Metabolites from the marine-derived fungus Chromocleista sp. isolated from a deep-water sediment sample collected in the gulf of Mexico

Article abstract of DOI:10.1021/np058113p

As part of our ongoing chemical investigation of biologically active metabolites from marine fungi, three new compounds, p-hydroxyphenopyrrozin (1) and diketopiperazines (3, 4), have been isolated from the marine-derived fungus Chromocleista sp. In addition, the fungus gave the known compound phenopyrrozin (2), four known diketopiperazines (6-9), N-acetyltryptamine (10), and agathic acid (11). Another new diketopiperazine (5) was separated and identified as a decomposition product of 3 and 4. The structures of the new metabolites were determined on the basis of mass spectroscopy, NMR experiments, and derivatization methods. The absolute configuration of 1 was determined by X-ray crystallography studies.

Full text of DOI:10.1021/np058113p

580
J. Nat. Prod. 2006, 69, 580-584
Metabolites from the Marine-Derived Fungus Chromocleista sp. Isolated from a Deep-Water
Sediment Sample Collected in the Gulf of Mexico
Young Chul Park, Sarath P. Gunasekera,* Jose V. Lopez, Peter J. McCarthy, and Amy E. Wright
DiVision of Biomedical Marine Research, Harbor Branch Oceanographic Institution, 5600 U.S. 1 North, Fort Pierce, Florida 34946
ReceiVed October 24, 2005
As part of our ongoing chemical investigation of biologically active metabolites from marine fungi, three new compounds,
p-hydroxyphenopyrrozin (1) and diketopiperazines (3, 4), have been isolated from the marine-derived fungus
Chromocleista sp. In addition, the fungus gave the known compound phenopyrrozin (2), four known diketopiperazines
(6-9), N-acetyltryptamine (10), and agathic acid (11). Another new diketopiperazine (5) was separated and identified
as a decomposition product of 3 and 4. The structures of the new metabolites were determined on the basis of mass
spectroscopy, NMR experiments, and derivatization methods. The absolute configuration of 1 was determined by X-ray
crystallography studies.
The chemical diversity of secondary metabolites reported to date
has shown that marine microbes are an excellent resource for the
discovery of potential new drugs.1,2 Furthermore, marine-derived
fungi have been shown to be a potential source for secondary
metabolites with interesting bioactivities.3,4 In our continuing search
for new metabolites from marine-derived fungi, we have isolated
a new 7a(S)-p-hydroxyphenopyrrozin (1) and two new diketopip-
erazines (3, 4) along with their diketopiperazine decomposition
product (5) from the cultured fungus Chromocleista sp. isolated
from a marine sediment sample collected at a depth of 70 m in the
Gulf of Mexico. In addition, the same fungus yielded the known
compounds phenopyrrozine (2),5 a radical scavenger,6 cyclo(L-Pro-
L-Tyr)7 (6), cyclo(L-Pro-L-Phe)8 (7), cyclo(L-Pro-L-Val)9 (8), and
cyclo(L-Pro-D-Leu)10 (9), Nb-acetyltryptamine (10),11 and agathic
acid (11).12,13 The structures of all of the known compounds have
been confirmed by comparison with spectroscopic data reported in
the literature.
Results and Discussion
In this paper, we report the isolation and structure elucidation
of new compounds 1, 3, 4, and 5. The fungal cell mass and added
resin were extracted with a mixture of CH2Cl2/MeOH (1:1) to give
a crude extract. The EtOAc-soluble fraction of the crude extract
was subjected to silica gel column chromatography using a heptane/
EtOAc/MeOH (80:20:0 f 0:0:100) gradient to give seven fractions.
Further chromatography of the second fraction gave nine subfrac-
tions. Repeated chromatography of these subfractions gave com-
pounds 1-4 and 6-11. Compound 5 was isolated as a decompo-
sition product of compounds 3 and 4.
