Antifungal activity of artemisinin derivatives

Article abstract of DOI:10.1021/np050074u

A series of 29 artemisinin derivatives (2-30), including four new compounds (16-18, 20), together with artemisinin (1), artemisinic acid (31), and arteannuin B (32), were tested for antifungal activity against two opportunistic pathogens, Candida albicans

Full text of DOI:10.1021/np050074u

1274
J. Nat. Prod. 2005, 68, 1274-1276
Antifungal Activity of Artemisinin Derivatives
Ahmed M. Galal,*,† Samir A. Ross,‡,§ Melissa Jacob,‡ and Mahmoud A. ElSohly‡,
College of Pharmacy, Cairo University, Kasr El-Eini Street, 11562, Cairo, Egypt, and National Center for Natural Products
Research, Department of Pharmacognosy, and Department of Pharmaceutics, School of Pharmacy, University of Mississippi,
University, Mississippi 38677
Received March 3, 2005
A series of 29 artemisinin derivatives (2-30), including four new compounds (16-18, 20), together with
artemisinin (1), artemisinic acid (31), and arteannuin B (32), were tested for antifungal activity against
two opportunistic pathogens, Candida albicans and Cryptoccocus neoformans. Of all the compounds tested,
anhydrodihydro-artemisinin (3) demonstrated more potent antifungal activity against C. neoformans than
amphotericin B. Also, â-arteether (7) and R-arteether (8) showed marked activity against C. neoformans.
Against C. albicans, the overall antifungal effect of these compounds was weak or negligible. Derivatives
2-30 were prepared according to literature procedures.
Chart 1
During the past two decades, the incidence of fungal
infections, in particular those associated with immuno-
compromised patients, has increased dramatically.1 Cryp-
tococcus neoformans is the cause of the most common life-
threatening opportunistic fungal infections in patients with
HIV/AIDS.2 Although the occurrence of C. neoformans
among this group of patients has decreased in the past few
years due to the introduction of triple HIV therapy, the
incidence remains high, particularly in developing coun-
tries.3 Cryptococcosis caused by C. neoformans involves
infection of the central nervous system, which is often
manifested as meningitis, and is now seen most often in
patients with AIDS, of whom 2-20% develop this condi-
tion.4 In addition, candidiasis caused by Candida albicans
is also one of the most frequent (though uncommonly life-
threatening) fungal infections attacking persons with HIV/
AIDS.5 On the other hand, development of resistance,
particularly with suppressed immunity, is a challenging
problem in the treatment of fungal infections.6As part of a
continuing search for new, safer, and more effective anti-
fungal drugs acting on the opportunistic pathogens C.
albicans and C. neoformans, we herein report on the in
vitro antifungal activity of a series of artemisinin deriva-
tives against these organisms. A series of 29 artemisinin
derivatives was prepared for this study. With the exception
of compounds 16, 17, 18, and 20, which are new, the
remainder of compounds 2-30 are known. The structures
of the new compounds were determined on the basis of
spectroscopic data interpretation, as shown in the Experi-
mental Section. The relative stereochemistry at C-16 and
C-19 of compounds 17 and 18 was established on the basis
of NOESY data. In compound 17, the R-oriented H-12
displayed a NOESY correlation with H-16, and the â-ori-
ented H-15 correlated with H-19, suggesting similar stereo-
orientations. In compound 18, the â-oriented H-15 showed
NOESY correlations with both H-16 and H-19; thus H-16
and H-19 were assigned as having â-orientation.The anti-
fungal activity of compounds 1-32 was evaluated against
two opportunistic pathogens, C. albicans and C. neofor-
mans, with amphotericin B as a positive control, following
standard procedures.7 The results are shown in Table 1.
Compounds 2, 3, 7, and 8, which all contain a peroxide
group, were the most active derivatives, as shown in Table
1. Compound 4, obtained as a mixture of two epimers,8 was
inactive, although it has a peroxide group.
The stereoisomers 17 and 18 exhibited almost identical
antifungal activity with IC50 values of 1.0 and 0.9 µg mL-1
,
* To whom correspondence should be addressed. Tel: 9661 4677191.
