JBIR-56 and JBIR-57, 2(1 H)-pyrazinones from a marine sponge-derived streptomyces sp. SpD081030SC-03

Article abstract of DOI:10.1021/np200386c

Strain SpD081030SC-03, representing a novel species of Streptomyces, was isolated from a marine sponge. Two 3,5,6-trisubstituted 2(1H)-pyrazinones, JBIR-56 (1) and JBIR-57 (2), were isolated from a culture of SpD081030SC-03. The planar structures of 1 and

Full text of DOI:10.1021/np200386c

ARTICLE
pubs.acs.org/jnp
JBIR-56 and JBIR-57, 2(1H)-Pyrazinones from a Marine
Sponge-Derived Streptomyces sp. SpD081030SC-03
Keiichiro Motohashi,† Kennichi Inaba,† Shinichiro Fuse,‡ Takayuki Doi,§ Miho Izumikawa,†
Shams Tabrez Khan,† Motoki Takagi,*,† Takashi Takahashi,‡ and Kazuo Shin-ya*,^
†Biomedicinal Information Research Center (BIRC), Japan Biological Informatics Consortium (JBIC), 2-4-7 Aomi, Koto-ku,
Tokyo 135-0064, Japan
‡Department of Applied Chemistry, Tokyo Institute of Technology, 2-12-1, Ookayama, Meguro-ku, Tokyo 152-8552, Japan
§Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aza-Aoba, Aramaki, Aoba, Sendai 980-8578, Japan
^Biomedicinal Information Research Center (BIRC), National Institute of Advanced Industrial Science and Technology (AIST),
2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan
S Supporting Information
b
ABSTRACT: Strain SpD081030SC-03, representing a novel species of Streptomyces, was isolated
from a marine sponge. Two 3,5,6-trisubstituted 2(1H)-pyrazinones, JBIR-56 (1) and JBIR-57
(2), were isolated from a culture of SpD081030SC-03. The planar structures of 1 and 2 were
assigned on the basis of extensive NMR and MS analyses. In addition, analyses of the methylated
derivative of 1 confirmed a 3,5,6-trisubstituted 2(1H)-pyrazinone moiety. The absolute
configurations of the amino acid residues were determined by application of Marfey’s method.
Because 1 did not appear to comprise the normal connection of amino acid units, we confirmed
its structure by the total synthesis of 1. Biosynthetically, 1 consists of a unique skeleton connected
to the peptide chain at C-5 of the pyrazinone ring.
icroorganisms from marine habitats,1,2 especially actino-
bacteria, constitute a promising untapped resource of novel
M
compounds; therefore, these organisms are currently receiving a
great deal of attention. When compared to higher organisms,
microorganisms can be easily maintained under laboratory condi-
tions, ensuring a constant and inexpensive supply of their secondary
metabolites. Moreover, many compounds originally believed to be
produced by marine organisms have been found to be produced by
host-associated microorganisms.3 A significant body of work on the
isolation of actinobacteria from marine habitats has emerged in the
last 10 years, and screening of these organisms has yielded several
novel bioactive compounds.4ꢀ6 Our group has recently engaged in
the isolation of microorganisms from marine sources, including
fungi and actinobacteria. We have discovered novel compounds
produced by newly identified species of marine-derived actinomy-
cetes including the anthracyclines, tetracenoquinocin and
5-iminoaranciamycin,7 tetrapeptides JBIR-34 and JBIR-35,8 a novel
salicylamide, JBIR-58,9 a new diterpene, JBIR-65,10 and JBIR-66.11
In the course of our screening for new metabolites, we successfully
isolated two new peptides, JBIR-56 (1) and JBIR-57 (2), from the
culture broth of a new species (SpD081030SC-03) of Streptomyces
associated with an unidentified marine sponge. Here, the fermenta-
tion, isolation, and structure elucidation of 1 and 2 and the total
synthesis of 1 are described.
based on its 16S rRNA gene sequence.12 Comparison of almost
the entire length (1507 bp) of the 16S rRNA with sequences in the
Eztaxon-type strain database13 revealed that the closest phyloge-
netic neighbor of the strain was Streptomyces lucensis NBRC
13056T (AB184280), with a sequence identity of only 97.6%.
The low identity with previously reported sequences indicates that
this strain is a novel species in the genus Streptomyces.12
The strain was cultured at 27 ꢀC for five days by rotary shaking
in baffled 500 mL Erlenmeyer flasks containing 100 mL of
production medium. Compounds 1 and 2 were then recovered
from the supernatant of the fermentation broth using HP-20
resin, after which they were purified by sequential chromatogra-
phy utilizing MPLC and HPLC.
