Structure and total synthesis of aspernigerin: A novel cytotoxic endophyte metabolite

Article abstract of DOI:10.1002/chem.200501423

Aspernigerin (1), a novel cytotoxic alkaloid consisting of an unprecedented structural framework has been isolated from the extract of a culture of Aspergillus niger IFB-E003, an endophyte in Cyndon dactylon. Its structure was elucidated on the basis of comprehensive NMR spectral analysis and confirmed by single-crystal X-ray analysis. Aspernigerin (1) has been shown to be cytotoxic to the tumor cell lines nasopharynyeal epidermoid KB, cervical carcinoma Hela, and colorectal carcinoma SW1116 with corresponding IC₅₀ values of 22, 46, and 35 UM, respectively. A feasible total synthetic route for aspernigerin (1) has been established for further pharmacological research.

Full text of DOI:10.1002/chem.200501423

FULL PAPER
DOI: 10.1002/chem.200501423
Structure and Total Synthesis of Aspernigerin: A Novel Cytotoxic Endophyte
Metabolite
Li Shen, Yong-Hao Ye, Xiao-Ting Wang, Hai-Liang Zhu,* Chen Xu, Yong-Cun Song,
Hai Li, and Ren-Xiang Tan*[a]
Abstract: Aspernigerin (1), a novel cy-
totoxic alkaloid consisting of an unpre-
cedented structural framework has
been isolated from the extract of a cul-
ture of Aspergillus niger IFB-E003, an
endophyte in Cyndon dactylon. Its
structure was elucidated on the basis of
comprehensive NMR spectral analysis
and confirmed by single-crystal X-ray
analysis. Aspernigerin (1) has been
shown to be cytotoxic to the tumor cell
lines nasopharynyeal epidermoid KB,
cervical carcinoma Hela, and colorectal
carcinoma SW1116 with corresponding
IC50 values of 22, 46, and 35 mm, respec-
tively. A feasible total synthetic route
for aspernigerin (1) has been establish-
ed for further pharmacological re-
search.
Keywords: aspernigerin · cytotoxic ·
endophyte · structure elucidation ·
total synthesis
Introduction
curiosity to include endophytes, a large population of poorly
investigated microbes, which have been reasoned to be
unique, at least with regards to the fact that they spend their
life spans in a symbiotic manner inside the living normal
plant tissue where they could have “gene recombinations”
with the host species.[5]
A fast-growing wealth of reports has affirmed that cancer
has been and will continue to be one of the major life-
threatening diseases in both developed and developing
countries owing principally to the shortage of efficacious
tumor-eradicating/suppressing agents. Comparable to taxol
(a plant secondary metabolite detectable in most Taxus spe-
cies), some microbe-generated natural products were proven
to be quite unique in treating/preventing cancerous disor-
ders.[1] Whilst current chemical and biological interests are
being focused on early microbial secondary metabolites,
such as prodigiosin derivatives,[2] it has been postulated that
microorganisms collected from previously unexplored habi-
tats may provide access to a large family of efficient produc-
ers of novel lead structures for new antitumor agents.[3] As a
follow up to our characterization of tumor suppression relat-
ed metabolites from marine microbes,[4] we expanded our
Aspergillus niger IFB-E003 was isolated as an endophyte
from the healthy leaves of Cynodon dactylon, a salty soil
borne grass belonging to the family Graminae. Previously,
this endophytic strain was disclosed to be a rich source of bi-
oactive naphtha-pyrone framed polyphenols.[6] Surprisingly,
the combined mother liquor left upon fractionations of
those phenolic metabolites from the extract was demonstrat-
ed to contain basic substances with pronounced cytotoxic ac-
tivity. Subsequent attention to the cytotoxic fraction led to
the full characterization of an active alkaloid with an unpre-
cedented structural framework, trivially named aspernigerin
(1,4-bis[2-(3,4-dihydro-2H-quinolin-l-yl)-2-oxoethyl]pipera-
zine 1). This paper is dedicated to the structure elucidation,
cytotoxicity, and total synthesis of this novel antitumor en-
dophyte metabolite.
