Varioxepine a, a 3H-oxepine-containing alkaloid with a new oxa-cage from the marine algal-derived endophytic fungus Paecilomyces variotii

Article abstract of DOI:10.1021/ol502329k

A new 3H-oxepine-containing alkaloid, varioxepine A (1), characterized by a structurally unprecedented condensed 3, 6, 8-trioxabicyclo[3.2.1]octane motif, was isolated from the marine algal-derived endophytic fungus Paecilomyces variotii. Due to the low proton/carbon ratio, the unambiguous assignment of the planar structure and relative configuration was precluded by NMR experiments and solved by single crystal X-ray analysis. The absolute configuration was established by DFT conformational analysis and TDDFT-ECD calculations. Compound 1 inhibited plant pathogenic fungus Fusarium graminearum.

Full text of DOI:10.1021/ol502329k

Letter
pubs.acs.org/OrgLett
Varioxepine A, a 3H‑Oxepine-Containing Alkaloid with a New Oxa-
Cage from the Marine Algal-Derived Endophytic Fungus
Paecilomyces variotii
Peng Zhang,†,‡ Attila Mandi,§ Xiao-Ming Li,† Feng-Yu Du,† Jia-Ning Wang,†,‡ Xin Li,†,‡ Tibor Kurtan,*,§
́
́
and Bin-Gui Wang*,†
†Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Nanhai Road 7, Qingdao
266071, People’s Republic of China
‡University of Chinese Academy of Sciences, Yuquan Road 19A, Beijing 100049, People’s Republic of China
§Department of Organic Chemistry, University of Debrecen, P.O. Box 20, 4010 Debrecen, Hungary
S
* Supporting Information
ABSTRACT: A new 3H-oxepine-containing alkaloid, varioxepine
A (1), characterized by a structurally unprecedented condensed
3,6,8-trioxabicyclo[3.2.1]octane motif, was isolated from the marine
algal-derived endophytic fungus Paecilomyces variotii. Due to the
low proton/carbon ratio, the unambiguous assignment of the planar
structure and relative configuration was precluded by NMR
experiments and solved by single crystal X-ray analysis. The
absolute configuration was established by DFT conformational
analysis and TDDFT-ECD calculations. Compound 1 inhibited
plant pathogenic fungus Fusarium graminearum.
xepine- or 3H-oxepine-containing diketopiperazine alka-
loids are new members of a rarely reported class of
from marine red alga, showing antimicrobial activity. Com-
pound 1 is a 3H-oxepine-containing alkaloid having a
condensed 3,6,8-trioxabicyclo[3.2.1]octane unit, which has
not been reported yet in natural products. Structure elucidation
for 1 proved to be difficult, particularly for the central parts
(rings A and B), due to a low proton/carbon ratio and lack of
descriptive 2D NMR signals. Herein, the isolation, structure
elucidation including the determination of absolute config-
uration, bioactivity evaluation, and possible biogenetic pathway
of compound 1 are described.
O
natural products.1 This class of alkaloids generally possesses an
oxepine−pyrimidinone−ketopiperazine tricyclic core derived
from different amino acids. Although a very limited number of
natural products containing these structural features are
described,2 there is increasing fascination with their structural
determination, bioactivity, and biogenesis. Due to various
biological activities such as antiplasmodial, anti-inflammatory,
growth inhibitory, and transcriptional activation on LXRα
(Liver X Receptor),2 the oxepine-containing natural products
have attracted much attention from synthetic chemists.3
Varioxepine A (1)5 was initially obtained as a yellow
amorphous solid. Its molecular formula, C26H29N3O5, was
established from a prominent pseudomolecular ion peak at m/z
464.2184 [M + H]+ (calcd 464.2180) in its HRESIMS,
implying the presence of 14 degrees of unsaturation.