The structures of 1 and 2 were primarily elucidated by
spectroscopic methods, including 2D NMR. The molecular
formula of JBIR-56 (1) was established as C19H30N4O5 based
on the HRESIMS data ([M + H]+, m/z 395.2294). The peptide
1
structure of 1 was evident from the H and 13C NMR data
recorded in DMSO-d6 as shown in Table 1. The absorptions at
’ RESULTS AND DISCUSSION
Streptomyces sp. SpD081030SC-03 isolated from a marine
sponge was identified as a new species of the genus Streptomyces
Received: May 6, 2011
Published: July 05, 2011
r
Copyright
2011 American Chemical Society and
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3380 and 1660 cmꢀ1 in the IR spectrum of 1 revealed the
presence of hydroxy, amide, and carbonyl functional groups.
Together with these IR absorptions, typical NMR chemical shifts
for amide and R-methine resonances (δC 174.0 and 171.4, δC/H
50.7/4.52 and 48.1/4.12, respectively) indicated that 1 is a
peptide compound. Detailed structural information was obtained
from the HSQC, HMBC, and DQF-COSY spectra of 1
(Figure 1a). The sequence from an amino proton 20-NH (δH
8.18) to the doublet methyl protons H3-50 (δH 0.89) through an
R-methine proton H-20 (δH 4.52, δC 50.7), methylene protons
H2-30 (δH 1.52), and a methine proton H-40 (δH 1.56), which
was in turn coupled to doublet methyl protons H3-60 (δH 0.88),
was established on the basis of the DQF-COSY spectrum. Long-
range couplings of both H-20 and H-30 to a carbonyl carbon C-10
(δC 171.4) observed in the HMBC spectrum indicated the
presence of a leucine residue. Similarly, an alanine unit was
assigned on the basis of interpretation of the DQF-COSY and
HMBC spectroscopic data. Long-range couplings from the
amide proton 200-NH (δH 8.34) to the carbonyl carbon C-10
and the R-methine carbon C-200 (δC 48.1) proved a Leu-Ala unit
as shown in Figure 1a.
The leucine residue was further connected to two carbons
(δC 163.3 and 120.3) on the basis of HMBC correlations with
the 20-NH. The δC 163.3 chemical shift suggested that leucine
was involved in an amide bond with a conjugated acid. Additional
ethyl (δH 2.99, 2.93, 1.13) and isopropyl (δH 3.22, 1.15) spin
systems were also apparent from the DQF-COSY spectrum. The
methylene protons of the ethyl group had HMBC correlations to
carbons at δC 145.9 and 120.3, whereas the methyl protons showed
correlations to carbons at δC 145.9 and 23.2. These correlations
suggested a 2,3-disubstituted pentenoic acid fragment connected to
the leucine. The isopropyl group had HMBC correlations to two
carbons (δC 158.7 and 155.9) (Figure 1a). The remaining atoms
required by the molecular formula were two nitrogens, an oxygen, and
a hydrogen. While there were several ways to combine these elements,
a weak correlation from the methylene protons of the ethyl group to
the carbon at δC 155.9 was consistent with only two reasonable
structures, a 3,5,6-trisubstituted 2(1H)-pyrazinone or a 3,5,6-trisub-
stituted 4(1H)-pyridazinone. Carbon chemical shift predictions were
in much better agreement with the pyrazinone structure.
1
Table 1. H (600 MHz) and 13C (150 MHz) NMR Spectro-
scopic Data for JBIR-56 (1) and JBIR-57 (2) in DMSO-d6
JBIR-56 (1)
JBIR-57 (2)
1H
1H
In order to provide additional support for this structure,
chemical modification was conducted as follows: 1 was treated
with methyl iodide and potassium carbonate in DMF to yield
1-methyl-JBIR-56 methyl ester (3) as the main derivative. The
molecular formula of 3 was established as C21H34N4O5 based on
the HRESIMS data ([M + H]+, m/z 423.2601). The appearance
of two new signals (δC/H 30.6/3.60 and 52.7/3.70 ppm)
position 13C
(J in Hz)
position 13C
(J in Hz)
2
155.9
158.7
120.3
145.9
2
155.8
158.3
120.9
140.8
3
3
5
5
6
6
7
29.5 3.22, q (6.6)
19.9 1.15, d (6.6)
19.8 1.15, d (6.6)
163.3
7
29.5 3.21, q (6.8)
19.9 1.15, d (6.8)
19.8 1.15, d (6.8)
163.6
1
compared to 1 was observed in the H and 13C NMR spectra
8
8
of 3. The structure of 3, including signals of a methoxy group, 100-
O-CH3 (δC/H 52.7/3.70), and an N-methyl group, 1-CH3 (δC/H
30.6/3.60), was confirmed by analysis of the DQF-COSY,
9
9
10
11
10
1
13
23.2 2.99, dq (12.6, 7.2) 11
2.93, dq (12.6, 7.2)
16.7 2.53, s
HSQC, and HMBC spectra (Figure 1b). Each Hꢀ C long-
range correlation from the 100-O-CH3 to an ester carbonyl
carbon, C-100 (δC 174.7), from the N-methyl proton, 1-CH3, to
an amide carbonyl carbon, C-2 (δC 156.4), and an aromatic
quaternary carbon, C-6 (δC 147.2), confirmed the 3,5,6-trisub-
stituted 2(1H)-pyrazinone moiety (Figure 1b). Taken together,
the structure of 1 was proposed.