[a] L. Shen, Y.-H. Ye, X.-T. Wang, Prof. Dr. H.-L. Zhu, Dr. C. Xu,
Dr. Y.-C. Song, H. Li, Prof. Dr. R.-X. Tan
Institute of Functional Biomolecules
State Key Laboratory of Pharmaceutical Biotechnology
Nanjing University, Nanjing 210093 (P. R. China)
Fax : (+86)25-8330-2728
E-mail: zhuhl@nju.edu.cn
rxtan@nju.edu.cn
Supporting information (Figures S1–S9 and Table S1) for this article
is available on the WWW under http://www.chemeurj.org/ or from
the author.
Chem. Eur. J. 2006, 12, 4393 – 4396
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
4393

Results and Discussion
3.28 (s) and 53.0/2.56 ppm (s) with an integration ratio of
1:2. These spectral data, along with its molecular formula
and the remaining unsaturation degrees (see above), could
only be explained by assuming the presence of a piperazine
ring with both nitrogen atoms bridged through a methylene
group to the carbonyl of the N-acyl-1,2,3,4-tetrahydroquino-
line residue. Moreover, this assumption was reinforced by
the HMBC correlations of H-12 to C-11 and C-14/15, and
the NOE cross-peak between H-12 and H-14/15. Thus, the
structure of the cytotoxic alkaloid was determined to be 1,4-
bis[2-(3,4-dihydro-2H-quinolin-l-yl)-2-oxoethyl]piperazine
(1). For simplicity, we have trivially named the alkaloid as-
pernigerin.
Aspernigerin (1) was afforded as colorless crystals. Its mo-
lecular formula C26H32N4O2, necessitating 13 degrees of un-
saturation, was evidenced from the protonated molecular
ion at m/z 433.2605 (calculated for the formula of molecular
mass m/z 433.2598) in its HR (+)-ESI mass spectrum. How-
ever, the 13C NMR spectrum of 1 produced a total of only
twelve carbon resonance lines, implying that it was a highly
symmetric molecule consisting most probably of two identi-
cal motifs, each containing a pair of equivalent carbons. A
1
correlative interpretation of its IR, H and 13C NMR (Figur-
es S1 and S2 see Supporting Information), and DEPT spec-
tra suggested that each motif was composed of a 1,2-disub-
stituted benzene nucleus, an amine, an amide carbonyl, and
six methylenes, two sets of which correspond altogether to
ten degrees of unsaturation. The three that were left had to
be compensated for by creating three more rings for the
structure of 1. With this in mind, subsequent scrutiny of its
2D NMR spectra (HMBC, HMQC, COSY, and NOESY,
Figures S3–S6), allowing for the unambiguous assignment of
all carbon and proton signals, generated the structural as-
sumption for aspernigerin (1) on the basis of the following
spectral features.
Amide-containing molecules might show trans/cis equili-
briums in solution, which could be assigned by NMR techni-
ques.[7,8] As for aspernigerin (1), a broadened H-8 singlet at
d=7.45 ppm meant that the amide C=O must be oriented
preferably towards the phenyl ring (Scheme 1).[8c] Further-
The COSY correlations of the H-3 quintuplet with a pair
of triplets produced by H-2 and H-4 revealed the
Scheme 1. Amide-bond rotation of aspernigerin (1).
ꢀ
ꢀ
CH2CH2CH2 moiety isolated by heteroatom(s) and/or
quaternary carbon(s). However, the magnitude of the C-2/
H-2 shift values (dC/dH =43.4/3.80 ppm) confirmed the an-
chorage of 2-methylene on a more electron-poor nitrogen
atom. Along with the discernible NOE effect and allyl-like
coupling of H-4 with H-5, the HMBC correlations of H-3 to
C-10, of H-4 to C-5, and of H-5 to C-4 highlighted the con-
nection of 4-methylene to C-10 (dC =133.0 ppm) in the ben-
zene nucleus. These observations, in conjunction with the
HMBC correlations of H-2 to C-9 and C-11 (dC =139.0
and 169.0 ppm, respectively), disclosed the presence of the
N-acyl-1,2,3,4-tetrahydroquinoline residue. Subtracting sig-
more, the splitting pattern of the 2,3,4-methylene proton
pairs, which appeared as triplet, quintuplet, and triplet, re-
spectively, highlighted that the reduced ring of the tetrahy-
droquinoline moieties was not in a rigid half-chair confor-
mation, but in a rapidly inverting half-chair conformation.[9]
The anticipated conformation of 1 in solution was contrary
to that of some previously recognized analogues.[8c,d]
Accordingly, X-ray crystallographic analysis of this metab-
olite was highly desired for the structural and conformation-
al assignment of 1. A colorless single crystal of 1 obtained
from its MeOH solution was found to be suitable for the X-
ray diffraction analysis and the data subsequently obtained
confirmed the structure elucidated for 1 (Figures 1 and S9).
