Torrubiellutins A-C, from insect pathogenic fungus Torrubiella luteorostrata BCC 12904

Article abstract of DOI:10.1016/j.tet.2009.05.070

Investigation of the insect pathogenic fungus Torrubiella luteorostrata led to the isolation of three new macrocyclic torrubiellutins A-C (1-3) and the known pyrone diterpene 4. Structures were elucidated by spectroscopic data including 1D, 2D NMR, and MS

Full text of DOI:10.1016/j.tet.2009.05.070

Tetrahedron 65 (2009) 6069–6073
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Tetrahedron
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Torrubiellutins A–C, from insect pathogenic fungus Torrubiella
luteorostrata BCC 12904
*
Pattama Pittayakhajonwut , Atit Usuwan, Chakapong Intaraudom, Punsa Khoyaiklang,
Sumalee Supothina
National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Paholyothin Road, Klong Luang, Pathumthani 12120 Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Investigation of the insect pathogenic fungus Torrubiella luteorostrata led to the isolation of three new
macrocyclic torrubiellutins A–C (1–3) and the known pyrone diterpene 4. Structures were elucidated by
spectroscopic data including 1D, 2D NMR, and MS spectral data. The absolute stereochemistry was
determined by chemical means using Mosher reactions and Marfey’s reagent, together with NOESY
spectral data. Torrubiellutin C showed biological activities against KB, MCF-7, NCI-H187, and Vero cell
Received 18 February 2009
Received in revised form 4 May 2009
Accepted 21 May 2009
Available online 3 June 2009
lines with IC50 varying from 0.78 to 4.36
flammatory activity with IC50 values of 3.49 and 1.21
m
g/mL, while compound 4 exhibited antimalaria and anti-in-
g/mL, respectively.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
m
Torrubiella luteorostrata
Macrocyclic compounds
Torrubiellutin
Pyrone diterpene
Antimalarial
Anti-inflammatory
1. Introduction
Compound 1, obtained as a colorless solid, the molecular for-
mula of which was determined as C26H37NO5 based on HRESIMS
Insect fungi have consistently been shown to be a good source of
bioactive metabolites.1 Several compounds isolated from insect fungi
in our BIOTEC Culture Collection were described together with their
biological activities. As a part of our on-going search for bioactive
substances from microorganisms, the crude extracts of the insect
fungus Torrubiella luteorostrata BCC 12904 exhibited biological ac-
tivity against human breast cancer (MCF-7) and human epidermoid
showing m/z peak at 466.2577 [MþNa]þ,
D
þ1.3 mmu. 13C NMR
spectrum together with DEPT-135 spectral data gave 24 signals of 6
methyl, 1 methylene, 13 methine, and 4 quaternary carbons. Two
aromatic methine signals at dC 128.2 and 129.3 contained two
carbons each. COSY and HMBC spectra gave, respectively, 1H–1H
and 1H–13C correlations as shown in Table 1. The spectral in-
formation gave the partial structures from C-1 to C-11 and from
C-14 to aromatic ring. The correlation of methyl at dH 2.79 to the C-1
in HMBC spectrum indicated an amide linkage. In addition, the
methine at H-11 (dH 5.05), suggesting a connection to an oxygen,
together with the remaining carbon at dC 170.4 (C-13) led to the
chemical structure of compound 1, named as torrubiellutin A.
Torrubiellutin A (1) is a macrocyclic compound containing an
amino acid unit. The configuration of amino acid, N-methyl-phe-
nylalanine (NMePhe), was determined by acid hydrolysis of 1 with
6 N HCl and then derivatization with Marfey’s reagent (FDAA).3,4
carcinoma (KB) with IC50 values of 1.03–10.37 and 1.27–15.52 mg/mL,
respectively. Further investigation led to the isolation of new mac-
rocyclic torrubiellutins A–C (1–3), and the pyrone diterpene 4. Bio-
logical activity of isolated compounds was also evaluated.
2. Results and discussion
The extract obtained from culture broth was subjected to
Sephadex LH20 chromatography followed by either silica column
or reversed-phase HPLC to afford pure torrubiellutins A (1) and B
(2). In addition, purification of the mycelium extract yielded zeorin
[C30H52O2, 215–217 ꢀC],2 torrubiellutins B (2) and C (3), and pyrone
diterpene (4).