12
14.0 1.13, t (7.2)
Leu
10
Leu
10
171.4
171.6
20
30
50.7 4.52, dd (13.8, 8.4) 20
42.6 1.52, dd (13.8, 6.0) 30
50.6 4.53, dd (14.8, 8.4)
42.6 1.52, dd (14.8, 6.2)
24.7 1.56, q (6.2)
23.3 0.89, d (6.2)
22.4 0.88, d (6.2)
8.16, d (8.4)
The molecular formula of JBIR-57 (2) was determined to be
C18H28N4O5 on the basis of the HRESIMS data ([M + H]+, m/z
381.2130), which showed the disappearance of a methyl or a
methylene group from 1. The IR and UV spectra of 2 were similar
to those of 1. In addition, most of the NMR spectroscopic data
for 2 were closely related to those of 1; however, the ethyl moiety
consisting of the triplet methyl protons H3-12 and the methylene
protons H2-11 in 1 was replaced by a singlet methyl signal (δH
2.53, δC 16.7). These collective spectroscopic data demonstrated
that 2 was a C-6 methyl analogue of 1.
40
50
60
20-NH
Ala
100
24.7 1.56, q (6.0)
23.1 0.89, d (6.0)
22.4 0.88, d (6.0)
8.18, d (8.4)
40
50
60
20-NH
Ala
100
174.0
174.1
200
48.1 4.12, dq (7.2, 6.6) 200
47.8 4.14, dq (7.0, 6.2)
17.7 1.24, d (7.0)
8.41, br s
300
200-NH
17.7 1.23, d (7.2)
8.34, br s
300
200-NH
1
1
Figure 1. Key correlations observed in 2D NMR spectra of 1 (a) and 3 (b) (bold lines show Hꢀ H DQF-COSY results, and arrows show
HMBC results).
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Scheme 1. Total Synthesis of JBIR-56
The absolute configuration of 1 was defined by Marfey’s
method.14 The acid hydrolysate of 1 was reacted with FDAA
(Marfey’s reagent) and then analyzed on the basis of the UV
absorption at 340 nm and positive mode ESI-LC-MS monitoring
using an analytical reversed-phase UPLC system equipped with
an ODS column. All derivatives were identified on the basis of
comparison of their retention times, molecular formulas, and UV
spectra with those of standard amino acids derived from FDAA
conjugated compounds. The configurations of the Ala and Leu
residues were determined to be L and D, respectively. Overall, the
absolute configuration of 1 was assigned as 20R,200S. Similarly,
the absolute configuration of 2 was determined to be the same as
that of 1.
separated by silica gel column chromatography. The structures of
both isomers 8a and 8b were confirmed by 2D NMR techniques
such as HMBC. Further transformations were then conducted by
employing isomer 8a. An isopropyl group was introduced to
the 3-position of 8a and the benzyl group was removed to
yield 6-ethyl-3-isopropyl-2(1H)-pyrazinone (10).15 Iodination
of compound 10 at the 5-position gave 11. Compound 11 was
treated with two equivalents of n-BuLi to generate a dianion. The
in situ generated dianion was subsequently treated with chlor-
oformic acid methyl ester to give the 5-acylated product.16
Hydrolysis of the methyl ester 12 provided the desired 6-ethyl-
3-isopropyl-2(1H)-pyrazinone-5-carboxylic acid (13). Coupling
ofthe carboxylic acid13 andthe dipeptide14 was conducted inthe
presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hy-
drochloride (EDCI HCl). Acidic removal of the tert-butyl group
3
’ TOTAL SYNTHESIS OF JBIR-56
gave the desired product 1 in good yield. The structureof synthetic
1 was confirmed by the NMR spectra and HRESIMS data.
Although the NMR data of synthetic 1 were in close agreement
with those of 1 produced by Streptomyces sp. SpD081030SC-03,
there was a minute difference in the chemical shifts of the Ala
residuebetweenthe synthetic and natural product(see Supporting
Information, Table S1). Thus, a mixture (1:1) of synthetic and
natural products was analyzed by NMR. The NMR data showed
that the chemical shifts of the synthetic and natural product were
indistinguishable. In addition, other resulting spectroscopic data,
Because 1 was not composed of typical amino acid units and
this skeleton is the first example in both natural and synthetic
compounds, we confirmed its structure by the total synthesis of 1.
