Shornephine A: Structure, chemical stability, and P-glycoprotein inhibitory properties of a rare diketomorpholine from an Australian marine-derived Aspergillus sp.

Article abstract of DOI:10.1021/jo501501z

Chemical analysis of an Australian marine sediment-derived Aspergillus sp. (CMB-M081F) yielded the new diketomorpholine (DKM) shornephine A (1)together with two known and one new diketopiperazine (DKP), 15b-β-hydroxy-5-N-acetyladreemin (2), 5-N-acetyladreemin (3), and 15b-β-methoxy-5-N-acetyladreemin (4), respectively. Structure elucidation of 1.4 was achieved by detailed spectroscopic analysis, supported by chemical degradation and derivatization, and biosynthetic considerations. The DKM (1)underwent a facile (auto) acid-mediated methanolysis to yield seco-shornephine A methyl ester (1a). Our mechanistic explanation of this transformation prompted us to demonstrate that the acid-labile and solvolytically unstable DKM scaffold can be stabilized by N-alkylation. Furthermore, we demonstrate that at 20 μM shornephine A (1)is a noncytotoxic inhibitor of P-glycoprotein-mediated drug efflux in multidrug-resistant human colon cancer cells.

Full text of DOI:10.1021/jo501501z

Article
pubs.acs.org/joc
Open Access on 08/26/2015
Shornephine A: Structure, Chemical Stability, and P‑Glycoprotein
Inhibitory Properties of a Rare Diketomorpholine from an Australian
Marine-Derived Aspergillus sp.
Zeinab G. Khalil, Xiao-cong Huang, Ritesh Raju, Andrew M. Piggott, and Robert J. Capon*
†
Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072, Australia
*
S Supporting Information
ABSTRACT: Chemical analysis of an Australian marine
sediment-derived Aspergillus sp. (CMB-M081F) yielded the
new diketomorpholine (DKM) shornephine A (1) together
with two known and one new diketopiperazine (DKP), 15b-β-
hydroxy-5-N-acetyladreemin (2), 5-N-acetyladreemin (3), and
1
5b-β-methoxy-5-N-acetyladreemin (4), respectively. Structure
elucidation of 1−4 was achieved by detailed spectroscopic
analysis, supported by chemical degradation and derivatization,
and biosynthetic considerations. The DKM (1) underwent a facile (auto) acid-mediated methanolysis to yield seco-shornephine
A methyl ester (1a). Our mechanistic explanation of this transformation prompted us to demonstrate that the acid-labile and
solvolytically unstable DKM scaffold can be stabilized by N-alkylation. Furthermore, we demonstrate that at 20 μM shornephine
A (1) is a noncytotoxic inhibitor of P-glycoprotein-mediated drug efflux in multidrug-resistant human colon cancer cells.
INTRODUCTION
■
Diketopiperazines (DKPs) are well represented among natural
products, particularly fungal metabolites, with structural
diversity extending across fused heterocycles, prenylation,
polythio-bridging, dimerization, nitration, halogenation, oxida-
tion, and more. Typically viewed as products of nonribosomal
1
2
peptide synthases, the recent discovery of a DKP cyclase
suggests a more complex biosynthetic landscape. Indeed, the
diverse structural and biological properties exhibited by natural
DKPs have attracted much attention, encouraging efforts to
probe biosynthetic pathways, develop innovative syntheses, and
3
apply DKPs in human and animal health. The 2010 release by
Pfizer Animal Health of a new class of anthelmintic inspired by
the Penicillium DKP paraherquamide (6) (the first in over two
4
decades) is illustrative of this potential. By contrast, reports of
the closely related diketomorpholine (DKM) motif are rare,
with accounts of the DKM scaffold being rare across natural
products, synthetic, and medicinal chemistry.
Figure 1. Aspergillus sp. (CMB-M081F) metabolites 1−4 and
methanolysis product 1a.
This report describes our investigation of an Australian
marine sediment-derived Aspergillus sp. (CMB-M081F), leading
to the isolation of a new DKM, shornephine A (1), plus its
methanolysis product seco-shornephine A methyl ester (1a),
and three biosynthetically related DKPs, 15b-β-hydroxy-5-N-
acetyladreemin (2), 5-N-acetyladreemin (3), and 15b-β-
methoxy-5-N-acetyladreemin (4) (Figure 1). Structures were
assigned to 1−4 on the basis of detailed spectroscopic analysis,
chemical degradation and derivatization, and consideration of
biosynthetic relationships. To explore the mechanism behind
the solvolysis of 1, we reviewed all known DKM natural
products (7−16) (Figure 5), and assessed the chemical stability
of a range of synthetic DKMs (17−26) (Figure 6). We also
investigated the biological properties of 1 against prokaryotic,
eukaryotic, and mammalian cells.
