Cyclopeptides with anti-inflammatory activity from seeds of Annona montana

Article abstract of DOI:10.1021/np8001282

Four new cyclopeptides, cyclomontanins A-D (1-4), annomuricatin C (5), and (+)-corytuberine were isolated from a methanol extract of Annona montana seeds. Their structures were elucidated by 2D NMR analysis, ESIMS/MS fragment evidence, and chemical means. The structure of 1 was confirmed by synthesis. Compounds 1, 3, and 4 exhibited anti-inflammatory activity in vitro using the J774.1 macrophage model.

Full text of DOI:10.1021/np8001282

J. Nat. Prod. 2008, 71, 1365–1370
1365
Cyclopeptides with Anti-inflammatory Activity from Seeds of Annona montana
Pei-Hsuan Chuang,† Pei-Wen Hsieh,‡ Yu-Liang Yang,§ Kuo-Feng Hua,§ Fang-Rong Chang,† Jentaie Shiea, Shih-Hsiung Wu,§
,|
and Yang-Chang Wu*,†,
Graduate Institute of Natural Products, Kaohsiung Medical UniVersity, Kaohsiung 807, Taiwan, Republic of China, Graduate Institute of
Natural Products, Chang Gung UniVersity, Tao-Yuan 333, Taiwan, Republic of China, Institute of Biological Chemistry, Academia Sinica,
Taipei 115, Taiwan, Republic of China, National Sun Yat-Sen UniVersity-Kaohsiung Medical UniVersity Joint Research Center,
Kaohsiung 804, Taiwan, Republic of China, and Center of Excellence for EnVironmental Medicine, Kaohsiung Medical UniVersity,
Kaohsiung 807, Taiwan, Republic of China
ReceiVed February 26, 2008
Four new cyclopeptides, cyclomontanins A-D (1-4), annomuricatin C (5), and (+)-corytuberine were isolated from
a methanol extract of Annona montana seeds. Their structures were elucidated by 2D NMR analysis, ESIMS/MS fragment
evidence, and chemical means. The structure of 1 was confirmed by synthesis. Compounds 1, 3, and 4 exhibited anti-
inflammatory activity in Vitro using the J774.1 macrophage model.
Annonaceae is a large family of tropical and subtropical trees
and shrubs, comprising 130 genera and about 2300 species.1,2 Many
natural products, including alkaloids,3–6 ent-kauranes,7,8 flavonoids,9,10
annonaceous acetogenins,2 and cyclopeptides,11 have been isolated
from annonaceous plants. These products show biological effects
such as cytotoxicity,2,12 antimalarial,2,3 pesticidal,2,13 antiplatelet,9
anti-inflammatory,7,8 and vasorelaxant activities.14 Recently, we
reported two new cyclopeptides, cyclosquamosins H and I, from
the seeds of Annona squamosa and found that cyclosquamosin D
showed anti-inflammatory activity using the in Vitro J774.1
macrophages model.15
Annona montana Macf. (Annonaceae), also called mountain
soursop, is distributed mainly in tropical America, the West Indies,
and southern Taiwan.1 No cyclopeptides have previously been
reported from this species. In our continuing research on annona-
ceous cyclopeptides, four new cyclopeptides, 1-4, one known
cyclopeptide, annomuricatin C (5), and one known alkaloid, (+)-
corytuberine (6), were obtained from a methanol extract of A.
montana seeds. Compound 5 was isolated from this plant for the
first time.16 All structures were confirmed by spectroscopic and
chemical evidence. The structure of 1 was also established by total
synthesis using a solid-phase synthetic method.
Lipopolysaccharide (LPS) is an outer membrane component of
Gram negative bacteria that binds to toll-like receptor 4 (TLR4).
LPS stimulation effects severe biological responses within host cells,
including fever, procoagulant activity, septic shock, and death.17–19
However, the stimulation of LPS involved in nitrogen intermediates,
prostaglandins, and cytokines such as tumor necrosis factor-R (TNF-
R), interleukin-1 (IL-1), and IL-6, both in ViVo and in Vitro, is
mediated through activation of the host immune and inflammatory
cells. TNF-R and IL-6 are mediators in the inflammatory response
involved in host defense.17,20–23 In this study we investigated the
anti-inflammatory activity of cyclopeptides using LPS- and Pam3Cys-
stimulated macrophages, an in Vitro model.
at m/z 762.3555 [M + Na]+ (calcd 762.3551). The IR absorptions
at 3315, 1659, and 1522 cm-1 were attributed to amino, amide
carbonyl, and aromatic groups, respectively. The 1H spectrum had
eight amide NH signals and an indole NH at δ 8.37 (d), 8.56 (s),
8.73 (d), 8.76 (d), 9.16 (d), 9.28 (d), 9.42 (s), 10.34 (dd), and 11.75
(d), respectively, and the 13C NMR spectrum (pyridine-d5) showed
eight amide carbonyls at δ 169.6, 170.0, 172.0, 172.4, 172.6, 172.8,
172.9, and 174.8, indicating that 1 was a peptide. Marfey’s method
indicated that the amino acids all had L-configuration.7,8 Complete
assignments for 1H and 13C NMR signals (Table 1) were assigned
using a combination of 2D NMR experiments (COSY, TOCSY,
and HMQC spectra).