The synthetic route for 1 is illustrated in Scheme 1. Initially,
monoalkylation of a 2,6-dichloropyrazine (4) and the subsequent
introduction of a benzyloxy group at the C-2 position were
conducted to afford 2-benzyloxy-6-ethylpyrazine (6).15 N-Oxi-
dation at the C-4 position, followed by chlorination, gave a
mixture of regioisomers 8a and 8b. These isomers could be
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including the (ꢀ) sign and magnitude of the specific rotation of
synthetic 1, were identical to those of natural 1. On the basis of
these findings, we validated the structure of 1.
subsequently applied to a Diaion HP-20 column, and the column was
then washed with 20% aqueous MeOH and eluted with 100% MeOH.
After evaporation of the 100% MeOH eluent in vacuo, the resultant
residue (611 mg) was subjected to reversed-phase MPLC (Purif-Pack
ODS-100) using an aqueous MeOH linear gradient system (20ꢀ100%
MeOH), and fractions including major metabolites were collected by
LC-MS monitoring. The 80% MeOH eluate (9.32 mg) was then
subjected to preparative reversed-phase HPLC using an L-column2
ODS column (20 i.d. ꢁ 150 mm) developed with 65% aqueous MeOH
containing 0.1% formic acid (flow rate 10 mL/min) to give JBIR-56
(1, tR = 13.7 min, 2.41 mg) and JBIR-57 (2, tR = 11.3 min, 0.21 mg).
JBIR-56 (1): colorless oil; [R]25D ꢀ11 (c 0.1, MeOH); UV (MeOH)
’ CONCLUSION
We isolated two new peptides, JBIR-56 (1) and JBIR-57 (2),
from the culture broth of a new sponge-derived species of
Streptomyces, SpD081030SC-03. The methylated derivative (3)
of 1 helped to confirm the part of these compounds that was
most intriguing structurally, the 3,5,6-trisubstituted 2(1H)-pyr-
azinone moiety. Although 3,6-disubstituted 2(1H)-pyrazinone
derivatives with an isopropyl group at the C-3 position such as
argvalin17 and phevalin18 have been isolated from Streptomyces
spp., there have been no reports of 2(1H)-pyrazinones with a
peptide chain connected at the C-5 position, as in 1. Accordingly,
the unique structures of 1 and 2 may have been biosynthesized by
a novel nonribosomal peptide synthetase. Thus, investigations of
the detailed biosynthesis of 1 are now underway.
λmax (log ε) 262 (3.97), 312 (3.66) nm; IR (KBr) νmax 3380, 1660 cmꢀ1
;
1H NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6),
see Table 1; HRESIMS m/z 395.2294 [M + H]+ (calcd for
C19H31N4O5, 395.2294).
JBIR-57 (2): colorless oil; [R]25D ꢀ12 (c 0.1, MeOH); UV (MeOH)
λmax (log ε) 263 (4.00), 310 (3.67) nm; IR (KBr) νmax 3380, 1650 cmꢀ1
;
1H NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz, DMSO-d6)
see Table 1; HRESIMS m/z 381.2130 [M + H]+ (calcd for
C18H29N4O5, 381.2138).
’ EXPERIMENTAL SECTION
Methylation of 1. Compound 1 (4.0 mg) from a large-scale culture
(4 L) was dissolved in 400 μL of DMF and mixed with 0.4 mg of K2CO3 and
50 μL of methyl iodide. The solution was then allowed to stand at 50 ꢀC for
12 h. Next, the mixture was diluted with MeOH, evaporated in vacuo, and
subjected to preparative reversed-phase HPLC using a PEGASIL ODS
column(20i.d.ꢁ 150 mm) developed with 70% aqueous MeOH (flow rate
10 mL/min) to give 1-methyl-JBIR-56 methyl ester (3, tR = 11.6 min,
2.2 mg). The structure of 3 was confirmed by LC-MS and NMR analyses:
colorless oil; UV (MeOH) λmax (log ε) 266 (3.98), 322 (3.68) nm; 1H
NMR (600 MHz, CDCl3) δH 8.17 (1H, d, J = 7.2 Hz, 20-NH), 6.94 (1H, d,
J = 7.1 Hz, 200-NH), 4.57 (1H, m, H-200), 4.52 (1H, m, H-20), 3.70 (3H, S,
100-O-CH3), 3.60 (3H, s, 1-N-CH3), 3.42, 3.34 (2H, m, H-11), 3.41 (1H, m,
H-7), 1.71 (1H, m, H-40), 1.85, 1.67 (2H, m, H-30), 1.41 (3H, d, J = 7.2 Hz,
H-300), 1.30 (3H, t, J = 7.2 Hz, H-12), 1.23 (3H, d, J = 6.5 Hz, H-8), 1.22
(3H, d, J = 6.5 Hz, H-9), 0.98 (3H, d, J = 6.5 Hz, H-50), 0.95 (3H, d, J = 6.5
Hz, H-60); 13C NMR (150 MHz, CDCl3) δC 173.5 (C, C-100), 171.9 (C,
C-10), 165.4 (C, C-10), 158.4 (C, C-3), 156.4 (C, C-2), 147.2 (C, C-6),
121.7 (C, C-5), 52.7 (CH3, 100-O-CH3), 51.9 (CH, C-20), 48.3 (CH, C-200),
40.5 (CH2, C-30), 30.9 (CH, C-7), 30.6 (CH3, 1-N-CH3), 25.3 (CH, C-40),
23.3 (CH3, C-50), 22.4 (CH2, C-11), 22.2 (CH3, C-60), 20.2 (CH3, C-8),
20.1 (CH3, C-9), 18.6 (CH3, C-300), 12.9 (CH3, C-12); HRESIMS m/z
423.2601 [M + H]+ (calcd for C21H35N4O5, 423.2607).
