Antiprotozoal and antimicrobial compounds from the plant pathogen septoria pistaciarum

Article abstract of DOI:10.1021/np200940b

Four new 1,4-dihydroxy-5-phenyl-2-pyridinone alkaloids, 17-hydroxy-N-(O-methyl)septoriamycin A (1), 17-acetoxy-N-(O-methyl)septoriamycin A (2), 13-(S)-hydroxy-N-(O-methyl)septoriamycin A (3), and 13-(R)-hydroxy-N-(O- methyl)septoriamycin A (4), together with the known compounds (+)-cercosporin (5), (+)-14-O-acetylcercosporin (6), (+)-di-O-acetylcercosporin (7), lumichrome, and brassicasterol, were isolated from an ethyl acetate extract of a culture medium of Septoria pistaciarum. Methylation of septoriamycin A (8) with diazomethane yielded three di-O-methyl analogues, two of which existed as mixtures of rotamers. We previously reported antimalarial activity of septoriamycin A. This compound also exhibited significant activity against Leishmania donovani promastigotes. Compounds 5-7 showed moderate in vitro activity against L. donovani promastigotes and chloroquine-sensitive (D6) and -resistant (W2) strains of Plasmodium falciparum, whereas compound 5 was fairly active against methicillin-sensitive and methicillin-resistant strains of Staphylococcus aureus. Compounds 5-7 also displayed moderate phytotoxic activity against both a dicot (lettuce, Lactuca sativa) and a monocot (bentgrass, Agrostis stolonifera) and cytotoxicity against a panel of cell lines.

Full text of DOI:10.1021/np200940b

Article
pubs.acs.org/jnp
Antiprotozoal and Antimicrobial Compounds from the Plant
Pathogen Septoria pistaciarum
Mallika Kumarihamy,†,⊥ Shabana I. Khan,†,⊥ Melissa Jacob,† Babu L. Tekwani,† Stephen O. Duke,‡
Daneel Ferreira,†,⊥ and N. P. Dhammika Nanayakkara*,†
†National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, and ⊥Department of Pharmacognosy,
School of Pharmacy, The University of Mississippi, University, Mississippi 38677, United States
‡Natural Products Utilization Research Unit, USDA-ARS, University, Mississippi 38677, United States
S
* Supporting Information
ABSTRACT: Four new 1,4-dihydroxy-5-phenyl-2-pyridinone
alkaloids, 17-hydroxy-N-(O-methyl)septoriamycin A (1), 17-
acetoxy-N-(O-methyl)septoriamycin A (2), 13-(S)-hydroxy-N-
(O-methyl)septoriamycin A (3), and 13-(R)-hydroxy-N-(O-
methyl)septoriamycin A (4), together with the known
compounds (+)-cercosporin (5), (+)-14-O-acetylcercosporin
(6), (+)-di-O-acetylcercosporin (7), lumichrome, and brassi-
casterol, were isolated from an ethyl acetate extract of a culture
medium of Septoria pistaciarum. Methylation of septoriamycin
A (8) with diazomethane yielded three di-O-methyl analogues,
two of which existed as mixtures of rotamers. We previously
reported antimalarial activity of septoriamycin A. This
compound also exhibited significant activity against Leishmania
donovani promastigotes. Compounds 5−7 showed moderate in
vitro activity against L. donovani promastigotes and chloroquine-sensitive (D6) and -resistant (W2) strains of Plasmodium
falciparum, whereas compound 5 was fairly active against methicillin-sensitive and methicillin-resistant strains of Staphylococcus
aureus. Compounds 5−7 also displayed moderate phytotoxic activity against both a dicot (lettuce, Lactuca sativa) and a monocot
(bentgrass, Agrostis stolonifera) and cytotoxicity against a panel of cell lines.
A (8), the major 2-pyridinone alkaloid in this extract, showed
hytotoxins from plant pathogenic fungi may act as
Pinhibitors of plant-like metabolic pathways in the significant antileishmanial activity in addition to its reported
antiplasmodial activity. The pigments were identified as three
known perylenequinones, (+)-cercosporin (5),4 (+)-14-O-
acetylcercosporin (6),4 and (+)-di-O-acetylcercosporin (7).5
These pigments have previously been identified as phytotoxins
produced by a number of phytopathogenic Cercospora species
and have been linked to their pathogenicity.6 Their biosynthesis
appeared to be controlled by numerous environmental and
physiological factors, and the presence of even small amounts
of certain compounds in the medium was found to have a
strong stimulatory or inhibitory effect on their production.6,7
Their ability to generate reactive oxygen species in the presence
of light has been attributed to their phytotoxic activity.6
Cercosporin and its esters have also been reported to have
antibacterial and antifungal8 activities as well as growth-
inhibitory effects on lettuce4 and tomato seeds.8 In this study,
the perylenequinones showed antileishmanial, antiplasmodial,
and cytotoxic activities in addition to antibacterial and
antifungal activities. Two more known compounds, lumi-
chrome9 and brassicasterol,10 were also isolated and identified.