RESULTS AND DISCUSSION
■
Aspergillus sp. (CMB-M081F), isolated from a marine sediment
collected in 2007 at an intertidal depth of 1 m near Shorncliffe,
Queensland, Australia, was noteworthy in that cultivations were
especially slow at producing secondary metabolites. For
example, HPLC-DAD-ESIMS analysis of EtOAc extracts
derived from 2-week agar plate cultivations failed to detect
Received: July 8, 2014
Published: August 26, 2014
©
2014 American Chemical Society
8700
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Table 1. H NMR (600 MHz) and 13C (150 MHz) Data of Shornephine A (1) and seco-Shornephine A Methyl Ester (1a)
1
a
a
pos
δH, mult (J in Hz) for 1 in CDCl3
δC
pos
δH, mult (J in Hz) for 1a in DMSO
δC
1
2
3
167.7
57.3
36.9
1
172.5
52.1
49.8
4.32, d (11.1)
1-OMe
3.38, s
a 3.26, d (13.7)
2
3.79, ddd (9.7, 8.0, 5.4)
7.35, d (8.0)
b 2.81, dd (13.7, 11.1)
2-NH
3
4
4
5
6
7
8
6
9
9
1
1
1
1
1
1
88.5
a 2.32, dd (14.2, 5.4)
b 2.24, dd (14.2, 9.7)
32.8
-OH
2.07, br s
131.5
117.1
117.3
121.2
117.1
141.4
4
55.8
130.0
117.0
121.4
115.6
141.5
6.91, d (7.7)
5
b
e
e
6.69 , m
6
6.56, d (7.4)
b
6.67 , m
7
6.78, dd (8.0, 7.4)
6.70, d (8.0)
6.91, d (7.7)
8
9
-OH
d
9-OH
10
9.50, br s
0
135.9
131.0
1-NH
6.34, s
11-NH
12
10.20, br s
2
3
4
5
94.9
44.9
179.7
42.3
13
6.39, dd (17.3, 10.6)
a 5.18, d (17.3)
b 5.11, d (10.6)
1.38, s
144.1
113.1
14
6.04, dd (17.4, 10.8)
a 5.07, dd (10.8, 0.6)
b 4.99, dd (17.4, 0.6)
1.01, s
143.5
113.8
15
1
1
1
2
3
6
7
′
′
′
22.9
25.8
16
22.1
21.8
1.38, s
17
0.93, s
165.9
78.5
1′
2′
2′-OH
3′
173.5
72.5
4.76, dd (8.8, 1.6)
a 3.32, d (15.1)
3.90, dd (9.6, 3.3)
5.53, br s
34.4
b 2.92, dd (15.1, 8.8)
a 2.79, dd (13.8, 3.3)
b 2.61, dd (13.8, 9.6)
40.4
4
5
6
7
′
136.2
126.7
129.3
128.5
4′
138.8
129.7
128.2
126.3
b
f
f
′/9′
′/8′
′
7.20
5′/9′
6′/8′
7′
7.22, d (7.2)
b
7.20
7.26, ddd (7.2, 7.2, 0.6)
b
7.20
7.18, td (7.2, 0.6)
e,f
a
13
b,c
d
C NMR assignments supported by gHSQC and gHMBC data. Overlapping signals. Not observed. Assignments are interchangeable.
any secondary metabolites, with peaks corresponding to 1−4
only appearing post 5-weeks (Supporting Information, Figure
S1). To investigate 1−4 further, we subjected a scaled up (15
plate) cultivation to solvent extraction, partitioning, trituration,
and C reversed-phase HPLC (H O/MeOH) fractionation, to
for 10 DBE and requiring that 1a be tricyclic. Further analysis
of the NMR data revealed COSY correlations diagnostic for five
isolated spin systems (Figure 2) and H NMR resonances
1
8
2
yield 1a and 2−4, with the unexpected isolation of 1a (m/z
489) rather than 1 (m/z 457) being attributed to methanolysis
during fractionation. Detailed spectroscopic analysis readily
identified 2 (C H N O , Δmmu +1.3) and 3 (C H N O ,
28
28
4
4
28 28
4
3
Δmmu −0.4) as the known DKP fungal metabolites 15b-β-
5
6
hydroxy-5-N-acetylardeemin and 5-N-acetylardeemin, respec-
tively, with 4 (C H N O , Δmmu +0.4) being identified as
2
9
30
4
4
the new homologue 15b-β-methoxy-5-N-acetylardeemin (Sup-
porting Information, Figures S10−S15). Supportive of this
latter assignment, the NMR (CDCl ) data for 4 exhibited
3
resonances for a 15b-β-OMe moiety (δH 2.70; δC 52.1),
positioned by HMBC correlations from the OMe to C-15β, and
ROESY correlations from the OMe to H-16α and H-17. A
detailed account of the structure elucidation, chemistry, and
biology of 1 and its methanolysis product 1a is presented
below.
Figure 2. Diagnostic 2D NMR correlations for 1 and 1a.
indicative of a methyl ester (δH 3.38) and two isolated
HRESI(+)MS analysis of 1a returned an adduct ion ([M +
deshielded exchangeable protons (δ 9.50 and 10.20). A series
H
+
Na] ) consistent with a molecular formula (C H N O ,
of HMBC correlations permitted assembly of the complete
planar structure for 1a as shown (Figure 2). On the basis of this
assignment, we hypothesized that 1a was a methanolysis artifact
of solvolytically unstable DKM natural product (i.e.,
shornephine A (1)).
26
30
2
6
Δmmu +0.6) requiring 13 double-bond equivalents (DBE).