General Experimental Procedures. Optical rotations were mea-
sured using a Horiba SEPA-300 polarimeter. UV and IR spectra were
measured with a Beckman Coulter DU730 UV/vis spectrophotometer and
a Horiba FT-720 spectrophotometer, respectively. NMR spectra were
collected using a JEOL model EX-270, JEOL model ECP-400, or a Varian
NMR System 600 NB CL in DMSO-d6 (δC 39.7, δH 2.49 ppm), CD3OD
(δC 49.3, δH 3.30 ppm), or CDCl3 (δC 77.0, δH 7.25 ppm), with the residual
solvent peak serving as the internal standard. HRESIMS data were recorded
using a Waters LCT-Premier XE mass spectrometer. Reversed-phase MPLC
was conducted on a Purif-Pack ODS-100 column (Shoko Scientific).
Analytical reversed-phase HPLC was conducted using an L-column2 ODS
column (4.6 i.d. ꢁ 150 mm; Chemical Evaluation and Research Institute) in
conjunction with a Waters 2996 photodiode array detector and a Waters
3100 mass detector. Analytical reversed-phase UPLC (Waters, ACQUITY)
was performed using a BEH ODS column (2.1 i.d. ꢁ 50 mm; Waters) in
conjunction with a Waters ACQUITY UPLC photodiode array eλ detector
and an LCT-Premier XE mass spectrometer. Preparative reversed-phase
HPLC was conducted using an L-column2 ODS column (20 i.d. ꢁ 150 mm)
or a PEGASIL ODS (20 i.d. ꢁ 150 mm, Senshu Scientific) in conjunction
with a Hitachi High Technologies L-2455 photodiode array detector. Other
reagents and solvents were of the highest grade available.
Determination of Amino Acid Configuration. A sample of 1
or 2 (1.0 mg) was hydrolyzed in 6 N HCl at 110 ꢀC for 12 h. After
concentration to dryness, the residue was dissolved in 10 mL of
EtOAcꢀH2O (1:1). The amino acid mixture was then recovered in
the aqueous layer. After drying the aqueous layer in vacuo, it was
dissolved in 5% NaHCO3 (500 μL), and FDAA (Marfey’s reagent, 0.2
mg) in acetone (500 μL) was added. The mixture was then heated in an
oil bath at 80 ꢀC for 3 h. The reaction products were subsequently
analyzed by UPLC (Waters, ACQUITY) as follows: Waters BEH ODS
column (2.1 i.d. ꢁ 50 mm, flow rate of 0.3 mL/min) developed with an
aqueous MeCN containing 0.1% formic acid linear gradient system
(10ꢀ100% MeCN, 7 min). The retention times of the FDAA derivatives
were determined by monitoring UV absorption at 340 nm and the
positive mode of ESIMS. The retention times of the standard FDAA
derivatives were as follows: L-Leu 3.37 min, D-Leu 3.73 min, L-Ala 2.47
min, and D-Ala 2.70 min. The retention times of the FDAA derivatives of
1 and 2 are Ala unit (2.47 min) and Leu unit (3.73 min).
Isolation and Identification of the Bacterium. Streptomyces
strain SpD081030SC-03 was isolated from a marine sponge, Demospon-
giae, collected in Ishigaki City, Okinawa Prefecture, Japan. This strain was
isolated using starch casein nitrate agar plates and a simple dilution plating
method.19 To identify the strain, almost complete 16S rRNA gene
sequences were determined (DDBJ accession number AB539002) and
compared to previously published sequences using EzTaxon.13
Fermentation. Streptomyces sp. SpD081030SC-03 was cultivated in
15 mL test tubes that each contained 5 mL of a seed medium consisting of
1.0% starch (Kosokagaku), 1.0% Polypepton (Nihon Pharmaceutical),
1.0% molasses (Dai-Nippon Meiji Sugar), and 1.0% meat extract (Extract
Ehlrich, Wako Pure Chemical Industry) (pH 7.2). The test tubes were
shaken on a reciprocal shaker (355 rpm) at 27 ꢀC for two days. Aliquots
(2.5 mL) of the broth were then transferred to 500 mL baffled Erlenmeyer
flasks containing 100 mL of a production medium consisting of 2.5% starch,
1.5% soybean meal (Nisshin Oillio), 0.2% dry yeast (Mitsubishi Tanabe
Pharma), 0.4% CaCO3 (Kozaki Pharmaceutical), and 1.0% Diaion HP-20
resin (Mitsubishi Chemical) (pH 6.2) and cultured on a rotary shaker
(180 rpm) at 27 ꢀC for five days.