The sample was co-injected with
NMePhe ( - and -forms) and analyzed by reversed-phase HPLC.
The result indicated the -form of NMePhe. The molecule has
L-FDAA derivatives of standard
D
L
L
stereogenic centers at C-4, C-5, C-8, C-9, C-10, and C-11. The abso-
lute configurations at C-5 and C-9 were assigned by employing the
Mosher method.5 Treatment of 1 with R- and S-MTPACl afforded
compounds 5 and 6, respectively. The differences in chemical shift
values (DdR–S) of diester derivatives 5b and 6b (given in Fig. 1) were
* Corresponding author. Tel.: þ66 25646700x3559; fax: þ66 25646632.
E-mail address: pattamap@biotec.or.th (P. Pittayakhajonwut).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.05.070

6070
P. Pittayakhajonwut et al. / Tetrahedron 65 (2009) 6069–6073
Table 1
conformation shown in Figure 2. The absolute configuration at C-11
was assigned as S- based on the evidence from NOESY correlation
between H-3 and H-10 and the coupling constant of 3.7 Hz be-
tween H-10 and H-11. The dihedral angle of ꢁ85.8ꢀ was also cal-
culated based on the conformation shown in Figure 2. Therefore, six
stereogenic centers can now be assigned as 4R, 5S, 8S, 9R, 10R, and
11S, respectively. The chemical structure of torrubiellutin A (1) can
be depicted as shown in Figure 3.
1D and 2D NMR assignment of torrubiellutin A in acetone-d6
dH (int., mult., J in Hz)a
dC
COSY
HMBC (H/C#)
b
No.
1
2
3
4
5
6
7
8
d
166.5
119.2
149.1
39.5
d
d
6.28 (1H, d, 15.0)
7.07 (1H, dd, 15.0, 10.3)
2.68–2.71 (1H, m)
3.94 (1H, d, 5.1)
d
d
1, 4
1
18
6, 7
d
d
H-5
d
77.7
136.0
122.8
34.2
d
Compound 2, a colorless solid, gave similar 1H NMR and 13C
NMR spectral data to compound 1. The 1H NMR spectrum dis-
played two additional methyl protons at dH 2.11 and 2.16, which
correlated in the HMBC spectrum to the carbonyl esters at dC
170.26 and 169.20, respectively. The data indicated the presence
of two additional acetyl groups in the molecule. Also the lower
field shift of two methine protons at dH 4.72 (H-9) and 5.07 (H-5)
5.66 (1H, dd, 11.0, <1)
2.58–2.62 (1H, m)
d
5, 19
d
H-7
d
9
3.15 (1H, ddd, 11.0, 9.3, 1.1) 76.4
7
d
10
11
13
14
16
1.56–1.58 (1H, m)
5.05 (1H, dq, 6.5, 3.7)
d
41.3
d
72.6
170.4
67.7
35.2
d
d
d
d
3.74 (1H, dd, 10.5, 4.4)
3.24 (1H, dd, 13.8, 4.4),
3.33 (1H, dd, 13.8, 10.5)
2.79 (3H, s)
1.18 (3H, d, 7.0)
1.56 (3H, d, 0.9)
1.01 (3H, d, 6.8)
0.69 (3H, d, 6.8)
1.10 (3H, d, 6.5)
d
d
d
10, 20, 60
H-14
(
Dd¼1.57 and 1.13 ppm, respectively) suggested the two acetyl
17
18
19
20
21
22
10
38.2
18.8
14.5
17.8
9.3
d
1
groups being substituted at H-9 and H-5, respectively. HRESIMS
H-4
d
3, 4, 5
5, 6, 7
7, 8, 9
9, 10, 11
10, 11
d
confirmed the molecular formula C30H41O7N showing m/z peak at
550.2784 [MþNa]þ,
D
þ0.9 mmu. The methine protons at H-5
H-8
H-10
H-11
d
and H-9 also appeared as a singlet and a double doublet (J¼11.5
and 2.4 Hz), respectively. Irradiation at H-10 resulted in a cou-
pling constant of 2.4 Hz, suggesting a gauche conformation
between H-8 and H-9. The result indicated an angle distortion of
H-8 and H-9 in order to avoid steric effect from the acetyl group.
