Acid-catalyzed synthesis of 10-substituted triazolyl artemisinins and their growth inhibitory activity against various cancer cells

Article abstract of DOI:10.1016/j.bmcl.2010.05.074

A diastereomeric and regioisomeric library of 10-substituted triazolyl artemisinin compounds (6a-6h, 7a-7h, and 8a-8h) with a potent growth inhibitory activities against various cancer cell lines was established. These compounds were synthesized by a reaction with dihydroartemisinin (2) and various substituted triazoles (5a-5h) in methylene chloride using a BF₃Et₂O catalyst. Most of the compounds exhibited a strong potency in the submicromolar range, and, in particular, 6f, 7f, and 8f, which have a pentylphenyltriazole moiety, proved to be promising candidates for preclinical trials.

Full text of DOI:10.1016/j.bmcl.2010.05.074

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4112–4115
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Acid-catalyzed synthesis of 10-substituted triazolyl artemisinins
and their growth inhibitory activity against various cancer cells
a,
Sangtae Oh a, Woon-Seob Shin b, Jungyeob Ham c, , Seokjoon Lee
*
*
a Department of Basic Science, Kwandong University College of Medicine, Gangneung 210-701, Republic of Korea
b Department of Microbiology, Kwandong University College of Medicine, Gangneung 210-701, Republic of Korea
c Korea Institute of Science and Technology, Gangneung Institute, Gangneung 210-340, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A diastereomeric and regioisomeric library of 10-substituted triazolyl artemisinin compounds (6a–6h,
7a–7h, and 8a–8h) with a potent growth inhibitory activities against various cancer cell lines was estab-
lished. These compounds were synthesized by a reaction with dihydroartemisinin (2) and various substi-
tuted triazoles (5a–5h) in methylene chloride using a BF3Et2O catalyst. Most of the compounds exhibited
a strong potency in the submicromolar range, and, in particular, 6f, 7f, and 8f, which have a pentylphe-
nyltriazole moiety, proved to be promising candidates for preclinical trials.
Received 30 March 2010
Revised 3 May 2010
Accepted 18 May 2010
Available online 9 June 2010
Keywords:
Artemisinin
Ó 2010 Elsevier Ltd. All rights reserved.
Triazoyl artemisinin
Anticancer
Diastereomer
Regioisomer
Since naturally occurring endoperoxide sesquiterpene lactone
artemisinin (1) isolated from Artemisia annua L. has been used as
an important lead compound for antimalarial drug development,1
semisynthetic antimalarial agents including artemether, arteether,
artesuic acid, and artelinic acid synthesized from dihydroartemisi-
nin (2) are now being used in clinical treatments due to their ther-
apeutic efficacy and non-toxicity.2–5 Although 2 has been proven to
exhibit neurotoxicity in an animal model,6 clinical trials have been
carried out to unearth the anticancer, antiviral, immunosuppres-
sive, and antifungal properties of various artemisinin analogs7.
We have recently reported that 10-substituted triazolyl artem-
isinins (4) synthesized via Cu(I)-catalyzed Huisgen 1,3-cycloaddi-
tion of diastereomeric 10-azidoartemisinins (3) with various
alkynes exhibit strong growth inhibitory activity against cancer
cell lines (Fig. 1).8 Although we have found some promising anti-
cancer drug candidates by Huisgen 1,3-cycloaddition, we could
not synthesize all possible diastereomers, only those with a phe-
nyltriazolyl substituent. The three diastereomers obtained with a
phenyltriazolyl group showed strong growth inhibitory effects
against cancer cell lines. Therefore, development of a novel syn-
thetic method for establishing a possible diastereomeric 10-
substituted triazolyl artemisinin library is essential. As mentioned
in our recent report,8 changes in reaction conditions, such as the
copper catalyst and solvent system, are not possible; therefore,
we altered the synthesis strategy. Instead of Huisgen 1,3-cycload-
dition of 3 with alkynes to yield a series of structure 4, substi-
tuted 1,2,3-triazoles are introduced in the C-10 anomeric
position of 2 under acidic conditions (Fig. 2). Rapid equilibration
of the oxonium intermediate and 1,2,3-triazoles under acidic con-
ditions result in epimerization at the C-9 and C-10 positions of 2
such that it can form possible two diastereomers (6 and 8) and
one regioisomer (7).9,10 Thus, diastereomeric and regiomeric
derivatives of 2 will have the substituted triazol moieties at the
C-10 position.
