Synthesis of novel 1,2,3-triazole based artemisinin derivatives and their antiproliferative activity

Article abstract of DOI:10.1039/c7nj04271j

Two series of novel 1,2,3-triazole based artemisinin derivatives were designed, synthesized via a copper(i)-catalyzed azide alkyne cycloaddition (CuAAC) reaction and investigated for their antiproliferative activity by MTT assay against various human canc

Full text of DOI:10.1039/c7nj04271j

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Synthesis of novel 1,2,3ꢀtriazole based artemisinin derivatives and their
antiproliferative activity
Deepak Singh Kapkoti a#, Shilpi Singh b#, Suaib Luqmanb* and Rajendra Singh Bhakunia*
aMedicinal Chemistry Department, CSIRꢀCentral Institute of Medicinal and Aromatic Plants,
Lucknowꢀ226015, India;
bMolecular Bioprospection Department, CSIRꢀCentral Institute of Medicinal and Aromatic
Plants, Lucknowꢀ226015, India;
#Authors contributed equally
*Corresponding authors. Tel: +915222718622; Fax: 915222342666
Eꢀmail addresses: rs.bhakuni@cimap.res.in; bhakunirs2000@gmail.com (R. S. Bhakuni),
s.luqman@cimap.res.in (S. Luqman)
1

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Abstract
Two series of novel 1,2,3ꢀtriazole based artemisinin derivatives were designed,
synthesized via copper(I)ꢀcatalyzed azide alkyne cycloaddition (CuAAC) reaction and
investigated for their antiproliferative activity by MTT assay against various human
cancer cell lines. In series 1, compounds 9, 17, 18, and 22 were showed better
antiproliferative activity against all the tested cell lines as compared to
dihydroartemisinin (DHA, 5). Compound 9 and compound 17 were the most active,
with IC50 range from 4.06 to 36.65 ꢁM and 7.16 to 28.21 ꢁM respectively. Compound 9
showed potent antiproliferative activity against the A431 cell lines with IC50 4.06 ꢁM
and compound 17 displayed potent activities against A549 cell line with IC50 7.16 ꢁM.
In series 2, compounds 24, 27 and 28 showed better activity than other derivatives.
Active compounds 9 and 17 showed significant (p<0.05) cell cycle arrest at G2/M
phase and apoptosis in skin and lung cancer cells. These molecules significantly
(p<0.05) induce the ROS formation in tested cell lines. Furthermore the toxicity study
on human erythrocyte revealed that these molecules are nonꢀtoxic even at the higher
tested concentration (100 µg/ml). Some 3αꢀhydroxydeoxydihydroartemisininꢀtriazole
derivatives (11a, 14a, 15a, 17aꢀ22a) were also synthesized along with their peroxy
counterparts. The results antiproliferative activity revealed that except compound 11a,
all the compounds with peroxy functionality are more active than their 3α ꢀ
hydroxydeoxy counterparts.
Keywords: Artemisinin derivatives; 1,2,3ꢀTriazole; Synthesis; Antiproliferative
activity; Cell Cycle analysis; ROS; Osmotic fragility
2

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1. Introduction
Artemisinin (1) (qinghaosu), a wellꢀknown antimalarial compound was isolated from the
plant Artemisia annua L. Asteraceae. It is a sesqueterpene lactone endoperoxide having 1,2,4
trioxane system which is essential component for its antimalarial activity.1,2 Currently,
artemisinin and its semisynthetic derivatives such as artemether (2), arteether (3), and
artesunate (4) (Figure 1) have been effectively used for malaria treatment. These derivatives
are active against both uncomplicated and severe forms of malaria and finally converted to
dihydrortemisinin (5), the active metabolite of artemsinin inside the body.3,4
Figure 1. Artemisinin and their clinically used derivatives for malaria treatment.
The anticancer property of artemisinin (1) was firstly documented by Woerdenbag et al. in
1993.5 Interestingly, it was found that the potent anticancer activity of artemisinin (1) was
also associated with the presence of intact endoperoxide bond. The absence of endoperoxide
moiety does not completely abolish the antitumor activity but significantly reduces it as
compared to those compounds with the intact endoperoxide bond.6ꢀ9 These residual
anticancer activity suggesting that artemisinin compounds may also follow peroxideꢀ
independent mechanism.7 In a general concurrence, free iron or heme/hemeꢀbound proteins
have been responsible for the bioreductive activation of artemisinin.10ꢀ12 It has been supposed
that iron activated artemisinin damage the cancer cells by the formation of highly alkylating
carbon centered radicals and reactive oxygen species (ROS),9,13 which is responsible for
selective action of artemisinin (1) on cancer cells by induced apoptosis, growth arrest,
decrease cell proliferation, DNA damage and reducing angiogenesis.14 It was found that
artemisinin (1) and its derivatives were active against both the drug sensitive and resistant
cancer cell lines.15ꢀ17 These compounds are active against highly metastatic and aggressive
cancers.14,18 Furthermore, artemisinin derivatives also show synergistic interaction with other
anticancer drugs with minimum toxicity towards normal cells.19ꢀ21 Antitumor activity of
3

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several artemisinin derivatives including both the monomers and dimers has been document
in vitro and animal model.22ꢀ24
1,2,3ꢀtriazoles are the nitrogen heterocycles posseses diverse range of pharmacological
activities including antiproliferative, antimicrobial, antiꢀinflammatory, antiretroviral,
anticonvulsant and transforming growth factor β1 type 1 receptor inhibition.25,26 These
compounds are generally stable to hydrolysis in acidic and basic condition as well as to
oxidoꢀreductive environment which make them stable to metabolic degradation and indicates
their high aromatic stabilization. 1,2,3ꢀTriazole moieties have a very high dipole moment
(about 5 Debye) and are capable of hydrogen bonding which is favourable condition for
binding with the biomolecular targets and also improves their solubility.27,28 There are
number of drugs in market having 1,2,3ꢀtriazole moiety such as cefatrizine (antibiotic),29
tazobactam (antibiotic),29 rufinamide (anticonvulsant)30 and many of them are in different
phase of clinical trials and may become drugs in future such as carboxyamidotriazole or CAI
(anticancer),31 tertꢀbutyldimethylsilylspiroaminooxathioledioxide or TSAO (HIV reverse
transcriptase inhibitor)32 (Figure 2).
Figure 2. Potential drugs based on 1,2,3ꢀtriazole system.
In the present study, we have designed and synthesized two series of novel artemisinin
triazole based derivatives and evaluated for their antiproliferative activity against the various
organ specific cell lines. Further the mechanism of potential leads was evaluated via cell
cycle analysis and ROS production. Furthermore, the toxicity of the potential leads was also
estimated by Osmotic fragility assay.
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2. Result and discussion
2.1. Chemistry
We have targeted to synthesized two series of novel artemisininꢀazole derivatives. In the first
series, synthesized derivatives have artemisinin part attached to the 4 position of 1,2,3ꢀ
triazole system and in second series, artemisinin part was attached to 1 position of 1,2,3ꢀ
triazole system (Figure 3).
Figure 3. Structures of target molecules.
The synthesis of the series 1 derivatives of artemisinin is depicted in Scheme 1. Artemisinin
(1) was firstly reduced into DHA (5) by using NaBH4 as a reducing agent in methanol at 0 to
ꢀ5 ºC temperature. Then the propargyl derivative of artemisinin (6) was synthesized by the
reaction of DHA (5) with propargyl alcohol in the presence of BF3.OEt2 as a catalyst in dry
ether at 0ꢀ5 ºC temperature. 1,2,3ꢀtriazole based artemsinin derivatives were synthesized by
Copper(I)ꢀcatalyzed azide alkyne cycloaddition (CuAAC) reaction which is the most used
click reaction. Targeted compounds (9ꢀ22) were synthesized by the CuAAC reaction between
the compound (6) and appropriate aromatic azides (8aꢀn) were carried out in the presence of
CuSO4.5H2O and sodium ascorbate in dichloromethane and water (5:3). The aromatic azides
(8aꢀh) were synthesized by the diazotization of corresponding aromatic amines (7aꢀh) with
sodium nitrite followed by the reaction with sodium azide as outline in Scheme 2.
Further the ortho and para substituted azidoacetophenones (8eꢀg) obtained by the above
reaction were treated with 2ꢀ and 4ꢀsubstituted trifluoromethyl benzaldehyde to form 2ꢀand 4ꢀ
substituted azido chalcones (8iꢀn) via the base catalysed Claisen Schmidt condensation
reaction (Scheme 2). Generally it was found that 2ꢀtrifluoromethyl substituted azido
chalcones were formed easily in good yield but the synthesis of 4ꢀtrifluoromethyl substituted
azido chalcones led to the different side products along with the desired chalcone derivatives.
So in that case amino substituted chalcone derivatives were firstly synthesized by Claisen
Schmidt reaction of different amino acetophenone with 4ꢀ(trifluoromethyl)benzaldehyde,
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Comp
R
Comp
Comp
R
9
14, 14a
19, 19a
20, 20a
21, 21a
22, 22a
10
15, 15a
16
11, 11a
12
17, 17a
18, 18a
13
º
Scheme 1. Reagents and conditions: a) NaBH4, MeOH, 0ꢀ5 C, b) Propargyl alcohol,
BF3Et2O; c) RN3, CuSO4.5H2O, Sodium ascorbate in DCM:H2O (5:3).
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Scheme 2. Reagents and conditions: a) NaNO2, HCl, NaN3, H2O:CH3COOH, b) RCHO,
KOH, MeOH
then these 4ꢀtrifluoromethyl substituted amino chalcones were converted into the
corresponding azides by the diazotization with sodium nitrite followed by the reaction with
sodium azide. Synthesis of amino chalcones derivatives from amino acetophenones
sometimes required the high temperature (refluxing condition) to complete the reaction.
Series 2 contains mostly dimers (29ꢀ33) and some monomers (27, 28) derivatives of
artemisinin were synthesized according to the Scheme 3. Both the monomers and dimers
derivatives of artemisinin were prepared by the CuAAc reaction of mono and dipropargyl
ether derivatives of different benzene diols (26aꢀf) with 12ꢀβꢀazidoethoxydihydroartemisinin
(24). The etherification of benzene diols were accomplished by its reaction with propargyl
bromide in the presence of K2CO3 as base in acetone under refluxing condition (Scheme 4).
For
the
synthesis
of
12ꢀβꢀazidodihydroartemisinin
(24),
12ꢀβꢀ(2ꢀ
bromoethoxy)dihydroartemisinin (23a or 12ꢀβꢀ(2ꢀiodoethoxy)dihydroartemisinin, 23b) was
firstly prepared by the etherification of DHA with 2ꢀbromoethanol (or 2ꢀiodoethanol, in case
of compound 23b) in the presence of BF3.OEt2 as catalyst in dry ether then 12ꢀβ(2ꢀ
bromoethoxy)dihydroartemisinin (23a) was reacted with sodium azide in DMSO to afford the
12ꢀβꢀazidodihydroartemisinin.
The
CuAAC
reaction
between
the
12ꢀβꢀ
azidodihydroartemisinin and different mono and dipropagyl ether derivatives of benzene
diols give monomers and dimers derivatives of artemisinin respectively.
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Compounds
R
Compounds
R
27
28
31
ꢀH
ꢀCHO
ꢀH
32
33
ꢀCHO
ꢀCOOH
º
Scheme 3. Reagents and conditions: a) NaBH4, MeOH, 0 to ꢀ5 C; b) Bromoethanol (or
Iodoethanol), BF3Et2O; c) NaN3, DMSO; d) mono and di propargyl ether derivatives of
benzene diol, CuSO4.5H2O, Sodium ascorbate .