Results and Discussion
Cyclomontanin A (1) was obtained as a white powder. The
molecular formula was determined to be C35H49N9O9 by HRESIMS
* To whom correspondence should be addressed. Tel: (+886)-(0)7-312-
1101, ext. 2197. Fax: (+886)-(0)7-311-4773. E-mail: yachwu@kmu.edu.tw.
† Graduate Institute of Natural Products, Kaohsiung Medical University.
‡ Chang Gung University.
§ Academia Sinica.
National SunYat-Sen University-Kaohsiung Medical University Joint
Research Center.
| Center of Excellence for Environmental Medicine, Kaohsiung Medical
University.
The sequence and connectivity of the amino acid residues were
deduced by ESIMS/MS analysis (Figure 1) and HMBC experiment
10.1021/np8001282 CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/08/2008

1366 Journal of Natural Products, 2008, Vol. 71, No. 8
Chuang et al.
Table 1. 1H and 13C NMR Data of 1 and 2 (pyridine-d5)
1
2
δH (mult., J in Hz)
δC (mult.)
δH (mult., J in Hz)
δC (mult.)
Gly1
Pro2
CdO
NH
169.7 (s)
Gly1
Pro2
CdO
NH
169.8 (s)a
10.34 (dd,8.0,4.4)
3.59-3.99 (m)
10.35 (br s)
3.87-3.97 (m)
4.77 (dd,16.8,8.4)
43.8 (t)
43.8 (t)
4.78 (dd,16.8,8.4)
CdO
172.6 (s)
61.8 (d)
29.5 (t)
24.8 (t)
CdO
172.6 (s)a
61.8 (d)
29.6 (t)
R
ꢀ
γ
4.41 (t,8.4)
2.03 (m)
1.30 (m)
1.71 (m)
3.59-3.99 (m)
R
ꢀ
γ
4.46 (t,8.4)
1.96 (m)
1.30 (m)
1.73-1.87 (m)
3.61-3.82 (m)
24.8 (t)
δ
48.7 (t)
170.0 (s)
δ
48.6 (t)
Thr3
Trp4
CdO
NH
R
Thr3
CdO
NH
R
170.0 (s)a
8.37 (d,9.6)
5.41 (t,8.8)
4.54 (m)
8.28 (d,9.6)
5.41 (t,8.8)
4.57 (m)
57.8 (d)
69.0 (d)
20.2 (q)
172.9 (s)
58.0 (d)
69.4 (d)
20.1 (q)
172.9 (s)a
ꢀ
γ
CdO
NH
R
ꢀ
γ
1.63 (d,6.4)
1.64 (d,6.0)
Kyn4
CdO
NH
R
8.76 (d,7.2)
5.73 (td,10.4,4.4)
3.59-3.99 (m)
8.91 (d,9.6)
6.01 (td,9.6,3.2)
3.59-3.99 (m)
55.6 (d)
29.4 (t)
51.6 (d)
42.4 (t)
ꢀ
Ar
ꢀ
109.9 (s)
123.5 (d)
111.9 (d)
119.0 (d)
121.5 (d)
119.1 (d)
137.3 (s)
128.9 (s)
γCdO
Ar
199.5 (s)
117.8 (s)
132.0 (d)
115.1 (d)
134.6 (d)
117.5 (d)
152.4 (s)
7.52 (d,2.0)
7.55 (d,7.6)
7.18 (t, 7.6)
7.25 (t,7.6)
7.83 (d,7.6)
7.86 (d,8.0)
6.50 (dd,8.0)
7.23-7.28 (m)
6.92 (t,8.0)
NH2
7.80 (br s)
11.75 (d,1.6)
Ala5
Asn6
CdO
NH
R
172.8 (s)
Ala5
Asn6
CdO
NH
R
172.8 (s)a
9.28 (d,3.2)
4.47 (m)
1.13 (d,7.2)
9.48 (br s)
4.57 (m)
1.35 (d,7.2)
52.9 (d)
16.6 (q)
172.0 (s)
53.1 (d)
17.0 (q)
ꢀ
ꢀ
CdO
NH
R
CdO
NH
R
172.0 (s)a
9.16 (d,5.2)
5.05 (d,2.8)
3.59-3.99 (m)
9.13 (d,4.8)
5.05 (m)
3.61-3.97 (m)
51.1 (d)
36.0 (t)
174.8 (s)
51.3 (d)
36.3 (t)
174.9 (s)
ꢀ
ꢀ
γCdO
NH2
γCdO
NH2
8.56 (s)
9.42 (s)
8.75 (m)
9.41 (s)
Leu7
CdO
NH
R
172.4 (s)
Leu7
CdO
NH
R
172.4 (s)a
8.73 (d,7.2)
5.41 (t,8.8)
1.71 (m)
1.91 (m)
1.91 (m)
8.75 (m)
5.4 (t,8.8)
1.73-2.10 (m)
1.96 (m)
0.89 (d,6.0)
0.90 (d,6.0)
54.3 (d)
44.4 (t)
54.4 (d)
44.2 (t)
25.0 (d)
22.4 (s)
22.4 (s)
ꢀ
ꢀ
γ
γ
δ
24.9 (d)
22.3 (s)
22.5 (s)
δ
0.89 (d,6.0)
0.91 (d,6.0)
a Interchangeable.