In order to prepare a complete library, 4-substituted-1-H-tria-
zoles (5a–5h) were synthesized via Huisgen 1,3-cycloaddition of
various alkynes and azidotrimethylsilane (TMSN3) (1.5 equiv) with
5 mol% of CuI in a mixture of DMF and methanol (5:1) shown in
Scheme 1.11,12 Acid (BF3Et2O, 0.8 equiv)-catalyzed reactions with
2 and 4-substituted-1-H-triazoles (5a–5h) in methylene chloride
resulted in a complex mixture of diastereomers and regioisomers,
which included 9-epi-10b-(4-substituted triazolyl) artemisinin
(6a–6h), 10
a-(5-substituted triazolyl) artemisinin (7a–7h), and
10a-(4-substituted triazolyl) artemisinin (8a–8h) (Scheme 1). Con-
trary to our expectation that epimerization under acidic condition
would result in the formation of the diastereomer 4-substituted-
10b-pyrazolylartemisinin (9) along with the diastereomers 6, 7,
and 8, diastereomer 9 was not found in the mixture of products.
In order to synthesize 9, we replaced the Lewis acid BF3Et2O with
other acids, such as SnCl4, p-toluenesulfonic acid, TiCl4, and AlCl3,
* Corresponding authors. Tel.: +82 33 650 7112; fax: +82 33 650 7299 (J.H.); tel.:
+82 33 649 7454; fax: +82 33 641 1074 (S.L.).
E-mail addresses: ham0606@kist.re.kr (J. Ham), sjlee@kwandong.ac.kr, sjlee@
kd.ac.kr (S. Lee).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.074

S. Oh et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4112–4115
4113
H
N
H
H
H
O
O
O
O
O
O
O
O
O
O
H
O
O
O
H
H
H
10
O
O
O
N
O
OH
N3
3
N
4
1
2
R
Figure 1. Artemisinin and its derivatives.
H
N
O
N
N
O
O
O
acid
R
R
catalyst
H+
O
H+
+
+
+
N
R
N
N
N
N
N
OH
N
N
N
N
N
R
R
O
N
6
7
8
H
Figure 2. Addition of a 1,2,3-triazolyl substituent to the anomeric position of dihydroartemisinin (2).
Table 1
but these attempts were unsuccessful. We confirmed the stereo-
chemistry according to each diastereomeric type on the basis of
the H-9/H-10 coupling constants obtained by 1H nuclear magnetic
resonance (NMR) and the chemical shifts of the 9-methyl group in
the 13C NMR spectra of 6a, 7a, and 8a.13 6a and 8a were deter-
mined to be the same compounds which was synthesized by direct
Cu(I)-catalyzed Huisgen 1,3-cycloaddition of 10-azidoartemisinin
(3) and phenylacetylene (a) in our previous report.8 When the 1H
NMR and 13C NMR spectra of 7a was compared with those of 6a
and 8a, we found that compound 7a was the only unknown prod-
uct synthesized via the new synthetic method.14 The H-9/H-10
coupling constant (J = 10.7 Hz) and chemical shift (12.2 ppm in
13C NMR) of the 9-methyl group of 7a showed that 7a, like 8a, pos-
Yields and ratios of 10-substituted triazolyl artemisinins synthesized via an acid-
catalyzed reaction
Yielda (%)
Ratiob
Yielda (%)
Ratiob
6
7
8
6
7
8
a
b
c
79
65
78
87
4.3
2.1
1
7.1
5.2
4.3
6.7
1
1
2.4
2.4
e
f
g
h
80
64
67
75
1
5.6
4.3
5.6
7.8
3.2
1
1
3.6
2.1
2.4
d
1
1
a
Isolated yield.
b
Ratios of each product were calculated by integrating the H-9 peak in the
1H NMR of non-purified product mixtures.
sesses 9b-methyl and 10a-triazolyl groups. However we finally dif-
and 8a is a 1,4-disubstituted triazole. These two compounds (7a
and 8a) share a regioisomeric relationship as shown in Scheme 1.
