Cycloheptapeptides from Cordyceps sp.
Journal of Natural Products, 2007, Vol. 70, No. 10 1603
µM, while cordyheptapeptide B (16) was inactive at 10 µg/mL (12
µM). In contrast, both cyclic peptides showed moderate cytotoxicity.
IC50 values of 15 against KB, BC, NCI-H187, and Vero cells were
(1H, s, 9′-O H), 9.47 (1H, s, 9-O H), 6.45 (2H, d, J ) 1.8 Hz, H-7 and
H-7′), 6.11 (1H, d, J ) 1.8 Hz, H-5′), 5.98 (1H, d, J ) 1.8 Hz, H-5),
5
4
4
.16 (1H, d, J ) 15.7 Hz, Ha-1), 4.75 (1H, d, J ) 15.7 Hz, Ha-1′),
.72 (1H, d, J ) 15.7 Hz, Hb-1), 4.63 (1H, d, J ) 15.7 Hz, Hb-1′),
.02 (3H, s) and 4.00 (3H, s) (8-OCH and 8′-OCH ), 3.80 (1H, s,
3 3
0.78, 0.20, 0.18, and 14 µM, respectively. Corresponding IC50 values
of 16 were 2.0, 0.66, 3.1, and 1.6 µM.
H-4), 3.77 (1H, s, H-4′), 3.66 (1H, m, H-3′), 3.64 (1H, m, H-3), 3.46
3H, s, 6-OCH ), 1.23 (3H, d, J ) 6.5 Hz, H-11), 1.21 (3H, d, J ) 6.5
(
3
Experimental Section
13
Hz, H-11′); C NMR (CDCl , 125 MHz) δ 157.7 (s) 157.6 (s) and
3
General Experimental Procedures. Melting points were measured
with an Electrothermal IA9100 digital melting point apparatus and are
uncorrected. Optical rotations were measured with a JASCO P-1030
digital polarimeter. UV spectra were recorded on a Varian CARY 1E
UV–visible spectrophotometer. FT-IR spectra were recorded on a
Bruker VECTOR 22 spectrometer. NMR spectra were recorded on a
Bruker AV500D spectrometer. ESI-TOF mass spectra were measured
with a Micromass LCT mass spectrometer.
Fungal Material. Cordyceps species used in this study were isolated
on Coleoptera larvae collected in Doi Innthanon National Park, Chiang
Mai Province, Thailand, by Ms. Kanoksri Tasanatai of the BIOTEC.
These fungi were deposited in the BIOTEC Culture Collection (BCC)
as BCC 16173 and BCC 16176 on November 12, 2004.
157.4 (s) (C-6, C-8, and C-8′), 154.5 (s, C-6′), 149.9 (s) and 149.8 (s)
(C-9 and C-9′), 136.1 (s, C-10a), 136.1 (s, C-10a′), 135.0 (s, C-4a),
134.8 (s, C-4a′), 123.9 (s, C-10), 123.1 (s, C-10′), 113.3 (s) and 113.2
(s) (C-9a and C-9a′), 110.3 (s, C-8a), 109.8 (s, C-8a′), 101.8 (d, C-5′),
98.4 (d, C-7), 98.1 (d, C-7′), 97.9 (d, C-5), 74.0 (d, C-3′), 73.8 (d,
C-3), 66.5 (d, C-4), 66.5 (d, C-4′), 65.0 (t, C-1), 64.5 (t, C-1′), 56.3 (q)
and 56.2 (q) (8-OCH
16.8 (q) (C-11 and C-11′); HRMS (ESI-TOF) m/z 587.1902 [M + Na]
(calcd for C31 10Na, 587.1893).