Crystallization of compound 1 in MeOH furnished colorless
crystals, mp 231-233 °C and specific rotation [R]25D +34.0 (c 0.34,
MeOH). High-resolution FABMS of 1 revealed a molecular ion
peak at m/z 232.0979 for M + H+ corresponding with the molecular
H-7a), 3.34 (1H, m, H-5â), 3.24 (1H, m, H-5R), 2.29 (1H, m, H-7â),
formula C13H13NO3 and eight degrees of unsaturation. IR spectral
absorptions at 1676 and 3188 cm-1 indicated the presence of a
conjugated carbonyl group and hydroxyl functionality. The 13C and
DEPT NMR spectra revealed the presence of three methylene
carbons (δC 42.9, 32.0, and 29.3), two aromatic methine carbons
each accounting for two carbon atoms (δC 129.4, 116.3), one
phenolic carbon (δC 158.3), one aliphatic methine (δC 63.7), three
quaternary olefinic carbons (δC 141.9, 127.4, and 125.6), and a
carbonyl carbon (δC 173.7). The 1H NMR spectrum revealed signals
for seven protons observed at δ 4.35 (1H, dd, J ) 10.5, 5.7 Hz,
2.22 (2H, m, H-6), and 1.13 (1H, m, H-7R). The COSY spectrum
displayed the correlations for geminal couplings in H2-5, H2-6, and
H2-7 and correlations between H2-5 and H2-6, H2-6, and H2-7,
indicating consecutive methylene connectivities. The second set of
proton signals at δ 7.52 (2H, dd, J ) 8.5 Hz, H-2′ and H-6′) and
6.75 (2H, dd, J ) 8.5 Hz, H-3′ and H-5′) indicated the presence of
a para-substituted benzene ring. HMBC correlations from δH 3.24
(H-5R) and δH 3.34 (H-5â) to C-3, C-6, C-7, and C-7a determined
the position of the carbonyl group. Additional HMBC correlations
revealed connectivities from the methine signal at δH 4.35 (H-7a)
to C-1, C-2, C-3, C-6, and C-7. The combination of the above data
established a bicyclic system in the molecular structure. The HMBC
correlations from δH 7.52 (H-2′ and H-6′) to C-1 and C-4′ connected
* To whom correspondence should be addressed. Tel: 772-465-2400.
Fax: 772-461-2221. E-mail: sgunaseker@hboi.edu.
10.1021/np058113p CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/30/2006

Metabolites from the Fungus Chromocleista sp.
Journal of Natural Products, 2006, Vol. 69, No. 4 581
correlation between H2-8 and C-6, C-7, and C-9. Combination of
these data assigned the hydroxyl group to position C-6 of the proline
moiety. The HMBC correlations between H-3 (δ 4.25 in 3 and 4.21
in 4) and C-2, C-5, C-10, and C-1′ and other correlations described
above established a phenylalanine-proline diketopiperazine molec-
ular structures for 3 and 4. The absolute configuration of C-3 of
the phenylalanine residue in compound 3 was determined by
Marfey’s method.15 HPLC analysis of the FDAA derivative of 3
gave the same retention time as the derivative prepared from an
authentic (L)-phenylalanine, and therefore, the L configuration was
assigned to the phenylalanine residue. Compounds 3 and 4 when
stored separately in CDCl3 at room temperature gradually decom-
posed to give compound 5. The high-resolution FABMS of 5
revealed a molecular ion peak at m/z 243.0832 ([M + H]+),
supporting the molecular formula C14H14N2O2 with nine degrees
of unsaturation, and indicated a difference in elements H2O (18
1
amu) from 3 and 4. Analysis of the H, 13C, and HMQC NMR
spectra of 5 showed the presence of two amide carbonyl carbons
(δC 161.7 and 157.1), two olefinic carbons (δC 132.6, C and 119.1,
CH) and six aromatic carbons (δC-1′ 135.0, δC-2′,6′ 129.6, δC-3′,5′
129.0, and δC-4′ 127.6). These data together with three ring systems
accounted for all nine degrees of unsaturation in the molecule.