The cross-peak correlation from H-9 to H3-20 was also observed
in NOESY spectrum. Therefore, the chemical structure of com-
pound 2, named as torrubiellutin B, was established as shown in
Figure 3. Although, torrubiellutin B was isolated as a major
product, it could not be obtained as a single crystal for X-ray
crystallographic study.
12.6
139.6
20/60 7.19–7.30 (2H, m)
129.3ꢂ2 H-30, H-50
40, 16
30/50 7.19–7.30 (2H, m)
128.1ꢂ2 H-20, H-40, H-60 10
40
7.19–7.30 (1H, m)
126.1
d
d
a
Recorded at 500 MHz.
Recorded at 125 MHz.
b
calculated in order to assign the absolute configurations at C-5 and
C-9 as S- and R-, respectively. Due to the limited amount of com-
pound 1, the absolute configurations of C-4, C-8, C-10, and C-11
were determined by NOESY spectral data and coupling constant
values (J). In the presence of D2O, H-5 and H-9 appeared as a singlet
and double doublet (J¼11.0, <1 Hz), respectively. The former
indicated no coupling between H-4 and H-5. In addition, the con-
formation of H-4 and H-5 was restricted by the configuration of two
olefins at C-2 and C-6. The olefinic proton at H-2 coupled to H-3
with a coupling constant of 15.0 Hz indicated a trans-configuration
and the cross-peak correlation showing in NOESY spectrum
between H-7 and H3-19 indicated a cis-configuration of the olefin at
C-6. Moreover, the NOESY spectral data displayed cross-peak cor-
relations from H-2 to H-4; H-3 to H-10; H-4 to H3-19, suggesting R-
configuration at C-4. The double doublet of H-9 (J¼11.0, <1 Hz) was
changed to a doublet with a coupling constant of 11.0 Hz after ir-
radiation at H-8. Thus, it was implied that the coupling constant
between H-9 and H-10 is 11.0 Hz and that between H-8 and H-9 is
small (J<1 Hz). Together with the cross-peak in NOESY spectrum,
which correlated H-9 to H3-21; H-8 to H3-19 and H3-21, this
indicated an anti-relationship between H-9 and H-10 and a syn-
relationship between H-8 and H-9. The absolute configurations at
C-8 and C-10 can therefore be assigned as S- and R-, respectively.
The coupling constant agreed with the dihedral angle of 95.2ꢀ be-
tween H-8 and H-9 as calculated from the most favorable
Figure 2. 3D structural conformation of torrubiellutin A generated by MM2 force field
calculations for energy minimization from modeling program Chem3D Ultra 9.0 with
the observed NOE correlations (arrows).
+0.17
Compound 3, named as torrubiellutin C, was obtained as
a colorless solid. 1H NMR and 13C NMR spectral data are similar to
those of 1 except for an extra methyl group at dH 2.17, which
correlated in HMBC to the carbonyl ester at dC 171.27. This in-
dicated the presence of an acetyl group in the molecule. HRESIMS
determined the molecular formula C28H39O6N, showing m/z peak
-0.01
+0.01
H
CH3
+0.07
CH3
H
O
O
-0.02
+0.05
CH3
OR
+0.13
H
O
CH3
OR
-0.18
-0.07
+0.04
N
H
H3C
+0.10
+0.02
CH3
at 508.2669 [MþNa]þ,
D
ꢁ0.6 mmu. HMBC spectral data showed
+0.03
correlations from H-9 to C-6; H3-18 to C-3, C-4 and C-5; H3-19 to
H-5 and H-6; H3-20 to H-7 and H-8. The lower field shift of H-9 at
dH 4.77 (Dd¼1.62 ppm) indicated the presence of an acetyl group
at C-9. Coupling patterns of H-5 and H-9 were also similar to those
of compound 2. H-5 appeared as a singlet and H-9 appeared as
5b) R1 = R2 = S-MTPA
6b) R1 = R2 = R-MTPA
Figure 1. Dd values (Dd
¼
dR dS) obtained from 5b and 6b.
