The application of tandem aza-wittig reaction to synthesize artemisinin-guanidine hybrids and their anti-tumor activity

Article abstract of DOI:10.1002/ardp.201000363

Three series of novel artemisinin-guanidine hybrids 4a-4f, 8a-8h and 9a-9h have been facilely synthesized via four-component reaction (aza-Wittig reaction) and evaluated for their anti-tumor activities against A549, HT-29 and MDA-MB-231 cell lines in vitro. All of the tested compounds showed enhanced anti-tumor activities with IC₅₀ values ranging from 0.02 μM to 12.0 μM as compared to DHA (dihydroartemisinin). Among them, artemisinin derived dimers, compounds 9b (IC₅₀ = 0.05 μM), 9d (IC₅₀ = 0.06 μM) and 9f (IC₅₀ = 0.02 μM) were found to be most active against HT29 cells. An efficient method to generate three series of novel artemisinin derivates using multi-component reaction was developed. From the preliminary results we can conclude that artemisinin guanidine hybrids may be promising candidates for new anticancer agents. Copyright

Full text of DOI:10.1002/ardp.201000363

Arch. Pharm. Chem. Life Sci. 2011, 344, 631–638
631
Full Paper
The Application of Tandem Aza-Wittig Reaction to
Synthesize Artemisinin–Guanidine Hybrids and
Their Anti-Tumor Activity
Lijun Xie, Yanfang Zhao, Xin Zhai, Peng Li, Chun Liu, Yangxiong Li, and Ping Gong
Key Laboratory of Original New Drugs Design and Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical University, P.R. China
Three series of novel artemisinin–guanidine hybrids 4a–4f, 8a–8h and 9a–9h have been facilely
synthesized via four-component reaction (aza-Wittig reaction) and evaluated for their anti-tumor
activities against A549, HT-29 and MDA-MB-231 cell lines in vitro. All of the tested compounds showed
enhanced anti-tumor activities with IC50 values ranging from 0.02 mM to 12.0 mM as compared to DHA
(dihydroartemisinin). Among them, artemisinin derived dimers, compounds 9b (IC50 ¼ 0.05 mM), 9d
(IC50 ¼ 0.06 mM) and 9f (IC50 ¼ 0.02 mM) were found to be most active against HT29 cells.
Keywords: Anti-tumor activity / Artemisinin / aza-Wittig reaction / Guanidine
Received: November 25, 2010; Revised: February 22, 2011; Accepted: March 2, 2011
DOI 10.1002/ardp.201000363
Introduction
these reported compounds are used in practice because of
their low-efficiency in vivo. Thus, it is still necessary to search
and discover novel water-soluble artemisinin derivatives with
better anti-tumor activity. The guanidine group, a small and
water-soluble backbone, has been an attractive pharmaco-
logical scaffold present in several anti-tumor drugs [15–17].
It was worthwhile that guanidine moiety was introduced as
water solubilizing functional group into artemisinin nucleus
via aza-Wittig reaction using hybrid approach. These novel
artemisinin analogs may possess excellent anti-tumor
activity with increasing bioavailability in terms of water
solubility or stability.
Despite the large number of anticancer drugs available for
medical use, the treatment of cancer remains a challenging
therapeutic problem because of the several side effects of
traditional chemotherapy and the emergence of resistance
associated with the molecularly targeted anti-tumor drugs
[1]. In order to develop novel more effective and reliable anti-
tumor agents that circumvent these limitations, the search
for novel anti-tumor agents has turned to natural sources, in
particular plants used in traditional folk medicines [2].
Artemisinin, a sesquiterpene endoperoxide isolated from
Artemisia annua L., is being widely used to treat malaria [3].
Recently, artemisinin and its derivatives also showed potent
and broad anti-tumor properties in various human cancer
cell lines [4–9]. Despite their obvious value, however, the
chemotherapeutic value of them is limited to a great extent
by their low solubility or short plasma half-life. To overcome
these shortcomings, many metabolically stable or water-
soluble artemisinin derivatives were prepared by replace-
ment of oxygen at C-10 with carbon or nitrogen, some of
them showed good anti-tumor activity against different
cancer cell lines in vitro [10–14]. Unfortunately, only few of
Prompted by these observations, we have designed three
series of artemisinin–guanidine hybrids which were facilely
synthesized via tandem aza-Wittig reaction (Fig. 1). The
target compounds 4a–4f were obtained by bonding
guanidine moiety into the artemisinin nucleus at the C-10
position directly, expected to be more stable toward metab-
olism process. The artemisinin analogs of 8a–8h were also
prepared to explore whether the length of the spacer
between artemisinin nucleus and guanidine moiety has an
effect on their activities, in which the guanidine group was
combined into the artemisinin nucleus through an oxyethyl-
flexible linkage. Moreover, literature data report that some
novel artemisinin-derived trioxane dimmer showed more
potent anti-tumor activity than corresponding monomer
[18], thus dimers 9a–9h were further synthesized through
an alkylguanidine linkage. All the prepared compounds were
Correspondence: Ping Gong, 103 Wenhua Road, Shenhe District,
110016 Shenyang, Liaoning, P.R. China.
E-mail: gongpinggp@126.com
Fax: þ86 24 2398-6429
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Arch. Pharm. Chem. Life Sci. 2011, 344, 631–638
indicated as 10b by the coupling constants (J ¼ 3.4 Hz)
between 9-H and 10-H and chemical shift of H-10
1
(4.77 ppm) in H-NMR.
The azido compound 6 was transformed to 2-(10b-dihy-
droartemisinoxy) ethyl amine 7 via Staudinger reduction.
Compounds 9a–9h were prepared using the same procedure
as synthesizing compounds 4a–4f by tandem aza-Wittig reac-
tion involving compound 6, the triphenyl phosphine, appro-
priately substituted isocyanatobenzene and compound 7.
Biological results and discussion
All the compounds 4a–4f, 8a–8h and 9a–9h were evaluated
for their anti-tumor activities on human non-small-cell lung
cancer cell line (A549), human colorectal cancer cell line
(HT-29) and human breast cancer cell line (MDA-MB-231) by
the MTT assay, using dihydroartemisinin (DHA) as positive
control. And the results expressed as IC50 were summarized
in Table 1. The IC50 values are the average of at least two
independent experiments.
Figure 1. Structure of artemisinin and the target compounds.
screened for their anti-tumor activities against A549, HT-29
and MDA-MB-231 cell lines in vitro.
As shown in Table 1, all of the tested compounds showed
moderate to excellent anti-tumor activities against three
cancer cell lines. In general, most of the prepared compounds
showed moderate activity against MDA-MB-231 cells at single-
digit mM range. Furthermore, all compounds, with the excep-
tion of 4a–4c and 8g, the activity of them against A549 were 2-
to 4-fold more potent than against MDA-MB-231 cells, which
displayed good selectivity for inhibition of A549 cell line.
