
alyst provided compound 8b.22) The coupling reaction between using lithium bromide provided bromide 19 (79% yield).
compounds 7b and 8b using 1-(3-dimethylaminopropyl)- Bromide substitution of compound 19 with mercaptopyridine
3-ethylcarbodiimide hydrochloride (EDC) and 4-dimethyl- in the presence of potassium carbonate gave compound 20
aminopyridine (DMAP) as previously presented afforded (85% yield). A controlled ozonolysis of hybrid 14 provided the
benzoylated lipids 9a, 10, and 11a.22) Silyl ether deprotection isolable ozonide 21 (75% yield). Direct one-step oxidation of
of compounds 9a and 11a using tetrabutylammonium fluoride compound 14 using sodium periodate and potassium perman-
gave compounds 9b (94% yield) and 11b (49% yield) as link- ganate gave acid 22 (52% yield).
ers, respectively.
The synthesis of further deoxoartemisinin–glycolipid hy-
The route used for the preparation of deoxoartemisinin– brids from glycolipids 9b and 11b are shown in Chart 4. The
glycolipid hybrids 12, 13, and 14 according to the previously coupling reaction between carboxymethyldeoxoartemisinin
described method is illustrated in Chart 3.22) Coupling of (5) and glycolipid 9b using EDC and DMAP provided the
carboxymethyl deoxoartemisinin (5) with functionalized dau- 4-(hydroxymethyl)benzoic acid-linked hybrids 23 and 24
mone precursor 7b by esterification using EDC and DMAP (55%). Coupling between compound (5) and glycolipid 11b
afforded compounds 12 (22% yield), 13 (20% yield), and 14 using the same conditions provided hybrid 25 (61% yield).
(32% yield). The absolute configurations of C1-H and C2-H
In vitro anticancer activities of deoxoartemisinin–glycolipid
of L-rhamnose of the hybrid 12 and C3-H and C4-H of that hybrids 16–23 and 25, along with daumone (1), artemisinin
of the hybrid 13 were assigned as cis diequatorial and trans (2), and artemisinin derivative 6 were tested against the cancer
diaxial as shown in stuctures 12 and 13, respectively. In order cell lines MDA-MB-231, MCF-7, A549, HSC-2, and Ca.9-22
to determine the effect of functional groups in the glycolipid using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetra-
portion of the hybrid on anticancer activity, we synthesized a zolium bromide (MTT) colorimetric method. The results of
compound library from compounds 13 and 14. Controlled hy- these assays are summarized in Table 1. The drug standard
droboration and subsequent oxidation of compound 13 using used for comparison was cisplatin. Under our conditions,
9-BBN and H2O2–NaOH provided alcohol 15 (70% yield). artemisinin (2) and its derivative (6) as well as daumone (1)
Mesylation of compound 15 as previously described followed failed to show notable activity at concentrations below 50µM.
by addition of sodium azide provided hybrid azide 16 (57%). However, deoxoartemisinin–glycolipid hybrids showed more
A click reaction between azide 16 and 4′-(2-propynyloxy)- potent activity than either glycolipid or artemisinin alone.
acetophenone initiated by CuSO4·5H2O and sodium ascorbate Among the hybrid compounds, 17 and 18 did not exhibit no-
provided triazole 17 (32% yield). Ozonolysis of compound 13 table anticancer activity. An alcohol or triazole function seems
followed by reduction with sodium borohydride gave alcohol to interfere with the anticancer activity in these cancer cell
18 (75% yield). Mesylation of compound 18 using mesylate lines. However, the other hybrids exhibited good anticancer
chloride with triethylamine as base followed by substitution activity, even better than cisplatin in specific cell lines. The
Reagents and conditions: Yields: (a) KMnO4, NaIO4, H2O–acetone (1:1), rt, 12h, 99%; (b) H2SO4, MeOH, N2, reflux, 9h, 97%.
Chart 1
Reagents and conditions: Yeilds: (a) NaOMe, amberlite resin (H+), MeOH, rt, 12h, 91%; (b) imidazole, TBDMSCI, DMF, rt, 4h, 31%; (c) EDC, DMAP, DMF, rt, 24h, 9a
(10%), 10 (19%), 11a (27%); (d) TBAF, THF–H2O (20:1), rt, 24h, 9b (74%), 11b (49%).
Chart 2

448
Vol. 62, No. 5
4-(methoxy)benzoic acid linker between deoxoartemisinin and larly the dimeric hybrids 21 and 22 with superior IC50 values
the glycolipid in hybrid 25 increased the anticancer activity, of 10µM. For the breast (MCF-7) cancer cell line, hybrids 19
with the metastatic breast cancer cell line (MDA-MB-231) in and 20 showed greater than 5 times higher anticancer activity
which the cytotoxicity was approximately five times greater than cisplatin. In the lung cancer cell line (A549), hybrids 16,
than cisplatin with a IC50 value of 11.8µM. For the metastatic 19, and 20 showed 5 to 6 times more potent anticancer activ-
breast cancer cell line, all of the hybrid compounds 16, 20, 21, ity than the reference at concentrations of 9.7, 8.1, and 6.8µM,
22, and 25 showed better activity than the reference, particu- respectively. For the oral squamous (mouth origin) carcinoma
Reagents and conditions: Yields: (a) EDC, DMAP, DMF, rt, 12h, 12 (22%), 13 (20%) and 14 (34%); (b) 9-BBN, THF, rt, 1h, 70%; (c) MsCl, Et3N, CH2Cl2, 10min, NaN3,
DMF, 60°C, 10h, 57%; (d) CuSO4·5H2O, NaAsc, CH2Cl2–H2O (1:1, v/v), rt, 1h, 32%; (e) O3, CH2Cl2 −78°C, 5min, NaBH4, THF–MeOH (1:1, v/v), 0°C, 8h, 60%; (f)
MsCl, Et3N, CH2Cl2, rt, 10min, LiBr, acetone, reflux, 10h, 79%; (g) K2CO3, percaptopyridine, acetone, reflux, 10h, 85%; (h) O3, CH2Cl2, −78°C, 5min, 75%; (i) NaIO4,
acetone–H2O (1:1, v/v), rt, 4h, 52%.
Chart 3

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449
Reagents and conditions: Yields: (a) EDC, DMAP, DMF, rt, 24h, 23 (17%), 24 (55%); (b) EDC, DMAP, DMF, rt, 24h, 61%.
