11-Hydroxymonocerin from the plant endophytic fungus Exserohilum rostratum

Article abstract of DOI:10.1021/np8004024

A new analogue of monocerin, 11-hydroxymonocerin (2), along with monocerin (1) and 12-hydroxymonocerin (3) were isolated from cultures of Exserohilum rostratum, a fungal strain endophytic in Stemona sp. The structure of 2 was determined by analysis of NMR and MS data and by comparison of spectroscopic data to those of 1. Monocerin (1) and 11-hydroxymonocerin (2) displayed activity against Plasmodium falciparum (K1, multidrug-resistant strain) with IC ₅₀ values of 0.68 and 7.70 μM, respectively. None of the compounds were cytotoxic against any of the tumor cell lines tested.

Full text of DOI:10.1021/np8004024

J. Nat. Prod. 2008, 71, 1657–1659
1657
11-Hydroxymonocerin from the Plant Endophytic Fungus Exserohilum rostratum
Ruengrit Sappapan,† Damrong Sommit,‡ Nattaya Ngamrojanavanich,§ Somchai Pengpreecha,§ Suthep Wiyakrutta,
Nongluksna Sriubolmas,*,| and Khanitha Pudhom*,§
Program in Biotechnology, Faculty of Science, Chulalongkorn UniVersity, Bangkok 10330 Thailand, Department of Chemistry, Faculty of
Science, Mahanakorn UniVersity of Technology, Bangkok 10530, Thailand, Research Centre for Bioorganic Chemistry, Department of
Chemistry, Faculty of Science, Chulalongkorn UniVersity, Bangkok 10330 Thailand, Department of Microbiology, Faculty of Science, Mahidol
UniVersity, Bangkok 10400, Thailand, and Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn UniVersity,
Bangkok 10330, Thailand
ReceiVed July 3, 2008
A new analogue of monocerin, 11-hydroxymonocerin (2), along with monocerin (1) and 12-hydroxymonocerin (3)
were isolated from cultures of Exserohilum rostratum, a fungal strain endophytic in Stemona sp. The structure of 2 was
determined by analysis of NMR and MS data and by comparison of spectroscopic data to those of 1. Monocerin (1) and
11-hydroxymonocerin (2) displayed activity against Plasmodium falciparum (K1, multidrug-resistant strain) with IC50
values of 0.68 and 7.70 µM, respectively. None of the compounds were cytotoxic against any of the tumor cell lines
tested.
Endophytic fungi have proven to be a rich source of bioactive
secondary metabolites.1-5 Exserohilum rostratum has been isolated
from both marine and terrestrial sources and is known as a marine
invertebrate pathogen6,7 and a cause of foot and root diseases,8
and its chemical study has led to the identification of phytotoxins
useful as herbicides.9 In the course of our search for biologically
active metabolites from endophytic fungi from Thai medicinal
plants, a subculture of an isolate of E. rostratum, obtained from
leaves of a Stemona sp., was cultivated in yeast extract sucrose
(YES) broth. An EtOAc extract of the culture showed significant
antimalarial activity against Plasmodium falciparum (K1, multidrug-
resistant strain). Herein, we report the isolation and structural
elucidation of a new isocoumarin derivative, 11-hydroxymonocerin
(2), together with known monocerin (1) and 12-hydroxymonocerin
(3). The compounds were evaluated for antiplasmodal (potential
antimalarial) and cytotoxic activities. This is a first report of the
potential antimalarial activity of monocerin (1) and its analogues.
The culture broth of E. rostratum grown in YES broth for 3
weeks was extracted with EtOAc, and the crude extract was purified
3.97 and 3.91). Analysis of 13C NMR and HSQC experiments
by column chromatography to yield compounds 1-3.
revealed the presence of a conjugated ester carbonyl (δC 167.6)
Monocerin (1), the major metabolite, was characterized by
strongly hydrogen-bonded with an OH, six aromatic carbons of
analyses of its spectroscopic data and by comparison with data
reported in the literature.10 Monocerin has been isolated as an
antifungal, insecticidal, and phytotoxic secondary metabolite from
several fungal species including Helminthosporium monoceras,11
Exserohilum turcum,12-14 Fusarium larVarum,15,16 and Micro-
dochium bolleyi.10
which three were oxygenated (δC 158.7, 156.2, and 137.4), and
one protonated (δC 104.6), four oxymethine (δC 81.8, 80.9, 75.0,
and 74.6), two methylene (δC 35.9 and 25.9), and a methyl (δC
1
9.9) carbon. The H and 13C NMR spectra were very similar to
those of monocerin (1), except for the marked differences in
chemical shift values corresponding to position 11. In the 1H NMR
spectrum of 2, the signal attributable to an oxygen-bearing methine
at δH 3.53 replaced those corresponding to the methylene signal of
1 at δH 1.55 and 1.66.
