Neonectrolide A, a new oxaphenalenone spiroketal from the fungus Neonectria sp.

Article abstract of DOI:10.1021/ol302979f

Neonectrolide A (1), an oxaphenalenone spiroketal with the previously undescribed (5,8′-dimethyl-5′-oxo-3a′,4,5,5′-tetrahydro- 3H,3′H-spiro[furan-2,2′-isochromeno[3,4,5-def]chromene]-3′-yl) but-3-enoic acid skeleton, was isolated from cultures of the fungus Neonectria sp. Its absolute configuration was assigned by electronic circular dichroism (ECD) calculations. The skeleton of an oxaphenalenone fused with a 1,6-dioxaspiro[4.5]decane moiety in 1 could be derived from the coisolated putative precursors, corymbiferan lactone E (2) and 3-dehydroxy-4-O- acetylcephalosporolide C (3).

Full text of DOI:10.1021/ol302979f

ORGANIC
LETTERS
XXXX
Vol. XX, No. XX
000–000
Neonectrolide A, a New Oxaphenalenone
Spiroketal from the Fungus Neonectria sp.
Jinwei Ren,†,r Fan Zhang,†,r Xiangyu Liu,‡ Li Li,§ Gang Liu,*,† Xingzhong Liu,† and
Yongsheng Che*,^
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy
of Sciences, Beijing 100190, People’s Republic of China, College of Resources &
Environment, Huazhong Agricultural University, Wuhan 430070, People’s Republic
of China, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking
Union Medical College, Beijing 100050, People’s Republic of China, and Beijing Institute
of Pharmacology & Toxicology, Beijing 100850, People’s Republic of China
cheys@im.ac.cn; liug@im.ac.cn
Received October 30, 2012
ABSTRACT
Neonectrolide A (1), an oxaphenalenone spiroketal with the previously undescribed (5,80-dimethyl-50-oxo-3a0,4,5,50-tetrahydro-3H,30H-spiro[furan-
2,20-isochromeno[3,4,5-def]chromene]-30-yl)but-3-enoic acid skeleton, was isolated from cultures of the fungus Neonectria sp. Its absolute
configuration was assigned by electronic circular dichroism (ECD) calculations. The skeleton of an oxaphenalenone fused with a 1,6-
dioxaspiro[4.5]decane moiety in 1 could be derived from the coisolated putative precursors, corymbiferan lactone E (2) and 3-dehydroxy-4-O-
acetylcephalosporolide C (3).
Oxaphenalenones have been isolated frequently from
fungi and plants.1ꢀ10 The notable structural feature for
this class of natural products is the presence of either a
benzo[de]isochromen-1(3H)-one or a benzo[de]chromen-
2(3H)-one skeleton, in which the naphthalene unit fused
with the δ-lactone moiety in A or B mode of junction
(Figure 1), respectively. Oxaphenalenones are an impor-
tant class of compounds showing various biological ef-
fects. Examples include bacillosporins AꢀC, the dimeric
oxaphenalenones isolated from the fungus Talaromyces
bacillisporus as mycotoxins and antibacterial agents;3 con-
ioscleroderolide, an antibacterial and cytotoxic metabolite
from a marine-derived fungus Coniothyrium cereale;2
2-(40-hydroxyphenyl)-naphthalic anhydride, a phytoalexin
from the unripe green fruit banana Musa acuminata;7 and
scleroderolide, a cholesteryl ester transfer protein (CEPT)
inhibitor from a Penicillium sp. FO-5637.10
† Institute of Microbiology.
‡ Huazhong Agricultural University.
§ Institute of Materia Medica.
^ Beijing Institute of Pharmacology & Toxicology.
r These authors contributed equally to this work.
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r
10.1021/ol302979f
XXXX American Chemical Society