Compounds 9a–9h exhibited excellent anti-tumor activities
against HT-29 cells with IC50 values in the tens-digit nM
range, which were 10- to 102-fold more active than those
of the corresponding compounds 4a–4f and 8a–8h.
Chemistry
The synthetic routes of target compounds 4a–4f, 8a–8h and 9a–
9h are illustrated in Scheme 1. 10b-Azidodihydroartemisinin 2
and 10a-azidodihydroartemisinin 3 were obtained by treat-
ing compound 1 with trimethylsilyl chloride and sodium
azide under the catalysis of potassium iodide at room
temperature for 28 h according to a modified Haynes’
method [14]. The a- and b-isomers appeared as two distinct
spots on TLC and were separated by column chromatography,
with b-isomers as the major products. Subsequently, the
target compounds 4a–4c with b at the C-10 position (10b)
were prepared by tandem aza-Wittig reaction of compound 2
[19], the triphenyl phosphine, substituted isocyanatobenzene
and appropriate secondary alkylamine. To investigate the
impact of the C-10 configuration of target compounds on
bioactivities, compounds 4d–4f with a at the C-10 position
(10a) were also prepared from compound 3 using the same
way as compounds 4a–4c. In our previous report, the stereo-
chemistry of compound 2 had already been determined by
single crystal X-ray diffraction studies [20]. The configuration
of compound 3 at the C-10 position was confirmed as 10a by
the H-9/H-10 coupling constant (J ¼ 10.1 Hz) and a chemical
shift H-10 (4.86 ppm), which was also consistent with data of
compounds 3 in related literature [11].
It was very interesting that compounds 4a–4e bearing a
same molecular structure but only different C-10 stereochem-
istry showed significant difference in inhibitory effect
against A549 cells. The anti-tumor activity of compounds
4d, 4e was 2–10 fold more active than those of related com-
pounds 4a–4c. This data demonstrated that the compounds
with 10b were favored over compounds with 10a on A549
cell line. In addition, it is worth to mention that the anti-
tumor potencies of compounds 8a–8h against different three
cancer cell lines were only comparative to those of com-
pounds 4a–4f. The results indicated that the introduction
of oxyethyl between artemisinin nucleus and guanidine
moiety has little impact on their anti-tumor activities.
The data in Table 1 indicated that dimers 9a–9h had excel-
lent anti-tumor activity to HT29 cells and good activity to
A549 cells but moderate activity to MDA-MB-231 cells, which
revealed selective toward HT29 cell type. In particular, three
of the compounds 9a–9h (9b, 9d and 9f) showed IC50 values in
the nM range against HT29 cells. It was worth noting that the
most promising compound, 9f (IC50 ¼ 0.24 mM, 0.02 mM,
2-(10b-Dihydroartemisinoxy) ethyl bromide 5 was prepared
according to a reported procedure in [21], followed by treat-
ing with sodium azide to give 2-(10b-dihydroartemisinoxy)
ethyl azide 6. Finally, the products 8a–8h were obtained from
6 in a similar manner as described for the preparation of 4a–
4f. The configuration of compound 5 at the C-10 position was
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Arch. Pharm. Chem. Life Sci. 2011, 344, 631–638
Novel Artemisinin-Guanidine Hybrids
633
Scheme 1. Synthesis of the target compounds.
1.40 mM), was 32.5-, 6.0 ꢀ 102- and 6.5- times more active
than DHA (IC50 ¼ 7.80 mM, 12 mM, 9.10 mM), respectively. As
expected, the point of view that artemisinin-derived trioxane
dimmer usually showed more potent anti-tumor activity
than corresponding monomer was further proved in this
paper.
screening, all of the targeted compounds were more potent
in anti-tumor activity than DHA, suggesting that the presence
of a guanidine moiety significantly enhanced their anti-
tumor activity to three cancer cell lines. Compounds 9b,
9d and 9f of dimers, were found to be most potent against
HT29 cell as compared to other compounds. From these
preliminary results we can conclude that artemisinin guani-
dine hybrids may be promising candidates for new anti-
cancer agents. Taking them as lead, further studies are
In summary, we have developed an efficient method to
generate three series of novel artemisinin derivates using
a four-component reaction. Through anti-tumor activity
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Table 1. Anti-tumor activity of the compounds against A549, HT-29 and MDA-MB-231 cell lines in vitro
Compd.
No.
IC50 (mM)a
MDA-MBA-231 b
Compd.
IC50 (mM)a
MDA-MBA-231 b
A549b
HT-29 b
No.
A549b
HT-29 b
4a
4b
4c
4d
4e
4f
8a
8b
8c
8d
8e
DHAd
1.20
6.53
3.18
0.56
0.13
0.60
n.d. c
0.59
0.58
1.30
0.45
7.80
5.35
6.81
4.24
1.92
1.61
2.0
2.64
1.00
1.10
1.48
1.52
12.0
0.77
3.04
4.70
6.30
n.d c
1.40
1.61
1.10
3.60
n.d. c
6.45
9.10
8f
8g
8h
9a
9b
9c
9d
9e
9f
0.65
1.34
0.69
0.72
0.64
0.17
0.68
0.36
0.24
n.d. c
0.98
3.93
n.d.c
1.36
0.10
0.05
0.24
0.06
0.40
0.02
0.09
0.10
1.05
0.83
2.03
1.17
2.08
n.d.c
1.18
1.57
1.42
1.33
1.26
9g
9h
a
IC50: Concentration of the compound (mM) producing 50% cell growth inhibition after 72 h of drug exposure, as determined by the
MTT assay. Each experiment was run at least two times, and the results are presented as average values.b A549, non-small-cell lung
adenocarcinoma cell line; HT-29, human colon cancer cell line; MDA-MB-231, human breast cancer cell line.c n.d.: not
determined.d Used as a positive control.
Compound 2 as white solid: M.p.: 101–1038C; MS (ESI) m/z:
332.2 (M þ Na)þ; 1H-NMR (300 MHz, DMSO-d6) d: 5.76 (s, 1H), 5.54
(d, J ¼ 3.6 Hz, 1H), 2.46 (m, 1H), 2.26–2.12 (m, 1H), 2.07–1.96
(m, 1H), 1.87–1.78 (m, 1H), 1.77–1.65 (m, 1H), 1.58 (m,2H), 1.48–
1.33 (m, 3H), 1.31 (s, 3H), 1.16 (m, 1H), 0.89 (d, J ¼ 6.3 Hz, 3H),
0.84 (d, J ¼ 7.3 Hz, 3H).
currently underway for establishing a library that includes
more diastereomers and finding better analogues as potent
anticancer agents.
Experimental
Compound 3: M.p.: 41–438C; MS (ESI) m/z: 332.2 (M þ Na)þ;
1H-NMR (300 MHz, DMSO-d6) d: 5.53 (s, 1H), 4.86 (d, J ¼ 10.1 Hz,
1H), 2.29–2.06 (m, 2H), 2.05–1.94 (m, 1H), 1.87–1.76 (m, 1H), 1.65–
1.33 (m, 5H), 1.30 (s, 3H), 1.24–1.10 (m, 1H), 0.89 (d, J ¼ 6.2 Hz,
3H), 0.81 (d, J ¼ 7.1 Hz, 3H).