The 13C NMR (DMSO-d ) data for 1a (Table 1) revealed
6
resonances for three ester/amide carbonyls (δ 172.5, 173.5
C
2
and 179.7) and 14 sp carbons (δ 113.8 to 143.5), accounting
C
8
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To test this hypothesis, we repeated the cultivation and
employed an alternative isolation strategy, involving sequential
trituration with hexane and CH Cl , followed by HPLC with an
to direct solvolysis as no trace of the methanolysis product 1c
could be detected (Figure 3).
This mechanistic proposal is interesting on two levels. First,
it highlights a simple nonenzymatic transformation that could
have implications in our understanding of the biosynthesis (and
2
2
H O/MeCN gradient, to yield 1−4. HRESI(+)MS analysis of 1
2
+
returned an adduct ion ([M + Na] , C H N O , Δmmu
0.3) consistent with a DKM, while 1D NMR (CDCl ) data
2
5
26
2
5
7
−
(
biomimetic synthesis) of DKPs such as notoamide C (5), itself
3
4
Table 1) confirmed replacement of the methyl ester and 2′-H
biosynthetically related to paraherquamide (6) (Figure 4), a
hydroxyl methine resonances in 1a, with a deshielded 2′-H
lactone methine (δ 4.76) in 1. As predicted, exposure of 1 to
H
MeOH resulted in methanolysis to 1a. Further examination of
the 2D NMR data for 1 revealed an ABC heterocyclic ring
system similar to that exhibited by cometabolite ardeemins 2−
4, but bearing 4-OH, 9-OH, and a C-12 reverse isoprene
moiety. Diagnostic ROESY correlations positioned H-2, H-2′,
H-3α, 4-OH, H -16, and H -17 on the same (α) face of the
3
3
heterocyclic scaffold and defined the relative configuration for 1
Figure 2). Analytical acid hydrolysis of 1 (50 μg) followed by
esterification of the hydrolysate with (R)-α-methoxy-α-
trifluoromethyl)phenylacetic acid (R-Mosher acid) returned
an ester identical with that obtained from authentic (S)-
phenyllactic acid (Supporting Information, Figure S2). The
observations outlined above confirmed the structures for 1 and
(
Figure 4. Biosynthetic relationship between notoamide C (5) and
paraherquamide (6).
(
2
,8−10
topic that has attracted much recent attention.
Second, it
1a as indicated (Figure 1). As the mechanism behind the
suggests that with suitable functionalization (e.g., 1), the DKM
moiety can be rendered stable to direct solvolysis, which has
implications for the possible use of DKMs in drug discovery.
To explore this latter issue further, we reviewed the literature
on known DKM natural products, noting it was limited to the
methanolysis of 1 to 1a was not immediately obvious, we
elected to explore this matter further. We hypothesize (Figure
3) that 1 undergoes an auto (C-9 phenol) acid-catalyzed
dehydration to yield a C-4 carbocation, which engages in a 1,2-
1
1
sigmatropic rearrangement and H O addition to yield seco-
2
fungal metabolites lateritin (7) from Gibberella lateritium,
12
shornephine A (1b). Our failure to detect 1b suggests that this
intermediate is solvolytically very unstable and rapidly trans-
forms to the methyl ester 1a. Curiously, 1 appeared to be stable
bassiatin (8) from Beauveria bassiana (K-717), three
13
homologous DKMs 9−11 from Fusarium sporotrichioides,
14
the DKM 12 from the Thai Sea hare Bursatella leachii, the
fungal mollenines A and B (13 and 14) from Eupenicillium
molle, and javanicunines A and B (15 and 16) from
15
16
Eupenicillium javanicum (Figure 5). On close examination,
we noted that 7−16 were all N-alkylated, and none were
solvolytically unstable. Consistent with these observations,
while shornephine A (1) is N-alkylated and stable to direct
solvolysis (i.e., does not form 1c), the seco form 1b is not N-
alkylated and is rapidly transformed into 1a. To test the
hypothesis that solvolytically unstable DKMs can be stabilized
by N-alkylation, we prepared the synthetic DKMs 17−24 and
the N-methylated analogues 25 and 26. As predicted, on
exposure to MeOH, 17−24 underwent rapid (t1 < 10 min)
/2
methanolysis, while 25 and 26 proved stable even after 48 h.
The Aspergillus sp. (CMB-M081F) metabolites 1−4 and the
methanolysis artifact 1a were not cytotoxic (IC > 30 μM)
5
0
against Gram-negative bacteria Escherichia coli (ATCC 11775)
and Pseudomonas aeruginosa (ATCC 10145), Gram-positive
bacteria Staphylococcus aureus (ATCC 9144 and ATCC 25923)
and Bacillus subtilis (ATCC 6633 and ATCC 6051), the fungus
Candida albicans (ATCC 90028), or human colon (SW620 and
SW620 Ad300) or cervical (KB-3−1 and KB-V1) cancer cell
lines (Supporting Information, Figures S36 and S38).
Systemic administration of chemotherapeutic agents (anti-
cancer drugs) is often used for the treatment of human cancers.
