Diketopiperazines from the Cordyceps-colonizing fungus Epicoccum nigrum

Article abstract of DOI:10.1021/np900654a

New diketopiperazines, epicoccins E-H (1-4) and diphenylalazines A (5) and B (6), have been isolated from the solid-substrate fermentation culture of the Cordyceps-colonizing fungus Epicoccum nigrum. The structures of 1-6 were determined primarily by NMR

Full text of DOI:10.1021/np900654a

J. Nat. Prod. 2009, 72, 2115–2119
2115
Diketopiperazines from the Cordyceps-Colonizing Fungus Epicoccum nigrum
†
†
‡
§
†
‡
†
,†
Huijuan Guo, Bingda Sun, Hao Gao, Xulin Chen, Shuchun Liu, Xinsheng Yao, Xingzhong Liu, and Yongsheng Che*
Key Laboratory of Systematic Mycology & Lichenology, Institute of Microbiology, Chinese Academy of Sciences,
Beijing 100190, People’s Republic of China, Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan
UniVersity, Guangzhou 510632, People’s Republic of China, and State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese
Academy of Sciences, Wuhan 430071, People’s Republic of China
ReceiVed October 17, 2009
New diketopiperazines, epicoccins E-H (1-4) and diphenylalazines A (5) and B (6), have been isolated from the
solid-substrate fermentation culture of the Cordyceps-colonizing fungus Epicoccum nigrum. The structures of 1-6 were
determined primarily by NMR experiments, and the structure of 1 was confirmed by X-ray crystallography. The absolute
configurations of 3 and 5 were assigned using the modified Mosher (3) and Marfey’s (5) methods, respectively. Compounds
3
-5 showed inhibitory effects on HIV-1 replication in C8166 cells.
Diketopiperazines (DKPs) are an important class of compounds
displaying a variety of biological effects, such as antimicorbial,
1-3
herbicidal, antiviral, immunosuppressive, and antitumor activities.
Naturally occurring DKPs have frequently been isolated from fungal
sources. Examples include golmaenone, a radical scavenger from
4
a marine-derived Aspergillus sp., the rostratins, cytotoxic DKP
5
disulfides from a marine-derived Exserohilum rostratum, the
epicorazines, antibacterial metabolites from a basidiomycete
6
Stereum hirsutum, and the verticillins, dimeric DKPs from a
7
marine-derived Penicillium sp.
Cordyceps sinensis (Berk.) Sacc. (Anamorph: Hirsutella sinensis;
8
Clavicipataceae), known as Chinese caterpillar fungus or “Dong
Chong Xia Cao” (caterpillar-in-winter, herb-in-summer), is the
combination of the fungus and the dead caterpillar larva of the moth
Hepilus spp. It has been known and used for centuries in traditional
9
Chinese medicine (TCM). In nature, it is found only at high
altitudes on the Himalayan Plateau and is therefore difficult to
harvest. Chemical investigations of the fungal species that colonize
the fruiting body of C. sinensis (Cordyceps-colonizing fungi) in
our laboratory have led to the isolation of structurally diverse and
carbons including two ketone ones. These data accounted for all
of the H and C resonances for 1 and revealed structural similarity
1
13
1
0-12
biologically active metabolites.
agents from this unique fungal source, the fungus Epicoccum nigrum
XZC04-CS-302) isolated from a sample of C. sinensis collected
In a search for anti-HIV-1
Table 1. NMR Spectroscopic Data (500 MHz, DMSO-d
6
) for 1
(
and 2
in Linzhi, Tibet, People’s Republic of China, was grown in a solid-
substrate fermentation culture. Its organic solvent extract showed
inhibitory effects on HIV-1 replication in C8166 cells. Fractionation
of the extract afforded six new DKPs, named epicoccins E-H
1
2
position
δ
C
, mult.
δ
H
(J in Hz)
δ
C
, mult.
H
δ (J in Hz)
1
2
160.8, qC
70.5, qC
43.2, CH
162.9, qC
74.2, qC
45.7, CH
(
1-4) and diphenylalazines A (5) and B (6), together with the
3
a
2
2.76, br d (12)
2
2.54, d (12)
known epicoccins A (7), B (8), and D (9), which were previously
isolated by us from a different strain of Cordyceps-colonizing E.
nigrum. Details of the isolation, structure elucidation, and anti-
3b
4
2.94, dd (12, 8.0)
44.9, CH 3.04, t (8.0)
207.2, qC
2.88, br d (11)
3.16, dd (11, 5.0)
41.4, CH 3.73, t (5.0)
65.3, CH 3.95, dd (5.0, 2.5) 64.8, CH 4.59, br d (3.0)
2.76, dd (12, 8.0)
43.4, CH 3.08, t (8.0)
207.9, qC
2.44, br d (18)
3.05, d (18)
45.4, CH 3.72, br d (10)
1
1
5
6
6
7
8
9
1
2
3
a
b
41.5, CH
2
38.0, CH
2
HIV-1 activity of these metabolites are reported herein.