Compound 2 was isolated as a white solid and was optically
active. The molecular formula was determined to be C16H20H7 by
analysis of its HRESIMS (m/z 347.1128 [M + Na]+, ∆ -0.1 mmu),
implying seven double-bond equivalents. The IR spectrum showed
a strong broadened OH absorption band at 3431 cm-1 and
characteristic absorption bands for ester carbonyl (1665 cm-1) and
Treatment of 2 with acetic anhydride resulted in formation of
1
diacetate 5, and the H NMR spectrum of 5 revealed two acetate
1
aromatic ring (1521, 1455, and 1276 cm-1) groups. The H NMR
methyl singlets at δH 2.09 and 2.40, respectively. The shift in the
signal corresponding to H-11 (δH 4.92) indicated that one OH group
was attached to C-11 in 2. The proposed structure was confirmed
by a proton spin system from H-4 to H3-13, established by 1H-1H
correlation observed in the COSY spectrum and by HMBC
correlations (Figure 1) of H-4 to δC 130.6 (C-4a), 101.9 (C-8a),
104.5 (CH-5), and 80.9 (CH-3) as well as H-3 to δC 38.9 (CH2-9).
The full assignments and connectivity were determined by 1H-1H
COSY correlations as indicated by bold lines and HMBC correla-
tions shown by arrows (Figure 1). By analysis of NOESY data,
the compound exhibited NOEs between H-3 and H-4 and between
spectrum showed a chelated OH signal (δH 11.24, OH-8) and signals
for an aromatic proton (δH 6.61), four oxygen-bearing methine
groups (δH 5.10, 4.63, 4.02, and 3.53), and two OCH3 groups (δH
* To whom correspondence should be addressed. Tel: 66-2-218-7639.
Fax: 66-2-218-7598. E-mail: Khanitha.P@chula.ac.th.
† Program in Biotechnology, Chulalongkorn University.
‡ Mahanakorn University of Technology.
§ Faculty of Science, Chulalongkorn University.
Mahidol University.
| Faculty of Pharmaceutical Sciences, Chulalongkorn University.
10.1021/np8004024 CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/06/2008

1658 Journal of Natural Products, 2008, Vol. 71, No. 9
Notes
NMR spectra were recorded on a Varian YH400 spectrometer at 400
1
MHz for H NMR and at 100 MHz NMR for 13C NMR using TMS
(tetramethylsilane) as internal standard.
Isolation of Endophytic Fungi. Healthy leaves and roots of a
Stemona sp. were collected from Amphur Bangban, Ayutthaya Province,
Thailand, in June 2007. Plant samples were washed in tap water and
air-dried. The cleaned leaf and root fragments were surface-sterilized
as described by Schulz and co-workers19 with some modifications. Plant
fragments were sequentially immersed in 70% EtOH for 1 min, 6%
NaOCl solution for 5 min, and sterile distilled H2O for 1 min (two
times). Then, the surface-sterilized fragments were cut into small pieces
(ca. 5 mm in length) using a sterile blade and placed on sterile water
agar plates for further incubation at 30 °C. The hyphal tip of the
endophytic fungus growing out from the plant tissue was cut by a sterile
pipet and transferred onto a potato dextrose agar (PDA) plate. After
incubation at 30 °C for 7-14 days, culture purity was determined from
colony morphology.
Figure 1. Key HMBC and COSY correlations for 2.
Identification of Endophyte. The fungal endophyte isolate (stem3)
was identified on the basis of both morphology of the fungus grown
on banana leaf agar at 25 °C and analysis of the DNA sequences of
the ITS region of the rRNA gene. The fungus grew on PDA as brown
filamentous colonies, and they developed characteristic brown spores.