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Scheme 4. Reagents and conditions: a) Propargyl bromide, K2CO3, acetone, reflux.
The stereochemistry (α or β orientation) of Cꢀ12 position of all the synthesized derivatives of
artemisinin were determined with the help of 1H NMR spectroscopy. The Hꢀ12 proton/s gave
a doublet at ~4.90 ppm with the small coupling constant (J~3.5 Hz) which indicated the α
orientation of Hꢀ12 proton33ꢀ35 so all the synthesized derivatives are 12ꢀβ isomers. The high
coupling constant values (= ~15.5 Hz) of αꢀβ unsaturated double bond of the compounds
having chalcone moiety (17ꢀ22) indicated the E (trans) isomerism for olefinic double bond.
During the synthesis of artemisininꢀtriazole derivatives, small amount of 3αꢀ
hydroxydeoxydihydroartemisinin ether azole derivatives were also formed whose
concentration were increased by increasing the mole equivalent concentration of catalyst
(CuSO4.5H2O and sodium ascorbate). At 0.5 mole equivalent CuSO4.5H2O and 1.5 mole
equivalent of sodium ascorbate, peroxy derivatives were completely converted into 3ꢀ
hydroxydeoxydihydroartemisinin derivatives. They were generally more polar than their
peroxy counterpart in TLC analysis. HRMS data indicated that the 3ꢀ
hydroxydeoxyartemisinin derivatives have the same mass as peroxy derivatives, suggested
1
that they have the same molecular formula. H NMR spectra showed a new broad singlet or
sometimes doublet peak with small coupling constant (~1.5ꢀ2.5 Hz) at around 3.56 ppm
suggested that the presence of hydroxyl group. The small coupling constant values of Cꢀ3
proton/s indicated its equatorial (β) orientation thus the hydroxyl group has α (axial)
orientation.36ꢀ39 13C and DEPTꢀ135 spectra showed new peak at ~69.50 ppm in place of CH2
of Cꢀ3 carbon peak at ~36.20 ppm indicated that the CH2 group of Cꢀ3 has been hydroxylated
into CHOH group at Cꢀ3 position. The NMR data of 3αꢀhydroxydeoxydihydroartemisininꢀ
triazoles derivatives showed quite similarity with those reported for 3αꢀ
hydroxydeoxyartemisinin.36 A plausible mechanism of seteroselective alpha hydroxylation of
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Cꢀ3 position of artemisinin derivatives has given in figure 4. Some of the 3ꢀ
hydroxydeoxyartemisinin triazole derivatives (11a, 14a, 15a, 17aꢀ22a) have been synthesized
along with their peroxy counterparts to know the effect of endoperoxide bond on
antiproliferative activity.
Figure 4. Plausible mechanism of alpha hydroxylation at Cꢀ3 position.
2.2. Artemisinin derivatives inhibited the cell proliferation in various cancer cell lines
Antiproliferative effect of artemisinin derivatives series 1 and 2 were analysed using MTT
assay in various organ specific cancer cell lines namely K562 (human erythromyeloblastoid
leukemia cell line), PCꢀ3 (human prostate carcinoma), A431 (human squamous carcinoma),
MDAMBꢀ231 (human breast carcinoma), COLOꢀ205 (human colon carcinoma), A549
(human lung carcinoma) and one normal cell line, HEKꢀ293 (human kidney cells). In series
1, Compound 9, 15, 17 and 22 were able to inhibit the proliferation of colon, skin, leukaemia,
breast, lung and prostate cancer cells with the IC50 ranged from 4.06ꢀ89.85 µM (Table 1).
Compound 10, 21 and 22a were reducing the proliferation of skin and breast cells with the
IC50 ranged from 17.97ꢀ52.86 µM whereas compound 21a inhibits the proliferation of A431
and PCꢀ3 cell line with the IC50 57.18 and 25.31 µM respectively. Compound 11a and 13
were selectively inhibiting the skin carcinoma with the IC50 values 68.27 µM and 43.42 µM
respectively. Compound 17a showed selectivity for breast cancer cells with IC50 of 43.19 µM
while rest of the molecules are also inhibit the proliferation of cells with percent inhibition
ranged from 3.30–49.60% (Figure 5). In series 2, among all the synthesized compounds only
compound 27 was displayed broad spectrum efficiency by inhibiting the growth of lung,
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Figure 5. Antiproliferative effect of artemisninꢀtriazole derivatives (Series 1) on various
organ specific cell line at higher tested concentration of 50 µg/ml. ART: Artemisnin, DHA:
dihyroartemisnin, DOX: Doxorubicin used as positive control. ART. All values are mean ±
SD (n=3).*Comparison of test with artemisnin (Parent). **p<0.05
Figure 6. Antiproliferative effect of artemisninꢀtriazole derivatives (seriesꢀ2) on various
organ specific cell lines at higher tested concentration of 50µg/ml. ART: Artemisnin, DHA:
dihyroartemisnin, DOX: Doxorubicin used as positive control. All values are mean ± SD
(n=3).*Comparison of test with artemisnin (Parent). **p<0.05
colon, breast, leukaemia, skin and prostate cancer cells with IC50 ranged from 16.20ꢀ59.93
µM while compound 28 hamper the proliferation of prostate, leukaemia, lung and colon
cancer cells (21.45ꢀ77.30 µM). Compound 29 was found to be selective for breast (49.98
µM) and lung (20.95 µM) cancer cells whereas rest was inhibited the proliferation up to
47.85 % (Figure 6). Interestingly, all the synthesized derivatives of series 1 and series 2 were
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Table 1. Antiproliferative potential of Arteimisinin and their derivatives
IC50 (ꢁM)
Compound
Series 1
K562
PCꢀ3
A431
MDAMBꢀ231 COLOꢀ205 A549
HEKꢀ293
***/###
###
***/###
###
###
***/###
Compound 9
11.34
10.36
ꢀ
ꢀ
ꢀ
4.06
15.43
36.65
12.43
ꢀ
ꢀ
ꢀ
ꢀ
###
Compound 10
Compound 11a
Compound 13
Compound 15
Compound 17
ꢀ
ꢀ
ꢀ
52.81
68.27
43.42
73.87
20.93
44.04
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
###
###
###
###
78.37
28.21
21.67
11.55
89.85
13.35
45.57
13.28
44.11
###
###
***/###
7.16
###
###
###
Compound 17a
Compound 18
Compound 21
ꢀ
69.67
ꢀ
ꢀ
ꢀ
43.19
27.32
17.97
ꢀ
50.58
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
###
###
21.12
ꢀ
37.30
47.25
49.49
57.03
###
Compound 21a
ꢀ
25.31
17.14
ꢀ
57.18
31.97
37.37
42.27
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
###
###
###
Compound 22
Compound 22a
Compound 24
Series 2
Compound 27
Compound 28
Compound 29
DHA
44.17
ꢀ
52.65
56.59
39.20
37.80
32.43
ꢀ
25.93
ꢀ
44.42
###
###
100.22
68.81
106.75
###
###
###
###
###
###
59.93
53.38
ꢀ
42.44
ꢀ
17.98
77.30
ꢀ
52.22
47.67
1.43
25.71
ꢀ
38.23
ꢀ
28.72
60.46
ꢀ
16.20
21.45
20.95
43.43
ꢀ
ꢀ
ꢀ
ꢀ
77.19
ꢀ
7.17
###
ꢀ
49.48
50.99
39.03
5.13
15.74
68.82
7.81
56.23
ꢀ
5.39
ART
DOX
11.99
6.36
DHAꢀ dihyroarteimisnin, ARTꢀ Arteimisinin, DOXꢀdoxorubicin. Data represents mean value of two independent evaluations in triplicates.***Comparison between DOX and
compounds. ### Comparison between DHA and synthesized derivatives
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nontoxic against the normal cell line kidney cells except compound 24 and DHA with IC50
values 106.75 and 77.19 ꢁM respectively. Compound 9 (IC50 range from 4.06 to 36.65 ꢁM)
and 17 (IC50 range from 7.16 to 28.21 ꢁM) were the most active, almost 1.5 to 28 times
active than parent compound DHA (IC50 range from 42.44 to 115.74 ꢁM). Compound 9 has
the highest activity against skin carcinoma cell line with IC50 4.06 ꢁM and 17 has the highest
activity against lung carcinoma with IC50 7.16 ꢁM therefore these compounds were selected
for further mechanistic evaluation.
Regarding the SAR of synthesized derivatives, in Series 1, compound 9 having
phenolic OH group at para position of benzene ring, exhibited the highest antiproliferative
activity as compared to the other derivatives. Introduction of OCH3, CN, NO2 group at para
position and COCH3 groups at three different positions (ortho, meta and para) led the
reduction in antiproliferative activity relative to that of compound 9, expect compound 15
having COCH3 group at meta position showed comparable activity against the PCꢀ3 and
COLOꢀ205 cell lines. Comparing ortho, meta and para isomers, 14, 15, and 16 respectively,
compound 14 was found to be the most active among them. Chalcones (17ꢀ22) with ortho and
para substitution on A ring are generally more active than with meta substituted compounds.
Compound 17 with ortho substitution at both the ring (A & B ring) is the most active. A
comparison of antiproliferative activity of 3ꢀhydroxydeoxydihydroartemisinin ether azole
derivatives (11a, 14a, 15a, 17aꢀ22a) with derivatives with peroxy functionality suggested
that compounds with intact endoperoxide bonds are more active than their deoxy counterparts
expect the compound 11a showed better activity than compound 11. Our finding showed the
strong agreement with the previous reports, where the anticancer activity of artemisinin
derivatives were attributed to the presence of endoperoxide linkage and the absence of
endoperoxide bond does not completely abolish the anticancer activity but significantly
reduce it.6ꢀ9 Compound 11a might be follow peroxide independent mechanism. In Series 2,
monomers were found to be more potent than their dimers. Compounds 27, 28 and
intermediate 24 were more active than other derivatives.
2.3. Compound 9 and 17 arrests G2/M phase and induce apoptosis in A431 and A549
cell line
The effect of most active compound 9 on A431 and 17 on A549 have been studied by cell
cycle analysis using PI labelling which is used for pseudohypodiploid cells quantification.