yielded the fragment of N-terminal dipeptide [Gly-Pro]. Another
series of adjacent H ions (i.e., Y ion-H2O) at m/z 566, 471, 340,
and 269 were obtained from N-terminal to C-terminal of the
breaking Thr-Pro amide bond, corresponding to the successive loss
of [Gly-Pro], Leu, Asn, and Ala, yielding the fragment of C-terminal
Figure 1. ESIMS/MS fragments of 1.
(Figure 2). The protonated molecular ion [M + H]+ of 1 (m/z 740)
was subjected to ESIMS/MS. The ring opening began at the Thr-
Pro amide bond and gave a series of adjacent B ions at m/z 638,
453, 382, 268, and 155 corresponding to the successive loss of
Thr, Trp, Ala, Asn, and Leu from C-terminal to N-terminal and
Figure 2. Significant TOCSY, HMBC, and ROESY correlations
of 1.

Cyclopeptides from Seeds of Annona montana
Journal of Natural Products, 2008, Vol. 71, No. 8 1367
Scheme 1. Synthetic Route for 1a
a Reagents and conditions: (a) Fmoc-Leu-OH, DIEA, DCM, room temperature, 2 h; (b) 20% piperidine in DMF; (c) Fmoc-Gly-OH, HBTU, HOBt, DMF, room
temperature, 3 h; (d) Fmoc-Pro-OH, HBTU, HOBt, DMF, room temperature, 3 h; (e) Fmoc-Thr(tBu)-OH, HBTU, HOBt, DMF, room temperature, 3 h; (f) Fmoc-
Trp(Boc)-OH, HBTU, HOBt, DMF, room temperature, 3 h; (g) Fmoc-Ala-OH, HBTU, HOBt, DMF, room temperature, 3 h; (h) Fmoc-Asn(Trt)-OH, HBTU, HOBt,
DMF, room temperature, 3 h; (i) AcOH/TFE/DCM, room temperature, 2 h; (j) HATU, DIEA, DCM, room temperature, 24 h; (k) TFA/TIS/H2O, room temperature,
1 h.
dipeptide [Trp-Thr]-H2O. These results suggested that the partial
amino acid sequence of 1 was Thr-Trp-Ala-Asn-Leu. The sequence
of 1 was determined by HMBC and ROESY correlations (Figure
2), which elucidated the amino acid sequence of 1 as Gly1-Pro2-
Thr3-Trp4-Ala5-Asn6-Leu7. In addition, the difference of 13C NMR
chemical shifts of Pro2 (∆Cꢀ-Cγ) 7.6 ppm) suggested that the amide
bond in the Pro2 residue was trans.24 Thus, the structure of 1 was
assigned as cyclo(-Gly1-transPro2-Thr3-Trp4-Ala5-Asn6-Leu7-).
The ESIMS/MS data of 2 at m/z 744 [M + H]+ indicated
preferential opening at the Thr-Pro amide bond level to obtain one
series of adjacent B ions (at m/z 643, 453, 382, 268, and 155) and
one series of adjacent H ions (Y ion-H2O at m/z 571, 458, and 291).
The amino acid residues were lost in the order Thr, Kyn, Ala, Asn,
Leu, and the terminal dipeptide [Gly-Pro] sequentially from the
C-terminal to N-terminal. Furthermore, the ions at m/z 545, 356,
and 284 were attributed to the fragments [Kyn-Ala-Asn-Leu-Gly],
[Ala-Asn-Leu-Gly], and [Asn-Leu-Gly], respectively. The ion at
m/z 425 indicated the fragment [Pro-dehydroThr-Kyn-Ala]-NH.