Comparison of 1H NMR spectra of 3 and 5 indicated the expected
close similarities and that the C-6 methylene group (δ 1.93, 2.31)
in 3 has been substituted by a new olefinic signal appearing at δ
6.15. Similarly, the 13C spectrum of 5 showed the presence of two
new olefinic signals instead of C-6 hydroxyl and C-7 methylene
carbons present in compounds 3 and 4. The HMBC correlations
between the newly formed olefinic proton at δ 6.15 (1H, t, J ) 2.8
Hz, H-7) and C-6 olefinic carbon (δ 132.7, C), C-5 amide carbon
(δ 157.1, CdO), C-8 (δ 27.8) methylene carbon, and C-9 (δ 45.4)
methylene carbon established the position of the double bond in
the proline residue. These data established the structure of dike-
topiperazine 5 as cyclo(6,7-en-Pro-L-Phe) and the structures of
diketopiperazines 3 and 4 as C-6 epimers of cyclo(6-Hyp-L-Phe).
Figure 1. Computer-generated perspective drawing of the X-ray
model of compound 1.
the p-hydroxyphenyl group to the bicyclic system. To determine
the absolute stereochemistry at the chiral center 7a, this compound
was crystallized for X-ray studies. The structure of compound 1
was confirmed as 5,6,7,7a(S)-tetrahydro-2-hydroxy-1-(p-hydroxy-
phenyl)-3H-pyrrolizin-3-one (Figure 1) by single-crystal X-ray
diffraction.14
Comparison of the NMR and mass spectroscopic data of 2 with
those of compound 1 and with literature reported data5,6 identified
compound 2 as 5,6,7,7a-tetrahydro-2-hydroxy-1-phenyl-3H-pyr-
rolizin-3-one. This compound was previously isolated from a
Penicillium sp., and its absolute configuration at 7a was not reported
The absolute configuration at C-6 in compounds 3 and 4 was
not determined by Marfey’s method because the hydroxyproline
residue decomposed under acidic conditions. Therefore, the ster-
eochemistry at C-6 of compounds 3 and 4 was determined on the
basis of proton-proton coupling constants observed between the
C-3 methine proton and nonequivalent C-10 benzylic protons in
the phenylalanine residue. The use of coupling constants to
determine the stereochemistry in similar ABX type systems in
diketopiperazines is described in the literature.16-21 The proton-
proton coupling constants of H-3 and benzylic H-10a and H-10b
in these publications. The specific rotation value of [R]25 -10.2
D
reported for this known compound5 and a similar specific rotation
value of [R]25D -13.7 observed for compound 2 suggested the same
7a-chiral center for both isolates. The positive specific rotation value
of [R]25 +34.0 observed for 1 with 7a(S) configuration thus
D
suggested an opposite 7a(R) configuration for 2, and therefore, the
structure of 2 was confirmed as 5,6,7,7a(R)-tetrahydro-2-hydroxy-
1-phenyl-3H-pyrrolizin-3-one.