Treatment of compound 9 with IC50 and double of IC50 for 24 h significantly altered the
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phases of cell cycle (Figure 7A). The percentage of subꢀdiploid cells was increased after the
treatment of compound 9 at both the tested concentration with the 6.1 fold and 5.6 fold
respectively in A431 cells. Simultaneous reduction of cells at G0/G1 phase was also observed
with compound 9 (1.8 to 2.0 fold) at both concentrations with respect to control but there is
no significant alteration observed in S phase. Compound 9 significantly increases the number
of cells at G2/M phase to 2.5 fold at 4.06 µM and 2.9 fold at 8.12 µM concentration
compared to control (Figure 7B). Likewise compound 17 in A549 cells at both the
concentration IC50 (7.16 µM) and double of IC50 (14.32 µM) increase subꢀdiploid cells 3.4
fold and 6.8 fold respectively. Compound 17 decrease the cells 2.23ꢀ2.27 fold in G0/G1phase
at both the concentration with compare to control whereas no significant alteration was found
in Sꢀphase (Figure 8A). Compound 17 significantly increase to 1.6 fold with 7.16 µM and 1.2
fold with 14.32 µM compared to control at G2/M phase (Figure 8B). Interestingly, at IC50
(7.16 µM) concentration, compound 17 significantly arrest the G2/M phase with increase in
apoptosis whereas at double of IC50 (14.32 µM), cells are arrested in G2/M phase but
decrease in the apoptotic cells. From the results, we can conclude that the compounds 9 and
17 inhibited the cell proliferation via the increase in apoptosis and cell cycle arrest at G2/M
phase. Artemisinin compounds have been reported to exert both the cytostatic and cytotoxic
effects on cancer cells and induced growth arrest in cell cycle phases (A1, A2).19,40 Our
findings are similar with the Yao et al., 2008 and Jiao et al., 2007, who have reported that
DHA arrest the cell cycle at G2/M phase against the leukemia (K562 cells) and ovarian
cancer cells.41,42 In another report, 200 µM DHA induced apoptosis in leukemia cells.21
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Figure 7. Effect of compound 9 on cell cycle in A431 cell line. Cells were treated with
compound 9 at 4.06 µM (IC50) and 8.16 µM (double of IC50) concentrations for 24 h. Cells
were collected and stained with Propidium iodide (PI) for flow cytometric analysis. (A)
Represent the pictorial graphs obtained from cell cycle analysis using FACS Diva software
(B) Represents percent population in different phase of cell cycle after the treatment with
compound 9. The result is one representative example of two separate experiments.
*Comparison of test with control. **p<0.05
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Figure 8. Cell cycle analysis of compound 17 in A549 cell line. Cells were treated with
compound 17 at 7.16 µM (IC50) and 14.32 µM (double of IC50) concentrations for 24 h. Cells
were collected and stained with Propidium iodide (PI) for flow cytometric analysis. (A)
Represent the pictorial graphs obtained from cell cycle analysis using FACS Diva software
(B) Represents percent population in different phase of cell cycle after the treatment with
compound 17. The result is one representative example of two separate experiments.
*Comparison of test with control.
Figure 9. Compound 9 induced ROS generation in skin carcinoma (A431). (A) Represents
the images obtained during recordings of DCF fluorescence intensity reflecting the ROS
level. (B) Represents percent population of DCF positive cells shifted towards green channel
and nonꢀDCF positive cells. The result is one representative example of two separate
experiments. *Comparison of test with control.
2.4. Compound 9 and 17 induced ROS formation in A431 and A549 cell line
ROS involved in various stages in the apoptotic pathway such as caspase activation, release
of mitochondrial apoptogenic factors, DNA damage etc.43,44 Therefore we further investigate
whether compound 9 in A31 and Compound 17 in A549 exert its apoptotic effect by inducing
the ROS, the ROS levels was measured using DCFDA dye. In flow cytometric analysis, the
ROS level at IC50 (4.06 µM) and double of IC50 (8.12 µM) of compound 9 was significantly
increased which indicated by number of DCF positive cells (Figure 9A & B). The DCF
positive cells were 28.7% and 45.2% at 4.06 µM and 8.12 µM respectively with respect to
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control (14.3%). Similarly Compound 17 in A549 cells at both the concentration IC50
(7.16µM) and double of IC50 (14.32 µM) increased ROS level 23.2% and 38.8% respectively
in compare to control (15.4%, Figure 10A & B). Mercer et al., 2007 and 2011 reported that
reactive oxygen species and free radicals (especially highly alkylated carbon centered free
radical) released from iron activated artemisinin which leads to cell alterations.9,13 Results
showed that both the compounds 9 and 17 are able to induce the ROS production therefore
might be responsible for apoptosis. Previous reports also in accordance to our findings, Xu et
al., 2011 & Bostwick et al., 2000 reported that increased oxidative stress due to ROS
production associated with the artemisinin cytotoxicity and might be responsible for the
selective action of artemisinin on cancer cells.14,45
Figure 10. Compound 17 induced ROS generation in lung cancer cells (A549). (A)
Represents the images obtained during recordings of DCF fluorescence intensity reflecting
the ROS level. (B) Represents percent population of DCF positive cells shifted towards green
channel and nonꢀDCF positive cells. The result is one representative example of two separate
experiments. *Comparison of test with control
2.5. Compound 9 and 17 were nonꢀtoxic to human erythrocytes
The toxicity of the both the potent compounds 9 and 17 were also determined with
erythrocyte osmotic fragility evaluation (Figure 11). Both the compounds 9 and 17 were
found to be nonꢀtoxic to human erythrocytes even at higher concentrations (100 µg/ml) with
lower MEF50 0.57 and 0.55 respectively with respect to PDT (MEF50 0.68; Table 2).
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Figure 11. Erythrocyte osmotic fragility curve of Artemisnin derivatives compound 9 and 17,
Curcumin and Podophyllotoxin (PDT).
Table 2. Mean Erythrocyte Fragility (MEF) of active derivatives of Arteimisinin
(Compound 9 & 17)
Samples
Concentration
100µg/ml
100µg/ml
ꢀ
MEF50
0.57
0.55
Compound 9
Compound 17
Control
0.58
100µg/ml
100µg/ml
0.67
0.51
Podophyllotoxin
Curcumin
3. Conclusion
In conclusion, two novel series of artemisininꢀ1,2,3ꢀtriazole based derivatives have been
synthesized and explored as potent antiproliferative agents. The compound 9 and 17
exhibited potent antiproliferative activity. Both the compounds induced apoptosis along with
the cell cycle arrest. The active compounds 9 and 17 are also able to induce ROS in tested
cell lines which suggested that the inhibition cellular proliferation by compound 9 and 17
possibly linked with their ROS generation. Interestingly, both the compounds were nonꢀtoxic
for normal kidney cells (HEKꢀ293). Additionally, Erythrocyte osmotic fragility assay
confirms the nonꢀtoxic nature of both the compounds 9 and 17 at higher concentration of 100
µg/ml. This study may contribute to further design and development of potential artemisininꢀ
triazole based derivatives in future with higher therapeutic index. Since toxicity is the major
concern for existing anticancer drugs and our results suggested that compounds 9 and 17 are
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non toxic at higher concentration so these compounds could be taken up as a lead for further
evaluation and validation against skin and lung cancer.
4. Experimental:
4.1. General materials & methods
Melting points were recorded on a Toshniwal melting point apparatus and are uncorrected.
NMR spectra were recorded on a Bruker Avance, 500 MHz instrument using CDCl3, Acetoneꢀ
d6 as a solvent and TMS as an internal standard. The chemical shift values (δ) are determined in
ppm and coupling constants values are reported in Hz. HRMS spectra were recorded on Agilent
6545 QꢀTOF LC/MS mass spectrometer. TLC analyses were carried out on Merck
aluminum sheet TLC plates using silica gel coated with flourescent indicator F254 plates. The
compounds on TLC plates were visualized by vanillin–sulfuric acid spraying reagent. Reaction
products were purified by column chromatography using 60ꢀ120 mesh size Silicaꢀgel. The
1
structures of synthesized compounds were established by their H, 13C, DEPTꢀ135 and COSY
NMR spectroscopic analysis. All the chemicals and dried solvents used for the reaction work
were purchased from Sigma Aldrich India Ltd and Meck India Ltd and used without further
purification. DMEM (Dulbecco’s modified essential eagle medium), RPMIꢀ1640() and FBS
(fetal bovine serum) were purchased from Gibco, India. MTT dye, trypsinꢀEDTA, Propidium
iodide (PI), 2',7'ꢀdichlorofluorescin diacetate (DCFDA), antibiotic/antimycotic (Ab/Am)
Sodium diꢀhydrogen phosphate (NaH2PO4), Disodium hydrogen phosphate (Na2HPO4) and
phosphate buffer saline (PBS) were obtained from Sigma Aldrich, India Ltd. Sodium
bicarbonate, Heparin HEPES were acquired from Himedia, India. Solvents such as Dimethyl
sulphoxide (DMSO), ethanol, formaldehyde, isopropanol were purchased from Merck, India
Ltd.
4.2. Synthesis of 12ꢀβꢀ(prop 2 yn 1 yloxy)ꢀdihydroartemisinin
To a solution of DHA (5 gram, 17.59 mmol) in dry Et2O at 0 ºC temperature was added 1.97
gram, 35.18 mmol of propargyl alcohol and 8 ml of BF3.OEt2 dropwise. The reaction mixture
was stirred at same temperature for 6 hrs. After completion of reaction, the reaction mixture
was washed with 5 % NaHCO3 solution, then water and brine solution, dried over anhydrous
Na2SO4. The organic layer was concentrated in vaccum. The residue was purified by column
chromatography using 60ꢀ120 mesh size silica gels as absorbent.
12ꢀβꢀ(prop 2 yn 1 yloxy)ꢀdihydroartemisinin: White crystalline solid; yield 82%; mp
1
95ꢀ97 ºC; H NMR (500 MHz, CDCl3): δ=5.40 (1H, s, Hꢀ5), 4.96 (1H, d, J=3.5 Hz, Hꢀ12),
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4.30 (1H, d, J=1.5 Hz, Hꢀ1b’), 4.29 (1H, d, J=1.5 Hz, Hꢀ1a’), 2.65 (1H, m, Hꢀ11), 2.38 (1H,
s, Hꢀ3’), 1.43 (3H, s, H3ꢀ15), 0.93 (3H, d, J=6.5 Hz, H3ꢀ14), 0.92 (3H, d, J=2.5 Hz, H3ꢀ13);
13C NMR (125 MHz, CDCl3): δ=104.16 (Cꢀ4), 100.61 (Cꢀ12), 88.07 (Cꢀ5), 81.06 (Cꢀ6),
79.77 (Cꢀ2’), 73.97 (Cꢀ3’), 54.96 (Cꢀ1’), 52.54 (Cꢀ1), 44.34 (Cꢀ7), 37.41 (Cꢀ10), 36.40 (Cꢀ3),
34.61 (Cꢀ9), 30.59 (Cꢀ11), 26.14 (Cꢀ15), 24.68 (Cꢀ2), 24.45 (Cꢀ8), 20.34 (Cꢀ14), 12.81 (Cꢀ
13).
4.3. General procedure for synthesis of aromatic azides, 8aꢀh
7.40 mmol of aromatic amines were dissolved in 5 ml of concentrated HCl and 5 ml of
CH3COOH solution and the reaction mixture was kept at 0ºC. Then the solution of 11.10
mmol of sodium nitrite (NaNO2) dissolved in 5 ml water was added dropwise into the
reaction mixture. A reddish brown coloured gas was evolved. The reaction mixture was
stirred for 30 minutes at 0ºC. Then 14.8 mmol of Sodium azide (NaN3) dissolved in 5ml of
water was added into the reaction mixture dropwise. After that reaction mixture was stirred
for 2ꢀ4 hrs at room temp. After completion of reaction, the reaction mixture was extracted
with dichloromethane and the combined organic layer was washed with water, then brine
solution and dried over anhydrous sodium sulphate. The crude product was obtained almost
as a single product and used in further step without purification.
4.4. General procedure for the synthesis of different substituted azido chalcones, 8iꢀ8n:
For the synthesis of different substituted azidochalcones, 6.21 mmol of 2, 3 & 4 substituted
azido acetophenones were dissolved in 10 ml of methanol at room temperature and 6.83
mmol of 2 or 4ꢀsubstituted trifluoromethyl benzaldehyde was added into the reaction mixture.