While clinically successful, this mode of treatment is
compromised by multidrug resistant (MDR) cancers that
exhibit either high intrinsic or acquired resistance to multiple
chemotherapeutic agents. Factors that contribute to MDR
include overexpression of membrane spanning adenosine
triphosphate binding cassette (ABC) transporter proteins
such as P-glycoprotein (P-gp) and associated accelerated drug
efflux. Although P-gp inhibitors offer the prospect of reversing
Figure 3. Proposed mechanism for methanolysis of 1.
8
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related synthetic DKMs we employed a Calcein AM assay. In
this assay, a nonfluorescent reagent calcein AM diffuses into the
cellular cytoplasm of P-gp overexpressing human colon cancer
(
SW620 Ad300) cells, where it undergoes hydrolysis to yield
the fluorescent dye calcein. Significantly, calcein AM is a P-gp
substrate and the hydrolyzed product calcein is not. In response
to functioning P-gp, calcein AM is effluxed prior to hydrolysis,
leading to reduced intracellular fluorescence. In the presence of
a P-gp inhibitor, calcein AM efflux is blocked and calcein AM
undergoes hydrolysis to calcein, leading to increased intra-
cellular fluorescence. Intracellular calcein fluorescence is
quantified by cell flow cytometry to arrive at a fluorescence
arbitrary ratio (FAR), which measures intracellular calcein
fluorescence in cells exposed to a putative P-gp inhibitor, with
that from cells not exposed to an inhibitor. The larger the FAR
17
value then (in principle) the more effective the P-gp inhibitor.
Importantly, all test DKMs were determined to be stable to
solvolysis for the 45 min duration of the calcein AM assay.
Using this approach we established the P-gp inhibitory
properties of 1 (FAR 35.5), 19 (FAR 52.9), 23 (FAR 41.5)
and 24 (FAR 40.5) (positive control verapamil FAR = 72.5)
(
Supporting Information, Figure S37). This observation raises
the prospect that the hitherto largely overlooked DKM scaffold
may be engineered to deliver a clinically useful inhibitor of P-gp
mediated drug efflux. If achieved, such an outcome would
greatly improve the prognosis for MDR cancer chemotherapy.
EXPERIMENTAL SECTION
■
Collection and Isolation of Aspergillus sp. (CMB-M081F).
Strain CMB-M081F was isolated from marine sediment collected
collected in 2007 at an intertidal depth of 1 m near Shorncliffe,
Queensland, Australia. The freshly collected sediment was sealed in a
Falcon tube (50 mL) and transferred at rt to the laboratory, where it
was stored in the dark at −30 °C for 1 week. A sample (1 g) was
thawed, suspended in sterile 0.9% saline (8 mL), and subjected to
heat-shock (60 °C for 30 min), and an aliquot (100 μL) was used to
prepare three 10-fold serial dilutions. Aliquots (50 μL) from the saline
solution and serial dilutions were applied to M1 agar plates
Figure 5. Known DKM natural products 7−16.
(
(
comprising 2% agar in artificial ocean sea salt (3.3%; 25 mL), starch
1%), yeast extract (0.4%), peptone (0.2%), and rifampicin (0.0005%)
and incubated at 27 °C for 4−5 weeks. Pure strains of individual
bacterial and fungal colonies, including CMB-M081F, were obtained
by standard microbiological techniques and were grown to dense
colonies on single agar plates. Taxonomic analysis identified CMB-
M081F as an Aspergillus sp. (Supporting Information, section 1.1).
Analytical Cultivation and Chemical Analysis of Aspergillus
sp. (CMB-M081F). A single colony of Aspergillus sp. (CMB-M081F)
applied to an M1 agar plate was incubated at 27 °C for 5 weeks, after
which the agar was diced and extracted with EtOAc (100 mL), and the
organic phase was concentrated in vacuo. The extract (5.6 mg) was
analyzed by HPLC-DAD-ESI(±)MS (Zorbax SB-C 5 μm 150 × 4.6
8
mm column, 1 mL/min gradient elution from 90% H O:MeCN to
2
1
00% MeCN over 15 min with isocratic 0.05% HCO H modifier) to
2
+
reveal noteworthy peaks at 11.1 min (m/z 435 [M + H] , 1), 12.2 min
+
+
(
m/z 485 [M + H] , 2), 12.4 min (m/z 469 [M + H] , 3), and 12.8
+
min (m/z 499 [M + H] , 4) (Supporting Information, Figure S1).
Preparative Cultivation and Fractionation of Aspergillus sp.
(CMB-M081F). Method 1: Fifteen M1 agar plates were cultivated and
processed as described above to yield an extract (83.7 mg) that was
sequentially triturated (10 mL aliquots) to yield hexane (33.8 mg),
CH Cl (25.9 mg) and MeOH (7.8 mg) soluble fractions. The CH Cl
2
2
2
2
solubles were fractionated by semipreparative reversed-phase HPLC
Figure 6. Synthetic DKMs 17−26: (a) HBTU, DIPEA, DMF, rt, h;
(
Zorbax SB-C 5 μm 250 × 9.4 mm column, 3 mL/min gradient
(
b) p-TsOH, toluene, microwave 140 °C, 300 W, 3 min.