Chemistry
Melting points were obtained on a Bu¨chi Melting Point B-540
apparatus (Bu¨chi Labortechnik, Flawil, Switzerland) and were
uncorrected. Mass spectra (MS) were taken in ESI mode on
Agilent 1100 LC-MS (Agilent, Palo Alto, CA, USA). Proton (1H)
nuclear magnetic resonance spectroscopy was performed using
Bruker ARX-300, 300 MHz spectrometers (Bruker Bioscience,
Billerica, MA, USA) with TMS as an internal standard. IR spectra
(KBr disks) were recorded with a Bruker IFS 55 instrument
(Bruker). Elemental analysis was determined on a Carlo-Erba
1106 Elemental analysis instrument (Carlo Erba, Milan, Italy).
Unless otherwise noted, all the materials were obtained from
commercially available sources and were used without further
purification.
Preparation of 2-(10b-dihydroartemisinoxy)
ethyl bromide 5
BF3 ꢁ Et2O (16 mL) was added to a solution of dihydroartemisinin
(20.0 g, 70.4 mmol) and 2-bromoethanol (13.1 g, 105.6 mmol) in
100 mL of CH2Cl2 at 08C. The mixture was stirred at room
temperature until the reaction completed. The reaction mixture
was washed with a saturated NaHCO3 solution, water and brine.
The organic layer was dried and concentrated. The residue was
recrystallized from petroleum ether to give compound 5 (12.7 g,
46.3%) as white crystals.
M.p.: 155–1588C; MS (ESI, m/z): 413.2 (M þ Na)þ; 1H-NMR
(300 MHz, DMSO-d6) d: 5.43 (s, 1H), 4.77 (d, J ¼ 3.4 Hz, 1H),
3.94 (td, J ¼ 9.3, 4.1 Hz, 1H), 3.78–3.58 (m, 3H), 2.47–2.36 (m,
1H), 2.25–2.13 (m, 1H), 2.05–1.95 (m, 1H), 1.89–1.75 (m, 2H), 1.61
(m, 2H), 1.44–1.31 (m, 3H), 1.29 (s, 3H), 1.22–1.06 (m, 2H), 0.90 (d,
J ¼ 6.5 Hz, 3H), 0.87 (d, J ¼ 7.5 Hz, 3H).
Preparation of 10b-azidodihydroartemisinin 2 and
10a-azidodihydroartemisinin 3
Trimethylchlorosilane (300 mmol, 38.1 mL) was added gradually
to the mixture solution of dihydroartemisinin (200 mmol,
56.8 g) and sodium azide(300 mmol, 19.5 g) in dry dichlorometh-
ane (300 mL), and then sodium iodide (5 mmol, 3.0 g) was
added to the reaction mixture at 0–58C. The reaction mixture
was stirred at room temperature for 28 h. The mixture was
quenched with a saturated NaHCO3 solution (100 mL) and
diluted with dichloromethane. The organic phase was washed
with brine, then collected and dried overnight over anhydrous
Na2SO4. After filtering, the solvent was evaporated under
reduced pressure. The crude mixture was purified by column
chromatography (silica, 1%–5% EtOAc/hexanes) to furnish the
compound 2 (29.0 g, 47%) and compound 3 (3.0 g, 5%).
Preparation of 2-(10b-dihydroartemisinoxy) ethyl azide 6
NaN3 (4.0 g, 60.2 mmol) was added to a stirred solution of com-
pound 5 (8.0 g, 20.5 mmol) and sodium iodide (0.15 g, 1 mmol)
in DMF (60 mL). The reaction mixture was heated to 608C for
3–5 h. The mixture was poured into ice water, stirred for 1 h and
separated by filtration to give white compound 6 (7.1 g, 98%).
M.p.: 86–888C; MS (ESI, m/z): 376.2 (M þ Na)þ; 1H-NMR
(300 MHz, DMSO-d6) d: 5.39 (s, 1H), 4.76 (d, J ¼ 3.3 Hz, 1H),
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Arch. Pharm. Chem. Life Sci. 2011, 344, 631–638
Novel Artemisinin-Guanidine Hybrids
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3.91–3.81 (m, 1H), 3.56–3.34 (m, 3H), 2.47–2.37 (m, 1H), 2.25–2.13
(m, 1H), 2.06–1.94 (m, 1H), 1.87–1.48 (m, 4H), 1.45–1.32 (m, 3H),
1.29 (s, 3H), 1.14 (m, 1H), 0.90 (d, J ¼ 3.8 Hz, 3H), 0.88 (d,
J ¼ 4.9 Hz, 3H).
for C27H38F3N3O5 (in %): C, 59.88; H, 7.07; N, 7.76. Found: C, 59.97;
H, 7.10; N, 7.68.
(Z)-N-(10a-Deoxoartemisinin)-N0-(4-(trifluoromethoxy)-
phenyl)morpholine-4-carboxamidine 4c
Preparation of 2-(10b-dihydroartemisinoxy) ethyl amine 7
A 100-mL round-bottomed flask was charged with compound 6
(6.0 g, 16.9 mmol) and THF (60 mL). To this solution, triphenyl
phosphine (7.2 g, 27.3 mmol) was added slowly, and the reaction
mixture was stirred for 2 h at 608C. Several drops of water
(1.0 mL, 55.5 mmol) were added, and the resulting suspension
was stirred for 6 h. The mixture was concentrated under reduced
pressure. The crude mixture was purified by flash column
chromatography (silica gel, dichloromethane/methanol, 200:1)
to afford the desired compound 7 (4.1 g, 73.8%) as yellow oil.
Yield: 31%. M.p.: 103–1058C; MS (ESI, m/z): 556.3 (M þ H)þ; IR (KBr)
cmꢂ1: 3415.6, 2927.6, 1627.6, 1510.0, 1452.1, 1380.1, 1258.4,
1201.2, 1162.9, 1038.3, 926.5, 854.5, 608.4, 548.8; 1H-NMR
(300 MHz, CDCl3) d: 7.19 (d, J ¼ 9.0 Hz, 2H), 7.01
(d, J ¼ 9.0 Hz, 2H), 5.36 (s, 1H), 4.80 (d, 7.4 Hz, 1H), 3.85–3.68
(m, 4H), 3.57–3.43 (m, 2H), 3.37–3.27 (m, 2H), 2.60–2.44 (m, 1H),
2.38 (m, 1H), 2.06 (m, 1H), 1.98–1.83 (m, 1H), 1.48 (s, 3H), 0.97
(d, J ¼ 6.1 Hz, 3H), 0.86 (d, J ¼ 6.0 Hz, 3H); Anal. calcd.
for C27H36F3N3O6 (in %): C, 58.37; H, 6.53; N, 7.56. Found; C,
58.41; H, 6.55; N, 7.52.
