940 Journal of Natural Products, 2005, Vol. 68, No. 6
Notes
40% B in 30 min), with the retention time (tR) as specified for
each compound.
collected by centrifugation, washed with H2O (3 mL × 3), and
dissolved in MeOH/CH2Cl2 (1/2). To this solution was added
Si gel (2 g), and the solvents were evaporated. The Si gel was
placed on the top of a Si gel column, and the product was
eluted with MeOH/CH2Cl2 (0/100 f 2/98 f 5/95) to give pure
1 (20 mg, 56%) as a white fluffy solid. A sample of the latter
material (5 mg) was crystallized from MeOH/CHCl3/H2O: mp
256-257 °C; UV (MeOH) λmax 194, 249, 337; 1H NMR (600
MHz, MeOD/CDCl3/D2O, 50/50/5 v/v/v) δ 9.37 (1H, s, H-7), 7.69
(2H, AA′BB′, apparent J ) 8.7 Hz, H-3′′ H-5′′), 6.82 (2H,
AA′BB′, apparent J ) 8.7 Hz, H-2′′ H-6′′), 3.77 (3H, s, Me-1),
3.56 (3H, s, Me-3), 3.52 (2H, t, J ) 6.6 Hz, H-1′), 3.44 (2H, t,
J ) 6.6 Hz, H-4′), 1.75 (4H, br m, H-2′ H-3′); 13C NMR (150.9
MHz, DMSO-d6) δ 169.5 (C-7 ′′), 163.6 (C-9), 161.7 (C-4), 161.0
(C-1′′), 151.3 (C-2), 150.1 (C-8a), 148.4 (C-7), 140.8 (C-6), 129.7
(C-3′′, C-5′′), 126.1 (C-4′′), 125.9 (C-4a), 115.7 (C-2′′, C-6′′), 40.1
(C-4′), 40.0 (C-1′), 30.1 (C-3), 29.4 (C-1), 27.4, 27.3 (C-2′, C-3′);
ESIMS m/z 427.1 [M + H]+, 449.2 [M + Na]+, (negative mode)
425.1 [M - H]-; HRMS m/z 426.1643 (calcd for C20H22N6O5
426.1652); anal. C 56.18%, H 5.42%, N 19.37%, calcd for
C20H22N6O5 C 56.33%, H 5.20%, N 19.71%; RP HPLC tR 20
min.
Biological Material. Crude extract from leeches was
obtained as previously described.7 A total of 300 g of cut heads
from frozen L. nilotica was extracted first with 94% ethyl
alcohol at room temperature (3 × 1.5 L) and subsequently with
H2O (3 × 1.2 L). The combined EtOH and H2O fractions were
directly used in the next step.
Isolation of 1 and 2 from the Crude Extract. The crude
extract (obtained from 300 g of leeches) was evaporated to
dryness. The residue (0.8 g) was redissolved in MeOH/H2O,
1/1 (100 mL), Si gel (15 g) was added, and the solvents were
evaporated. The resulting powder was applied to a Si gel (30
g) column. Elution with EtOAc/MeCN/H2O (first from 100/0/0
to 75/25/0 and then from 120/90/10 to 20/130/50) afforded a
crude mixture of 1 and 2. Purification of the latter mixture
with semipreparative RP HPLC (Platinum, 10.0 × 250 mm, 5
µm particle size, Alltech) applying an appropriate gradient of
MeCN in 0.1% aqueous TFA afforded pure 1 (210 µg) and 2
(196 µg) as white amorphous solids.
