Novel cyclopeptide and unique flavone from Desmos rostrata. Total synthesis of desmorostratone

Article abstract of DOI:10.1016/j.tet.2009.06.017

Two new compounds, a cyclic peptide desmocyclopeptide (1) and a special flavone desmorostratone (2) were isolated from the stem bark of Desmos rostrata, along with two known compounds, desmosdumotins B (3) and C (4). Their structures were established on the basis of the spectral data, including mass spectrometry and 2D NMR. The total synthesis of desmorostratone (2) was performed in order to confirm its structure as well as that of desmosdumotin C (4), which was a tautomeric mixture in the solution. Finally, cytotoxity of these compounds were evaluated. Desmosdumotin C (4) displayed a moderate inhibition activity against KB cell line with an IC₅₀ of 19.2 μM, whereas the other products showed a weak inhibition against the same cell line target.

Full text of DOI:10.1016/j.tet.2009.06.017

Tetrahedron 65 (2009) 7171–7176
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Novel cyclopeptide and unique flavone from Desmos rostrata. Total
synthesis of desmorostratone
,
b
b
c
Ngoc Tuan Nguyen a, Van Cuong Pham a , Marc Litaudon , Françoise Gueritte , Bernard Bodo ,
*
´
Van Tuyen Nguyen a, Van Hung Nguyen a
,
*
a Institute of ChemistrydVietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet Road, Cau Giay, Hanoi, Viet Nam
b Institut de Chimie des Substances Naturelles, CNRS, 91198 Gif-sur-Yvette cedex, France
c Laboratoire de Chimie et Biochimie des Substances NaturellesdCNRS, Muse´um National d’Histoire Naturelle, 63 rue Buffon, 75005 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new compounds, a cyclic peptide desmocyclopeptide (1) and a special flavone desmorostratone (2)
were isolated from the stem bark of Desmos rostrata, along with two known compounds, desmosdu-
motins B (3) and C (4). Their structures were established on the basis of the spectral data, including mass
spectrometry and 2D NMR. The total synthesis of desmorostratone (2) was performed in order to confirm
its structure as well as that of desmosdumotin C (4), which was a tautomeric mixture in the solution.
Finally, cytotoxity of these compounds were evaluated. Desmosdumotin C (4) displayed a moderate
Received 23 December 2008
Received in revised form 20 May 2009
Accepted 4 June 2009
Available online 10 June 2009
inhibition activity against KB cell line with an IC50 of 19.2
inhibition against the same cell line target.
m
M, whereas the other products showed a weak
Ó 2009 Elsevier Ltd. All rights reserved.
8'
9'
6'
1. Introduction
5'
3
O
O
4'
4
5
2
1
H
O
O
8
3'
N
In the course of a screening program on the flora of Vietnam, the
plant Desmos rostrata P.T. Li (basionym: Dasymaschalon rostratum,
Annonaceae) was selected for its cytotoxicity against KB cell line (15%
1'
7'
2'
9
6
7
NH
O
OH
OH
O
HN
4a
inhibition at the concentration of 1
mg/mL for the stem bark extract).
O
O
N
We have previously isolated and characterized from this plant several
antiplasmodial alkaloids, containing the tetrahydroisoquinoline
motif.1 Analysis of the CH2Cl2 fraction of the stem bark of D. rostrata
led to the isolation of two new compounds, a cyclic peptide desmo-
cyclopeptide (1) and an unique flavone type desmorostratone (2),
together with two known compounds, desmosdumotins B (3)2 and C
(4).3a,b Cyclopeptide compounds are usually found in the seed of the
Annonaceae plants and rarely observed from stem bark. Desmos-
dumotin C (4) has been previously reported from Desmos dumosus
and Campomanesia lineatifolia as a single tautomer, whereas it was
isolated from D. rostrata in a tautomeric mixture of the two isomers as
indicated by its 1H NMR spectrum.
N
O
H
O
desmocyclopeptide (1)
O
4b
desmosdumotin C (4)
3'
4'
2'
12
8
O
13 O
9
5'
1'
O
O
2
7
6'
3
6
10
11
4
5
O
O
OH
O
desmorostratone (2)
desmosdumotin B (3)
In order to confirm the structures of desmorostratone (2) and
desmosdumotin C (4), their total syntheses were attempted, starting
from 1-acetyl-2,4,6-trihydroxybenzene. Cytotoxicity of these com-
pounds was evaluated on KB cells. However, they showed a moder-
ate or weak cytotoxicity.
