Diketopiperazine derivatives from the marine-derived actinomycete Streptomyces sp. FXJ7.328

Article abstract of DOI:10.3390/md11041035

Five new diketopiperazine derivatives, (3Z,6E)-1-N-methyl-3-benzy lidene-6-(2S-methyl-3-hydroxypropylidene)piperazine-2,5-dione (1), (3Z,6E)-1-N-methyl-3-benzylidene-6-(2R-methyl-3-hydroxypropylidene)piperazine-2, 5-dione (2), (3Z,6Z)-3-(4-hydroxybenzylid

Full text of DOI:10.3390/md11041035

Mar. Drugs 2013, 11, 1035-1049; doi:10.3390/md11041035
OPEN ACCESS
Marine Drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Communication
Diketopiperazine Derivatives from the Marine-Derived
Actinomycete Streptomyces sp. FXJ7.328
1
2
1
1
1
2,
Pei Wang , Lijun Xi , Peipei Liu , Yi Wang , Wei Wang , Ying Huang * and
1
,
Weiming Zhu *
1
Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and
Pharmacy, Ocean University of China, Qingdao 266003, China;
E-Mails: wangpei850212@163.com (P.W.); liupeipei@ouc.edu.cn (P.L.);
wangyi0213@hotmail.com (Y.W.); wwwakin@ouc.edu.cn (W.W.)
2
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of
Sciences, Beijing 100101, China; E-Mail: xilijun1002@163.com
*
Authors to whom correspondence should be addressed; E-Mails: weimingzhu@ouc.edu.cn (W.Z.);
huangy@im.ac.cn (Y.H.); Tel./Fax: +86-532-82031268 (W.Z.); +86-10-64807311 (Y.H.).
Received: 16 January 2013; in revised form: 6 March 2013 / Accepted: 6 March 2013 /
Published: 28 March 2013
Abstract: Five new diketopiperazine derivatives, (3Z,6E)-1-N-methyl-3-benzy
lidene-6-(2S-methyl-3-hydroxypropylidene)piperazine-2,5-dione (1), (3Z,6E)-1-N-methyl-
3
-benzylidene-6-(2R-methyl-3-hydroxypropylidene)piperazine-2,5-dione (2), (3Z,6Z)-3-
4-hydroxybenzylidene)-6-isobutylidenepiperazine-2,5-dione (3), (3Z,6Z)-3-((1H-imidazol-
-yl)-methylene)-6-isobutylidenepiperazine-2,5-dione (4), and (3Z,6S)-3-benzylidene-6-
2S-but-2-yl)piperazine-2,5-dione (5), were isolated from the marine-derived actinomycete
Streptomyces sp. FXJ7.328. The structures of 1–5 were determined by spectroscopic
analysis, CD exciton chirality, the modified Mosher’s, Marfey’s and the C Marfey’s
(
5
(
3
methods. Compound 3 showed modest antivirus activity against influenza A (H1N1) virus
with an IC50 value of 41.5 ± 4.5 μM. In addition, compound 6 and 7 displayed potent
anti-H1N1 activity with IC50 value of 28.9 ± 2.2 and 6.8 ± 1.5 μM, respectively. Due to the
1
3
lack of corresponding data in the literature, the C NMR data of (3Z,6S)-3-benzylidene-6-
isobutylpiperazine-2,5-dione (6) were also reported here for the first time.
Keywords: Streptomyces; diketopiperazine derivatives; antivirus activity; H1N1

Mar. Drugs 2013, 11
. Introduction
Marine actinomycetes are a rich source of bioactive compounds with new structures [1], e.g., the
1036
1
cytotoxic thiocoraline from Micromonospora sp. L-13-ACM2-092 [2], and the anti-inflammatory
cyclomarins A–C from Streptomyces sp. CNB-382 [3]. As one of the most important classes of
bioactive compounds, alkaloids have received much attention. As part of our ongoing research on
bioactive alkaloids with new structures from marine-derived actinomycetes [4–7], Streptomyces sp.
FXJ7.328 was isolated from marine sediment, and was found to produce alkaloids by TLC visualizing
with Dragendorff’s reagent in a saline culture. The ethyl acetate extract of fermentation broth exhibited
antivirus activity against H1N1 influenza virus at 100 μg/mL and displayed a series of peaks with UV
absorptions at 220 and 340 nm similar to those of diketopiperazine derivatives such as albonoursin and
(
3Z,6S)-3-benzylidene-6-isobutylpiperazine-2,5-dione by HPLC-UV analysis [8]. Chemical investigation
of the extract resulted in the isolation and identification of five new diketopiperazine derivatives
Figure 1), namely (3Z,6E)-1-N-methyl-3-benzylidene-6-(2S-methyl-3-hydroxypropylidene)-piperazine-
(
2
2
,5-dione (1), (3Z,6E)-1-N-methyl-3-benzylidene-6-(2R-methyl-3-hydroxypropylidene)-piperazine-
,5-dione (2), (3Z,6Z)-3-(4-hydroxybenzylidene)-6-isobutylidenepiperazine-2,5-dione (3), (3Z,6Z)-3-
(
(
(1H-imidazol-5-yl)methylene)-6-isobutylidenepiperazine-2,5-dione (4), and (3Z,6S)-3-benzylidene-6-
2S-but-2-yl)piperazine-2,5-dione (5). In addition, five known analogues were also isolated and their
structures were identified as (3Z,6S)-3-benzylidene-6-isobutylpiperazine-2,5-dione (6) [8], albonoursin
7) [8–10], (3Z,6E)-1-N-methyl-3-benzylidene-6-isobutylidenepiperazine-2,5-dione (8) [9,11], (3Z,6S)-
-benzylidene-6-isopropylpiperazine-2,5-dione (9) [12], and (3E,6E)-1-N-methyl-3-benzylidene-6-
(
3
isobutylidenepiperazine-2,5-dione (10) [13], respectively, by comparing their NMR data and specific
rotation (Supplementary Information) with those reported in the literatures. Compounds 3, 6 and 7
displayed activity against influenza A (H1N1) virus with the IC50 values of 41.5 ± 4.5, 28.9 ± 2.2 and
6
.8 ± 1.5 μM, respectively.
Figure 1. Chemical structures of compounds 1–10 from Streptomyces sp. FXJ7.328.
O
O
O
13
11
13
11
1
3
11
9
N
1
1
N
3
NH
1
3
1
3
17 HN
5
15
HN
7
5
1
7
HN
5
10
15
1
R 15
7
7
7
O
R
O
R
O
0
10
9
2: R=OH
9
3
: R=OH; 7: R=H
1
1
1
1
: R=OH; 8: R=H
a: R=S-MPTA
b: R=R-MPTA
2
2
2
a: R=S-MPTA
b: R=R-MPTA
1
5
c: R=p-BrC H CO (unprepared)
6
4
2
c: R=p-BrC H CO
O
6
4
2
13
17
3
13
11
O
O
NH
1
O
3
13
11
11
1
1
N
1
1
3
15
HN
17
5
R1
9
NH
NH
3
1
7
3
1
HN
5
N
15
17 HN
5
7
O
R2
7
NH HN
5
15
9
7
10
O
6
: R =H R =
1
2
O
O
10
10
9
4
5
9
: R =R =CH
3
10
1
2