Chart 4
Table 1. Cytotoxic Activities of Deoxoartemisinin–Glycolipid Hybrids
(1, 2, 6, 16–23, 25) against Five Human Cancer Cell Lines
Conclusion
In conclusion, ten new non-acetal deoxoartemisinin–glyco-
lipid hybrids were synthesized, which showed potent anti-oral
and anti lung cancer activity. It is also noteworthy that the
hybrid compounds generally showed dramatic increases in
anticancer activity compared with the non-coupled molecules
daumone (1) or artemisinin (2) and also compared with cispla-
tin. The deoxoartemisinin–glycolipid hybrids deserve further
evaluation as possible anti-oral and anti-lung cancer drug
candidates.
IC50 values (µM)
Compounds
MDA-
MB-231
MCF-7
A549
HSC-2 Ca.9-22
Artemisinin (1)
>50
>50
>50
21.7
>50
>50
>50
48.0
6.4
>50
>50
>50
>50
>50
>50
9.7
>50
>50
>50
>50
>50
>50
Daumone (2)
6
16
32.9
5.4
33.7
17
>50
>50
>50
>50
8.1
>50
>50
>50
>50
34.6
29.0
>50
>50
>50
>50
18
Experimental
19
9.2
8.8
>50
46.4
>50
5.4
Chemistry All commercial reagents and solvents were
used as received without further purification unless otherwise
indicated. Reaction solvents were distilled from calcium hy-
dride for dichloromethane and from sodium metal and benzo-
phenone for tetrahydrofuran. Air- and moisture-sensitive reac-
tions were carried out in oven dried sealed glassware under a
positive pressure of dry nitrogen. The reactions were moni-
tored and the Rf values were determined using analytical thin
layer chromatography (TLC) with Merck silica gel 60 and F254
precoated plates (0.25-mm thickness). Spots on the TLC plates
were visualized using ultraviolet light (254nm) and a basic
20
6.8
5.4
16.0
5.5
21
22
43.9
20.7
>50
>50
>50
6.7
23
>50
11.8
>50
>50
>50
11.3
25
>50
>50
Cisplatine
8.1
MDA-MB-231: metastatic breast cancer cells (estrogen receptor-negative); MCF-7:
breast cancer cells (estrogen receptor-positive); A549: lung cancer cells; HSC-2: oral
squamous carcinoma cells (gingival origin); Ca.9-22: oral squamous carcinoma cell
(mouth origin).
cell line (Ca.9-22), deoxoartemisinin–glycolipid hybrids 16, potassium permanganate solution or cerium sulfate/ammo-
19, and 20 showed anticancer activity with IC50 values be- nium dimolybdate/sulfuric acid solution followed by heating
tween 34.6 and 29.0µM, which reflect weaker potency than on a hot plate. Flash column chromatography was performed
1
cisplatin. However, for the oral squamous (gingival origin) with Merck silica gel 60 (230–400 mesh). H-NMR spectra
carcinoma cell line (HSC-2), the mono or di-deoxoartemis- were recorded on Bruker DRX-250 or Bruker DRX-400 NMR
inin–glycolipid hybrids 16, 19, 20, and 22 with IC50 values spectrometers. Proton chemical shifts are reported in ppm
below 5.5µM, generally showed greater than two fold anti-oral (δ) relative to internal tetramethylsilane (TMS, δ 0.00). Data
cancer activity compared to cisplatin. As expected, the anti- were reported as follows: chemical shift (δ, ppm), integra-
cancer activities of the hybrids were much more potent than tion, multiplicity (s=singlet, brs=broad singlet, d=doublet,
the individual components artemisinin or glycolipid. However, t=triplet, q=quartet, and m=multiplet), coupling constants
the aromatic ring-linked hybrids (23, 25) exhibited weaker (J, Hz). 13C-NMR spectra were recorded on Bruker DRX-250
activities than non-linked types. The chemical functions from (63MHz) or Bruker DRX-400 (100MHz) NMR spectrometers
the glycolipid terminal double bond seem to play an important with complete proton decoupling. Carbon chemical shifts were
role in enhancing anticancer activity. The best results were ob- reported in ppm (δ) relative to TMS with the respective sol-
tained with azide, bromide, thiol, trioxolane, and acid groups.
vent resonance as the internal standard (CDCl3, δ 77.0ppm).
High resolution-mass spectrometer (HR-MS) analyses were

450
Vol. 62, No. 5
recorded on a JEOL JMS-700 analyzer spectrometer (FAB). m/z 567.9899, Found 567.9833. Anal. Calcd for C31H49O9: C,
Matrix-assisted
laser
desorption/ionization-time-of-flight 65.84; H, 8.67. Found: C, 65.79; H, 8.66. Compound 14. [α]D20
1
(MALDI-TOF) masses were recorded on an Applied Biosys- +47 (c=0.1, CHCl3); H-NMR (250MHz, CDCl3) δ: 5.72–5.92
tems 4700 proteomics analyzer spectrometer.
(1H, m), 5.29–5.37 (2H, m), 4.83–5.08 (4H, m), 4.75 (3H, m),
Synthesis of 10β-(Methylethanoate) Deoxoartemisinin 3.66–3.96 (2H, m), 2.58–2.88 (4H, m), 2.42–2.59 (2H, m),
6
To a stirred solution of carboxylic acid 522) (600mg, 2.23–2.41 (2H, m), 1.41 (6H, s), 1.24–2.21 (30H, m), 1.20 (3H,
2.32mmol) in 40mL of MeOH under nitrogen H2SO4 (150 µL, d, J=6.0Hz), 1.12 (3H, d, J=5.8Hz), 0.97 (6H, d, J=5.4Hz),
2.78mmol) was added. The mixture was stirred and heated to 0.87 (6H, d, J=7.3Hz). 13C-NMR (63MHz, CDCl3) δ: 170.6,
reflux for 9h. After completion (TLC), the mixture was cooled 138.9, 114.4, 103.8, 103.3, 92.9, 88.9, 88.8, 80.9, 80.8, 77.5,
to room temperature and EtOAc (40mL) was added. The reac- 77.0, 76.5, 72.5, 71.7, 71.1, 70.7, 69.9, 66.7, 52.3, 44.3, 37.3,
tion mixture was treated with a saturated solution of NaHCO3 36.8, 36.5, 36.4, 35.5, 34.4, 33.9, 29.6, 26.0, 25.7, 25.1, 24.8,
until basic. The aqueous phase was extracted with EtOAc 20.2, 19.2, 17.7, 14.1, 13.2; MALDI-TOF MS: Found 897.5077
(3×20mL) and the combined organic extracts were washed [M+Na]+. Calcd 874.5123 for C48H74O14.