apicoplast, a chloroplast-like organelle, essential for the survival
of plasmodium species.1 Previously we reported2 the isolation
and identification of the 2-pyridinone alkaloid septoriamycin A3
(8) and three of its derivatives from the causative agent of
septoria leaf spot disease in pistachio, Septoria pistaciarum
(Ascomycetes), as part of our program to identify antimalarial
compounds from plant pathogenic fungi. Although we detected
several minor alkaloids of the same class in the original extract,
their structural elucidation was not possible due to insufficient
quantities. In order to isolate the minor compounds, we
recultured the same fungus under the identical conditions at a
larger scale. In the ethyl acetate extract of this fermentation
culture broth, a series of pigments were detected with 2-
pyridinone alkaloidal architecture. The extract showed
herbicidal, antimicrobial, antiplasmodial, and antileishmanial
activities albeit with low selectivity indices. From this extract,
four more new minor septoriamycin A analogues, 17-hydroxy-
N-(O-methyl)septoriamycin A (1), 17-acetoxy-N-(O-methyl)-
septoriamycin A (2), 13-(S)-hydroxy-N-(O-methyl)-
septoriamycin A (3), and 13-(R)-hydroxy-N-(O-methyl)-
septoriamycin A (4), in addition to the parent and its
previously reported analogues were identified. Septoriamycin
Received: December 2, 2011
Published: April 24, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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confirming that compound 1 was 17-hydroxy-N-(O-methyl)-
septoriamycin A (Figure 1).
RESULTS AND DISCUSSION
■
Fractionation of an EtOAc extract of a culture medium of S.
pistaciarum by Sephadex LH-20 gel column chromatography
followed by purification using silica gel and RP C18
chromatography afforded four minor 2-pyridinone alkaloids
(1−4) in addition to the known septoriamycin A (8) and its
three derivatives,2 three known perylenequinones, (+)-cerco-
sporin (5), (+)-14-O-acetylcercosporin (6), and (+)-di-O-
acetylcercosporin (7), lumichrome, and brassicasterol.
The molecular formula of compound 1 was determined as
C23H31NO5 by HRESIMS. UV maxima (205.0, 240.9, and
297.0 nm) and IR absorptions (3400, 3207, 1647, and 1555
cm−1) of 1 were consistent with those of a 2-pyridinone
Figure 1. COSY, HMBC, and ROESY correlations of compound 1.
The HRESIMS data established the molecular formula of
2
1
1
moiety. The aromatic regions of the H NMR and COSY
spectra were similar to those observed for 5-phenyl-2-
pyridinones previously isolated from this species and consisted
of a one-proton singlet and an A2B2C spin system due to a
monosubstituted phenyl group.2 The aliphatic region exhibited
two oxymethine and two methyl doublets, indicating the
presence of a 2,4-dimethyltetrahydropyran moiety and an N-
methoxy group. HMBC correlations of H-2′ and H-6′ (δH 7.43)
with C-3′ and C-5′ (δC 128.6), C-4′ (δC 127.8), C-1′ (δC 133.3),
and C-5 (δC 114.5) and those of H-6 (δH 7.44) with C-2 (δC
158.0), C-4 (δC 161.9), C-5 (δC 114.5), and C-1′ (δC 133.3)
supported the partial structure of the substituted pyridinone
ring moiety. HMBC correlations of the H-7 oxymethine
doublet (δH 4.73) with C-4 (δC 161.9), C-2 (δC 158.0), C-3 (δC
111.6), C-11 (δC 86.3), C-9 (δC 36.4), C-8 (δC 39.8), and C-15
(δC 17.8) supported the partial structure of the tetrahydropyran
compound 2 as C25H33NO6. The H and 13C NMR spectra
were similar to those of 1 except for the presence of additional
resonances [(δH 2.06) δC 21.1 (CH3), δC 171.2 (CO)]
originating from an O-acetyl group. HMBC correlations
between the C-12 oxymethylene group and the acetyl carbonyl
carbon indicated that compound 2 was 17-O-acetyl-N-(O-
methyl)septoriamycin A. The remaining HMBC and COSY
3
correlations were identical to those of compound 1. Both J7,8
and 3J10,11 values were 10.2 Hz, indicating that these protons are
trans-diaxially oriented. ROESY correlations of compound 1
(Figure 1) and 2 were identical to those observed for
septoriamycin A (8), suggesting that these two compounds
had the same relative configurations. Since we have previously
assigned the absolute configuration of septoriamycin A on the
basis of X-ray diffraction data,2 and all these compounds
presumably share a common biosynthetic origin, compounds 1
and 2 also have a 7R, 8R, 10S, 11R, and 12R absolute
configuration. It is further supported by their dextrorotatory
specific rotations.
1
moiety. The major difference in the rest of the H NMR
resonances of compound 1 and the reported2 N-(O-methyl)-
septoriamycin A (9) is the replacement of the methyl doublet
in the chain attached to C-11 of the tetrahydropyran ring by
two diastereotopic oxymethylene hydrogens. This indicated
that compound 1 was the C-17-oxygenated analogue of 9. The
COSY spectrum of compound 1 displayed cross-peaks between
the C-17 oxymethylene (δH 3.60, 3.49) and C-12 methine
proton (δH1.87). In the HMBC spectrum the oxymethylene
protons showed cross-peaks with C-11 (δC 86.3), C-12 (δC
36.5), and C-13 (δC 22.3), and the H-11 oxymethine doublet at
δH 3.36 with C-7 (δC 81.2), C-9 (δC 36.4), C-12 (δC 36.5), C-
13 (δC 22.3), C-16 (δC 12.6), and C-17 (δC 63.6), further
The HRESIMS data of 3 established its molecular formula as
C23H31NO5. Comparison of the NMR spectra of 3 with those
of 8 showed that the major difference was the replacement of a
methyl triplet (δH 0.89) of the latter by a methyl doublet (δH
1.16) and an oxymethine doublet (δH 3.78) in the former.