Moreover, this crystallographic determination revealed that
the piperazine at the center of the molecule was in a chair-
conformation with the two carbonyl groups stretching to-
wards two opposite directions.
nals due to the residue from the H and 13C NMR spectra of
1 produced only two methylene resonances at dC/dH =60.7/
1
Abstract in Chinese:
Figure 1. X-ray crystallographic structure of 1.
4394
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Total Synthesis of Aspernigerin
FULL PAPER
The conformation of the amide in 1 determined by its
NMR spectral data acquired in CDCl3 was not completely
consistent with that from its single-crystal X-ray diffraction,
highlighting that its conformation is highly state-dependent.
This is a reasonable assumption as the trans/cis rotation
equilibrium codepends on temperature[8a,10] and the sol-
vent.[7a,8b,11] Obviously, the CDCl3 used in this case might be
the appropriate solvent for the preferred rotamer (corre-
sponding to that with carbonyls orienting towards the
phenyl ring) of aspernigerin (1).
production of this endophyte metabolite, we have also de-
veloped a feasible total synthetic route for 1. In addition to
its antitumor activity, comparable to that of 5-fluorouracil,
aspernigerin (1) possesses a unique structural framework,
not yet encountered in any presently available anticancer
drug or investigated cytotoxic chemicals. Accordingly, this
work is of particularly high significance for the research and
development of a new family of antitumor drugs with such
structural character, the biosynthetic pathway of which pro-
vides a big mystery for biochemists.
The in vitro cytotoxicity of aspernigerin (1) was evaluated
against three human tumor cell lines, and as a result it was
found to be significantly cytotoxic against the human tumor
cell lines, nasopharynyeal epidermoid KB, cervical carcino-
ma Hela, and colorectal carcinoma SW1116, with the corre-
sponding IC50 (the inhibition concentration at which 50%
survival of cells was allowed) values of 22, 46, and 35 mm, re-
spectively. The IC50 values of 5-fluorouracil (a clinically pre-
scribed antitumor drug coassayed as a positive control)
against the three tumor cell lines were 14, 115, and 42 mm,
respectively.
To develop a feasible route both for the construction of
the novel framework and for enough material of 1 required
for the pharmacological research, total synthesis of asperni-
gerin (1) was carried out based on the retrosynthetic analy-
sis illustrated in Scheme 2. Starting from commercially avail-
Experimental Section
General: Melting points were measured on a Boetius micromelting point
apparatus and are uncorrected. IR spectra were recorded on KBr disks
by using a Nexus 870 FTIR spectrometer, and UV spectra on a U-3000
UV-VIS spectrometer. NMR spectra were acquired in CDCl3 on Bruker
DRX-500 and DPX-300 spectrometers by using solvent signal and TMS
as internal standards. The EIMS spectrum was recorded on a VG-ZAB-
HS mass spectrometer, and high-resolution electrospray ionization mass
spectrometry (HR-ESI-MS) spectrum on a Mariner Mass 5304 instru-
ment. ELISA plate reader was obtained from Sunrise (USA). Silica gel
(200–300 mesh) for column chromatography and silica GF254 for TLC
were produced by Qingdao Marine Chemical Company (China). Sepha-
dex LH-20 was purchased from Pharmacia Biotech (Sweden). All chemi-
cals used in the study were of analytical grade.
Microorganism: The isolation and identification of the endophytic fungal
strain A. niger IFB-E003 have been
described in our previous paper.[6]
Extraction and isolation: Cultivation
of strain IFB-E003 and fractionation
for naphtha-pyrones have been previ-
ously detailed.[6] The combined mother
liquor left therefrom, after having
been tested for recognition of cytotox-
ic compounds, was rechromatographed
by using silica-gel column chromatog-
raphy (200 g silica gel; CHCl3/MeOH
100:0!100:16). By using TLC moni-
toring, the elutes produced (500 mL
each) were combined into six frac-
tions, which were assessed for cytotox-
icity. The active fraction (fraction 2)
was further fractionated over silica gel
(CHCl3/MeOH 100:0!100:16) to
afford three parts (fractions 2–1–frac-
tions 2–3). Repeated gel filtration of
Scheme 2. Total synthetic route for aspernigerin (1). Reagents and conditions: a) BrCH2COOH, aqueous
NaOH solution (10%), RT, 1 h, 80%; b) HCl (37%, several drops), RT, stirring, 10 min, 74.6%; c) SOCl2,
DMF, CH2Cl2, reflux, 4 h; d) 1,2,3,4-tetrahydroquinoline, DMAP, pyridine, CH2Cl2, 08C!RT, 4 h, 47.3%
(steps c and d). DMF=N,N-dimethylformamide; DMAP=4-dimethylaminopyridine.
