Y. Zang et al. / Phytochemistry 119 (2015) 70–75
73
OH
4
OH
CH3
O
CH3
O
HO
O
OH
15
HO
O
OH
11
3
O
O
O
O
O
2
CH3
1
9
10
OH
OH
OH
O
O
HO
O
OH
HO
O
OH
O
O
O
O
11
12
Fig. 4. Structures of compounds 9–12. Key 1H–1H COSY (bold bonds) and 1H–13C HMBC correlations (arrows) for compound 9.
transesterification with sodium ethanolate of 9 to afford ethyl
3-hydroxybutyrate 12 which was analyzed by GC on chiral column.
The retention time of 12 was thus compared to racemic mixture
and to the (S) enantiomer obtained by baker’s yeast reduction of
ethyl acetoacetate (Wipf et al., 1983). The absolute configurations
of talapolyester G 9 was thus determined as (11R, 15R) which is
in accordance with the previously described 15G256 polyesters.
We thus isolated some new compounds from the strain
T. stipitatus ATCC10500, cultivated in PDB liquid medium, never
described until now. In our hands, out of all the liquid or solid
growth media we have tested, these compounds could only be iso-
lated from the PDB liquid medium. Thus, this work is another
example of the deep dependence of fungal metabolic expression
on culture conditions (the OSMAC strategy). Again, the plasticity
of the production of chemically diverse compounds by a given
strain highlights the biotechnological value of filamentous fungi.
All compounds were evaluated for their antiplasmodial activity
against the chloroquine-resistant P. falciparum (FcB1 strain).
Compound 7 displayed the best inhibition against P. falciparum
spectra were recorded in MeOH using a JASCO J-810. IR spectra were
taken on a Shimadzu FTIR-8400S Infrared spectrophotometer. UV
spectra were recorded on a Kontron Uvikon 9X3W Double Beam
UV/vis spectrophotometer (Bioserv, France). Mass spectra were
recorded on an API Q-STAR PULSAR i of Applied Biosystem. For the
CID spectra, the collision energy was 40 eV and the collision gas
was nitrogen. All NMR experiments were recorded on Bruker
Avance III HD 400 MHz and 600 MHz spectrometers (Wissembourg,
France) equipped with a BBFO Plus Smartprobe and a triple reso-
nance TCI cryoprobe, respectively. Chemical shifts are expressed
in d (ppm), and are referenced to the residual non-deuterated sol-
vent signals. Preparative HPLC was performed on an Agilent system
and an Agilent PrepHT XDB-C18 column (21.2 ꢁ 150 mm i.d.; 5
USA). Column chromatography (CC) was performed using silica gel
(Lichroprep RP-18, 40–63 m, Merck KGaA, Germany).
lm;
l
4.2. Fermentation, extraction and isolation
T. stipitatus ATCC 10500 strain was grown in sterilized liquid
media PDB at 27 °C under agitation on a rotary shaker (150 rpm).
After 14 days of cultivation, the culture was filtered to separate
the mycelium and the filtrate. The culture filtrate (3 L) was then
extracted by ethyl acetate (3 ꢁ 1 L) and the combined organic
phases were dried and gave, after removal of the solvent under
vacuum, 1.3 g crude extract.
with an IC50 value of 19 lM. Interestingly, this compound was
not cytotoxic on HeLa cells as well as on 3T3-L1 preadipose cell
lines model from mouse embryo.
3. Conclusion
This crude extract was subjected to preparative HPLC which
afforded compound 6 (5.2 mg) and 7 (4.5 mg) (gradient from 10%
to 60% acetonitrile in H2O for 42 min; flow rate: 20.0 mL/min; 6,
tR = 13.0 min; 7, tR = 14.0 min). Subfractions were further purified
by preparative HPLC (from 10% to 30% MeCN in H2O for 33 min,
flow rate: 20.0 mL/min) and afforded pure compounds 5 (2.1 mg,
tR = 15.4 min), 8 (1.2 mg, tR = 14.0), 1 (13.3, mg, tR = 14.0) and 9
(4.8 mg, tR = 21.9).
Chemical investigation of the fungus T. stipitatus ATCC10500 in
PDB liquid medium led to the isolation of the four new talaroe-
namines BꢀE. Interestingly, talaroenamine A had previously been
isolated from the strain of T. stipitatus
DtropC but never from a
wild-type strain. In the course of this study, a new polyester was
also identified. All the compounds were evaluated for their
antiplasmodial activity and some of them displayed a modest
activity against P. falciparum (IC50: 19.2 lM) without noticeable
toxicity against preadipocytes and Hela cells (IC50 > 100 lM).
4.2.1. Talaroenamine B (5): Pale yellow oil; [a D20
MeOH); ECD
]
ꢀ19.7 (c 0.0033,
4. Experimental
(c 0.390 ꢁ 10ꢀ3 mol/L, MeOH, 20 °C) kmax
(De) 230 (0.16), 330
(1.3), 372 (ꢀ1.12); UV (MeCN) kmax nm (log
e): 240 (3.5), 375
4.1. General
(3.9); IR (NaCl) mmax cmꢀ1: 3419, 3252, 2983, 2926, 1687, 1616,
1566, 1504, 1456, 1398, 1338, 1307, 1184, 1080, 1024, 837, 738,
688; 1H and 13C NMR data (DMSO-d6) see Table 1; (ꢀ) HR-ESI-MS:
m/z 256.0980 [MꢀH]ꢀ.
Optical rotations were determined using a Perkin Elmer 341
Polarimeter and the [
values are given in deg cm2 gꢀ1. ECD
a]
D