ꢁ

P. Pittayakhajonwut et al. / Tetrahedron 65 (2009) 6069–6073
6071
O
CH3
CH3
Torrubiellutin C exhibited cytotoxicity against KB, MCF-7, NCI-
H187, and Vero cell lines with IC50 varying from 0.78 to 4.36 g/
mL, while torrubiellutins A and B were inactive against cancerous
cell lines (KB, MCF-7, NCI-H187) but showed weak cytotoxicity
against Vero cell line with IC50 values of 12.33 and 43.62 mg/mL,
respectively. Compound 4 displayed antimalaria and anti-in-
O
21
20
m
CH3
CH3
R
11
9
R
OH
O
H3C
O
S
OR2
7
S
22CH3
O
19
14
N
16
CH3
HO
5
S
H
R
OR1
CH3
flammatory activity against COX-2 with IC50 values of 3.49 and
1.21 mg/mL, respectively.
1
H3C
3
CH3
18
17
1) R1 = R2 = H
2) R1 = R2 = COCH3
4
CH3
H3C
4. Experimental
3) R1 = H, R2 = COCH3
5a) R1 = S-MTPA, R2 = H
5b) R1 = R2 = S-MTPA
6a) R1 = R-MTPA, R2 = H
6b) R1 = R2 = R-MTPA
4.1. General experimental procedures
Mps were determined on an Electrothermal IA9100 digital
melting point apparatus and are uncorrected. UV spectra were
recorded on a Cary 1E UV–visible spectrophotometer in 95%
EtOH. IR spectra were taken on a Bruker VECTOR 22 FT-IR spec-
trometer. Specific rotations were measured in either 95% EtOH or
MeOH using a JASCO P-1030 polarimeter. NMR spectra, including
COSY, NOESY, DEPT, HMQC, and HMBC experiments, were
recorded on a Bruker AV500D NMR spectrometer (1H at 500 MHz
and 13C at 125 MHz). High resolution ESIMS were performed on
a Micromass LCT mass spectrometer; accurate mass was de-
termined by lock mass calibration. High performance liquid
chromatography (HPLC) was performed on a Dionex, UltiMate
3000. Column Chromatography was performed using Merck silica
gel (230–400 mesh ASTM).
Figure 3. Chemical structures of compounds 1–5.
double doublet with coupling constants of 11.5 and 2.4 Hz. NOESY
spectral data provided the same information as in compound 2.
Thus, the chemical structure of torrubiellutin C can be depicted as
shown in Figure 3.
29
Compound 4 was obtained as colorless solid, [
a
]
ꢁ62.8 (c 0.10,
D
MeOH).6 1H NMR and 13C NMR spectra recorded in mixture of CDCl3
and CD3OD were consistent with those given in the patent,6 known
as BR-050. Those data in DMSO-d6 were also reported in the ex-
perimental section (4.2.4).
4.2. Fungus material, extraction, and isolation
Torrubiellutins A and B were inactive against KB, MCF-7, NCI-
H187 cell lines and malaria, Plasmodium falciparum K1 strain, but
showed cytotoxicity against Vero cell line with IC50 values of 12.33
T. luteorostrata Zimm. was isolated from
a scale insect
Homoptera sp. from the underside of an unidentified leaf col-
lected at Doi Inthanon National Park, Chiang Mai, Thailand. The
fungus was identified by Dr. N.L. Hywel-Jones and registered at
BIOTEC Culture Collection (BCC) as BCC 12904. The fungus was
grown on a surface at 25 ꢀC in 20ꢂ1 L Erlenmeyer flasks, con-
taining 250 mL medium, which consists of 3% sucrose, 2% malt
extract, 0.2% bacto-peptone, 0.1% yeast extract, 0.05% KCl, 0.05%
MgSO4$7H2O, 0.05% KH2PO4. After 26 days, the mycelium was
separated by filtration and culture broth was extracted thrice
with equal volume of EtOAc. A gum (0.47 g) obtained from the
culture broth was subjected to Sephadex LH20 column chroma-
tography, eluted with 100% MeOH, to give three fractions. Each
fraction was further purified by either silica column, using EtOAc
and hexane (3:2) as eluent, or reverse-phase HPLC, using
LiChroCART 250-10 LiChrospher100 RP-18 column eluted with
55% CH3CN in water, to yield torrubiellutin B (2, 62.5 mg) and
torrubiellutin A (1, 5.9 mg). Mycelium was macerated in MeOH
(two days) followed by CH2Cl2 (two days). The solvents were
then combined, evaporated, and extracted with EtOAc (ꢂ3). A
gum (5.63 g) was obtained and separation was conducted by
using Sephadex LH20 column chromatography (eluted with 100%
MeOH) followed by a silica column (eluted with EtOAc and
hexane; 2:3) to give five fractions. The first and second fractions
were further purified by another silica column using EtOAc/
hexane (2:3) as eluent to yield zeorin (83.1 mg). Torrubiellutin B
(2, 0.23 g) was obtained from the third fraction. The fourth
fraction (0.45 g) was purified by a Sephadex LH20 column (eluted
with 100% MeOH) followed by a silica column (eluted with EtOAc
and hexane; 2:3), to provide torrubiellutins B (2, 0.18 g) and C (3,
13.1 mg), respectively. The fifth fraction gave a colorless solid of
compound 4 (0.30 g).