These changes could be attributed to the substitution of one of
the C-13 diastereotopic methylene hydrogens in 8 with a
hydroxy group, indicating that compound 3 is the 13-hydroxy
analogue of N-(O-methyl)septoriamycin A (9). The methyl
doublet at δH 0.93 (17-CH3) showed HMBC cross-peaks with
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Figure 2. Rotamer representation of compounds 3 and 4.
C-11 (δC 88.9), C-12 (δC 41.4), and C-13 (δC 68.9), and the
oxymethine multiplet at δH 3.78 with C-11 (δC 88.9), C-12 (δC
41.4), and C-17 (δC 15.9), confirming the C-13 location of the
secondary hydroxy group, and thus, compound 3 was 13-
hydroxy-N-(O-methyl)septoriamycin A.
correlations, to assign the C-13 absolute configuration of
compounds 3 and 4. An observed J12,13 value of 7.2 Hz for 3
3
suggested a dihedral angle of ca. 30° or 150° between H-12 and
H-13. Eight rotamers are possible for 3 with these dihedral
angles for the S (3a−3d) and R (3e−3h) epimers (Figure 2).
Observed ROESY correlations between H-12 and H-13, H-13
and CH3-17, and CH3-14−CH3-17 ruled out all conformers
except 3a as the probable most abundant rotamer for
compound 3 in solution, indicating a 13S absolute config-
uration. Similarly, an observed 3J12,13 of 1.0 Hz for 4 supported
the fact that the dihedral angle between H-12 and H-13 was
90°, as shown in Figure 2 for the 13S (4a, 4b) and R (4c, 4d)
epimers. In the ROESY spectrum, H-12 showed correlation
with H-13 and CH3-14 and the absence of interaction between
CH3-14 and CH3-17, indicating 4a as the dominant rotamer
and, hence, a 13R absolute configuration.
ROESY data and 1H NMR coupling constants of compound
3 showed close correlations to those reported2 for septor-
iamycin A (8). Our attempts to determine the absolute
configuration at C-13 by Mosher analysis were unsuccessful.
Treatment of compound 3 with the R- and S-Mosher’s acid
chlorides afforded a mixture not worth resolving. Methylation
prior to acylation also yielded a mixture of products. The
absolute configuration of compound 3 was proposed by a
3J12,13-based comparison of compounds 3 and 4 (vide infra).
Compound 4 had the same molecular formula, C23H31NO5,
as that of 3 based on HRESIMS data. The 1H NMR spectra of
these compounds were similar except for the downfield shift of
the oxymethine proton (δH 4.20) and the upfield shift of a
methyl doublet (δH 1.00). The COSY and HMBC spectra of
compound 4 showed the same correlations as those observed
for compound 3, suggesting that both compounds have the
same gross structure. Identical coupling constants and ROESY
correlations for the protons in the tetrahydrofuran moiety
indicated that they had the same relative configuration except at
C-13. Since compounds 3, 4, and 8 presumably share a
common biosynthetic origin, compounds 3 and 4 also have a
7R, 8R, 10S, 11R, 12R absolute configuration. As described
earlier for compound 3, our attempts to determine the absolute
configuration of the C-13 stereogenic center by Mosher analysis
were unsuccessful. Thus, we used a J-based approach relying on
1H NMR coupling constants combined with ROESY
Methylation of compounds 3 and 4 with diazomethane
afforded several products. Treatment of septoriamycin A (8)
with diazomethane as a model gave three products, whereas
methylation with MeI and Cs2CO3 afforded a single compound,
which was identified as analogue 9. The products of
diazomethane methylation of septoriamycin A were separated
by chromatography. All these products had the same molecular
formula, C24H33NO4, by HRESIMS, suggesting that they were
1
di-O-methyl derivatives. The H NMR spectrum of the least
polar compound (10a) was similar to that of 9 except for the
presence of an additional O-methyl resonance in the former.