2. Results and discussion
Compound 1 was obtained as white solid (mp 261–262 ꢀC) and
20
optically active [
a]
ꢁ126.7 (c 2, CHCl3). In its HR-ESI mass spec-
D
trum, the protonated molecular ion was observed at m/z 486.2727
[MþH]þ (calcd 486.2716 for C25H36N5O5), suggesting the molecular
formula C25H35N5O5. The peptide nature of 1 was suggested by the
* Corresponding authors. Tel.: þ84 4 7564995; fax: þ84 4 8361283.
E-mail addresses: phamvc@ich.vast.ac.vn (V.C. Pham), hungnv@ich.vast.ac.vn
(V.H. Nguyen).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.06.017

7172
N.T. Nguyen et al. / Tetrahedron 65 (2009) 7171–7176
molecular formula itself and from elucidation of its NMR spectra.
Analysis of the 1H and 13C NMR spectral data for 1, with the aid of
1H–1H-COSY, HSQC, HMBC and NOESY spectra, indicated the pres-
ence of an alanine, a glycine, a proline, an isoleucine and a phe-
nylalanine (Table 1). The determination of the peptide sequence
was established from the connectivity of the carbonyl of residue (i)
and the NH proton of residue (iþ1) on the HMBC spectrum as fol-
lows: the correlations of the carbonyl at dC 173.5 of the Ala1 with its
appropriate amino acid standards, that secured the
for Ala, Ile, Phe and Pro.
L-configuration
Compound 2 was isolated as yellow solid (mp 199–201 ꢀC).
The HR-ESIMS of 2 showed the pseudomolecular ion [MþH]þ at
m/z 311.1279 (calcd 311.1283 for C19H19O4), suggesting the
molecular formula C19H18O4. This indicated eleven degrees of
unsaturation. The 1D NMR spectra (1H and 13C) displayed the
signals of three methyl groups, a methoxy, an sp3 quaternary
carbon, six sp2 methines and eight sp2 quaternary carbons,
including two carbonyl groups and two aromatic oxygenated
carbons as assigned from their chemical shifts (Table 2). In the
1H–1H-COSY spectrum, the correlations of the two equivalent
protons H-20 and H-60 (dH 7.76) with the two other equivalent
ones, H-30 and H-50 (dH 7.53) which in turn were correlated to
H-40 (dH 7.56). This indicated the presence of a phenyl ring (C-
ring).
Ha
proton at dH 4.37, as well as to the NH proton of the Gly2 at dH
7.78 were observed. The carbonyl group of the Gly2 at dC 168.5 was
not correlated to an amide proton, suggesting its connection with
the Pro3. Cross-peaks of the carbonyl of the Pro3 at dC 172.1 with its
Ha
proton at dH 4.21 and NH proton (dH 7.12) of the Ile4 were pre-
sented. In addition, the carbonyl of Ile4 at dC 172.3 was correlated
with its H
proton at dH 4.18 and NH proton of Phe5 at dH 7.56. Fi-
nally, 2J-HMBC correlation of the carbonyl of Phe5 at dC 172.3 with
its H
proton (dH 4.64) and NH proton of Ala1 (dH 8.25) were noted.
a
a
Analysis of HMBC spectrum revealed the correlations of H-3
(
dH 6.82) with C-10 (dC 130.6), C-2 (dC 161.2), the carbonyl C-4 (dC
Complete analysis of the HMBC spectrum determined the structure
of 1 as drawn in Figure 1. The NOESY spectrum of 1 depicted the
175.9) and C-10 (dC 116.0). Furthermore, cross-peaks of C-2 with
H-20 and H-60 (dH 7.76) were observed. This observation indicated
the linkages of C-2 with C-10 and C-4 with C-10. On the other
hand, the two superposed methyl groups CH3-12 and CH3-13
were correlated to C-9 (dC 176.9), C-8 (dC 43.0) and C-7 (dC 170.2).