Mar. Drugs 2013, 11
1037
2
. Results and Discussion
2
.1. Structure Elucidation
The ethyl acetate extract of the fermentation broth of Streptomyces sp. FXJ7.328 was subjected to
extensive chromatographic separations over silica gel, Sephadex LH-20 and by HPLC to yield the new
compounds 1–5 and the known analogues 6–10.
Compounds 1 and 2 were at first isolated as a racemic mixture with an excess of one enantiomer
and thought to be a pure compound according to their identical NMR data and a small negative
specific rotation ([α] −5.8). When (R)-Mosher’s acyl chloride was used to determine the absolute
D
1
configuration, the Mosher’s ester was found to be a separable mixture according to H NMR spectrum
and HPLC profile. A chiral HPLC column was used to separate the enantiomeric mixture of 1 and 2,
which yielded compounds 1 and 2 in an approximate ratio of 2:1 (Table S37). The planar structures of
1
and 2 were established based on MS and NMR data of the racemic mixture. HRESIMS gave an ion
+
peak at m/z 287.1387 [M + H] (calcd. for C H N O 287.1390), corresponding to the molecular
16
19
2
3
18 2 3
formula C16H N O . Analysis of the 1D NMR data (Table 1) revealed one methyl (δH/C 1.01/17.9),
3
2
one N-methyl (δH/C 3.17/31.3), one sp oxygenated methylene (δH/C 3.36/66.7), seven sp methines, one
3
sp methine (δH/C 3.59/34.9), and two signals (δ
C
158.5, 158.9) interpreted as conjugated amide
1
carbonyls. H NMR signals at δ
H
7.52 (2H, d, J = 7.7), 7.40 (2H, t, J = 7.7) and 7.31 (1H, t, J = 7.2)
revealed the presence of a monosubstituted benzene nucleus that was further identified as a
dehydro-phenylalanine (deh-Phe) unit from the key HMBC correlations of H-13/17 (δ 7.52) to C-11
116.1) and C-15 (δ 128.7), and of H-11 (δ 6.75) to C-13 (δ 129.9) and C-2 (δ
COSY correlations of H-7/H-8/H-9 and H-8/H-10 combined with the key HMBC correlations from
H-7 (δ 5.56) to C-5 (δ 158.9) and C-6 (δ 130.4), and from N-CH (δ 3.17) to C-6 (δ 130.4)
suggested another amino acid unit, N-methyl dehydro-5-hydroxyleucine. The key HMBC correlation
H
1
1
(
δ
C
C
H
C
C
158.5). H- H
H
C
C
3
H
C
of N-CH
3
to C-2 finally established the conjugate diketopiperazine (DKP) structure of 1 and 2 (Figure 2).
6
The NOE correlation from N-CH
3
to H-7 indicated the E-configuration of the Δ double bond. The
3
,11
Z-configuration of Δ
because of the deshielding effect of the 2-carbonyl group, as e.g., δH-11 6.85, 6.40 and 6.74 for
3Z,6E)-1-N-methyl-3-benzylidene-6-isobutylidenepiperazine-2,5-dione [11], (3E,6E)-1-N-methyl-3-
double bond could be deduced from the relative downfield shift of H-11
(
benzylidene-6-isobutylidenepiperazine-2,5-dione [13], and (3Z,6Z)-3-benzylidene-6-isobutylidene
piperazine-2,5-dione [10], respectively. The S-configuration of 1 was determined by analysis of
exciton chirality CD of its p-bromobenzoate (1c) [14]. The stable conformers of the p-bromobenzoates
of 1c and its enantiomer 2c (unprepared) were obtained by HyperChem Release 7.5 software [4]
(Figure 3). The transition dipole orientations of two chromophores, the conjugated DKP core and the
p-bromobenzoate in 1c and 2c, were oriented in counterclockwise and clockwise manners, which
should result in negative and positive Cotton effects at long wavelength, respectively. The measured
negative CD effect of 1c at λext 313 nm (Δε −5.2) indicates the S-configuration of 1c (Figure 3). This
deduction was further validated by modified Mosher’s method for primary alcohols [15,16]. When 1
reacted with R- and S-MTPA chloride, the S- (1a) and R-MTPA esters (1b) were obtained,
respectively. The chemical shift difference between two methylene protons of C-9 in S-MTPA ester 1a
is larger than that in R-MTPA ester 1b (Δδ 0.08 vs. 0.01), indicating S-configuration of C-8 in 1. Thus,