able piperazine and 1,2,3,4-tetrahydroquinoline, 1 could be
fractions 2–2 (cytotoxic) over Sephadex LH-20 (CHCl3/MeOH 1:1) pro-
duced aspernigerin (1, 93 mg). Colorless crystal; m.p. 186–1888C; for 1H
and 13C NMR data (CDCl3) see Table 1; IR (KBr): n˜ =2950, 2805, 1639,
1582, 1490, 1454, 1391, 1015, 845, 778 cmꢀ1; UV/Vis (MeOH): lmax (log
e)=246 (4.23), 215 nm (4.37); EIMS (70 eV): m/z (%): 432 [M]+ (9), 300
(3), 272 (100), 217 (9), 172 (62), 111 (79), 56 (46), 42 (51); HR-ESI-MS:
m/z: calcd for C26H32N4O2: 433.2598; found: 433.2605 [M+H]+.
synthesized in four steps in good yields. The synthetic mate-
rial was identical to the natural metabolite (1, Figure S8).
Regarding the manipulation of the reaction, it was notewor-
thy that addition of a catalytic amount of DMAP was re-
quired, which allowed the completion of the reaction at
room temperature within 3–4 h.
Crystal structure measurement of aspernigerin (1): Crystal structure de-
termination was carried out on a Nonius CAD4 diffractometer equipped
with graphite-monochromated MoKa (l=0.71073) with Lorentz polariza-
tion and absorption corrections for a crystal (0.25”0.20”0.20 mm) of the
title compound. The intensities were collected at 293 K by using w scan
mode with variable scan speed. A total of 11639 reflections were collect-
ed in the range of q=2.37–25.99o, of which 2238 were independent. The
1831 observable reflections with Iꢁ2s(I) were used in the structure solu-
tion and refinements. The structure was solved by direct methods and re-
fined on F2 by full-matrix least-squares methods by using SHELX-97.[12]
Conclusion
We have characterized a novel potent antitumor metabolite,
aspernigerin (1), from A. niger IFB-E003, an endophytic
fungus residing in the healthy leaves of C. dactylon. For a
chemical generation of the novel framework and a scaled-up
Chem. Eur. J. 2006, 12, 4393 – 4396
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
4395

R.-X. Tan, H.-L. Zhu et al.
Table 1. NMR spectral data for aspernigerin (1) in CDCl3.
and pyridine (1.2 mL, 14.8 mmol) were
added sequentially to the suspension,
kept in an ice bath, followed by the
dropwise addition of 1,2,3,4-tetrahy-
droquinoline (394 mg, 2.96 mmol). The
reaction mixture was stirred for 4 h at
room temperature. After evaporation
of the solvent, the remaining mixture
was subjected to silica-gel column
C no.
dC [ppm]
dH [ppm], mult., int., J [Hz]
COSY
Key HMBC correlations
2/2’
3/3’
4/4’
5/5’
6/6’
7/7’
8/8’
9/9’
43.4 CH2
24.0 CH2
26.8 CH2
128.5 CH
125.3 CH
126.1 CH
124.4 CH
139.0 C
3.80, t, 4H, 6.5
1.96, q, 4H, 6.5
2.72, brt, 4H, 6.5
7.13, m, 2H
7.09, m, 2H
7.14, m, 2H
H-3
C-3/3’, 4/4’, 9/9’, 11/11’
C-2/2’, 4/4’, 10/10’
C-3/3’, 5/5’, 9/9’
C-4/4’, C-7/7’
C-5/5’, 8/8’,10/10’
C-5/5, 8/8’, 9/9’
H-2, H-4
H-3, H-5
H-4
7.45, brs, 2H
chromatography
(CHCl3/MeOH
100:1!100:8). After evaporation of
the solvent from the elute, the afford-
ed residue was subjected to gel filtra-
tion over Sephadex LH-20 (CHCl3/
CH3OH 1:1) to give 1 (152 mg, 47.3%
yield, two steps).