1-N-(4-Hydroxybenzoyl)-1,4-diaminobutane (8). Pu-
trescine (6) (0.88 g, 10 mmol) was dissolved in DMF (50 mL),
and a mixture of BOP (1.1 g, 2.5 mmol) and p-hydroxybenzoic
acid (0.28 g, 2 mmol) in DMF (10 mL) was added. The
precipitation of a white crystalline material resulted. The
mixture was heated to 60 °C to obtain a clear solution. After
2 h at that temperature the solution was cooled in ice, filtered,
and concentrated in high vacuum. The oily, slightly yellowish
residue was dissolved in 1 N HCl (50 mL), evaporated to
dryness, redissolved in 1 N HCl (20 mL), and applied to a
Dowex-H+ column (4 × 10 cm). The resin was washed with
H2O (300 mL), MeOH (300 mL), and H2O (400 mL), and then
the product was eluted with 25% NH4OH/H2O (1/7). Evapora-
tion of the solvents resulted in a white amorphous solid, which
was subjected to column chromatography on Si gel. Elution
with MeOH/CH2Cl2/NH4OH (2/4/1) gave 8 as a white crystal-
line solid, which was dried, dissolved in H2O, and applied to a
Dowex-2 (OH-) column (2 × 10 cm). The column was washed
with H2O (400 mL), 25% NH4OH (300 mL), and H2O to
neutrality. The product was eluted with 1 N HCl (500 mL)
and next with H2O/MeOH (1/1, 200 mL). Pure 8 was thus
obtained as the hydrochloride salt (110 mg, 22%): 1H NMR
(300 MHz, MeOD/CDCl3/D2O, 50/50/5 v/v/v) δ 7.7 (2H, AA′BB′,
apparent J ) 8.8 Hz, H-3′ H-5′), 6.83 (2H, AA′BB′, apparent
J ) 8.8 Hz, H-2′ H-6′), 3.37 (2H, t, J ) 6.4 Hz, H-1), 2.97 (2H,
t, J ) 7.1 Hz, H-4), 1.68 (4H, br m, H-2, H-3); 13C NMR (50.1
MHz, CD3OD) δ 170.8 (C-7′), 162.5 (C-1′), 130.5 (C-3′, C-5′),
124.4 (C-4′), 116.1 (C-2′, C-6′), 40.4, 40.2 (C-1, C-4), 27.0, 25.6
(C-2, C-3); ESIMS m/z 209.2 (M + H)+, 231.1 (M + Na)+;
HRMS m/z 208.1207 (calcd for C11H16N2O2, 208.1212).
1-N-(4-Hydroxybenzoyl)-1,5-diaminopentane (9). Com-
pound 9 was prepared from cadaverine (7) (10 mmol, 1.2 mL)
and p-HBA (0.28 g, 2 mmol) as described for 8 [yield 78 mg
(15%) as the hydrochloride salt]: 1H NMR (300 MHz, MeOD/
CDCl3/D2O, 50/50/5 v/v/v) δ 7.57 (2H, AA′BB′, apparent J )
8.8 Hz, H-3′ H-5′), 6.66 (2H, AA′BB′, apparent J ) 8.8 Hz,
H-2′ H-6′), 3.32 (2H, t, J ) 6.4 Hz, H-1), 2.95 (2H, t, J ) 7.3
Hz, H-5), 1.60 (4H, br m, H-2 H-4), 1.41 (2H, br m, H-3); 13C
NMR (50.1 MHz, CDCl3) δ 170.6 (C-7′), 159.6 (C-1′), 130.1 (C-
3′, C-5′), 125.8 (C-4′), 115.8 (C-2′, C-6′), 40.1, 40.0 (C-1, C-5),
28.7, 27.1 (C-2, C-4), 23.7 (C-3); ESIMS m/z 223.1 (M + H)+,
245.1 (M + Na)+; HRMS m/z 222.1631 (calcd for C12H18N2O2,
222.1638).
1,3-Dimethyllumazine-7-carboxylic acid (4-(4-hydroxy-
benzoylamino)butyl)amide (12). 12 was prepared and
purified as described for 1 from 1,3-dimethyllumazine-7-
carboxylic acid8 (11) (20 mg, 85 µmol). Yield: 23 mg (65%),
white solid. An analytical sample was crystallized from MeOH/
CHCl3/H2O to give colorless needles: mp 239-240 °C; 1H NMR
(600 MHz, DMSO-d6) δ 9.14 (1H, br t, NH), 9.00 (1H, s H-6),
8.28 (1H, br t, NH), 7.66 (2H, AA′BB′, apparent J ) 8.6 Hz,
H-3′′ H-5′′), 6.75 (2H, AA′BB′, apparent J ) 8.6 Hz, H-2′′ H-6′′),
3.62 (3H, s, Me-1), 3.35 (2H, br dd, H-1′), 3.32 (3H, s, Me-3),
3.23 (2H, br dd, H-4′), 1.45 (4H, br m, H-2′ H-3′); 13C NMR
(150.9 MHz, DMSO-d6) δ 166.5 (C-7′′), 162.3 (C-9), 160.3 (C-
1′′), 159.7 (C-4), 150.9 (C-2), 147.1 (C-8a), 146.0 (C-7), 137.7
(C-6), 130.3 (C-4a), 129.4 (C-3′′, C-5′′), 125.5 (C-4′′), 115.1 (C-
2′′, C-6′′), 40.1 (C-4′), 40.0 (C-1′), 29.6 (C-3), 28.9 (C-1), 27.1,
27.0 (C-2′, C-3′); ESIMS m/z 427.1 (M + H)+, 449.2 (M + Na)+;
(negative mode) 425.1 (M - H)-; HRMS m/z 426.1649 (calcd
for C20H22N6O5 426.1652); anal. C 56.13%, H 5.43%, N 19.45%,
calcd for C20H22N6O5 C 56.33%, H 5.20%, N 19.71%; RP HPLC
tR 19 min.