1
MS (ESI, m/z): 328.1 (M þ H)þ; H-NMR (300 MHz, DMSO-d6) d:
5.34 (s, 1H), 4.69 (d, J ¼ 3.4 Hz, 1H), 3.71–3.54 (m, 1H), 3.30 (dt,
J ¼ 9.8, 5.7 Hz, 1H), 2.73–2.62 (m, 2H), 2.47–2.33 (m, 1H), 2.25–
2.12 (m, 1H), 2.07–1.92 (m, 1H), 1.86–1.78 (m, 1H), 1.78–1.66
(m, 2H), 1.63–1.33 (m, 3H), 1.29 (s, 3H), 1.21–1.07 (m, 1H), 0.89
(d, J ¼ 6.3 Hz, 3H), 0.85 (d, J ¼ 7.4 Hz, 3H).
(Z)-N-(10b-Deoxoartemisinin)-N0-(4-(trifluoromethoxy)-
pheny)morpholine-4-carboxamidine 4d
Yield: 38%. M.p.: 109–1128C; MS (ESI, m/z): 556.3 (M þ H)þ; IR (KBr)
cmꢂ1: 3425.5, 2930.5, 1627.9, 1508.1, 1450.2, 1381.5, 1261.3,
1201.8, 1164.4, 1037.8, 925.9, 856.1, 619.1, 549.8; 1H-NMR
(300 MHz, CDCl3) d: 7.19 (d, J ¼ 8.6 Hz, 2H), 7.01 (d,
J ¼ 8.9 Hz, 2H), 5.44 (s, 1H), 5.10 (d, J ¼ 9.6 Hz 1H), 3.86–3.64
(m, 4H), 3.58–3.43 (m, 2H), 3.44–3.24 (m, 2H), 2.55–2.44 (m, 1H),
2.37 (m, J ¼ 14.4, 2.8 Hz, 1H), 2.1–1.99 (m, 1H), 1.97–1.83 (m, 1H),
1.48 (s, 3H), 0.98 (d, J ¼ 5.9 Hz, 3H). 0.88 (d, J ¼ 7.1 Hz, 3H); Anal.
calcd. for C27H36F3N3O6 (in %): C, 58.37; H, 6.53; N, 7.56. Found: C,
58.29; H, 6.56; N, 7.51.
General procedure for the preparation of compounds 4a–4f
The triphenylphosphine (0.24 g, 0.93 mmol) was dissolved in
10 mL of dry THF and then added dropwise to a solution of
10 mL of dry THF containing of azide 2 or azide 3 (0.29 g,
0.93 mmol) at 608C and the mixture was stirred for 1 h. The
appropriately substituted isocyanatobenzene was added to form
carbodiimide analogs as a highly reactive intermediate, which
subsequently undergoes nucleophilic addition when secondary
alkylamine was added to the reaction mixture drop-wise, the
reaction mixture was stirred at room temperature for 1 h. The
reaction mixture was concentrated under reduced pressure
and purified by flash column chromatography (silica gel,
dichloromethane/methanol, 100:1) to afford the target com-
pounds 4a–4f.
(Z)- N-(10b-Deoxoartemisinin)-N0-(4-(trifluoromethoxy)-
phenyl)piperidine-1-carboxamidine 4e
Yield: 39%. M.p.: 100–1028C; MS (ESI, m/z): 554.2 (M þ H)þ; IR (KBr)
cmꢂ1: 3416.6, 2926.8, 1627.9, 1513.8, 1452.2, 1380.9, 1257.8,
1201.3, 1162.4, 1038.9, 926.6, 878.6, 608.4, 486.3; 1H-NMR
(300 MHz, CDCl3) d: 7.21 (d, J ¼ 8.7 Hz, 2H), 7.00 (d,
J ¼ 9.0 Hz, 2H), 5.42 (s, 1H), 5.02 (d, J ¼ 9.1 Hz, 1H), 3.53–3.40
(m, 2H), 3.38–3.21 (m, 2H), 2.68–2.52 (m, 1H), 2.42–2.27 (m, 1H),
2.10–1.96 (m, 1H), 1.95–1.82 (m, 1H), 1.80–1.52 (m, 11H), 1.49
(s, 3H), 0.95 (d, J ¼ 6.0 Hz, 3H), 0.93 (d, J ¼ 5.7 Hz, 3H); Anal.
calcd. for C28H38F3N3O5 (in %): C, 60.75; H, 6.92; N, 7.59. Found: C,
60.81; H, 6.93; N, 7.43.
(Z)-N-(10a-Deoxoartemisinin)-N0-(4-(trifluoromethoxy)-
phenyl)piperidine-1-carboxamidine 4a
Yield: 36%. M.p.: 101–1038C; MS (ESI, m/z): 554.3 (M þ H)þ; IR (KBr)
cmꢂ1: 3417.1, 2932.7, 1628.0, 1510.0, 1449.7, 1380.7, 1263.4,
1202.1, 1165.1, 1037.0, 926.0, 856.4, 617.1, 549.9; 1H-NMR
(300 MHz, CDCl3) d: 7.23 (d, J ¼ 9.0 Hz, 2H), 7.00
(d, J ¼ 9.0 Hz, 2H), 5.44 (s, 1H), 5.08 (d, J ¼ 10.9 Hz, 1H), 3.48
(s, 2H), 3.39–3.20 (m, 2H), 2.66 (m, 1H), 2.34 (td, J ¼ 14.0, 3.8 Hz,
1H), 2.06 (dq, J ¼ 17.7, 14.7 Hz, 1H), 1.95–1.82 (m, 1H), 1.80–1.51
(m, 11H), 1.49 (s, 3H), 1.47–1.28 (m, 3H), 1.28–1.14 (m, 2H), 0.96
(d, J ¼ 5.5 Hz, 3H), 0.93 (d, J ¼ 4.5 Hz, 3H); Anal. calcd.
for C28H38F3N3O5 (in %): C, 60.75; H, 6.92; N, 7.59. Found: C,
60.81; H, 6.91; N, 7.53.
(Z)-N-(10b-Deoxoartemisinin)-N0-(2-chloro-5-
(trifluoromethyl)phenyl)pyrrolidine-1-carboxamidine 4f
Yield: 48%. M.p.: 101–1038C; MS (ESI, m/z): 558.1 (M þ H)þ; IR (KBr)
cmꢂ1: 3421.6, 2928.3, 1627.8, 1511.5, 1451.6, 1381.3, 1264.4,
1201.1, 1163.4, 1038.5, 926.0, 859.6, 618.4, 536.3; 1H-NMR
(300 MHz, DMSO-d6) d: 7.68–7.55 (m, 2H), 7.42 (d, J ¼ 8.2 Hz,
1H), 5.26 (s, 1H), 4.70 (d, J ¼ 9.3 Hz, 1H), 3.56–3.34 (m, 4H),
2.47–2.40 (m, 1H), 2.18 (td, J ¼ 13.9, 3.3 Hz, 1H), 2.06–1.93
(m, 1H), 1.94–1.72 (m, 4H), 1.67–1.39 (m, 4H), 1.30 (s, 3H), 0.88
(d, J ¼ 6.0 Hz, 3H), 0.75 (d, J ¼ 7.1 Hz, 3H); Anal. calcd.
for C27H35ClF3N3O4 (in %): C, 58.11; H, 6.32; N, 7.53; Found: C,
58.23; H, 6.26; N, 7.51.