Results and Discussion
60.6, CH 4.67, d (8.0)
157.4, qC
62.3, CH 4.46, br d (8.0)
159.9, qC
′
Epicoccin
E
(1) was assigned the molecular formula
′
74.0, qC
51.9, CH
69.7, qC
41.6, CH
C H N O S (11 degrees of unsaturation) on the basis of its
18 18 2 7 2
′a
2
2.87, br d (11)
2.99, dd (11, 7.0)
45.1, CH 3.08, t (7.0)
203.3, qC
53.7, CH 3.18, d (5.0)
2
2.55, d (12)
2.78, dd (12, 8.0)
38.8, CH 2.69, t (8.0)
61.0, CH 4.27, dd (8.0, 5.0)
1.93, dd (14, 8.0)
2.69, dd (14, 8.0)
42.4, CH 3.35, d (8.0)
+
1
HRESIMS (m/z 461.0448 [M + Na] ; ∆ +0.5 mmu). The H and
C NMR data of 1 (Table 1) suggested the presence of three
3′b
4′
1
3
5
6
6
7
8
9
′
exchangeable protons (δ 5.56, 5.74, and 6.24, respectively), three
′a
′b
′
36.5, CH
2
methylenes, nine methines (five bonded to oxygen or nitrogen),
3
two heteroatom-bonded sp quaternary carbons, and four carbonyl
78.1, CH 4.39, t (5.0)
63.3, CH 4.44, dd (7.0, 5.0) 66.7, CH 3.78, br d (3.0)
60.6, CH 4.62, t (7.0)
6.24, d (2.5)
′
′
57.5, CH 4.33, br d (8.0)
6.04, d (3.0)
*
To whom correspondence should be addressed. Tel/Fax: +86 10
OH-8
OH-5′
OH-7′
OH-8′
8
2618785. E-mail: cheys@im.ac.cn.
†
4.80, d (5.0)
Institute of Microbiology.
Jinan University.
Wuhan Institute of Virology.
‡
5.74, d (5.0)
5.56, d (4.0)
§
5.80, d (3.0)
1
0.1021/np900654a  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/17/2009

2
116 Journal of Natural Products, 2009, Vol. 72, No. 12
Guo et al.
) for 3
Table 2. NMR Spectroscopic Data (500 MHz, DMSO-d
and 4
6
3
4
position
δ
C
, mult.
δ
H
(J in Hz)
δ
C
, mult.
H
δ (J in Hz)
1
2
3
3
4
5
6
6
/1′
165.6, qC
71.6, qC
34.1, CH
165.5, qC
71.4, qC
34.1, CH
/2′
a/3′a
b/3′b
/4′
/5′
a/6′a
b/6′b
2
2.27, dd (13, 8.0)
2.76, brd (13)
2
2.32, dd (13, 8.5)
2.74, br d (13)
44.0, CH 2.93, t (8.0)
207.7, qC
33.8, CH
43.5, CH 2.91, t (8.5)
207.3, qC
43.2, CH
2
2.20, dt (17, 4.0)
2.59, ddd
2
2.39, dd (17, 4.0)
2.59, dd (17, 10)
(
17, 12, 6.0)
Figure 1. Thermal ellipsoid representation of 1.
7
a/7′a
25.8, CH
2
1.88, dd (17, 12)
65.7, CH 4.13, ddd
(10, 5.0, 4.0)
1
1
1
to those of both epicoccins B (8) and D (9). Specifically, the H
7
8
9/9′
b/7′b
/8′
2.12, dd (17, 4.0)
1
3
and C NMR chemical shifts for the left portion of 1 were nearly
identical to those of the symmetrical 9, whereas those for the right
portion of 1 closely matched the right half of 8. These observations
64.7, CH 4.30, dd (5.5, 3.0) 68.3, CH 4.27, m
63.4, CH 4.28, d (8.0, 5.5) 63.2, CH 4.32, dd (8.5, 4.0)
14.2, CH
10/10′
OH-7/7′
OH-8/8′
3
1.90, s
14.1, CH
3
1.92, s
1
1
5.08, d (5.0)
5.30, d (3.0)
were supported by relevant H- H COSY and HMBC correlations,
leading to assignment of the gross structure of 1 as shown.
The relative configuration of 1 was assigned on the basis of its
5.30, d (3.0)
1
1
1
H- H coupling constants and by comparison of its H NMR data
mmu). Analysis of its H and 13C NMR data (Table 2) revealed
1
with those of the known precedents 8 and 9. The same small
coupling constant (8.0 Hz) observed between H-4 and H-9 as in 8
and 9 suggested their cis relationship, whereas a coupling constant
of 5.0 Hz between H-7 and H-8 indicated that they were pseu-
doequatorially oriented with respect to the cyclohexanone ring.
Absence of coupling between H-8 and H-9 implied that they were
nearly perpendicular to each other. Thus, the relative configuration
for the left portion of 1 was assigned to be the same as that of 9.
Similar assignment was made for the right portion of 1, which
possesses the same relative configuration as the right portion of 8.