with brine (20mL), dried over MgSO4 and concentrated in
Synthesis of Deoxoartemisinin–Glycolipid Hybrid 16
reduced pressure. The product was purified by flash column Under N2 atmosphere, a 9-BBN solution in tetrahydrofuran
chromatography on silica gel with hexane–EtOAc (3:2, v/v) (THF) (0.5M, 0.16mmol) was added to the hybrid 13 (47.0mg,
as eluant to give compound 6 (770mg, 97%) as a colorless 0.083mmol) and the mixture was stirred for 1h at room tem-
oil. Compound 6: [α]D20 +143 (c=0.03, CHCl3); 1H-NMR perature. After the conversion was completed (TLC), H2O2–
(250MHz, CDCl3) δ: 5.21 (1H, s), 4.70 (1H, m), 3.59 (3H, s), NaOH (3N) (1:1, v/v) solution was added until pH 7–8. EtOAc
2.67–2.46 (2H, m), 2.34 (1H, dd, J=15.1, 3.9Hz), 2.21 (1H, td, (10mL) was added and the organic layer was washed with
J=13.7, 3.6Hz), 2.02–1.34 (7H, m), 1.30 (3H, s), 1.28–1.04 (3H, NaHCO3 (10mL) and brine (10mL). The organic layer was
m), 0.85 (3H, d, J=5.8Hz), 0.76 (3H, d, J=7.6Hz); 13C-NMR dried over MgSO4, filtered and concentrated under reduced
(63MHz, CDCl3) δ: 172.0, 103.1, 89.1, 80.8, 71.4, 52.1, 51.8, pressure. The product was purified by silica gel column chro-
44.1, 37.4, 36.4, 35.8, 34.3, 29.7, 25.9, 24.7, 24.6, 20.1, 12.9; matography using hexane–EtOAc (1:1, v/v) to give compound
MALDI-TOF MS: Found 363.1594 [M+Na]+. HR-MS (FAB) 15 (33.1mg, 0.058mmol, 70%). Mesylate chloride (5.4L,
Calcd for C18H29O6 [M+H]+ m/z 341.1964, Found 341.1965. IR. 0.069mmol) was added to a stirred solution of compound 15
Synthesis of Deoxoartemisinin–Glycolipid Hybrids 12, (33.1mg, 0.058mmol) and triethylamine (16.0L, 0.116mmol) in
13, and 14 To a stirred solution of 12-carboxymethyldeoxo- dry CH2Cl2 (5mL) at room temperature. When TLC showed
artemisin 522) (0.48g, 1.47mmol), EDC (2.82g, 14.71mmol) that starting material was completely disappeared (10min), the
and DMAP (1.80g, 14.71mmol) in N,N-dimethylformamide reaction was quenched by addition of water (10mL). The or-
(DMF) (10mL), compound 7b1) (0.38g, 1.47mmol) was added ganic layer was extracted with EtOAc (3×10mL), washed with
and stirred at room temperature for 12h. After completion brine (10mL), dried over MgSO4, filtered and the filtrate con-
(TLC), the reaction mixture was quenched with slow addition centrated under reduced pressure. NaN3 (7.5mg, 0.116mmol)
of satured citric acid (10mL). The organic phase was extracted in DMF (5mL) was added to the crude, stirred and warmed at
with EtOAc (3×10mL) and washed with brine (3×10mL). 60°C for 10h. On completion (TLC), the reaction mixture was
The organic layer was dried over MgSO4, filtered and con- washed with NH4Cl (10mL). The organic layer was extracted
centrated in reduced pressure. The product was purified by with EtOAc (3×10mL), dried over MgSO4, filtered and evapo-
flash columns chromatography on silica gel with EtOAc as rated under reduced pressure. The product was purified by sili-
eluant to give compound 12 (0.18g, 0.32mmol, 22%), com- ca gel column chromatography using hexane–EtOAc (1:1, v/v)
pound 13 (0.16g, 0.28mmol, 20%), and compound 14 (0.22g, as eluant to give compound 16 (20.2mg, 0.033mmol, 57%) as
0.25mmol, 34%). Compound 12: [α]D20 +14 (c=0.1, CHCl3); a colorless syrup. Compound 16: [α]D20 −81 (c=0.08, CHCl3);
1H-NMR (250MHz, CDCl3) δ: 0.87 (d, J=7.58Hz, 3H), 0,96 1H-NMR (250MHz, CDCl3) δ: 5.31 (1H, s), 4.97–4.84 (1H, m),
(d, J=5.37, 3H), 1.07–1.24 (m, 4H), 1.12 (d, J=6.32Hz, 3H), 4.80–4.69 (2H, m), 3.95–3.72 (3H, m), 3.27 (2H, t, J=6.79Hz),
1.21 (d, J=6.00Hz, 3H), 1.40 (s, 3H), 1.48–2.23 (m, 17H), 2.83–2.61 (2H, m), 2.47 (1H, dd, J=15.0, 4.3Hz), 2.32 (1H, td,
2.24–2.56 (m, 2H), 2.60–2.84 (m, 2H), 3.72–3.85 (m, 2H), 3.89 J=13.8, 3.8Hz), 2.18–1.42 (22H, m), 1.39 (3H, s), 1.21 (3H, d,
(m, 1H), 4.67–4.82 (m, 1H), 4.71 (s, 1H), 4.83–4.92 (m, 1H), J=6.3Hz), 1.12 (3H, d, J=6.0Hz), 0.96 (3H, d, J=5.8Hz), 0.86
4.95 (d, J=10.23Hz, 1H), 5.00 (d, J=16.94Hz, 1H), 5.32 (s, (3H, d, J=7.6Hz); 13C-NMR (63MHz, CDCl3) δ: 170.9, 103.4,
1H), 5.82 (tdd, J=16.94, 10.23Hz, 1H): IR (KBr, cm−1) νmax 96.4, 89.1, 81.0, 72.1, 71.8, 70.4, 69.0, 67.4, 52.4, 51.6, 44.4,
3473, 2927, 2873, 2360, 2342, 1736, 1463, 1373, 1202, 1130, 37.6, 37.2, 36.6, 34.6, 32.2, 29.8, 29.3, 29.0, 26.8, 26.1, 25.7,
1103, 1039; HR-MS (FAB) Calcd for C31H50NaO9 [M+Na]+ 24.8, 20.3, 19.1, 17.8, 13.2; MALDI-TOF MS: Found 632.3568
m/z 589.3353, Found 589.3377. Anal. Calcd for C31H50O9: C, [M+Na]+. HR-MS (FAB) Calcd for C31H52N3O9 [M+H]+ m/z
65.72; H, 8.83. Found: C, 65.74; H, 8.91. Compound 13. [α]D20 610.3704, Found 610.3694.