Compound 2: pale yellow solid; [R]
NMR (CDCl , 500 MHz) δ 9.53 (1H, s) and 9.47 (1H, s) (9-OH and
3 3 3
and 8′-OCH ), 55.2 (q, 6-OCH ), 16.9 (q) and
+
32
H O
2
6
1
D
-69 (c 0.05, MeOH); H
3
9′-OH), 6.49 (1H, d, J ) 2.0 Hz, H-7′), 6.48 (1H, d, J ) 2.1 Hz, H-7),
5.94 (1H, d, J ) 2.0 Hz, H-5′), 5.89 (1H, d, J ) 2.1 Hz, H-5), 5.22
(1H, d, J ) 15.9 Hz) and 5.18 (1H, d, J ) 15.8 Hz) (Ha-1 and Ha-1′),
4.92 (1H, d, J ) 15.9 Hz) and 4.80 (1H, d, J ) 15.8 Hz) (Hb-1 and
Fermentation of BCC 16173, Extraction, and Isolation.
Cordyceps sp. BCC 16173 was maintained on potato dextrose agar at
2
5 °C. The agar was cut into pieces (1 × 1 cm) and inoculated into 6
250 mL Erlenmeyer flasks containing 25 mL of potato dextrose broth
PDB; potato starch 4.0 g, dextrose 20.0 g, per liter). After incubation
Hb-1′), 4.09 (3H, s) and 4.07 (3H, s) (8-OCH
(2H, br s, H-4 and H-4′), 3.67–3.66 (2H, m, H-3 and H-3′), 3.47 (3H,
s, 6-OCH ), 1.24 (3H, d, J ) 6.4 Hz) and 1.23 (3H, d, J ) 6.4 Hz)
(H-11 and H-11′); C NMR (CDCl
3 3
and 8′-OCH ), 3.88
×
(
3
1
3
at 25 °C for 7 days on a rotary shaker (200 rpm), each primary culture
was transferred into a 1 L Erlenmeyer flask containing 250 mL of the
same liquid medium (PDB) and incubated at 25 °C for 7 days on a
rotary shaker (200 rpm). Each 25 mL portion of the secondary cultures
3
, 125 MHz) δ 157.9 (s) 157.7 (s)
and 157.5 (s) (C-6, C-8, and C-8′), 153.9 (s, C-6′), 149.9 (s) and 149.7
(s) (C-9 and C-9′), 137.6 (s, C-4a), 137.6 (s, C-4a′), 135.6 (s, C-10a),
135.6 (s, C-10a′), 123.6 (s) and 123.0 (s) (C-10 and C-10′), 114.1 (s)
and 113.8 (s) (C-9a and C-9a′), 110.4 (s, C-8a), 110.4 (s, C-8a′), 100.8
(d, C-5′), 98.1 (d, C-7), 97.6 (d, C-7′), 97.3 (d, C-5), 73.4 (d) and 732
(d) (C-3 and C-3′), 65.6 (d) and 66.5 (d) (C-4 and C-4′), 65.1 (t) and
(
in 6 flasks) was transferred into 60 × 1 L Erlenmeyer flasks each
containing 250 mL of yeast extract sucrose medium (YES; yeast extract
0 g, sucrose 150 g, per liter), and final fermentation was carried out
2
at 25 °C for 24 days under static conditions. The cultures were filtered
to separate mycelia and filtrate. Mycelia were macerated in MeOH (2.5
64.5 (t) (C-1 and C-1′), 56.4 (q) and 56.3 (q) (8-OCH
55.2 (q, 6-OCH ), 17.1 (q) and 17.0 (q) (C-11 and C-11′); HRMS (ESI-
TOF) m/z 587.1909 [M + Na] (calcd for C31
Compound 3 (6′-O-desmethyl ES-242-2): pale yellow solid; mp
3 3
and 8′-OCH ),
3
+
L, rt, 2 days), then filtered. To the filtrate were added H
2
O (200 mL)
32
H O10Na, 587.1893).