Then 62.10 mmol of KOH was added and the reaction mixture was stirred for 2ꢀ4 hrs. After
completion of reaction, the resulting mixture was poured into ice –cold water and acidified by
addition of 1 N HCl. The crude product was extracted with dichloromethane (50 ml x 3) and
the organic layer was washed with cold water, dried over anhydrous Na2SO4 and concentrated
in vacuo. The residue was chromatographed on 60ꢀ120 mesh size silica gels to get the desired
products for further reaction.
Synthesis of chalcones with 4ꢀtrifluoromethyl benzaldehyde gave different side product so in
that case chalcones were formed by the Clasien Schmidt reaction of different amino
substituted acetophenones with 4ꢀtrifluoromethyl benzaldehydes followed by the
diazotisation and then azide formation of the product as the procedure described for the
synthesis of aromatic azides.
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4.5. General procedure for the synthesis of Series 1 type of artemisininꢀazole
derivatives, 9ꢀ22
A mixture of Compound 6 (0.621 mmol), appropriate aromatic azide (8aꢀ8i, 0.931 mmol) and
hexadecyltrimethylammonium bromide (0.310 mmol) was taken in dichloromethane (5ml)
and stirred for 5 min at room temperature. CuSO4.5H2O (0.0621 mmol, 10 mol%) and
sodium ascorbate (0.156 mmol, 25 mol%) were dissolved in 3 ml of water and added to the
reaction mixture. The resulting solution was stirred for 1ꢀ2 hrs at room temperature. After
completion of reaction, the reaction mixture was extracted with dichloromethane (25ml x 3
times) and organic layer was washed with water, dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The crude product was purified on silica gel (60ꢀ120
mesh size) column chromatography using EtOAc/Hexane as eluent to afford the target
compounds (comp 9 to 22). For the synthesis of 3αꢀhydroxydeoxydihydroartemisinin
derivatives (11a, 14a, 15a, 17aꢀ22a), the same procedure as described above was followed
except 0.310 mmol of CuSO4.5H2O and 0.776 mmol of sodium ascorbate were used.
4ꢀ(4ꢀ(12ꢀβꢀdihydroartemisinoxymethyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀyl)phenol (9): White solid;
1
yield 66%; mp 125ꢀ128 ºC; H NMR (500 MHz, CDCl3): δ=7.84 (1H, s, Hꢀ3’), 7.56 (2H, m,
Hꢀ5’, Hꢀ9’), 7.01 (2H, m, Hꢀ6’, Hꢀ8’), 6.72 (1H, brs, OH, D2O exchangeable), 5.46 (1H, s, Hꢀ
5), 4.99 (1H, d, J=12.5 Hz, Hꢀ1b’), 4.95 (1H, d, J=3.5 Hz, Hꢀ12), 4.74 (1H, d, J=12.5 Hz, Hꢀ
1a’), 2.67 (1H, m, Hꢀ11), 1.45 (3H, s, H3ꢀ15), 0.92 (1H, d, J=6.5 Hz, H3ꢀ14), 0.90 (3H, d,
J=7.5 Hz, Hꢀ13).13C NMR (125 MHz, CDCl3): δ=156.75 (Cꢀ7’), 145.34 (Cꢀ2’), 130.25 (Cꢀ
4’), 122.49 (Cꢀ5’, Cꢀ9’), 121.13 (Cꢀ3’), 116.42 (Cꢀ6’, Cꢀ8’), 104.25 (Cꢀ4), 101.74 (Cꢀ12),
88.06 (Cꢀ5), 81.17 (Cꢀ6), 61.51 (Cꢀ1’), 52.54 (Cꢀ1), 44.39 (Cꢀ7), 37.36 (Cꢀ10), 36.43 (Cꢀ3),
34.56 (Cꢀ9), 30.87 (Cꢀ11), 26.14 (Cꢀ15), 24.68 (Cꢀ2), 24.47 (Cꢀ8), 20.32 (Cꢀ14), 12.99 (Cꢀ
13); HRMS (ESI) calcd for C24H31N3O6 [M + H]+ 458.2291, found 458.2289.
4ꢀ(12ꢀβꢀdihydroartemisinoxymethyl)ꢀ1ꢀ(4ꢀmethoxyphenyl)ꢀ1Hꢀ1,2,3ꢀtriazole (10): Light
1
brown solid; yield 67%; mp 84ꢀ86 ºC; H NMR (500 MHz, CDCl3): δ=7.85 (1H, s, Hꢀ3’),
7.60 (2H, d, J=8.5 Hz, Hꢀ5’, Hꢀ9’), 7.00 (2H, d, J=8.5 Hz, Hꢀ6’, Hꢀ8’), 5.43 (1H, s, Hꢀ5),
4.98 (1H, d, J=12.5 Hz, Hꢀ1b’), 4.95 (1H, d, J=3.5 Hz, Hꢀ12), 4.73 (1H, d, J=12.5 Hz, Hꢀ1a’),
3.85 (3H, s, OCH3), 2.67 (1H, m, Hꢀ11), 1.44 (3H, s, H3ꢀ15), 0.91 (6H, m, H3ꢀ13, H3ꢀ14); 13C
NMR (125 MHz, CDCl3): δ=159.82 (Cꢀ7’), 145.54 (Cꢀ2’), 130.57 (Cꢀ4’), 122.23 ( Cꢀ5’, Cꢀ
9’), 120.96 (Cꢀ3’), 114.77 (Cꢀ6’, Cꢀ8’), 104.15 (Cꢀ4), 101.81 (Cꢀ12), 88.01 (Cꢀ5), 81.13 (Cꢀ
6), 61.71 (Cꢀ1’), 55.64 (OCH3), 52.55 (Cꢀ1), 44.41 (Cꢀ7), 37.37 (Cꢀ10), 36.43 (Cꢀ3), 34.58
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(Cꢀ9), 30.87 (Cꢀ11), 26.17 (Cꢀ15), 24.68 (Cꢀ2), 24.47 (Cꢀ8), 20.32 (Cꢀ14), 13.01 (Cꢀ13);
HRMS (ESI) calcd for C25H33N3O6 [M+H]+ 472.2448, found 472.2439.
4ꢀ(12ꢀβꢀdihydroartemisinoxymethyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀyl)benzonitrile (11): White solid;
1
yield 68%; mp 159ꢀ161 ºC; H NMR (500 MHz, CDCl3): δ=8.03 (1H, s, Hꢀ3’), 7.92 (2H, d,
J=8.5 Hz, Hꢀ6’, Hꢀ8’), 7.84 (2H, d, J=8.5 Hz, Hꢀ5’, Hꢀ9’), 5.42 (1H, s, Hꢀ5), 5.00 (1H, d,
J=12.5 Hz, Hꢀ1b’), 4.96 (1H, d, J=3.5 Hz, Hꢀ12), 4.77 (1H, d, J=12.5 Hz, Hꢀ1a’), 2.68 (1H,
m, Hꢀ11), 1.47 (3H, s, H3ꢀ15), 0.92 (6H, m, H3ꢀ13, H3ꢀ14); 13C NMR (125 MHZ, CDCl3):
δ=146.60 (Cꢀ2’), 139.86 (Cꢀ4’), 133.94 (Cꢀ6’, Cꢀ8’), 120.60 (Cꢀ3’), 120.50 ( Cꢀ5’, Cꢀ9’),
117.70 (CN), 112.42 (Cꢀ7’), 104.24 (Cꢀ4), 102.04 (Cꢀ12), 88.03 (Cꢀ5), 81.09 (Cꢀ6), 61.57 (Cꢀ
1’), 52.49 (Cꢀ1), 44.33 (Cꢀ7), 37.40 (Cꢀ10), 36.39 (Cꢀ3), 34.54 (Cꢀ9), 30.85 (Cꢀ11), 26.17 (Cꢀ
15), 24.68 (Cꢀ2), 24.48 (Cꢀ8), 20.32 (Cꢀ14), 13.01 (Cꢀ13); HRMS (ESI) calcd for
C25H30N4O5 [M+H]+ 467.2294, found 467.2275.
4ꢀ(12ꢀβꢀ(3αꢀhydroxyꢀdihydroartemisinoxy)methyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀyl)benzonitrile
1
(11a): White solid; yield 64%; mp 178ꢀ180 ºC; H NMR (500 MHz, CDCl3): δ=7.99 (1H, s,
Hꢀ3’), 7.91 (2H, dd, J=8.5, 2.0 Hz, Hꢀ6’, Hꢀ8’), 7.83 (2H, dd, J=8.5, 2.0 Hz, Hꢀ5’, Hꢀ9’), 5.31
(1H, s, Hꢀ5), 5.00 (1H, d, J=12.5 Hz, Hꢀ1b’), 4.93 (1H, d, J=4.5 Hz, Hꢀ12), 4.72 (1H, d,
J=12.5 Hz, Hꢀ1a’), 3.57 (1H, m, Hꢀ3), 2.49 (1H, m, Hꢀ11), 1.34 (3H, s, H3ꢀ15), 0.94 (3H, d,
J=6.5 Hz, H3ꢀ14), 0.87 (3H, d, J=7.5 Hz, H3ꢀ13); 13C NMR (125 MHz, CDCl3): δ=146.70 (Cꢀ
2’), 139.83 (Cꢀ4’), 133.95 (Cꢀ6’, Cꢀ8’), 120.56 (Cꢀ5’, Cꢀ9’), 120.23 (Cꢀ3’), 117.69 (CN),
112.43 (Cꢀ7’), 108.07 (Cꢀ4), 99.55 (Cꢀ12), 93.90 (Cꢀ5), 84.12 (Cꢀ6), 69.58 (Cꢀ3), 61.49 (Cꢀ
1’), 42.43 (Cꢀ1), 40.63 (Cꢀ7), 34.88 (Cꢀ10), 34.69 (Cꢀ9), 30.34 (Cꢀ2, Cꢀ11), 25.06 (Cꢀ8),
20.98 (Cꢀ14), 18.81 (Cꢀ15), 12.29 (Cꢀ13); HRMS (ESI) calcd for C25H30N4O5 [M+H]+
467.2294, found 467.2293.
4ꢀ(12ꢀβꢀdihydroartemisinoxymethyl)ꢀ1ꢀ(4ꢀnitrophenyl)ꢀ1Hꢀ1,2,3ꢀtriazole (12): Yellow
1
solid; yield 67%; mp 128ꢀ130 ºC; H NMR (500 MHz, Acetone d6): δ=8.76 (1H, s, Hꢀ3’),
8.47 (2H, dd, J=7.0, 1.2 Hz, Hꢀ5’, Hꢀ9’), 8.24 (2H, dd, J=7.0, 1.2 Hz, Hꢀ6’, Hꢀ8’), 5.47 (1H,
s, Hꢀ5), 4.97 (1H, d, J=12.5 Hz, Hꢀ1b’), 4.92 (1H, d, J=3.5 Hz, Hꢀ12), 4.73 (1H, d, J=12.5
Hz, Hꢀ1a’), 2.65 (1H, m, Hꢀ11), 1.33 (3H, s, H3ꢀ15), 0.92 (3H, d, J=6.5 Hz, H3ꢀ14), 0.89 (3H,
d, J=7.5 Hz, H3ꢀ13); 13C NMR (125 MHz, Acetone d6): δ=148.23 (Cꢀ7’), 147.36 (Cꢀ2’),
142.51 (Cꢀ2’), 126.39 (Cꢀ6’, Cꢀ8’), 122.84 (Cꢀ3’), 121.64 ( Cꢀ5’, Cꢀ9’), 104.57 (Cꢀ4), 102.06
(Cꢀ12), 88.64 (Cꢀ5), 81.62 (Cꢀ6), 61.54 (Cꢀ1’), 53.65 (Cꢀ1), 45.50 (Cꢀ7), 38.15 (Cꢀ10), 37.25
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(Cꢀ3), 35.53 (Cꢀ9), 31.83 (Cꢀ11), 26.30 (Cꢀ15), 25.63 (Cꢀ2), 25.31 (Cꢀ8), 20.69 (Cꢀ14), 13.32
(Cꢀ13); HRMS (ESI) calcd for C24H30N4O7 [M+H]+ 487.2193, found 487.2196.
