Synthesis of Distamycin A and 2640 Analogues
J. Am. Chem. Soc., Vol. 122, No. 27, 2000 6393
4-[[[4-[[(4-tert-Butyloxycarbonyl)amino-1-methylpyrrol-2-yl]car-
bonyl]amino-1-methylpyrrol-2-yl]carbonyl]amino-1-methyl-2-(((car-
bonyl)amino)propio-3-nitrile)pyrrole (4). Tripyrrole 3 (70 mg, 0.14
mmol, 1 equiv) in THF/MeOH (3:1, 2 mL) was treated with a solution
of LiOH (24 mg, 0.56 mmol, 4 equiv) in H2O (0.5 mL). The solution
was warmed at 60 °C for 5 h and then diluted with EtOAc (20 mL)
and H2O (20 mL). The layers were separated, and the aqueous layer
was brought to pH 3 with 10% aqueous HCl. The resulting slurry was
extracted with EtOAc (4 × 20 mL), and the combined organic extracts
were dried (Na2SO4), filtered, and concentrated. The crude acid (30
mg, 0.062 mmol) in DMF (1 mL) was treated with EDCI (23 mg, 0.12
mmol, 2 equiv) and DMAP (19 mg, 0.16 mmol, 2.5 equiv), followed
by 3-aminopropionitrile (13 mg, 0.12 mmol, 2 equiv). The reaction
mixture was stirred for 14 h at 25 °C and then diluted with EtOAc (20
mL) and washed with 10% aqueous HCl (3 × 20 mL) and saturated
aqueous NaHCO3 (3 × 20 mL). The organic phase was dried (Na2-
SO4), filtered, and concentrated to afford 4 (31 mg, 95%) as a yellow
the assay of a library of compounds against a library of DNA
hairpin oligonucleotides, with automation of the assay, provides
qualitative and/or quantitative information on the binding of all
library members against a library of available sequences. Studies
on extensions of this work are in progress and will be disclosed
in due time.
Experimental Section
Methyl 4-[[(4-tert-Butyloxycarbonyl)amino-1-methylpyrrol-2-yl]-
carbonyl]amino-1-methylpyrrole-2-carboxylate (2). Initial condi-
tions: a solution of 1a (250 mg, 1.05 mmol, 1 equiv) and 1b (200 mg,
1.05 mmol, 1 equiv) in DMF (5 mL) was treated with EDCI (403 mg,
2.1 mmol, 2 equiv) and DMAP (320 mg, 2.6 mmol, 2.5 equiv) and the
resulting solution was stirred for 14 h at 25 °C. The reaction mixture
was poured into EtOAc (50 mL) and washed with 10% aqueous HCl
(3 × 50 mL) and saturated aqueous NaHCO3 (3 × 50 mL). The organic
phase was dried (Na2SO4), filtered, and concentrated to provide 2 (350
mg, 89%) as a tan foam. For optimized large scale:41 A solution of 1a
(3.8 g, 15.8 mmol, 1 equiv) and 1b (3.0 g, 15.8 mmol, 1 equiv) in
DMF (40 mL) and CH2Cl2 (10 mL) was treated with EDCI (4.5 g,
23.5 mmol, 1.5 equiv) and DMAP (2.3 g, 18.9 mmol, 1.2 equiv), and
the resulting solution was stirred for 18 h at 25 °C. The reaction mixture
was poured into EtOAc (60 mL) and washed with 10% aqueous HCl
(3 × 50 mL) and saturated aqueous NaHCO3 (3 × 50 mL). The organic
phase was dried (Na2SO4), filtered, and concentrated to provide 2 (5.8
1
solid: mp 170-172 °C; H NMR (DMSO-d6, 500 MHz) δ 9.93 (s,
1H), 9.87 (s, 1H), 9.10 (s, 1H), 8.35 (t, 1H, J ) 5.6 Hz), 7.23 (d, 1H,
J ) 1.5 Hz), 7.22 (d, 1H, J ) 1.5 Hz), 7.00 (d, 1H, J ) 1.5 Hz), 6.93
(d, 1H, J ) 1.9 Hz), 6.89 (s, 1H), 6.84 (s, 1H), 3.84 (s, 3H), 3.81 (s,
6H), 3.40 (q, 2H, J ) 6.2 Hz), 2.73 (t, 2H, J ) 6.4 Hz), 1.46 (s, 9H);13C
NMR (acetone-d6, 125 MHz) 162.6, 160.0, 159.6, 153.8, 124.5, 124.0,
123.8, 123.6 (2C), 123.5, 119.3, 119.2, 119.1, 117.8, 104.8, 104.7,
103.9, 79.3, 36.6 (2C), 36.5, 36.1, 28.5, 18.6; IR (film) νmax 3318, 2978,
1698, 1644, 1586, 1537 cm-1; MALDIHRMS (DHB) m/z 559.2387
(M + Na+, C26H32N8O5 requires 559.2393).