Mar. Drugs 2013, 11
1038
the structure of 1 was clearly elucidated as (3Z,6E)-1-N-methyl-3-benzylidene-6-(2S-methyl-3-hydoxy
propylidene)-piperazine-2,5-dione. Compound 2 showed the opposite specific rotation and the opposite
chemical shift difference between the two methylene protons of C-9 in S- and R-MTPA esters (2a and 2b),
indicating R-configuration of 2. So compound 2 was identified as (3Z,6E)-1-N-methyl-3-benzylidene-
6
-(2R-methyl-3-hydroxypropylidene)piperazine-2,5-dione.
1
13
Table 1. H and C NMR Data for 1–5 (600 and 150 MHz, DMSO-d , δ values).
6
1
and 2
3
4
5
Position
δH, mult.
δH, mult.
(J in Hz)
10.24, s
δH, mult.
(J in Hz)
δH, mult.
(J in Hz)
8.47, s
δ
C
, type
δ
C
, type
δ
C
, type
C
δ , type
(
J in Hz)
1
2
3
4
5
6
7
8
9
31.3, NCH
158.5, qC
126.5, qC
3
3.17, s
158.0, qC
125.3, qC
157.8, qC
125.2, qC
161.0, qC
127.3, qC
9.82, s
9.93, s
158.9, qC
130.4, qC
157.4, qC
125.2, qC
156.8, qC
125.9, qC
166.8, qC
60.2, CH 3.86, t, (3.3)
40.9, CH 1.80, m
129.7, CH 5.56, d, (9.9)
125.1, CH 5.66, d, (10.4)
23.9, CH 2.93, m
125.5, CH 5.68, d, (9.9)
24.4, CH 2.95, m
34.9, CH
66.7, CH
17.9, CH
3.59, m
3.36
24.8, CH
15.3, CH
12.1, CH
2
3
3
1.46, m; 1.18, m
0.91, d, (7.1)
0.86, t, (7.7)
2
3
22.2, CH
3
3
0.96, d, (6.5)
0.96, d, (6.5)
22.8, CH
3
3
0.97, d, (6.6)
0.97, d, (6.6)
1
0
1
2
1.01, d, (7.1)
22.2, CH
22.8, CH
1
1
116.1, CH 6.75, s
133.9, qC
115.0, CH 6.66, s
123.9, CH
105.1, CH 6.60, s
137.0, qC
114.6, CH 6.66, s
133.9, qC
1
1
3/17 129.9, CH 7.52, d, (7.7)
4/16 129.2, CH 7.40, t, (7.7)
130.9, CH 7.36, d, (8.5)
115.6, CH 6.79, d, (8.5)
157.5, qC
119.8, CH 7.52, s
129.3, CH 7.45, d, (7.7)
129.7, CH 7.39, t, (7.7)
128.5, CH 7.29, t, (7.7)
1
5
128.7, CH 7.31, t, (7.2)
137.1, CH 7.94, s
Figure 2. Selected 2D NMR correlations for 1–5.
Figure 3. The stable conformers of 1c and 2c and the measured CD curve for
p-bromobenzoate 1c.
2
5
0
2
10
0
CD[mdeg]
-10
-
20
25
-
190
300
400
Wavelength [nm]

Mar. Drugs 2013, 11
1039
The molecular formula of compound 3 was determined to be C H N O based on HRESIMS with
1
5
16
2
3
+
a peak at m/z 273.1231 [M + H] (calcd. for C15
H
17
N
2
O
3
273.1234). The similarity of the 1D NMR
1
spectra to those of albonoursin (7) [8,9] indicated a conjugated DKP as well. The difference of H and
1
3
C NMR data between 3 and 7 pointed to a p-hydroxy substituted phenyl system in 3 instead of the
benzene ring in 7, which explains the obvious upfield shifts of H-13/17 (δ
H
1
7.36), H-14/16 (δ
H
6.79)
1
and C-14/16 (δ
C
115.6) by the electron-donor effect of the hydroxy group. H- H COSY correlations
between H-13/17 and H-14/16 and the key HMBC correlations from H-11 (δ 6.66) to C-13/17
H
C C C C
(δ 130.9) and C-2 (δ 158.0), from H-13/17 to C-15 (δ 157.5), and from H-14/16 to C-12 (δ 123.9)
1
1
further supported the existence of a dehydro-tyrosine (deh-Tyr) unit. The H- H COSY correlations
from H-7 (δ 5.66) to H-9/10 (δ 0.96) through H-8 (δ 2.93) along with the HMBC correlations from
H
H
H
C
H-7 to C-5 (δ 157.4) supported the existence of a dehydro-leucine (deh-Leu) unit. The NOE
3
,11
correlation between H-4 (δ 9.82) and H-13/17 indicated the Z-configuration of the Δ double bond.
H
The NOE correlations of H-1 (δ 10.24) to H-8 and H-9/10 combined with the relative downfield shift
H
6
of H-7 revealed Z-configuration of the Δ double bond. Therefore, compound 3 was determined to be
(
3Z,6Z)-3-(4-hydroxybenzylidene)-6-isobutylidenepiperazine-2,5-dione.
Compound 4 was found to have the molecular formula of C H N O from the HRESIMS peak at
1
2
14
4
2
+
m/z 247.1189 [M + H] (calcd. for C12
H
15
N
4
O
2
247.1190). 1D NMR (Table 1) and 2D NMR (Figure 2)
2
data disclosed the same Z-deh-Leu unit as in compound 3. The remainder C
6
H
5
N
3
O displayed three sp
2
methine signals at δH/C 6.60/105.1, 7.94/137.1 and 7.52/119.8, two sp quaternary carbon signals at δ
C
1
25.2 and 125.9, and one amide carbonyl signal at δ
to C-13 (δ 119.8) and C-12 (δ 137.0), of H-13 (δ
C-2 (δ
C
157.8. The HMBC correlations of H-15 (δ
H
7.94)
C
C
H
7.52) to C-15 (δ 137.1), and of H-11 (δ 6.60) to
C
H
C
157.8) and C-12 suggested a dehydro-histidine (deh-His) unit. The Z-configurations of both
3
,11
6
Δ
and Δ double bonds were deduced from the relative downfield shifts of H-11 and H-7 consistent
H
with those of (3Z,6R)-3-((1H-imidazol-5-yl)methylene)-6-isopropylpiperazine-2,5-dione (δ 6.51) [17]
and compound 3, respectively. Compound 4 was therefore elucidated as (3Z,6Z)-3-((1H-imidazol-5-yl)
methylene)-6-isobutylidenepiperazine-2,5-dione.
The molecular formula of C15
H
18
19
N
2
O
2
was assigned to 5 according to the HRESIMS peak at m/z
259.1441), indicating an isomer of 6. The 1D NMR (Table 1)
+
2
59.1439 [M + H] (calcd. for C15
H
N
2
O
2
spectra were very similar to those of 6 [8] except for the leucine moiety signals, suggesting that a
deh-Phe unit was also presented in the structure of 5. The NOE correlation from H-4 to H-13/17 and
3
,11
the relative downfield shift of H-11 accounted for the Z-configuration of the Δ double bond [8]. The
1
main differences of the H NMR spectra are a methyl triplet in 5 replacing the methyl doublet in 6;
further, a distinct split methylene signal in 5 substitutes the overlapped methylene proton signal in 6.
These observations combined with the separate downfield and upfield shifts of methine and methylene
carbon signals revealed the existence of an isoleucine moiety in 5, which was further confirmed by the
1
1
H- H COSY correlations of H-1/H-6/H-7/H-8/H-9 and H-7/H-10 (Figure 2) and the key HMBC
correlation of H-10 to C-6. The absolute configuration of the isoleucine moiety was determined by
Marfey’s method [18] combined with C Marfey’s method [19,20]. The 1-fluoro-2,4-dinitrophenyl-5-
3
L-alanine amide (FDAA) derivatives of the acid hydrolysates of 5 and four authentic isoleucine
samples (L-, L-allo-, D- and D-allo-) were prepared. HPLC analysis over ODS column (Figure S38)
revealed that acid hydrolysates of 5 displayed the same retention time (t
R
23.06 min) as the authentic
28.11 min) and
L-Ile (t 23.06 min) and L-allo-Ile (t 23.06 min) but were different from D-Ile (t
R
R
R