The 13C NMR spectrum of this compound showed changes in
resonances in the pyridone ring. A downfield shift of C-4 (δC
177) indicated that the carbonyl group now resided at this
carbon, suggesting 10a was N,2-di-O-methylseptoriamycin A. A
hypsochromic shift of the UV absorption (λmax 278.9 nm) when
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1
Table 1. H and 13C NMR Data for Compounds 1−4 in CDCl3−Methanol-d4
1
2
3
4
b
a
b
a
b
a
b
a
position
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
2
3
4
5
6
7
8
9
158.0
111.6
161.9
114.5
133.0
81.2
158.0
111.4
161.8
114.3
133.1
81.3
158.0
112.0
161.6
114.5
132.9
80.8
158.2
112.0
161.7
114.8
132.8
80.9
7.43, s
7.44, s
7.42, s
7.43, s
4.73, d (10.2)
1.87, m
4.74, d (10.2)
1.91, m
4.70, d (10.2)
1.91, m
4.66, d (10.2)
1.91, m
39.8
36.4
36.2
36.7
36.4
1.34, q (12.6)
1.98, brd (13.2)
1.69, m
36.5
1.30, q (12.6)
2.00, (overlap)
1.64, m
42.6
1.06, q (12.6)
1.80, dt (13.8, 3.6)
2.03, m
42.2
1.16, q (12.0)
1.84, dt (13.2, 3.6)
1.89, m
10
11
12
13
14
15
16
17
35.8
86.3
36.5
22.3
12.6
17.8
17.1
63.6
36.0
86.2
37.0
22.3
12.6
17.7
17.0
65.3
34.1
88.9
41.4
68.9
23.1
17.9
17.7
15.9
32.9
88.5
38.7
66.0
22.8
17.9
17.7
10.5
3.36, d (10.2)
1.87, m
3.33, d (10.2)
2.00, m
3.14, d (10.2)
2.05, m
3.12, dd (10.2, 2.8)
1.75, m
1.02, m, 1.58, m
0.88, t (7.2)
0.91, d (6.6)
0.95, d (6.6)
3.49, dd (10.6, 3.6)
3.60, dd (10.6, 3.6)
1.02, m, 1.55, m
0.89, t (7.2)
0.91, d (7.2)
0.97, d (7.2)
3.91, dd (11.2, 5.4)
3.98, dd (11.6, 4.5)
3.78, m (7.2, 7.2)
1.16, d (6.6)
0.85, d (6.6)
0.83, d (6.6)
0.93, d (7.2)
4.20, m (6.6, 1.0)
1.16, d (6.3)
0.87, d (6.6)
0.84, d (6.6)
1.01, d (7.2)
1′
133.3
129.2
128.6
127.8
133.4
129.3
128.6
127.9
133.4
129.2
128.5
127.7
133.7
129.4
128.6
127.8
2′, 6′
3′, 5′
4′
7.44, d (7.3)
7.39, t (7.2)
7.32, t (7.2)
9.63, s
7.44, d (7.3)
7.40, t (7.3)
7.34, t (7.3)
9.47, s
7.43, d (7.3)
7.38, t (7.8)
7.32, t (7.2)
9.63, s
7.43, d (7.3)
7.36, d (7.2)
7.30, t (7.2)
9.65, s
OH
OCH3
COCH3
COCH3
65.0
4.04, s
65.0
21.1
4.06, s
65.0
4.04, s
65.0
4.05, s
2.06, s
171.2
a
b
1H NMR spectra recorded at 600 MHz. 13C NMR spectra recorded at 100 MHz.
1
Table 2. H and 13C NMR Data Methylated Compounds 10a, 10b, and 10c in CDCl3−Methanol-d4
10a
10b
10c
b
a
b
a
b
a
position
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
2
3
4
5
6
7
8
9
155.0
119.5
177.0
134.6
132.7
78.4
159.2
124.3
164.9
117.2
133.4
79.4
157.2
122.8
164.8
115.9
134.0
79.5
158.0
125.6
155.7
128.5
139.6
79.4
156.0
125.3
157.0
128.5
139.7
80.7
7.51, s
7.44, s
7.49, s
8.15, s
8.14, s
4.63, d (10.2)
2.0, m
4.56, d (10.2)
2.3, m
4.21, d (10.2)
2.3, m
4.33, d (10.4)
2.44, m
4.33, d (10.4)
2.52, m
36.3
33.8
33.8
32.6
32.9
43.4
1.78, m
43.2
43.1
1.79, m
1.84, m
43.1
43.1
1.86, m
1.86, m
1.02, m
1.04, m
1.06, m
0.96, m
0.96, m
10
11
12
13
33.7
89.2
34.2
22.8
1.69, m
32.3
89.0
36.1
22.6
31.9
88.9
36.7
21.9
1.72, m
1.72, m
32.4
89.8
36.0
22.5
32.5
90.0
36.0
22.5
1.88, m
1.88, m
2.92, d (12.0)
1.54, m
2.97, d (12.0)
1.57, m
2.87, d (12.0)
1.57, m
2.92, d (10.0)
1.60, m
2.94, d (10.0)
1.60, m
1.54, m
1.53, m
1.53, m
1.5, m
1.5, m
0.82, m
0.85, m
0.85, m
1.1, m
1.1, m
14
13.0
17.6
0.84, t (7.2)
0.68, d (6.6)
0.77, d (6.6)
0.89, d (6.6)
12.7
17.5
12.4
17.8
0.84, t (6.6)
0.80, d (6.6)
0.75, d (6.6)
0.91, d (6.6)
0.84, t (6.6)
0.80, d (6.6)
0.68, d (6.6)
0.91, d (6.6)
12.9
17.6
12.8
17.6
0.87, t (6.6)
0.81, d (6.6)
0.64, d (6.4)
0.90, d (6.6)
0.87, t (6.6)
0.81, d (6.6)
0.67, d (6.6)
0.90, d (6.6)
15
16
17.7
17.3
17.6
17.7
17.8
17
17.0
16.8
17.0
17.2
17.2
1′
126.6
128.9
128.3
127.8
66.8
133.8
128.6
128.4
127.6
64.5
133.8
128.5
128.5
127.5
64.5
133.0
129.0
128.9
129.8
61.7
133.0
128.9
128.7
129.8
62.1
2′, 6′
3′, 5′
4′
7.56, d (7.2)
7.33, t (7.2)
7.25, t (7.2)
4.01, s
7.43, d (7.2)
7.39, t (7.2)
7.34, t (7.2)
4.08, s
7.43, d (7.2)
7.39, t (7.2)
7.34, t (7.2)
4.08, s
7.48, d (6.8)
7.42, t (6.8)
7.39, t (7.3)
3.36, s
7.48, d (7.5)
7.42, d (7.5)
7.39, t (7.3)
3.37, s
OCH3
OCH3
64.4
4.0, s
61.5
61.2
4.01, s
3.34, s
60.6
60.4
4.16, s
4.14, s
a
b
1H NMR spectra recorded at 600 MHz. 13C NMR spectra recorded at 100 MHz.