(Z)-3-(10a-Deoxoartemisinin)-2-(4-(trifluoromethoxy)-
phenyl)diethylguanidine 4b
Yield: 45%. M.p.: 93–958C; MS (ESI, m/z): 542.3 (M þ H)þ; IR (KBr)
cmꢂ1: 3422.0, 2930.1, 1625.5, 1509.5, 1454.7, 1382.3, 1264.8,
1202.1, 1161.9, 1017.9, 925.3, 878.5, 616.9, 521.5; 1H-NMR
(300 MHz, CDCl3) d: 7.21 (d, J ¼ 8.6 Hz, 2H), 7.01 (d,
J ¼ 8.9 Hz, 2H), 5.39 (s, 1H), 5.02 (d, J ¼ 10.2 Hz, 1H), 3.70–3.55
(m, 2H), 3.41–3.13 (m, 2H), 2.68–2.53 (m, 1H), 2.35 (td, J ¼ 14.1,
3.9 Hz, 1H), 2.09–1.97 (m, 1H), 1.89 (m, 2H), 1.48 (s, 3H), 1.28–1.26
(m, 2H), 1.25–1.15 (m, 6H), 0.94 (d, J ¼ 6.3 Hz, 6H); Anal. calcd.
General procedure for the preparation of compounds 8a–8h
The foregoing method for the preparation of the 4a–4f was
applied to prepare the 8a–8h except that azide 7 was used instead
of azide 2 or azide 3.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

636
L. Xie et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 631–638
7.32 (s, 1H), 5.41 (s, 1H), 4.84 (d, J ¼ 3.4 Hz, 1H), 4.05–3.93 (m, 1H),
3.84–3.64 (m, 3H), 3.45–3.32 (m, 4H), 2.73–2.61 (m, 1H), 2.37
(td, J ¼ 14.1, 3.8 Hz, 1H), 2.08–1.98 (m, 1H), 1.94–1.83 (m, 1H),
1.72–1.56 (m, 8H), 1.40 (s, 3H), 1.28–1.17 (m, 2H), 0.94
(d, J ¼ 6.3 Hz, 3H), 0.91 (d, J ¼ 7.4 Hz, 3H); Anal. calcd.
for C30H42F3N3O5 (in %): C, 61.95; H, 7.28; N, 7.22. Found: C,
61.96; H, 7.33; N, 7.14.
(E)-2-(3-Bromophenyl)-3-[2-(10b-dihydroartemisinoxy)-
ethyl]diethylguanidine 8a
Yield: 33%. M.p.: 91–938C; MS (ESI, m/z): 580.1 (M þ H)þ; IR (KBr)
cmꢂ1: 3415.3, 2927.2, 1633.2, 1513.5, 1461.6, 1380.3, 1201.1,
1163.4, 1037.7, 927.0, 851.7, 6124.2.538.0; 1H-NMR (300 MHz,
CDCl3) d: 7.19–7.09 (m, 3H), 6.94 (d, J ¼ 6.1 Hz, 1H), 5.36
(s, 1H), 4.79 (d, J ¼ 3.3 Hz, 1H), 3.96–3.84 (m, 1H), 3.69–3.56
(m, 1H), 3.44–3.27 (m, 6H), 2.73–2.58 (m, 1H), 2.37 (td,
J ¼ 14.0, 3.7 Hz, 1H), 2.10–1.99 (m, 1H), 1.96–1.83 (m, 1H),
1.78–1.46 (m, 5H), 1.43 (s, 3H), 1.19 (t, J ¼ 7.1 Hz, 6H), 0.97
(d, J ¼ 6.0 Hz, 3H), 0.86 (d, J ¼ 7.3 Hz, 3H); Anal. calcd.
for C28H42BrN3O5 (in %): C, 57.93; H, 7.29; N, 7.24. Found: C,
58.07; H, 7.24; N, 7.21.
(E)-2-(3-Chlorophenyl)-3-[2-(10b-dihydroartemisinoxy)
ethyl]diethylguanidine 8f
Yield: 78%. M.p.: 96–988C; MS (ESI, m/z): 536.4 (M þ H)þ; IR (KBr)
cmꢂ1: 3415.3, 2930.0, 1637.6, 1598.1, 1452.5, 1380.1, 1201.9,
1201.3, 1164.84, 1037.9, 902.6, 816.3, 534.8; 1H-NMR
(300 MHz, CDCl3) d: 7.28 (d, J ¼ 16.5 Hz, 1H), 7.21–7.05
(m, 3H), 5.41 (s, 1H), 4.83 (d, J ¼ 3.4 Hz, 1H), 4.01–3.89 (m, 1H),
3.86–3.76 (m, 1H), 3.75–3.61 (m, 2H), 3.56–3.38 (m, 4H), 2.75–2.55
(m, 1H), 2.37 (td, J ¼ 14.2, 3.8 Hz, 1H), 2.04 (dq, J ¼ 15.2, 4.5,
3.2 Hz, 1H), 1.96–1.82 (m, 1H), 1.65–1.43 (m, 4H), 1.41 (s, 3H), 1.24
(t, J ¼ 7.1 Hz, 6H), 0.95 (d, J ¼ 4.9 Hz, 3H), 0.93 (d, J ¼ 5.9 Hz, 3H);
Anal. calcd. for C28H42ClN3O5 (in %): C, 62.73; H, 7.90; N, 7.84.
Found: C, 62.70; H, 7.95; N, 7.79.
(E)-N0-(3-Bromophenyl)-N-(2-(10b-dihydroartemisinoxy)-
ethyl)morpholine-4-carboxamidine 8b
Yield: 42%. M.p.: 86–888C; MS (ESI, m/z): 594.1 (M þ H)þ; IR (KBr)
cmꢂ1: 3415.5, 2930.6, 1647.3, 1598.1, 1452.5, 1380.5, 1261.3,
1201.3, 1164.84, 1038.8, 922.6, 826.1, 617.1, 544.8; 1H-NMR
(300 MHz, CDCl3) d: 7.18–7.01 (m, 3H), 6.82 (d, J ¼ 6.9 Hz, 1H),
5.33 (s, 1H), 4.75 (d, J ¼ 3.4 Hz, 1H), 3.96–3.81 (m, 1H), 3.81–3.63
(m, 4H), 3.59–3.46 (m, 1H), 3.37–3.20 (m, 6H), 2.70–2.54 (m, 1H),
2.38 (td, J ¼ 14.2, 3.9 Hz, 1H), 2.10–1.99 (m 1H), 1.98–1.82 (m, 1H),
1.74–1.47 (m, 4H), 1.44 (s, 3H), 0.97 (d, J ¼ 5.8 Hz, 3H), 0.75
(d, J ¼ 7.3 Hz, 3H); Anal. calcd. for C28H40BrN3O6 (in %): C,
56.56; H, 6.78; N, 7.07. Found: C, 56.60; H, 6.69; N, 7.03.