1
+22 (c=0.1, CHCl3); H-NMR (250MHz, CDCl3) δ: 0.87 (d,
Synthesis of Deoxoartemisinin–Glycolipid Hybrid 17
J=7.58Hz, 3H,) 0.97 (d, J=5.69Hz, 3H), 1.11 (d, J=6.00Hz, 4′-(Propagyloxy)acetophenone (2.9mg 0.016mmol) was added
3H), 1.16–1.27 (m, 4H), 1.28 (d, J=5.69Hz, 3H), 1.41 (s, 3H), to a solution of 16 (10.0mg, 0.016mmol), CuSO4·5H2O (4.1mg,
1.49–2.23 (m, 17H), 2.25–2.49 (m, 2H), 2.61–2.83 (m, 1H), 0.016mmol) and sodium ascorbate (6.5mg, 0.033mmol) in
3.58–3.85 (m, 3H), 3.63–3.71 (m, 1H), 4.70–4.82 (m, 1H), CH2Cl2–H2O (5mL, 1:1, v/v) for 1h. On completion (TLC),
4.73 (s, 1H), 4.91 (br s, 1H), 4.94 (d, J=10.19Hz, 1H), 5.00 brine was added (10mL) and the organic phase was extracted
(d, J=16.98Hz, 1H), 5.34 (s, 1H), 5.70–5.93 (tdd, J=16.98, with EtOAc (3×10mL), dried over MgSO4, filtered and the
10.19Hz, 1H). IR (KBr, cm−1) νmax 2924, 2851, 2369, 2337, filtrate was concentrated under reduced pressure. The product
1734, 1456, 1368: HR-MS (FAB) Calcd for C31H49O9 [M+H]+ was purified by silica gel column chromatography using hex-

May 2014
451
ane–EtOAc (1:1, v/v) as eluant to give 17 (4mg, 0.005mmol, (2H, t, J=6.9Hz), 2.83–2.59 (2H, m), 2.45 (1H, dd, J=15.0,
32%). Compound 17: [α]D20 −32 (c=0.06, CHCl3); 1H-NMR 4.3Hz), 2.31 (1H, td, J=14.4, 3.6Hz), 2.18–1.40 (20H, m),
(250MHz, CDCl3) δ: 8.00–7.91 (2H, m), 7.62 (1H, s), 7.08–7.00 1.38 (3H, s), 1.20 (3H, d, J=6.3Hz), 1.12 (3H, d, J=6.2Hz),
(2H, m), 5.31 (1H, s), 5.28 (2H, s), 4.97–4.83 (1H, m), 0.95 (3H, d, J=5.7Hz), 0.85 (3H, d, J=7.4Hz); 13C-NMR
4.80–4.68 (2H, m), 4.37 (2H, t, J=7.0Hz), 3.92–3.70 (3H, m), (63MHz, CDCl3) δ: 170.9, 103.4, 96.4, 89.1, 81.0, 71.9, 71.8,
2.81–2.62 (2H, m), 2.55 (3H, s), 2.53–2.43 (1H, m), 2.39–2.25 70.4, 68.9, 67.4, 52.4, 44.4, 37.6, 37.1, 36.6, 34.6, 33.9, 32.8,
(1H, m), 2.16–1.43 (22H, m), 1.38 (3H, s), 1.20 (3H, d, 32.1, 29.7, 28.3, 26.0, 24.9, 24.8, 20.3, 19.1, 17.8, 13.2; MALDI-
J=6.2Hz), 1.11 (3H, d, J=6.0Hz), 0.95 (3H, d, J=4.4Hz), 0.85 TOF MS: Found 655.3136 [M+Na]+. HR-MS (FAB) Calcd for
(3H, d, J=7.1Hz); 13C-NMR (100MHz, CDCl3) δ: 196.7, 170.7, C30H50BrO9 [M+H]+ m/z 633.2638, Found 633.2636.
162.0, 143.4, 130.7, 130.6, 122.6, 114.4, 103.3, 96.3, 88.9, 80.9,
71.9, 71.7, 70.2, 68.8, 67.3, 62.1, 52.2, 50.5, 44.2, 37.4, 37.0,
Synthesis of Deoxoartemisinin–Glycolipid Hybrid 20
solution of compound 19 (27.0mg, 0.043mmol),
A
36.5, 36.4, 34.4, 32.0, 30.3, 29.7, 29.6, 29.0, 26.4, 25.9, 25.4, K2CO3 (17.7mg 0.128mmol) and mercaptopyridine (4.7mg
24.7, 20.2, 19.0, 17.7, 13.1; MALDI-TOF MS: Found 806.3607 0.043mmol) in acetone (10mL) was stirred at reflux. On
[M+Na]+. HR-MS (FAB) Calcd for C42H62N3O11 [M+H]+ m/z completion (TLC), brine (10mL) was added. The organic layer
784.4384, Found 784.4388.
Synthesis of Deoxoartemisinin–Glycolipid Hybrid 18
was extracted with EtOAc (3×10mL), dried over MgSO4,
filtered and the filtrate was concentrated under reduced pres-
A
solution of compound 13 (100mg, 0.176mmol) in dry CH2Cl2 sure. The product was purified by silica gel column chroma-
(10mL) was flushed with N2 and then subjected to a steady tography using hexane–EtOAc (1:1, v/v) to give compound
stream of O3 at −78°C until the solution became saturated 20 (24.2mg, 0.036mmol, 85%) as a colorless syrup. [α]D20 −45
and appeared blue. Excess O3 was flushed out from the solu- (c=0.1, CHCl3); 1H-NMR (250MHz, CDCl3) δ: 8.44–8.37
tion with N2 and the solvent removed under reduced pressure. (1H, m), 7.50–7.41 (1H, m), 7.19–7.12 (1H, m), 6.95 (1H, ddd,
THF–MeOH (20mL, 1:1, v/v) was added and the solution J=7.27, 5.0, 1.0Hz), 5.30 (1H, s), 4.89 (1H, m), 4.79–4.67 (2H,
was carefully treated with excess of sodium tetrahydroborate m), 3.94–3.71 (3H, m), 3.16 (2H, t, J=7.4Hz), 2.81–2.59 (2H,
(66.0mg, 1.76mmol) at 0°C for 8h. The resulting mixture was m), 2.44 (1H, J=15.0, 4.4Hz), 2.39–2.24 (1H, m), 2.18–1.41
concentrated under reduced pressure, followed by addition of (20H, m), 1.38 (3H, s), 1.20 (3H, d, J=6.3Hz), 1.11 (3H, d,
water (5mL) and EtOAc (10mL). The organic layer was ex- J=6.0Hz), 0.94 (3H, d, J=5.8Hz), 0.84 (3H, d, J=7.4Hz);
tracted by EtOAc (3×10mL), dried over MgSO4, filtered and 13C-NMR (63MHz, CDCl3) δ: 170.8, 149.4, 135.8, 122.2, 119.2,
solvent evaporated under reduced pressure. The crude prod- 103.2, 96.3, 89.0, 80.8, 71.9, 71.6, 70.3, 68.8, 67.2, 52.3, 44.3,
uct was purified by flash silica gel column chromatography 37.4, 37.0, 36.5, 36.4, 34.5, 32.0, 30.0, 29.6, 29.4, 28.9, 25.9,
using hexane–EtOAc (3:2, v/v) as eluant to give compound 18 25.3, 24.7, 24.7, 20.2, 19.0, 17.7, 13.0; MALDI-TOF MS: Found
(54mg, 0.095mmol, 60%) as a colorless syrup. Compound 18. 686.3982 [M+Na]+. HR-MS (FAB) Calcd for C35H54NO9S
[α]D20 −13 (c=0.1, CHCl3); H-NMR (250MHz, CDCl3), δ: 5.29 [M+H]+ m/z 664.3519, Found 664.3522.