In a search for new cytotoxic metabolites from rarely
studied fungi inhabiting unique environments, a strain of
Neonectria sp. isolated from a soil sample that was col-
lected from the Qinghai-Tibetan plateau (N: 28ꢀ270, E:
97ꢀ020), Chayu, Tibet, People’s Republic of China, was
chemically investigated. Although the Neonectria is a com-
mon fungal genus, its chemistry remained largely unex-
plored.11 Fractionation of an EtOAc extract prepared
from a solid-substrate fermentation culture afforded
neonectrolide A (1), an oxaphenalenone spiroketal with
the new skeleton of 4,5-dihydro-3H,30H-spiro[furan-2,20-
isochromeno[3,4,5-def]chromen]-50(3a0H)-one. Two new
metabolites, corymbiferan lactone E (2) and 3-dehydroxy-
4-O-acetylcephalosporolide C (3), were also isolated as the
putative biosynthetic precursors of 1. Details of the struc-
ture elucidation and cytotoxicity of 1ꢀ3, as well as plau-
sible biogenesis of 1 are reported herein.
Figure 1. Two modes of junction in oxaphenalenones.
C-4, and of C-1 and C-3 to the same oxygen atom,
establishing a δ-lactone unit (ring C) fused with the
naphthalene at C-11/C-11a/C-4. In turn, HMBC correla-
tions from H-40, H-50, H2-70, and H2-80 to C-60 located C-60
between C-50 and C-70. Considering the doubly oxygenated
nature of C-60, and the chemical shifts for C-5 (δC 150.6)
and C-90 (δC 76.9), the two C-60 bondedoxygen atoms were
individually attached to C-5 and C-90 to form a 1,6-
dioxaspiro[4.5]decane moiety to satisfy the unsaturation
requirement of 1, even though no additional evidence for
these linkages were provided by the HMBC data. Collec-
tively, these data (Figure 2) permitted assignment of the
planar structure of 1.
Table 1. NMR Spectroscopic Data for 1 in CDCl3
Neonectrolide A (1) was assigned a molecular formula
of C25H26O8 (13 degrees of unsaturation) by HRESIMS
(m/z 477.1525 [M þ Na]þ; Δ ꢀ0.5 mmu). Analysis of its
NMR data (Table 1) revealed one exchangeable proton
(δH 11.64), four methyl groups (two methoxys), three
methylenes, three methines (two oxymethines), 12 aromatic/
olefinic carbons with four protonated, one doubly oxyge-
nated sp3 quaternary carbon (δC 111.3), and two car-
boxylic carbons (δC 170.9 and 171.9, respectively). The
b
pos.
δHa (J in Hz)
δC
HMBC (H f C#)
1
3
4
5
6
7
170.9
72.9
5.99, d (6.0)
1, 4, 5, 11a, 40, 50
98.1
150.6
95.7
6.31, s
6.76, s
3, 4, 5, 7, 7a
160.0
113.6
147.7
118.2
163.2
98.2
7a
8
1
1Hꢀ H COSY NMR data of 1 defined the two isolated
9
1, 7a, 10, 11, 12
spin systems of C-70ꢀC-100 and C-20ꢀC-3 (via C-50), and
the latter was attached to the C-10 methyl formate on the
basis of HMBC correlationsfrom H2-20 and H3-110 toC-10.
HMBC cross peaks from H-9 to C-7a, C-10, C-11, and
C-12, H-6 to C-4, C-5, C-7, and C-7a, H3-12 to C-7a, C-8,
and C-9, and from the intramolecularly hydrogen-bonded
phenolic proton at 11.64 ppm to C-9, C-10, and C-11, plus
chemical shift (δC 132.7) consideration of the remaining
aromatic carbon (C-11a) in 1, a naphthalene unit (rings A
and B) was established with a methyl and a hydroxy group
located at C-8 and C-10, respectively. A weak, but distinct
four-bond W-type correlation from H-9 to C-1 connected
the C-1 carboxylic carbon (δC 170.9) to C-11,12 whereas
that of H3-13 with C-7 indicated that the C-13 methoxy
unit is attached to C-7. Further correlations from H-3 to
C-1, C-4, C-5, and C-11a enabled the connections of C-3 to
10
11
11a
12
13
10
132.7
25.4
2.80, s
3.94, s
7a, 8, 9
7
55.5
171.9
37.8
20
3.09, mc;
10, 30, 40
3.01, dd (17.0, 8.0)
5.97, mc
30
40
50
60
70
80
90
128.9
127.2
47.5
111.3
36.2
30.9
76.9
21.0
51.9
10, 20, 50
3, 20, 50, 60
3, 4, 30, 60
5.44, dd (15.5, 10.0)
3.08, dd (10.0, 6.0)
2.25, m
50, 60, 80, 90
60, 70, 90, 100
2.30, m; 1.62, m
4.46, m
100
110
OH-10
1.25, d (6.0)
3.68, s
80, 90
10
11.64, s
9, 10, 11
a Recorded at 500 MHz. b Recorded at 125 MHz. c Mutiplicity due to
signal overlapping.
(11) Shiono, Y.; Shimanuki, K.; Hiramatsu, F.; Koseki, T.; Tesuya,
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Liu, X.; Liu, G.; Che, Y. Org. Lett. 2012, 14, 3320–3323.
The C-30/C-40 olefin was assigned E-geometry on the
basis of the large (15.5 Hz) coupling constant observed
B
Org. Lett., Vol. XX, No. XX, XXXX