The last carbon C-7 was further correlated with the methoxy
group (dH 3.96) and CH3-11 (dH 1.98) which showed 3J-HMBC
correlation to the carbonyl C-5 (dC 183.6). These data indicated
that C-8 was bonded to both CH3-12 and CH3-13 and C-6 linking
to CH3-11. The quaternary carbon C-10 was thus linked to both C-
5 and C-9, forming the A-ring. The chemical shifts of C-9 and C-2
suggested that they were linked to an oxygen atom. Taking into
account the molecular formula of 2, determined that C-2 was
linked to C-9 through the oxygen atom O-1. These data permitted
establishing the structure of 2 as drawn in Figure 2. This unique
flavone was isolated for the first time from natural source and
named desmorostratone. This compound was previously de-
scribed as synthetic one, forming during the synthesis of des-
mosdumotin B.7
strong spatial interactions of H
that the amide bond between Gly2–Pro3 was trans.4 The dNN(i,iþ1)
and da
sequential connectivity from Ala1 to Phe5 was clearly
a d
of Gly2 with H of Pro3, indicating
N(i,iþ1)
observed in the NOESY spectrum as indicated in Figure 1. The
amino acids residue and the sequence of 1 were confirmed by
fragmentation of [MþH]þ ion at m/z 486. A series of adjacent an ion
peaks were observed at m/z 311, 240, 183 and 86 corresponding to
the successive loss of Phe, Ala, Gly and Pro. A second series of peaks
at m/z 339, 268, 211 and 114 were assigned to adjacent bn ion (loss
of Phe, Ala, Gly and Pro). This new pentacyclopeptide 1 is named
desmocyclopeptide. The structure of 1 is very close to that of
pseudostellarin A which has been previously reported from the
roots of Pseudostellaria heterophylla.5
Ala1
O
H
N
O
Phe5
H
N
Gly2
NH
O
O
3'
N
2'
4'
N
trans
13
12
H
C
Ile4
Pro3
O
8
5'
O
O
B
7
2
1'
9
6'
A
3
6
Figure 1. Selected HMBC (/) and NOESY (4) correlations of 1.
10
4
5
11
The absolute configuration of the chiral amino acids in desmo-
cyclopeptide (1) was determined by HPLC analysis of the acid hy-
drolysate derivatised with Marfey’s reagent,6 and comparison with
O
O
Figure 2. Keys HMBC correlations of 2.
Table 1
NMR data for desmocyclopeptide (1) (CDCl3, 1H: 500.13 MHz, 13C: 125.76 MHz)
Residue
Ala1
dC
dH m (J, Hz)
Residue
-CH2
dC
dH m (J, Hz)
Residue
Phe5
dC
dH m (J, Hz)
g
25.1
d
1.99 m
2.12 m
3.48 ddd (6.7, 6.7, 9.5)
3.97 ddd (7.0, 7.0, 9.5)
a
b
-CH
-CH3
49.5
15.8
173.5
4.37 m
1.31 d (7.5)
a
b
-CH
-CH2
57.0
37.2
d
4.64 m
3.18 dd (7.0, 13.7)
3.26 dd (9.0, 13.7)
d
-CH2
46.9
d
C]O
NH
8.25 d (7.5)
C]O
172.1
10-Phe
20,60-Phe
30,50-Phe
40-Phe
C]O
136.8
129.4
128.4
126.8
172.3
Gly2
Ile4
7.22 m
7.20 m
7.19 m
a
-CH2
42.5
d
3.56 dd (3.2, 15.5)
4.35 dd (5.7, 15.5)
a-CH
b-CH
g-CH2
58.7
35.7
24.9
d
4.18 dd (8.5, 8.5)
1.88 m
0.97 m
1.40 m
0.81 d (6.5)
0.83 t (7.5)
C]O
NH
168.5
7.78 dd (3.2, 5.7)
NH
7.56 d (7.5)
Pro3
g
0-CH3
15.3
10.9
172.3
a
b
-CH
-CH2
62.5
29.7
d
4.21 dd (6.0, 8.0)
1.96 m
2.29 m
d-CH3
C]O
NH
7.12 d (8.5)

N.T. Nguyen et al. / Tetrahedron 65 (2009) 7171–7176
7173
Table 2
isomers 4a and 4b with a ratio of 2.5:1, respectively. This result
confirmed the structure of 4 and indicated that the two enolic
forms 4a and 4b were stable enough at room temperature and
could be identified by NMR. Oxidative cyclization of 4 was firstly
carried out with DMSO/I2 at 100 ꢀC, but the desired compound 2
was not observed. This reaction was then performed with addition
of a small amount of H2SO4 and heated at 80 ꢀC.7 Under this con-
dition, only desmosdumotin B (3) was obtained with 71% yield. An
alternative approach was the cyclization of 2 in basic media with
concurrent oxidation (Scheme 2). However, the methoxy group was
cleaved off and two flavanones, 7 and 8 were obtained under this
reaction condition. These results indicated that the methoxy group
in the structure of 4 (or even in the structures of the cyclized
products) was unstable in both acidic and basic media. This ob-
servation suggested that the oxidative cyclization of 4 should be
carried out under mild conditions. Finally, desmorostratone (2) was
obtained in good yield by treatment of 4 with SeO2 in dime-
thylsulfate at 70 ꢀC. Comparison of NMR data of the synthetic
compound with the natural one revealed that they were identical.