A GenBank search for sequences similar to their ITS region revealed
Exserohilum rostratum in the family Pleosporaceae as the closest
matches, with sequence identity of 99%. The culture of isolate stem3
(accession number EU571210) has been deposited at the Department
of Microbiology, Mahidol University, Thailand.
Figure 2. ∆δ values (in ppm) ) δS - δR for (S)- and (R)-MTPA
esters in 2a and 2b.
Table 1. In Vitro Antimalarial Activity of Compounds 1, 2, 4,
and 5 against P. falciparum
Fermentation, Extraction, and Isolation. The endophytic fungus
Exserohilum rostratum was cultured in 1000 mL Erlenmeyer flasks
(×25) containing 200 mL of yeast extract sucrose (YES) broth at 30
°C for 21 days under static conditions. The fungal cells were separated
from the broth by filtration, and a culture broth was subsequently
extracted with EtOAc (equal volume × 3), yielding 4.10 g of crude
extract. The extract was subjected to SiO2 column chromatography (CC)
eluted with hexane-EtOAc and MeOH-CH2Cl2 mixtures of increasing
polarity to afford eight fractions (I-VIII). Fraction II was rechromato-
graphed over silica gel eluted with EtOAc-hexane (2:3) to afford
monocerin (1, 764.2 mg). Fraction IV was further subjected to flash
CC on silica gel (benzene-EtOAc, 1:1) to yield compound 2 (165.4
mg). Fraction V was rechromatographed by flash CC on SiO2 eluted
with benzene-EtOAc (3:2), followed by preparative TLC (MeOH-
CH2Cl2, 1:19) to give compound 3 (3.6 mg).
compound
IC50 (µM)
1
2
4
5
0.68
7.70
0.82
9.10
dihydroartemisinina
a Standard antimalarial drug.
4 × 10-3
H-4 and H-10, indicating that these protons are all on the same
face of the ring system.
The absolute configuration of 2 was assigned by application of
the modified Mosher method.17,18 Treatment of 2 with (S)- and
(R)-MTPA Cl afforded the (R)-MTPA ester (2a) and (S)-MTPA
ester (2b), respectively. The difference in chemical shift values (∆δ
) δS - δR) for the diastereomeric esters 2b and 2a was calculated
in order to assign the absolute configuration at C-11. Calculation
for all relevant signals suggested the R absolute configuration at
C-11, as shown in Figure 2. Therefore, compound 2 was determined
to be 11(R)-hydroxymonocerin.
Compound 3 had the same molecular formula as 2, C16H20H7.
Comparison of the optical rotation and its NMR spectroscopic data
with those in the literature10 indicated that compound 3 is 12(R)-
hydroxymonocerin.
Monocerin (1), 11-hydroxymonocerin (2), and their acetylated
products 4 and 5 were evaluated for antiplasmodial activity against
the multidrug-resistant K1 strain of Plasmodium falciparum.
Monocerin exhibited antiplasmodial activity (IC50 value of 0.68
µM). Activity of the 11-hydroxy analogue (2) was 10-fold lower,
indicating that an additional OH group in the n-propyl chain reduces
the activity (Table 1). Conversion of the OH groups to acetyl esters
(4 and 5) did not show any significant effect on their activity. The
compounds were also tested for cytotoxicity against five human
tumor cell lines: BT474, CHAGO, Hep-G2, KATO-3, and SW-
620. None were cytotoxic at a concentration of 20 µg/mL.
Monocerin (1): colorless oil; 1H NMR (400 MHz, CDCl3) δ 11.25
(1H, s, 8-OH), 6.57 (1H, s, H-5), 5.03 (1H, brs, H-3), 4.52 (1H, brs,
H-4), 4.08 (1H, m, H-10), 3.92 (3H, s, 6-OCH3), 3.86 (3H, s, 7-OCH3),
2.58 (1H, m, H-9ꢀ), 2.12 (1H, dd, J ) 14.4, 5.2 Hz, H-9R), 1.66 (1H,
m, H-11a), 1.55 (1H, m, H-11b), 1.38 (2H, m, H2-12), 0.88 (3H, t, J )
7.0 Hz, H3-13); 13C NMR (100 MHz, CDCl3) δ 167.8 (C, C-1), 158.7
(C, C-6), 156.2 (C, C-8), 137.2 (C, C-7), 131.2 (C, C-4a), 104.9 (CH,
C-5), 101.9 (C, C-8a), 81.3 (CH, C-3), 78.7 (CH, C-10), 74.7 (CH,
C-4), 60.7 (CH3, 7-OCH3), 56.2 (CH3, 6-OCH3), 39.0 (CH2, C-9), 38.0
(CH2, C-11), 19.1 (CH2, C-12), 13.9 (CH3, C-13); HRESIMS m/z
331.1151 [M + Na]+ (calcd for C16H20O6 Na, 331.1158).