On the basis of the stereo- and regio-chemistry of the three prod-
ucts, we summarized the yields and ratios for each product in Ta-
ferentiated these two compounds by comparing the nuclear
Overhauser effect (NOE) between H-9 and the triazole methine
proton. In the 1D-NOE experiment, 7a did not exhibit an NOE while
8a did; thus, we concluded that 7a is a 1,5-disubstituted triazole
Cl
CH3
F
H
N
a
e
b
f
d
h
c
g
(a)
N
R:
R
+
TMSN3
F
F
F3C
N
a - h
R
C5H11
OCH3
5a - 5h
H
N
H
O
O
H
N
H
O
O
O
O
O
O
H
N
N
N
O
O
O
(b)
O
+
+
H
2
+
H
H
H
O
O
N
O
O
R
R
5a - 5h
N
N
N
N
N
N
N
N
N
R
R
R
9
6a - 6h
7a - 7h
8a - 8h
Scheme 1. Reagents and conditions: (a) azidotrimethylsilane (1.5 equiv), CuI (5 mol%), DMF/MeOH = 5:1, reflux, 24 h. Isolated yields: 5a, 85%; 5b, 66%; 5c, 89%; 5d, 75%; 5e,
61%; 5f, 91%; 5g, 70%; 5h, 79%; (b) triazole (5a–5h, 1 equiv), BF3Et2O (0.8 equiv) methylene chloride, rt, 24 h.

4114
S. Oh et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4112–4115
Table 2
Growth inhibition assay with various cancer cell lines
a
Growth inhibitory concentration of the derivatives against cancer cells (GI50 , lM)
DLD-1
U-87
HeLa
SiHa
A172
B16
a
6
7
8
0.28
0.60
0.93
0.15
0.20
0.50
0.03
0.04
0.58
0.14
0.14
0.73
0.11
0.15
0.57
0.20
0.22
0.78
b
6
7
8
0.16
0.29
0.40
1.34
0.11
0.28
0.73
0.04
0.11
1.58
0.19
0.12
1.74
0.10
0.09
0.70
0.11
0.12
c
6
7
8
2.15
0.31
0.48
0.63
0.09
0.16
0.41
0.06
0.05
0.96
0.08
0.14
0.56
0.08
0.13
0.84
0.77
0.11
d
6
7
8
1.17
0.04
0.29
0.35
0.10
0.25
0.21
0.06
0.09
0.41
0.16
0.24
0.33
0.13
0.31
0.28
0.10
0.21
e
6
7
8
1.07
0.17
2.38
0.53
0.08
0.29
0.35
0.07
0.19
0.62
0.08
0.32
0.40
0.08
0.29
0.48
0.08
0.34
f
6
7
8
0.84
0.22
0.14
0.27
0.09
0.07
0.24
0.04
0.09
0.44
0.10
0.08
0.26
010
0.07
0.34
0.08
0.07
g
6
7
8
0.22
0.88
0.49
0.07
0.23
0.39
0.10
0.09
0.13
0.07
0.24
0.36
0.07
0.21
0.30
0.10
0.29
0.13
h
6
7
8
1.28
0.17
0.30
0.45
0.17
0.13
0.24
0.08
0.05
0.46
0.07
0.12
0.54
0.10
0.14
0.70
0.10
0.13
Taxol
0.01
0.02
0.02
0.03
0.01
0.01
a
GI50 values were calculated by non-linear regression analysis using the GraphPad Prism software (R2 > 0.95).
ble 1. In general, the yield of 1,5-disubstituted triazole type artem-
isinins (7) is higher than that of the others (6 and 8).15
be analyzed in preclinical trials to evaluate their in vivo anticancer
activity and as well as toxicity.
In order to evaluate the growth inhibitory effect of the members
of the synthetic 10-substituted triazolyl artemisinin library, we
examined the effect of these derivatives against cancer cell lines
such as DLD-1, U-87, HeLa, SiHa, A172, and B1616 using the MTT
colorimetric method.17 These results are summarized in Table 2.
The growth inhibitory activity of the compounds (6a, 8a–8d,
and 8f–8h) reported in our previous manuscript were re-evaluated
in the same batch system used for obtaining the novel synthetic
artemisinin library. Although there were some unintentional devi-
ations in the DLD-1 cell lines, most of the antiproliferation activi-
ties of the previous compounds observed in this test were in
accordance with those observed in previous screening test results.8
The two newly obtained artemisinin group, namely, 9-epi-10b-(4-
Acknowledgment
This work was supported by a National Research Foundation of
Korea Grant funded by the Korean Government (2009-0073025).