NMR data for desmorostratone (2): (CDCl3, 298 K)
Carbon
dC
dH m (J, Hz)
Carbon
dC
dH m (J, Hz)
2
3
4
5
6
7
8
9
161.2
113.1
175.9
183.6
119.2
170.2
43.0
176.9
116.0
130.6
d
20
125.6
129.3
131.7
129.3
125.6
10.0
24.5
24.5
62.2
7.76 m (1.0, 7.5)
7.53 m
7.56 m
7.53 m
7.76 m (1.0, 7.5)
1.98 s
1.64 s
1.64 s
3.96 s
6.82 s
d
30
40
d
50
d
60
d
11
12
13
OMe
d
d
10
10
d
d
The two known compounds, desmosdumotins B (3)2 and C (4)3a,b
were also isolated from this plant. Their structures were established
from the spectral data analysis. However, the 1H NMR spectrum of 4
showed two nearly similar sets of signal with a ratio of 2.5:1. This
suggested the presence of two isomers. Analysis of 2DNMR spectra of
this mixture allowed assignment of NMR data for the two isomers 4a
and 4b (Scheme 1). The two enolic forms were determined by HMBC
correlations of their enolic protonwith C-8 in 4a and with C-50 for 4b.
As mentioned above, desmosdumotin C (4) has been previously
reported from D. dumosus and C. lineatifolia as single tautomer 4a. In
order to confirm the structures of desmorostratone (2) and des-
mosdumotin C (4), their total syntheses were accomplished.
O
O
OH
O
desmosdumotin B (3, 71%)
HO
OH
Ac
HO
O
O
O
Me3SiCHN2
MeCN/MeOH
MeI
SeO2, Me2SO4
ter-BuOK, MeOH
Ac
4
OH
5
OH
6 (72%)
O
O
desmorostratone (2, 65%)
O
O
O
O
O
O
O
O
PhCHO
50% aq. NaOH/EtOH
Ac
+
O
O
OH
O
O
OH
OH
8 (42%)
9 (30%)
4 (91%)
(60%)
Scheme 2. Synthesis of desmorostratone (2).
O
O
O
OH
In order to understand the formation of desmorostratone (2)
and related compounds such as desmosdumotins B (3) and C (4) in
the plants, their biosynthesis pathways were proposed (Scheme 3).
As similar with the well-known biosyhthesis of chalcones, cinna-
moyl-ScoA (11) is resulted from phenylalanine (10). Condensations
of 11 with three malonate units following by intramolecular cycli-
zation afford the triketone 12. C-Methylation of 12 yields the
champanone B (13), which has recently isolated from C. line-
atifolia.3b Cyclization of 13 provides the champanone C (14), pre-
viously characterized from D. dumosus and C. lineatifolia.3b,10
Oxidation of 14 yields desmosdumotin B (3). On the other hand,
champanone (13) could be O-methylated affording desmosdumo-
tin C (4), which should be further cyclized to give compound 16.
Finally, oxidation of 16 finishes the biosynthesis of desmoros-
tratone (2). However, desmorostratone (2) could be also derived
from desmosdumotin B (3) by O-methyaltion of its tautomer 15
(Scheme 3). The presence of desmosdumotin C, champanone B (13)
and especially champanone C (14) in the plants supports for the
hypothesis that C-methylation process is probably occurred before
intramolecular cyclization of 13 and 14.
O
OH
4a
O
O
4b
Scheme 1. Synthesis of desmosmodutin C (4).
The syntheses of desmosdumotin B (3)7 and derivatives of
desmosdumotin C8a,b have been previously described, starting from
2,4,6-trihydroxyacetophenone. During the synthesis of 3, desmoro-
stratone (2) has been obtained as side product with low yield. Re-
cently, a number of desmosdumotin B derivatives have been also
prepared and some of them showed interesting anti tumors ac-
tivity.9 Our effort in this work was studying and optimization of the
synthetic conditions to obtain desmorostratone (2) in high yield.
The synthesis of desmosdumotin C (4) was performed by using
the modified method previously illustrated.6,7 Methylation of 3
with MeI in the presence of NaOCH3 gave 6 in low yield (35%).
However, when using t-BuOK as a base, compound 6 was obtained
in higher yield (72%). Exposure of 6 to a solution of Et2O containing
excess of trimethylsilanediazomethane at ꢁ18 ꢀC yielded 7 in 60%.