Mar. Drugs 2013, 11
1040
D-allo-Ile (t
and L-allo-Ile were further analyzed by C
hydrolysates of 5 was the same as for the authentic L-Ile (t
R
28.11 min). The FDAA derivatives of the acid hydrolysates of 5 and the authentic L-Ile
HPLC column (Figure S39). The retention time of the acid
38.50 min), but different from the
3
R
authentic L-allo-Ile (t 37.49 min). Thus, the isoleucine moiety in 5 was unambiguously identified as
R
L-Ile, and the structure of compound 5 was elucidated as (3Z,6S)-3-benzylidene-6-(2S-but-2-yl)
piperazine-2,5-dione.
2
.2. The Postulated Biosynthesis Pathway of Compounds 1–10
Compounds 1–10 were postulated to be produced biogenetically from the amino acid pathway
(
Figure 4). Cyclic condensation between Phe and Leu formed cyclo(Phe-Leu) that underwent
dehydrogenation in Phe moiety to form compounds 6 and 7. The N-methylation of 6 produced an
un-isolated intermediate (a) that further underwent another dehydrogenation in the Leu moiety to form
compound 8. The oxidation of 8 at the same homoallylic positions produced compounds 1 and 2. By a
similar biosynthetic pathway, compounds 3–5 and 9 were produced from the cyclic condensation and
successive dehydration of Tyr with Leu, His with Leu, Phe with Ile, and Phe with Val, respectively.
The dehydrogenation is favored to form Z-products because of the steric hindrance of the carbonyl
oxygen, while the dehydrogenation of Leu moiety in the N-Me substituted compounds only produced
the E-products due to the strong steric hindrance by N-Me. So, compound 10 could be an artifact
produced from the photo-isomerization of compound 8 under light during the extraction and the
subsequent isolation steps.
Figure 4. The postulated biosynthetic pathway of 1–3 and 5–8.
3
: R =OH, R =H, R =Me
1 2 3
NH2 R3
7
: R =R =H, R =Me
1
2
3
O
O
HO C
2
[
-H]
O
R2
CO H
2
NH R3
[-H]
HN
NH R3
NH2
R1
R1
HN
R2
R1
R =H: Phe; R =OH: Tyr
1
1
O
R2
5
6
: R =R =H, R =Me
1 3 2
O
[Me]
: R =R =H, R =Me
1
2
3
[-H]
N
[O]
R =H
2
R =Me
3
1
1
and 2
8
R =H
HN
O
a
2
.3. The Bioactivities of Compounds 1–10 from Streptomyces sp. FXJ7.328
Compounds 1 and 3–10 were tested for antivirus effects on H1N1 by the CPE inhibition assay [21,22],
separately. The IC50 values of 1, 3–10 and ribavirin (positive control) were 75.5 ± 2.2, 41.5 ± 4.5,
2.6 ± 3.9, 106.5 ± 4.2, 28.9 ± 2.2, 6.8 ± 1.5, 94.5 ± 3.0, 113.8 ± 4.9, 156.6 ± 4.0, and 38.8 ± 1.5 μM,
6
respectively. Except for compounds 3, 6 and 7, the other compounds were inactive (IC50 > 50 μM)
against H1N1 influenza virus, indicating that both (Z)-deh-Phe and Leu or (Z)-deh-Leu moieties are
necessary for anti-H1N1 activity (Figure 5). The dehydrogenation of Leu increases the activity, while
the hydroxylation of deh-Phe or deh-Leu, the inversion of double bonds in deh-Phe and deh-Leu, and

Mar. Drugs 2013, 11
1041
the N -methylation reduce the activity. In addition, the new compounds 1–5 were tested for
1
cytotoxicity against HL-60 and K562 cell lines by MTT method [23], and A549 cell lines by SRB
method [24] and for anti-inflammatory effects by inhibition of LPS-mediated NF-κB transcription
activity in RAW264.7 cells [25]. The antimicrobial activities of compounds 1–10 against Escherichia
coli, Enterobacter aerogenes, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus, and
Candida albicans were also evaluated by 2-fold dilution method [26]. The results (Table S1,
Supplementary Information) showed that new compounds 1–5 did not exhibit cytotoxicity (IC > 100 μM)
50
and anti-inflammatory effects (IC50 > 10 μM); compounds 1–10 did not show antimicrobial effects as
well (MIC > 100 μg/mL). Although dehydro-DKPs have been reported to display diverse bioactivities,
such as inhibition of protein tyrosine kinase [27], cell cycle arrest [28], inhibition of blood platelet
aggregation [29], anti-bacteria [30], antitumor [30] and anti-inflammation [31], the antivirus effect on
H1N1 was reported here for the first time.
Figure 5. The structure-activity relationship (SAR) of compounds 3–7 and 9 for anti-H1N1
viral activity.
O
O
O
NH
HN
H
HN
NH
O
5
9
increase anti-H1N1 activity
O
NH
NH
HN
O
O
6
increase anti-H1N1 activity
HN
O
7
decrease anti-H1N1 activity
O
O
O
HN
N
NH HN
NH
HN
HO
O
3
4
3
. Experimental Section
.1. General Experimental Procedures
Optical rotations were obtained on a JASCO P-1020 digital polarimeter. UV spectra were measured
3
on a Beckman DU 640 spectrophotometer. IR spectra were recorded on a Nicolet Nexus 470
spectrophotometer as KBr disks. CD spectra were collected using a JASCO J-715 spectropolarimeter.
NMR data of 1, 2 and 4–6 were measured on a JEOL JNM-ECP 600 spectrometer, and chemical shifts