of compound 3 (δ 4.25H-3 (3JH-3/H-10a ) 9.9 Hz and 3JH-3/H-10b
)
Compounds 3 and 4 were identified as two new diketopiperazine
epimers that bear a hydroxyl group at the quaternary carbon atom
(C-6) of the proline residues. High-resolution FABMS of 3
supported the molecular formula C14H16N2O3 (m/z 243.1197 [M +
H - H2O]+), and low-resolution ESIMS of 4 supported the
molecular formula C14H16N2O3 (m/z 261.2 [M + H]+). Both
compounds 3 and 4 displayed the typical 13C NMR chemical shift
values observed for a diketopiperazine ring system, which included
the characteristic amide groups at C-2 (δC 168.1 and 167.7) and
C-5 (δC 166.1 and 165.6), methine residue at C-3 (δC 55.8 and
59.3), and aliphatic quaternary carbon at C-6 (δC 87.6 and 86.5),
respectively. The 13C chemical shift values for C-6 in 3 (δC 87.6)
and in 4 (δC 86.5) suggested the presence of a hydroxyl group
attached to this position. The COSY spectra of 3 and 4 indicated
the presence of three consecutive methylene groups characteristic
of a proline residue in both compounds. Similarly, the presence of
five aromatic protons and a benzylic group in both compounds (see
Table 1) indicated the presence of a phenylalanine residue. The
HMBC spectrum showed correlations between H2-7 of the proline
residue and the remaining methylene carbons C-8 and C-9 of the
proline residue and to C-6 (δ 87.6 in 3 and 86.5 in 4) and C-5 (δ
166.1 in 3 and 165.6 in 4). Similarly, the HMBC spectrum showed
3
3.6 Hz), 2.73H-10a (2JH-10a/H-10b ) 14.3 Hz and JH-10a/H-3 ) 9.9
Hz), and 3.62H-10b (2JH-10b/H-10a ) 14.3 Hz and 3JH-10b/H-3 ) 3.6
Hz)) indicated a dihedral angle of 180° (trans) for H3-C-C-H10a
and a dihedral angle of 69° (gauche) for H3-C-CH10b (Figure 2).
These coupling constant values are in agreement with the known
cyclo(L-Pro-L-Phe) reported in the literature.16 In contrast, the
proton-proton coupling constants of H-3 and benzylic H-10 of
3
compound 4 (δ 4.21H-3 (3JH-3/H-10a ) 4.0 Hz and JH-3/H-10b
)
8.1 Hz), 3.17H-10a (2JH-10a/H-10b ) 13.6 Hz and JH-10a/H-3 ) 4.0
Hz), and 3.27H-10b (2JH-10b/H-10a ) 13.6 Hz and 3JH-10b/H-3 ) 8.1
Hz)) indicated a dihedral angles of 50° (gauche) for H3-C-C-
H10a and a dihedral angle of 166° (trans) for H3-C-C-H.10b
Similarly, these coupling constants are in agreement with the known
cyclo(D-Pro-L-Phe) reported in the literature.17 These data confirmed
the structures of diketopiperazines 3 and 4 as cyclo(D-6-Hyp-L-
Phe) and cyclo(L-6-Hyp-L-Phe), respectively. Related diketopip-
erazines with a (L)-trans-4-Hyp residue and a (D)-cis-4-Hyp residue
have been reported from the marine sponge of the family Jaspidae16
and the marine yeast Aureobasidium pullulans,17 respectively.
3
The four new compounds were screened for their bioactivity
against the bacterium Staphylococcus aureus and the fungus

582 Journal of Natural Products, 2006, Vol. 69, No. 4
Park et al.
Table 1. NMR Spectral Data of 3-5 in CDCl3 (1H, 500 MHz; 13C, 125 MHz)
3
4
5
position
2
δH (mult; J in Hz)
δC
δH (mult; J in Hz)
δC
δH (mult; J in Hz)
δC
168.1 (C)
55.8 (CH)
167.7 (C)
59.3 (CH)
161.7 (C)
58.5 (CH)
4.25 (dd, 9.9, 3.6)
5.78 (s)
4.21 (dd, 8.1, 4.0)
6.17 (s)
4.34 (dd, 8.7, 4.0)
5.89 (s)
NH
5
6
166.1 (C)
87.6 (C)
36.8 (CH2)
165.6 (C)
86.5 (C)
36.8 (CH2)
157.1 (C)
132.7 (C)
119.1 (CH)
7
1.93 (m)
1.98 (m)
6.15 (t, 2.8)
2.31 (m)
2.22 (m)
8