The gross structure and the relative configuration of 1 were
confirmed by single-crystal X-ray diffraction analysis, and a
perspective ORTEP plot is shown in Figure 1. The presence of
one methyl group, three methylenes, three methines (one of which
was oxygenated), and two carbonyl carbons (δ 165.6 and 207.7,
C
respectively). These data accounted for only half of its elemental
composition, suggesting its symmetrical nature. Interpretation of
1
1
the H- H COSY NMR data identified an isolated proton spin
system, which was C-6/6′-C-9/9′-C-4/4′-C-3/3′ (including OH-
8
/8′). HMBC correlations from H-3a/3′a to C-1/1′ and C-5/5′ and
from H-6b/6′b to C-5/5′ established the C-3/3′-C-6/6′ fragment,
completing the cyclohexanone ring. Upon extensive analysis of its
2
D NMR data, the symmetrical 6-5-6-5-6 DKP skeleton was
established for 3, but without the cross-ring bridges as found in 1
or 2. The chemical shift of C-10/10′ (δ /δ 1.90/14.2) and the
HMBC cross-peak from H -10/10′ to C-2/2′ indicated that C-10/
0′ was connected to the sulfur atom attached to C-2/2′, which is
H
C
13
3
two sulfur atoms in 1 and the value of the Flack parameter (0.045)
1
also allowed assignment of the absolute configurations of all the
stereogenic centers in 1 as 2R,4R,7R,8R,9S,2′R,4′R,6′S,7′R,8′R,9′S.
The molecular formula of epicoccin F (2) was established as
1
4
closely related to that of exserohilone (10), except that the olefin
unit in 10 was reduced in 3.
C
18
H
20
N
2
O
6
S
3
(10 degrees of unsaturation) by HRESIMS (m/z
The relative configuration of 3 was also assigned by analysis of
+
1
1
4
79.0376 [M + Na] ; ∆ +0.5 mmu). The presence of three sulfur
its H- H coupling constants and NOESY data. The 8.0 Hz vicinal
coupling constant for H-4/4′ and H-9/9′ revealed their cis relation-
ship, compared to the trans configuration deduced from the large
coupling constant of 12.6 Hz for corresponding protons in exse-
atoms in this formula implied that 2 could possess the same sulfur
linkages as in epicoccin A (7). Indeed, the NMR data of 2 (Table
1
) revealed nearly identical structural features to those of 7, except
that one carbonyl carbon (δ 207.8) was replaced by an oxymethine
/δ 4.27/61.0) in 2, suggesting that the C-10 ketone functionality
14
rohilone (10). No coupling was observed between H-8/8′ and H-9/
9′, indicating that both protons are pseudoequatorially oriented.
(δ
H
C
was reduced. This observation was consistent with the fact that
NOESY correlations of H -10/10′ with H-3b/3′b and of H-3a/3′a
3
the molecular weight of 2 is two mass units higher than that of 7
with H-4/4′ indicated that H -10/10′ was opposite H-4/4′. Further
3
1
1
1
14
and was confirmed by H- H COSY correlations of H-5′ with H-4′,
OH-5′, and H-6′, as well as HMBC cross-peaks from H-4′ and H-6′a
to C-5′. Therefore, the gross structure of epicoccin F was established
as depicted in 2.
comparison of the H NMR data of 3 with those of 10 and
rostratin B established its relative configuration as shown.
5
The absolute configuration of 3 was determined using the modified
15,16
Mosher method.
Treatment of 3 with (S)- and (R)-MTPA Cl
The relative configuration of 2 was deduced from 1H- H
coupling constants and NOESY data. The coupling patterns for
relevant protons on the left portion of 2 were identical to their
counterparts on the left portion of 7, indicating that the left halves
of both metabolites possess the same relative configuration. The
1
afforded the (R)-MTPA (3a) and (S)-MTPA esters (3b), respectively.
The difference in chemical shift values (∆δ ) δ - δ ) for the dia-
S
R
stereomeric esters 3b and 3a was calculated to assign the 8S absolute
configuration. Therefore, the 1R,4S,5S,8S,9S,1′R,4′S,5′S,8′S,9′S ab-
solute configuration was proposed for 3 on the basis of the ∆δ
results summarized in Figure 2.
8.0 Hz coupling constant observed for H-4′ and H-9′ revealed their
cis relationship, whereas the lack of coupling between H-4′ and
H-5′, H-7′ and H-8′, and H-8′ and H-9′ indicated that the vicinal
angle between the protons in each pair was close to 90°. In addition,
NOESY correlation of H-9′ with OH-5′ placed H-5′ on the face
opposite that of H-4′ and H-9′. Considering the absolute configu-
ration established for 1 by X-ray data, the additional stereogenic
center C-5′ in 2 was deduced to have the R configuration.