1
(1H, s), 4.95–4.82 (1H, m), 4.76–4.66 (2H, m), 3.93–3.72 (3H,
Synthesis of Deoxoartemisinin–Glycolipid Hybrid 21
A
m), 3.63 (2H, t, J=6.6Hz), 2.80–2.60 (2H, m), 2.45 (1H, dd, solution of compound 14 (50.0mg, 0.057mmol) in dry CH2Cl2
J=15.0, 4.4Hz), 2.30 (1H, td, J=13.8, =3.7Hz), 2.14–1.40 (20H, (10mL) was flushed with N2 and then subjected to a steady
m), 1.37 (3H, s), 1.19 (3H, d, J=6.3Hz), 1.10 (3H, d, J=6.2Hz), stream of O3 at −78°C until the solution became saturated and
0.94 (3H, d, J=5.8Hz), 0.84 (3H, d, J=7.6Hz); 13C-NMR appeared blue. After the reaction completed, excess O3 was
(63MHz, CDCl3) δ: 170.7, 103.3, 96.2, 88.9, 80.8, 71.9, 71.8, flushed out from the solution with N2 and the solvent removed
70.5, 68.9, 67.3, 62.9, 52.4, 44.4, 37.6, 37.2, 36.6, 34.6, 32.8, under reduced pressure. The product was purified by silica
32.1, 29.7, 26.0, 25.8, 25.6, 24.8, 20.3, 19.1, 17.8, 13.2; MALDI- gel column chromatography using hexane–EtOAc (3:2, v/v)
TOF MS: Found 593.3291 [M+Na]+. HR-MS (FAB) Calcd for as eluant to give compound 21 (39.3mg, 0.043mmol, 75%).
1
C30H51O10 [M+H]+ m/z 571.3482, Found 571.3470.
[α]D20 +50 (c=0.1, CHCl3); H-NMR (250MHz, CDCl3) δ: 5.33
Synthesis of Deoxoartemisinin–Glycolipid Hybrid 19 (1H, s), 5.32 (1H, s), 5.21 (1H, s), 5.14 (1H, d, J=4.8Hz), 5.05
Mesylate chloride (8µL, 0.104mmol) was added to a stirred (1H, s), 4.99–4.85 (2H, m), 4.84–4.66 (3H, m), 3.95–3.80 (1H,
solution of compound 18 (54.0mg, 0.095mmol) and trieth- m), 3.78–3.65 (1H, m), 2.89–2.61 (4H, m), 2.57–2.42 (2H, m),
ylamine (27µL, 0.189mmol) in dry CH2Cl2 (5mL) at room 2.41–2.23 (2H, m), 2.22–1.46 (30H, m), 1.41 (6H, s), 1.20 (3H,
temperature. When TLC showed that starting material was d, J=6.2Hz), 1.12 (3H, d, J=6.0Hz), 0.97 (6H, d, J=5.7Hz),
completely disappeared (10min), the reaction was quenched 0.87 (6H, d, J=7.4Hz); 13C-NMR (63MHz, CDCl3) δ: 170.9,
by addition of water (10mL). The organic layer was washed 170.6, 103.6, 103.3, 103.3, 94.0, 93.4, 88.9, 88.8, 80.8, 72.2,
with brine (10mL), dried over MgSO4, filtered and the filtrate 71.8, 71.6, 70.8, 69.9, 66.9, 52.3, 52.2, 44.3, 44.2, 37.4, 37.3,
concentrated under reduced pressure. The crude was added 36.8, 36.4, 35.4, 34.4, 31.1, 29.6, 29.5, 25.9, 25.4, 24.7, 24.6,
to a stirred solution of LiBr (24.7mg 0.284mmol) in acetone 24.5, 23.8, 20.2, 19.0, 17.7, 13.2, 13.1; HR-MS (FAB) Calcd for
(5mL) and the mixture was refluxed. On completion (TLC), C48H74O17 [M+H]+ m/z 923.5004, Found 923.5002.
the reaction was quenched with brine (10mL). The organic
Synthesis of Deoxoartemisinin–Glycolipid Hybrid 22 In
layer was extracted with EtOAc (3×10mL), dried over MgSO4, a stirred solution of compound 14 (100mg, 0.114mmol) in
filtered and the filtrate was concentrated under reduced pres- acetone–water (1:1, v/v, 20mL) was added sodium periodate
sure. The product was purified by silica gel column chroma- (16mg, 0.075mmol) and potassium permanganate (72mg,
tography using hexane–EtOAc (1:1, v/v) to give compound 0.457mmol) at room temperature. After completion (4h, TLC),
19 (47.1mg, 0.074mmol, 79%) as a colorless syrup. [α]D20 +40 the solvent was evaporated. Water (10mL) and EtOAc (10mL)
1
(c=0.02, CHCl3); H-NMR (250MHz, CDCl3) δ: 5.30 (1H, s), were added and the organic phase was extracted with EtOAc
4.96–4.83 (1H, m), 4.79–4.67 (2H, m), 3.93–3.71 (3H, m), 3.41 (3×10mL). The crude product was purified by flash silica gel

452
Vol. 62, No. 5
column chromatography using hexane–EtOAc (1:1, v/v) as organic phase was dried over MgSO4, filtered and the solvent
eluant to compound 22 (52.7mg, 0.059mmol, 52%). [α]D20 +69 was evaporated under reduced pressure. The product was
1
(c=0.06, CHCl3); H-NMR (250MHz, CDCl3) δ: 5.41 (1H, s), purified by flash column chromatography on silica gel using
5.33 (1H, s), 5.03–4.69 (5H, m), 3.99–3.68 (2H, m), 2.92–2.61 EtOAc as eluant to give compound 9b (24.8mg, 0.063mmol,
(4H, m), 2.61–2.23 (6H, m), 2.22–1.46 (28H, m), 1.41 (6H, s), 74%). Compound 9b. 1H-NMR (400MHz, CDCl3) δ: 8.01
1.24 (3H, d, J=6.8Hz), 1.12 (3H, d, J=5.7Hz), 0.97 (6H, d, (2H, d, J=8.1Hz), 7.42 (2H, d, J=8.1Hz), 5.75–5.90 (1H, m),
J=5.4Hz), 0.88 (6H, d, J=6.6Hz); 13C-NMR (63MHz, CDCl3) 4.91–5.12 (3H, m), 4.87 (1H, s), 4.77 (2H, brs), 3.61–3.89 (3H,
δ: 170.9, 170.6, 103.8, 103.3, 92.9, 88.8, 88.7, 80.9, 80.8, 72.5, m), 2.13–2.26 (2H, m), 1.94–2.13 (2H, m), 1.68–1.82 (1H, m),
71.7, 71.1, 70.7, 69.9, 66.9, 52.3, 44.3, 37.7, 36.8, 36.5, 36.4, 35.5, 1.53–1.68 (1H, m), 1.44 (4H, m), 1.32 (3H, d, J=5.9Hz), 1.15
34.4, 33.9, 29.6, 26.0, 25.7, 25.1, 24.8, 20.2, 19.2, 17.7, 14.1, 13.2; (3H, d, J=5.9Hz). MALDI-TOF MS: Found 415.2137 [M+
MALDI-TOF MS: Found 915.5246 [M+Na]+. HR-MS (FAB) Na]+. Calcd 392.2227 for C22H32O6.