between H-30 and H-40.13 NOESY correlations of H2-70
with H-30 and H-40 were used to place these protons on the
same face of ring D, whereas those of H-90 with H-50
revealed their spatial proximity (Figure 2). The absolute
configuration of 1 was deduced by comparison of the
experimental and simulated electronic circular dichroism
(ECD) spectra generated by time-dependent density
functional theory (TDDFT).14 Considering the above-
mentioned NOESY data, one of the four stereoisomers,
(3S,50R,60R,90R)-1, (3R,50S,60S,90S)-1, (3S,50S,60S,90S)-1,
and (3R,50R,60R,90R)-1, should represent the actual con-
figuration of 1. Since the 6/6/6/5 ring system in 1
was relatively rigid, which would significantly affect the
CD property, whereas the conformationally flexible side
chain had insignificant effect on the CD spectrum of 1, a
angle of 47.0ꢀ between H-3 and H-50 (Figure S21, Support-
ing Information), corresponding to a 3JHH value of 6.0 Hz
from the Karplus equation (3JHH = A þ B cosΦ þ C
cos2Φ; A = 7, B = ꢀ1, C = 5, Φ = dihedral angle).15,16
Whereas the dihedral angle between H-3 and H-50 was
calculated as 174.4ꢀ in conformer 4d (Figure S22, Support-
ing Information), with a theoretical 3JHH value of 13 Hz,
the actual 3JHH value observed between H-3 and H-50 in 1
was 6.0 Hz, matching that calculated in 4b, supporting the
absolute configuration deduced from the ECD spectra.
Therefore, 1 was deduced to have the 3R, 50S, 60S, and 90S
absolute configuration.
Compound 2 gave a pseudomolecular ion [M þ H]þ
peak at m/z 261.0755 by HRESIMS, consistent with the
molecular formulaC14H12O5. Its 1H and 13C NMR spectra
showed resonances for two exchangeable protons (δH
10.12 and 11.99, respectively), two methyl groups (one
methoxy), one oxymethylene, 10 sp2 carbons with two
protoned, and one carboxylic carbon (δC 170.3). Analysis
of its NMR data revealed structural similarity to corymbi-
feran lactone A,1 except that the C-7 methoxy, C-5 hydro-
xy, and C-4 hydroxymethyl group in corymbiferan lactone
A were replaced by a hydroxy, a methoxy, and a methyl
group in 2, respectively, which were supported by relavant
HMBC data, completing the planar structure of 2 as
shown. (Note: A different numbering system was used
for 2, in which C-7, C-5, and C-4 in corymbiferan lactoneA
corresponded to C-5, C-7, and C-8 in 2, respectively.)
Compound 3 was assigned the molecular formula
C12H18O5 by HRESIMS (m/z 265.1056 [M þ Na]þ; Δ
ꢀ0.4mmu). Its 1H and 13C NMR datawereconsistentwith
those for two methyls, five methylenes, two oxymethines,
two carboxylic carbons (δC 172.1 and 170.0), and a ketone
carbon (δC 208.4). The NMR data of 3 were nearly
identical to those of cephalosporolide C,17 both having
the 10-methyloxecane-2,7-dione moiety. Interpretation of
the 2D NMR data of 3established its structure as 3-dehydroxy-
4-O-acetylcephalosporolide C. The relative configura-
tion of 3 was deduced by NOED data. Upon irradiation
of H-4, enhancement was observed for H-7b in the NOE
difference spectrum of 3, whereas enhancement was
observed for H-7a upon irradiation of H-9, suggesting a
trans relationship between H-4 and H-9.
Figure 2. Selected key HMBC and NOESY correlations of 1.
simplified structure 4 was used for ECD calculations
(Figure 3). A systematic conformational analysis was
performed for 4aꢀ4d by the Molecular Operating Environ-
ment (MOE) software package using the MMFF94 mole-
cular mechanics force field calculation. The MMFF94
conformational search followed by reoptimization using
TDDFT at B3LYP/6-31G(d) basis set level afforded three
lowest-energy conformers for enantiomers 4a and 4b and
two for 4c and 4d, respectively (Figures S7 and S8, Sup-
porting Information). The overall calculated ECD spectra
of 4aꢀ4d were then generated by Boltzmann-weighting of
the conformers. The absolute configuration of 1 was
extrapolated by comparison of the experimental and cal-
culated ECD spectra of 4aꢀ4d (Figure 3). The experimen-
tal CD spectrum of 1 was nearly identical to the calculated
ECD spectrum of (3R,50S,60S,90S)-4 (4b), both showing
positive Cotton effects (CEs) in 230ꢀ265 nm, and negative
CEs in the regions of 270ꢀ295 and 295ꢀ400 nm (Figure 3).
The energy-minimized conformer of 4b showed a dihedral
The absolute configuration of 3 was assigned using the
modified Mosher’s method on the semisynthetic product 5
(Figure S25, Supporting Information).18 Specifically,
treatment of 3 with NaOHꢀMeOH afforded 5, and sub-
sequent treatment of 5 with (S)- and (R)-MTPA Cl
afforded the R- (5a) and S-MTPA (5b) esters, respectively.
The difference in chemical shift values (Δδ = δS ꢀ δR) for
the diastereomeric esters 5b and 5a was calculated to assign
the 4R absolute configuration (Figure S25, Supporting
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Org. Lett., Vol. XX, No. XX, XXXX
C