2-Chloro-6-ethylpyrazine (5). A 1.0 M THF solution of ethyl
magnesium bromide (16.1 mL, 16.1 mmol) was slowly added to 2,6-
dichloropyrazine (4) (2.0 g, 13.4 mmol) and iron acetylacetonate
(237 mg, 0.671 mmol) in a mixed solution of THF (50 mL) and
Isolation. The fermentation broth (2 L) was separated into the
mycelial cake and supernatant by centrifugation. The supernatant was
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N-methyl pyrrolidone (NMP, 5.0 mL) at 0 ꢀC. After 30 min, the reaction
was quenched with saturated NaHCO3(aq), and the product was then
extracted with EtOAc three times. The combined organic extracts were
subsequently washed with brine, dried over anhydrous MgSO4, filtered,
and concentrated under reduced pressure. Next, the residue was purified
by column chromatography (hexaneꢀEtOAc) to give 5 (836 mg, 5.86
mmol) in 44% yield as a colorless powder: mp >300 ꢀC; IR (neat) νmax
3057, 2930, 1726, 1520, 1375 cmꢀ1; 1H NMR (270 MHz, CDCl3) δH
8.41(1H, s), 8.36 (2H, s), 2.83 (2H, q, J= 7.6Hz), 1.33 (3H, t, J =7.6 Hz);
13C NMR (67.5 Hz, CDCl3) δC 159.0, 148.6, 141.8, 141.4, 28.2, 13.2;
HRESIMS m/z [M + H]+ 143.0372 (calcd for C6H8ClN2, 143.0376).
2-Benzyloxy-6-ethylpyrazine 4-Oxide (7). A 60% oil disper-
sion of NaH (50 mg, 1.26 mmol) and benzyl alcohol (0.130 mL,
1.26 mmol) in dimethoxyethane (DME, 4.0 mL) were added to a DME
(4.0 mL) solution of the pyrazine 5 (100 mg, 0.701 mmol) at 45 ꢀC. The
mixture was then stirred for 18 h, after which it was quenched with
saturated NaHCO3(aq). The product was subsequently extracted and
purified using the above procedure to give 6 (142 mg, 0.662 mmol) in
94% yield as a colorless oil. Next, sodium perborate monohydrate
(413 mg, 4.14 mmol) was added to an acetic acid (30 mL) solution of
the pyrazine 6 (740 mg, 3.45 mmol) at 80 ꢀC. The mixture was then
stirred for 26 h, after which the reaction mixture was quenched with
saturated NaOH(aq), and the product was extracted and purified using
the procedure described above to give 7 (621 mg, 2.70 mmol) in 78%
yield as a colorless oil: IR (neat) νmax 3446, 3089, 2973, 1599, 1521,
1446 cmꢀ1; 1H NMR (270 MHz, CDCl3) δH 7.70 (1H, s), 7.68 (1H, s),
7.40ꢀ7.43 (2H, m), 7.36ꢀ7.39 (3H, m), 5.42 (2H, s), 2.69 (2H, q, J =
7.6 Hz), 1.29 (3H, t, J = 7.6 Hz); 13C NMR (67.5 Hz, CDCl3) δC 162.5,
158.5, 135.8, 128.5, 128.3, 128.3, 126.4, 120.3, 68.7, 28.4, 12.4; HRE-
SIMS m/z [M + H]+ 231.1129 (calcd for C13H15N2O2, 231.1134).