and 43.66 mg/mL, respectively. While torrubiellutin C, having one
acetyl group at C-9, exhibited strong cytotoxicity against KB, MCF-
7, NCI-H187, and Vero cell lines with IC50 varying from 0.78 to
4.36
activity with an IC50 value of 3.49
anti-inflammatory against COX-2 with IC50 value of 1.21
m
g/mL (see Table 2). Compound 4 also displayed antimalarial
g/mL and also exhibited strong
g/mL,
m
m
while the inflammatory activity against COX-1 was inactive. This is
the first report of antimalarial and anti-inflammatory activity of
BR-050. Compound 4 was reported to inhibit bone resorption and
is useful for diseases with abnormal bone metabolism.6 In addi-
tion, the diterpenoids with a pyrone moiety, such as sesquicillin,7
pycnophorin8 exhibited various biological activities including
insecticidal, anticancer, antihypertensive, anti-inflammatory, an-
tibacterial activities, etc.
Table 2
Biological activity of compounds 1–4
Compound Cytotoxicity (IC50
MCF-7a KBa
,
m
g/mL)
Antimalarialc Anti-inflammatoryd
(IC50 g/mL) (IC50 g/mL)
,
m
, m
NCI-H187a Verob
1
2
3
4
Inactive Inactive Inactive
Inactive Inactive Inactive
12.33 Inactive
43.62 Inactive
4.36 Inactive
0.69 3.49
nt
nt
nt
0.78
6.17
1.30
0.85
1.32
2.58
COX-1¼inactive,
COX-2¼1.21
nt¼not tested.
a
IC50>20
IC50>50
IC50>20
IC50>10
m
g/mL was inactive.
g/mL was inactive.
b
c
m
m
m
g/mL was inactive.
g/mL was inactive.
d
3. Conclusion
Three macrocyclic torrubiellutins A–C (1–3) together with two
known pyrone diterpene (4) and zeorin have been isolated from
the insect pathogenic fungus T. luteorostrata BCC 12904.
4.2.1. Torrubiellutin A (1)
27
Colorless solid; mp 96 ꢀC; [
a]
ꢁ103.7 (c 0.1255, EtOH); IR nmax
D
(CHCl3): 3397, 2970, 2927, 1738, 1652, 1601, 1487, 1454, 1410, 1351,

6072
P. Pittayakhajonwut et al. / Tetrahedron 65 (2009) 6069–6073
MeOH
1277, 1204, 1088, 991, 754, 702 cmꢁ1; UV
l
max
(log
3): 215.7
4.3. Determination of absolute stereochemistry
(4.03) nm; 1H and 13C NMR data in acetone-d6 given in Table 1;
HRESIMS: calcd for C26H37NO5Na: 466.2564; found: m/z 466.2577
[MþNa]þ.
4.3.1. Hydrolysis of torrubiellutin A (1) with 6 N HCl
Torrubiellutin A (1, 2.0 mg) was heated at 110 ꢀC with 6 N HCl
(500 mL) for 15 h. The reaction was evaporated to dryness under
vacuum and the resulting material was derivatized with Marfey’s
reagent.