Correlation of HMBC and ROESY spectra proved that the sequence
of 2 was cyclo(-Gly1-transPro2-Thr3-Kyn4-Ala5-Asn6-Leu7-). Ac-
cording to the negative CD bands at 222.3 and 198.9 nm, compound
2 was proposed to have an R-helix conformation.28 The amino acid
sequence of 2 was similar to that of 1, except for Kyn and Trp.
The molecular formula of 3 was established as C51H67N13O12
by HR-ESIMS, which showed a pseudomolecular ion peak at m/z
1054.5106 [M + H]+ (calcd 1054.5110). IR absorptions at 3291,
1670, and 1524 cm-1 indicated the presence of amino, amide
carbonyl, and aromatic groups, respectively.
In accordance with the same amino acid sequence as mentioned
above, cyclomontanin A (1) was synthesized using Fmoc/t-Bu
chemistry and a 2-chlorotrityl chloride resin as solid support
(Scheme 1).25,26 The knit sequence was coupled with hydroxyben-
zotriazole/O-(benzotriazol-1-yl)-N,N,N′,N′ -tetramethyluronium
hexafluorophosphate (HOBt/HBTU) in N,N-dimethylformamide
(DMF), and the cyclization was allowed to proceed in solution using
O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluo-
rophosphate (HATU) and diisopropylethylamine (DIEA) in dichlo-
romethane (DCM). After the side-chain protecting groups were
removed with 95% aqueous trifluoroacetic acid (TFA), the crude
cyclopeptide was purified by preparative RP-HPLC, which yielded
1, and was identified on the basis of ESIMS/MS and 1H NMR data.
1
Since the H NMR spectrum of 3 showed better resolution at
320 K than at room temperature, the following NMR experiments
were carried out under this condition. However, correlations of 2D
NMR spectra were difficult to interpret, especially HMBC and
ROESY; thus the sequence of 3 was elucidated by ESIMS/MS data.
ESIMS/MS on the [M + H]+ ion of 3 yielded preferential ring
opening at the Phe-Asn amide bond and gave relative B ions of
peptide fragments at m/z 1055, 939, 841, 704, 509, 443, and 342;
one series of adjacent H ions at m/z 921, 823, 687, 572, and 425;
and one series of adjacent A ions at m/z 910, 813, 562, 413, and
314. On the basis of the fragment data, amino acid residues were
lost sequentially from the C-terminal to N-terminal, corresponding
to the successive loss of Asn, Val, His, Asn, Phe, Thr, and the
terminal tripeptide [Pro-Pro-Phe]. Opening at the same amide bond
level, a series of Y ions at m/z 713, 612, and 465 agreed with
successive loss of [Phe-Pro-Pro], Thr, and Phe from N-terminal to
Cyclomontanin B (2) was obtained as a pale yellow powder and
showed a pseudomolecular ion peak at m/z 766.3495 [M + Na]+
(calcd 766.3500) on HR-ESIMS, corresponding to the molecular
formula C35H49N9O9. IR absorptions indicated the presence of amine
(3316 cm-1), amide carbonyl (1662 cm-1), and aromatic groups
(1521 cm-1). Ten amide (NH) and eight amide carbonyl signals
were observed in its 1H and13C NMR spectra (Table 1), indicating
a peptide. Analysis of COSY, TOCSY, and HMQC spectra
demonstrated that the amino acid residues were Gly, Pro, Thr, Ala,
Asn, Leu, and Kyn (kynurenine; a tryptophan metabolite27). Using
Marfey’s method, the amino acid residues were established to have
L-configurations.7,8 The chemical shift difference between Cꢀ and
Cγ of Pro2 was 4.8 ppm; thus, the Pro2 residue in 2 had trans
geometry.24

1368 Journal of Natural Products, 2008, Vol. 71, No. 8
Table 2. 1H and 13C NMR Data of 3 (320 K; pyridine-d5)
Chuang et al.
δH (mult., J in Hz)
δC (mult.)
δH (mult., J in Hz)
δC (mult.)