Fax: 9661 46577245. E-mail: amgalalv@yahoo.com.
† Cairo University.
respectively, against Cryptococcus neoformans. However,
both compounds were inactive against Candida albicans.
Among the deoxy-artemisinin derivatives studied, four
compounds, 21, 22, 25, and 27, exhibited marginal activity.
‡ National Center for Natural Products Research.
§ Department of Pharmacognosy, University of Mississippi.
Department of Pharmaceutics, University of Mississippi.
10.1021/np050074u CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/12/2005

Notes
Journal of Natural Products, 2005, Vol. 68, No. 8 1275
Chart 2
C. albicans are yeasts, the disparity in activity could be
explained by the artemisinin derivatives inhibiting molec-
ular targets or processes only present in C. neoformans.
Moreover, all of these compounds are only fungistatic, not
fungicidal, at the highest test concentration of 50 µg mL-1
used, suggesting a mechanism of action other than fungi-
cidal drugs such as amphotericin B and caspofungin. On
the basis of the observation that polymorphonuclear leu-
kocytes kill C. neoformans, at least in part via generation
of fungicidal oxidants,11 one might speculate on the mode
of action of this class of compounds as the liberation of free
radicals that interfere with the growth of these organ-
isms.12,13 It is hoped that these preliminary results will
stimulate the further study of the potential of artemisinin
derivatives as antifungal agents.
Experimental Section
General Experimental Procedures. Melting points were
recorded on an Electrothermal 9100 instrument. Optical
rotations were recorded at ambient temperature using a
JASCO DIP 370 digital polarimeter. IR spectra were obtained
using an ATI Mattson Genesis Series FTIR spectrometer. The
NMR spectra were recorded on Bruker Avance DRX 400 and
500 spectrometers using the solvent peak as reference. 2D
NMR data were measured with standard pulse programs and
acquisition parameters. HRESIMS were obtained on a Bruker
BioAPEX 30es ion cyclotron high-resolution HPLC-FT spec-
trometer by direct injection onto an electrospray interface.
Test Compounds. Compounds 2-30 were prepared fol-
lowing literature procedures,14-20 while artemisinin (1), arte-
misinic acid (31), and arteanuine B (32) were isolated from
Artemisia annua, as previously reported.21 Compound 33 was
prepared according to a literature procedure.16 Reactions were
run in oven-dried round-bottomed flasks. Diethyl ether was
distilled from sodium benzophenone ketyl and stored prior to
use under an atmosphere of argon. All dihydroxy alcohols were
dried by Al2O3-grade Ι prior to use. All other compounds were
purchased from Aldrich Chemical Co. and used without further
purification. Column chromatography was performed using
flash silica gel (Merck, particle size 230-400 mesh). Analytical
thin-layer chromatography (TLC) was performed with silica
gel 60 F254 plates (Merck, 250 µm thickness). Visualization was
accomplished by spraying with p-anisaldehyde spray reagent
followed by heating using a hot-air gun.
Compound Preparation. Preparation of Compound
16. To a stirred solution of 33 (75 mg, 0.27 mmol) in dry ether
(15 mL) and ethylene glycol (52 mg) was added BF3‚OEt2 (18
µL). The reaction mixture was left for 24 h, after which it was
quenched with 25 mL of 2% aqueous NaHCO3, and extracted
with ether (50 mL × 3). The combined ether extract was
washed with water, dried over Na2SO4, and concentrated
under reduced pressure. Column chromatography of the oily
crude product using a gradient of EtOAc in hexane (20% f
50%) afforded 16 (10 mg, gum): [R]D +160.7° (c 0.028, MeOH);
IR (film) νmax 3436 (br, OH), 2923, 2873, 1454, 1378, 1103,
1006, 986 cm-1; 1H NMR (C5D5N, 400 MHz) δ 5.93 (1H, s, H-5),
5.54 (1H, s, H-12), 5.12 (1H, s, H-13a), 4.94 (1H, s, H-13b),
4.12 (1H, t, J ) 5.2 Hz, H-16a), 4.00 (2H, q, J ) 4.9 Hz, H-17a,
H-17b), 3.79 (1H, q, J ) 5.1 Hz, H-16b), 2.32 (1H, m, H-7),
1.42 (3H, s, Me-15), 1.20 (2H, m, H-1, H-10), 0.76 (3H, d, J )
5.4 Hz, Me-14); 13C NMR (C5D5N, 100 MHz) δ 143.8 (C, C-11),
114.5 (CH2, C-13), 104.0 (C, C-4), 101.6 (CH, C-12), 88.3 (CH,
C-5), 81.5 (C, C-6), 66.6 (CH, C-16), 61.6 (CH2, C-17), 52.2 (CH,
C-1), 48.5 (CH, C-7), 37.3 (CH, C-10), 36.7 (CH2, C-3), 34.4
(CH2, C-9), 31.7 (CH2, C-8), 24.7 (CH2, C-2), 26.1 (CH3, C-15),
20.2 (CH3, C-14); HRESIFTMS m/z 349.1637 [M + Na]+ (calcd
for C17H26O6Na, 349.1621); Rf 0.17 (hexane-EtOAc, 1:1).