been previously reported, which incorporated either
a 30,3a0,4,5,50,60,6a0,70-octahydro-3H-spiro[furan-2,20-
pyrano [2,3,4-de]chromene]19ꢀ21 or a 1,2,40,50-tetrahydro-
30H-spiro[benzo[f]chromene-3,20-furan] core,22 compound
1 possesses the previously undescribed (5,80-dimethyl-50-
oxo-3a0,4,5,50-tetrahydro-3H,30H-spiro[furan-2,20-isochro-
meno[3,4,5-def]chromene]-30-yl)but-3-enoic acid skeleton
originated from fusion of an oxaphenalenone and an 1,6-
dioxaspiro[4.5]decane unit. Biosythetically, compound 3,
the 10-membered lactone with the C-9 methyl group, could
be derived from a 10-carbon phenol via a serious of
oxadative reactions.23 In addition, compound 1 could be
generated from the coisolated 2 and 3 via the reaction
cascades as illustrated in the hypothetical biosynthetic path-
ways (Scheme 1).
Scheme 1. Hypothetical Biosynthetic Pathways for 1
Figure 3. The experimental CD spectrum of 1 in MeOH and the
calculated ECD spectra of 4aꢀ4d. Structures 4aꢀ4d represent
four possible stereoisomers of 4.
Acknowledgment. We thank Mr. Zhufeng Geng of
Analytical and Testing Center, Beijing Normal University
for conducting NMR experiments. Financial support
from the National Natural Science Foundation of China
(30925039 and 81001380), the Beijing Natural Science Foun-
dation (5111003), and the Ministry of Science and Technol-
ogy of China (2012ZX09301-003 and 2009CB522300) is
gratefully acknowledged.
Information). Therefore, the 4R and 9S absolute config-
uration was proposed for 3.
Compounds 1ꢀ3 were tested for cytotoxicity against
human tumor cell lines, HeLa, A549, HCT116, and
T24. Compounds 1 and 3 were cytotoxic to T24 cells,
showing IC50 values of 47.1 and 19.0 μM, respectively (the
positive control cisplatin showed an IC50 value of 22.1 μM),
whereas 2 did not show detectable activity at 50 μg/mL.
Compound 1 is a new member of the oxaphenalenone-
derived natural products. Although several synthetic com-
pounds with partial structural similarity to 1 have
Supporting Information Available. Experimental pro-
1
cedures, characterization data, H and 13C APT NMR
spectra of 1ꢀ3, and UV and CD calculations for 1. This
material is available free of charge via the Internet at
http://pubs.acs.org.
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The authors declare no competing financial interest.
D
Org. Lett., Vol. XX, No. XX, XXXX