Phe1
CdO
NH
R
173.9 (s)
Phe5
CdO
NH
R
171.8 (s)a
8.77 (m)
5.05 (m)
3.19 (m)
3.48 (m)
9.57 (br s)
5.28 (m)
3.73 (m)
56.6 (d)
38.0 (t)
57.2 (d)
35.1 (t)
ꢀ
ꢀ
Ar
139.2 (s)
130.5 (d)
128.5 (d)
126.5 (d)
173.6 (s)
Ar
138.4 (s)
129.6 (d)
128.8 (d)
126.9 (d)
170.9 (s)
58.9 (d)
28.6 (t)
7.51 (d, 7.5)
7.29 (t, 7.5)
7.16 (m)
7.34 (d, 7.5)
7.23 (t, 7.5)
7.16 (m)
Asn6
CdO
NH
R
Pro2
CdO
R
ꢀ
8.52 (m)
5.28 (m)
3.19 (m)
3.30 (m)
3.63 (m)
1.67 (m)
1.89 (m)
1.75 (m)
1.89 (m)
3.63 (m)
3.68 (m)
51.9 (d)
38.3 (t)
ꢀ
γ
δ
25.2 (t)
48.0 (t)
γCdO
NH2
CdO
NH
R
173.2 (s)a
171.8 (s)a
His7
9.49 (m)
5.23 (m)
3.63 (m)
3.68 (m)
Pro3
CdO
NH
R
171.3 (s)a
61.4 (d)
30.8 (t)
55.7 (d)
27.6 (t)
ꢀ
4.35 (m)
1.89 (m)
Ar
136.0 (s)
118.3 (d)
134.4(d)
ꢀ
7.41 (br s)
8.58 (br s)
7.70 (br s)
2.58 (m)
1.53 (m)
1.75 (m)
3.30 (m)
3.48 (m)
NH
CdO
NH
R
ꢀ
γ
γ
δ
22.3 (t)
47.1 (t)
Val8
Asn9
170.5 (s)
7.95 (d, 8.5)
4.95 (t, 8.0)
2.28 (m)
1.11 (d, 6.5)
1.21 (d, 6.5)
56.2 (d)
32.7 (d)
18.4 (q)
19.6 (q)
172.6 (s)
Thr4
CdO
NH
R
ꢀ
γ
172.2 (s)
8.27(br s)
4.47 (t, 5.0)
4.36 (m)
62.5 (d)
66.9 (d)
21.2 (q)
CdO
NH
R
9.26 (d, 6.5)
5.68 (m)
3.33 (m)
1.32 (d, 6.5)
52.7 (d)
39.2 (t)
ꢀ
γCdO
NH2
173.9 (s)a
a Interchangeable.
C-terminal and yielded the fragment of C-terminal tetrapeptide
[Asn-His-Val-Asn]. Furthermore, another series of adjacent Y ions
at m/z 955, 858, 758, 612, and 496 were obtained from N-terminal
to C-terminal of the breaking Pro-Phe amide bond, corresponding
to the successive loss of Pro, Pro, Thr, Phe, and Asn and yielding
the C-terminal tertapeptide [His-Val-Asn-Phe]. Thus, the structure
of 3 was established as cyclo(-Phe1-Pro2-Pro3-Thr4-Phe5-Asn6-His7-
Val8-Asn9-). The differences in the 13C NMR chemical shifts of
Pro2 (∆Cꢀ-Cγ ) 3.4 ppm) and Pro3 (∆Cꢀ-Cγ ) 8.5 ppm) suggested
that the amide bonds in the Pro2 and Pro3 residues in 3 were trans
and cis, respectively.24 Finally, based on Marfey’s analysis, each
amino acid residue configuration in 3 was L.7,8
IR absorptions at 3321 and 1676 cm-1 indicated amide bonds
in the structure of 4. The molecular formula was determined to be
C34H48N8O9 by HR-ESIMS. Analysis of 2D NMR spectra, COSY,
TOCSY, and HMQC demonstrated that the amino acid residues of
4 were Pro × 2, Ala, Tyr, Leu, Gly, and Asn, and all were L
configuration based on Marfey’s analysis.7,8
Tyr5-Ala6-Asn7). The trans amide bond in the Pro2 residue was
indicated by the 13C NMR chemical shift of Pro2 (∆δCꢀ-Cγ ) 8.6
ppm).24
Annomuricatin C (5) was initially isolated from A. muricata seeds
in 2004.16 According to this report, the major conformation (>85%,
318 K, in DMSO-d6) of 5 was determined and was found to be
cytotoxic against KB cells with an IC50 of 1.0 µM. However, our
compound 5 showed a 2:1 ratio between major/minor conformations
(318 K, in DMSO-d6) and showed no cyctotoxicity against Hep
G2, Hep 3B, MDA-MB-231, MCF-7, A549, and Ca9-22 cell lines.
In order to investigate whether cyclopeptides 1, 3, 4, and 5
exhibited immunomodulation activity in cultured murine macroph-
age J774A.1 cells, the cells were pretreated with various cyclo-
peptides (30 µg/mL) for 30 min at 37 °C, followed by LPS
challenge for 6 h. DMSO-treated J774A.1 cells were used as
controls. LPS was noted to stimulate significant levels of TNF-R
and IL-6 secretions; in cyclopeptide 1, 3, 4, and 5 pretreated cells,
LPS-induced secretion of TNF-R and IL-6 was decreased by about
50-80% (Figure 3).