11-Hydroxymonocerin (2): white solid; mp 118-121 °C; [R]20
D
+50.0 (c 0.1, in EtOH); UV (EtOAc) λmax (log ε) 308 (3.78), 273 (4.23)
nm; IR (KBr) νmax 3431, 2930, 1665, 1455, 1276, 1117 cm-1; 1H NMR
(400 MHz, CDCl3) δ 11.24 (1H, s, 8-OH), 6.61 (1H, s, H-5), 5.10
(1H, brs, H-3), 4.63 (1H, brs, H-4), 4.02 (1H, m, H-10), 3.97 (3H, s,
6-OCH3), 3.91 (3H, s, 7-OCH3), 3.53 (1H, brs, H-11), 2.57 (1H, m,
H-9ꢀ), 2.27 (1H, dd, J ) 14.4, 5.0 Hz, H-9R), 1.53 (1H, m, H-12a),
1.45 (1H, m, H-12b), 1.02 (3H, t, J ) 6.8 Hz, H3-13); 13C NMR (100
MHz, CDCl3) δ 167.6 (C, C-1), 158.7 (C, C-6), 156.2 (C, C-8), 137.4
(C, C-7), 130.6 (C, C-4a), 104.6 (CH, C-5), 101.9 (C, C-8a), 81.8 (CH,
C-10), 80.9 (CH, C-3), 75.0 (CH, C-11), 74.6 (CH, C-4), 60.8 (CH3,
7-OCH3), 56.3 (CH3, 6-OCH3), 35.9 (CH2, C-9), 25.9 (CH2, C-12),
9.9 (CH3, C-13); HRESIMS m/z 347.1106 [M + Na]+ (calcd for
C16H20O7 Na, 347.1107).
Experimental Section
Acetylation of 1 and 2. Acetic anhydride (0.3 mL) and DMAP
(catalytic amount) were added to a solution of monocerin (1) (10 mg)
in pyridine, and the mixture was left stirring at room temperature for
5 h. After removing the solvent under reduced pressure, acetate 4 was
General Experimental Procedures. Optical rotations were measured
on a Perkin-Elmer 341 polarimeter using a sodium lamp at wavelength
589 nm, and UV data were recorded on a Varian 50Probe UV/vis
spcectrophotometer. Melting points were measured using a Fisher-Johns
melting point apparatus. IR spectra were recorded on a Perkin-Elmer
model 1760X Fourier transform infrared spectrophotometer. HRESIMS
spectra were obtained using a Micromass LCT mass spectrometer. The
purified by CC on silica gel with EtOAc-hexane (3:1); 95% yield (10.8
1
mg): colorless oil; [R]20 -3.0 (c 0.1, in EtOH); H NMR of 4 (400
D
MHz, CDCl3) δ 6.89 (1H, s, H-5), 4.96 (1H, brs, H-3), 4.54 (1H, d,
J ) 3.2 Hz, H-4), 4.11 (1H, m, H-10), 3.94 (3H, s, 6-OCH3), 3.82

Notes
Journal of Natural Products, 2008, Vol. 71, No. 9 1659
(3H, s, 7-OCH3), 2.51 (1H, m, H-9ꢀ), 2.39 (3H, s, 8-OCOCH3), 2.10
(1H, dd, J ) 14.4, 5.6 Hz, H-9R), 1.65 (1H, m, H-11a), 1.53 (1H, m,
H-11b), 1.34 (2H, brm, H2-12), 0.88 (3H, t, J ) 7.2 Hz, H3-13); 13C
NMR (100 MHz, CDCl3) δ 168.8 (C, 8-OCOCH3), 159.5 (C, C-1),
157.5 (C, C-6), 145.5 (C, C-8), 142.6 (C, C-7), 132.2 (C, C-4a), 109.8
(C, C-8a), 109.5 (CH, C-5), 79.4 (CH, C-3), 78.4 (CH, C-10), 74.0
(CH, C-4), 60.7 (CH3, 7-OCH3), 55.8 (CH3, 6-OCH3), 38.5 (CH2, C-9),
37.6 (CH2, C-11), 20.5 (CH3, 8-OCOCH3), 18.6 (CH2, C-12), 13.5 (CH3,
C-13); HRESIMS m/z 373.1260 [M + Na]+ (calcd for C18H22O7 Na,
373.1263).