Condensation of 7 with benzaldehyde at room temperature pro-
vided compound 4 in good yield (91%). 1H NMR spectrum of the
synthetic compound was identical with that of the natural one
isolating from D. rostrata and showed the presence of the two
3. Biological evaluation
In vitro cytotoxicity of the isolated compounds in human tumor
cell lines (KB) was assessed as previously described.11a,b IC50 values

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N.T. Nguyen et al. / Tetrahedron 65 (2009) 7171–7176
COOH
COOH
O
O
O
HO
O
3X
HOOC
COSCoA
C-methylation
COSCoA
CoAS
NH2
cyclization
O
OH
12
O
OH
phenyallanine (10)
cinnamoyl-SCoA (11)
champanone B (13)
O
O
O
O
O
O
HO
O
tautomerisation
cyclization
oxidation
OH
OH
O
O
champanone C (14)
desmosdumotin B (3)
15
O-methylation
MeO
O
O
O
O
O
O
O-methylation
cyclization
oxidation
13
O
OH
desmosdumotin C (4)
Scheme 3. Proposed biosynthesis pathway for desmorostratone (2) and related compounds.
O
O
O
16
desmorostratone (2)
defined, as the concentration required inhibiting cell growth by
50%, were determined after 72 h drug exposures. The results in-
dicated that only desmosdumotin C (4) showed a moderate cyto-
1651, 1531, 1437 cmꢁ1; HRMS (ESI): [MþH]þ, found 486.2727.
C25H36N5O5 requires 486.2716; ESI-MS/MS on [MþH]þ ion, m/z:
486, 458, 339, 311, 268, 240, 211, 183, 114, 86; NMR data see Table 1.
toxic activity with an IC50 of 19.2
mM. The other compounds (1–3)
presented very weak cytotoxicity on the same cell line target even
when they were tested at the concentration of 25 mM.
4.4. Desmorostratone (2): C19H18O4
Yellow solid (n-hexane/EtOAc), mp 199–201 ꢀC; Rf 0.29 (n-hex-
4. Experimental section
4.1. General
ane/EtOAc: 9:1); UV (MeOH) lmax (log 3) 213.4 (2.44), 253.0 (2.85),
274.9 (2.89); nmax (KBr) 1675, 1632, 1451, 1405, 1123 cmꢁ1; HRMS
(ESI): [MþH]þ, found 311.1279. C19H19O4 requires 311.1283; NMR
data see Table 2.
Analytical TLC was performed with 0.25 mm silica gel 60 plates.
Plates were developed by spraying with 5% H2SO4 in EtOH followed
by heating on a hot plate. 1H NMR spectra were recorded at
500.13 MHz and reported in ppm using tetramethylsilane
(0.00 ppm) or solvent (CDCl3: 7.24 ppm) as an internal standard.
Data are reported as s¼singlet, d¼doublet, m¼multiplet, br¼broad;
coupling constant(s) in Hz. 13C NMR spectra were recorded at
4.5. Desmosdumotin B (3): C18H16O4
Yellow solid (n-hexane/EtOAc), mp 215–216 ꢀC; Rf 0.75 (CH2Cl2/
MeOH: 98:2); nmax (KBr) 3435, 2934, 1685, 1635, 1602, 1555, 1446,
1427, 1294,1161 cmꢁ1; 1H NMR (500.13 MHz, CDCl3): dH 13.05 (1H, s,
chelated-OH), 7.81 (2H, m, J 7.0 Hz, H-20 and H-60), 7.61 (1H, m, J
7.0 Hz, H-40), 7.57 (2H, m, H-30 and H-50), 6.89 (1H, s, H-3),1.88 (3H, s,
Me-11),1.59 (6H, s, Me-12 and Me-13); 13C NMR (125.76 MHz, CDCl3):
dC 196.2(C-7),180.8(C-4),174.1(C-9),164.3(C-2),163.5(C-5),132.6(C-
40),130.0(C-10),129.5 (C-30 andC-50),126.0(C20 andC-60),110.5(C-10),
110.2 (C-3), 108.6 (C-6), 47.3 (C-5), 25.3 (C-12 and C-13), 7.0 (C-11);
HRMS (ESI): [MþH]þ, found 297.1121. C18H17O4 requires 297.1127.
125.76 MHz with chemical shifts reported in ppm (d) in reference to
the solvent peak of CDCl3 77.16). The ESIMS spectra were mea-
(d
sured by Electrospray MS in the positive mode.