1.86 (m)
3.64 (m)
2.73 (dd, 14.3, 9.9)
3.62 (dd, 14.3, 3.6)
19.9 (CH2)
45.4 (CH2)
41.0 (CH2)
1.94 (m)
3.66 (m)
3.17 (dd, 13.6, 4.0)
3.27 (dd, 13.6, 8.1)
19.1 (CH2)
45.3 (CH2)
41.0 (CH2)
2.67 (m)
3.96 (m)
2.98 (dd, 13.7, 8.7)
3.34 (dd, 13.6, 4.0)
27.8 (CH2)
45.4 (CH2)
41.2 (CH2)
10
1′
2
3′
4′
5′
6′
135.7 (C)
135.7 (C)
135.7 (C)
7.23 (d, 7.3)
7.35 (t, 7.3)
7.29 (t, 7.3)
7.35 (t, 7.3)
7.23 (d, 7.3)
129.3 (CH)
129.2 (CH)
127.6 (CH)
129.2 (CH)
129.3 (CH)
7.23 (d, 7.3)
7.35 (t, 7.3)
7.29 (t, 7.3)
7.35 (t, 7.3)
7.23 (d, 7.3)
129.8 (CH)
129.2 (CH)
127.6 (CH)
129.2 (CH)
129.8 (CH)
7.18 (d, 7.3)
7.31 (t, 7.3)
7.26 (t, 7.3)
7.31 (t, 7.3)
7.18 (d, 7.3)
129.6 (CH)
129.0 (CH)
127.6 (CH)
129.0 (CH)
129.6 (CH)
this strain goes beyond the scope of this paper (http://www.biology-
.duke.edu/fungi/mycolab/primers.htm), followed by BLAST queries
against GenBank, which resulted in a closest match to C. malacheita
(97%). The fungus is maintained on Emerson’s YpSs agar prepared
with 75% artificial seawater and has been deposited into the Harbor
Branch Marine Microbial Culture Collection. For chemical investigation
the strain was cultured for 7 days in SYZ broth (15 g of soluble starch,
2 g of yeast extract, 4 g of NZ-amine, 2 g of dextrose, 4 g (dry weight)
of Amberlite XAD16 resin, 750 mL of artificial seawater, 250 mL of
deionized water) at 25 °C and 210 rpm as 4 × 1 L batches in triple-
baffled Fernbach flasks. The cell mass and resin were collected by
centrifugation (8500g, 15 min).
Figure 2. Proposed conformation models of the C-3-C-10 bond
in compounds 3 and 4.
Candida albicans. Compound 1 showed a minimum inhibition
concentration of 25 µg/mL against C. albicans. Compounds 3-5
showed no activity at concentrations up to 50 µg/mL. None of the
compounds showed activity against S. aureus when tested at 50
µg/mL. The cytoxicity of these compounds was evaluated against
a panel of cancer cell lines: P388 murine leukemia, A549 human
lung adenocarcinoma, PANC-1 human pancreatic cancer, and NCI-
ADR-RES tumor cells. All new compounds failed to register any
cytotoxicity at a concentration of 5 µg/mL.
Extraction and Isolation. The cultured fungal cell mass and
Amberlite XAD-16 resin were filtered and then extracted five times
with 50% CH2Cl2/MeOH (1 L). The organic extracts were combined
and dried over anhydrous MgSO4 and concentrated in vacuo to afford
a dark brown extract (7.3 g). The extract was partitioned five times
with 70% EtOAc/H2O (1L). The organic layer was dried with anhydrous
MgSO4 and concentrated in vacuo to yield 1.35 g of an EtOAc-soluble
fraction. The EtOAc-soluble fraction was chromatographed on a column
of SiO2 gel using a heptane/EtOAc/MeOH (80:20:0 f 0:0:100) step
gradient system to give seven fractions. The second fraction (248.6
mg), eluted with heptane/EtOAc/MeOH (60:40:0), was rechromato-
graphed on a column of SiO2 gel using a heptane/EtOAc/MeOH (50:
50:0 f 0:95:5) step gradient system to provide nine subfractions. The
combined fractions 7 and 8 (83.8 mg) eluted with EtOAc/MeOH (95:
5) were further subjected to normal-phase Sep-Pack silica cartridge
(Waters Corp., Milford, MA) separation with 3% MeOH/CH2Cl2 to
yield 5,6,7,7a(S)-tetrahydro-2-hydroxy-1-(p-hydroxyphenyl)-3H-pyr-
rolizin-3-one (1) as a white solid (52 mg, 0.7% of crude extract wt)