In a similar fashion, compound 2 (10 mg), acetic anhydride (0.3
mL), and DMAP (catalytic amount) in pyridine (1 mL) were allowed
to react at room temperature for 5 h, and the reaction mixture was
processed as described above for 4 to afford diacetate 5 (12.1 mg, 96%
yield): [R]20D +24.0 (c 0.1, in EtOH); 1H NMR of 5 (400 MHz, CDCl3)
δ 6.90 (1H, s, H-5), 4.98 (1H, brs, H-3), 4.92 (1H, m, H-11), 4.59
(1H, d, J ) 3.6 Hz, H-4), 4.26 (1H, m, H-10), 3.97 (3H, s, 6-OCH3),
3.84 (3H, s, 7-OCH3), 2.48 (1H, m, H-9ꢀ), 2.40 (3H, s, 11-OCOCH3),
2.21 (1H, dd, J ) 14.4, 6.0 Hz, H-9R), 2.09 (3H, s, 8-OCOCH3), 1.62
(2H, m, H2-12), 0.87 (3H, t, J ) 7.6 Hz, H3-13); 13C NMR (100 MHz,
CDCl3) δ 171.5 (C, 11-OCOCH3), 169.5 (C, 8-OCOCH3), 160.0 (C,
C-1), 158.0 (C, C-6), 146.0 (C, C-8), 143.2 (C, C-7), 132.2 (C, C-4a),
110.4 (C, C-8a), 110.0 (CH, C-5), 78.9 (CH, C-3), 78.7 (CH, C-10),
74.7 (CH, C-11), 74.4 (CH, C-4), 61.2 (CH3, 7-OCH3), 56.3 (CH3,
6-OCH3), 35.6 (CH2, C-9), 23.7 (CH2, C-12), 21.0 (CH3, 8-OCOCH3),
21.0 (CH3, 11-OCOCH3), 9.9 (CH3, C-13); HRESIMS m/z 431.1321
[M + Na]+ (calcd for C20H24O9 Na, 431.1318).
CHAGO (lung carcinoma), Hep-G2 (hepatocarcinoma), KATO-3
(gastric carcinoma), and SW-620 (colon carcinoma), using the MTT
[3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide] colori-
metric method.22
Acknowledgment. Financial support from The Asahi Glass Founda-
tion, Japan, and The 90th Anniversary of Chulalongkorn University
Fund (Ratchadaphisek Somphot Endowment Fund) is gratefully
acknowledged. We also thank the National Center of Excellence for
Petroleum, Petrochemicals, and Advanced Materials, NCE-PPAM, for
partial support and Ms. S. Harntanong, Program in Biotechnology,
Faculty of Science, Chulalongkorn University, for cytotoxicity assays.
References and Notes
(1) Schulz, B.; Boyle, C.; Draeger, S.; Rommert, A.-K.; Krohn, K. Mycol.
Res. 2002, 106, 996–1004.
(2) Strobel, G. A. Microbes Infect. 2003, 5, 535–544.
(3) Wijeratne, E. M. K.; Paranagama, P. A.; Marron, M. T.; Gunatilaka,
M. K.; Arnold, A. E.; Gunatilaka, A. A. L. J. Nat. Prod. 2008, 71,
218–222.
(4) Wu, S.-H.; Chen, Y.-W.; Shao, S.-C.; Wang, L.-D.; Li, Z.-Y.; Li, S.-
L.; Huang, R. J. Nat. Prod. 2008, 71, 731–734.
(5) Ding, G.; Liu, S.; Guo, L.; Zhou, Y.; Che, Y. J. Nat. Prod. 2008, 71,
615–618.
(6) Alker, A. P.; Smith, G. W.; Kim, K. Hydrobiologia 2001, 460, 105–
111.
(7) De, R.; Purkait, R.; Pal, A. K.; Purkayastha, R. P. Indian J. Exp. Biol.
1999, 37, 706–709.