4.2. Plant material and extraction
The plant D. rostrata P.T. Li was collected in Hatinh-Vietnam in
March 2004 and a specimen (VN 1273) was deposited at the In-
stitute of Ecology and Biological ResourcesdVASTdVietnam. Dried
and ground stem bark (1.0 kg) of D. rostrata were extracted with
MeOH at room temperature for three times (3ꢂ4 L). The solvent
was removed under diminished pressure. The crude MeOH extract
was suspended in water and extracted successively with
n-hexane and then with CH2Cl2. The CH2Cl2 extract solution was
concentrated under diminished pressure and the residue (5.3 g)
was purified by repeated open column chromatography over silica
gel, eluted with a mixture of CH2Cl2/MeOH to afford desmocyclo-
peptide (1, 25 mg), desmorostratone (2, 16 mg), desmosdumotin B
(3, 125 mg) and desmosdumotin C (4, 86 mg).
4.6. Desmosdumotin C (4): C19H20O4
Yellow solid (n-hexane/EtOAc), mp 93–94 ꢀC; Rf 0.52 (n-hexane/
EtOAc: 9:1); nmax (KBr) 3434, 2934, 1664, 1616, 1573, 1519, 1419,
1202, 1116 cmꢁ1; 4a: 1H NMR (500.13 MHz, CDCl3): dH 19.14 (1H, s,
chelated-OH), 8.26 (1H, d, J 16.0 Hz, H-8), 7.86 (1H, d, J 16.0 Hz, H-7),
7.59 (1H, m, H-4), 7.32 (4H, m, H-2, H-3, H-5 and H-6), 3.88 (3H, s,
OMe), 1.93 (3H, s, Me-70), 1.31 (6H, s, Me-80 and Me-90); 13C NMR
(125.76 MHz, CDCl3): dC 198.0 (C-60), 192.4 (C-20), 187.2 (C-9), 176.6
(C-40),144.8 (C-7),135.3 (C-1),130.6 (C-3 and C-5),129.0 (C-4),128.9
(C-2 and C-6), 123.3 (C-8), 113.6 (C-30), 106.6 (C-10), 62.1 (OMe), 50.4
(C-50), 24.3 (Me-80 and Me-90), 9.8 (Me-70); 4b: 1H NMR
(500.13 MHz, CDCl3): dH 18.72 (1H, s, chelated-OH), 8.47 (1H, d, J
16.0 Hz, H-8), 7.88 (1H, d, J 16.0 Hz, H-7), 7.61 (1H, m, H-4), 7.32 (4H,
m, H-2, H-3, H-5, H-6), 3.82 (3H, s, OMe), 1.88 (3H, s, CH3-70), 1.40
(6H, s, CH3-80, CH3-90); 13C NMR (125.76 MHz, CDCl3): dC 201.6 (C-60),
189.5 (C-9), 186.2 (C-20), 171.0 (C-40), 145.4 (C-7), 135.2 (C-1), 130.7
(C-3 and C-5),129.0 (C-2 and C-6), 123.7 (C-8), 118.5 (C-30), 108.4 (C-
4.3. Desmocyclopeptide (1): C25H35N5O5
White solid (Et2O/EtOH), mp 261–262 ꢀC; Rf 0.37 (CH2Cl2/
20
MeOH: 92/8); [
a]
ꢁ126.7 (c 2, CHCl3); UV (MeOH) lmax (log
3)
D
241.6 (2.98), 283.0 (2.45); nmax (KBr) 3542, 3477, 3341, 2969, 2876,

N.T. Nguyen et al. / Tetrahedron 65 (2009) 7171–7176
7175
10), 61.9 (OMe), 46.1 (C-50), 24.4 (Me-80 and Me-90), 10.2 (Me-70);
50), 126.0 (C-20 and C-60), 105.7 (C-6), 103.2 (C-10), 81.2 (C-2), 48.5
(C-8), 41.7 (C-3), 24.8 (Me), 24.7 (Me), 6.7 (Me); HRMS (ESI):
[MþH]þ, found 299.1279. C18H19O4 requires 299.1283; 9: Rf 0.56
(CH2Cl2/MeOH: 97:3); 1H NMR (500.13 MHz, CDCl3): dH 15.80 (s,
chelated-OH, 1H); 7.47–7.39 (5H, m, phenyl ring), 5.32 (1H, dd, J 3.5,
11.0 Hz, H-2), 3.03 (1H, dd, J 11.0, 18.0 Hz, H-3ax), 2.92 (1H, dd, J 3.5,
HRMS (ESI): [MþH]þ, found 313.1446. C19H21O4 requires 313.1440.