and hexane (600 mL). The aqueous MeOH layer was concentrated under
reduced pressure. The residue was dissolved in EtOAc, washed with
2
6
190–192 °C (dec); [R]
D
+50 (c 0.05, MeOH); IR (KBr) νmax 3407,
-1 1
H
(
(
2
O, and concentrated under reduced pressure to leave a brown solid
4.60 g). This crude extract was subjected to column chromatography
CC) on silica gel (5.5 × 20 cm, MeOH/CH Cl , step gradient elution
1737, 1625, 1364, 1230, 1095 cm ; H NMR (CDCl , 500 MHz) δ
3
9.48 (1H, s) and 9.47 (1H, s) (9-OH and 9′-OH), 6.45 (1H, d, J ) 2.2
Hz, H-7′), 6.44 (1H, d, J ) 2.2 Hz, H-7), 5.89 (2H, m, H-5 and H-5′),
5.41 (2H, m, H-4 and H-4′), 5.28 (1H, d, J ) 15.6 Hz) and 5.23 (1H,
d, J ) 15.6 Hz) (Ha-1 and Ha-1′), 4.87 (1H, d, J ) 15.6 Hz) and 4.85
2
2
from 0:100 to 15:85) to obtain seven fractions (1–7): 1 (1780 mg), 2
77 m g), 3 (23 mg), 4 (20 mg), 5 (19 mg), 6 (81 mg), and 7 (352 mg).
Fraction 7 contained mostly 15, which was further purified by silica
gel CC (elution with MeOH/CH Cl , 252 mg). Each of the other
fractions was repeatedly fractionated by CC on silica gel (MeOH/
CH Cl ) and preparative HPLC using a reversed-phase column (Nova-
Pak HR C18, 2.5 × 10 cm, 6 µm; mobile phase MeCN/H O, 45:55 or
0:60; flow rate 8 mL/min) to furnish 14 compounds: 1 (54.9 mg, from
fraction 1), 2 (1.7 mg, from fraction 6), 3 (37.2 mg, from fraction 1),
(
(1H, d, J ) 15.6 Hz) (Hb-1 and Hb-1′), 4.07 (3H, s, 8′-OCH
(3H, s, 8-OCH ), 3.97 (1H, m) and 3.95 (1H, m) (H-3 and H-3′), 3.42
(3H, s, 6-OCH ), 1.16 (3H, s) and 1.14 (3H, s) (4-OCOCH and 4′-
OCOCH ), 1.10 (3H, d, J ) 6.4 Hz) and 1.09 (3H, d, J ) 6.4 Hz)
(H-11 and H-11′); C NMR (CDCl
COCH ), 169.0 (s, 4′-OCOCH ), 157.5 (s, C-8′), 157.1 (s, C-8), 156.9
3
), 4.05
2
2
3
3
3
2
2
3
1
3
2
3
, 125 MHz) δ 169.0 (s, 4-O-
4
3
3
(s, C-6), 153.1 (s, C-6′), 149.7 (s, C-9), 149.7 (s, C-9′), 135.5 (s, C-10a),
135.5 (s, C-10a′), 131.1 (s) and 130.9 (s) (C-4a and C-4a′), 125.0 (s)
and 124.3 (s) (C-10 and C-10′), 115.6 (s) and 115.3 (s) (C-9a and C-9a′),
110.4 (s) and 110.1 (s) (C-8a and C-8a′), 102.2 (d, C-5′), 98.7 (d, C-5),
97.9 (d, C-7), 97.2 (d, C-7′), 73.3 (d) and 73.2 (d) (C-3 and C-3′), 66.7
4
6
(13.7 mg, from fractions 1 and 2), 5 (36.2 mg, from fractions 2 and
), 6 (ES-242-4; 330.2 mg, from fractions 1 and 2), 7 (ES-242-5; 48.9
mg, from fraction 1), 8 (19.5 mg, from fractions 2, 3, and 4), 9 (ES-
42-2; 523 mg, from fractions 1 and 2), 10 (ES-242-3; 28.3 mg), 11
ES-242-1; 31.5 mg) and 12 (24.9 mg) from fraction 1, 13 (5.0 mg,
2
(
(d, C-4), 66.7 (d, C-4′), 65.0 (t, C-1), 65.0 (t, C-1′), 56.3 (q, 8-OCH
56.3 (q, 8′-OCH ), 55.1 (q, 6-O CH ), 19.2 (q) and 19.0 (q) (4-OCOCH
and 4′-OCOCH ), 16.8 (q) and 16.7 (q) (C-11 and C-11′); HRMS (ESI-
TOF) m/z 671.2112 [M + Na] (calcd for C35
3
),
from fraction 6), and 14 (ES-242-8; 4.9 mg, from fraction 5).