8
elution from 90% H O/MeOH to 100% MeOH over 30 min) to yield
2
seco-shornephine A methyl ester (1a) (t = 23.9 min, 1.5 mg, 1.8%),
R
the MDR phenotype, no P-gp inhibitors have yet advanced to
the clinic. To evaluate the P-gp inhibitory properties of 1 and
15b-β-hydroxy-5-N-acetylardeemin (2) (t = 25.5 min, 1.9 mg, 2.2%),
R
1
7
5-N-acetylardeemin (3) (t = 27.0 min, 2.1 mg, 2.5%), and 15b-β-
R
8
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methoxy-5-N-acetylardeemin (4) (tR = 28.0 min, 2.5 mg, 3.0%).
Method 2: Ten M1 agar plates were cultivated and processed as
described above to yield an extract (60.2 mg) that was sequentially
partitioned into hexane (2 mg) and CH Cl (60 mg) soluble fractions.
CDCl ) δ 167.1 (C-1), 166.3 (C-1′), 135.4 (C-4′), 135.1 (C-4), 128.8
3
(C-5/9), 128.3 (C-5′/9′), 127.8−130.8 (C-6/7/8/6′/7′/8′), 78.1 (C-
2′), 54.7 (C-2), 39.0 (C-3), 38.1 (C-3′); HRMS(ESI-TOF) m/z [M +
+
+
H] calcd for C H NO 296.1281, found 296.1277.
2
2
18 18
3
The CH Cl solubles were fractionated by semipreparative reversed-
cyclo-(L-Tryptophan-L-mandelic acid) (19): colorless oil (4.0 mg,
2
2
75%); [α]D23 −5 (c 0.14, CHCl ); H NMR (600 MHz, CDCl ) δ
1
phase HPLC (Zorbax SB-C 5 μm column 250 × 9.4 mm column, 3
8
3
3
mL/min gradient elution from 90% H O/MeCN to 100% MeCN over
8.12 (br s, H-9), 7.47 (d, J = 8.4 Hz, H-10), 7.04−7.47 (m, H-5/6/7/
8/5′/6′/7′), 5.77 (s, H-2′), 4.41 (br d, J = 10.5 Hz, H-2), 3.47 (dd, J =
14.8, 3.4 Hz, H-3a), 3.24 (br s, 2-NH), 2.87 (dd, J = 14.8, 10.5 Hz, H-
2
3
1
5
0 min) to yield shornephine A (1) (t = 23.9 min, 2.0 mg, 1.8%),
R
5b-β-hydroxy-5-N-acetylardeemin (2) (t = 25.5 min, 1.5 mg, 2.2%),
R
3b); 13C NMR (150 MHz, CDCl ) δ 167.1 (C-1′), 166.2 (C-1), 136.6
-N-acetylardeemin (3) (t = 27.0 min, 1.9 mg, 2.5%), and 15b-β-
R
3
methoxy-5-N-acetylardeemin (4) (tR = 28.0 min, 1.5 mg, 3.0%).
(C-8a), 134.5 (C-3′), 129.5 (C-5′/7′), 129.0 (C-6′), 127.1 (C-4a),
127.1 (C-4′/8′), 126.8 (C-5), 126.7 (C-6), 124.0 (C-7), 118.7 (C-10),
111.7 (C-8), 108.8 (C-4), 79.5 (C-2′), 54.7 (C-2), 29.4 (C-3);
(
Note: All % yields were determined on a mass-to-mass measure
against the crude EtOAc extract.)
+
+
Characterization of Aspergillus sp. (CMB-M081F) Metabo-
HRMS(ESI-TOF) m/z [M + Na] calcd for C H N O Na
19 16 2 3
lites and Solvolysis Products. Shornephine A (1): pale yellow oil;
343.1053, found 343.1055.
[
α]D22 +22 (c 0.05, CHCl ); UV (MeCN) λ (log ε): 210 (4.54),
cyclo-(L-Tryptophan-L-phenyllactic acid) (20): colorless oil (4.0
3
max
mg, 75%); [α]D23 −76 (c 0.05, CHCl ); H NMR (600 MHz, CDCl )
1
2
42 (3.79), 301 (3.37) nm; NMR (CDCl ) see Table 1 and
3
3
3
Supporting Information Table S1 and Figures S6 and S7; HRMS(ESI-
δ 8.12 (br s, H-9), 7.48 (d, J = 7.6 Hz, H-5), 7.35 (d, J = 7.3 Hz, H-8),
7.35 (t, J = 7.3 Hz, H-6′/8′), 7.28 (m, H-5′/9′), 7.26 (m, H-7′), 7.23
(dd, J = 7.6, 7.3 Hz, H-7), 7.14 (dd, J = 7.6, 7.6 Hz, H-6), 6.90 (br s,
H-10), 5.67 (br s, 2-NH), 5.05 (dd, J = 5.3, 4.0 Hz, H-2′), 4.26 (br d, J
= 10.1 Hz, H-2), 3.31 (m, H-3a), 3.28 (m, H-3b), 3.27 (dd, J = 14.4,
+
+
TOF) m/z [M + Na] calcd for C H N O Na 457.1734, found
25
26
2
5
4
57.1731.
seco-Shornephine A methyl ester (1a): pale yellow oil; [α] 23 −73
D
(c 0.02, CHCl ); UV (MeCN) λ (log ε): 218 (3.94), 256 (3.75),
3 max
13
3
10 (3.59) nm; NMR (DMSO-d ) see Table 1 and Supporting
4.0 Hz, H-3′a), 3.15 (dd, J = 14.4, 5.3 Hz, H-3′b); C NMR (150
6
Information Table S2 and Figures S8−S9; HRMS(ESI-TOF) m/z [M
MHz, CDCl ) δ 166.8 (C-1), 166.3 (C-1′), 137.1 (C-8a), 135.1 (C-
3
+
+
+
Na] calcd for C H N O Na 489.2009, found 489.2015.