Mar. Drugs 2013, 11
1042
were recorded as δ values. NMR spectra of 1a, 1b, 1c, 2a, 2b, 3, and NOESY spectra of all the
compounds were recorded on a Bruker Avance 600 spectrometer. HRESIMS measurements were
taken on a Q-TOF ULTIMA GLOBAL GAA076 LC mass spectrometer. Semipreparative HPLC was
performed using an ODS column (YMC-pak ODS-A, Kyoto, Japan, 10 × 250 mm, 5 μm, 4.0 mL/min)
and chiral separation was performed on chiral column (CHIRALPAK AY-H, Kyoto, Japan,
4
.6 × 150 mm, 0.5 mL/min) by HPLC. Marfey’s analysis and C Marfey’s analysis were implemented
3
using ODS column (YMC-pak ODS-A, 4.6 × 250 mm, 5 μm, 1.0 mL/min) and C column (Agilent
3
3
Zorbax StableBond C , Palo Alto, CA, USA, 4.6 × 150 mm, 5 μm, 1.0 mL/min), respectively. TLC
and column chromatography (CC, 2.5 × 103 cm) were performed on plates precoated with silica gel
GF254 (10–40 μm, Qingdao Marine Chemical Factory, Qingdao, China), and over Sephadex LH-20
(
Amersham Biosciences, Uppsala, Sweden), respectively. Vacuum-liquid chromatography (VLC,
× 40 cm) utilized silica gel (200–300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) and
7
RP-18 (40–63 μm, Merck, Darmstadt, Germany). Sea salt used was made by the evaporation of
seawater collected in Laizhou Bay (Weifang Haisheng Chemical Factory, Shangdong, China). Glucose
(
Shanghai Huixing Biochemical Reagent Co., Ltd., Shanghai, China); beef extract, yeast extract and
peptone (Beinjing Shuangxuan Microbe Culture Medium Products Factory, Beijing, China); soluble
starch (Beijng Aoboxing Universeen Bio-Tech Co., Ltd., Beijing, China); K HPO (Tianjin Kermel
2
4
4
Chemical Reagent Co., Ltd., Tianjin, China); MgSO (Shanghai Chemical Reagent Research Institute,
Shanghai, China), and CaCO (Tianjijn Bodi Chemical Co., Ltd., Tianjin, China).
3
3
.2. Actinomycete Material
The actinomycete strain Streptomyces sp. FXJ7.328 was isolated from coastal sediment collected at
Huanghai beach (E 121.706°, N 39.007°), Dalian, China in January 2009. The sediment sample was
dried at room temperature, suspended in sterile distilled water, serially diluted, heated in a water bath
at 55 °C for 10 min, and spread-plated on oatmeal agar plates (ISP3, medium 3 of the International
Streptomyces Project) [32]. After four weeks of incubation at 28 °C, the strain was purified on yeast
extract-malt extract agar (ISP2, medium 2 of the International Streptomyces Project) [32], and was
identified as a member of the genus Streptomyces on the basis of 16S rRNA gene sequence analysis.
Genomic DNA isolation, PCR amplification of 16S rRNA gene and sequence alignment of the strain
were performed as described previously [33]. Its 16S rRNA gene sequence (GenBank access No.
JF346514) showed 99% similarity with type strains of Streptomyces albus subsp. albus (AB184781),
Streptomyces almquistii (AB184258), Streptomyces flocculus (DQ442498), Streptomyces gibsonii
(
NR_041180) and Streptomyces rangoonensis (NR_041110). The producing strain was prepared on
ISP3 medium and stored in Huang’s Lab at 4 °C.
3
.3. Fermentation and Extraction
The spores of Streptomyces sp. FXJ7.328 were directly cultured in 500 mL Erlenmeyer flasks
containing 150 mL fermentation media consisted of 2% glucose, 0.3% beef extract, 1% yeast extract,
% soluble starch, 1% peptone, 0.05% K HPO , 0.05% MgSO , 0.2% CaCO , and 3.3% sea salt
1
2
4
4
3
(
pH 7.0). The cultures were incubated on a rotatory shaker at 180 rpm at 28 °C for eight days. The
whole fermentation broth (120 L) was divided into three equal parts that were extracted three times

Mar. Drugs 2013, 11
1043
with equal volumes of EtOAc separately. The EtOAc solutions were combined and evaporated under
reduced pressure to give a dark brown gum (32.5 g).
3.4. Purification and Identification
The EtOAc extract (32.5 g) was subjected to SiO VLC eluting with CH Cl -petroleum ether
2
2
2
(0%–100%), and then with MeOH-CH
2 2
Cl (0%–50%), to give nine fractions (Fr.1–Fr.9). Fraction 3
(
3.82 g) was separated into three subfractions by gel filtration over Sephadex LH-20 with
CH OH/CH Cl (1:1). Fraction 3-2 (442 mg) was further subjected to HPLC separation eluting with
3
2
2
7
0% MeOH to yield 7 (5.5 mg, t
R R
= 7.20 min, 0.046 mg/L), 8 (20 mg, t = 11.5 min, 0.17 mg/L), and
1
0 (3 mg, t = 9.5 min, 0.025 mg/L). Fraction 4 (3.2 g) and Fraction 5 (2.78 g) were both separated
R
into five parts (Fr.4-1–Fr.4-5 and Fr.5-1–Fr.5-5) by RP-18 column chromatography, eluting with
CH OH/H O (5%–100%). Fraction 4-2 (512 mg) was further separated by Sephadex LH-20 with
CH OH/CH Cl (1:1) to give four subfractions (Fr.4-2-1–Fr.4-2-4). Compound 3 (7 mg, 0.058 mg/L)
3
2
3
2
2
was purified from the precipitate of Fr.4-2-2 (135 mg) after washing with MeOH, and compound 9
6 mg, t = 7.4 min, 0.05 mg/L) was obtained from Fr.4-2-3 (163 mg) by HPLC purification eluting
(
R
with 60% MeOH. Fraction 4-4 (332.7 mg) was separated by Sephadex LH-20 with CH
1:1) to afford four subfractions (Fr.4-4-1–Fr.4-4-4). Fr.4-4-2 (94 mg) was also submitted to HPLC
purification on ODS column eluting with 70% MeOH to yield a racemic mixture of 1/2 with an excess
of one enantiomer (11 mg, t = 5.59 min, 0.092 mg/L) which showed two peaks (1:2) by HPLC
3 2 2
OH/CH Cl
(
R
analysis on a chiral column (CHIRALPAK AY-H, Figure S37, Supplementary Information). Then the
chiral separation by HPLC (Amylose tris(5-chloro-2-methylphenylcarbamate)) with EtOH yielded 1
(
7 mg, 12.12 min, 0.058 mg/L) and 2 (3 mg, 8.82 min, 0.025 mg/L). Then Fr.4-4-3 (120 mg) was
further subjected to HPLC separation eluted with 60% MeOH to yield 5 (3 mg, t = 10.4 min,
.025 mg/L) and 6 (4.7 mg, t = 11.64 min, 0.039 mg/L). Fraction 5-2 (245 mg) was further purified
by Sephadex LH-20 with MeOH to produce four subfractions (Fr.5-2-1–Fr.5-2-4). Compound 4 (2.5 mg,
R
0
R
t
R
= 14.7 min, 0.021 mg/L) was obtained from Fr.5-2-3 (63 mg) by HPLC with 45% MeOH.
2
5
Compound 1: Yellow solid; UV (MeOH) λmax (log ε): 238 (4.10), 323 (4.51) nm; [α]
−28
D
(
c 0.05, CH OH), IR (KBr) ν 3496, 3208, 3073, 3022, 1675, 1617, 1494, 1454, 1376, 1033, 997,
3
−
max
1
1
13
7
63, 688 cm ; H and C NMR data, Table 1.
2
5
Compound 2: Yellow solid; UV (MeOH) λmax (log ε): 238 (4.10), 323 (4.51) nm; [α]
D
+28
(
3
c 0.05, CH OH), IR (KBr) νmax 3496, 3208, 3073, 3022, 1675, 1617, 1494, 1454, 1376, 1033, 997,
−1
1
13
7
63, 688 cm ; H and C NMR data, Table 1.
Compound 3: Yellow amorphous solid; UV (MeOH) λmax (log ε): 248 (3.75), 347 (4.10) nm; IR
(
KBr) νmax 3183, 3073, 2960, 2867, 1681, 1639, 1606, 1541, 1421, 1359, 1275, 1173, 1024, 998, 829,
−1
1
13
+
7
61 cm ; H and C NMR data, Table 1; HRESIMS m/z 273.1231 [M + H] (calcd. for
273.1234).
15 17 2 3
C H N O
Compound 4: Yellow solid; UV (MeOH) λmax (log ε): 250 (3.71), 347 (4.25) nm; IR (KBr) νmax
−
1
1
13
2
973, 2937, 2879, 1708, 1638, 1577, 1459, 1379, 1273, 1122, 1092, 1025, 960 cm ; H and C NMR
+
data, Table 1; HRESIMS m/z 247.1189 [M + H] (calcd. for C12
H
15
N
4
O
2
247.1190).