The ESIMS/MS data on the [M + H]+ of 4 showed preferential
ring opening at the Pro-Asn amide bond and gave relative Y ions
of peptide fragments at m/z 614, 557, 446, 348, 186, and 115,
together with a series of adjacent B ions at m/z 598, 268, and 155,
corresponding to the successive loss of Pro, Gly, Leu, Pro, Tyr,
and Ala from N-terminal to C-terminal and yielded a fragment of
Asn. Furthermore, one series of adjacent A ions at m/z 569, 500,
335, 240, and 127 and adjacent X ions series at m/z 585, 472, and
142 were obtained from C-terminal to N-terminal of the breaking
of the same amide bond, corresponding to the successive loss of
Asn, Ala, Tyr, Pro, Leu, Gly, and yielded Pro. Therefore, the
structure of 4 was determined to be cyclo-(Pro1-Gly2-Leu3-Pro4-
To further investigate the dose-dependent activity of cyclopep-
tides 1, 3, 4, and 5, J774A.1 cells were pretreated with various
concentrations of cyclopeptide 1, 3, 4, or 5 for 30 min, followed
by stimulation with LPS or Pam3Cys, a synthetic component
binding to TLR2, for an additional 6 h. Cyclomontanin A (1)
showed significant inhibition of TNF-R and IL-6 production at 1
and 3 µg/mL, respectively, even within LPS- or Pam3Cys-
stimulated J774A.1 cells. Specifically, cyclomontanin C (3) showed
a marked inhibition of TNF-R and IL-6 production in LPS- or
Pam3Cys-stimulated J774A.1 cells at 3-50 µg/mL. Cyclomontanin
D (4) and annomuricatin C (5) decreased the secretion of TNF-R
and IL-6 in LPS-stimulated cells at 30 µg/mL. On stimulation with

Cyclopeptides from Seeds of Annona montana
Journal of Natural Products, 2008, Vol. 71, No. 8 1369
Table 3. 1H and 13C NMR Data of 4 (pyridine-d5)
δH (mult., J in Hz)
and annomuricatin C (5) exhibited a dose-response relationship
with IL-6 secretion in Pam3Cys-stimulated cells.
δC (mult.)
172.4 (s)a
In conclusion, we isolated and elucidated four novel cyclopep-
tides (1-4) and demonstrated that cyclomontanin A (1), cyclomon-
tanin C (3), cyclomontanin D (4), and annomuricatin C (5) exhibited
anti-inflammatory effects within the LPS- or Pam3Cys-stimulated
J774A.1 cells. These finding are worthy of further research to
determine the mechanisms of these anti-inflammatory effects and
to suggest additional synthetic analogues of cyclopeptides 1, 3, 4,
and 5 for further drug development investigation.
Pro1
CdO
R
4.49 (m)
64.7 (d)
30.2 (t)
26.4 (t)
ꢀ
2.26 (m), 2.05 (m)
1.92 (m), 1.69 (m)
3.80 (m), 3.63 (m)
γ
δ
CdO
NH
R
48.3 (t)
Gly2
Leu3
170.7 (s)a
10.04 (d, 6.0)/9.40 (br s)
4.93 (m)/4.49 (m)
3.75 (m)/4.20 (m)
43.7 (t)/44.2 (t)
172.5 (s)a
CdO
NH
R
8.01 (m)
Experimental Section
4.93 (m)
54.8 (d)
General Experimental Procedures. Optical rotations were measured
with Jasco DIP 370 and P-1020 digital polarimeters. IR and UV spectra
were obtained on Mattson Genesis II infrared and Jasco UV-530
ultraviolet spectrophotometers, respectively. The CD spectra were
measured on a Jasco J-720 spectropolarimeter. NMR (400 and 500
MHz, using pyridine-d5 as solvent) spectra were collected on Varian
Unity 400 MHz NMR, Varian Unity INOVA-500 NMR, and Bruker
AVANCE II 500 NMR spectrometers. Low-resolution and high-
resolution ESIMS were recorded on a Bruker APEX II spectrometer
(FT-ICR/MS, FTMS). ESIMSn were collected on a MicroMass Q-TOF
ultima global mass spectrometer or a Bruker Esquire 3000 ion trap
mass spectrometer. Shimadzu LC-10AT pumps, Shimadzu SPD-10A
UV-vis detector, Ascentis C18 (250 × 4.6 mm i.d.; 5 µm)/preparative
Ascentis C18 (250 × 21.2 mm i.d.; 5 µm) columns, and Develosil
C30-UG-5 (250 × 4.6 mm i.d.; 5 µm)/preparative Develosil C30-UG-5
(250 × 20 mm i.d.; 5 µm) columns were employed for HPLC.
