Y. Kobayashi et al. / Bioorg. Med. Chem. 7 (1999) 2073±2081
2079
Consequently, the erythrocytes undergo colloid-osmotic
lysis. This mode of action of LGA on erythrocytes
accompanied by cell invagination is the ®rst reported
example for natural defense substances.
14-[N-(tert-Butoxycarbonyl)-ꢀ-alanyl]-4,10-bis[(2S)-2-
methylbutyryl]-4,10,14-triazatetradecanal (8). To a solu-
tion of 7 (320 mg, 0.535 mmol) in THF (15 mL) and
MeOH (0.1 mL) was added LiBH (60 mg, 2.8 mmol).
4
After stirring at room temperature overnight, 1 M aqu-
eous potassium sodium tartrate (30 mL) was added.
Extraction with CHCl and concentration aorded a
Experimental
Chemistry
3
crude residue, which was then dissolved in dry CH Cl
2
2
(
added Et N (0.59 mL, 4.2 mmol) and SO -pyridine
10 mL) and DMSO (5 mL). To this solution were
General methods. 13CH I was purchased from Shoko
3
3
3
ꢀ
0 C for 2 h and then at room temperature for 1 h, the
Tsusho, Tokyo, Japan. All other chemicals were pur-
chased from Kanto Chemicals, Tokyo, Japan. NMR
spectra were recorded on a Bruker AM-500 spectro-
complex (307 mg, 1.93 mmol) at 0 C. After stirring at
ꢀ
reaction mixture was diluted with EtOAc (30 mL), and
washed successively with H O, 5% aqueous KHSO ,
saturated aqueous NaHCO , and brine. Concentration
1
13
meter at 500 MHz for H, 125 MHz for C with CHCl3
3
2
4
1
at 7.24 ppm, CHD OD at 3.30 ppm, CDCl at 77.0
2
3
3
1
3
ppm, or CD OD at 49.0 ppm. NMR data are those of
3
and ¯ash chromatography on silica gel (10 g, CHCl3:
MeOH, 95:5) aorded 225.4 mg (76%) of 8 as a color-
equilibrated mixtures of conformers due to amide/ure-
thane bond rotation. Reactions requiring anhydrous con-
ditions were carried out in dry, freshly distilled solvents
under a nitrogen or argon atmosphere.
2
4
ꢀ
less oil: [a] +23.4 (c 1.39, MeOH); IR (®lm) 3315,
d
�
1 1
1710, 1625 cm ; H NMR (CDCl , 500 MHz) d 9.7±9.6
3
(m, 1H), 7.2±7.0 (br, 1H), 5.4±5.3 (br, 1H), 3.62±3.30
(m, 12H), 2.77±2.60 (m, 2H), 2.55±2.40 (m, 2H), 2.35
(m, 2H), 1.80±1.45 (m, 8H), 1.36 (m, 2H), 1.35 (brs,
Isolation and puri®cation of LGA (1). LGA was isolated
from the skin mucus of Aulacocephalus temmincki, a
species of soap®sh (Grammistidae), as reported pre-
9H), 1.02 (m, 6H), 0.80 (m, 6H); 13C NMR (CDCl , 125
3
MHz) 200.8, 199.4, 177.6, 176.8, 176.5, 176.5, 171.7,
156.0, 79.0, 48.4, 47.3, 45.5, 43.9, 43.0, 42.4, 40.5, 37.4,
37.3, 37.3, 37.2, 36.8, 36.2, 35.5, 29.6, 29.4, 29.2, 28.4,
27.3, 27.3, 24.1, 18.0, 17.9, 17.8, 17.7, 12.1, 12.1, 12.0.
2
1
viously. Brie¯y, the acetone extract of mucus scraped
from the ®sh skin was partitioned between water and
ethyl acetate, and the ethyl acetate layer was fractionated
through an alumina column (chloroform:methanol, 98:2
to 92:8) followed by repeated reversed-phase HPLC on
ODS to yield LGA (0.5% yield based on wet mucus).
8,14-Bis[(2S)-2-methylbutyryl]-4,8,14-triaza-17-hepta-
decanolactam (2). A solution of aldehyde 8 (28.9 mg,
0
.052 mmol) in dry CH Cl (1 mL) and TFA (1 mL)
2 2
N-(tert-Butoxycarbonyl)-ꢀ-alanine (6). b-Alanine (1.78
was stirred at room temperature for 20 min. After
removal of the solvents, the residue was dissolved in dry
MeOH (10 mL). MS3A (1 g) and Et N (14 mL) were
g, 20 mmol) was dissolved in dioxane:H O (2:1) (60
mL). To this solution were added 1 M aqueous NaOH
2
3
(
20 mL) and Boc O (4.7 mL, 20 mmol). The reaction
2
added to give pH 7. After stirring at room temperature
for 45 min, NaBH CN (12 mg, 0.19 mmol) was added.
mixture was stirred at room temperature for 30 min.