Mar. Drugs 2013, 11
1044
2
5
Compound 5: White solid; UV (MeOH) λmax (log ε): 229 (4.05), 302 (4.17) nm; [α] −36 (c 0.66,
D
CH
33, 800, 760, 619 cm ; H and C NMR data, Table 1; HRESIMS m/z 259.1439 [M + H] (calcd. for
C H N O 259.1441).
3
OH), IR (KBr) νmax 3168, 3077, 3042, 2961, 2867, 1680, 1646, 1546, 1426, 1392, 1357, 1092,
−1
1
13
+
9
1
5
19
2
2
2
5
1
(
3Z,6S)-3-Benzylidene-6-isobutylpiperazine-2,5-dione (6): [α]
600 MHz, DMSO-d ): δ 8.58 (1H, s, NH-1), 9.98 (1H, s, NH-4), 3.94 (1H, m, H-6), 1.58 (2H, m,
H-7), 1.80 (1H, m, H-8), 0.89 (3H, d, J = 6.1, H-9/10), 0.88 (3H, d, J = 5.5, H-9/10), 6.67 (1H, s,
D
−54 (c 0.3, DMSO); H NMR
(
6
1
3
H-11), 7.48 (2H, d, J = 7.7, H-13/17), 7.39 (2H, t, J = 7.7, H-14/16), 7.30 (1H, t, J = 7.7, H-15). C
NMR (150 MHz, DMSO-d
6
): δ 161.0 (C
q
, C-2), 127.3 (C
q
, C-3), 168.1 (C
q
, C-5), 54.2 (CH, C-6),
4
4.2 (CH , C-7), 24.1 (CH, C-8), 23.3 (CH , C-9), 22.6 (CH , C-10), 114.9 (CH, C-11), 133.9 (C ,
2
3
3
q
C-12), 129.2 × 2 (CH, C-13/17), 129.8 × 2 (CH, C-14/16), 128.5 (CH, C-15). HRESIMS m/z 259.1456
+
[
M + H] (calcd. for C15
H
19
N
2
O
2
259.1441).
3
.5. Preparation of p-Bromobenzoate (1c) of Compound 1 [14]
Compound 1 (2 mg, 6.99 μmol) was dissolved in 1 mL of CH Cl , and triethylamine (10 μL) and
2
2
p-bromobenzoyl chloride (20 mg, 92.2 μmol) were added. The mixture was stirred for 7 h at room
temperature. Then 2 mL of H O were added and the solution was extracted three times with CH Cl
5 mL each). The CH Cl solutions were combined and evaporated under reduced pressure to give a
gum. p-Bromobenzoate 1c (2 mg, 4.27 μmol, 7.9 min, 61% yield) was obtained by HPLC purification
eluting with 85% (MeOH-H O).
2
2
2
(
2
2
2
2
5
p-Bromobenzoate (1c): Yellow solid; [α]
D
−50 (c 0.1, CH
3
OH); CD (c 0.11, MeOH) λext (Δε)
): δ 3.18 (3H, s, 1-NCH ),
1
3
5
13 (−5.2), 254 (+4.78), 208 (−1.73). H NMR (600 MHz, DMSO-d
6
3
.57 (d, J = 9.5 Hz, H-7), 4.09 (m, H-8), 4.20 (dd, J = 7.3, 10.3 Hz, H-9a), 4.28 (dd, J = 6.0, 10.4 Hz,
-10), 6.71 (s, H-11), 7.53 (2H, d, J = 7.6 Hz, H-13/17), 7.38 (2H, t,
J = 7.5 Hz, H-14/16), 7.30 (t, J = 7.4 Hz, H-15), 7.86 (2H, d, J = 8.5 Hz, H-2′/6′ in p-BrC H CO -),
H-9b), 1.14 (3H, d, J = 6.7 Hz, CH
3
6
4
2
13
7
1
3
.67 (2H, d, J = 8.5 Hz, H-2′/6′ in p-BrC
6
H
4
CO
2
6 3
-). C NMR (150 MHz, DMSO-d ): δ 31.3 (CH ,
-NCH ), 158.5 (C , C-2), 126.0 (C , C-3), 158.8 (C
3
q
q
q
, C-5), 131.1 (C , C-6), 130.4 (CH, C-7),
q
1.4 (CH, C-8), 69.3 (CH , C-9), 17.5 (CH , C-10), 116.6 (CH, C-11), 133.5 (C , C-12), 129.7 × 2
2
3
q
(
CH, C-13/17), 129.1 × 2 (CH, C-14/16), 128.0 (CH, C-15), 165.6 (C
29.4 (C , C-1′ in p-BrC CO -), 132.3 × 2 (CH, C-2′/6′ in p-BrC CO
p-BrC CO -), 127.9 (C
q
, -CO
2
6 4 2
- in p-BrC H CO -),
1
q
6
H
4
2
6
H
4
2
-), 131.5 × 2 (CH, C-3′/5′ in
6
H
4
2
q
, C-4′ in p-BrC
6
H
4
CO
2
-). ESIMS m/z 469.1 and 471.1; HRESIMS m/z
+
79
4
69.0755 [M + H] (calcd. for C23
H
22 BrN
2
O
4
469.0758).
3
.6. Preparation of S-MTPA and R-MTPA Esters 1a, 1b, 2a, and 2b of Compounds 1 and 2 [15,16]
Compound 1 (1 mg, 3.50 μmol) was dissolved in 500 μL of anhydrous pyridine and 4-dimethyl-
aminopyridine (3 mg, 24.6 μmol) and (R)-MTPACl (10 μL) were added. The reaction was stirred for
2 h at room temperature. Then 1 mL of H O was added, and the solution was extracted three times
1
2
2 2 2 2
with CH Cl (5 mL each). After removal of CH Cl under reduced pressure, the residue was purified
by semipreparative HPLC (70% MeOH-H O) to yield (S)-MTPA ester 1a (1.2 mg, 2.39 μmol,
2
R
t = 27.11 min, 68% yield). By the same procedure, (R)-MTPA ester 1b (1.1 mg, 2.19 μmol,