Plant Material. Seeds of A. montana were offered from the Taitung
District Agricultural Research and Extention Station (TDARES),
Taitung, Taiwan in September, 2006. The plant material was identified
by Dr. Ching-Shan Yang (TDARES). A voucher specimen is deposited
at the Graduate Institute of Natural Products (Annona-05), Kaohsiung
Medical University, Kaohsiung, Taiwan.
ꢀ
3.07 (m)/2.91 (m)
2.51 (m)
1.19 (d, 6.0)
1.18 (d, 6.0)
32.5 (t)/30.9 (t)
32.9 (d)/33.3 (d)
19.2 (q)/19.0 (q)
20.5 (q)/21.8 (q)
172.1 (s)a
γ
δ
Pro4
Tyr5
CdO
R
4.49 (m)
63.5 (d)/62.9 (d)
30.1 (t)
25.6 (t)
ꢀ
2.05 (m), 1.92 (m)
1.69 (m), 1.56 (m)
3.68 (m), 3.59 (m)
γ
δ
CdO
NH
R
48.6 (t)
173.6 (s)a
9.21 (br s)
5.38 (m)
3.39 (dd, 13.0, 4.0), 3.27 (m)
58.9 (d)
40.2 (t)
ꢀ
Ar
130.9/130.1 (s)
131.0/131.3 (d)
116.2/116.3 (d)
157.3/157.9 (s)
172.2 (s)a
7.98 (m)/7.98 (m)
7.08 (d, 7.5)/7.19 (m)
Ala6
CdO
NH
R
8.91 (br s)
4.84 (m)
1.82 (d, 8.0)
51.4 (d)/53.2 (d)
18.3 (q)/18.2 (q)
173.9 (s)a
ꢀ
Asn7 CdO
NH
R
8.68 (br s)
5.28 (m)
3.65 (m)/3.12 (dd, 13.0, 4.0)
Extraction and Isolation. The air-dried seeds (ca. 1.4 kg) of A.
montana were crumbled, then extracted with MeOH at room temper-
ature. The MeOH extract (122.5 g) was partitioned between n-hexane
and 20% aqueous MeOH to yield n-hexane and 20% MeOH(aq) layers.
The MeOH(aq) layer (105.8 g) was further partitioned between
n-butanol and H2O to give n-butanol, insoluble, and water layers. The
n-butanol layer (60.8 g) was separated on Diaion HP-20 with a gradient
of 10% to 100% MeOH and acetone to yield five fractions (A-E).
Fraction C (2.7 g) was separated using a flash column with 85%
MeCN(aq) to afford three subfrations (C-1-3). Subfraction C-1 was
further separated on a flash column eluting with 85% MeCN(aq) to
afford five subfrations (C-1-1-5). Subfractions C-1-2 and C-1-4 were
purified by an open column and HPLC (Develosil C30-UG-5, 70%
MeCN(aq) with 0.5% TFA) to yield 6 (18.2 mg) and 3 (3.1 mg),
respectively. Subfraction C-2 was purified by preparative reversed-phase
HPLC (Ascentis C18 column, 75% MeCN(aq)) to obtain 1 (44.3 mg)
and 2 (4.3 mg). Subfraction C-3 was separated to four fractions (C-
3-1-4) by preparative RP-HPLC (Ascentis C18 column, 65% MeC-
N(aq)) and than separated by HPLC (Develosil C30-UG-5 column, 75%
MeCN(aq) with 0.5% TFA) to afford 3 (126.9 mg), 4 (15.6 mg), and
5 (14.4 mg).
56.2 (d)
36.8 (t)
ꢀ
γCdO
NH2
176.8 (s)a
a Interchangeable.
Cyclomontanin A (1): white powder; [R]26 -37.7 (c 0.0017,
D
MeOH); UV (MeOH) λmax (log ε) 210 (4.50), 218 (4.50), 279 (3.66)
nm; CD (c ) 1.7 × 10-4 M, MeOH) λmax (∆ε) 252.2 (+1.67), 219.2
(-1.37), 197.5 (-7.72) nm; IR (neat) νmax 3315, 1659, 1622, 1522,
1
1458 cm-1; H and 13C NMR data, see Table 1; ESIMS/MS m/z 638,
566, 471, 453, 382, 340, 269, 268, 155; HRESIMS m/z 762.3555 [M
+ Na]+ (calcd for C35H49N9O9Na, 762.3551).
Cyclomontanin B (2): pale yellow powder; [R]26D -60.0 (c 0.006,
MeOH); UV (MeOH) λmax (log ε) 210 (4.54), 228 (4.33), 259 (3.71),
363 (3.49) nm; CD (c ) 1.5 × 10-4 M, MeOH) λmax (∆ε) 275.6
(+1.24), 249.8 (+1.40), 222.3 (-4.80), 198.9 (-4.46) nm; IR (neat)
Figure 3. Anti-inflammatory effects of cyclopeptides 1, 3, 4, and
5 at 30 µg/mL toward LPS-stimulated J774A.1 cells. *p < 0.05
versus DMSO.