Preparation of Compounds 17 and 18. In a 100 mL
round-bottomed flask were introduced dihydroartemisinin (2)
(850 mg, 3.0 mmol) and dry ether (25 mL). The mixture was
stirred at room temperature, and cyclohexane-1,4-diol (mixture
of cis and trans) (170 mg) was added. To the stirred solution,
Table 1. Antifungal Activity of Artemisinin Derivatives
IC50/MIC (µg mL-1
)
compound
C. albicans
C. neoformans
1
8.0/50
9.0/-a
7.0/50
-
2.0/50
2
0.09/12.5
0.045/0.195
0.6/25
3
5
6
-
1.5/-
7
15/50
3.0/-
-/-
0.085/3.13
0.045/1.56
0.045/-
0.6/-
8
9
15
16
17
18
19
20
22
-/-
-/-
3.0/-
-/-
1.0/-
-/-
0.9/-
-/-
0.087/-
0.31/-
-/-
30.0/-
0.1/0.15
6.0/-
0.35/0.625
amphotericin B
a - ) inactive at highest test concentration of 50 µg mL-1
.
The rest of the compounds were totally inactive. In addi-
tion, the two aza-deoxyartemisinin derivatives 29 and 30
were also inactive. Compound 28, which carries a per-
oxide function, was weakly active, which suggests that
the presence of the typical tetracyclic trioxane skeleton
of artemisinin is crucial for activity. The requirement of
the peroxide function as part of the typical tetracyclic
trioxane skeleton of artemisinin and its derivatives for the
exhibition of antifungal activity was also observed as an
essential functionality for their antimalarial and cytotoxic
activities.9,10
It is interesting to note the inactivity of most of these
compounds for C. albicans. While both C. neoformans and

1276 Journal of Natural Products, 2005, Vol. 68, No. 8
Notes
BF3.OEt2 (570 µL) was then added by hypodermic syringe. The
stirring was continued for 80 min, after which time the
reaction was quenched and worked up as usual to leave a
gummy residue (1.13 g). The residue was loaded onto a silica
gel column (flash type, 270-400 mesh, 170 g) and eluted with
increasing amounts of EtOAc in hexane (15 f 50%). Fractions
of 5 mL were collected and similar fractions were pooled by
guidance of TLC to afford two compounds, 17 and 18. Com-
pound 17 (184 mg, gum): [R]D +180° (c 0.080, CHCl3); IR (film)
νmax 3478 (br, OH), 2937, 2870, 1447, 1364, 1193, 1136, 1098,
1029, 993 cm-1; 1H NMR (CDCl3, 500 MHz) δ 5.42 (1H, s, H-5),
4.87 (1H, d, J ) 3.4 Hz, H-12), 3.83 (1 H, m, H-16), 3.69 (1 H,
m, H-19), 2.62 (1H, m, H-11), 1.45 (1H, m, H-7), 1.42 (3H, s,
Me-15), 1.32 (1H, m, H-10), 1.23 (1H, m, H-1), 0.95 (3H, d, J
) 6.2 Hz, Me-14), 0.90 (3H, d, J ) 7.3 Hz, Me-13); 13C NMR
(CDCl3, 125 MHz) δ 104.4 (C, C-4), 100.3 (CH, C-12), 88.5 (CH,
C-5), 81.6 (C, C-6), 71.6 (CH, C-16), 69.2 (CH, C-19), 53.0 (CH,
C-1), 44.9 (CH, C-7), 37.9 (CH, C-10), 36.9 (CH2, C-3), 35.1
(CH2, C-9), 31.24 (CH, C-11), 31.2 (CH2, C-20), 30.7 (CH2,