4ꢀ(4ꢀ(12ꢀβꢀdihydroartemisinoxymethyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀyl)picolinonitrile (13): Brown
solid; yield 69%; mp 140ꢀ142 ºC; 1H NMR (500 MHz, CDCl3): δ=9.14 (1H, d, J=3.5 Hz, Hꢀ
5’), 8.34 (1H, dd, J=8.5, 2.5 Hz, Hꢀ8’), 8.12 (1H, s, Hꢀ3’), 7.90 (1H, d, J=8.5 Hz, Hꢀ9’), 5.40
(1H, s, Hꢀ5), 5.00 (1H, d, J=12.5 Hz, Hꢀ1b’), 4.92 (1H, d, J=3.5 Hz, Hꢀ12), 4.81 (1H, d,
J=12.5 Hz, Hꢀ1a’), 2.68 (1H, m, Hꢀ11), 1.46 (3H, s, H3ꢀ15), 0.92 (6H, m, H3ꢀ13, H3ꢀ14); 13C
NMR (125 MHz, CDCl3): δ=147.21 (Cꢀ2’), 142.25 (Cꢀ7’), 135.33 (Cꢀ4’), 133.28 (Cꢀ6’),
129.32 (Cꢀ8’), 128.08 ( Cꢀ5’), 120.56 (Cꢀ3’), 116.33 (CN), 104.27 (Cꢀ4), 102.31 (Cꢀ12),
88.05 (Cꢀ5), 81.05 (Cꢀ6), 61.65 (Cꢀ1’), 52.48 (Cꢀ1), 44.30 (Cꢀ7), 37.42 (Cꢀ10), 36.37 (Cꢀ3),
34.53 (Cꢀ9), 30.87 (Cꢀ11), 26.14 (Cꢀ15), 24.67 (Cꢀ2), 24.48 (Cꢀ8), 20.29 (Cꢀ14), 12.98 (Cꢀ
13); HRMS (ESI) calcd for C24H29N5O5 [M + H]+ 468.2247, found 468.2242.
1ꢀ(2ꢀ(4ꢀ(12ꢀβꢀdihydroartemisinoxymethyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀyl)phenyl)ethanone (14):
1
Yellow solid; yield 65%; mp 40ꢀ42 ºC; H NMR (500 MHz, CDCl3): δ= 7.82 (1H, s, Hꢀ3’),
7.70 (1H, dd, J=7.5, 1.5 Hz, Hꢀ6’), 7.64 (1H, dd, J=7.5, 1.5 Hz, Hꢀ8’), 7.58 (1H, dd, J=7.5,
1.5 Hz, Hꢀ7’), 7.48 (1H, dd, J=7.5, 1.5 Hz, Hꢀ9’), 5.43 (1H, s, Hꢀ5), 5.01 (1H, d, J=12.5 Hz,
Hꢀ1b’), 4.97 (1H, d, J=3.5 Hz, Hꢀ12), 4.78 (1H, d, J=12.5 Hz, Hꢀ1a’), 2.66 (1H, m, Hꢀ11),
2.17 (3H, s, COCH3), 1.44 (3H, s, H3ꢀ15), 0.92 (3H, d, J=6.5 Hz, H3ꢀ14), 0.90 (3H, d, J=7.5
Hz, H3ꢀ13); 13C NMR (125 MHz, CDCl3): δ=199.52 (Cꢀ10), 145.90 (Cꢀ2’), 136.44 (Cꢀ4’),
134.49 (Cꢀ5’), 131.87 (Cꢀ8’), 129.88 (Cꢀ7’), 128.97 (Cꢀ6’), 125.68 (Cꢀ9’), 124.05 (Cꢀ3’),
104.17 (Cꢀ4), 101.85 (Cꢀ12), 88.02 (Cꢀ5), 81.12 (Cꢀ6), 61.58 (Cꢀ1’), 52.56 (Cꢀ1), 44.39 (Cꢀ
7), 37.37 (Cꢀ10), 36.43 (Cꢀ3), 34.59 (Cꢀ9), 30.85 (Cꢀ11), 29.18 (COCH3), 26.16 (Cꢀ15),
24.67 (Cꢀ2), 24.47 (Cꢀ8), 20.32 (Cꢀ14), 13.00 (Cꢀ13). HRMS (ESI) calcd for C26H33N3O6
[M+H]+ 484.2448, found 484.2450.
1ꢀ(2ꢀ(4ꢀ(12ꢀβꢀ(3αꢀhydroxyꢀdihydroartemisinoxy)methyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀ
1
yl)phenyl)ethanone (14a): Brown viscous liquid; yield 62%; H NMR (500 MHz, CDCl3):
δ=7.80 (1H, s, Hꢀ3’), 7.69 (1H, dd, J=7.5, 1.5 Hz, Hꢀ6’), 7.61 (1H, dd, J=7.5, 1.5 Hz, Hꢀ8’),
7.58 (1H, dd, J=7.5, 1.5 Hz, Hꢀ7’), 7.46 (1H, dd, J=7.5, 1.5 Hz, Hꢀ9’), 5.29 (1H, s, Hꢀ5), 4.97
(1H, d, J=12.5 Hz, Hꢀ1b’), 4.92 (1H, d, J=4.5 Hz, Hꢀ12), 4.73 (1H, d, J=12.5 Hz, Hꢀ1a’), 3.56
(1H, d, J=2.5 Hz, Hꢀ3), 2.52 (1H, m, Hꢀ11), 2.16 (3H, s, COCH3), 1.55 (3H, s, H3ꢀ15), 0.92
(3H, d, J=7.5 Hz, H3ꢀ14), 0.85 (3H, d, J=6.5 Hz, H3ꢀ13); 13C NMR (125 MHz, CDCl3):
δ=199.52 (C=O), 145.99 (Cꢀ2’), 136.42 (Cꢀ4’), 134.46 ( Cꢀ5’), 131.89 (Cꢀ8’), 129.89 (Cꢀ7’),
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128.94 (Cꢀ6’), 125.72 (Cꢀ9’), 123.99 (Cꢀ3’), 108.04 (Cꢀ4), 99.83 (Cꢀ12), 93.73 (Cꢀ5), 84.20
(Cꢀ6), 69.61 (Cꢀ3), 61.62 (Cꢀ1’), 42.54 (Cꢀ1), 40.68 (Cꢀ7), 34.77 (Cꢀ10), 34.74 (Cꢀ9), 30.35
(Cꢀ2), 30.32 (Cꢀ11), 29.18 (COCH3), 25.02 (Cꢀ8), 21.02 (Cꢀ14), 18.81 (Cꢀ15), 12.38 (Cꢀ13);
HRMS (ESI) calcd for C26H33N3O6 [M+H]+ 484.2448, found 484.2441.
1ꢀ(3ꢀ(4ꢀ(12ꢀβꢀdihydroartemisinoxymethyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀyl)phenyl)ethanone (15):
Light brown solid; yield 66%; mp 115ꢀ118 ºC; 1H NMR (500 MHz, CDCl3): δ= 8.29 (1H, dd,
J=2.4, 1.2 Hz, Hꢀ5’), 8.04 (1H, s, Hꢀ3’), 8.02 (2H, m, Hꢀ7’, Hꢀ9’), 7.65 (1H, t, J=8.0 Hz, Hꢀ
8’), 5.44 (1H, s, Hꢀ5), 5.01 (1H, d, J=13.0 Hz, Hꢀ1b’), 4.98 (1H, d, J=3.5 Hz, Hꢀ12), 4.77
(1H, d, J=13.0 Hz, Hꢀ1a’), 2.69 (1H, m, Hꢀ11), 2.68 (3H, s, COCH3), 1.46 (3H, s, H3ꢀ15),
0.92 (6H, m, H3ꢀ13, H3ꢀ14); 13C NMR (125 MHz, CDCl3): δ=196.66 (Cꢀ10), 146.18 (Cꢀ2’),
138.55 (Cꢀ6’), 137.53 (Cꢀ4’), 130.23 (Cꢀ8’), 128.43 (Cꢀ7’), 124.85 (Cꢀ9’), 120.83 (Cꢀ3’),
119.75 ( Cꢀ5’), 104.23 (Cꢀ4), 102.05 (Cꢀ12), 88.03 (Cꢀ5), 81.12 (Cꢀ6), 61.74 (Cꢀ1’), 52.54
(Cꢀ1), 44.39 (Cꢀ7), 37.39 (Cꢀ10), 36.42 (Cꢀ3), 34.56 (Cꢀ9), 30.88 (Cꢀ11), 26.76 (Cꢀ15,
COCH3), 24.69 (Cꢀ2), 24.49 (Cꢀ8), 20.32 (Cꢀ14), 13.02 (Cꢀ13); HRMS (ESI) calcd for
C26H33N3O6 [M+H]+ 484.2448, found 484.2446.
1ꢀ(3ꢀ(4ꢀ(12ꢀβꢀ(3αꢀhydroxyꢀdihydroartemisinoxy)methyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀ
1
yl)phenyl)ethanone (15a): Brown solid; yield 63%; mp 90ꢀ92 ºC; H NMR (500 MHz,
CDCl3): δ=8.26 (1H, s, Hꢀ3’), 8.01 (1H, s, Hꢀ5’), 7.99 (2H, dd, J=8.0, 1.5 Hz, Hꢀ7’, Hꢀ9’),
7.63 (1H, dd, J=6.5, 6.5 Hz, Hꢀ8’), 5.31 (1H, s, Hꢀ5), 4.98 (1H, d, J=12.5 Hz, Hꢀ1b’), 4.92
(1H, d, J=4.0 Hz, Hꢀ12), 4.70 (1H, d, J=12.5 Hz, Hꢀ1a’), 3.56 (1H, d, J=1.5 Hz, Hꢀ3), 2.52
(1H, m, Hꢀ11), 1.59 (3H, s, H3ꢀ15), 0.92 (6H, m, H3ꢀ13, H3ꢀ14); 13C NMR (125 MHz,
CDCl3): δ=196.74 (C=O), 146.20 (Cꢀ2’), 138.51 (Cꢀ6’), 137.46 (Cꢀ4’), 130.25 (Cꢀ8’), 128.43
(Cꢀ7’), 124.79 (Cꢀ9’), 120.65 (Cꢀ3’), 119.70 ( Cꢀ5’), 108.05 (Cꢀ4), 99.54 (Cꢀ12), 93.85 (Cꢀ5),
84.14 (Cꢀ6), 69.57 (Cꢀ3), 61.55 (Cꢀ1’), 42.44 (Cꢀ1), 40.65 (Cꢀ7), 34.84 (Cꢀ10), 34.69 (Cꢀ9),
30.35 (Cꢀ2, Cꢀ11), 26.75 (COCH3), 25.05 (Cꢀ8), 20.98 (Cꢀ14), 18.80 (Cꢀ15), 12.32 (Cꢀ13);
HRMS (ESI) calcd for C26H33N3O6 [M+H]+ 484.2448, found 484.2451.