4.2.2. Torrubiellutin B (2)
25
Colorless solid; mp 107 ꢀC; [
a
]
ꢁ132.3 (c 0.1165, EtOH); IR nmax
D
(KBr): 3468, 2974, 2934, 1742, 1658, 1618, 1453, 1375, 1239, 1086,
MeOH
1021, 930, 752, 702 cmꢁ1; UV
l
(log
3
): 216.6 (4.37), 252.8
4.3.2. Derivatization of amino acid with Marfey’s (FDAA)
reagent and HPLC analysis
max
(3.97) nm; 1H and 13C NMR data in acetone-d6 given in Table 3;
HRESIMS: calcd for C30H41NO7Na: 550.2775; found: m/z 550.2784
[MþNa]þ.
The crude product obtained by following the procedure de-
scribed in Section 4.3.1 was allowed to react with 50
fluoro-2, 4-dinitrophenyl-5- -alanine amide) in acetone (10 mg/
1 mL) and 1 M NaHCO3 (100 L). The mixture was warmed at
60 ꢀC for 15 min. After cooling to room temperature, the mixture
was neutralized with 2 N HCl (50 L). The solution was diluted
L) and subjected to HPLC analysis (15
mL L-FDAA (1-
L
4.2.3. Torrubiellutin C (3)
m
26
Colorless solid; mp 79–80 ꢀC; [
a
]
ꢁ29.3 (c 0.1235, EtOH); IR
D
nmax (KBr): 3444, 2926, 1739, 1655, 1608, 1454, 1357, 1240, 1083,
m
EtOH
1015, 979, 752, 702 cmꢁ1; UV
l
(log
3
): 217.7 (4.12) nm; 1H
with 50% CH3CN (100
injection).
m
mL
max
and 13C NMR data in CDCl3 given in Table 3; HRESIMS: calcd for
C28H39NO6Na: 508.2675; found: m/z 508.2669 [MþNa]þ.
The L-FDAA derivatives of NMePhe (D- and L-forms) were also
prepared as previously described. Standards NMePhe was prepared
by stirring Fmoc-NMePhe (20 mg) in piperidine and DMF for
30 min at rt. The mixture was evaporated and water (1.5 mL) was
added. The solution was washed twice with EtOAc. Then the water
was evaporated to obtain NMePhe.
4.2.4. BR-050 (4)
Colorless solid; mp 171.6–172.5; C27H40O4; 1H NMR (500 MHz,
DMSO-d6)
d 0.67 (s, 3H), 0.86 (s, 3H), 1.09–1.14 (2H, m), 1.23 (dq,
1H, J¼12.9, 4.4 Hz), 1.45 (dt, 1H, J¼8.6, <1 Hz), 1.50–1.58 (m, 3H),
1.60 (s, 3H), 1.63 (dd, 1H, J¼11.8, 2.4 Hz), 1.65 (s, 3H), 1.71 (td, 1H,
J¼11.5, 4.3 Hz), 1.83 (s, 3H), 1.89–1.94 (m, 2H), 1.98 (dt, 1H, J¼11.6,
<1 Hz), 2.05 (dd, 1H, J¼11.6, 4.3 Hz), 2.11 (s, 3H), 2.35 (ddd, 1H,
J¼11.6, 5.4, 5.4 Hz), 2.44 (dd, 1H, J¼13.2, 4.3 Hz), 2.68 (dd, 1H,
J¼13.2, 12.4 Hz), 3.35 (m, interfered with water in DMSO-d6, 1H),
4.13 (d, 1H, J<1 Hz), 4.32 (d, 1H, J¼5.1 Hz, OH), 4.44 (t, 1H,
J<1 Hz), 5.11 (t, 1H, J¼7.1 Hz), 10.06 (s, 1H, OH); 13C NMR
The FDAA derivatives of hydrolysate of 1 and standards NMePhe
(
D
- and
m
L-forms) were analyzed by HPLC using Nova-Pak C18 column
(4
m, 3.9ꢂ150 mm2) with 30% CH3CN in water containing 0.05%
formic acid, flow rate 0.6 mL/min, 35 ꢀC and detected at 340 nm.