(E)-N0-(3-Chlorophenyl)-N-(2-(10b-dihydroartemisinoxy)-
ethyl)-4-methylpiperazine-1-carboxamidine 8g
Yield: 78%. M.p.: 71–738C; MS (ESI, m/z): 563.4 (M þ H)þ; IR (KBr)
cmꢂ1: 3417.6, 2928.5, 1628.2, 1511.5, 1451.6, 1381.3, 1264.4,
1201.1, 1163.4, 1038.1, 937.0, 856.7, 633.4; 1H-NMR (300 MHz,
CDCl3) d: 7.24 (d, J ¼ 7.9 Hz, 1H), 7.12–6.91 (m, 3H), 5.40 (s, 1H),
4.82 (d, J ¼ 3.2 Hz, 1H), 4.03–3.88 (m, 1H), 3.80–3.64 (m, 1H),
3.63–3.51 (m, 2H), 3.48–3.32 (m, 4H), 2.72–2.60 (m, 1H), 2.53–2.44
(m, 4H), 2.37–2.31(m, 1H), 2.32 (s, 3H), 2.11–1.97 (m, 1H), 1.97–
1.83 (m, 1H), 1.76–1.46 (m, 5H), 1.42 (s, 3H), 0.96 (d, J ¼ 6.2 Hz,
3H), 0.88 (d, J ¼ 7.3 Hz, 3H); Anal. calcd. for C29H43ClN4O5 (in %):
C, 61.85; H, 7.70; N, 9.95. Found: C, 61.88; H, 7.65; N, 9.92.
(E)-N0-(4-(Trifluoromethyl)phenyl)-N-(2-(10b-
dihydroartemisinoxy)ethyl)pyrrolidine-1-carboxamidine 8c
Yield: 42%. M.p.: 97–998C; MS (ESI, m/z): 568.2 (M þ H)þ; IR (KBr)
cmꢂ1: 3414.4, 2931.1, 1629.8, 1510.0, 1447.6, 1380.4, 1268.7,
1202.1, 1165.1, 1038.4, 925.0, 856.4, 617.1, 559.6; 1H-NMR
(300 MHz, CDCl3) d: 7.58 (d, J ¼ 6.2 Hz, 2H), 7.33
(d, J ¼ 7.5 Hz, 2H), 5.41 (s, 1H), 4.82 (d, J ¼ 1.5 Hz, 1H), 3.96–
3.79 (m, 2H), 3.78–3.43 (m, 6H), 2.76–2.60 (m, 1H), 2.45–2.29
(m, 1H), 2.09–1.83 (m, 5H), 1.38 (s, 3H), 0.95 (d, J ¼ 6.1 Hz, 3H),
0.93 (d, J ¼ 7.8 Hz, 3H); Anal. calcd. for C29H40F3N3O5 (in %): C,
61.36; H, 7.10; N, 7.40. Found; C, 61.50; H, 7.03; N, 7.29.
(E)-N0-(4-(Trifluoromethoxy)phenyl)-N-(2-(10b-
dihydroartemisinoxy)ethyl)piperidine-1-carboxamidine 8h
Yield: 78%. M.p.: 93–958C; MS (ESI, m/z): 598.4 (M þ H)þ; IR (KBr)
cmꢂ1: 3415.1, 2928.3, 1629.6, 15291.2, 1450.8, 1383.5, 1383.3,
1
(E)-N0-(3-(Trifluoromethyl)phenyl)-N-(2-(10b-
dihydroartemisinoxy)ethyl)-4-methylpiperazine-1-
1105.6, 1038.6, 928.9, 793.2, 481.3; H-NMR (300 MHz, CDCl3) d:
7.23 (d, J ¼ 9.0 Hz, 2H), 7.19 (d, J ¼ 9.0 Hz, 2H), 5.42 (s, 1H), 4.84
(d, J ¼ 3.0 Hz, 1H), 4.08–3.92 (m, 1H), 3.86–3.63 (m, 3H), 3.52–3.29
(m, 4H), 2.72–2.61 (m, 1H), 2.42–2.31 (m, 1H), 2.09–1.97 (m, 10H),
1.97–1.81 (m, 1H), 1.75–1.48 (m, 10H), 1.40 (s, 3H), 1.31–1.19
(m, 1H), 0.94 (d, J ¼ 4.9 Hz, 3H), 0.92 (d, J ¼ 6.3 Hz, 3H); Anal.
calcd. for C30H42F3N3O6 (in %): C, 60.29; H, 7.08; N, 7.03. Found: C,
60.30; H, 7.16; N, 7.06.
carboxamidine 8d
Yield: 78%. M.p.: 81–838C; MS (ESI, m/z): 597.2 (M þ H)þ; IR (KBr)
cmꢂ1: 3415.6, 2928.7, 1629.2, 1511.5, 1451.6, 1381.3, 1264.4,
1201.1, 1163.4, 1038.1, 937.0, 858.7, 614.4; 1H-NMR (300 MHz,
CDCl3) d: 7.42 (t, J ¼ 7.8 Hz, 1H), 7.31–7.16 (m, 3H), 5.37 (s, 1H),
4.79 (d, J ¼ 3.3 Hz, 1H), 3.97–3.87 (m, 1H), 3.71–3.44 (m, 3H),
3.44–3.26 (m, 3H), 2.69–2.59 (m, 1H), 2.52–2.35 (m, 5H), 2.32 (s,
3H), 2.09–1.98 (m, 1H), 1.95–1.84 (m, 1H), 1.42 (s, 3H), 1.32–1.18
(m, 2H), 0.96 (d, J ¼ 6.0 Hz, 3H), 0.81 (d, J ¼ 7.3 Hz, 3H); Anal.
calcd. for C30H43F3N4O5 (in %): C, 60.39; H, 7.26; N, 9.39. Found: C,
60.55; H, 7.23; N, 9.23.
General procedure for the preparation of compounds 9a–9h
The foregoing method for the preparation of the 8a–8h was
applied to prepare the 9a–9h except that compound 7 was used
instead of secondary amines.