1
4′), 131.3 (C-5′/9′), 128.6 (C-7′), 126.8 (C-4a), 126.8 (C-6′/8′),
124.0 (C-10), 123.6 (C-7), 120.6 (C-6), 119.0 (C-5), 112.5 (C-8),
109.4 (C-4), 79.6 (C-2′), 53.6 (C-2), 38.6 (C-3′), 30.1 (C-3);
2
6
30
2
6
5
,6
23
5b-β-Hydroxy-5-N-acetyladreemin (2):. pale yellow oil; [α]D
−
19 (c 0.05, MeOH); NMR (CDCl ) see Supporting Information
3
+
+
+
Table S3 and Figures S10−S11; HRMS(ESI-TOF) m/z [M + Na]
HRMS(ESI-TOF) m/z [M + H] calcd for C H N O 335.1390,
20 19 2 3
+
calcd for C H N O Na 507.2003, found 507.2016.
found 335.1376.
28
28
4
4
5
,6
23
5
-N-Acetyladreemin (3):. pale yellow oil; [α]D −21 (c 0.05,
cyclo-(L-Alanine-L-mandelic acid) (21): colorless oil (2.5 mg, 62%);
[α]D23 +29 (c 0.05, CHCl ); H NMR (600 MHz, CDCl ) δ 7.38 (m,
1
MeOH); NMR (CDCl ) see Supporting Information Table S4 and
3
3
3
+
Figures S12−S13; HRMS(ESI-TOF) m/z: [M + Na] calcd for
H-4′/8′), 7.28−7.35 (m, H-5′/6′/7′), 5.11 (s, H-2′), 4.50 (m, H-2),
+
13
C H N O Na 491.2054, found 491.2050.
3.42 (br s, 2-NH), 1.48 (d, J = 6.8 Hz, H-3); C NMR (150 MHz,
2
8
28
4
3
23
1
5b-β-Methoxy-5-N-acetyladreemin (4): pale yellow oil; [α]D
CDCl ) δ 175.8 (C-1), 173.8 (C-1′), 138.9 (C-3′), 126.8 (C-4′/8′),
3
−
16 (c 0.05, MeOH); UV (MeCN) λmax (log ε) 219 (3.97), 259
126.1−130.2 (C-5′/6′/7′), 75.7 (C-2′), 49.7 (C-2), 19.1 (C-3);
+
+
(
3.78), 307 (3.62) nm; NMR (CDCl ) see Supporting Information
HRMS(ESI-TOF) m/z [M + Na] calcd for C H NO Na 228.0631,
11 11 3
3
+
Table S5 and Figures S14−S15; HRMS(ESI-TOF) m/z [M + Na]
calcd for C H N O Na 521.2159, found 521.2163.
found 228.0633.
+
29
30
4
4
cyclo-(L-Alanine-L-phenyllactic acid) (22): colorless oil (3.1 mg,
69%); [α]D23 +85 (c 0.05, CHCl ); H NMR (600 MHz, CDCl ) δ
1
Synthesis of DKMs 17−26. The DKMs 17−26 were all prepared
using a common two-step method. Step 1: An amino acid methyl ester
3
3
7.19−7.31 (m, H-5′/6′/7′/8′/9′), 4.45 (br s, H-2), 4.29 (br s, H-2′),
(
1 equiv) was treated with HBTU (1.2 equiv) and DIPEA (2.8 equiv)
4.22 (br s, 2-NH), 3.14 (d, J = 13.3 Hz, H-3′a), 2.85 (dd, J = 13.3, 7.6
1
3
in the presence of either (S)-phenyllactic acid (1 equiv) or (S)-
mandelic acid (1 equiv), in anhydrous DMF (5 mL). The resulting
reaction mixture was stirred at rt for 3 h under argon, concentrated in
vacuo, and partitioned between EtOAc (2 × 50 mL) and 1 M HCl (50
mL), and the combined organic layers were dried with anhydrous
Hz, H-3′b), 1.33 (br s, H-3); C NMR (150 MHz, CDCl ) δ 174.7
3
(C-1), 174.5 (C-1′), 136.9 (C-4′), 127.2−129.3 (C-5′/6′/7′/8′/9′),
72.8 (C-2), 50.2 (C-2′), 40.5 (C-3′), 17.8 (C-3); HRMS(ESI-TOF)
+
+
m/z [M + Na] calcd for C H NO Na 242.0788, found 242.0790.