(8) Giha, O. H. Niger. J. Plant Prot. 1977, 3, 167–177.
(9) Tan, R. X.; Jensen, P. R.; Williams, P. G.; Fenical, W. J. Nat. Prod.
2004, 67, 1374–1382.
(10) Zhang, W.; Krohn, K.; Draeger, S.; Schulz, B. J. Nat. Prod. 2008,
71, 1078–1081.
(11) Aldridge, D. C.; Turner, W. B. J. Soc., Sect. C 1970, 18, 2598–2600.
(12) Robenson, D. J.; Strobel, G. A. Agric. Biol. Chem. 1982, 46, 2681–
2683.
Preparation of (R)-MTPA Ester (2a) and (S)-MTPA Ester (2b).
A reaction mixture of 2 (2 mg), (S)- or (R)-MTPA Cl (20 µL), and
DMAP (catalytic amount) in pyridine (0.25 mL) was stirred at room
temperature overnight. After removing the solvent under reduced
pressure, the (R)- and (S)-MTPA esters (2a and 2b) were purified by
mini-column chromatography on silica gel with EtOAc (1:4).
1
Compound 2a: H NMR of 2R-MTPA ester (400 MHz, CDCl3) δ
(13) Cuq, F.; Herrmann-Gorline, S.; Klabe, A.; Rossignol, M.; Petitprez,
M. Phytochemistry 1993, 34, 1265–1270.
(14) Lim, C.-H. Agric. Chem. Biotechnol. (Engl. Ed.) 1999, 42, 45–47.
(15) Grove, J. F.; Pople, M. J. Chem. Soc., Perkin Trans. 1 1979, 2048–
2051.
6.57 (1H, s, H-5), 5.25 (1H, m, H-11), 5.03, (1H, m, H-3), 4.37 (1H,
d, J ) 3.6 Hz, H-4), 4.24 (1H, m, H-10), 3.98 (3H, s, 6-OCH3), 3.91
(3H, s, 7-OCH3), 2.56 (1H, m, H-9ꢀ), 2.15 (1H, dd, J ) 14.0, 6.2 Hz,
H-9R), 2.10 (1H, m, H-12a), 1.74 (1H, m, H-12b), 1.17 (3H, t, J ) 7.2
Hz, H3-13).
(16) Claydon, N.; Grove, J. F.; Pople, M. J. InVertebr. Pathol. 1979, 33,
1
364–367.
Compound 2b: H NMR of 2S-MTPA ester (400 MHz, CDCl3) δ
(17) Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512–519.
(18) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Org. Chem.
1991, 56, 1296–1298.
(19) Schulz, B.; Sucker, J.; Aust, H. J.; Krohn, K.; Ludewig, K.; Jone,
P. G.; Doring, D. Mycol. Res. 1995, 99, 1007–1015.
(20) Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D.
Antimicrob. Agents Chemother. 1979, 16, 710–718.
(21) Trager, W.; Jensen, J. B. Science 1976, 193, 673–675.
(22) Twentyman, P. R.; Luscombe, M. A. Br. J. Cancer. 1987, 56, 279–
285.
6.58 (1H, s, H-5), 5.23 (1H, m, H-11), 4.97, (1H, m, H-3), 4.50 (1H,
d, J ) 3.2 Hz, H-4), 4.12 (1H, m, H-10), 3.96 (3H, s, 6-OCH3), 3.94
(3H, s, 7-OCH3), 2.29 (1H, m, H-9ꢀ), 2.12 (1H, m, H-12a), 2.04 (1H,
dd, J ) 14.2, 5.8 Hz, H-9R), 1.92 (1H, m, H-12b), 1.26 (3H, t, J ) 7.0
Hz, H3-13).
Biological Assays. Antimalarial activity in vitro was determined by
means of the microculture radioisotope technique based on the method
decribed by Desjardins.20 The parasite P. falciparum (K1, multidrug-
resistant strain) was cultured continuously according to the method of
Trager and Jensen.21 An IC50 value of 4.0 × 10-3 µM (n ) 3) was
observed for the positive control, dihydroartemisinin. Cytotoxicity was
assessed against human cell cultures, BT474 (breast carcinoma),
(23) Carmichael, J.; DeGraff, W. G.; Gazdar, A. F.; Minna, J. D.; Mitchell,
B. Cancer Res. 1987, 47, 936–942.
NP8004024