1
g, 97%): mp 78-79 °C; H NMR (DMSO-d6, 500 MHz) δ 10.07 (s,
1H), 9.87 (s, 1H), 7.46 (s, 1H), 7.11 (s, 1H), 6.96 (s, 1H), 6.90 (s, 1H),
3.88 (s, 3H), 3.84 (s, 3H), 3.73 (s, 3H); 13C NMR (acetone-d6, 125
MHz) 161.9, 159.5, 153.8, 124.1, 123.9, 123.8, 121.3, 120.0, 117.8,
108.9 (2C), 103.9, 79.2, 51.1, 36.7, 36.5, 28.5; IR (film) νmax 3328,
2954, 1694, 1644, 1586 cm-1; MALDIHRMS (DHB) m/z 399.165 (M
+ Na+, C18H24N4O5 requires 399.1644).
Distamycin A Hydrochloride. A solution of nitrile 4 (12 mg, 0.022
mmol) in anhydrous EtOH (0.3 mL) was treated with 8.0 N HCl/EtOH
(1 mL) at 0 °C for 30 min and then slowly warmed to 25 °C and stirred
for 2 h. The solvent was removed under a stream of N2, and the residue
was washed with Et2O (3 mL) and dried in vacuo for 30 min. The
resulting solid was taken up in EtOH (0.3 mL) and treated with 7%
NH3/EtOH (1 mL) at 25 °C. After 1 h the reaction was concentrated to
a tan solid and dried under reduced pressure for 1 h. The crude amidine
was dissolved in MeOH (0.2 mL) and cooled to -40 °C. The solution
was treated with a solution containing N-formylimidazole, prepared
by treating carbonyldiimidazole (18 mg, 0.11 mmol) in THF (0.4 mL)
with a solution of formic acid (4.3 mL, 0.11 mmol) in THF (0.4 mL)
at 25 °C for 15 min. The reaction mixture was stirred at -40 °C for 1
h and then concentrated to a volume of 0.2 mL. The product was
precipitated with EtOAc (1 mL) and collected by filtration. The crude
product was dissolved in cold i-PrOH (2 mL) containing decolorizing
carbon (100 mg), stirred at 0 °C for 30 min, filtered, and concentrated
to a light yellow solid. The solid material was taken up EtOAc/acetone/
MeOH/0.01 N HCl (5:3:1:1, 2 mL) and stirred with SiO2 for 30 min
and then filtered through Celite to remove traces of NH4Cl and afford
pure distamycin A (4.9 mg, 45%) identical in all respects with authentic
Methyl 4-[[[4-[[(4-tert-Butyloxycarbonyl)amino-1-methylpyrrol-
2-yl]carbonyl]amino-1-methylpyrrol-2-yl]carbonyl]amino-1-meth-
ylpyrrole-2-carboxylate (3). Initial conditions: a sample of 2 (50 mg,
0.13 mmol, 1 equiv) was treated with 4.0 N HCl/EtOAc (1 mL). The
reaction mixture was stirred at 25 °C for 30 min, then concentrated
and dried under reduced pressure for 1 h. EDCI (50 mg, 0.27 mmol, 2
equiv), DMAP (33 mg, 0.27 mmol, 2 equiv), and 1a (63 mg, 0.27
mmol, 2 equiv) were added to a solution of the crude amine in DMF
(1 mL). The reaction mixture was stirred for 3 h at 25 °C, diluted with
EtOAc (10 mL) and washed with 10% aqueous HCl (3 × 10 mL) and
saturated aqueous NaHCO3 (3 × 10 mL). The organic phase was dried
(Na2SO4), filtered, and concentrated to a yellow solid. The solid material
was suspended in 1:1 MeOH/10% NaOH (20 mL) and stirred for 30
min at 25 °C to decompose small amounts of contaminate symmetrical
anhydride. The solution was then poured into EtOAc (20 mL) and
washed with NaHCO3 (3 × 20 mL). The organic phase was dried (Na2-
SO4), filtered, and concentrated to provide 3 (47 mg, 73%) as a yellow
foam. For optimized large scale:41 dipyrrole 2 (2.8 g, 7.43 mmol, 1
equiv) was treated with 4.0 N HCl/EtOAc (20 mL). The reaction
mixture was stirred at 25 °C for 30 min and then concentrated and
dried under reduced pressure. EDCI (2.1 g, 11.1 mmol, 1.5 equiv),
DMAP (1.1 g, 9.0 mmol, 1.2 equiv), and 1a (2.0 g, 8.2 mmol, 1.1
equiv) were added to a solution of the crude amine in DMF (100 mL).