References and notes
1. Klayman, D. L. Science 1985, 228, 1049.
2. Brewer, T. G.; Peggins, J. O.; Grate, S. J.; Petras, J. M.; Levine, B. S.; Weina, P. J.;
Swearengen, J.; Heiffer, M. H. Trans. R. Soc. Trop. Med. Hyg. 1994, 88, 33.
3. Lin, A. J.; Lee, M.; Klayman, D. L. J. Med. Chem. 1989, 32, 1249.
4. Lin, A. J.; Klayman, D. L.; Milhous, W. K. J. Med. Chem. 1987, 30, 2147.
5. Lin, A. J.; Miller, R. E. J. Med. Chem. 1995, 38, 764.
6. Genovese, R. F.; Newman, D. B.; Brewer, T. G. Pharmacol. Biochem. Behav. 2000,
67, 37.
7. Lee, S. Mini-Rev. Med. Chem. 2007, 7, 411.
substituted triazolyl) artemisinins (6a–6h) and 10
triazolyl) artemisinins (7a–7h), showed a similar potency as
10 -(4-substituted triazolyl) artemisinin (8a–8h) at submicromo-
a-(5-substituted
8. Cho, S.; Oh, S.; Um, Y.; Jung, J.-H.; Ham, J.; Shin, W. S.; Lee, S. Bioorg. Med. Chem.
Lett. 2009, 19, 382.
a
lar concentrations. Based on this preliminary structure–activity
relationship, there were a little differences of growth inhibitory
effect according to the stereo- and regio-chemistry. As far as we
9. Chorki, F.; Crousse, B.; Bonnet-Deipon, D.; Bégué, J. P.; Brigaud, T.; Portella, C.
Tetrahedron Lett. 2001, 42, 1487.
10. Chorki, F.; Grellepois, F.; Crousse, B.; Ourévitch, M.; Bonnet-Deipon, D.; Bégué,
J. P. J. Org. Chem. 2001, 66, 7858.
are concerned, the 10a-(5-substituted triazolyl) artemisinin group
11. Jin, T.; Kamijo, S.; Yamamoto, Y. Eur. J. Org. Chem. 2004, 3789.
12. The procedure for the synthesis of 4-phenyl-1-H-1,2,3-triazole (5a):
Ethynylbenzene (500 mg, 4.9 mmol) and azidotrimethylsilane (847 mg,
7.4 mmol) were added to a solution of DMF and MeOH (6 mL, 5:1) under N2
atmosphere. After adding the CuI (46.7 mg, 0.25 mmol), the reaction mixture
was refluxed for 12 h, which was monitored by TLC, and then quenched with
water (10 mL). The mixture was extracted with dichloromethane (50 mL, two
times), dried with MgSO4, and then evaporated under reduced pressure. The
residue was purified on silica-gel chromatography with ethyl acetate/hexane
(5:2) to afford 5a.
(7a–7h) is more or less as potent as the other groups (6 and 8).
Although at this stage we cannot comment on the activity differ-
ence with changes in functional group, three compounds, namely,
6f, 7f, and 8f, with a pentylbenzene group were found to be the
most active anticancer molecules.