Mar. Drugs 2013, 11
1045
t = 24.74 min, 63% yield), (S)-MTPA ester 2a (0.8 mg, 1.59 μmol, t = 24.74 min, 45% yield) and
R
R
(
R)-MTPA ester 2b (0.9 mg, 1.79 μmol, t
R
= 27.11 min, 51% yield) were obtained from the
reaction of 1 and 2 (1 mg, 3.50 μmol each) with (S)-MTPACl, (R)-MTPACl and (S)-MTPACl
(
10 μL each), respectively.
1
(
S)-MTPA ester 1a: White solid. H NMR (600 MHz, DMSO-d ): δ 3.07 (3H, s, 1-NCH ),
6
3
3
.99 (m, H-8), 1.06 (3H, d, J = 6.8 Hz, H-10), 5.51 (d, J = 9.6 Hz, H-7), 6.76 (s, H-11), 4.36 (dd,
J = 5.6, 10.5 Hz, H-9a), 4.28 (dd, J = 7.4, 10.4 Hz, H-9b). 7.51 (2H, d, J = 7.5 Hz, H-13/17), 7.41 (2H,
+
t, J = 7.6 Hz, H-14/16), 7.32 (t, J = 7.4 Hz, H-15). HRESIMS m/z 525.1612 [M + Na] (calcd. for
C
26
H
25
F
3
N O
2 5
Na 525.1608).
1
(
R)-MTPA ester 1b: White solid. H NMR (600 MHz, DMSO-d ): δ 3.12 (3H, s, 1-NCH ), 3.99
6
3
(m, H-8), 1.07 (3H, d, J = 6.8 Hz, H-10), 5.51 (d, J = 9.6 Hz, H-7), 6.76 (s, H-11), 4.33 (2H, d, J = 6.5 Hz,
H-9), 7.50 (2H, d, J = 7.6 Hz, H-13/17), 7.41 (2H, t, J = 7.6 Hz, H-14/16), 7.32 (t, J = 7.4 Hz, H-15).
+
HRESIMS m/z 525.1613 [M + Na] (calcd. for C H F N O Na 525.1608).
2
6
25
3
2
5
1
(
S)-MTPA ester 2a: White solid. H NMR (600 MHz, DMSO-d
): δ 3.12 (3H, s, 1-NCH ), 3.99
6 3
(m, H-8), 1.07 (3H, d, J = 6.8 Hz, H-10), 5.51 (d, J = 9.6 Hz, H-7), 6.76 (s, H-11), 4.33 (2H, d, J = 6.5 Hz,
H-9), 7.50 (2H, d, J = 7.6 Hz, H-13/17), 7.41 (2H, t, J = 7.6 Hz, H-14/16), 7.32 (t, J = 7.4 Hz, H-15).
+
HRESIMS m/z 525.1607 [M + Na] (calcd. for C H F N O Na 525.1608).
2
6
25
3
2
5
1
(
R)-MTPA ester 2b: White solid. H NMR (600 MHz, DMSO-d
6 3
): δ 3.07 (3H, s, 1-NCH ), 3.98
(
m, H-8), 1.06 (3H, d, J = 6.8 Hz, H-10), 5.51 (d, J = 9.6 Hz, H-7), 6.76 (s, H-11), 4.35 (dd,
J = 5.6, 10.5 Hz, H-9a), 4.27 (dd, J = 7.4,10.4 Hz, H-9b), 7.50 (2H, d, J = 7.5 Hz, H-13/17), 7.41 (2H, t,
+
J = 7.6 Hz, H-14/16), 7.32 (t, J = 7.4 Hz, H-15). HRESIMS m/z 525.1606 [M + Na] (calcd. for
26 25 3 2 5
C H F N O Na 525.1608).
3
.7. Preparation of FDAA Derivatives of the Acid Hydrolysates of 5 and Four Authentic Isoleucine
Samples (L-, L-allo-, D- and D-allo-) and Marfey’s Analysis [18] and C Marfey’s Analysis [19,20]
3
Compound 5 (1 mg, 3.88 μmol) was dissolved in 6 M HCl (1 mL) in a sealed tube and the mixture
was heated at 105 °C for 17 h. Then the solution was cooled and evaporated to dryness. The residue,
L-Ile, L-allo-Ile and D-Ile, D-allo-Ile, was dissolved in H
2
O (250 μL each), respectively. 50 μL of each
(40 μL). The
solution was treated with 200 μL of 1% FDAA in acetone followed by 1.0 M NaHCO
3
reaction was maintained 1 h at 45 °C and then quenched by addition of 2.0 M HCl (10 μL). The
corresponding FDAA derivatives of hydrolysate of 5, L-Ile, L-allo-Ile, D-Ile and D-allo-Ile were
analyzed by ODS HPLC column maintained at 30 °C using the following programs: solvent A,
H
2
O + 0.2% TFA; solvent B, MeCN; linear gradient, 0 min 25% B (75% A), 40 min 60% B (40% A),
45 min 100% B ; UV detection at 340 nm. The retention times for the FDAA derivatives of the
hydrolysate of 5, L-Ile, L-allo-Ile, D-Ile and D-allo-Ile were 23.06, 23.06, 23.06, 28.11 and 28.11 min,
respectively (Figure S38). The FDAA derivatives of the hydrolysate of 5, L-Ile, and L-allo-Ile were
o
further analyzed by C
3
HPLC column maintained at 50 C. The column was developed with a linear
gradient of 15%–60% MeOH/water (+isocratic 5% of a 1% formic acid solution in MeCN) over
5 min with UV detection at 340 nm. The retention times for the FDAA derivatives of the hydrolysate
of 5, standard L-Ile, and L-allo-Ile, were 38.50, 38.50, and 37.49 min, respectively (Figure S39).
5