(E)-N0-(3-(Trifluoromethyl)phenyl)-N-(2-(10b-
2-(3-Chlorophenyl)-1,3-bis[2-(10b-dihydroartemisinoxy)-
dihydroartemisinoxy)ethyl)piperidine-1-carboxamidine 8e
Yield: 78%. M.p.: 93–958C; MS (ESI, m/z): 582.3 (M þ H)þ; IR (KBr)
cmꢂ1: 3412.1, 2929.2, 1632.6, 1599.2, 1449.8, 1384.1, 1333.3,
1105.6, 982.9, 799.5, 471.1; 1H-NMR (300 MHz, CDCl3) d: 7.52
(d, J ¼ 8.2 Hz, 1H), 7.51–7.44 (m, 1H), 7.38 (d, J ¼ 6.8 Hz, 1H),
ethyl]guanidine 9a
Yield: 39%. M.p.: 98–1008C; MS (ESI, m/z): 790.3 (M þ H)þ; IR (KBr)
cmꢂ1: 3421.1, 2943.3, 2875.8, 1636.1, 1468.3, 1382.4, 1191.3,
1103.8, 1029.2, 983.6, 875.9, 619.6, 498.5; 1H-NMR (300 MHz,
DMSO-d6) d: 7.39 (s, 1H), 7.31–6.99 (m, 3H), 5.33 (s, 2H), 4.72
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Arch. Pharm. Chem. Life Sci. 2011, 344, 631–638
Novel Artemisinin-Guanidine Hybrids
637
(d, J ¼ 3.1 Hz, 2H), 3.88–3.73 (m, 2H), 3.55–3.37 (m, 6H), 2.46–2.34
(m, 2H), 2.19 (td, J ¼ 13.6, 3.2 Hz, 2H), 2.07–1.94 (m, 2H), 1.90–
1.74 (m, 2H), 1.75–1.45 (m, 10H), 1.29 (s, 6H), 0.90 (d, J ¼ 6.0 Hz,
6H), 0.85 (d, J ¼ 7.3 Hz, 6H); Anal. calcd. for C41H60ClN3O10 (in %):
C, 62.30; H, 7.65; N, 5.32. Found: C, 62.35; H, 7.68; N, 5.24.
2-(2-Chloro-5-(trifluoromethyl)phenyl)-1,3-bis[2-(10b-
dihydroartemisinoxy)ethyl]guanidine 9f
Yield: 43%. M.p.: 106–1088C; MS (ESI, m/z): 858.2 (M þ H)þ; IR (KBr)
cmꢂ1: 3414.3, 2950.3, 2874.4, 1624.2, 1479.5, 1379.8, 1323.6,
1138.8, 1104.9, 1029.6, 984.2, 875.3, 825.5, 663.2, 538.4;
1H-NMR (300 MHz, CDCl3) d: 7.22 (s, 1H), 7.17 (d, J ¼ 8.3 Hz,
1H), 7.01 (d, J ¼ 8.0 Hz, 1H), 5.37 (s, 2H), 4.81 (d, J ¼ 3.3 Hz,
2H), 4.00–3.88 (m, 2H), 3.68–3.60 (m, 2H), 3.48–3.40 (m, 4H),
2.73–2.61 (m, 2H), 2.38 (td, J ¼ 14.0, 3.9 Hz, 2H), 2.05 (m, 2H),
1.98–1.84 (m, 2H), 1.78–1.53 (m, 8H), 1.43 (s, 6H), 0.98
(d, J ¼ 5.9 Hz, 6H), 0.86 (d, J ¼ 7.3 Hz, 6H); Anal. calcd.
for C42H59ClF3N3O10 (in %): C, 58.77; H, 6.93; N, 4.90. Found:
C, 58.79; H, 6.96; N, 4.83.
2-(2,4-Dichlorophenyl)-1,3-bis[2-(10b-
dihydroartemisinoxy)ethyl]guanidine 9b
Yield: 34%. M.p.: 100–1028C; MS (ESI, m/z): 825.6 (M þ H)þ; IR (KBr)
cmꢂ1: 3412.3, 2948.6, 2873.7, 1627.9, 1469.9, 1379.4, 1158.7,
1103.6, 1029.3, 985.5, 875.1, 824.7, 723.0, 541.7; 1H-NMR
(300 MHz, CDCl3) d: 7.49 (d, J ¼ 8.0 Hz, 1H), 7.41 (s, 1H), 7.20
(d, J ¼ 8.0 Hz, 1H), 5.36 (s, 2H), 4.79 (d, J ¼ 3.2 Hz, 2H), 4.01–3.87
(m, 2H), 3.75–3.58 (m, 2H), 3.56–3.45 (m, 4H), 2.73–2.56 (m, 2H),
2.38 (td, J ¼ 13.8, 3.2 Hz, 2H), 2.10–2.00 (m, 2H), 1.98–1.84
(m, 2H), 1.79–1.55 (m, 10H), 1.42 (s, 6H), 0.98 (d, J ¼ 5.6 Hz,
6H), 0.81 (d, J ¼ 7.2 Hz, 6H); Anal. calcd. for C41H59Cl2N3O10
(in %): C, 59.70; H, 7.21; N, 5.09. Found: C, 59.83; H, 7.23; N,
5.01.
2-(3-Fluorophenyl)-1,3-bis[2-(10b-
dihydroartemisinoxy)ethyl]guanidine 9g
Yield: 28%. M.p.: 108–1108C; MS (ESI, m/z): 774.3 (M þ H)þ; IR (KBr)
cmꢂ1: 3422.0, 2924.3, 2874.5, 1635.5, 1450.3, 1383.9, 1193.3,
1104.7, 1029.6, 984.8, 875.2, 825.0, 619.1, 484.0; 1H-NMR
(300 MHz, CDCl3) d: 7.24 (m, J ¼ 7.7 Hz, 1H), 6.78 (m, J ¼ 18.3,
9.2 Hz, 3H), 5.39 (s, 2H), 4.80 (d, J ¼ 3.4 Hz, 2H), 4.03–3.84 (m, 2H),
3.63 (m, 2H), 3.50–3.42 (m, 4H), 2.74–2.58 (m, 2H), 2.38 (td,
J ¼ 14.4, 3.9 Hz, 2H), 2.12–1.98 (m, 2H), 1.91 (m, 2H), 1.80–1.46
(m, 10H), 1.41 (s, 6H), 0.98 (d, J ¼ 5.9 Hz, 6H), 0.85 (d, J ¼ 9.0 Hz,
6H); Anal. calcd. for C41H60FN3O10 (in %): C, 63.63; H, 7.81; N, 5.43.
Found: C, 63.70; H, 7.78; N, 5.33.