In conclusion, we have established a 10-substituted triazolyl
artemisinins library (6a–6h, 7a–7h, and 8a–8h) comprising com-
pounds that strongly inhibit the growth of cancer cell lines, such
as DLD-1, U-87, HeLa, SiHa, A172, and B16. These compounds could
be synthesized by an acid-catalyzed reaction with 2 and various
substituted triazoles (5a–5h) in methylene chloride at room tem-
perature. Compounds 6f, 7f, and 8f, which have a pentylphenyltriaz-
ole moiety, exhibit potent activity; therefore, these compounds will
13. Lee, S.; Oh, S. Tetrahedron Lett. 2002, 43, 2891.
14. The procedure for the synthesis of 10-(phenyltriazolyl) artemisinin (6a, 7a, and
8a): To a solution of dihydroartemisinin (2, 1.0 g, 3.5 mmol) and 4-phenyl-1H-
1,2,3-triazole (a, 0.51 g, 3.5 mmol) in dichloromethane (85 mL) was added
BF3Et2O (0.40 g, 2.8 mmol) at room temperature, and then stirred for 12 h. If
starting materials were disappeared on TLC, the reaction mixture was diluted
with water (100 mL), and extracted with dichloromethane (50 mL, three times)
dried with MgSO4, and then evaporated under reduced pressure. The residue

S. Oh et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4112–4115
4115
was purified on silica-gel chromatography with ethyl acetate/hexane (5:2) to
afford 6a, 7a, and 8a. The spectral data of 6a and 8a were previously reported
in Ref. 8. 7a: 1H NMR (300 MHz, CDCl3) d 7.95 (1H, s, triazole), 7.82 (2H, d,
J = 6.9 Hz, phenyl), 7.42 (2H, d, J = 7.3 Hz, phenyl), 7.37 (1H, t, J = 7.3 Hz,
phenyl), 5.80 (1H, d, J = 10.7 Hz, H-10), 5.57 (1H, s, H-12), 3.42 (1H, m), 2.42
(1H, td, J = 14.6, 4.1 Hz), 2.06 (1H, m), 1.42 (3H, s, 3-CH3), 1.00 (3H, d, J = 5.9 Hz,
9-CH3), 0.69 (3H, d, J = 7.1 Hz, 6-CH3) ppm; 13C NMR (75 MHz, CDCl3) d 148.0,
132.2, 128.8, 128.5, 126.1, 104.6, 92.2, 88.6, 79.8, 51.6, 45.4, 37.3, 36.2, 34.0,
31.6, 25.8, 21.7, 20.2, 12.2 ppm.
(3H, d, J = 5.88 Hz, 9-CH3), 0.66 (3H, d, J = 7.14 Hz, 6-CH3) ppm; 13C NMR
(75 MHz, CDCl3) d 145.0, 134.9, 132.1, 128.7, 126.2, 104.7, 92.3, 88.7, 79.8, 51.6,
45.4, 37.4, 36.2, 34.0, 32.0, 25.9, 24.6, 21.7, 20.2, 12.0 ppm; 6f: 1H NMR
(300 MHz, CDCl3) d 7.91 (1H, s, triazole), 7.74 (1H, d, J = 8.04 Hz, phenyl), 7.24
(1H, d, J = 8.25 Hz, phenyl), 6.47 (1H, d, J = 10.26 Hz, H-10), 5.65 (1H, s, H-12),
2.63 (1H, t, J = 7.5 Hz, ben-CH2–), 2.57 (1H, m), 2.37 (1H, td, J = 12.99, 3.84 Hz),
1.49 (3H, s, 3-CH3), 0.99 (3H, d, J = 3.84 Hz, 9-CH3), 0.97 (3H, d, J = 6.96 Hz,
6-CH3), 0.89 (3H, t, J = 6.78 Hz, CH3) ppm; 13C NMR (75 MHz, CDCl3) d 148.5,
143.5, 131.8, 128.8, 127.6, 126.2, 102.8, 91.1, 89.5, 82.4, 51.2, 47.1, 39.9, 37.3,