and 5,6,7,7a(S)-tetrahydro-2-hydroxy-1-phenyl-3H-pyrrolizin-3-one (2)
as a white solid (14 mg, 0.2% of crude extract). The third fraction
(142.1 mg) was rechromatographed on a column of silica gel with a
heptane/EtOAc/MeOH (30:70:0 f 0:90:10) gradient system to provide
six subfractions. These subfractions on further purification by HPLC
(SiO2, 5 µm, 250 × 10 mm) with 3% MeOH/CH2Cl2 gave cyclo(L-6-
Hyp-L-Phe) (3) as a white solid (0.8 mg, 0.01% of crude extract),
cyclo(D-6-Hyp-L-Phe) (4) as a white solid (1.8 mg, 0.02% of crude
extract), and compounds 6-11. Compounds 3 and 4 in CDCl3 on
leaving at room temperature in the NMR tube gradually decomposed
to give cyclo(6,7-en-Pro-L-Phe) (5) as a pure compound.
Experimental Section
General Experimental Procedures. The melting point measured
with a Gallenkamp melting point apparatus is uncorrected. Optical
rotations were recorded on a Jasco DIP-370 digital polarimeter. UV
spectra were measured with a Hitachi U-3010 spectrophotometer. IR
spectra were obtained on a Midac M-1200 with Galactic GRAMS/386
software. 1D and 2D NMR spectra were measured on a Bruker AMX-
1
500 instrument. The H NMR chemical shifts (referenced to CDCl3
observed at δ 7.24) were assigned using a combination of data from
COSY and HMQC experiments. Similarly, 13C NMR chemical shifts
(referenced to CDCl3 observed at δ 77.0 and CD3OD observed at δ
49.0) were assigned on the basis of DEPT and HMQC experiments.
Low-resolution mass spectra ESI-MS were obtained on a Finnigan LTQ
LC-MS with an electrospray ionization detector. The HRMS were
obtained on a Finnigan MAT95Q mass spectrometer at the Spectro-
scopic Services Group, University of Florida, Gainesville, FL.
Isolation and Cultivation of Fungal Strain R721. The fungal strain
R721 was isolated from a sediment sample collected on August 17,
2001, by a manned submersible (Clelia) at a depth of 70 m, from the
Gulf of Mexico (latitude 26°15.87′ N; longitude 83°42.40′ W). The
fungus was isolated using solid agar methods with starch casein medium
consisting of the following: 10 g of starch, 1 g of casein, 1 mL of
trace metal solution,22 15 g of agar, 750 mL of artificial seawater, and
250 mL of deionized water. R721 was identified by partial sequence
analysis of the 28S large subunit rRNA gene using LROR and LR7
primers derived from the R. Vilgalys laboratory. A close taxonomic
relationship likely exists between the two fungal genera Chromocleista
and Penicillium, since both belong in the family Trichocomaceae, order
Eurotiales. Although our 28S rRNA analysis gives only limited
identification for fungal isolate R721, full taxonomic identification of
5,6,7,7a(S)-Tetrahydro-2-hydroxy-1-(p-hydroxyphenyl)-3H-pyr-
rolizin-3-one (1): [R]25 +34.0 (c 0.34, MeOH); UV (MeOH) λmax
D
(log ꢀ) 198 (4.5), 222 (1.3), 307 (1.8) nm; IR (KBr) νmax 3188, 1676,
1
1519, 1427, 1361 cm-1; H NMR (CD3OD, 500 MHz) δ 7.52 (2H, d,
J ) 8.5 Hz, H-2′ and H-6′), 6.75 (2H, d, J ) 8.5 Hz, H-3′ and H-5′),
4.35 (1H, dd, J ) 10.5, 5.7 Hz, H-7a), 3.34 (1H, m, H-5â), 3.24 (1H,
m, H-5R), 2.29 (1H, m, H-7â), 2.22 (2H, m, H-6), 1.13 (1H, m, H-7R);
13C NMR (CD3OD, 125 MHz) δ 173.7 (C, C-3), 158.3 (C, C-4′), 141.9
(C, C-2), 129.4 (CH, C-2′ and C-6′), 127.4 (C, C-1), 125.6 (C, C-1′),
116.3 (CH, C-3′ and C-5′), 63.7 (CH, C-7a), 42.9 (CH2, C-5), 32.0

Metabolites from the Fungus Chromocleista sp.