4.6.1. Preparation of 6
A
solution of 2,4,6-trihydroxyacetophenone (5, 982 mg,
5.8 mmol) and potassium tert-butoxide (2.4 g, 21.4 mmol) in an-
hydrous MeOH (70 mL) containing methyliodide (1.2 mL,
19.2 mmol) was heated to reflux for 7 h. The reaction mixture was
cooled to 0 ꢀC and acidified with 1 N HCl aqueous solution, then
extracted three times with EtOAc (3ꢂ30 mL). The combined organic
layers were washed with water (3ꢂ50 mL), dried over Na2SO4 and
concentrated under diminished pressure. The residue was chro-
matographed on silica gel colunm, eluted with n-hexane/EtOAc
(9:1) to provide 6 (876 mg, 72%). Yellow solid, mp 164–165 ꢀC (n-
hexane/EtOAc); Rf 0.45 (CH2Cl2/MeOH: 97:3); nmax (KBr) 3123, 1665,
18.0 Hz, H-3eq), 1.86 (3H, s, Me),1.42 (3H, s, Me),1.40 (3H, s, Me); 13
C
NMR (125.76 MHz, CDCl3): dC 201.8 (C-5), 198.1 (C-7), 182.9 (C-4),
161.3 (C-9), 138.1 (C-10), 129.0 (C-30 and C-50), 128.9 (C-40), 125.8 (C-
20 and C-60), 107.4 (C-8), 101.5 (C-10), 76.1 (C-2), 52.4 (C-6), 38.3 (C-
3), 25.5 (Me), 23.1 (Me), 7.9 (Me); HRMS (ESI): [MþH]þ, found
299.1286. C18H19O4 requires 299.1283.
4.6.5. Preparation of desmorostratone (2)
A solution of 4 (100 mg, 0.32 mmol) in Me2SO4 (10 mL) was
stirred at 70 ꢀC for 1 h, and then SeO2 was added and continuously
stirred for additional 3 h. The reaction mixture was extracted with
Et2O (3ꢂ15 mL). The combined organic extracts were washed with
water, dried over Na2SO4 and concentrated under diminished
pressure. The residue was chromatographed on silica gel column,
eluted with CH2Cl2/MeOH (20:1, v/v), recrystallization from
n-hexane/EtOAc gave desmorostratone (2) (66 mg, 65%). Yellow
solid (n-hexane/EtOAc), mp 197–199 ꢀC; NMR-spectra are identical
with those of the natural compound.
1596, 1532, 1378, 1338, 1278 cmꢁ1 1H NMR (500.13 MHz, DMSO-
;
d6): dH 18.93 (1H, br s, chelated-OH), 2.47 (3H, s, Me–Ac),1.78 (3H, s,
Me), 1.28 (6H, s, 2ꢂMe); 13C NMR (125.76 MHz, DMSO-d6): dC 199.8,
196.5, 189.1, 175.1, 113.8, 106.1, 47.6, 24.7, 7.7; HRMS (ESI): [MþH]þ,
found 211.0975. C11H15O4 requires 211.0970.
4.6.2. Preparation of 7
To a solution of 6 (545 mg, 2.6 mmol) in a mixture of CH3CN/
MeOH (2:1; 7.5 mL), was added slowly a solution of (CH3)3SiCHN2
in diethyl ether (5 mL, 10 mmol) at ꢁ18 ꢀC and the resulting mix-
ture was stirred for 3 h. Acetic acid was then added to destroy the
excess of (CH3)3SiCHN2. The mixture was concentrated under di-
minished pressure and the residue was purified by silica gel column
chromatography, eluted with n-hexane/EtOAc (9:1) to afford 7
(352 mg, 60%) as yellow oil. Rf 0.63 (CH2Cl2/MeOH: 97:3); nmax (KBr)
3112,1661,1602,1526,1374,1331,1279 cmꢁ1; 1H NMR (500.13 MHz,
CDCl3) (mixture of two tautomers): dH 18.91 (s) and 18.13 (s) (2:1,
chelated-OH), 3.94 (s) and 3.87 (s) (2:1, OMe), 2.70 (s) and 2.61 (s)
(1:2, C(O)Me), 1.97 (s) and 1.91 (s) (2:1, Me), 1.44 (s) and 1.33 (s)
(2:1, 2ꢂMe); 13C NMR (125.76 MHz, CDCl3): dC 199.6, 199.1, 192.5,
192.1, 189.2, 189.5, 173.1, 172.4, 118.1, 117.3, 107.6, 107.2, 61.9, 61.7,
46.5, 46.3, 24.6, 24.5, 10.7, 10.2; HRMS (ESI): [MþH]þ, found
225.1122. C12H17O4 requires 225.1127.
4.6.6. Peptide hydrolysis
Desmocyclopeptide (1) sample (3.0 mg) was dissolved in 6 N
HCl (1 mL) and heated at 110 ꢀC for 24 h. The solvent was removed
under reduced pressure and the resulting material was subjected to
further derivatisation.