Epicoccin H (4) was also obtained as a colorless powder. It was
assigned the molecular formula C20H N O S (nine degrees of
26 2 8 2
unsaturation) on the basis of HRESIMS analysis (m/z 509.1023
+
[M + Na] ; ∆ +0.5 mmu). The NMR data of 4 (Table 2) revealed
structural features nearly identical to those of 3, except that the
C-7 methylene (δ
thine (δ /δ 4.13/65.7) in 4. This was supported by H- H COSY
correlations of OH-7/7′ (δ 5.08) with H-7/7′ and HMBC cross-
peaks from OH-7/7′ to C-6/6′, C-7/7′, and C-8/8′. On the basis
H
C
/δ 1.88, 2.12/25.8) was replaced by an oxyme-
1
1
Epicoccin G (3) was obtained as a colorless powder. It was
H
C
assigned the molecular formula C20
unsaturation) by HRESIMS (m/z 477.1125 [M + Na] ; ∆ +0.5
H
26
N
2
O
6
S
2
(nine degrees of
H
+

Diketopiperazines from the Fungus Epicoccum nigrum
Journal of Natural Products, 2009, Vol. 72, No. 12 2117
to 5, except that the aromatic proton (δ
H
7.29) attached to C-11′
9.74) in 5. The
H- H coupling patterns observed for H-9′ and H-10′, and H-12′
(
δ
C
127.89) was replaced by a phenolic proton (δ
H
1
1
and H-13′, were indicative of a p-substituted aryl ring, permitting
assignment of the gross structure of 6 as shown. The Phe unit in 6
was assigned the L-configuration by comparison of the specific
2
5
25
rotation of 6 ([R]
D
+334) with that of 5 ([R]
D
+332).
Compounds 1-6 were tested for in vitro anti-HIV-1 activity.
Compounds 3-5 showed inhibitory effects on HIV-1 replication
in C8166 cells, with EC50 values of 13.5, 42.2, and 27.9 µM,
respectively (the CC50 values for these compounds were all greater
than 100 µM; the positive control indinavir showed an EC50 value
of 8.2 nM).
Figure 2. ∆δ values (in ppm) ) δ
S)-MTPA esters 3a and 3b.
S
R
- δ obtained for (R)- and
(
Table 3. NMR Spectroscopic Data (400 MHz, DMSO-d
and 6
6
) for 5
Epicoccins E (1) and F (2) are new DKPs possessing the
characteristic internal sulfur bridge(s). Although another class of
DKPs, the epipolythiodioxopiperazines (ETPs), possess an internal
5
6
position
δ
C
, mult.
δ
H
(J in Hz)
C
δ , mult.
δ
H
(J in Hz)
1
8
disulfide bridge on the DKP ring, 1 and 4 incorporated the rare
1
2
3
5
6
8.55, br s
8.40, br s
cross-ring sulfur bridges significantly different from those of the
161.76, qC
131.44, qC
166.93, qC
162.5, qC
129.5, qC
167.1, qC
1
1
common ETPs, with epicoccins A-D as the only examples.
However, 1 differs from its most closely related known precedent
9 by virtue of the presence of a sulfur bridge linking C-2′ and C-6′
instead of C-2′ and C-7′ in 9, whereas 2 differs from 7 by having
56.35, CH 4.34, t (5.1)
63.5, CH 4.26, t (5.0)
3.11, dd (14, 5.1) 30.5, CH 3.16, dd (14, 5.0)
3.09, dd (14, 5.1) 3.16, dd (14, 5.0)
7
7
8
9
1
1
1
1
1
7
8
9
1
1
1
1
a
b
39.52, CH
2
2
a C-5′-OH instead of the C-5′ ketone functionality. Epicoccins G
135.61, qC
135.8, qC
14
(
3) and H (4) are closely related to exserohilone (10), but differ
129.82, CH 7.18, d (7.6)
128.35, CH 7.26, t (7.6)
126.86, CH 7.14, t (7.6)
128.35, CH 7.27, t (7.6)
129.82, CH 7.16, d (7.6)
129.7, CH 7.17, d (7.4)
128.3, CH 7.25, t (7.4)
127.0, CH 7.17, t (7.4)
128.3, CH 7.25, t (7.4)
129.7, CH 7.17, d (7.4)
34.6, CH 2.69, s
3
118.5, CH 6.64, s
124.0, qC
131.5, CH 6.91, d (8.4)
115.0, CH 6.75, d (8.4)
157.8, qC
115.0, CH 6.75, d (8.4)
131.5, CH 6.91, d (8.4)
9.74, br s
in having a cyclohexenone moiety rather than a cyclohexanone ring.
Diphenylalazines A (5) and B (6) are DKPs possessing a Phe and
a dehydro N-Me-Phe unit, respectively, and are closely related to
0
1
2
3
4
′
′
′
0′
1′
2′
3′
19
XR335 isolated from Streptomyces spp., but differ in the identity
of the substituents on the aryl ring of the dehydro N-Me-Phe unit.
It is generally accepted that the natural DKPs seem to be
produced via a nonribosomal pathway, catalyzed by the nonribo-
34.94, CH
3
2.62, s
117.47, CH 6.72, s
133.79, qC
129.31, CH 7.02, d (7.4)
127.99, CH 7.36, t (7.4)
127.89, CH 7.29, t (7.4)
127.99, CH 7.34, t (7.4)
129.31, CH 7.02, d (7.4)
2
0-22
somal peptide synthetases (NRPSs).
Recently, a new family
of enzymes named cyclodipeptide synthases (CDPSs), unrelated
to the NRPSs, were reported to be involved in biosynthesis of
the antibacterial agent albonoursin isolated from Streptomyces
OH-11′
22,23
noursei.