Calcd for C47H73O16 [M+H]+ m/z 893.4899, Found 893.4873.
Synthesis of Deoxoartemisinin–Glycolipid Hybrids 23
Synthesis of Glycolipid Derivatives 9a,b, 10, and 11a,b and 24 To a stirred solution of 5 (23.6mg, 0.07mmol), EDC
A solution of compound 8b22) (86mg, 0.32mmol), EDC (139mg, 0.72mmol) and DMAP (88.0mg, 0.72mmol) in DMF
(618mg, 3.2mmol) and DMAP (394mg, 3.2mmol) in DMF (5mL), compound 9b (28.4mg, 0.07mmol) was added at room
(5mL) was combined with compound 7b1) (100mg, 0.39mmol) temperature and the reaction mixture was stirred for 12h.
at room temperature. The reaction mixture was stirred for After completion (TLC), the reaction mixture was quenched
24h. After completion (TLC), the reaction mixture was with slow addition of satured citric acid (5mL), extracted with
quenched with slow addition of saturated citric acid (10mL), EtOAc (3×10mL) and washed with brine (3×10mL). The or-
extracted with EtOAc (3×5mL) and washed with brine ganic layer was dried over MgSO4, filtered and concentrated
(10mL). Organic layers were combined and dried over MgSO4, under reduced pressure. The product was purified by flash col-
filtered and the solvent evaporated under reduced pressure. umns chromatography on silica gel with hexane–EtOAc (3:2,
The crude products were purified by flash column chromatog- v/v) as eluant to give compound 23 (8.6mg, 0.012mmol, 17%)
raphy on silica gel using hexane–EtOAc (2:2, v/v) as eluant and compound 24 (27.6mg, 0.039mmol, 55%). Compound
1
to give compound 9a (16mg, 0.03mmol, 10%), compound 23: [α]D20 +30 (c=0.1, CHCl3); H-NMR (250MHz, CDCl3) δ:
10 (23.1mg, 0.3mmol, 19%) and compound 11a (43.3mg, 8.05 (2H, d, J=8.1Hz), 7.45 (2H, d, J=8.1Hz), 5.83 (1H, tdd,
1
0.08mmol, 27%) as white powders. Compound 9a. H-NMR J=6.6, 17.0, 10.2Hz), 5.35 (1H, s), 5.23 (2H, brs), 5.13–4.91
(400MHz, CDCl3) δ: 8.01 (2H, d, J=8.1Hz), 7.39 (2H, d, (3H, m), 4.90–4.68 (3H, m), 4.05–3.87 (1H, m), 3.86–3.74 (1H,
J=8.1Hz), 5.74–5.90 (1H, m), 4.91–5.22 (3H, m), 4.84–4.91 m), 3.86–1.44 (24H, m), 1.41 (3H, s), 1.32 (3H, d, J=6.6Hz),
(1H, m), 4.80 (2H, s), 3.63–3.87 (3H, m), 2.15–2.26 (1H, m), 1.16 (3H, d, J=6.0Hz), 0.96 (3H, d, J=5.5Hz), 0.87 (3H, d,
1.94–2.13 (3H, m), 1.55–1.71 (2H, m), 1.36–1.52 (4H, m), 1.32 J=7.4Hz); 13C-NMR (63MHz, CDCl3) δ: 171.3, 165.4, 141.3,
(3H, d, J=5.9Hz), 1.15 (3H, d, J=6.0Hz), 0.95 (9H, s), 0.11 138.9, 129.9, 129.5, 127.5, 114.4, 103.2, 93.4, 89.0, 72.0, 80.8,
(6H, s). MALDI-TOF MS: Found 529.3077 [M+Na]+. Calcd 71.9, 71.7, 69.5, 68.4, 52.2, 65.6, 44.1, 37.4, 36.9, 36.4, 36.0,
1
506.3149 for C28H46O6Si. Compound 10. H-NMR (400MHz, 34.4, 33.7, 32.7, 29.7, 28.8, 26.0, 25.2, 24.6, 20.1, 19.1, 17.7, 13.0.
CDCl3) δ: 8.07 (2H, d, J=8.1Hz), 8.01 (2H, d, J=8.2Hz), MALDI-TOF MS: Found 701.4027 [M+H]+. HR-MS (FAB)
7.38–7.45 (4H, m), 5.84 (1H, m), 5.11–5.24 (2H, m), 4.92–5.11 Calcd for C40H631N2O10 [M+H]+ m/z 701.3901, Found 701.3897.