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compared to 9 (λmax 295 nm)11 and COSY and HMBC
compounds 5−7 showed nonspecific moderate phytotoxic
activity toward both bentgrass (A. stolonifera) and lettuce (L.
sativa cv. L., iceberg) in the presence of light (Table 4). General
1
correlations further supported this structure. The H and 13C
NMR spectra of compound 10b indicated that it existed as a
3:1 mixture of two rotamers about the C-3−C-7 bond. Their
1H and 13C NMR spectra were similar to those of compound 9
but had an additional methoxy resonance. HMBC correlations
showed that the additional methoxy group correlated to C-4,
indicating that 10b was N,4-di-(O-methyl)septoriamycin A. At
a
Table 4. Phytotoxic Activity of Compounds 5−7
compound
concentration (mM)
lettuce
bentgrass
5
6
7
1.87
1.73
1.62
3
2
3
2
4
4
1
100 °C in pyridine, duplicated H NMR resonances coalesced
but were restored to the original ratio when the temperature
returned to ambient conditions. Atropisomers of 3-cyclohexyl-
or 3-cycloheptyl-N,4-dihydroxy-2-pyridone alkaloids have pre-
viously been isolated from several fungal species.11−15 Even
though a number of 3-pyrano-N,4-dihydroxy-2-pyridone
alkaloids similar to septoriamycin A analogues have been
identified16 from fungal sources, none have been reported to
exist as rotamers. The introduction of a 4-methoxy group
appears to cause restricted rotation about the C-3−C-7 bond,
giving rise to rotamers. The most polar compound (10c)
a
Concentration (mM) = 1 mg/mL. Ranking based on scale of 0 to 5:
0 = no effect; 5 = no growth.
phytotoxcity of phytotoxins from host-specific pathogens is
very common. Biosynthesis of cercosporin (5) appeared to be
controlled by numerous environmental and physiological
factors, and its production has been linked to the pathogenicity
of fungi.6,7 The possible mechanism of phytotoxic activity of
this type of compound has previously been attributed to their
ability to generate reactive oxygen species in the presence of
light.6 This suggested that the selective inhibition of the plant-
like metabolic pathways in the apicoplast of malaria parasite1 is
not responsible for the observed antimalarial activity of
compounds 5−7. Septoriamycin A (8), with demonstrated
antiplasmodial and antifungal activities,2 exhibited significant
antileishmanial activity, with an IC50 of 0.11 μM and an IC90 of
0.29 μM (Table 5), and was more potent than the positive
1
showed ionic characteristics. H and 13C NMR data indicated
that the compound also existed as a 3:1 mixture of two
diastereomeric rotamers. HMBC correlations showed that the
methoxy resonance correlated to C-2 and C-4, suggesting that
10c is the 2,4-di-O-methylpyridinone-N-oxide analogue of
septoriamycin A. The presence of the 4-methoxy group appears
to cause restricted rotation about the C-3−C-7 bond. At 100
°C in pyridine duplicated 1H NMR resonances coalesced. Even
though the original ratio of rotamers reappeared at ambient
temperature, some decomposition of 10c was also observed.
The known perylenequinones, (+)-cercosporin (5),4 (+)-14-
O-acetylcercosporin (6),4,5 and (+)-di-O-acetylcercosporin
(7),5 lumichrome,9 and brassicasterol10 were identified by
comparing their spectroscopic data with literature data. The
absolute configurations of 5−7 were determined by comparing
experimental and reported electronic circular dichroism
data.17,18 The helicity of compounds 5−7 was confirmed as
M (aS), and the absolute configuration of both C-14 and C-17
as R.19
Table 5. Antileishmanial Activity of Compounds 5−8
b
c
compound
IC50, μM
IC90, μM
5
6
7
8
1.14
1.7
2.81
8.5
3.1
9.7
0.11
2.9
0.29
5.58
0.38
a
pentamidine
a
amphotericin B
0.18
a
b
Positive controls. IC50: concentration causing 50% growth
c
inhibition. IC90: concentration causing 90% growth inhibition.
Compounds 1−4 showed no antimicrobial, antifungal,
antiprotozoal, phytotoxic, or cytotoxic activity in vitro.
Compounds 5−7 showed moderate in vitro antiplasmodial
activity (Table 3) but were cytotoxic to Vero cells. Their low
selectivity indices (ratio of cytotoxicity vs antiplasmodium
activity) preclude them as antimalarial drug leads. Even though
the plant pathogen S. pistaciarum is host-specific to pistachio,
controls pentamidine and amphotericin B. Compounds 5−7
also showed significant antileishmanial activity, with IC50 values
of 1.14, 1.7, and 3.1 μM, respectively (Table 5).
Compound 5 was moderately active against both methicillin-
sensitive and methicillin-resistant Staph. aureus, with MIC
values 2.5 and 5.0 μg/mL (4.7 and 9.4 μM), respectively. In the
same assay, the positive control, ciprofloxacin, had a MIC value
of 0.37 μg/mL (1.11 μM) against both methicillin-sensitive and
methicillin-resistant Staph. aureus.