3
3
3
Fermentation of BCC 16176, Extraction, and Isolation. BCC
3
+
1
6176 was fermented and extracted in the same way and scale as
described for BCC 16173. The mycelium extract (2.73 g) was subjected
to column chromatography on silica gel (5.5 × 20 cm, MeOH/CH Cl
step gradient elution from 0:100 to 20:80) to separate into 12 fractions
36
H O12Na, 671.2104).
2
7
Compound 4: pale yellow solid; mp 178–180 °C (dec); [R]
D
+49
2
2
,
(c 0.05, MeOH); UV (MeOH) λmax (log ε) 236 (4.72), 294 sh (4.14),
308 sh (4.10), 354 (3.89) nm; IR (KBr) νmax 3385, 1625, 1361, 1156,
1090, 978, 829 cm ; H NMR (CDCl , 500 MHz) δ 9.61 (1H, d, J )
3
-
1 1
(
(
1–12): 1 (128 mg), 2 (1193 mg), 3 (33 mg), 4 (79 mg), 5 (18 mg), 6
11 mg), 7 (222 mg), 8 (303 mg), 9 (49 mg), 10 (41 mg), 11 (38 mg),
1.6 Hz, 9-OH), 9.51 (1H, br s, 9′-OH), 6.78 (1H, s, H-7′), 6.48 (1H, d,
J ) 2.1 Hz, H-7), 6.41 (1H, s, H-10′), 6.02 (1H, d, J ) 2.1 Hz, H-5),
5.09 (1H, m, Ha-1), 5.07 (1H, dd, J ) 15.7, 2.9 Hz, Ha-1′), 4.82 (1H,
dd, J ) 15.4, 2.0 Hz, Hb-1), 4.71 (1H, d, J ) 15.7 Hz, Hb-1′), 4.14
and 12 (110 mg). Fraction 1 was mainly 9 (ES-242-2). Fraction 2 was
chromatographed on silica gel (4.0 × 20 cm, MeOH/CH
2
Cl , step
2
gradient elution from 0:100 to 15:85) to furnish 10 (ES-242-1, 5.3 mg,
fraction 2-1), 9 (ES-242-2, 793 mg, fraction 2-2), and a peptide-
containing subfraction (7.9 mg, fraction 2-9). The subfraction 2-9 was
(3H, s, 8′-OCH
(1H, br s, H-4), 3.75 (1H, m, H-3), 3.72 (1H, m, H-3′), 3.45 (3H, s,
6-OCH ), 1.32 (3H, d, J ) 6.3 Hz, H-11′), 1.25 (3H, d, J ) 6.4 Hz,
3 3
), 4.08 (3H, s, 8-OCH ), 4.03 (1H, br s, H-4′), 3.88
subjected to preparative HPLC (MeCN/H
2
O ) 40:60) to yield 16 (3.3
3
1
3
mg). Additional 16 (3.0 mg) was obtained from fraction 3 by silica gel
CC and preparative HPLC. Fractions 4 and 9 were combined and
purified by CC on silica gel to obtain 15 (603 mg).
3
H-11); C NMR (CDCl , 125 MHz) δ 157.8 (s, C-6), 157.3 (s, C-8),
157.4 (s, C-8′), 152.7 (s, C-6′), 150.4 (s, C-9), 149.6 (s, C-9′), 137.4
(s, C-4a), 137.0 (s, C-4a′), 136.2 (s) and 135.6 (s) (C-10a and C-10a′),
120.1 (s, C-10), 116.1 (d, C-10′), 113.7 (s, C-9a′), 113.2 (s, C-9a),
111.5 (s, C-5′), 110.7 (s, C-8a), 110.2 (s, C-8a′), 98.5 (d, C-7), 98.5
(d, C-7′), 98.2 (d, C-5), 73.7 (d) and 73.6 (d) (C-3 and C-3′), 68.1 (d,
Compound 1 (6′-O-desmethyl ES-242-4): pale yellow solid; mp
2
6
1
1
79–180 °C (dec); [R]
D
-7 (c 0.05, MeOH); IR (KBr) νmax 3406,
623, 1360, 1092, 984, 830 cm- ; H NMR (CDCl
1 1
, 500 MHz) δ 9.53
3