2-(4-Trifluoromethoxyphenyl)-1,3-bis[2-(10b-
dihydroartemisinoxy)ethyl]guanidine 9c
Yield: 41%. M.p.: 101–1048C; MS (ESI, m/z): 840.3 (M þ H)þ; IR (KBr)
cmꢂ1: 3416.5, 2949.3, 2875.1, 1644.9, 1508.6, 1380.9, 1255.5,
1104.2, 1029.4, 984.8, 875.4, 825.2, 539.1; 1H-NMR (300 MHz,
DMSO-d6) d: 7.32 (d, J ¼ 8.0 Hz, 2H), 7.14 (d, J ¼ 8.0 Hz, 2H),
5.34 (s, 2H), 4.72 (d, J ¼ 3.0 Hz, 2H), 3.90–3.74 (m, 2H), 3.59–
3.38 (m, 6H), 2.46–2.32 (m, 2H), 2.19 (td, J ¼ 13.4, 2.3 Hz, 2H),
2.08–1.93 (m, 2H), 1.89–1.74 (m, 2H), 1.75–1.45 (m, 8H), 1.28
(s, 6H), 0.89 (d, J ¼ 5.9 Hz, 6H), 0.83 (d, J ¼ 7.2 Hz, 6H); Anal.
calcd. for C42H60F3N3O11 (in %): C, 60.06; H, 7.20; N, 5.00. Found;
C, 60.02; H, 7.34; N, 4.89.
2-(4-Chlorophenyl)-1,3-bis[2-(10b-
dihydroartemisinoxy)ethyl]guanidine 9h
Yield: 37%. M.p.: 101–1038C; MS (ESI, m/z): 790.3 (M þ H)þ; IR (KBr)
cmꢂ1: 3419.5, 2932.5, 2873.4, 1633.2, 1468.6, 1381.1, 1195.6,
1104.3, 1029.5, 986.3, 873.6, 824.9, 519.1; 1H-NMR (300 MHz,
CDCl3) d: 7.36 (d, J ¼ 8.5 Hz, 2H), 7.19 (d, J ¼ 8.6 Hz, 2H), 5.34
(s, 2H), 4.80 (d, J ¼ 3.2 Hz, 2H), 3.96 (m, 2H), 3.74–3.62 (m, 2H),
3.61–3.47 (m, 4H), 2.75–2.62 (m, 2H), 2.38 (td, J ¼ 14.0, 3.6 Hz,
2H), 2.12–1.98 (m, 2H), 1.98–1.83 (m, 2H), 1.76–1.43 (m, 10H), 1.41
(s, 6H), 0.98 (d, J ¼ 5.7 Hz, 6H), 0.86 (d, J ¼ 7.0 Hz, 6H); Anal.
calcd. for C41H60ClN3O10 (in %): C, 62.30; H, 7.65; N, 5.32. Found:
C, 62.42; H, 7.63; N, 5.29.
2-(4-Trifluoromethylphenyl)-1,3-bis[2-(10b-
dihydroartemisinoxy)ethyl]guanidine 9d
Yield: 31%. M.p.: 99–1018C; MS (ESI, m/z): 824.3 (M þ H)þ; IR (KBr)
cmꢂ1: 3418.1, 2934.6, 2873.5, 1631.2, 1468.9, 1383.9, 1193.6,
1104.8, 1029.2, 984.3, 875.2, 825.4, 620.5, 539.4; 1H-NMR
(300 MHz, DMSO-d6) d: 7.49 (d, J ¼ 7.8 Hz, 2H), 6.96
(d, J ¼ 7.5 Hz, 2H), 5.32 (s, 2H), 4.69 (d, J ¼ 1.6 Hz, 2H), 3.84–
3.69 (m, 2H), 3.51–3.39 (m, 2H), 3.38–3.20 (m, 4H), 2.46–2.29
(m, 2H), 2.28–2.08 (td, J ¼ 12Hz, 2H), 2.04–1.95 (m, 2H), 1.87–
1.74 (m, 2H), 1.75–1.44 (m, 7H), 1.28 (s, 6H), 0.88 (d, J ¼ 5.5 Hz,
6H), 0.81 (d, J ¼ 7.1 Hz, 6H); Anal. calcd. for C42H60F3N3O10
(in %): C, 61.22; H, 7.34; N, 5.10. Found: C, 61.33; H, 7.31; N,
5.09.
Anti-tumor activity assay in vitro
The anti-tumor activities of compounds 4a–4f, 8a–8h and 9a–9h
were evaluated with A549, HT-29 and MDA-MB-231 cell lines by
the standard MTT assay in vitro, with DHA as the positive control.
The cancer cell lines were cultured in minimum essential
medium (MEM) supplement with 10% fetal bovine serum (FBS).
Approximately 4 ꢀ 103 cells, suspended in MEM, were plated
onto each well of a 96-well plate and incubated in 5% CO2 at 378C
for 24 h. The test compounds at indicated final concentrations
were added to the culture medium and the cell cultures were
continued for 72 h. Fresh MTT was added to each well at a
terminal concentration of 5 mg/mL and incubated with cells
at 378C for 4 h. The formazan crystals were dissolved in
100 mL DMSO each well, and the absorbency at 492 nm (for
absorbance of MTT formazan) and 630 nm (for the reference
wavelength) was measured with the ELISA reader. All of the
compounds were tested twice in each of the cell lines. The results
expressed as IC50 (inhibitory concentration 50%) were the aver-
ages of two determinations and calculated by using the Bacus
Laboratories Incorporated Slide Scanner (Bliss) software.
2-(3-Bromophenyl)-1,3-bis[2-(10b-
dihydroartemisinoxy)ethyl]guanidine 9e
Yield: 39%. M.p.: 100–1028C; MS (ESI, m/z): 834.6 (M þ H)þ; IR (KBr)
cmꢂ1: 3421.1, 2924.0, 2873.5, 1631.2, 1468.9, 1383.9, 1193.6,
1104.8, 1029.2, 984.3, 875.2, 825.4, 620.5, 539.4; 1H-NMR
(300 MHz, DMSO-d6) d: 7.25–7.14 (m, 3H), 6.93 (s, 1H), 5.32
(s, 2H), 4.70 (d, J ¼ 3.3 Hz, 2H), 3.86–3.73 (m, 2H), 3.49–3.40
(m, 2H), 3.35–3.20 (m, 4H), 2.45–2.33 (m, 2H), 2.19
(td, J ¼ 13.9, 3.5 Hz, 2H), 2.07–1.95 (m, 2H), 1.92–1.75 (m, 2H),
1.75–1.46 (m, 8H), 1.29 (s, 6H), 0.89 (d, J ¼ 6.1 Hz, 6H), 0.82
(d, J ¼ 7.4 Hz, 6H); Anal. calcd. for C41H60BrN3O10 (in %): C,
58.99; H, 7.24; N, 5.03. Found: C, 58.90; H, 7.37; N, 4.97.
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

638
L. Xie et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 631–638
This work was supported by a grant from the National S&T Major Project of
China (No. 2009ZX09301-012).
[10] G. H. Posner, I. H. Paik, S. Sur, A. J. McRiner, et al., J. Med. Chem.
2003, 46, 1060–1065.
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2009, 19, 382–385.
The authors have declared no conflict of interest.
[12] G. H. Posner, A. J. McRiner, I. H. Paik, S. Sur, et al., J. Med. Chem.
2004, 47, 1299–1301.
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ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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