(3H, m), 4.78–4.83 (4H, m), 4.06–4.17 (1H, m), 3.79–3.89 (1H, Compound 24: H-NMR (250MHz, CDCl3) δ: 0.81–0.91 (m,
m), 2.36–2.45 (1H, m), 2.16–2.26 (1H, m), 2.03–2.15 (2H, m), 5H) 0.96 (d, J=5.84Hz, 2H) 1.15 (d, J=6.00Hz, 2H) 1.32 (d,
1.56–1.68 (2H, m), 1.37–1.56 (4H, m), 1.28 (3H, d, J=5.6Hz), J=5.84Hz, 3H) 1.41 (s, 3H) 2.14–2.43 (m, 1H) 2.46–2.59 (m,
1.19 (3H, d, J=6.0Hz), 0.96 (9H, s), 0.95 (9H, s), 0.11 (6H, 1H) 2.69–2.85 (m, 1H) 3.60–3.87 (m, 1H) 4.79–4.90 (m, 2H)
s), 0.11 (6H, s). MALDI-TOF MS: Found 777.4221 [M+Na]+. 4.91–5.13 (m, 1H) 5.23 (d, J=4.90Hz, 2H) 5.27–5.32 (m, 1H)
Calcd 754.4323 for C42H66O8Si2. Compound 11a. 1H-NMR 5.36 (s, 1H) 5.72–5.92 (m, 1H) 7.46 (d, J=8.06Hz, 2H) 8.03
(400MHz, CDCl3) δ: 8.04 (2H, d, J=8.1Hz), 7.57 (2H, d, (d, J=8.21Hz, 2H): 13C-NMR (63MHz, CDCl3) δ: 13.04 17.71
J=7.3Hz), 5.75–5.92 (1H, m), 4.86–5.15 (3H, m), 4.75–4.83 19.07 20.12 24.65 25.19 28.77 29.66 32.74 33.70 34.39 36.94
(1H, m), 4.80 (2H, s), 3.63–3.91 (3H, m), 2.17–2.29 (1H, m), 37.40 44.11 52.19 65.59 68.41 69.54 71.70 71.87 72.03 80.83
1.96–2.15 (3H, m), 1.55–1.71 (2H, m), 1.35–1.54 (4H, m), 1.24 89.00 93.45 103.25 114.37 127.53 129.54 129.90 138.87 141.32
(3H, d, J=6.4Hz), 1.15 (3H, d, J=6.0Hz), 0.95 (9H, s), 0.11 165.36 171.34; MALDI-TOF MS: Found 723.3771 [M+Na]+.
(6H, s). MALDI-TOF MS: Found 529.3053 [M+Na]+. Calcd Calcd 700.3873 for C39H56O11.
1
506.3149 for C28H46O6Si. Compound 11b H-NMR (400MHz,
Synthesis of Deoxoartemisinin–Glycolipid Hybrid 25
CDCl3) δ: 8.03 (2H, d, J=8.0Hz), 7.58 (2H, d, J=7.2Hz), Tetrabutylammonium fluoride (32.8mg, 0.17mmol) was added
5.74–5.93 (1H, m), 4.87–5.14 (3H, m), 4.72–4.81 (1H, m), to a solution of compound 11a (15.9mg, 0.031mmol) in THF–
4.78 (2H, s), 3.61–3.89 (3H, m), 2.17–2.27 (1H, m), 1.97–2.31 water (5mL, 20:1 v/v). The mixture was stirred for 24h at
(3H, m), 1.54–1.71 (2H, m), 1.36–1.55 (4H, m), 1.23 (3H, d, room temperature. After completion (TLC), ether (10mL) and
J=6.5Hz), 1.16 (3H, d, J=6.0Hz). MALDI-TOF MS: Found water (10mL) were added and the organic phase was washed
415.2107 [M+Na]+. Calcd 392.2268 for C22H32O6.
with water (3×10mL) and brine (10mL). The organic phase
Tetrabutylammonium fluoride (89.0mg, 0.34mmol) was was dried over MgSO4, filtered and the solvent evaporated
added to a solution of compound 9a (43.3mg, 0.085mmol) under reduced pressure. The product was purified by flash
in THF–water (5mL, 20:1, v/v). The mixture was stirred column chromatography on silica gel using EtOAc as eluant
for 24h at room temperature. After completion (TLC), ether to give compound 11b (6.1mg, 0.015mmol, 49%). To a stirred
(10mL) and water (10mL) were added and the organic phase solution of compound 5 (5.0mg, 0.015mmol), EDC (29.3mg,
was washed with water (3×10mL) and brine (10mL). The 0.15mmol) and DMAP (18.7mg, 0.15mmol) in DMF (5mL),

May 2014
453
Solov’eva T. F., Ovodov Y. S., Deev V. V., Pimenov A. A., Carbo-
hydr. Res., 260, 73–82 (1994).
3) Reggiardo Z., Infect. Immun., 21, 914–917 (1978).
4) Amano K., Ribi E., Cantrell J. L., J. Biochem., 93, 1391–1399
(1983).
5) Kusama T., Soga T., Ono Y., Kumazawa E., Shioya E., Nakayama
K., Uoto K., Osada Y., Chem. Pharm. Bull., 39, 3244–3253 (1991).
6) Erukulla R. K., Zhou X., Samadder P., Arthur G., Bittman R., J.
Med. Chem., 39, 1545–1548 (1996).
7) Sonoda Y., Hada N., Kaneda T., Suzuki T., Ohshio T., Takeda T.,
Kasahara T., Biol. Pharm. Bull., 31, 1279–1283 (2008).
8) Maeda N., Yoshida H., Mizushina Y., Bioact. Foods Promot.
Health, 393–405 (2010).
compound 11b (6.0mg, 0.015mmol) was added at room tem-
perature and the mixture stirred for 12h. After completion
(TLC), the reaction mixture was quenched with slow addi-
tion of satured citric acid (5mL), extracted with ethyl acetate
(3×10mL) and washed with brine (3×10mL). The organic
layer was dried over MgSO4, filtered and concentrated under
reduced pressure. The product was purified by flash columns
chromatography on silica gel with hexane–EtOAc (3:2, v/v) as
eluant to give compound 25 (6.5mg, 0.009mmol, 61%). Com-
pound 25: [α]D20 −37 (c=0.03, CHCl3); 1H-NMR (250MHz,
CDCl3) δ: 8.01 (2H, d, J=8.1Hz), 7.45 (2H, d, J=8.1Hz),
5.94–5.73 (1H, m), 5.35 (1H, s), 5.29–4.74 (7H, m), 4.12–3.95
(1H, m), 3.91–3.58 (2H, m), 2.87–2.62 (2H, m), 2.52 (1H, dd,
J=15.0, 3.6Hz), 2.44–2.16 (2H, m), 2.15–1.43 (19H, m), 1.40
(3H, s), 1.23 (3H, d, J=6.6Hz), 1.15 (3H, d, J=6.0Hz), 0.96
(3H, d, J=5.5Hz), 0.86 (3H, d, J=7.4Hz); 13C-NMR (63MHz,
CDCl3) δ: 171.4, 165.3, 138.9, 138.2, 129.9, 129.8, 127.6,
114.4, 103.2, 96.2, 89.4, 80.9, 72.0, 71.7, 70.7, 68.9, 67.2, 65.6,
52.2, 44.1, 37.4, 37.2, 29.9, 29.7, 28.8, 26.0, 28.8, 26.0, 25.2,
9) Jung M., Lee Y., Moon H., Jung Y., Jung H., Oh M., Eur. J. Med.