10/10’
11/11’
12/12
14/14’, 15/15’
133.0 C
169.0 C
60.7 CH2
53.0 CH2
3.28, s, 4H
2.56, brs, 8H
C-11/11’, 14,14’/15,15’
All the nonhydrogen atoms were refined anisotropically. All the hydro-
gen atoms were placed in calculated positions and were assigned fixed
isotropic thermal parameters at 1.2 times the equivalent isotropic U of
the atoms to which they are attached and allowed to ride on their respec-
tive parent atoms. The contributions of these hydrogen atoms were in-
cluded in the structure-factors calculations. The refinement gave the final
Acknowledgements
This work was cosupported by the Natural Science Foundation of China
(Key Project: 20432030) and the Ministry of Science and Technology of
China (Marine-863 Grant: 2003 AA624010).
R1 =0.042
with
w=[s2(Fo)2 +(0.1(max(0,F2o)
C
AHCTREUNG
ACHTREUNG
gerin (1, H2O) are listed in Table S1.[13]
Cytotoxicity assay: The cytotoxicity was evaluated as described else-
where[14] with some modifications. Briefly, target tumor cells were grown
to log phase in RPMI 1640 medium supplemented with 10% fetal bovine
serum. After diluting to 2”104 cellsmLꢀ1 with the complete medium, the
obtained cell suspension (100 mL) was added to each well of 96-well cul-
ture plates. The subsequent incubation was permitted at 378C, 5% CO2
atmosphere for 24 h before the cytotoxicity assessments. Tested samples
at preset concentrations were added to 6 wells with 5-fluorouracil coas-
sayed as a positive reference. After 48 h exposure period, PBS (40 mL
containing 2.5 mgmLꢀ1 of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT)) was added to each well. After 4 h the medium
was replaced by DMSO (150 mL) to solubilize the purple formazan crys-
tals produced. The absorbance at 570 nm of each well was measured on
an ELISA plate reader. The IC50 value was defined as the concentration
at which 50% survival of cells was allowed.
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Total synthetic procedure for aspernigerin (1): Solvents were dried and
refined by the conventional procedure prior to use. DMF was distilled
under reduced pressure from magnesium sulfate, dried twice over molec-
ular sieves (4 Š), and stored over molecular sieves (4 Š). CH2Cl2 was dis-
tilled from P2O5. Pyridine was distilled from NaOH.
N,N’-1,4-Piperazinediacetic acid: Piperazine (150 mg, 1.74 mmol) and
bromoacetic acid (496 mg, 3.57 mmol) were dissolved in NaOH (10%,
10 mL) and stirred for 1 h at room temperature. Colorless needles
(namely, disodium N,N’-1,4-piperazinediacetate, 343 mg, 80% yield) were
formed and collected by filtration. The obtained needles were dissolved
in water and treated with several drops of HCl (37%) whilst stirring for
10 min at room temperature. The white precipitate produced was collect-
ed by filtration and dried in vacuo to give N,N’-1, 4-piperazinediacetic
acid (210 mg, 74.6%). M.p. 250–2528C; 1H NMR (D2O): d=3.81 (s, 4H;
2”CH2), 3.58 ppm (s, 8H; 4”CH2) (see Figure S7); ESIMS: m/z: 203.1
[M+H]+.
Aspernigerin (1): DMF (2 drops) and SOCl2 (0.22 mL, 2.96 mmol) were
added sequentially to a suspension of N,N’-1,4-piperazinediacetic acid
(150 mg, 0.74 mmol) in CH2Cl2 (10 mL), followed by stirring under reflux
until no more HCl was produced. Removal of the solvent and excess
SOCl2 in vacuo (<608C) provided N,N’-1,4-piperazinediacetyl chloride
which was then suspended in CH2Cl2 (10 mL). DMAP (40 mg, 0.3 mmol)
[14] C. J. Wang, J.-G. Delcros, J. Biggerstaff, O. Phanstiel, J. Med. Chem.
2003, 46, 2663–2671.
Received: November 17, 2005
Published online: March 23, 2006
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Chem. Eur. J. 2006, 12, 4393 – 4396