36.3, 35.7, 34.0, 31.7, 31.4, 31.1, 25.8, 24.7, 22.5, 19.9, 18.3, 14.0 ppm; 7f: 1H
NMR (300 MHz, CDCl3) d 7.93 (1H, s, triazole), 7.72 (1H, d, J = 8.22 Hz, phenyl),
7.24 (1H, d, J = 8.4 Hz, phenyl), 5.78 (1H, d, J = 10.62 Hz, H-10), 5.57 (1H, s,
H-12), 3.40 (1H, m), 2.63 (1H, t, J = 7.5 Hz, ben-CH2–), 2.43 (1H, td, J = 12.99,
3.84 Hz), 2.04 (1H, m), 1.42 (3H, s, 3-CH3), 1.00 (3H, d, J = 5.85 Hz, 9-CH3), 0.96
(3H, t, J = 6.96 Hz, CH3), 0.68 (3H, d, J = 6.93 Hz, 6-CH3) ppm; 13C NMR (75 MHz,
CDCl3) d 148.1, 143.5, 132.2, 128.9, 127.6, 126.1, 104.6, 92.9, 88.5, 79.8, 51.7,
45.4, 37.4, 36.2, 35.7, 34.1, 31.7, 31.5, 31.1, 25.9, 24.7, 22.5, 21.8, 20.2, 14.0,
12.3 ppm; 6g: 1H NMR (300 MHz, CDCl3) d 7.84 (1H, s, triazole), 7.74 (2H, d,
J = 8.76 Hz, phenyl), 6.96 (2H, d, J = 8.70 Hz, phenyl), 5.74 (1H, d, J = 11.94 Hz
H-10), 5.53 (1H, s, H-12), 3.83 (3H, s, –OCH3), 2.53 (1H, m), 1.49 (3H, s, 3-CH3),
1.00 (3H, d, J = 5.9 Hz, 9-CH3), 0.87 (3H, d, J = 7.1 Hz, 6-CH3) ppm; 13C NMR
(75 MHz, CDCl3) d 161.2, 148.2, 131.4, 127.0, 114.7, 104.5, 103.6, 101.4, 99.0,
92.1, 81.0, 60.4, 55.3, 52.0, 45.6, 37.1, 37.0, 32.5, 31.1, 26.8, 20.5, 15.7,
14.6 ppm; 7g: 1H NMR (300 MHz, CDCl3) d 7.89 (1H, s, triazole), 7.74 (2H, d,
J = 8.79 Hz, phenyl), 6.96 (2H, d, J = 8.76 Hz, phenyl), 5.80 (1H, d, J = 11.91 Hz,
H-10), 5.57 (1H, s, H-12), 3.85 (3H, s, –OCH3), 3.40 (1H, m), 1.43 (3H, s, 3-CH3),
1.00 (3H, d, J = 5.88 Hz, 9-CH3), 0.86 (3H, d, J = 7.14 Hz, 6-CH3) ppm; 13C NMR
(75 MHz, CDCl3) d 160.0, 147.9, 131.8, 127.5, 114.2, 104.6, 103.8, 101.5, 98.7,
90.6, 81.2, 60.4, 55.3, 51.7, 45.4, 37.4, 37.3, 32.9, 31.6, 26.2, 20.3, 14.2,
12.3 ppm; 6h: 1H NMR (300 MHz, CDCl3) d 7.93 (2H, s, triazole), 7.37 (1H, d,
J = 8.25 Hz, phenyl), 6.80 (1H, s, phenyl), 6.49 (1H, d, J = 10.26 Hz, H-10), 5.65
(1H, s, H-12), 2.55 (1H, m), 2.37 (1H, td, J = 12.99, 4.02 Hz), 1.49 (3H, s, 3-CH3),
1.00 (3H, d, J = 5.67 Hz, 9-CH3), 0.98 (3H, d, J = 6.96 Hz, 6-CH3) ppm; 13C NMR
(75 MHz, CDCl3) d 171.2, 165.1, 161.6, 132.1, 108.9, 102.9, 91.1, 89.8, 82.4, 60.4,
51.2, 47.1, 39.9, 37.3, 36.3, 34.0, 31.7, 25.8, 19.8, 18.2, 14.2 ppm; 7h: 1H NMR
(300 MHz, CDCl3) d 7.94 (1H, s, triazole), 7.35 (2H, d, J = 8.25 Hz, phenyl), 6.80
(1H, s, phenyl), 5.43 (1H, d, J = 11.73 Hz, H-10), 5.58 (1H, s, H-12), 3.4 (1H, m),
2.4 (1H, td, J = 14.46, 3.66 Hz), 1.49 (3H, s, 3-CH3), 1.01 (3H, d, J = 5.88 Hz,
9-CH3), 0.69 (3H, d, J = 7.14 Hz, 6-CH3) ppm; 13C NMR (75 MHz, CDCl3) d 171.2,
165.0, 161.8, 132.4, 109.1, 108.9, 104.7, 99.9, 92.3, 79.8, 60.4, 51.6, 45.3, 42.2,
37.4, 36.2, 34.0, 31.6, 20.2, 18.7, 12.3 ppm.