Chem., 44, 3120–3129 (2009).
10) Klayman D. L., Science, 228, 1049–1055 (1985).
11) Warhurst D. C., Infection, 27 (Suppl. 2), S55–S58 (1999).
12) Trape J. F., Am. J. Trop. Med. Hyg., 64, 12–17 (2001).
13) Marquiño W., Huilca M., Calampa C., Falconi E., Cabezas C.,
Naupay R., Ruebush T. K. II, Am. J. Trop. Med. Hyg., 68, 608–612
(2003).
24.7, 20.1, 19.1, 17.7, 13.1; MALDI-TOF MS: Found 723.3849 14) Lin A. J., Lee M., Klayman D. L., J. Med. Chem., 32, 1249–1252
[M+Na]+. HR-MS (FAB) Calcd for C39H57O11 [M+H]+ m/z
(1989).
15) Lin A. J., Klayman D. L., Milhous W. K., J. Med. Chem., 30, 2147–
701.3901, Found 701.3889.
2150 (1987).
Biology Materials Dulbecco’s modified Eagle’s medium
(DMEM), DMEM/F12, fetal bovine serum (FBS), RPMI1640
were purchased from Gibco BRL (Rockville, MD, U.S.A.).
3-(4,5-Dimethylthiazol-2-yl)-2,4-diphenyl-2H-tetrazolium bro-
16) Chen H.-H., Zhou H.-J., Wang W.-Q., Wu G.-D., Cancer Chemother.
Pharmacol., 53, 423–432 (2004).
17) Chen H.-H., You L.-L., Li S.-B., Cancer Lett., 211, 163–173 (2004).
18) Oh S., Jeong I., Ahn C., Shin W., Lee S., Bioorg. Med. Chem., 12,
mide (MTT), dimethyl sulfoxide (DMSO), cholera toxin, hy-
3783–3790 (2004).
drocortisone, insulin, transferrin, triiodothyronine (T3), and
cisplatin were obtained from Sigma-Aldrich Chem. Co. (St.
Louis, MO, U.S.A.).
19) Dell’Eva R., Pfeffer U., Vene R., Anfosso L., Forlani A., Albini A.,
Efferth T., Biochem. Pharmacol., 68, 2359–2366 (2004).
20) Jung M., Park N., Moon H., Lee Y., Chung W., Park K., Bioorg.
Med. Chem. Lett., 19, 6303–6306 (2009).
21) Jung M., Tak J., Chung W., Park K., Bioorg. Med. Chem. Lett., 16,
1227–1230 (2006).
22) Ricci J., Park J., Chung W.-Y., Park K.-K., Jung M., Bioorg. Med.
Chem. Lett., 20, 6858–6860 (2010).
23) Wartenberg M., Wolf S., Budde P., Grüenheck F., Acker H., Hesche-
ler J., Wartenberg G., Sauer H., Lab. Invest., 83, 1647–1655 (2003).
24) Jung M. W. O., Patent 2004078762 (2004).
Cell Viability Assay The viability of cancer cells was de-
termined via a MTT assay. MCF-7 and MDA-MB-231 breast
cancer cells were cultured in DMEM supplemented with 10%
FBS in a humidified atmosphere of 5% CO2 at 37°C, and A549
lung cancer cells were maintained in 10% FBS-RPMI1640
medium. HSC-2 and Ca9.22 oral cancer cells were cultured
in DMEM–F12 (3:1) supplemented with 10% FBS, 1×10−10
M
cholera toxin, 0.4mg/mL hydrocortisone, 5µg/mL insulin, 5µg/
mL transferrin and 2×10−11 M T3. The compounds were dis-
solved in DMSO and diluted with culture media. Cancer cells
(1.0×104 cells/mL) were seeded onto each well of a 96-well
plate with the respective media and incubated to adhere over-
night. The cells were then treated with various concentrations
of each compound in serum-free medium for 24h. Twenty
microliters of a MTT solution (5mg/mL) was added to each
well, and the cells were incubated for 4h at 37°C. The medium
was then removed, and 200µL of DMSO was added to each
well. The absorbance was determined at 570nm using a micro-
plate reader (Bio-Rad Laboratories, Hercules, CA, U.S.A.).
25) Jung M., Lee S., Ham J., Lee K., Kim H., Kim S. K., J. Med.
Chem., 46, 987–994 (2003).
26) Beekman A. C., Barentsen A. R. W., Woerdenbag H. J., Van Uden
W., Pras N., Konings A. W. T., El-Feraly F. S., Galal A. M., Wik-
ströem H. V., J. Nat. Prod., 60, 325–330 (1997).
27) Li Y., Shan F., Wu J. M., Wu G. S., Ding J., Xiao D., Yang W.
Y., Atassi G., Leonce S., Caignard D. H., Renard P., Bioorg. Med.
Chem. Lett., 11, 5–8 (2001).
28) Posner G. H., Paik I.-H., Sur S., McRiner A. J., Borstnik K., Xie S.,
Shapiro T. A., J. Med. Chem., 46, 1060–1065 (2003).
29) Lai H., Sasaki T., Singh N. P., Messay A., Life Sci., 76, 1267–1279
(2005).
30) Efferth T., Drug Resist. Updat., 8, 85–97 (2005).
31) Jung M., Lee K., Kim H., Park M., Curr. Med. Chem., 11, 1265–
1284 (2004).
32) Jung M., Lee S., Bioorg. Med. Chem. Lett., 8, 1003–1006 (1998).
33) Gediya L. K., Njar V. C. O., Expert Opin. Drug Discovery, 4,
1099–1111 (2009).
34) Chu Q., Amano O., Kanda Y., Kunii S., Wang Q., Sakagami H.,
Anticancer Res., 29, 5023–5031 (2009).
35) Chadwick J., Mercer A. E., Park B. K., Cosstick R., O’Neill P.,
Bioorg. Med. Chem., 17, 1325–1338 (2009).
Acknowledgments This study was supported by a Grant
of the Korean Health Technology R&D Project, the Ministry
of Health and Welfare, the Republic of Korea (Project No.
A121443). D. M., M. K., J. R., and S. J. received fellowships
from the BK21 program of the Ministry of Education and
Human Resources Development, Republic of Korea.
References
36) Jung M., Freitas A. C. C., McChesney J. D., ElSohly H. N., Hetero-
cycles, 39, 23–29 (1994).
1) Jeong P. Y., Jung M., Yim Y., Kim H., Park M., Hong E., Lee W.,
Kim Y., Kim K., Paik Y., Nature (London), 433, 541–545 (2005).
2) Gorbach V. I., Krasikova I. N., Luk’yanov P. A., Loenko Y. N.,