The sample was also co-injected with
NMePhe ( - and -forms). The retention times of FDAA derivatives
of NMe- -Phe and NMe- -Phe were 19.71 and 21.09 min,
respectively.
L-FDAA derivatives of
D
L
L
D
(125 MHz, DMSO-d6)
d 10.8 (CH3), 17.5 (CH3), 17.9 (CH3), 18.0
(CH3), 21.8 (CH2), 21.9 (CH2), 23.0 (CH2), 23.5 (CH3), 26.0 (CH3),
28.0 (CH2), 31.1 (C), 34.4 (C), 37.7 (C), 37.8 (C), 38.6 (C), 41.1 (C),
55.1 (C), 72.2 (C), 102.5 (C), 106.8 (C), 109.6 (C), 125.7 (C), 130.5
(C), 149.3 (C), 155.1 (C), 165.6 (C), 165.0 (C); ESIMS m/z: 451.28
[MþNa]þ.
4.3.3. Preparation of MTPA esters of compound 1
To a solution of torrubiellutin A (1, 1.08 mg) in pyridine (100 mL)
and CH2Cl2 (300
was left at rt for four days. After removal of solvent, the mixture was
m
L) was added (ꢁ)-MTPACl (40
mL). The solution
Table 3
1H NMR (500 MHz) and 13C NMR (125 MHz) assignments of torrubiellutins B and C
No.
2 (In acetone-d6)
3 (In CDCl3)
dH (int., mult., J in Hz)a
dC
dH (int., mult., J in Hz)a
dC
b
b
1
2
3
4
d
166.1
120.2
147.2
38.0
d
166.6
119.1
149.0
39.8
6.40 (1H, d, 15.1)
7.08 (1H, dd, 15.1, 10.3)
2.87–2.92 (1H, m)
5.07 (1H, s)
6.08 (1H, d, 15.1)
7.17 (1H, dd, 15.1, 10.2)
2.60–2.64 (1H, m)
4.01 (1H, s)
5
78.6
78.3
6
7
8
9
10
11
13
14
d
133.3
122.6
33.4
76.8
39.7
71.3
170.0
67.0
35.1
38.0
d
136.9
122.6
33.7
77.0
39.8
72.1
170.2
67.9
35.2
38.7
5.25 (1H, dd, 10.9, 1.3)
2.74–2.78 (1H, m)
4.72 (1H, dd, 11.5, 2.4)
1.76–1.79 (1H, m)
4.61 (1H, dq, 6.5, 3.5)
d
5.47 (1H, d, 10.8)
2.67–2.73 (1H, m)
4.77 (1H, dd, 11.5, 2.4)
1.72–1.76 (1H, m)
4.70 (1H, dq, 6.5, 3.5)
d
3.80 (1H, dd, 10.4, 4.6)
3.26 (1H, dd, 13.7, 4.6), 3.34 (1H, dd, 13.7, 10.4)
2.81 (3H, s)
3.54 (1H, dd, 9.5, 5.3)
3.36–3.40 (2H, m)
2.74 (3H, s)
16
17
18
19
20
21
1.10 (3H, d, 7.0)
1.66 (3H, d, 1.0)
0.79 (3H, d, 6.79)
0.76 (3H, d, 6.8)
1.05 (3H, d, 6.5)
d
18.2
14.3
16.9
9.0
1.29 (3H, d, 7.0)
1.58 (3H, d, 0.4)
0.90 (3H, d, 6.8)
0.72 (3H, d, 6.9)
1.12 (3H, d, 6.5)
d
19.1
15.3
17.7
9.7
22
13.0
13.6
10
139.4
129.3ꢂ2
128.3ꢂ2
126.2
19.95/169.2
19.90/170.3
139.2
128.4ꢂ2
128.5ꢂ2
126.4
20/60
7.20–7.31 (2H, m)
7.20–7.31 (2H, m)
7.20–7.31 (1H, m)
2.16 (3H, s)
7.20–7.25 (2H, m)
7.25–7.35 (2H, m)
7.20–7.25 (1H, m)
d
30/50
40
5-OCOCH3
9-OCOCH3
2.11 (3H, s)
2.17 (3H, s)
21.0/171.3

P. Pittayakhajonwut et al. / Tetrahedron 65 (2009) 6069–6073
6073
purified by HPLC, using SunFire Prep C18 OBD column (10
mm,
exhibited activity against COX-1 with IC50 value of 4–5
mg/mL and
19ꢂ250 mm2) and eluting with a gradient system varied from 30%
to 70% CH3CN in water over 20 min and then from 70% to 100%
CH3CN in water over 10 min, to afford S-MTPA esters 5a (0.60 mg)
and 5b (0.23 mg). Similarly, compound 1 (0.69 mg) was reacted
with (þ)-MTPACl and after purification by HPLC, the mixture
obtained R-MTPA esters 6a (0.40 mg) and 6b (0.22 mg).