Journal of Natural Products, 2006, Vol. 69, No. 4 583
(CH2, C-7), 29.3 (CH2, C-6); LRESIMS (positive ion) m/z 232.2 [M
+ H]+; HRFABMS m/z 232.0979 [M + H]+ (calcd for C13H14NO3,
232.0973).
Antimicrobial Assays. MICs were determined for C. albicans
(ATCC #44506) by standard microdilution broth method using Sab-
ouraud dextrose broth (SDB)23
as growth medium. MICs against S.
cyclo(D-6-Hyp-L-Phe) (3): [R]25 -26.1 (c 0.03, MeOH); UV
aureus (ATCC #29213) were determined using a standard microdilution
broth method using cation-supplemented Mueller-Hinton broth as
growth medium. All plates were incubated for 18 h at 37 °C.
D
(MeOH) λmax (log ꢀ) 198 (3.5), 257 (0.3) nm; LRESIMS (positive ion)
m/z 261.2 [M + H]+; HRFABMS (positive ion) m/z 243.1197 [M + H
- H2O]+ (calcd for C14H15N2O2, 243.1133).
Cytotoxicity Assays. The compounds were analyzed for their effects
on proliferation of P388 murine leukemia, A549 human lung adeno-
carcinoma, PANC-1 human pancreatic cancer, and NCI-ADR-RES
tumor cell lines. Assays were run in 96-well plates. After appropriate
incubation with compound (45 h for P388 and 72 h for mammalian
cell lines), cytotoxicity was measured spectrophotometrically using
MTT as the indicator. The details of the cytotoxicity assays were
described in an earlier publication.24
cyclo(L-6-Hyp-L-Phe) (4): UV (MeOH) λmax (log ꢀ) 200 (3.7), 257
(0.4) nm; LRESIMS (positive ion) m/z 261.2 [M + H]+, 299.3 [M +
K]+, 283.2 [M + Na]+.
cyclo(6,7-en-Pro-L-Phe) (5): [R]25 -31.9 (c 0.05, MeOH); UV
D
(MeOH) λmax (log ꢀ) 200 (3.7), 257 (0.5), 330 (0.2) nm; LRESIMS
(positive ion) m/z 243.2 [M + H]+; HRFABMS (positive ion) m/z
243.0832 [M + H]+ (calcd for C14H15N2O2, 243.1133).
1-Fluoro-2,4-dinitrophenyl-5-L-alaninamide (FDAA, Marfey’s
reagent) Derivatization and Absolute Stereochemistry. A solution
of diketopiperazine (3, 0.15 mg) in 6 N HCl (1 mL) was heated at 100
°C for 16 h. The excess HCl was removed under vacuum. The dry
hydrolyzate was placed in a 1 mL reacti-vial and treated with 1%
solution of FDAA (200 µL) in acetone followed by 1.0 M NaHCO3
(40 µL). The reaction mixture was heated at 45 °C for 1.5 h and cooled.
The mixture was acidified with 2.0 M HCl (20 µL). Likewise, standard
D- and L-phenylalanine were derivatized separately. The derivatized
hydrolyzate and standard amino acids were subjected to HPLC (RP-
C18, 10 µm, 4.6 mm × 250 mm) using the following gradient program.