12
13
3
cyclo-(L-Tyrosine-L-mandelic acid) (23): colorless oil (2.5 mg,
65%); [α]D23 +2 (c 0.2, CHCl ); H NMR (600 MHz, CDCl ) δ
1
MgSO and concentrated in vacuo. Step 2: The amide product from
4
3
3
step 1 (1 equiv) was treated with p-TsOH (1.5 equiv) in anhydrous
toluene (5 mL), heated in a microwave reactor at 140 °C/300 W for 3
min, and concentrated in vacuo, and the residue was purified by C8
7.38−7.41 (m, H-4′/5′/6′/7′/8′), 6.95 (d, J = 8.1 Hz, H-6/8), 6.76 (d,
J = 8.1 Hz, H-5/9), 5.82 (s, H-2′), 5.78 (br s, 7-OH), 4.76 (br s, 2-
NH), 4.36 (br d, J = 10.4 Hz, H-2), 3.21 (dd, J = 14.0, 3.4 Hz, H-3a),
2.57 (dd, J = 14.0, 10.4 Hz, H-3b); 13C NMR (150 MHz, CDCl ) δ
reversed-phase HPLC (H O/MeCN). Overall yields: 17 (75%), 18
2
3
(
72%), 19 (75%), 20 (75%), 21 (65%), 22 (65%), 23 (62%), 24
131.1 (C-6/8), 129.9 (C-5′/7′), 129.7 (C-6′), 127.0 (C-4′/8′), 116.7
(
69%), 25 (50%), and 26 (41%).
(C-5/9), 80.6 (C-2′), 55.4 (C-2), 38.8 (C-3); HRMS(ESI-TOF) m/z
+
+
Characterization of Synthetic DKMs 17−26. cyclo-(L-Phenyl-
[M + Na] calcd for C H N O Na 320.0893, found 320.0895.
17 15 1 4
23
D
alanine-L-mandelic acid) (17): colorless oil (3.0 mg, 75%); [α]
−
8
cyclo-(L-Tyrosine-L-phenyllactic acid) (24): colorless oil (3.1 mg,
1
23 1
74 (c 0.04, CHCl ); H NMR (600 MHz, CDCl ) δ 7.39 (m, H-4′/
65%); [α]D −89 (c 0.1, CHCl ); H NMR (600 MHz, CDCl ) δ
3
3
3
3
′), 7.37−7.42 (m, H-5′/6′/7′), 7.31 (m, H-6/8), 7.27 (m, H-7), 7.07
7.31 (t, J = 7.5 Hz, H-5′/9′), 7.23 (m, H-6′/8′), 7.23 (m, H-7′), 6.75
(d, J = 8.2 Hz, H-6/8), 6.67 (d, J = 8.2 Hz, H-5/9), 6.05 (s, 2-NH),
5.03 (dd, J = 5.0, 4.6 Hz, H-2), 4.01 (br d, J = 10.5 Hz, H-2′), 3.20 (dd,
J = 14.5, 4.6 Hz, H-3a), 3.09 (dd, J = 14.5, 5.0 Hz, H-3b), 2.90 (dd, J =
(
d, J = 7.1 Hz, H-5/9), 5.83 (s, H-2′), 4.41 (br d, J = 11.4 Hz, H-2),
3
.29 (dd, J = 13.9, 3.1 Hz, H-3a), 2.62 (dd, J = 13.9, 11.4 Hz, H-3b);
1
3
C NMR (150 MHz, CDCl ) δ 166.2 (C-1′), 134.7 (C-4), 134.1 (C-
3
1
3
3
4
′), 129.8 (C-5/6/8/9), 129.6 (C-5′/6′/7′), 128.4 (C-7), 127.1 (C-
14.1, 3.4 Hz, H-3′a), 1.43 (dd, J = 14.1, 10.5 Hz, H-3′b); C NMR
′/8′), 79.7 (C-2′), 55.4 (C-2), 39.3 (C-3); HRMS(ESI-TOF) m/z
(150 MHz, CDCl ) δ 170.6 (C-1′), 170.0 (C-1), 155.5 (C-7), 134.6
3
+
+
[
M + H] calcd for C H NO 282.1125, found 282.1117.
(C-4), 134.2 (C-4′), 131.1 (C-6/8), 130.6 (C-7′), 129.2 (C-5′/9′),
128.2 (C-6′/8′), 116.5 (C-5/9), 78.7 (C-2), 55.4 (C-2′), 38.6 (C-3′),
17
16
3
cyclo-(L-Phenylalanine-L-phenyllactic acid) (18): white solid (2.5
mg, 72%); [α]D23 −446 (c 0.05, CHCl ); H NMR (600 MHz,
1
38.2 (C-3); HRMS(ESI-TOF) m/z [M + Na] calcd for
+
3
+
CDCl ) δ 7.31 (d, J = 7.3 Hz, H-5′/9′), 7.18−7.27 (m, H-6/7/8/6′/
C H N O Na 334.1050, found 334.1044.