COX-2 with IC50 value of 9–10 mg/mL.
Acknowledgements
We are grateful to Bioresources Research Network (BRN),
National Center for Genetic Engineering and Biotechnology (BIO-
TEC), for the financial support.
4.4. Biological tests
Torrubiellutins A–C (1–3) and compound 4 were tested for
cytotoxicity against KB, MCF-7, NCI-H187, Vero cell lines, and
antimalarial activity against Plasmodium falciparum K1 strain
(multi-drug resistant strain). Only compound 4 was evaluated for
anti-inflammatory activity against COX-1 and COX-2. Cytotoxicity
against cancer cell lines, including KB (human epidermoid carci-
noma, ATCC CCL-17), MCF-7 (human breast cancer, ATCC HTB-22),
and NCI-H187 (human small cell lung cancer, ATCC CRL-5804) were
performed using the resazurin microplate assay,9 and against Vero
cell (African green monkey kidney fibroblasts, ATCC CCL-81) were
performed using the green fluorescent protein microplate assay
(GFPMA).10 Ellipticine and doxorubicin were used as references for
the cytotoxicity assay. Ellipticine displayed cytotoxicity against
MCF-7, KB, NCI-H187, and Vero cell lines with IC50 values of 0.57,
Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2009.05.070.
References and notes
1. Isaka, M.; Kittakoop, P.; Kirtikara, K.; Hywel-Jones, N. L.; Thebtaranonth, Y. Acc.
Chem. Res. 2005, 38, 813–823.
2. Tsuda, Y.; Isobe, K.; Fukushima, S.; Ageta, H.; Iwata, K. Tetrahedron Lett. 1967, 1,
23–28.
3. Bhushan, R.; Bru¨ckner, H. Amino Acids 2004, 27, 231–247.
4. Marfey, P. Carlsberg Res. Commun. 1984, 49, 591–596.
5. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113,
4092–4096.
6. Terumi, K.; Akiko, M.; Akira, K.; Tomoko, A.; Tsutomu, A.; Kazuhide, I.; Shigeo,
M., W.O. Patent 1995006646, 1995.
7. Uchida, R.; Imasato, R.; Yamaguchi, Y.; Masuma, R.; Shiomi, K.; Tomoda, H.;
Omura, S. J. Antibiot. (Tokyo) 2005, 58, 397–404.
8. Sassa, T.; Kato, H.; Kajiura, H. Tetrahedron Lett. 1986, 27, 2121–2124.
9. O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Eur. J. Biochem. 2000, 267, 5421–5426.
10. Changsen, C.; Franzblau, S. G.; Palittapongarnpim, P. Antimicrob. Agents Che-
mother. 2003, 47, 3682–3687.
11. Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D. Antimicrob. Agents
Chemother. 1979, 16, 710–718.
12. Kirtikara, K.; Swangkul, S.; Ballou, L. R. Inflamm. Res. 2001, 50, 327–332.
0.45, 0.18, and 0.44–0.46
biological activities against MCF-7, KB, and NCI-H187 with IC50
values of 0.19, 0.04–0.11, and 0.03 g/mL, respectively. Antimalarial
mg/mL, respectively. Doxorubicin showed
m
activity was performed using the microculture radio isotope tech-
nique described by Desjardins et al.11 Dihydroartemisinin was used
as reference and showed antimalarial activity with IC50 value of
3.7 nM. Anti-inflammatory was done employing the method de-
scribed by Kirtikara et al.12 Aspirin was used as reference and