C-21), 30.1 (CH2, C-18), 27.3 (CH2, C-17), 26.6 (CH3, C-15),
25.1 (CH2, C-8), 24.9 (CH2, C-2), 20.7 (CH3, C-14), 13.5 (CH3,
C-13); HRESIFTMS m/z 405.2226 [M + Na]+ (calcd for
C21H34O6 Na, 405.2253); Rf 0.17 (hexane-EtOAc, 6:4). Com-
pound 18 (110.5 mg, gum): [R]D +37.2° (c 0.086, CHCl3); IR
(film) νmax 3478 (br, OH), 2935, 2870, 1450, 1375, 1100, 1024,
984 cm-1; 1H NMR (CDCl3, 500 MHz) δ 5.40 (1H, s, H-5), 4.88
(1H, d, J ) 3.3 Hz, H-12), 3.70 (2H, m, H-16, H-19), 2.59 (1H,
m, H-11), 1.45 (1H, m, H-7), 1.40 (3H, s, Me-15), 1.30 (1H, m,
H-10), 1.25 (1H, m, H-1), 0.94 (3H, d, J ) 6.3 Hz, Me-14), 0.86
(3H, d, J ) 7.3 Hz, Me-13); 13C NMR (CDCl3, 125 MHz) δ 104.5
(C, C-4), 100.4 (CH, C-12), 88.4 (CH, C-5), 81.5 (C, C-6), 74.4
(CH, C-16), 69.6 (CH, C-19), 53.0 (CH, C-1), 44.9 (CH, C-7),
37.9 (CH, C-10), 36.8 (CH2, C-3), 35.1 (CH2, C-9), 31.0 (CH2,
C-18, C-20), 28.5 (CH2, C-17, C-21), 24.8 (CH2, C-2), 25.0 (CH2,
C-8), 26.6 (CH3, C-15), 20.7 (CH3, C-14), 13.4 (CH3, C-13);
HRESIFTMS m/z 405.2223 [M + Na]+ (calcd for C21H34O6 Na,
405.2253); Rf 0.10 (hexane-EtOAc, 6:4).
Antimicrobial Bioassay. Two opportunistic fungal strains
(C. albicans ATCC 90028 and C. neoformans ATCC 90113)
were used in the in vitro evaluation of antifungal activity.
Susceptibility testing was performed using a modified version
of the NCCLS methods. The microbial inocula were prepared
by diluting the subcultured organism in its incubation broth.
Test compounds were dissolved in DMSO, serially diluted
using normal saline, and transferred in duplicate to 96-well
microtiter plates. The microbial inoculum was added to achieve
a final volume of 200 µL and final concentrations starting with
50 µg/mL for pure compounds. Amphotericin B (ICN Biomedi-
cals, Aurora, OH) was used as a positive control, and blank
(media only) controls were added to each test plate. The plates
were read turbidimetrically at 630 nm using an EL-340
Biokinetics Reader (Biotek Instruments, Winooski, VT) prior
to and after incubation. The percent growth was calculated
and plotted versus concentration to afford the IC50/MIC.
Acknowledgment. The authors acknowledge the United
States Department of Agriculture, Agriculture Research Ser-
vices Specific Cooperative Agreement No. 58-6408-7-012, for
partial support of this work. The authors are also grateful to
Mr. F. T. Wiggers for the NMR measurements, Dr. C. Dunbar
for the HRESIFTMS data, and Ms. M. Logan for performing
the antimicrobial assays.
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1
1451, 1379, 1269, 1211, 1026, 932 cm-1; H NMR (C6D6, 400
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NP050074U