Table 3. Antiplasmodial Activity of Compounds 5−7 and
10c
The cytotoxic potential of compounds 5−7 was further
evaluated against a panel of human solid tumor cell lines (SK-
MEL, KB, BT-549, SK-OV-3) and pig kidney epithelial cells
(LLC-PK11) (Table 6). Moderate cytotoxicity was observed
against all the cell lines. The di-O-methyl derivatives of
septoriamycin A, 10a, 10b, and 10c, were also evaluated for
the above activities, and compound 10c exhibited weak
antiplasmodial activity (Table 3).
chloroquine-
sensitive (D6)
clone
chloroquine-
resistant (W2)
clone
cytotoxicity to Vero
cells
b
b
IC50,
c
b
IC50,
IC50,
μM
c
compound
μM
S.I.
μM
S.I.
5
1.08
2.78
2.75
6.76
0.03
0.02
4.8
1.9
1.62
3.12
1.94
6.51
0.31
0.01
3.2
5.24
5.21
4.53
6
1.7
2.3
7
1.6
d
10c
>1.1
>1.2
NC
a
chloroquine
NC
NC
EXPERIMENTAL SECTION
a
■
artemisinin
General Experimental Procedures. Optical rotations were
obtained using a Rudolph Research Analytical Autopol IV automatic
polarimeter model 589-546. Melting points were measured with a
Unimelt, Thomas-Hoover capillary melting point apparatus. UV and
a
b
Positive controls. IC50: concentration causing 50% growth
c
inhibition. S.I. (selectivity index) = IC50 for cytotoxicity/IC50 for
d
antiplasmodial activity. NC: not cytotoxic.
887
dx.doi.org/10.1021/np200940b | J. Nat. Prod. 2012, 75, 883−889

Journal of Natural Products
Article
stirred at room temperature for 5 h. The reaction mixture was filtered,
and the solvent was evaporated. The product was dissolved in CH2Cl2
and passed through a plug of Florisil to give N-(O-methyl)-
septoriamycin A (9).
Table 6. Cytotoxic Activity [IC50 (μM)] of Compounds 5−
a
7
SK-MEL
KB
BT-549
SK-OV-3
LLC-PK11
Methylation of Septoriamycin A (8) with Diazomethane. A
solution of 8 (60.0 mg) in MeOH was treated with excess
diazomethane in Et2O at 0 °C for 2 h. The solvent was evaporated,
and the mixture was separated by PTLC (40% EtOAc in hexanes) to
yield compounds 10a (8 mg), 10b, and 10c. Compounds 10b and 10c
were further purified by HPLC using a reversed-phase Luna C18
column (1 × 25 cm) with MeOH−H2O (92:8) as the mobile phase at
a flow rate of 4 mL/min, to give compounds 10b (5 mg) and 10c (6.0
mg).
5
3.6
3.8
4.9
1.6
7.1
8.5
8.7
2.6
10.8
8.6
3.7
4.0
4.8
1.5
3.9
10.1
9.4
6
7
8.7
b
doxorubicin
2.6
1.6
a
IC50 = concentration causing 50% growth inhibition. SK-MEL =
human malignant melanoma. KB = human epidermal carcinoma. BT-
549 = human breast carcinoma (ductal). SK-OV-3 = human ovary
carcinoma. LLC-PK11 = pig kidney epithelial. Positive control.
b
Compound 10a: amorphous powder; [α]26 +14 (c 0.5, MeOH);
D
UV (MeOH) λmax (log ε) 205.0 (3.75), 232.0 (3.68), 278.9 (3.41) nm;
IR spectra were determined on a Varian-50 Bio UV visible
spectrophotometer and a Bruker-Tensor-27 infrared spectrophotom-
eter, respectively. NMR spectra were recorded on a Varian-Mercury-
Plus-400 or Varian Unity-Inova-600 spectrometer using CDCl3 and
methanol-d4 unless otherwise stated. MS data were obtained from an
Agilent Series 1100 SL equipped with an ESI source (Agilent
Technologies, Palo Alto, CA, USA). Column chromatography and
preparative TLC were carried out using Merck silica gel 60 (230−400
mesh) and silica gel GF plates (20 × 20 cm, thickness 0.25 mm),
respectively. HPLC analysis was conducted on a Hewlett-Packard
Agilent 1100 with diode array detector.
1
IR (CHCl3) νmax 2959, 2929, 2873, 1627, 1547, 1469, 1046 cm−1; H
and 13C NMR data (see Table 2); HRESIMS [M + Na]+ m/z
422.2307 (calcd for [C24H33NO4 + Na]+, 422.2307).
Compound 10b: amorphous powder; UV (MeOH) λmax (log ε)
204.0 (3.39), 235.0 (3.18), 308.0 (2.65) nm; IR (CHCl3) νmax 2958,
2929, 1740, 1656, 1524, 1457 cm−1; 1H and 13C NMR data (see Table
2); HRESIMS [M + H]+ m/z 400.2472 (calcd for [C24H33NO4 + H]+,
400.2487).
Compound 10c: amorphous powder; UV (MeOH) λmax (log ε)
207.0 (2.58), 240.9 (2.47), 307.1 (1.70) nm; IR (CHCl3) νmax 2900,
2850, 1575, 1450 cm−1; 1H and 13C NMR data (see Table 2);
HRESIMS [M + Na]+ m/z 422.2279 (calcd for [C24H33NO4 + Na]+,
422.2307).