Even though some biosynthetic studies suggested that
the sulfur atoms in the ETPs may be derived from methionine,
cysteine, and sodium sulfate, the mechanism by which the sulfurs
are incorporated remains to be fully investigated. Presumably,
of these data, the structure of 4 was elucidated as shown, and
the absolute configuration of 4 was presumed to be the same as
that of 3.
1
8
the biosynthesis of epicoccins E-H proceeds in a manner similar
2
4
The molecular formula of diphenylalazine A (5) was determined
to that of other known precedents.
to be C19
29.1265 [M + Na] ; ∆ +0.1 mmu). Analysis of its H and
NMR data (Table 3) revealed one amide proton (δ 8.55), one
N-methyl group (δ /δ 2.62/34.94), one methylene, one methine
/δ 4.34/56.35), 14
18 2 2
H N O (12 degrees of unsaturation) by HRESIMS (m/z
+
1
13
3
C
Experimental Section
H
General Experimental Procedures. Optical rotations were measured
on a Perkin-Elmer 241 polarimeter, and UV data were recorded on a
Shimadzu Biospec-1601 spectrophotometer. IR data were recorded
using a Nicolet Magna-IR 750 spectrophotometer. H and C NMR
data were acquired with Bruker Avance-400, 500, and 600 spectrom-
H
C
indicative of an amino acid R-CH group (δ
H
C
aromatic/olefinic carbons (11 of which were protonated), and two
1
13
1
carboxylic carbons. The H NMR resonances for a cluster of 10
aromatic protons at δ
monosubstituted aryl rings. HMBC correlations from H
and C-13, H-6 to C-5 and C-8, and H-1 (N-H) to C-5 established
the phenylalanine (Phe) unit, whereas those from the olefinic proton
H
7.02-7.34 suggested the presence of two
-7 to C-9
H C 5 H
eters using solvent signals (DMSO: δ 2.50/δ 39.5; pyridine-d : δ
7.21, 7.58, 8.73) as references. The HMQC and HMBC experiments
were optimized for 145.0 and 8.0 Hz, respectively. ESIMS data were
2
plus
recorded on a Bruker Esquire 3000 spectrometer. HRESIMS data
were obtained using a Bruker APEX III 7.0 T spectrometer.
3
H-7′ to C-2 and C-9′ and from H -14 to C-3 identified the
Fungal Material. The culture of E. nigrum was isolated by Dr. Mu
Wang from a sample of C. sinensis (Clavicipataceae) collected in
Linzhi, Tibet, in March 2004. The isolate was identified by one of the
authors (B.S.) and assigned the accession no. XZC04-CS-302 in X.L.’s
culture collection at the Institute of Microbiology, Chinese Academy
of Sciences, Beijing. The isolate was cultured on slants of potato
dextrose agar (PDA) at 25 °C for 15 days. Agar plugs were used to
inoculate 250 mL Erlenmeyer flasks, each containing 50 mL of media
N-methylated 3,7′-dehydrophenylalanine moiety. Key HMBC cor-
relations from the R-proton of Phe (H-6) to C-2 and from the
N-methyl proton (H -14) of 3,7′-dehydrophenylalanine to the
3
carboxylic carbon of Phe (C-5) established the DKP core, thereby
completing the gross structure of 5.
1
7
Marfey’s method was applied to assign the absolute configu-
ration of the Phe residue resulting from acid hydrolysis of 5. HPLC
analysis of the 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA)
derivative of the acid hydrolysate of 5 gave the same retention time
as that prepared from a sample of authentic L-Phe. Therefore, the
Phe residue in 5 was assigned the L-configuration.
(
0.4% glucose, 1% malt extract, and 0.4% yeast extract), and the final
pH of the media was adjusted to 6.5 before sterilization. Flask cultures
were incubated at 25 °C on a rotary shaker at 170 rpm for five days.
Twenty 500 mL Erlenmeyer flasks, each containing 150 mL of liquid
media (2% maltose, 6% Glucidex 29, 0.7% peptone, 0.75% Pharma-
Diphenylalazine B (6) gave a pseudomolecular ion [M + Na]+
peak at m/z 345.1210 (∆ +0.5 mmu) by HRESIMS, consistent with
a molecular formula of C19
media, trace amounts of MgSO
4
·7H
2
O, CaCO
3
, FeSO
4
2
·7H O, and
ZnSO ·7H O; final pH 7.0) and 30 g of vermiculite, were individually
4
2
H
18
N
2
O
3
(12 degrees of unsaturation).
inoculated with 15 mL of the seed culture and incubated at 25 °C under
Analysis of its NMR data (Table 3) revealed its structural similarity
static conditions for 40 days.

2
118 Journal of Natural Products, 2009, Vol. 72, No. 12
Extraction and Isolation. The fermented material (3 L) was freeze-
Guo et al.
14, 8.0 Hz, H-3a/3′a), 2.53 (2H, m, H-6b/6′b), 2.38 (2H, m, H-6a/6′a),
dried and extracted with ethyl methyl ketone (MEK; 3 × 500 mL),
and the organic solvent was evaporated to dryness under vacuum to
afford the crude extract (6.0 g). The extract was fractionated by silica
gel VLC using petroleum ether-EtOAc gradient elution. The fractions
eluted with 25 (80 mg), 30 (100 mg), 60 (100 mg), 70 (100 mg), and
2.30 (2H, m, H-7a/7′a), 2.15 (6H, s, H -10/10′).