15. Spectral data for 6b: 1H NMR (300 MHz, CDCl3) d 7.91 (1H, s, triazole), 7.72 (2H,
d, J = 8.07 Hz, phenyl), 7.23 (2H, d, J = 8.61 Hz, phenyl), 6.49 (1H, d, J = 10.1 Hz,
H-10), 5.65 (1H, s, H-12), 3.40 (1H, m), 2.58 (1H, td, J = 10.1, 2.0 Hz), 2.38 (3H, s,
tosyl), 1.49 (3H, s, 3-CH3), 0.99 (3H, d, J = 7.0 Hz, 9-CH3), 0.73 (3H, d, J = 7.0 Hz,
6-CH3) ppm; 13C NMR (75 MHz, CDCl3) d 148.5, 131.8, 129.5, 127.9, 104.6, 91.1,
82.4, 51.2, 47.1, 45.4, 39.9, 37.3, 36.3, 36.2, 25.9, 25.8, 24.7, 24.6, 21.7,
12.4 ppm; 7b: 1H NMR (300 MHz, CDCl3) d 7.93 (1H, s, triazole), 7.70 (2H, d,
J = 8.0 Hz, phenyl), 7.23 (2H, d, J = 7.87 Hz, phenyl), 5.78 (1H, d, J = 10.6 Hz,
H-10), 5.57 (1H, s, H-12), 3.41 (1H, m), 2.38 (3H, s, tosyl), 1.41 (3H, s, 3-CH3),
1.00 (3H, d, J = 5.9 Hz, 9-CH3), 0.69 (3H, d, J = 7.1 Hz, 6-CH3) ppm; 13C NMR
(75 MHz, CDCl3) d 148.1, 138.5, 132.2, 129.5, 126.1, 104.6, 92.9, 91.0, 88.5, 79.8,
51.7, 45.4, 37.4, 36.2, 34.1, 31.8, 25.9, 21.3, 20.2, 18.4, 12.3 ppm; 6c: 1H NMR
(300 MHz, CDCl3) d 7.89 (1H, s, triazole), 7.81 (2H, m, phenyl), 7.12 (2H, t,
J = 7.87 Hz, phenyl), 6.49 (1H, d, J = 10.26 Hz, H-10), 5.64 (1H, s, H-12), 2.57
(1H, m), 2.37 (1H, m), 1.51 (3H, s, 3-CH3), 1.00 (3H, d, J = 5.9 Hz, 9-CH3), 0.94
(3H, d, J = 7.1 Hz, 6-CH3) ppm; 13C NMR (75 MHz, CDCl3) d 171.2, 131.7, 128.1,
128.0, 115.9, 115.7, 91.1, 89.6, 82.4, 60.4, 51.2, 47.1, 39.9, 37.3, 36.3, 34.0, 31.7,
25.8, 24.7, 21.1, 19.9, 18.2, 16.7, 15.3, 14.2 ppm; 7c: 1H NMR (300 MHz, CDCl3)
d 7.92 (1H, s, triazole), 7.74 (2H, m, phenyl), 7.12 (2H, t, J = 8.97 Hz, phenyl),
6.46 (1H, d, J = 9.9 Hz, H-10), 5.76 (1H, s, H-12), 3.41 (1H, m), 1.41 (3H, s, 3-
CH3), 1.00 (3H, d, J = 5.85 Hz, 9-CH3), 0.93 (3H, d, J = 5.67 Hz, 6-CH3) ppm; 13C
NMR (75 MHz, CDCl3) d 172.8, 132.0, 127.9, 127.6, 115.9, 115.6, 109.5, 104.6,
102.9, 99.9, 91.1, 86.6, 82.2, 79.8, 44.0, 42.2, 39.6, 37.3, 35.5, 34.1, 33.7, 31.2,
24.2, 22.1, 20.2, 18.7, 12.3 ppm; 6d: 1H NMR (300 MHz, CDCl3) d 7.94 (1H, s,
triazole), 7.70 (1H, d, J = 6.75 Hz, phenyl), 7.36 (1H, t, J = 8.04 Hz, phenyl), 7.33
(1H, d, J = 3.3 Hz, phenyl), 6.50 (1H, d, J = 10.23 Hz, H-10), 5.65 (1H, s, H-12),
2.57 (1H, m), 2.38 (1H, td, J = 12.99, 4.02 Hz), 1.49 (3H, s, 3-CH3), 0.99 (3H, d,
J = 5.67 Hz, 9-CH3), 0.98 (3H, d, J = 6.93 Hz, 6-CH3) ppm; 13C NMR (75 MHz,
CDCl3) d 147.1, 134.8, 132.0, 130.1, 128.6, 126.3, 124.3, 102.9, 91.9, 89.7, 82.4,
60.4, 51.2, 47.1, 39.9, 37.3, 36.3, 34.0, 31.7, 25.8, 24.7, 21.1, 19.8, 18.2,