Solvent A: H2O in 50 mM (Et3NH)3PO4 at pH 3.0. Solvent B:
acetonitrile. Linear gradient, 10% to 35% of B in 60 min, flow rate
from 1 mL/min to 1.5 mL/min under UV absorption at 340 nm. The
retention times for FDAA derivatives of standard L-phenylalanine,
diketopiperazine (3), and standard D-phenylalanine were observed as
35.52, 35.52, and 40.29 min, respectively.
Acknowledgment. We are grateful to J. H. Reibenspies, Department
of Chemistry, Texas A & M University, College Station, Texas, for
X-ray crystallography data, and J.H.R. would like to acknowledge the
National Science Foundation for funds provided to purchase the X-ray
diffractometers and crystallographic computing systems. We thank D.
Powell of the University of Florida for high-resolution mass spectral
measurements, K. Janda for fermentation of the fungal material, D.
Harmody for antifungal assays, and P. Linley for cytotoxicity assays.
We also thank A. Ledger for molecular identification of the fungal
strain. The work was supported by the Center of Excellence in
Biomedical and Marine Biotechnology. This publication represents
Harbor Branch Oceanographic Institution Contribution No. 1625.
Supporting Information Available: X-ray crystallography data for
compound 1. This material is available free of charge via the Internet
at http://pubs.acs.org.
Single-Crystal X-ray Structure Determination. A Leica MZ7
polarizing microscope was used to identify a suitable specimen from a
representative sampling of materials. A crystal of size 0.10 × 0.10 ×
0.01 mm3 of p-hydroxyphenopyrrozin (1) crystallized in MeOH was
fixed to a nylon loop, which in turn was fashioned to a copper-mounting
pin. The mounted crystal was then placed in a cold nitrogen stream
(Oxford) maintained at 110 K. A Bruker D8 GADDS general-purpose
three-circle X-ray diffractometer was employed for sample screening
and data collection. The goniometer was controlled using the GADDS
software suite (Microsoft Win 2000 operating system). The sample was
optically centered with the aid of a video camera such that no
translations were observed as the crystal was rotated through all
positions. The detector was set at 5.0 cm from the crystal sample
(MWPC Hi-Star detector, 512 × 512 pixel). The X-ray radiation
employed was generated from a Cu sealed X-ray tube (KR ) 1.54184
Å with a potential of 40 kV and a current of 40 mA) and filtered with
a graphite monochromator in the parallel mode (175 mm collimator
with 0.5 mm pinholes). A rotation exposure was taken to determine
crystal quality and the X-ray beam intersection with the detector. The
beam intersection coordinates were compared to the configured
coordinates, and changes were made accordingly. The rotation exposure
indicated acceptable crystal quality, and the unit cell determination was
undertaken. Sixty data frames were taken at widths of 0.5° with an
exposure time of 10 s. Over 200 reflections were centered, and their
positions were determined. These reflections were used in the autoin-
dexing procedure to determine the unit cell. A suitable cell was found
and refined by nonlinear least squares and Bravais lattice procedures
and reported in Table 1 of the Supporting Information. The unit cell
was verified by examination of the hkl overlays on several frames of
data, including zone photographs. No supercell or erroneous reflections
were observed. After careful examination of the unit cell, a standard
data collection procedure was initiated. This procedure consists in
collection of one hemisphere of data collected using omega scans,
involving the collection of 2520 0.5° frames at fixed angles for φ, 2θ,
and ø (2θ ) -28°, ø ) 54.73°, 2θ ) -90°, ø ) 54.73°), while varying
omega. Additional data frames were collected to complete the data set.
Each frame was exposed for 10 s. The total data collection was
performed for a duration of approximately 24 h at 110 K. No significant
intensity fluctuations of equivalent reflections were observed. R indices
for all data (R1 ) 0.0623, wR2 ) 0.1361) and final R indices [I >
2σ(I)] (R1 ) 0.0525, wR2 ) 0.1316) were determined. After data
collection, the crystal was measured carefully for size, morphology,
and color. These measurements are reported in Table 1 of the Supporting
Information.
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