Mar. Drugs 2013, 11
.8. Bioassays
Cytotoxicity was assayed by the MTT [23] and SRB method [24]. In the MTT assay, HL-60 cell
1046
3
and K562 cell line were cultured in RPMI-1640 supplemented with 10% FBS under a humidified
atmosphere of 5% CO and 95% air at 37 °C, 198 μL of cell suspensions with a density of
2
4
−1
4
.6 × 10 cells mL was plated in 96-well microtiter plates and incubated for 24 h. Then, 2 μL of the
test solutions in MeOH were added to each well and further incubated for 36 h. The MTT solution
20 μL, 5 mg/mL in IPMI-1640 medium) was then added to each well and incubated for 4 h. Old
(
medium containing MTT (150 μL) was then gently replaced by DMSO and pipetted to dissolve any
formazan crystals formed. Absorbance was then determined on a Spectra Max Plus plate reader at
5
2
70 nm. In the SRB assay, 200 μL of the A549 cell suspension was plated in 96-well plates at density of
5
−1
× 10 cell mL . Then, 2 μL of the test solutions (in MeOH) were added to each well and the culture
was further incubated for 24 h. The cells were fixed with 12% trichloroacetic acid and the cell layer
stained with 0.4% SRB. The absorbance of the SRB solution was measured at 515 nm. Adriamycin
was used as positive control (IC 0.652 μM, 0.645 μM and 0.080 μM for HL-60, K562 and A549
50
cell, respectively).
The antiviral activity against H1N1 was evaluated by the CPE inhibition assay [21,22]. Confluent
MDCK cell monolayers were firstly incubated with influenza virus (A/Puerto Rico/8/34 (H1N1),
PR/8) at 37 °C for 1 h. After removing the virus dilution, cells were maintained in infecting media
(
RPMI 1640, 4 μg/mL of trypsin) containing different concentrations of test compounds at 37 °C.
After 48 h incubation at 37 °C, the cells were fixed with 100 μL of 4% formaldehyde for 20 min at
room temperature. After removal of the formaldehyde, the cells were stained with 0.1% crystal violet
for 30 min. The plates were washed and dried, and the intensity of crystal violet staining for each well
was measured in a microplate reader (Bio-Rad, Hercules, CA, USA) at 570 nm. The IC50 was calculated
as the compound concentration required inhibiting influenza virus yield at 48 h post-infection by 50%.
Ribavirin was used as the positive control with an IC50 value of 38.8 μM.
The anti-inflammatory effects of compounds were assayed by inhibition of lipopolysaccharide
(
(
LPS)-mediated NF-κB transcriptional activity in RAW 264.7 cells [25]. RAW 264.7 cells
5
2.5 × 10 cells/well) were placed in a 24-well plate. The cells were then stably transfected with
pNF-κB-Luc expression plasmid (0.5 μg/well). Transfections were performed using lipofectamine
000 in accordance with the instructions of the manufacturer (Invitrogen, Carlsbad, NM, USA). Stably
2
transfected cells were pretreated 2 h with the test compounds and stimulated with 0.1 μg/mL of LPS for
an additional 4 h. The luciferase assay was performed with the aid of a Steady-Glo Luciferase assay
system in accordance with the instructions of the manufacturer (Promega, Madison, WI, USA).
The antimicrobial activities against E. coli, E. aerogenes, P. aeruginosa, B. subtilis, S. aureus, and
C. albicans were evaluated by 2-fold dilution method [26]. The tested strains were cultivated on LB
broth for bacteria and in YPD broth for C. albicans at 37 °C. The test compounds were dissolved in
DMSO at different concentrations from 100 to 0.78 μg/mL (from 6.25 to 0.025 μg/mL for the positive
controls) by the continuous 2-fold dilution methods. The minimum inhibitory concentrations (MICs)
were determined in 96-well plates, and each well contains 100 μL of contents composed of 20 μL of
5
inoculums (5 × 10 CFU/mL), test compounds and LB or YPD media. The microtiter plates were
incubated at 35 °C for 24 h and were examined for microbes’ growth by turbidity in daylight. The

Mar. Drugs 2013, 11
1047
MICs were defined as the lowest concentration at which no visible growth of microbes could be
observed. Ciprofloxacin lactate and ketoconazole were used as positive controls for E. coli, E. aerogenes,
P. aeruginosa, B. subtilis, S. aureus, and C. albicans with MIC values of 0.05, 0.19, 0.1, 0.39, 3.12 and
0
.025 μg/mL, respectively.
4
. Conclusions
Five new vinylidene substituted diketopiperazines (DKPs) 1–5 were isolated and their structures
including absolute configurations were determined. The new compound 3 displayed modest activity
and the known analogs 6 and 7 displayed potent activity against H1N1 virus with IC values of
5
0
41.5 ± 4.5, 28.9 ± 2.2 and 6.8 ± 1.5 μM, respectively. The results showed that both Z-deh-Phe and Leu
or Z-deh-Leu substitutions significantly increase the anti-H1N1 activity of DKPs, while E-isomerization
and hydroxylation of both deh-Phe and deh-Leu moieties, and N-methylation reduce the activity. The
substitutions of both deh-Phe or deh-Tyr with deh-His and Leu or deh-Leu with iso-Leu also reduce
13
the anti-H1N1 activity. In addition, the C NMR data of (3Z,6S)-3-benzylidene-6-isobutylpiperazine-2,
-dione (6) was reported here for the first time.
5
Acknowledgements
This work was financially supported by the grants from 973 Program of China (No. 2010CB833804),
from NSFC (Nos. 21172204, 30973680 & 30670219), from 863 Program of China (Nos. 2013AA092901,
2012AA092104 and 2011AA09070106), from the Knowledge Innovation Program of Chinese Academy
of Sciences (No. KSCX2-EW-J-6/KSCX2-EW-G-12B), and the Special Fund for Marine Scientific
Research in the Public Interest of China (No. 2010418022-3). The cytotoxic and anti-inflammatory
effects were assayed by J. Li’s group, OUC. The antiviral effect on H1N1 was assayed by Wei Wang,
one of the authors.
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