Fermentation, Extraction, and Isolation. The plant pathogenic
fungus Septoria pistaciarum Caracc. (ATCC 22201) was obtained from
the American Type Culture Collection (Manassas, VA, USA), and it
was grown as previously reported.2
Compound 5: red crystals; mp 239 °C (lit.17 240−241.5 °C); UV
(MeOH) λmax (log ε) 210.0 (3.71), 221.0 (3.72), 267.0 (3.55), 470.0
(3.42), 563.0 (2.98) nm; IR (CHCl3) νmax 3396, 2924, 1616, 1267
The oily extract (3.3 g) was chromatographed over Sephadex LH-20
and eluted with 80% MeOH in CHCl3 to give 16 fractions. Fractions
2−11, which showed antiplasmodial activity, were combined and
chromatographed over a reversed-phase C18 column eluting with a
gradient of 10% to 100% MeOH−H2O to yield eight fractions.
Subfractions 2, 3, and 4 were combined and separated on Sephadex
LH-20 (MeOH) to give compounds 1 (5.0 mg) and 8 (35 mg).
Subfraction 5 was chromatographed over a silica gel column using
CH2Cl2−hexanes as the eluent to give compound 2 (8.0 mg).
Combined fractions 6, 7, and 8 were chromatographed over a silica gel
column using CH2Cl2−hexanes as the eluent to give 10 subfractions.
Subfractions 2, 3, and 5 were combined and further separated using a
C18 reversed-phase HPLC column eluting with MeCN−H2O (1:1),
flow rate 3.5 mL/min, to give compounds 3 (3.5 mg) and 4 (8.0 mg).
Subfractions 6, 7, and 8 were combined and purified on Sephadex LH-
20 (MeOH) to give compounds 5 (14.0 mg), 6 (12.0 mg), and
lumichrome (3.0 mg). Subfractions 9 and 10 were combined and
chromatographed over a silica gel column using CH2Cl2−hexanes as
the eluent to give compound 7 (10.0 mg) and brassicasterol (8.0 mg).
1
cm−1; H and 13C NMR and CD data were consistent with those
reported.4,17,19
Compound 6: red crystals; mp 135 °C (lit.5 134 °C); UV (MeOH)
λmax (log ε) 222.0 (3.9), 269.0 (3.74), 471.0 (3.62), 563.0 (3.2) nm; IR
1
(CHCl3) νmax 3338, 1735, 1616, 1266 cm−1; H and 13C NMR and
CD data were consistent with those reported.4,5
Compound 7: red, amorphous powder; UV (MeOH) λmax (log ε)
222.0 (3.9), 270.0 (3.84), 470.1 (4.0), 563.3 (3.26) nm; IR (CHCl3)
1
νmax 2921, 1736, 1617, 1211 cm−1; H and 13C NMR and CD data
were consistent with those reported.5
Antiplasmodial Assay. The antiplasmodial activity was deter-
mined against D6 (chloroquine-sensitive) and W2 (chloroquine-
resistant) strains of Plasmodium falciparum in an in vitro assay as
described earlier.20 Artemisinin and chloroquine were included as the
drug controls, and IC50 values were computed from the dose−
response curves using Microsoft Excel software.
Phytotoxicity Assay. The bioassay for phytotoxicity was carried
out according to the procedure described by Dayan et al.21 using
bentgrass (Agrostis stolonifera) and lettuce (Lactuca sativa cv. L.,
iceberg), in 24-well plates. Test compounds (1 mg each) were
dissolved in 100 μL of acetone, and a 20 μL aliquot of each solution
was pipetted onto the filter paper and dried for 30 min by airflow in a
sterile biohazard hood. Water (200 μL) was added after placing the
dried and sample-impregnated filter paper in the well. The solvent
controls were treated identically, using the solvent described above.
Phytotoxicity was ranked visually. The ranking of phytotoxic activity
was based on a scale of 0 to 5, with 0 showing no effect and 5 no
growth.
Antileishmanial Assay. The in vitro antileishmanial activity of the
compounds was carried out on a culture of Leishmania donovani
promastigotes as described earlier.22 Pentamidine and amphotericin B
were used as standard antileishmanial agents. The IC50 values for each
compound were computed from the growth inhibition curve using
Microsoft Excel software.
Antimicrobial Assay. All organisms were obtained from the
American Type Culture Collection (Manassas, VA, USA) and included
the fungi Candida albicans ATCC 90028, Candida glabrata ATCC
90030, Candida krusei ATCC 6258, Cryptococcus neoformans ATCC
90113, and Aspergillus fumigatus ATCC 90906 and the bacteria
Compound 1: amorphous powder; [α]26 +112 (c 0.05, MeOH);
D
UV (MeOH) λmax (log ε) 205.0 (3.56), 240.9 (3.51), 297.0 (2.79) nm;
IR (CHCl3) νmax 3400, 3207, 2967, 2929, 1647, 1555, 1454, 1049
cm−1; 1H and 13C NMR data (see Table 1); HRESIMS [M − H]+ m/z
400.2118 (calcd for [C23H31NO5 − H]+, 400.2124).