As a continuation of our investigation on structurally novel
and biologically active metabolites from marine-derived fungi,4
a new 3H-oxepine-containing diketopiperazine-type alkaloid,
varioxepine A (1, Figure 1), was isolated from an extract of
Paecilomyces variotii EN-291, an endophytic fungus obtained
1
The H NMR and HSQC spectra (measured in CDCl3)
indicated the presence of 29 protons, including three aromatic
resonances comprising five protons (δH 7.14−7.22, H-2′−H-
6′), eight signals between δH 3.74 and 6.53 due to protons
deshielded as a result of unsaturation or attached heteroatoms
(H-8−H-11, H-15, H2-20, and H-21), four methyl groups [δH
1.54 (s, H3-23), 1.52 (s, H3-24), and 0.86 (d, J = 7.1 Hz, H3-18
and H3-19)], one methine signal at δH 3,22 (m, H-17), and one
methylene at δH 3.08/3.24 (H2-16) as well as an exchangeable
proton signal at δH 7.80 (NH-2). The 13C NMR spectroscopic
data, in conjunction with DEPT experiments, exhibited the
Received: August 6, 2014
Figure 1. Chemical structure of compound 1.
© XXXX American Chemical Society
A
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Organic Letters
Letter
presence of 26 resonances which were assigned to four methyl,
two methylene (with one oxygenated), 12 methine (with nine
aromatic/olefinic), and eight quaternary (with one carbonyl
and four aromatic/olefinic) carbons.
The placement of the NH unit was established by its
correlations to C-1, C-4, and C-15 in the HMBC spectrum
(Figure 2), while the characteristic signals for an α-carbon/
The lack of some key definitive 2D NMR correlations that
could be used to unequivocally elucidate the structure of 1
prompted us to make efforts toward a single crystal X-ray
analysis. With many attempts, some small but suitable crystals
were fortunately obtained by slow evaporation and slow
diffusion of H2O into a saturated mixture of CHCl3−MeOH
(1:1) after eight months.5 Once the X-ray structure could be
resolved, the planar structure and the (12R*,13S*,15S*,21R*)
relative configuration of 1 were confidently assigned (Figure 3).
Figure 2. Selected 2D NMR correlations of compound 1.
proton (CH-15, δC/H 56.1/4.56) and an amide carbon (C-1, δC
165.5) indicated the incorporation of an amino acid unit in the
molecule. Detailed analysis of the COSY and HSQC data led to
the identification of five spin systems, including an oxepin
residue containing four methine protons (H-8−H-11), an
isopropyl moiety (H-17−H-19), two −CH−CH2− units (H-15
and H2-16, and H2-20 and H-21), and a monosubstituted
phenyl group (H-2′−H-6′).
Figure 3. X-ray crystallographic structure of compound 1. (Note: A
different numbering system is used for the structure in the text.)
Further analysis of the 1D and 2D NMR data of 1 resulted in
the connection of most of the above substructures (Figure 2).
The 13C NMR chemical shift of C-15 (δC 56.1) indicated that
this carbon was attached to a nitrogen atom, and HMBC
correlations of NH-2, H-15, and H2-16 with the downfield sp2
carbon (C-1, δC 165.5) positioned this carbon at C-1. Further
HMBC correlations of H-15 and H-17 with C-4, in turn, led to
the completion of the ketopiperazine ring in the molecule. The
presence of the 3H-oxepine ring was deduced by the HMBC
correlations of H-8 with C-6, along with the consideration of
the downfield chemical shifts for C-6 (δC 154.4) and C-8 (δC
143.6), which suggested the connection of them via an oxygen
atom. The observed HMBC correlations of H-10 with C-12
and of H-11 with C-6 provided further information to confirm
the presence of the 3H-oxepine ring.
The fused 3,6,8-trioxabicyclo[3.2.1]octane moiety was
deduced from the relevant HMBC correlations of the
oxymethine proton H-21 with two oxygenated quaternary
carbons C-13 and C-22 as well as with the geminal methyl
groups CH3-23 and CH3-24. Furthermore, H-11 correlated
with the oxy-carbon C-13, while H-20 correlated with the
oxygenated carbons C-12 and C-22, indicating the part of the
oxa-cage unit, ring C, which was condensed with the pyrimidine
moiety via C-12 and C-13. However, the connection of C-13 to
C-22 via an oxygen atom, which is expected to form ring B as
another part of the oxa-cage unit, was not confidently assigned,
since the characteristic HMBC correlation could not be
detected. The remaining heteroatom (N-5) was assigned to
connect with C-4 and C-6 to form the tetrahydro-pyrimidine
ring, which accounted for the remaining unsaturation
equivalents. However, again no useful HMBCs could be
observed for the determination of the pyrimidine core
rendering the assignment of this unusual planar structure
somewhat ambiguous.