3
Another sample of 3 (1.0 mg, 0.002 mmol), (R)-MTPA Cl (10.0
µL, 0.052 mmol), and pyridine-d (0.5 mL) were processed as described
above for 3a to afford 3b: H NMR (pyridine-d , 500 MHz) δ 6.46
(2H, s, H-8/8′), 4.74 (2H, d, J ) 8.0 Hz, H-9/9′), 3.29 (2H, d, J ) 14
Hz, H-3b/3′b), 2.86 (2H, t, J ) 8.0 Hz, H-4/4′), 2.70 (2H, m, H-7b/
7′b), 2.62 (2H, m, H-6b/6′b), 2.61 (2H, m, H-6a/6′a), 2.49 (2H, dd, J
5
1
5
9
5% EtOAc (30 mg) were individually separated by Sephadex LH-20
column chromatography (CC) using MeOH as eluent. The resulting
subfractions were combined into six fractions and subjected to further
purification by semipreparative RPHPLC (Agilent ZORBAX SB-C18
column; 5 µm; 9.4 × 250 mm; 2 mL/min) using different gradients to
) 14, 8.0 Hz, H-3a/3′a), 2.30 (2H, m, H-7a/7′a), 2.15 (6H, s, H
10′).
3
-10/
2
5
Epicoccin H (4): colorless powder; [R]
D
-175.0 (c 0.09, MeOH);
afford epicoccins E (1; 5.0 mg; 60% MeOH in H
2
O over 5 min,
UV (MeOH) λmax (log ε) 230 (3.83) nm; IR (neat) νmax 3416, 2922,
-
1 1 13
followed by 60-85% over 30 min), F (2; 5.0 mg; 60% MeOH in H
over 5 min, followed by 60-85% over 25 min), G (3; 7.0 mg; 35%
MeOH in H
2
O
1707, 1645, 1418,1246, 1076 cm ; H and C NMR data, see Table
2; HMBC data (DMSO-d , 600 MHz) H-3a/3′a f C-1/1′, 2/2′, 4/4′,
5/5′, 10/10′; H-3b/3′b f C-2/2′, 4/4′, 5/5′, 9/9′, 10/10′; H-4/4′ f C-2/
2′, 3/3′, 5/5′, 9/9′; H -6/6′ f C-5/5′, 7/7′, 8/8′; OH-7/7′ f C-6/6′, 7/7′,
8/8′; OH-8/8′ f C-7/7′, 9/9′; H-9/9′ f C-4/4′, 5/5′, 7/7′, 8/8′; H -10/
10′ f C-2/2′; key NOESY correlations (DMSO-d , 600 MHz) H-4/4′
T H-3a/3′a, 9/9′; H -10/10′ T H-3b/3′b; HRESIMS m/z 509.1023
(calcd for C20 Na, 509.1028).
Diphenylalazine A (5): white powder; [R]
MeOH); UV (MeOH) λmax (log ε) 215 (3.78), 295 (3.72) nm; IR (neat)
6
2
O over 5 min, followed by 35-85% over 25 min), and H
4; 5.0 mg; same gradient as in purification of 3); diphenylalazines A
5; 20 mg; 50% MeOH in water over 5 min, followed by 50-90%
O over 5 min,
followed by 60-100% over 30 min); and the known compounds
epicoccins A (7; 3.2 mg; 40% MeOH in H O over 5 min, followed by
0-80% over 30 min), B (8; 10 mg; same gradient as in purification
of 7), and D (9; 10 mg; same gradient as in purification of 6).
Epicoccin E (1): colorless needles (MeOH-H O); mp 273-275
+68.0 (c 0.17, MeOH); UV (MeOH) λmax (log ε) 224 (3.04)
nm; IR (neat) νmax 3438, 2923, 1727, 1666, 1422, 1323, 1287, 1133
(
(
2
3
over 30 min) and B (6; 2.0 mg; 60% MeOH in H
2
6
3
2
26 2 8 2
H N O S
2
5
4
D
+332.0 (c 0.48,
-1 1
2
νmax 3242, 2953, 2920, 1686, 1628, 1542, 1493, 1076 cm ; H and
13
2
5
°
C; [R]
D
6
C NMR data, see Table 3; HMBC data (DMSO-d , 400 MHz) H-1
f C-3, 5; H-6 f C-2, 5, 7, 8; H-7a f C-5, 6, 8, 9, 13; H-9 f C-7,
11, 13; H-10 f C-8, 12; H-11 f C-9, 13; H-12 f C-8, 10; H-13 f
C-7, 9, 11; H-14 f C-3, 5; H-7′ f C-2, 9′; H-9′ f C-11′; H-10′ f
C-8′, 12′; H-11′ f C-10′, 13′; H-12′ f C-8′, 10′; H-13′ f C-9′, 10′;
-
1
1
13
6
cm ; H and C NMR data, see Table 1; HMBC data (DMSO-d ,
6
8
00 MHz) H-3a f C-2, 4, 5, 9; H-4 f C-2, 4, 8, 9; H-6a f C-5, 7,
; H-7 f C-2, 5, 9; H-3′a f C-2′, 5′, 9′; H-4′ f C-2′, 3′, 5′; H-6′ f
C-2′, 5′, 7′, 8′; OH-8 f C-7, 8, 9; OH-7′ f C-7′; OH-8′ f C-8′, 9′;
HRESIMS m/z 461.0448 (calcd for C18 Na, 461.0453).