2-Benzyloxy-3-chloro-6-ethylpyrazine (8a). Phosphorus oxy-
chloride (0.130 mL, 1.37 mmol) was added to a toluene (5.0 mL) and
DMF (0.50 mL) solution at 0 ꢀC. After 15 min, a DMF (3 mL) solution
of pyrazine 7 (158 mg, 0.686 mmol) was added, and the mixture was
stirred for three days at 0 ꢀC. The reaction was then quenched with
saturated NaHCO3(aq). This procedure afforded 8a (113 mg, 0.454
mmol) in 66% yield as a colorless oil. Regioisomer 8b was obtained in
16% yield (27.8 mg, 0.112 mmol) as a colorless oil. 8a: IR (neat) νmax
2974, 2939, 1531, 1413, 1351, 1098 cmꢀ1; 1H NMR (270 MHz, CDCl3)
δH 7.79 (1H, s), 7.48ꢀ7.51 (2H, m), 7.30ꢀ7.42 (3H, m), 5.48 (2H, s),
2.72 (2H, q, J = 7.6 Hz), 1.29 (3H, t, J = 7.6 Hz); 13C NMR (67.5 Hz,
CDCl3) δC 155.0, 154.0, 136.2, 134.6, 133.7, 128.4, 128.1, 128.0, 68.5,
(1H, s), 3.30ꢀ3.38 (1H, m), 2.51 (2H, q, J = 7.8 Hz), 1.23 (1H, t, J = 7.8
Hz), 1.18 (6H, d, J = 6.8 Hz); 13C NMR (100 Hz, CD3OD) δC 160.5,
157.2, 141.3, 120.7, 29.5, 23.3, 19.2, 12.0; HRESIMS m/z [M + H]+
167.1191 (calcd for C9H15N2O, 167.1184).
6-Ethyl-5-iodo-3-isopropyl-2-pyrazinone (11). N-Iodosucci-
nimide (67.0 mg, 0.297 mmol) was added to a DMF (2.0 mL) solution
of 10 (45.0 mg, 0.270 mmol) at room temperature. After the reaction
mixture was stirred for 15 h, the reaction was quenched with H2O
and the product was extracted with EtOAc and concentrated under
reduced pressure. The residue was then purified by preparative TLC
(hexaneꢀEtOAc, 7:3) to give 11 (62.9 mg, 0.215 mmol) in 80% yield as
a white powder: mp 167 ꢀC; IR (neat) νmax 2946, 1869, 1636, 1446,
1276 cmꢀ1; 1H NMR (270 MHz, CD3OD) δH 3.20ꢀ3.34 (1H, m), 1.19
(1H, t, J = 7.6 Hz), 1.17 (6H, d, J = 6.6 Hz); 13C NMR (100 Hz,
CD3OD) δC 161.5, 158.5, 146.0, 90.1, 31.0, 30.2, 20.5, 13.0; HRESIMS
m/z [M + H]+ 293.0153 (calcd for C9H14IN2O, 293.0151).
6-Ethyl-3-isopropyl-2-pyrazinon-5-ylcarboxylic Acid (13).
A 1.67 M hexane solution of n-butyllithium (0.260 mL, 0.428 mmol) was
added to a THF solution (3.0 mL) of 11 (50.0 mg, 0.171 mmol)
at ꢀ78 ꢀC. After 20 min, methyl choloroformate (32.0 μL, 0.410 mmol)
was added, and the mixture was stirred for 25 min at ꢀ78 ꢀC. The
reaction was then quenched with saturated NH4Cl(aq), after which the
product was extracted with EtOAc. The organic extract was subse-
quently washed with brine, dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure. The crude mixture was then used
without purification because it was difficult to separate the desired ester
moiety 12 from the protonated byproduct 10. A 1.0 M sodium
hydroxide solution (2.0 mL, 2.00 mmol) was then added to the crude
mixture containing 12 (45.4 mg) at reflux temperature. After 9 h, the
reaction was quenched with 1.0 M hydrochloric acid. The product was
then extracted with EtOAc, concentrated under reduced pressure, and
purified by preparative TLC (CHCl3ꢀMeOH, 9:1) to give carboxylic
acid 13 (16.2 mg, 0.0771 mmol) in 60% yield (2 steps) as a white
powder: mp 244 ꢀC; IR (neat) νmax 2967, 1655, 1562, 1366 cmꢀ1; 1H
NMR (270 MHz, CD3OD) δH 3.26 (1H, sep, J = 1.6 Hz), 2.91 (2H, q,
J = 7.8 Hz), 1.18ꢀ1.23 (9H, m); 13C NMR (100 Hz, CD3OD) δC 169.9,
160.9, 157.9, 146.6, 125.4, 31.6, 25.0, 20.1, 14.0; HRESIMS m/z
[M + H]+ 211.1076 (calcd for C10H15N2O3, 211.1083).