The X-ray structure of 1 confirmed the unique ring system
including the unusual oxa-cage moiety (Figure S1 in the
Supporting Information), which has not been reported among
natural products yet.
The relative configuration of 1 was also studied by 2D
ROESY measurements simultaneously with the X-ray analysis.
ROE correlations observed between H-15, H-21, and CH3-24
suggested the same spatial orientation of these groups. The
weak correlations observed from H-11 to H-15 and H3-24
indicated a cis relationship between H-11 and H-15. This
suggested that the pyrimidine ring is cis-fused with the 1,4-
dioxane ring (ring C) and trans-fused with the 1,4-dioxepine
ring (rings B + C).6 The phenyl protons showed ROE
correlations with protons of the C-3 isopropyl methyls as well
as with CH3-24, and these correlations must derive from
different conformers suggesting the axial orientation of the C-
15 benzyl group (vide infra). Next, 1D selective gradient
ROESY experiments were performed (Figure S2 in the
Supporting Information), and the proton signal of H-21 at δH
3.97 was irradiated (Table 1). The results corroborated the
previous finding of the 2D ROESY experiment, but they did
not bring further information.
In order to determine the absolute configuration of 1,
conformational analysis and TDDFT-ECD calculations were
performed on the arbitrarily chosen (12R,13S,15S,21R)
enantiomer of 1, which is an approach that has proved to be
an efficient tool for studying the stereochemistry of condensed
natural products.7 The ECD spectrum of 1 in acetonitrile
showed negative bands at 346 and 242 nm and positive ones at
298 and 213 nm (Figure 4). The DFT reoptimization of the
initial MMFF conformers of (12R,13S,15S,21R)-1 at the
B3LYP/6-31G(d) level in gas phase afforded three major
conformers (37.4%, 32.0%, and 26.4%) above 2% population
B
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Letter
a
Table 1. NMR Data of 1 in CDCl3 (δ in ppm)
no.
δH (mult., J [Hz])
δC
1
165.5 (s)
2 (NH)
7.80 (s)
3
120.3 (s)
119.5 (s)
154.4 (s)
143.6 (d)
102.6 (d)
127.9 (d)
128.3 (d)
76.0 (s)
4
6
8
6.53 (1H, d, 7.3)
5.25 (1H, t, 7.3)
Figure 5. Structures and populations of B3LYP/6-31G(d) conformers
of (12R,13S,15S,21R)-1.
9
10
11
12
13
15
16
5.96 (1H, dd, 9.9, 7.3)
5.82 (1H, d, 9.9)
phenyl ring was pointing away from the piperazine ring and this
conformer matched the X-ray geometry and it was responsible
for the NOE correlation between H3-24 and ortho phenyl
protons. The geometry of conformer C was quite similar to that
of conformer B, but the plane of the phenyl ring was rotated.
The TDDFT-ECD spectra of the conformers were calculated
with three functionals (B3LYP, BH&HLYP, PBE0) and the
TZVP basis set. The computed ECD spectra of all the
conformers reproduced well the experimental solution ECD
spectrum and the 346 nm weak positive Cotton effect (CE)
derived mostly from conformers B and C. Similarly, the
Boltzmann-averaged ECD spectra of (12R,13S,15S,21R)-1 gave
good agreement with the experimental curve with BH&HLYP/
TZVP showing the best match (Figure 4), which allowed the
determination of the absolute configuration as
(+)-(12R,13S,15S,21R)-1.