After evaporation of the solvent, the residue was adjus-
ted to pH 3 with 5% aqueous KHSO , and extracted
with EtOAc. Concentration and recrystallization from
hexane±EtOAc aorded 6 (3.25 g, 87%) as white nee-
3
The mixture was stirred at room temperature overnight,
and ®ltered through a Celite pad, followed by the addi-
tion of saturated aqueous NaHCO . After insoluble
4
3
materials were ®ltered o, the reaction mixture was
dried over Na SO , and the concentrated crude product
was chromatographed through an alumina column (neu-
1
dles: H NMR (CDCl , 500 MHz) d 11±8.5 (br, 1H),
3
3
2
4
13
.36 (br, 2H), 2.54 (br, 2H), 1.40 (brs, 9H); C NMR
(
CDCl , 125 MHz) 177.4, 156.0, 79.7, 35.9, 34.4, 28.3.
3
tral, activity II, 10 g, CHCl :MeOH, 9:1) and further
3
puri®ed by HPLC (SiO , YMC A-024, CHCl :MeOH:
3
2
Ethyl 14-[N-(tert-butoxycarbonyl)-ꢀ-alanyl]-4,10-bis[(2S)-
2-methylbutyryl]-4,10,14-triazatetradecanoate (7). To a
ꢁ
solution of crude 5 HCl (41.9 mg, 0.090 mmol) in dry
i-PrNH , 9:1:0.2) to aord 3.0 mg (14%) of 2 as a color-
2
1
less oil: H NMR (CDCl , 500 MHz) d 4.09 and 3.99
3
(q each, 2H, J=7 Hz), 3.45 (m, 2H), 3.40 (m, 2H), 3.35±
3.13 (m, 4H), 2.58 (m, 2H), 2.54±2.36 (m, 2H), 1.63±1.40
(m, 6H), 1.30 (m, 2H), 1.20 (m, 2H), 1.15 and 1.12 (t
each, 3H, J=7 Hz), 0.97 (m, 6H), 0.74 (m, 6H); MS (EI)
m/z (rel. intensity) 438 (32), 423 (7), 381 (13), 353 (100),
CH Cl (2 mL) were added Et N (15 mL, 0.11 mmol),
2
2
3
HOBt (21 mg, 0.14 mmol), WSCI (30 mg, 0.16 mmol),
and N-Boc-b-alanine (6) (23 mg, 0.12 mmol). After
stirring at room temperature overnight, the solvent was
evaporated. The crude residue was dissolved in EtOAc,
+
57 (87); HRMS (EI) m/z calcd for C H N O (M )
3
24
46
4
and washed successively with 5% aqueous KHSO , satu-
4
438.3570, found 438.3610.
rated aqueous NaHCO , and brine. Concentration and
3
column chromatography of the residue on silica gel (2 g,
CHCl :MeOH, 98:2) aorded 7 (35.3 mg, 65%) as a col-
Preparation of acetylated analogue (3) from LGA. To a
solution of LGA (295 mg, 0.5 mmol) in dry pyridine (250
3
2
7
ꢀ
732, 1713, 1641, 1633 cm ; H NMR (CDCl , 270
ꢀ
stirring at room temperature for 15 h, complete removal
orless oil: [a] +15.0 (c 0.753, CHCl ); IR (®lm) 3315,
mL) was added Ac O (10 mL, 100 mmol) at 0 C. After
d
3
2
�
1
1
1
3
MHz) d 7.03 (br, 1H), 4.13 and 4.09 (each q, 2H, J=7.0
Hz), 3.57 (m, 2H), 3.42±3.07 (m, 10H), 2.57 and 2.53 (each
t, 2H, J=7 Hz), 2.60±2.42 (m, 2H), 2.38 (t, 2H, J=7 Hz),
of the solvent and ¯ash chromatography on silica gel
(100 mg, CHCl :MeOH, 95:5) aorded 308 mg (97%) of
3
3 as a yellow oil. HRMS (FAB, 3-nitrobenzylalcohol
+
1
1
.70±1.50 (m, 8H), 1.39 (s, 9H), 1.25 (m, 2H), 1.25 and
.21 (each t, 3H, J=7.0 Hz), 1.07 (m, 6H), 0.85 (m, 6H).
matrix) m/z calcd for C H N O ([M+H] ) 633.5319,
4
3
7
69
4
1
found 633.5292. Acetyl signals in H NMR of the acetyl