X-ray Crystallographic Analysis of 1. Upon crystallization from
MeOH-H O (10:1) using the vapor diffusion method, colorless crystals
NOESY correlations (DMSO-d
H-6 T H-9, 13, 14; H -7 T H-1, 9, 13; H-9 T H-1, 6; H-14 T H-6,
12′, 13′; H-7′ H-9′; HRESIMS m/z 329.1265 (calcd for
Na, 329.1266).
Absolute Configuration of Diphenylalazine A (5). A solution
6
, 400 MHz) H-1 T H-7a, 7b, 9, 13;
H
18
N O
2
7
S
2
2
2
5
T
2
19 18 2 2
C H N O
1
7
were obtained for 1, and a crystal (0.20 × 0.18 × 0.18 mm) was
separated from the sample and mounted on a glass fiber. Data were
collected using a Bruker SMART 1000 CCD diffractometer with
graphite-monochromated Mo KR radiation, λ ) 0.71073 Å at 173(2)
K. Crystal data: C18
P2
of 5 (1.5 mg) in 6 N HCl (1 mL) was heated at 100 °C for 16 h. Upon
removal of excess HCl under vacuum, the hydrolysate was placed in
a 1 mL reaction vial and treated with 1% solution of 1-fluoro-2,4-
dinitrophenyl-5-L-alanine amide (FDAA; 200 µL) in acetone followed
H
20
N
2
O
8
S
2
, M ) 456.48, space group monoclinic,
1
; unit cell dimensions a ) 10.861(2) Å, b ) 6.3257(13) Å, c )
3
by 1.0 M NaHCO (40 µL). The reaction mixture was heated at 45 °C
3
3
13.029(3) Å, V ) 887.9(3) Å , Z ) 2, Dcalcd ) 1.707 mg/m , µ )
0.356 mm , F(000) ) 476. The structure was solved by direct methods
for 1.5 h, cooled to room temperature, and then acidified with 2.0 N
HCl (20 µL). In a similar fashion, standard D- and L-Phe were
derivatized separately. The derivatives of the hydrolysate and standard
amino acids were subjected to RPHPLC analysis (Kromasil C18 column;
10 µm, 4.6 × 250 mm; 1.0-1.5 mL/min) at 25 °C using the following
-1
2
6
using SHELXL-97 and refined using full-matrix least-squares dif-
ference Fourier techniques. All non-hydrogen atoms were refined with
anisotropic displacement parameters, and all hydrogen atoms were
placed in idealized positions and refined as riding atoms with the relative
isotropic parameters. Absorption corrections were applied with the
3 3 4
gradient program: solvent A, 50 mM (Et NH) PO at pH 3.0; solvent
B, acetonitrile; linear gradient, 10%-35% B in 60 min with UV
detection at 340 nm. The retention times for the FDAA derivatives of
5 hydrolysate, standard L-Phe, and D-Phe were 44.75, 44.75, and 49.65
min, respectively.
27
Siemens Area Detector Absorption Program (SADABS). The 10 618
measurements yielded 3947 independent reflections after equivalent data
were averaged, and Lorentz and polarization corrections were applied.
The final refinement gave R
Epicoccin F (2): colorless powder; [R]
UV (MeOH) λmax (log ε) 215 (3.34) nm; IR (neat) νmax 3418, 2931,
2
5
1
) 0.0324 and wR
2
) 0.0814 [I > 2σ(I)].
Diphenylalazine B (6): white powder; [R]
D
+334.0 (c 0.213,
2
5
D
+52.0 (c 0.08, MeOH);
MeOH); UV (MeOH) λmax (log ε) 205 (4.04), 228 (3.84), 310 (3.93)
-
1
nm; IR (neat) νmax 3262, 2954, 2929, 1678, 1623, 1588, 1077 cm
;
-
1
1
13
1
13
2
1
5
856, 1664, 1417, 1320, 1051 cm ; H and C NMR data, see Table
; HMBC data (DMSO-d , 600 MHz) H-3a f C-2, 5, 9; H-3b f C-4,
, H-4 f C-2, 3, H-6a f C-5, 7, 8; H-9 f C-2, 4, 5, 7, 8; H-3′a f
H and C NMR data, see Table 3; HMBC data (DMSO-d
H-6 f C-2, 5; H -7 f C-5, 6, 8; H-10, 12 f C-8; H-7′ f C-2, 9′;
H-9′ f C-11′; H-10′, 12′ f C-8′; H -14 f C-3, 5; HRESIMS m/z
345.1210 (calcd for C19 Na, 345.1215).