Compound 1 was hydrolyzed in 6 N HCl (aq.) at 110 °C for
24 h to afford L-phenylalanine with [α]D20 −30.2 (c 0.11, H2O),
which was determined by comparison with an authentic
standard.2f Given the relative configuration, this result
suggested (12R,13S,15S,21R) absolute configuration, which
was in accordance with the result of the TDDFT-ECD
calculations. It should be pointed out that the L-configuration
of our phenylalanine degradation product is the same as that
reported by Lu et al.2d and Li et al.2f for the related oxepine- or
3H-oxepine-containing diketopiperidine metabolites, but oppo-
site to that described by Lee and co-workers.1
111.0 (s)
56.1 (d)
35.8 (t)
4.56 (1H, dd, 9.6, 4.6)
3.08 (1H, dd, 13.5, 9.6)
3.24 (1H, m)
17
18
19
20
3.22 (1H, m)
25.0 (d)
20.1 (q)
20.6 (q)
63.1 (t)
0.86 (3H, d, 7.1)
0.86 (3H, d, 7.1)
3.74 (1H, d, 11.6)
3.95 (1H, m)
21
3.97 (1H, m)
79.6 (d)
81.4 (s)
22
23
1.54 (3H, s)
1.52 (3H, s)
22.0 (q)
28.6 (q)
136.8 (s)
129.4 (d)
128.3 (d)
126.6 (d)
24
1′
2′,6′
3′,5′
4′
7.14 (2H, m)
7.22 (2H, m)
7.15 (1H, m)
a
Recorded at 500 and 125 MHz for H and 13C NMR, respectively.
Data were assigned by DEPT and HSQC as well as COSY and HMBC
experiments.
1
Oxepine-containing diketopiperazine alkaloids are new
members of a rarely observed class of natural products
containing both oxepine (or 3H-oxepine) and diketopiperazine
ring systems. The biosynthesis and total synthesis of these
related metabolites have been reported previously.2c,3,8
Varioxepine A (1) may be biosynthesized by the condensation
of anthranilic acid I with diketopiperazine II to yield the
intermediate III, followed by oxidation of the benzene ring to
form the oxepine derivative V via epoxy precursor IV, and
subsequent epoxidation of V and simultaneous rearrangement
of the double bonds at N-2 and C-4 as well as the opening of
the epoxy ring resulted in the formation of VII via VI, as
illustrated in Scheme 1.2d With the aid of the prenyltransferase,
VII is prenylated at the C-12 hydroxyl group to yield the key
intermediate VIII, which can be regarded as the precursor of 1.
Finally, cyclization to form the trioxabicyclo[3.2.1]octane
subunit presumably occurred from the vicinal diol IX obtained
by dihydroxylation of the prenyl group in VIII.
Figure 4. Experimental ECD spectrum of 1 (black curve) in
acetonitrile compared with the Boltzmann-weighted BH&HLYP/
TZVP spectrum (blue curve) computed for the B3LYP/6-31G(d)
conformers of (12R13S,15S,21R)-1. Bars represent rotational strengths
of the lowest-energy conformer.
(Figure 5). In all the conformers, the dihydro-oxepine ring
adopted a twist-boat conformation with an axial C-12−C-11
bond, which moved the H-11 to the proximity of Hs-24 and the
equatorial H-15. Although the C-15 benzyl group had axial
orientation in the three conformers, the alignment of the
phenyl group was different. In the lowest-energy conformer
(conformer A), the phenyl group was pointing toward the C-3
isopropyl group below the piperazine ring, which was in
accordance with the observed NOE correlations between the
isopropyl methyl and para phenyl proton. In conformer B, the
Compound 1 was evaluated for antimicrobial activity against
several human- and aqua-pathogenic bacteria (Aeromonas
hydrophila, Escherichia coli, Micrococcus luteus, Staphylococcus
aureus, Vibrio anguillarum, V. harveyi, and V. parahemolyticus).
The results revealed that compound 1 has diverse antibacterial
activities with the MIC values ranging from 16 to 64 μg/mL
(Table S1 in the Supporting Information). Moreover, 1
C
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Letter
2075−2080. (f) Li, G. Y.; Yang, T.; Luo, Y. G.; Chen, X. Z.; Fang, D.