Compound 2: amorphous powder; [α]26 +101 (c 0.05, MeOH);
D
UV (MeOH) λmax (log ε) 204.0 (3.44), 240.0 (3.25), 295.9 (2.56) nm;
1
IR (CHCl3) νmax 3331, 2967, 2925, 1740, 1651, 1230, 1045 cm−1; H
and 13C NMR data (see Table 1); HRESIMS [M + H]+ m/z 444.2370
(calcd for [C25H33NO6 + H]+, 444.2386).
Compound 3: amorphous powder; [α]26 −38 (c 0.43, CH3OH);
D
UV (MeOH) λmax (log ε) 208.2 (3.94), 241.9 (3.92), 297.0 (3.19) nm;
IR (CHCl3) νmax 3405, 3201, 2965, 2929, 1644, 1551, 1455, 1219, 755
1
cm−1; H and 13C NMR data (see Table 1); HRESIMS [M + H +
Na]+ m/z 425.2172 (calcd for [C23H31NO5 + H + Na]+, 425.2178).
Compound 4: amorphous powder; [α]26 +130 (c 0.2, MeOH);
D
UV (MeOH) λmax (log ε) 207.1 (3.01), 241.0 (2.95), 295.1 (2.10) nm;
1
IR (CHCl3) νmax 3422, 3159, 2926, 1641, 1541, 1456 cm−1; H and
13C NMR data (see Table 1); HRESIMS [M + H + Na]+ m/z
425.2174 (calcd for [C23H31NO5 + H + Na]+, 425.2178).
Methylation of Septoriamycin A (8) with MeI. A mixture of MeI (4
mL), Cs2CO3 (10.0 mg), and compound 8 (10.0 mg) in acetone was
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dx.doi.org/10.1021/np200940b | J. Nat. Prod. 2012, 75, 883−889

Journal of Natural Products
Article
Staphylococcus aureus ATCC 29213, methicillin-resistant Staphylococcus
aureus ATCC 33591 (MRSA), Escherichia coli ATCC 35218,
Pseudomonas aeruginosa ATCC 27853, and Mycobacterium intra-
cellulare ATCC 23068. Susceptibility testing was performed using a
modified version of the CLSI methods23,24 as described by
Samoylenko et al.25 The drug controls ciprofloxacin (ICN
Biomedicals, Aurora, OH, USA) for bacteria and amphotericin B
(ICN Biomedicals) for fungi were included in each assay.
Cytotoxicity Assay for Mammalian Cells. In vitro cytotoxicity
was determined against a panel of mammalian cells that included
kidney fibroblast (Vero), kidney epithelial (LLC-PK11), malignant
melanoma (SK-MEL), oral epidermal carcinoma (KB), breast ductal
carcinoma (BT-549), and ovary carcinoma (SK-OV-3) cells as
described earlier.26 The number of viable cells was determined by
using Neutral Red dye, and IC50 values were obtained from dose−
response curves. Doxorubicin was used as a positive control.
(11) Cai, P.; Smith, D.; Cunningham, B.; Brown-Shimer, S.; Katz, B.;
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ASSOCIATED CONTENT
■
(19) Morgan, B. J.; Mulrooney, C. A.; Kozlowski, M. C. J. Org. Chem.
2010, 75, 44−56.
S
* Supporting Information
NMR spectra of compounds 1−4, 10a, 10b, and 10c. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(20) Bharate, S. B.; Khan, S. I.; Yunus, N. A. M.; Chauthe, S. K.;
Jacob, M. R.; Tekwani, B. L.; Khan, I. A.; Singh, I. P. Bioorg. Med.
Chem. 2007, 15, 87−96.
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26, 2079−2094.
(22) Machumi, F.; Samoylenko, V.; Yenesew, A.; Derese, S.; Midiwo,
J. O.; Wiggers, F. T.; Jacob, M. R.; Tekwani, B. L.; Khan, S. I.; Walker,
L. A.; Muhammad, I. Nat. Prod. Commun. 2010, 5, 853−858.
(23) NCCLS. Methods for Dilution Antimicrobial Susceptibility
Tests for Bacteria that Grow Aerobically M7-A5; National Committee
on Clinical Laboratory Standards, 2000; 20: (2).
(24) NCCLS. Reference Method for Broth Dilution Antifungal
Susceptibility Testing of Conidium- Forming Filamentous Fungi;
Proposed Standard, M38-P; National Committee on Clinical
Laboratory Standards, 1998; 18: (13).
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 1-662-915-1019. Fax: 1-662-915-1006. E-mail:
dhammika@olemiss.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Institutes of Health
(R21 A1061431-01 and R01 AI 27094) and, in part, by the
United States Department of Agriculture, ARS, Specific
Cooperative Agreement No. 58-6408-2-009 and US DoD
CDMRP Investigator Initiated Grant Award W81XWH-09-2-
0093. We thank Dr. B. Avula and Mr. F. T. Wiggers, NCNPR,
(25) Samoylenko, V.; Jacob, M. R.; Khan, S. I.; Zhao, J.; Tekwani, B.
L.; Midiwo, J. O.; Walker, L. A.; Muhammad, I. Nat. Prod. Commun.
2009, 4, 791−796.
(26) Mustafa, J.; Khan, S. I.; Ma, G.; Walker, L. A.; Khan, I. A. Lipids
2004, 39, 167−172.
1
University of Mississippi, for recording the MS and H NMR
spectra (600 MHz), and Ms. M. Wright, Mr. J. Trott, Mr. S.
Jain, and Mr. R. Johnson for biological testing.
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