6
, 400 MHz)
6
2
3
C-4′; H-3′b f C-2′, 5′; H-4′ f C-5′; H-6′a f C-5′, 7′, 9′; H-8′ f
C-2′, 7′, 9′; H-9′ f C-2′, 5′, 7′, 8′; OH-8 f C-9; OH-8′ f C-8′, 9′;
18 2 3
H N O
28
Anti-HIV Bioassays. Anti-HIV assays included cytotoxicity and
NOESY correlations (DMSO-d
m/z 479.0376 (calcd for C18
Epicoccin G (3): colorless powder; [R]
UV (MeOH) λmax (log ε) 224 (3.83) nm; IR (neat) νmax 3398, 2919,
6
, 600 MHz) H-9′ T OH-5′; HRESIMS
HIV-1 replication inhibition evaluations. Cytotoxicity was measured
4
H
20
N
2
O
6
S
3
Na, 479.0381).
by the MTT method as described in the literature. Cells (3 × 10 /well)
2
5
D
+69.0 (c 0.17, MeOH);
were seeded into a 96-well microtiter plate in the absence or presence
of various concentrations of test compounds in triplicate and incubated
-
1
1
13
2
851, 1699, 1651, 1543, 1405, 1193 cm ; H and C NMR data, see
Table 2; HMBC data (DMSO-d , 600 MHz) H-3a/3′a f C-1/1′, 2/2′,
/4′, 5/5′; H-6a/6′a f C-8/8′; H-6b/6′b f C-3/3′, 5/5′, 7/7′, 8/8′; H-7b/
′b f C-6/6′; H-9/9′ f C-4/4′, 7/7′, 8/8′; H -10/10′ f C-2/2′; NOESY
, 600 MHz) H-4/4′ T H-3a/3′a, 9/9′; H-6a/6′a
-10/10′ T H-3b/3′b; HRESIMS m/z 477.1125 (calcd for
Na, 477.1130).
Preparation of (R)-MTPA (3a) and (S)-MTPA Esters (3b). A
sample of 3 (1.0 mg, 0.002 mmol), (S)-MTPA Cl (10.0 µL, 0.052
mmol), and pyridine-d (0.5 mL) were allowed to react in an NMR
2
at 37 °C in a humid atmosphere of 5% CO . After a 4-day incubation,
6
cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) method. The concentration that
caused the reduction of viable cells by 50% (CC50) was determined. In
parallel with the MTT assay, a HIV-1 replication inhibition assay was
determined by p24 antigen capture ELISA. C8166 cells were exposed
to HIV-1 (MOI ) 0.058) at 37 °C for 1.5 h, washed with PBS to remove
4
7
3
correlations (DMSO-d
T H-8/8′; H
C H N O S
20 26 2 6 2
6
3
4
free viruses, and then seeded into a 96-well microtiter plate at 3 × 10
cells per well in the absence or presence of test compounds (indinavir
sulfate was used as positive control). After 4 days, the supernatant was
collected and inactivated by 0.5% Triton X-100. The supernatant was
diluted three times, added to the plate coating with anti-p24 McAb
(provided by Dr. Bin Yan, Wuhan Institute of Virology, Wuhan,
People’s Republic of China), and incubated at 37 °C for 1 h. After
washing five times with PBST, the HRP-labeled anti-p24 antibody
5
1
tube at ambient temperature for 6 days. The H NMR data of the
R-MTPA ester derivative (3a) were obtained directly on the reaction
1
mixture: H NMR (pyridine-d
(
(
5
, 500 MHz) δ 6.40 (2H, s, H-8/8′), 4.91
2H, d, J ) 8.0 Hz, H-9/9′), 3.33 (2H, d, J ) 18 Hz, H-3b/3b′), 3.07
2H, t, J ) 8.0 Hz, H-4/4′), 2.67 (2H, m, H-7b/7′b), 2.61 (2H, dd, J )

Diketopiperazines from the Fungus Epicoccum nigrum
provided by Dr. Bin Yan) was added and incubated at 37 °C for 1 h.
The plate was washed five times with PBST, followed by adding
o-phenylenediamine (OPD) reaction mixture. The assay plate was read
at 490 nm using a microplate reader within 30 min. The inhibition rate
and the EC50 based on p24 antigen expression level were calculated.
Journal of Natural Products, 2009, Vol. 72, No. 12 2119
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1364–1367.
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13) Flack, H. D. Acta Crystallogr. A 1983, 39, 876–881.
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Acknowledgment. We gratefully acknowledge financial support
from the Ministry of Science and Technology of China (2007AA021506
and 2009CB522302) and the National Natural Science Foundation of
China (30925039 and U0633008).
(
(
(
19) Bryans, J.; Charlton, P.; Robinson, I. C.; Collins, M.; Faint, R.; Latham,
Supporting Information Available: H and 13C NMR spectra of
epicoccins E-H (1-4) and diphenylalazines A (5) and B (6). This
material is available free of charge via the Internet at http://pubs.acs.org.
1
C.; Shaw, I.; Trew, S. J. Antibiot. 1996, 49, 1014–1021.
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application to the director, CCDC, 12 Union Road, Cambridge CB2
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1
EZ, UK (fax: +44 1223 336033 or e-mail: deposit@ccdc.cam.ac.uk).
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1
NP900654A