3
18 17
1
4
7
5
′/8′), 6.93 (d, J = 7.1 Hz, H-5/9), 5.67 (br s, 2-NH), 5.02 (dd, J =
.3, 4.1 Hz, H-2′), 4.11 (br d, J = 11.0 Hz, H-2), 3.20 (dd, J = 14.3, 4.1
cyclo-(N-Methyl-L-tyrosine-L-mandelic acid) (25): colorless oil (0.9
mg, 41%); [α]D23 +38 (c 0.05, CHCl ); H NMR (600 MHz, DMSO-
1
3
Hz, H-3′a), 3.08 (dd, J = 14.3, 5.3 Hz, H-3′b), 3.02 (dd, J = 13.9, 3.4
d ) δ 7.23 (m, H-5′/6′/7′), 7.05 (d, J = 7.4 Hz, H-4′/8′), 6.84 (d, J =
6
Hz, H-3a), 1.58 (dd, J = 13.9, 11.0 Hz, H-3b); 13C NMR (150 MHz,
8.1 Hz, H-6/8), 6.55 (d, J = 8.1 Hz, H-5/9), 5.26 (s, H-2′), 4.43 (dd, J
8
704
dx.doi.org/10.1021/jo501501z | J. Org. Chem. 2014, 79, 8700−8705

The Journal of Organic Chemistry
Article
=
1
10.1, 5.3 Hz, H-2), 2.95 (dd, J = 14.7, 5.3 Hz, H-3a), 2.64 (dd, J =
4.7, 10.1 Hz, H-3b), 2.60 (s, 2-Me); 13C NMR (150 MHz, DMSO-
d ) δ 172.2 (C-1′), 172.0 (C-1), 155.3 (C-7), 141.4 (C-3′), 129.8 (C-
ACKNOWLEDGMENTS
We thank S. Bates and R. Robey (NCE, NIH) for providing
SW620 and SW620 Ad300 cell lines and M. M. Gottesman
■
6
4
), 129.7 (C-6/8), 127.4 (C-4′/8′), 126.7−128.2 (C-5′/6′/7′), 115.1
(
NIH) for providing KB-3-1 and KB-V1 cell lines. Z.G.K. and
(
C-5/9), 70.5 (C-2′), 63.5 (C-2), 34.8 (C-3), 28.6 (2-Me);
+
+
X.-C.H. acknowledge the provision of UQ International
Postgraduate Scholarships, and R.R. acknowledges the
provision of an Australian Postgraduate Award. This research
was funded in part by the Institute for Molecular Bioscience,
The University of Queensland, and the Australian Research
Council (LP120100088).
HRMS(ESI-TOF) m/z [M + Na] calcd for C H NO Na
1
8
17
4
3
34.1050, found 334.1059.
cyclo-(N-methyl-L-tyrosine-L-phenyllactic acid) (26): colorless oil
1.1 mg, 50%); [α]D23 +37 (c 0.05, CHCl ); H NMR (600 MHz,
1
(
3
CDCl ) δ 7.29 (m, H-7′), 7.19 (m, H-6′/8′), 7.04 (d, J = 7.6 Hz, H-
3
5
(
′/9′), 6.97 (d, J = 8.3 Hz, H-6/8), 6.81 (d, J = 8.3 Hz, H-5/9), 5.23
br s, 7-OH), 4.83 (dd, J = 9.3, 3.0 Hz, H-2′), 4.28 (dd, J = 4.7, 4.7 Hz,
H-2), 2.96 (s, 2-NMe), 2.92 (dd, J = 14.7, 4.7 Hz, H-3a), 2.87 (dd, J =
REFERENCES
■
1
1
1
9
4.5, 3.0 Hz, H-3′a), 2.79 (dd, J = 14.7, 4.7 Hz, H-3b), 1.70 (dd, J =
(
1) Mootz, H. D.; Marahiel, M. A. Curr. Opin. Chem. Biol. 1997, 1,
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4.5, 9.3 Hz, H-3′b); C NMR (150 MHz, CDCl ) δ 164.8 (C-1′),
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5
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(
+
+
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19
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(
1
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(
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CH Cl (150 μL) were treated with 3 eq. of either (R)-α-methoxy-α-
trifluoromethylphenylacetic acid [R-MTPA-OH] or (S)-α-methoxy-α-
7
(
18
2
2
(
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(
(
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MeCN over 15 min followed by a 5 min hold at 100% MeCN, with
isocratic 0.05% HCO H modifier) (Supporting Information, Figure
S2).
2
2
2
8
2
2
Methanolysis of DKMs 7−26. Solutions of 7−26 (100 μg in 100
μL of MeOH) were treated as described above for the methanolysis of
1
(Supporting Information, Figures S4 and S5).
(
ASSOCIATED CONTENT
■
S.-i.; Hosoe, T.; Kawai, K.-i.; Yaguchi, T.; Fukushima, K. Heterocycles
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S
Supporting Information
2
(
*
Full tabulated 2D NMR data including HSQC, HMBC,
1
13
gCOSY, gROESY, and H and C NMR spectra, HPLC
chromatograms, taxonomic analysis, and bioassay data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(
2
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: r.capon@uq.edu.au.
Present Address
†
Department of Chemistry & Biomolecular Sciences, Macquar-
ie University, Sydney, NSW 2109, Australia.
Notes
The authors declare no competing financial interest.
8
705
dx.doi.org/10.1021/jo501501z | J. Org. Chem. 2014, 79, 8700−8705