1ꢀ(4ꢀ(4ꢀ(12ꢀβꢀdihydroartemisinoxymethyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀyl)phenyl)ethanone (16):
1
Light brown solid; yield 71%; mp 135ꢀ137 ºC; H NMR (500 MHz, CDCl3): δ=8.10 (2H, d,
J=8.5 Hz, Hꢀ6’, Hꢀ8’), 8.01 (1H, s, Hꢀ3’), 7.85 (2H, d, J=8.5 Hz, Hꢀ5’, Hꢀ9’), 5.41 (1H, s, Hꢀ
5), 4.99 (1H, d, J=12.5 Hz, Hꢀ1b’), 4.95 (1H, d, J=3.5 Hz, Hꢀ12), 4.75 (1H, d, J=12.5 Hz, Hꢀ
1a’), 2.65 (1H, m, Hꢀ11), 2.63 (3H, s, COCH3), 1.41 (3H, s, H3ꢀ15), 0.90 (6H, m, H3ꢀ13, H3ꢀ
14); 13C NMR (125 MHz, CDCl3): δ=196.55 (C=O), 146.26 (Cꢀ2’), 140.12 (Cꢀ7’), 136.85
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(Cꢀ4’), 130.10 (Cꢀ6’, Cꢀ8’), 120.63 (Cꢀ3’), 120.08 ( Cꢀ5’, Cꢀ9’), 104.21 (Cꢀ4), 102.00 (Cꢀ12),
88.03 (Cꢀ5), 81.11 (Cꢀ6), 61.64 (Cꢀ1’), 52.52 (Cꢀ1), 44.37 (Cꢀ7), 37.39 (Cꢀ10), 36.41 (Cꢀ3),
34.56 (Cꢀ9), 30.87 (Cꢀ11), 26.68 (COCH3), 26.17 (Cꢀ15), 24.68 (Cꢀ2), 24.48 (Cꢀ8), 20.32 (Cꢀ
14), 13.01 (Cꢀ13); HRMS (ESI) calcd for C26H33N3O6 [M+H]+ 484.2448, found 484.2441.
(E)ꢀ1ꢀ(2ꢀ(4ꢀ(12ꢀβꢀdihydroartemisinoxymethyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀyl)phenyl)ꢀ3ꢀ(2ꢀ
1
(trifluoromethyl)phenyl)propꢀ2ꢀenꢀ1ꢀone (17): Yellow solid; yield 64%; mp 95ꢀ97 ºC; H
NMR (500 MHz, CDCl3): δ= 7.77 (1H, s, Hꢀ3’), 7.72 (1H, m, Hꢀ6’), 7.68 (1H, dd, J=7.5, 1.5
Hz, Hꢀ15’, Hꢀ18’), 7.63 (1H, d, J=16.0 Hz, Hꢀ12’), 7.59 (1H, m, Hꢀ17’), 7.58 (1H, dd, J=8.0,
1.5 Hz, Hꢀ8’), 7.54 (1H, m, Hꢀ16’), 7.45 (2H, m, Hꢀ7’, Hꢀ9’), 6.53 (1H, d, J=16.0 Hz, Hꢀ11’),
5.37 (1H, s, Hꢀ5), 4.93 (1H, d, J=12.5 Hz, Hꢀ1b’), 4.85 (1H, d, J=3.5 Hz, Hꢀ12), 4.64 (1H, d,
J=12.5 Hz, Hꢀ1a’), 2.58 (1H, m, Hꢀ11), 1.43 (3H, s, H3ꢀ15), 0.90 (3H, d, J=6.5 Hz, H3ꢀ14),
0.80 (3H, d, J=7.0 Hz, H3ꢀ13); 13C NMR (125 MHZ, CDCl3): δ=192.34 (C=O), 146.08 (Cꢀ
2’), 140.56 (Cꢀ12’), 134.92, 134.80 (Cꢀ4’/Cꢀ13’), 132.92 (Cꢀ5’), 132.25, 132.00 (Cꢀ8’/Cꢀ
17’), 130.04 (Cꢀ6’), 129.80, 129.78, 129.09 (Cꢀ9’/Cꢀ16’/Cꢀ18’), 127.91 (Cꢀ15’), 126.20,
126.15 (Cꢀ7’/Cꢀ11’), 125.15 (CF3), 124.15 (Cꢀ3’), 104.14 (Cꢀ4), 101.82 (Cꢀ12), 87.95 (Cꢀ5),
81.04 (Cꢀ6), 61.66 (Cꢀ1’), 52.52 (Cꢀ1), 44.32 (Cꢀ7), 37.32 (Cꢀ10), 36.41 (Cꢀ3), 34.51 (Cꢀ9),
30.73 (Cꢀ11), 26.14 (Cꢀ15), 24.65 (Cꢀ2), 24.38 (Cꢀ8), 20.30 (Cꢀ14), 12.86 (Cꢀ13); HRMS
(ESI) calcd for C34H36F3N3O6 [M+H]+ 640.2634, found 640.2646.
(E)ꢀ1ꢀ(2ꢀ(4ꢀ(12ꢀβꢀ(3αꢀhydroxyꢀdihydroartemisinoxy)methyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀ
yl)phenyl)ꢀ3ꢀ(2ꢀ(trifluoromethyl)phenyl)propꢀ2ꢀenꢀ1ꢀone (17a): Yellow solid; yield 58%;
mp 53ꢀ55 ºC; 1H NMR (500 MHz, CDCl3): δ=7.75 (1H, s, Hꢀ3’), 7.72 (1H, dd, J=8.0, 1.5 Hz,
Hꢀ5’), 7.68 (2H, dd, J=8.0, 1.5 Hz, Hꢀ15’, Hꢀ18’), 7.64 (1H, d, J=16.0 Hz, Hꢀ12’), 7.63 (1H,
dd, J=7.5, 1.0 Hz, Hꢀ17’), 7.59 (1H, dd, J=8.0, 1.0 Hz, Hꢀ8’), 7.52 (1H, m, Hꢀ16’), 7.45 (2H,
m, Hꢀ7’, Hꢀ9’), 6.53 (1H, d, J=16.0 Hz, Hꢀ11’), 5.24 (1H, s, Hꢀ5), 4.90 (1H, d, J=12.5 Hz,
Hꢀ1b’), 4.82 (1H, d, J=4.0 Hz, Hꢀ12), 4.60 (d, J=12.5 Hz, Hꢀ1a’), 3.55 (1H, br s, Hꢀ3), 2.47
(1H, m, Hꢀ11), 1.57 (3H, s, H3ꢀ15), 0.83 (6H, m, H3ꢀ13, H3ꢀ14); 13C NMR (125 MHz,
CDCl3): δ=192.29 (C=O), 146.15 (Cꢀ2’), 140.63 (Cꢀ12’), 134.93, 134.85 (Cꢀ4’/Cꢀ13’),
132.96 (Cꢀ5’), 132.21, 132.00 (Cꢀ8’/Cꢀ17’), 130.02 (Cꢀ6’), 129.77 (Cꢀ16’), 129.07 (Cꢀ9’, Cꢀ
18’), 127.93 (Cꢀ15’), 126.18, 126.13 (Cꢀ7’/Cꢀ11’), 125.20 (CF3), 124.16 (Cꢀ3’), 108.03 (Cꢀ
4), 99.83 (Cꢀ12), 93.65 (Cꢀ5), 84.16 (Cꢀ6), 69.60 (Cꢀ3), 61.66 (Cꢀ1’), 42.53 (Cꢀ1), 40.63 (Cꢀ
7), 34.77 (Cꢀ10), 34.68 (Cꢀ9), 30.31 (Cꢀ2), 30.23 (Cꢀ11), 24.96 (Cꢀ8), 20.99 (Cꢀ14), 18.79
(Cꢀ15), 12.25 (Cꢀ13); HRMS (ESI) calcd for C34H36F3N3O6 [M+H]+ 640.2634, found
640.2635.
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(E)ꢀ1ꢀ(2ꢀ(4ꢀ(12ꢀβꢀdihydroartemisinoxymethyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀyl)phenyl)ꢀ3ꢀ(4ꢀ
(trifluoromethyl)phenyl)propꢀ2ꢀenꢀ1ꢀone (18): Yellow solid; yield 61%; mp 124ꢀ126 ºC;
1H NMR (500 MHz, CDCl3): δ=7.76 (1H, s, Hꢀ3’), 7.71 (2H, t, J=8.0 Hz, Hꢀ15’, Hꢀ16’), 7.63
(2H, t, J=8.0 Hz, Hꢀ7’, Hꢀ9’), 7.59 (2H, t, J=8.0 Hz, Hꢀ14’, Hꢀ18’), 7.45 (2H, d, J=7.0 Hz, Hꢀ
15’, Hꢀ17’), 7.35 (1H, d, J=16.0 Hz, Hꢀ12’), 6.60 (1H, d, J=16.0 Hz, Hꢀ11’), 5.37 (1H, s, Hꢀ
5), 4.92 (1H, d, J=12.5 Hz, Hꢀ1b’), 4.84 (1H, d, J=3.0 Hz, Hꢀ12), 4.64 (1H, d, J=12.5 Hz, Hꢀ
1a’), 2.58 (1H, m, Hꢀ11), 1.43 (3H, s, H3ꢀ15), 0.92 (3H, d, J=6.5 Hz, H3ꢀ14), 0.75 (3H, d,
J=7.5 Hz, H3ꢀ13); 13C NMR (125 MHz, CDCl3): δ=192.30 (Cꢀ10’), 146.00 (Cꢀ2’), 143.76 (Cꢀ
12’), 137.40 (Cꢀ13’), 135.15 (Cꢀ4’), 134.90 ( Cꢀ5’, Cꢀ16’), 132.05 (Cꢀ8’), 129.96, 129.73,
129.61 (Cꢀ6’/Cꢀ14’/Cꢀ18’), 128.52 (Cꢀ15’, Cꢀ17’), 126.97 (Cꢀ7’), 125.94, 125.91, 125.68 (Cꢀ
9’/Cꢀ11’/CF3), 124.40 (Cꢀ3’), 104.17 (Cꢀ4), 101.63 (Cꢀ12), 87.95 (Cꢀ5), 81.03 (Cꢀ6), 61.42
(Cꢀ1’), 52.49 (Cꢀ1), 44.35 (Cꢀ7), 37.36 (Cꢀ10), 36.39 (Cꢀ3), 34.53 (Cꢀ9), 30.70 (Cꢀ11), 26.13
(Cꢀ15), 24.65 (Cꢀ2), 24.40 (Cꢀ8), 20.32 (Cꢀ14), 12.87 (Cꢀ13); HRMS (ESI) calcd for
C34H36F3N3O6 [M+H]+ 640.2634, found 640.2632.