The reaction mixture was stirred for 3 h at 25 °C, diluted with EtOAc
(100 mL), and washed with 10% aqueous HCl (3 × 100 mL) and
saturated aqueous NaHCO3 (3 × 100 mL). The organic phase was dried
(Na2SO4), filtered, and concentrated to provide 3 (3.6 g, 96%) as a
1
material: mp 186-188 °C; H NMR (DMSO-d6, 500 MHz) δ 10.28
(s, 1H), 9.99 (s, 1H), 9.96 (s, 1H), 9.14 (br s, 2H), 8.93 (br s, 2H),
8.29 (t, 1H, J ) 5.6 Hz), 8.11 (s, 1H), 7.25 (s, 1H), 7.21 (d, 1H, J )
1.5 Hz), 7.20 (d, 1H, J ) 1.5 Hz), 7.06 (s, 1H), 6.95 (d, 1H, J ) 1.5
Hz), 6.94 (d, 1H, J ) 1.5 Hz), 3.83 (s, 6H), 3.80 (s, 3H), 3.51 (q, 2H,
J ) 6.0 Hz), 2.66 (t, 2H, J ) 6.4 Hz); 13C NMR (CD3OD, 125 MHz)
171.0, 164.4, 161.4, 161.2, 160.4, 127.7, 124.5, 123.9, 123.4, 123.3,
122.0, 120.9, 120.8, 119.9, 106.7, 106.5, 105.8, 37.5, 36.9, 36.8, 36.7,
34.4; IR (film) νmax 3299, 2975, 1634 cm-1; MALDIHRMS (DHB)
m/z 504.2078 (M +Na+, C22H27N9O4 requires 504.2084).
Ethidium Bromide Assay. DNA hairpin oligonucleotides42 were
purchased from Genbase Inc. (San Diego) as 880 µM (base pairs)
solutions in water and stored as stock solutions at -80 °C. Prior to
use, each oligonucleotide was diluted to 88 µM in water and stored at
0 °C for no longer than 2 days. Each well of a Costar black 96-well
plate was loaded with Tris buffer containing ethidium bromide (0.1 M
Tris, 0.1 M NaCl, pH 8, 0.44 × 10-5 M ethidium bromide final
concentration, 88 µL). To each well was added one hairpin oligonucle-
otide (10 µL, 0.88 × 10-5 M in DNA base pairs final concentration).
To each well was added distamycin A (2 µL of a 0.1 mM solution in
1
yellow foam: mp 131-133 °C; H NMR (DMSO-d6, 500 MHz) δ
9.26 (s, 1H), 9.23 (s, 1H), 8.13 (s, 1H), 7.48 (d, 1H, J ) 1.9 Hz), 7.21
(d, 1H, J ) 1.9 Hz), 6.97 (d, 1H, J ) 1.5 Hz), 6.92 (s, 1H), 6.90 (d,
1H, J ) 2.2 Hz), 6.77 (s, 1H), 3.92 (s, 3H), 3.91 (s, 3H), 3.89 (s, 3H),
3.74 (s, 3H), 1.44 (s, 9H); 13C NMR (acetone-d6, 125 MHz) 161.9,
159.6, 159.5, 153.8, 124.1, 123.8 (2C), 123.7, 121.3, 120.0, 119.1,
117.7, 109.9 (2C), 104.7, 103.8, 79.2, 51.1, 36.6 (2C), 36.5, 28.5; IR
(film) νmax 3314, 2954, 1694, 1644, 1584, 1552 cm-1; MALDIHRMS
(DHB) m/z 521.2123 (M + Na+, C24H30N6O6 requires 521.2124).
(42) The full set of 512 hairpin oligonucleotides may be purchased from
GenBase, 6450 Lusk Blvd, Suite E. 107, San Diego, CA 92121, telephone:
(41) The large-scale synthesis of 2 and 3 was optimized by Takahiro
Ishii.