Peptide 15. EDCI HCl (13.7 mg, 0.0714 mmol), 1-hydroxyben-
3
zotriazole hydrate (9.60 mg, 0.0714 mmol), and N,N-diisopropylethy-
lamine (22.0 μL, 0.157 mmol) were added to a DMF (2.0 mL) solution
of 13 (10.0 mg, 0.0476 mmol) at room temperature. After 10 min, H-D-
Leu-Ala-OtBu (14, 15.0 mg, 0.0571 mmol) was added, and the mixture
was stirred for 16 h at room temperature. The reaction was then
quenched with H2O, and the product was extracted with EtOAc,
concentrated under reduced pressure, and subjected to preparative
TLC (hexaneꢀEtOAc, 4:6) to give 15 (16.7 mg, 0.0371 mmol) in
74% yield as a white powder: mp 172 ꢀC; IR (neat) νmax 3299, 2935,
1650, 1731, 1506, 1370 cmꢀ1; 1H NMR (400 MHz, CD3OD) δH 4.57
(1H, t, J = 6.4 Hz), 4.24 (1H, q, J = 7.8 Hz), 3.29ꢀ3.31 (1H, m), 3.05
(2H, q, J = 7.8 Hz), 1.68ꢀ1.69 (3H, m), 1.42 (9H, s), 1.35 (3H, d, J =
7.3 Hz), 1.22ꢀ1.26 (9H, m), 0.97 (6H, t, J = 5.8 Hz); 13C NMR
(100 Hz, CD3OD) δC 174.5, 173.3, 165.9, 160.6, 158.0, 147.4, 82.6,
52.8, 50.4, 43.0, 31.1, 28.2, 26.3, 24.6, 23.4, 22.5, 20.2, 17.3,14.1;
HRESIMS m/z [M + H]+ 451.2920 (calcd for C23H39N4O5, 451.2920).
Synthesis of 1. TFA (9.0 mL) was added to 1.0 mL of an aqueous
solution of 15 (16.7 mg, 0.0371 mmol) at room temperature. After 16 h,
the TFA and H2O were removed by reduced pressure. The residue was
then purified by preparative TLC (CHCl3ꢀMeOH, 9:1) to give 1 (14.6
mg, 0.0370 mmol) in quantitative yield as a white powder: colorless oil;
[R]25D ꢀ11 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 262 (3.97), 312
(3.66) nm; 1H NMR (600 MHz, DMSO-d6) and 13C NMR (150 MHz,
DMSO-d6) see Supporting Information (Table S1); HRESIMS m/z
[M + H]+ 395.2285 (calcd for C19H31N4O5, 395.2294).
27.4, 13.1; HRESIMS m/z [M
+
H]+ 249.0790 (calcd for
C13H13ClN2O, 249.0795). 8b: IR (neat) νmax 2977, 2939, 1538, 1392,
1
1325, 1196 cmꢀ1; H NMR (270 MHz, CDCl3) δH 7.88 (1H, s),
7.44ꢀ7.47 (2H, m), 7.30ꢀ7.40 (3H, m), 5.39 (2H, s), 2.88 (2H, q, J =
7.6 Hz), 1.30 (3H, t, J = 7.6 Hz); 13C NMR (67.5 Hz, CDCl3) δC 158.4,
152.8, 138.8, 136.3, 131.1, 128.5, 128.3, 128.2, 68.4, 27.7, 11.5; HRE-
SIMS m/z [M + H]+ 249.0789 (calcd for C13H14ClN2O, 249.0795).
6-Ethyl-3-isopropyl-2-pyrazinone (10). A 0.75 M THF solu-
tion of isopropylmagnesium bromide (5.20 mL, 3.88 mmol) was slowly
added to pyrazine 8a (483 mg, 1.94 mmol) and iron acetylacetonate
(34.3 mg, 0.0970 mmol) in a mixed solution of THF (20 mL) and NMP
(2.0 mL) at 0 ꢀC. After 16 h, the reaction was quenched with 1.0 M
HCl(aq) and the product was extracted and purified using the above
procedure to give 9 (417 mg, 1.62 mmol) in 51% yield as a colorless oil.
Palladium on carbon (25.0 mg) was added to a MeOH (4.0 mL) solution
of the pyrazine 9 (100 mg, 0.390 mmol) under a H2 atmosphere at room
temperature. The mixture was stirred for 1 h, after which the reaction
mixture was filtered through a short pad of Celite, and the filtrate was
concentrated under reduced pressure. The residue was then purified by
preparative TLC (hexaneꢀEtOAc, 7:3) to give 10 (57.3 mg, 0.345
mmol) in 88% yield as a white powder: mp 92ꢀ94 ꢀC; IR (neat) νmax
2974, 2085, 1460, 1219 cmꢀ1; 1H NMR (400 MHz, CD3OD) δH 7.16
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dx.doi.org/10.1021/np200386c |J. Nat. Prod. 2011, 74, 1630–1635

Journal of Natural Products
ARTICLE
’ ASSOCIATED CONTENT
S
Supporting Information. NMR spectra of compounds
b
1, 2, and synthetic 1. This material is available free of charge via
the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: +81-3-3599-8304. Fax: +81-3-3599-8494. E-mail:
motoki-takagi@aist.go.jp (M.T.). Tel: +81-3-3599-8854. Fax:
+81-3-3599-8494. E-mail: k-shinya@aist.go.jp (K.S.).
’ ACKNOWLEDGMENT
This work was supported by a grant from the New Energy and
Industrial Technology Department Organization (NEDO). The
authors thank EIWEISS Chemical Corporation for generously
providing EDCI HCl .
3
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