(E)ꢀ1ꢀ(2ꢀ(4ꢀ(12ꢀβꢀ(3αꢀhydroxyꢀdihydroartemisinoxy)methyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀ
yl)phenyl)ꢀ3ꢀ(4ꢀ(trifluoromethyl)phenyl)propꢀ2ꢀenꢀ1ꢀone (18a): Yellow solid; yield 59%;
1
mp 68ꢀ70 ºC; H NMR (500 MHz, CDCl3): δ=7.75 (1H, s, Hꢀ3’), 7.70 (2H, m, Hꢀ6’, Hꢀ8’),
7.63 (2H, m, Hꢀ7’, Hꢀ9’), 7.58 (2H, d, J=8.0 Hz, Hꢀ14’, Hꢀ18’), 7.45 (2H, d, J=8.0 Hz, Hꢀ15’,
Hꢀ17’), 7.34 (1H, d, J=16.0 Hz, Hꢀ12’), 6.60 (1H, d, J=16.0 Hz, Hꢀ11’), 5.24 (1H, s, Hꢀ5),
4.88 (1H, d, J=13.0 Hz, Hꢀ1b’), 4.82 (1H, d, J=4.0 Hz, Hꢀ12), 4.60 (1H, d, J=13.0 Hz, Hꢀ1a’),
3.55 (1H, brs, Hꢀ3), 2.39 (1H, m, Hꢀ11), 1.55 (3H, s, H3ꢀ15), 0.84 (3H, d, J=6.5 Hz, H3ꢀ14),
0.80 (3H, d, J=7.5 Hz, H3ꢀ13); 13C NMR (125 MHz, CDCl3): δ=192.24 (C=O), 146.12 (Cꢀ2’),
143.69 (Cꢀ12’), 137.44 (Cꢀ13’), 135.18 (Cꢀ4’), 134.91 (Cꢀ5’, Cꢀ16’), 132.03 (Cꢀ8’), 129.94
(Cꢀ6’), 129.60 (Cꢀ14’, Cꢀ18’), 128.50 (Cꢀ15’, Cꢀ17’), 127.01 (Cꢀ7’), 125.90 (Cꢀ11’), 125.87
(CF3), 125.64 (Cꢀ9’), 124.33 (Cꢀ3’), 108.02 (Cꢀ4), 99.58 (Cꢀ12), 93.75 (Cꢀ5), 84.10 (Cꢀ6),
69.59 (Cꢀ3), 61.51 (Cꢀ1’), 42.46 (Cꢀ1), 40.61 (Cꢀ7), 34.80 (Cꢀ10), 34.68 (Cꢀ9), 30.32 (Cꢀ2),
30.24 (Cꢀ11), 24.96 (Cꢀ8), 20.96 (Cꢀ14), 18.79 (Cꢀ15), 12.19 (Cꢀ13); HRMS (ESI) calcd for
C34H36F3N3O6 [M+H]+ 640.2634, found 640.2635.
(E)ꢀ1ꢀ(3ꢀ(4ꢀ(12ꢀβꢀdihydroartemisinoxymethyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀyl)phenyl)ꢀ3ꢀ(2ꢀ
1
(trifluoromethyl)phenyl)propꢀ2ꢀenꢀ1ꢀone (19): Yellow solid; yield 62%; mp 90ꢀ92 ºC; H
NMR (500 MHz, CDCl3): δ=8.34 (2H, dd, J=2.0, 1.5 Hz, Hꢀ5’), 8.19 (1H, dd, J=15.5, 2.0 Hz,
Hꢀ12’), 8.07 (1H, brs, Hꢀ3’), 8.05 (2H, dd, J=7.5, 1.5 Hz, Hꢀ15’, Hꢀ18’), 7.86 (1H, d, J=7.5
Hz, Hꢀ8’), 7.74 (2H, d, J=7.5 Hz, Hꢀ7’), 7.69 (1H, m, Hꢀ17’), 7.63 (1H, m, Hꢀ9’), 7.52 (1H,
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m, Hꢀ16’), 7.45 (1H, d, J=15.5 Hz, Hꢀ11’), 5.45 (1H, s, Hꢀ5), 5.03 (1H, d, J=13.0 Hz, Hꢀ1b’),
4.98 (1H, d, J=3.5 Hz, Hꢀ12), 4.77 (1H, d, J=13.0 Hz, Hꢀ1a’), 2.68 (1H, m, Hꢀ11), 1.47 (3H,
s, H3ꢀ15), 0.92 (6H, m, H3ꢀ13, H3ꢀ14); 13C NMR (125 MHz, CDCl3): δ=188.89 (C=O),
146.20 (Cꢀ2’), 141.41 (Cꢀ12’), 139.19 (Cꢀ6’), 137.58 (Cꢀ4’, Cꢀ13’), 133.56 (Cꢀ14’), 132.24
(Cꢀ17’), 130.30 (Cꢀ8’), 128.64, 128.06 (Cꢀ16’/Cꢀ18’), 126.41, 126.36 (Cꢀ9’/Cꢀ15’), 124.74
(Cꢀ7’, CF3), 120.84, 120.22 (Cꢀ3’/Cꢀ5’/Cꢀ11’), 104.23 (Cꢀ4), 102.02 (Cꢀ12), 88.03 (Cꢀ5),
81.13 (Cꢀ6), 61.73 (Cꢀ1’), 52.53 (Cꢀ1), 44.37 (Cꢀ7), 37.38 (Cꢀ10), 36.40 (Cꢀ3), 34.55 (Cꢀ9),
30.88 (Cꢀ11), 26.16 (Cꢀ15), 24.68 (Cꢀ2), 24.49 (Cꢀ8), 20.33 (Cꢀ14), 13.04 (Cꢀ13); HRMS
(ESI) calcd for C34H36F3N3O6 [M+H]+ 640.2634, found 640.2630.
(E)ꢀ1ꢀ(3ꢀ(4ꢀ(12ꢀβꢀ(3αꢀhydroxyꢀdihydroartemisinoxy)methyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀ
yl)phenyl)ꢀ3ꢀ(2ꢀ(trifluoromethyl)phenyl)propꢀ2ꢀenꢀ1ꢀone (19a):Yellow solid; yield 58%;
1
mp 48ꢀ50 ºC; H NMR (500 MHz, CDCl3): δ= 8.31 (1H, dd, J=2.0, 1.5 Hz, Hꢀ5’), 8.17 (1H,
d, J=15.5 Hz, Hꢀ12’), 8.05 (1H, dd, 8.0, 2.5 Hz, Hꢀ15’), 8.04 (1H, s, Hꢀ3’), 8.02 (1H, dd,
J=8.0, 2.5 Hz, Hꢀ18’), 7.84 (1H, d, J=7.5 Hz, Hꢀ8’), 7.72 (1H, d, J=8.0 Hz, Hꢀ7’), 7.67 (1H,
m, Hꢀ17’), 7.62 (1H, dd, J=7.5, 2.1 Hz, Hꢀ9’), 7.52 (1H, m, Hꢀ16’), 7.44 (1H, d, J=15.5 Hz,
Hꢀ11’), 5.31(1H, s, Hꢀ5), 4.99 (1H, d, J=12.5 Hz, Hꢀ1b’), 4.92 (1H, d, J=4.0 Hz, Hꢀ12), 4.70
(1H, d, J=12.5 Hz, Hꢀ1a’), 3.19 (1H, brs, Hꢀ3), 2.53 (1H, m, Hꢀ11), 1.52 (3H, s, H3ꢀ15), 0.91
(3H, d, J=7.5 Hz, H3ꢀ14), 0.83 (3H, d, J=6.0 Hz, H3ꢀ13); 13C NMR (125 MHz, CDCl3):
δ=188.84 (C=O), 146.23 (Cꢀ2’), 141.36 (Cꢀ12’), 139.15 (Cꢀ6’), 137.52 (Cꢀ4’, Cꢀ13’), 133.57
(Cꢀ14’), 132.55 (Cꢀ17’), 130.95 (Cꢀ8’), 128.61, 128.05 (Cꢀ16’/Cꢀ18’), 126.36, 126.33 (Cꢀ
9’/Cꢀ15’), 125.68 (Cꢀ7’), 124.68 (CF3), 122.83 (Cꢀ5’), 120.65 (Cꢀ11’), 120.15 (Cꢀ3’), 108.05
(Cꢀ4), 99.53 (Cꢀ12), 93.84 (Cꢀ5), 84.13 (Cꢀ6), 69.54 (Cꢀ3), 61.07 (Cꢀ1’), 40.65 (Cꢀ7), 42.43
(Cꢀ1), 34.84 (Cꢀ10), 34.69 (Cꢀ9), 30.34 (Cꢀ11), 30.37 (Cꢀ2), 24.83 (Cꢀ8), 20.47 (Cꢀ14), 18.60
(Cꢀ15), 12.32 (Cꢀ13); HRMS (ESI) calcd for C34H36F3N3O6 [M+H]+ 640.2634, found
640.2629.
(E)ꢀ1ꢀ(3ꢀ(4ꢀ(12ꢀβꢀdihydroartemisinoxymethyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀyl)phenyl)ꢀ3ꢀ(4ꢀ
1
(trifluoromethyl)phenyl)propꢀ2ꢀenꢀ1ꢀone (20): Yellow solid; yield 58%; mp 69ꢀ71 ºC; H
NMR (500 MHz, CDCl3): δ=8.38 (1H, t, J=1.7 Hz, Hꢀ3’), 8.09 (2H, m, Hꢀ7’, Hꢀ9’), 8.03
(2H, dd, J=2.5 Hz, 2.0, Hꢀ5’), 7.77 (2H, d, J=8.0 Hz, Hꢀ15’, Hꢀ17’), 7.70 (2H, d, J=8.0 Hz,
Hꢀ14’, Hꢀ18’), 7.69 (1H, d, J=7.0 Hz, Hꢀ8’), 7.67 (1H, d, J=15.5 Hz, Hꢀ12’), 7.61 (1H, d,
J=15.5 Hz, Hꢀ11’), 5.45 (1H, s, Hꢀ5), 5.01 (1H, d, J=13.0 Hz, Hꢀ1b’), 4.97 (1H, d, J=3.5 Hz,
Hꢀ12), 4.78 (1H, d, J=13.0 Hz, Hꢀ1a’), 2.65 (1H, m, Hꢀ11), 1.47 (3H, s, H3ꢀ15), 0.92 (6H, m,
H3ꢀ13, H3ꢀ14); 13C NMR (125 MHz, CDCl3): δ=188.59 (C=O), 146.20 (Cꢀ2’), 143.99 (Cꢀ
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12’), 139.34 (Cꢀ6’), 137.87, 137.63 (Cꢀ4’/Cꢀ13’), 132.44 (Cꢀ16’), 130.33 (Cꢀ8’), 128.73 (Cꢀ
14’, Cꢀ18’), 128.48 (Cꢀ7’), 126.04, 126.01 (Cꢀ17’/Cꢀ15’), 125.98, 124.74 (Cꢀ9’/CF3), 123.39
(Cꢀ3’), 120.88, 120.18 (Cꢀ5’/Cꢀ11’), 104.23 (Cꢀ4), 101.99 (Cꢀ12), 88.03 (Cꢀ5), 81.12 (Cꢀ6),
61.66 (Cꢀ1’), 52.54 (Cꢀ1), 44.38 (Cꢀ7), 37.39 (Cꢀ10), 36.41 (Cꢀ3), 34.56 (Cꢀ9), 30.88 (Cꢀ11),
26.16 (Cꢀ15), 24.69 (Cꢀ2), 24.50 (Cꢀ8), 20.32 (Cꢀ14), 13.03 (Cꢀ13); HRMS (ESI) calcd for
C34H36F3N3O6 [M+H]+ 640.2634, found 640.2635.