14.2 ppm; 7d: 1H NMR (300 MHz, CDCl3) d 7.83 (1H, s, triazole), 7.68 (1H, d,
J = 6.96 Hz, phenyl), 7.36 (1H, t, J = 8.07 Hz, phenyl), 7.33 (1H, d, J = 3.3 Hz,
phenyl), 5.79 (1H, d, J = 10.62 Hz, H-10), 5.58 (1H, s, H-12), 3.41 (1H, m), 2.43
(1H, td, J = 13.92, 3.84 Hz), 2.05 (1H, m), 1.42 (3H, s, 3-CH3), 1.00 (3H, d,
J = 5.85 Hz, 9-CH3), 0.69 (3H, d, J = 9.84 Hz, 6-CH3) ppm; 13C NMR(75 MHz,
CDCl3) d 146.8, 134.8, 132.3, 132.0, 130.1, 128.6, 126.2, 124.2, 104.7, 92.3, 88.7,
79.8, 51.6, 45.4, 37.4, 36.2, 34.0, 31.6, 25.9, 24.6, 21.8, 20.2, 12.3 ppm; 6e: 1H
NMR (300 MHz, CDCl3) d 7.85 (1H, s, triazole), 7.77 (1H, d, J = 7.68 Hz, phenyl),
7.73 (1H, d, J = 7.5 Hz, phenyl), 7.63 (1H, t, J = 7.5 Hz, phenyl), 6.52 (1H, d,
J = 10.26 Hz, H-10), 5.66 (1H, s, H-12), 2.54 (1H, m), 2.37 (1H, m), 1.48 (3H, s,
16. DLD-1: human colorectal adenocarcinoma; U-87 and A172: human glioma;
HeLa, and SiHa: human cervical carcinoma; B16: mouse melanoma.
17. Mosmann, T. J. Immunol. Methods 1983, 65, 55. Growth inhibitory activity of the
artemisinin derivatives was evaluated with the MTT assay. Cancer cells were
plated in 96-well culture plates at a density of 5 Â 103 cells/well in a final
volume of 100 lL of DMEM containing 10% FBS, preincubated for 4 h, and
treated with serial concentrations of artemisinin derivatives for 72 h. After
treatment, the cells were incubated for 4 h at 37 °C with a solution of MTT at a
concentration of 1 mg/mL. The culture supernatant was aspirated and DMSO
3-CH3), 0.99 (3H, d, J = 4.95 Hz, 9-CH3), 0.97 (3H, d, J = 6.93 Hz, 6-CH3) ppm; 13
C
NMR (75 MHz, CDCl3) d 145.6, 134.7, 132.1, 128.7, 102.9, 91.2, 89.7, 82.4, 51.2,
47.0, 40.3, 37.3, 36.3, 34.0, 31.7, 25.8, 24.7, 19.8, 18.0, 14.2; 7e: 1H NMR
(300 MHz, CDCl3) d 7.86 (1H, s, triazole), 7.77 (1H, d, J = 7.68 Hz, phenyl), 7.71
(1H, d, J = 7.68 Hz, phenyl), 7.61 (1H, t, J = 6.78 Hz, phenyl), 7.51 (1H, t,
J = 7.35 Hz, phenyl), 5.81 (1H, d, J = 10.62 Hz, H-10), 5.58 (1H, s, H-12), 3.38
(1H, m), 2.25 (1H, td, J = 14.5, 3.84 Hz), 2.07 (1H, m), 1.44 (3H, s, 3-CH3), 1.00
(100 lL) was added to dissolve the formed formazan crystals. The plate was
then read at 570 nm in a microplate spectrophotometer (SpectraMax 250,
Molecular Devices, CA, USA). Each assay was performed in triplicate. GI50 was
calculated by non-linear regression analysis from a sigmoidal dose–response
curve using the GraphPad Prism software ver 3.0 (GraphPad Software, CA, USA)
when R2 > 0.