1
νmax 3316, 1662, 1521, 1450 cm-1; H and 13C NMR, see Table 1;
ESIMS/MS m/z 643, 571, 545, 458, 453, 425, 382, 356, 291, 284, 268,
155; HRESIMS m/z 766.3495 [M + Na]+ (calcd for C34H49N9O10Na,
766.3500).
Pam3Cys, cyclomontanin D (4) showed approximately 20%, 50%,
60%, 85%, and 90% inhibition of TNF-R production at 3, 5, 10,
30, and 50 µg/mL, respectively. Additionally, cyclomontanin D (4)
Cyclomontanin C (3): pale yellow power; [R]27 -70.0 (c 0.001,
D
MeOH); UV (MeOH) λmax (log ε) 212 (3.91), 254 (3.07) nm; CD (c )
1.4 × 10-4 M, MeOH) λmax (∆ε) 255.1 (+1.34), 230.8 (-8.95), 222.8

1370 Journal of Natural Products, 2008, Vol. 71, No. 8
Chuang et al.
(-9.96), 205.1 (-11.39) nm; IR (neat) νmax 3291, 1670, 1524, 1439
All values are given as means ( SD. Data analysis involved one-way
ANOVA with subsequent Scheffe’s test.
1
cm-1; H and 13C NMR, see Table 2; ESIMS/MS m/z 955, 939, 921,
910, 858, 841, 823, 813, 758, 713, 704, 687, 612, 572, 562, 509, 496,
465, 443, 425, 413, 342, 314; HRESIMS m/z 1054.5106 [M + H]+
(calcd. for C51H68N13O12, 1054.5110).
Acknowledgment. This work was supported by a grant from the
National Science Council of the Republic of China. The authors would
like to thank Dr. C.-S. Yang, Taitung District Agricultural Research
and Extension Station, Council of Agriculture, Taiwan, for collection
and identification of A. montana.
Cyclomontanin D (4): white power; [R]27D -41.2 (c 0.001, MeOH);
UV (MeOH) λmax (log ε) 216 (3.89), 255 (3.15), 278 (3.04) nm; CD
(MeOH) λmax (∆ε) 254.7 (+1.67), 221.2 (-10.23), 200.0 (+10.81) nm;
1
IR (neat) νmax 3321, 1676 cm-1; H and 13C NMR data, see Table 3;
Supporting Information Available: ESIMS/MS fragments for 2,
3, and 4. The dose-response results of cyclopeptides 1, 3, 4, and 5 on
TNF-R and IL-6 production within LPS- and Pam3Cys-stimulated
J774A.1 cells. This material is available free of charge via the Internet
at http://pubs.acs.org.
ESIMS/MS m/z 614, 598, 585, 569, 557, 500, 472, 446, 348, 335, 268,
240, 186, 155, 142, 127, 115; HRESIMS m/z 735.3438 [M + Na]+
(calcd. for C34H48N8O9Na, 735.3442).
Hydrolysis and Derivatization of 1-5 (Marfey’s Method).29,30
Compounds 1-5 (0.1 mg) were each dissolved in 6 N HCl (0.5 mL)
in a sealed tube and heated at 130 °C for 12-16 h. After cooling and
drying, the total hydrolysates were dissolved in 300 µL of 1 M NaHCO3
solution and reacted with 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide
(FDAA or Marfey reagent, 1% in acetone, 250 µL) at 50 °C for 1 h.
Finally, the Marfey’s derivatives were analyzed by HPLC (Ascentis
C18, 5 µm, 250 × 4.6 mm i.d.; MeCN/H2O (0.5% TFA) ) 30/70; UV
detection at 340 nm) and compared to Marfey’s derivatives of amino
acid standards.
References and Notes
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Synthesis of 1 (Solid-Phase Synthesis Method).25,26 2-Chlorotrityl
chloride resin was purchased from Aldrich. The Fmoc-L-amino acids
with or without other protecting groups (such as Boc, t-Bu, and trityl
groups) and the coupling reagents (HOBt, HBTU, HATU) were
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various concentrations of cyclopeptides 1, 3, 4, and 5 at 37 °C for 30
min, followed by stimulating with LPS (0.1 µg/mL) for an additional
6 h. TNF-R and IL-6 concentration in culture media were assayed by
enzyme-linked immunosorbent assay (ELISA). In addition, J774A.1
cells were challenged with Pam3Cys (1 µg/mL), and the effect of 1, 3,
4, and 5 on cytokine production was examined as explained above.
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