1,3-Dimethyllumazine-6-carboxylic acid (4-(4-hydroxy-
benzoylamino)pentyl)amide (2). 2 was prepared as de-
scribed for 1 employing 1-N-(4-hydroxybenzoyl)-1,5-diamino-
pentane (9) and 1,3-dimethyllumazine-6-carboxylic acid (10)
(20 mg, 85 µmol). Purified with column chromatography
eluting with MeOH/CH2Cl2 (0/100 f 2/98 f 5/95), appropriate
fractions were collected and evaporated and the residue was
washed with H2O (2 mL) to give 2 (14 mg, 40%) as a beige
amorphous powder: mp 268-270 °C (dec); UV (MeOH) λmax
194, 249, 337; 1H NMR (600 MHz, MeOD/CDCl3/D2O, 50/50/5
v/v/v) δ 9.36 (1H, s, H-7), 7.62 (2H, AA′BB′, apparent J ) 8.8
Hz, H-3′′ H-5′′), 6.79 (2H, AA′BB′, apparent J ) 8.8 Hz, H-2′′
H-6′′), 3.77 (3H, s, Me-1), 3.56 (3H, s, Me-3), 3.50 (2H, t, J )
6.8 Hz, H-1′), 3.39 (2H, t, J ) 6.8 Hz, H-5′), 1.71 (4H, br m,
H-2′, H-4′), 1.49 (2H, br m, H-3′); ESIMS m/z 441.4 (M + H)+,
463.2 (M + Na)+; (negative mode) 439.2 (M - H)-; anal. C
57.03%, H 5.57%, N 18.91%, calcd for C21H24N6O5 C 57.26%,
H 5.49%, N 19.08%; HRMS m/z 440.1816 (calcd for C21H24N6O5
440.1808); RP HPLC tR 22.4 min.
1,3-Dimethyllumazine-7-carboxylic acid (4-(4-hydroxy-
benzoylamino)pentyl)amide (13). 13 was prepared and
purified as described for 2, starting from 1,3-dimethyllu-
mazine-7-carboxylic acid (11) (5 mg, 21 µmol). This yielded 4
mg of 13 (43%) as an amorphous off-white powder: mp 262-
1,3-Dimethyllumazine-6-carboxylic acid (4-(4-hydroxy-
benzoylamino)butyl)amide (1). 1,3-Dimethyllumazine-6-
carboxylic acid8 (10) (20 mg, 85 µmol) was placed in an
Eppendorf test tube and was dissolved in a 0.25 M solution of
1-N-(4-hydroxybenzoyl)-1,4-diaminobutane (8) (0.5 mL, 125
µmol) in DMF. BOP (66 mg, 150 µmol) and DIPEA (90 µL, 0.5
mmol) were added, and the mixture was thoroughly mixed and
left for 2 h with occasional shaking. Transfer of the mixture
to a 10 mL round-bottom flask and evaporation of the solvent
was followed by addition of solid NaHCO3 (14 mg, 170 µmol)
and H2O (2 mL). The slightly brownish precipitate was
1
264 °C (signs of decomposition); H NMR (600 MHz, DMSO-
d6) δ 9.90 (1H, brs. OH); 9.11 (1H, br t, NH), 8.99 (1H, s H-6),
8.23 (1H, br t, NH), 7.63 (2H, AA′BB′, apparent J ) 8.6 Hz,
H-3′′ H-5′′), 6.73 (2H, AA′BB′, apparent J ) 8.6 Hz, H-2′′ H-6′′),
3.63 (3H, s, Me-1), 3.35 (2H, br dd, H-1′), 3.32 (3H, s, Me-3),
3.23 (2H, br dd, H-5′), 1.55 (4H, br m, H-2′ H-4′); 1.30 (2H, br
m, H-3′); 13C NMR (150.9 MHz, DMSO-d6) δ 166.3 (C-7′′), 162.0
(C-9), 160.1 (C-1′′), 159.6 (C-4), 150.7 (C-2), 147.0 (C-8a), 146.0
(C-7), 137.4 (C-6), 130.2 (C-4a), 129.7 (C-3′′, C-5′′), 125.7 (C-