(E)ꢀ1ꢀ(3ꢀ(4ꢀ(12ꢀβꢀ(3αꢀhydroxyꢀdihydroartemisinoxy)methyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀ
yl)phenyl)ꢀ3ꢀ(4ꢀ(trifluoromethyl)phenyl)propꢀ2ꢀenꢀ1ꢀone (20a): Yellow solid; yield 55%;
1
mp 52ꢀ54 ºC; H NMR (500 MHz, CDCl3): δ= 8.37 (1H, dd, J=2.0, 1.5 Hz, Hꢀ5’), 8.04 (1H,
brs, Hꢀ3’), 8.02 (2H, dd, J=8.0, 1.2 Hz, Hꢀ7’, Hꢀ8’), 7.89 (1H, d, J=15.5 Hz, Hꢀ12’), 7.77
(2H, d, J=8.0 Hz, Hꢀ14’, Hꢀ18’), 7.70 (2H, d, J=8.0 Hz, Hꢀ15’, Hꢀ17’), 7.62 (1H, d, J=15.5
Hz, Hꢀ11’), 5.33 (1H, s, Hꢀ5), 5.02 (1H, d, J=12.5 Hz, Hꢀ1b’), 4.95 (1H, d, J=4.5 Hz, Hꢀ12),
4.74 (1H, d, J=12.5 Hz, Hꢀ1a’), 3.55 (1H, m, Hꢀ3), 2.52 (1H, m, Hꢀ11), 1.55 (3H, s, H3ꢀ15),
0.96 (3H, d, J=7.5 Hz, H3ꢀ14), 0.87 (3H, d, J=6.5 Hz, H3ꢀ13). 13C NMR (125 MHz, CDCl3):
δ=188.58 (C=O), 146.26 (Cꢀ2’), 144.01 (Cꢀ12’), 139.32 (Cꢀ6’), 137.87, 137.59 (Cꢀ4’/Cꢀ13’),
132.44 (Cꢀ16’), 130.34 (Cꢀ8’), 128.73, 128.48 (Cꢀ14’/Cꢀ18’), 126.03, 126.00 (Cꢀ7’/Cꢀ15’/Cꢀ
17’), 124.85, 124.69 (Cꢀ9’/CF3), 123.37 (Cꢀ3’), 120.66, 120.13 ( Cꢀ5’/Cꢀ11’), 108.06 (Cꢀ4),
99.52 (Cꢀ12), 93.88 (Cꢀ5), 84.16 (Cꢀ6), 69.60 (Cꢀ3), 61.55 (Cꢀ1’), 42.46 (Cꢀ1), 40.67 (Cꢀ7),
34.86 (Cꢀ10), 34.71 (Cꢀ9), 30.37 (Cꢀ2), 30.35 (Cꢀ11), 25.07 (Cꢀ8), 20.98 (Cꢀ14), 18.80 (Cꢀ
15), 12.32 (Cꢀ13); HRMS (ESI) calcd for C34H36F3N3O6 [M+H]+ 640.2634, found 640.2632.
(E)ꢀ1ꢀ(4ꢀ(12ꢀβꢀdihydroartemisinoxymethyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀyl)phenyl)ꢀ3ꢀ(2ꢀ
1
(trifluoromethyl)phenyl)propꢀ2ꢀenꢀ1ꢀone (21): Yellow solid; yield 65%; mp 93ꢀ95 ºC; H
NMR (500 MHz, CDCl3): δ=8.16 (3H, m, Hꢀ6’, Hꢀ8’, Hꢀ12’), 8.07 (1H, s, Hꢀ3’), 7.91 (2H,
dd, J=7.5, 1.2 Hz, Hꢀ5’, Hꢀ9’), 7.84 (1H, m, Hꢀ15’), 7.73 (1H, dd, J=7.5, 1.5 Hz, Hꢀ18’), 7.62
(1H, m, Hꢀ17’), 7.52 (1H, m, Hꢀ16’), 7.44 (1H, d, J=15.5 Hz, Hꢀ11’), 5.44 (1H, s, Hꢀ5), 5.01
(1H, d, J=12.5 Hz, Hꢀ1b’), 4.97 (1H, d, J=3.5 Hz, Hꢀ12), 4.78 (1H, d, J=12.5 Hz, Hꢀ1a’), 2.66
(1H, m, Hꢀ11), 1.45 (3H, s, H3ꢀ15), 0.91 (6H, m, H3ꢀ13, H3ꢀ14); 13C NMR (125 MHZ,
CDCl3): δ=188.83 (Cꢀ10), 146.27 (Cꢀ2’), 141.05 (Cꢀ12’), 140.06 (Cꢀ7’), 137.41 (Cꢀ4’),
133.69 (Cꢀ13’), 132.20 (Cꢀ17’), 130.62 (Cꢀ16’), 130.47 (Cꢀ6’, Cꢀ8’), 130.01 (Cꢀ18’), 128.53
(Cꢀ14’), 128.00 (Cꢀ15’), 126.35 (CF3), 120.71, 120.17 (Cꢀ3’/Cꢀ11’), 120.66 ( Cꢀ5’, Cꢀ9’),
104.21 (Cꢀ4), 102.02 (Cꢀ12), 88.03 (Cꢀ5), 81.12 (Cꢀ6), 61.65 (Cꢀ1’), 52.52 (Cꢀ1), 44.37 (Cꢀ
7), 37.39 (Cꢀ10), 36.41 (Cꢀ3), 34.56 (Cꢀ9), 30.88 (Cꢀ11), 26.17 (Cꢀ15), 24.67 (Cꢀ2), 24.49
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(Cꢀ8), 20.31 (Cꢀ14), 13.01 (Cꢀ13); HRMS (ESI) calcd for C34H36F3N3O6 [M+H]+ 640.2634,
found 640.2629.
(E)ꢀ1ꢀ(4ꢀ(12ꢀβꢀ(3αꢀhydroxyꢀdihydroartemisinoxy)methyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀyl)phenyl)ꢀ
3ꢀ(2ꢀ(trifluoromethyl)phenyl)propꢀ2ꢀenꢀ1ꢀone (21a): Orange solid; yield 62%; mp 48ꢀ50
ºC; 1H NMR (500 MHz, CDCl3): δ=8.15 (3H, m, Hꢀ6’, Hꢀ8’, Hꢀ12’), 8.04 (1H, s, Hꢀ3’), 7.90
(2H, dd, J=7.5, 1.2 Hz, Hꢀ5’, Hꢀ9’), 7.83 (2H, d, J=7.5 Hz, Hꢀ15’, Hꢀ18’), 7.73 (1H, m, Hꢀ
17’), 7.52 (1H, m, Hꢀ16’), 7.41 (1H, d, J=15.5 Hz, Hꢀ11’), 5.31 (1H, s, Hꢀ5), 4.94 (1H, m, Hꢀ
1b’), 4.93 (1H, d, J=3.0 Hz, Hꢀ12), 4.73 (1H, brs, Hꢀ1a’), 3.57 (1H, br s, Hꢀ3), 2.49 (1H, m,
Hꢀ11), 1.52 (3H, s, H3ꢀ15), 0.92 (6H, m, H3ꢀ13, H3ꢀ14); 13C NMR (125 MHz, CDCl3):
δ=188.81 (C=O), 146.33 (Cꢀ2’), 141.05 (Cꢀ12’), 140.01 (Cꢀ7’), 137.42 (Cꢀ4’), 133.69 (Cꢀ13’,
Cꢀ14’), 132.20 (Cꢀ17’), 130.62 (Cꢀ16’), 130.47 (Cꢀ6’, Cꢀ8’), 130.01 (Cꢀ18’), 127.99 (Cꢀ15’),
125.92 (CF3), 120.13 ( Cꢀ5’, Cꢀ9’), 120.49, 119.86 (Cꢀ3’/Cꢀ11’), 108.06 (Cꢀ4), 99.50 (Cꢀ12),
93.88 (Cꢀ5), 84.13 (Cꢀ6), 69.55 (Cꢀ3), 61.47 (Cꢀ1’), 42.41 (Cꢀ1), 40.64 (Cꢀ7), 34.86 (Cꢀ10),
34.69 (Cꢀ9), 30.35 (Cꢀ2), 30.34 (Cꢀ11), 25.05 (Cꢀ8), 20.98 (Cꢀ14), 18.80 (Cꢀ15), 12.31 (Cꢀ
13); HRMS (ESI) calcd for C34H36F3N3O6 [M+H]+ 640.2634, found 640.2635.
(E)ꢀ1ꢀ(4ꢀ(4ꢀ(12ꢀβꢀdihydroartemisinoxymethyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀyl)phenyl)ꢀ3ꢀ(4ꢀ
(trifluoromethyl)phenyl)propꢀ2ꢀenꢀ1ꢀone (22): Yellow solid; yield 67%; mp 139ꢀ141 ºC;
1H NMR (500 MHz, CDCl3): δ=8.20 (2H, dd, J=7.0, 2.0 Hz, Hꢀ6’, Hꢀ8’), 8.07 (1H, s, Hꢀ3’),
7.94 (2H, dd, J=7.0, 2.0 Hz, Hꢀ5’, Hꢀ9’), 7.86 (1H, d, J=15.5 Hz, Hꢀ12’), 7.77 (2H, d, J=8.5
Hz, Hꢀ15’, Hꢀ17’), 7.69 (2H, d, J=8.5 Hz, Hꢀ14’, Hꢀ18’), 7.61 (1H, d, J=15.5, Hꢀ11’), 5.45
(1H, s, Hꢀ5), 5.02 (1H, d, J=13.0 Hz, Hꢀ1b’), 4.98 (1H, d, J=3.5 Hz, Hꢀ12), 4.78 (1H, d,
J=13.0 Hz, Hꢀ1a’), 2.66 (1H, m, Hꢀ11), 1.46 (3H, s, H3ꢀ15), 0.92 (6H, m, H3ꢀ13, H3ꢀ14); 13C
NMR (125 MHZ, CDCl3): δ=188.43 (Cꢀ10’), 146.29 (Cꢀ2’), 143.63 (Cꢀ12’), 140.12 (Cꢀ7’),
137.99, 137.54 (Cꢀ4’/Cꢀ13’), 132.35 (Cꢀ16’), 130.36 (Cꢀ6’, Cꢀ8’), 128.66 (Cꢀ14’, Cꢀ18’),
126.03, 126.00 (Cꢀ15’/Cꢀ17’), 123.52 (CF3), 120.67, 120.08 (Cꢀ3’/Cꢀ11’), 120.23 ( Cꢀ5’, Cꢀ
9’), 104.23 (Cꢀ4), 101.98 (Cꢀ12), 88.04 (Cꢀ5), 81.12 (Cꢀ6), 61.61 (Cꢀ1’), 52.51 (Cꢀ1), 44.36
(Cꢀ7), 37.40 (Cꢀ10), 36.41 (Cꢀ3), 34.56 (Cꢀ9), 30.87 (Cꢀ11), 26.19 (Cꢀ15), 24.68 (Cꢀ2), 24.50
(Cꢀ8), 20.33 (Cꢀ14), 13.03 (Cꢀ13); HRMS (ESI) calcd for C34H36F3N3O6 [M+H]+ 640.2634,
found 640.2631.
(E)ꢀ1ꢀ(4ꢀ(4ꢀ(12ꢀβꢀ(3αꢀhydroxyꢀdihydroartemisinoxy)methyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ1ꢀ
yl)phenyl)ꢀ3ꢀ(4ꢀ(trifluoromethyl)phenyl)propꢀ2ꢀenꢀ1ꢀone (22a): Yellow solid; yield 65%;
1
mp 110ꢀ112 ºC; H NMR (500 MHz, CDCl3): δ=8.20 (2H, dd, J=7.0, 2.0 Hz, Hꢀ6’, Hꢀ8’),
29