M.; Zhang, G. L. Org. Lett. 2009, 11, 3714−3717.
(3) Doveston, R. G.; Steendam, R.; Jones, S.; Taylor, R. J. K. Org.
Lett. 2012, 14, 1122−1125.
Scheme 1. Plausible Biosynthetic Pathway for Compound 1
(R1 = Phenyl, R2 = Isopropyl)
(4) (a) Meng, L. H.; Li, X. M.; Lv, C. T.; Li, C. S.; Xu, G. M.; Huang,
C. G.; Wang, B. G. J. Nat. Prod. 2013, 76, 2145−2149. (b) An, C. Y.;
Li, X. M.; Li, C. S.; Wang, M. H.; Xu, G. M.; Wang, B. G. Mar. Drugs
2013, 11, 2682−2694. (c) Du, F. Y.; Zhang, P.; Li, X. M.; Li, C. S.;
Cui, C. M.; Wang, B. G. J. Nat. Prod. 2014, 77, 1164−1169. (d) Zhang,
P.; Meng, L. H.; Mandi, A.; Kurtan, T.; Li, X. M.; Liu, Y.; Li, X.; Li, C.
́ ́
S.; Wang, B. G. Eur. J. Org. Chem. 2014, 4029−4036.
(5) Varioxepine A (1): initially obtained as a yellow amorphous solid
and then crystals were obtained after eight months from CHCl3−
MeOH−H2O (5:5:1), mp 72−73 °C; [α]2D0: +65 (c 0.20, MeOH); UV
(MeOH) λmax (log ε): 200 (4.18), 326 (3.82) nm; ECD (MeCN, l
[nm] (Δε), c = 1.51 × 10−3 M): 346 (−1.21), 320 sh (1.75), 298
(5.36), 261 sh (−18.37), 242 (−22.88), 213 (21.88), negative CE
1
below 197 nm. For H and 13C NMR, see Table 1; HRESIMS: m/z
464.2184 [M + H]+ (calcd 464.2180).
(6) Zhou, Y.; Man
E. G.; Lin, W. H.; Proksch, P.; Kurtan
2011, 6009−6019.
(7) (a) Zhou, Z. F.; Kurtan
Guo, Y. W. Org. Lett. 2014, 16, 1390−1393. (b) Liang, L. F.; Kurtan
T.; Mandi, A.; Yao, L. G.; Li, J.; Zhang, W.; Guo, Y. W. Org. Lett. 2013,
15, 274−277. (c) Che, Q.; Zhu, T. J.; Mandi, A.; Kurtan, T.; Li, J.; Gu,
́
di, A.; Debbab, A.; Wray, V.; Schulz, B.; Muller, W.
̈
́
, T.; Aly, A. H. Eur. J. Org. Chem.
́
́
, T.; Mandi, A.; Geng, M. Y.; Ye, B. P.;
́
,
́
́
́
exhibited potent inhibitory activity against the plant-pathogenic
fungus Fusarium graminearum, with an MIC value of 4 μg/mL.
Q. Q.; Li, D. H. Org. Lett. 2012, 14, 3438−3441.
(8) Doveston, R. G.; Taylor, R. J. K. Tetrahedron. Lett. 2012, 53,
2533−2536.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures, fungal material, extraction and
isolation, antimicrobial assay, Cartesian coordinates of the
computed conformers, details of computational methods and
copies of 1D and 2D NMR spectra of 1 are disclosed. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: wangbg@ms.qdio.ac.cn (B.-G.W.).
*E-mail: kurtan.tibor@science.unideb.hu (T.K.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support from the Natural Science Foundation of
China (31330009) and from the Ministry of Science and
Technology of China (2010CB833802 and 2013AA092901) is
gratefully acknowledged. T.K. and A.M. thank the Hungarian
National Research Foundation (OTKA K105871) for financial
support and the National Information Infrastructure Develop-
ment Institute (NIIFI 10038) for CPU time.
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