4.6.7. HPLC analysis of Marfey’s (FDAA) derivatives
The hydrolysate mixture (3.5 mg) or the amino acid standards
(0.5
mg) was dissolved in 0.1 mL of water and treated with 0.2 mL of
1% 1-fluoro-2,4-dinitrophenyl-5-
L-alaninamide (FDAA) in acetone
and 0.04 mL of 1.0 M sodium bicarbonate. The vials were heated at
50 ꢀC for 90 min and the contents after cooling at room tempera-
ture, were neutralised with 1 N HCl. After degassing an aliquot of
the FDAA derivative was diluted with MeOH and chromatographed
on a RP C-18 column (250ꢂ4.6 mm) by means of a linear gradient of
acetonitrile and water containing 0.05% trifluoroacetic acid from
20:80 to 50:50 in 20 min and then isocratic. The flow rate was 1 mL/
min and the absorbance detection was at 340 nm. The chromato-
gram was compared with those of standards treated in the same
conditions.
4.6.3. Preparation of desmosdumotin C (4)
A solution of 7 (330 mg, 1.47 mmol) in EtOH (15 mL) and 50%
KOH in water (10 mL), was treated with benzaldehyde (1.5 mL,
1.48 mmol). The resulting solution was stirred at room temperature
for 24 h. The reaction mixture was poured into 1 N HCl aqueous
solution, and then extracted with CH2Cl2. The organic extract was
washed with brine, dried over Na2SO4 and concentrated under
diminished pressure. The residue was chromatographed on silica
gel, eluted with n-hexane/EtOAc (9:1, v/v) to obtain 4 (417 mg, 91%)
as tautomeric mixtures 4a and 4b with a ratio of 2.5:1, respectively.
Yellow solid (n-hexane/EtOAc), mp 92–96 ꢀC; NMR-spectra are
identical with those of the natural compound.
Amino acids in desmocyclopeptide (1): Gly (13.8 min),
L
-Ala
-Phe (21.5 min). TR of
-Pro (15.5 min), -Ile
(14.9 min),
standards:
(21.0 min),
L
-Pro (15.5 min),
-Ala (14.9 min),
-Phe (21.5 min),
L
-Ile (21.0 min),
-Ala (16.1 min),
-Phe (23.9 min).
L
L
D
L
L
L
D
Acknowledgements
4.6.4. Preparation of 8 and 9
A solution of Me2SO4 (2 mL), containing 4 (20 mg, 0.06 mmol)
was treated with NaOH (2.56 mg, 0.06 mmol) at room temperature
for 12 h. The reaction solution was then neutralized with 1 N HCl
aqueous solution to pH w7 and then extracted with EtOAc
(3ꢂ10 mL). The solvent was removed under diminished pressure
and then the residue was purified by preparative TLC (30% EtOAc/n-
hexane) to provide 8 (8 mg, 42% yield) and 9 (5.7 mg, 30% yield). 8:
Rf 0.46 (CH2Cl2/MeOH: 97:3); 1H NMR (500.13 MHz, CDCl3): dH
11.61 (1H, s, chelated-OH), 7.48–7.40 (5H, m, phenyl ring), 5.58 (1H,
dd, J 3.5, 13.5 Hz, H-2), 3.10 (1H, dd, J 13.5, 17.0 Hz, H-3ax), 2.88 (1H,
dd, J 3.5, 17.0 Hz, H-3eq), 1.81 (3H, s, Me), 1.45 (3H, s, Me), 1.41 (3H, s,
Me); 13C NMR (125.76 MHz, CDCl3): dC 196.6 (C-7), 194.5 (C-4),
186.0 (C-9), 164.0 (C-5), 136.3 (C-10), 129.6 (C-40), 129.1 (C-30 and C-
TheauthorsthankMr. D. C.Dao, Mr. Q.B.Nguyen(VASTdVietnam)
for plant collection and botanical determination, Dr. G. Aubert (ICSN
Gif-sur-Yvette) for cytotoxicity assay and Mr. D. Lacroix (MNHN) for
HPLCanalysis of amino acids. The CNRSisgratefullyacknowledgedfor
financial support of the Franco-Vietnamese Cooperation program
(Study of the flora of Vietnam).
Supplementary data
1H and 13C NMR spectra of the isolated compounds. Supple-
mentary data associated with this article can be found in the online
version, at doi:10.1016/j.tet.2009.06.017.

7176
N.T. Nguyen et al. / Tetrahedron 65 (2009) 7171–7176
6. Marfey, R. Carlsberg Res. Commun. 1984, 49, 591–596.
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