Pyrrolobenzodiazepine-Minor-Groove-Binding Hybrids
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 25 5139
(2 equiv). The resulting mixture was stirred overnight as it
warmed to room temperature, acidified with 20% HCl to
pH ) 5, and then evaporated to dryness in a vacuum. The
residue was dissolved into a small volume of MeOH and then
ethyl ether was added to precipitate the crude product as a
brown solid: this procedure was repeated 5 times. The solid
residue was purified by column chromatography (CH2Cl2/
CH3OH 20%) and recrystallized (CH3OH/diethyl ether) to give
18-21.
1-Meth yl-4-[[2′-[[(11S,11a S)-11-h yd r oxy-7-m eth oxy-11,-
2,3,11a -t e t r a h yd r o-5H -5-oxop yr r olo[2,1-c][1,4]b e n zo-
d ia zep in -8-yl]oxy]eth yl]ca r boxa m id o]p yr r ole-2-ca r box-
a m id o]p r op ion a m id in e Hyd r och lor id e (22). White solid,
1
mp 227-230 °C. H NMR (DMSO-d6): δ 1.7 (m, 2H), 2.6 (m,
2H), 2.8 (m, 2H), 3.4-3.8 (m, 8H), 3.9 (s, 3H), 3.92 (s, 3H), 4.7
(s, 1H), 6.7 (s, 1H), 6.8 (s, 1H), 7.07 (s, 1H), 7.22 (s, 1H), 8.3
(br s, 1H), 8.7 (s, 1H), 8.77 (br s, 2H), 9.07 (br s, 2H), 9.5 (s,
1H). [R]D ) +6.31 (MeOH, c ) 0.52). TR (retention time) )
25
8.14 min. Anal. (C25H32ClN7O5): C, H, Cl, N.
3-[1-Meth yl-4-[[2′-[[(11S,11a S)-11-h yd r oxy-7-m eth oxy-
10-N-[[(tr ich lor oeth yl)oxy]car bon yl]-1,2,3,10,11,11a-h exa-
h yd r o-5H -5-oxop yr r olo[2,1-c][1,4]b en zod ia zep in -8-yl]-
oxy]e t h yl]ca r b oxa m id o]p yr r ole -2-ca r b oxa m id o]p r o-
p ion a m id in e Hyd r och lor id e (18). Yellow solid, mp 51-
53 °C. 1H NMR (DMSO-d6): δ 1.8-2.0 (m, 5H), 2.7 (t, 2H),
3.0-3.5 (m, 9H), 3.76 (s, 3H), 3.78 (s, 3H), 4.24 (br s, 2H), 4.61
(d, J ) 12 Hz, 1H); 5.09 (d, J ) 12 Hz, 1H); 5.49 (d, J ) 9.4
Hz, 1H), 6.79 (d, J ) 1.4 Hz, 1H), 6.85 (s, 1H), 7.07 (s, 1H),
7.151 (d, J ) 1.4 Hz, 1H); 8.32 (t, 1H), 8.76 (s, 1H), 9.08 (s,
1H), 10.14 (s, 1H). FAB-MS (MALDI-TOF): 704 [M + 1]+.
3-[1-Meth yl-4-[1-m eth yl-4-[[2′-[[(11S,11a S)-11-h yd r oxy-
7-m eth oxy-1,2,3,11a -tetr a h yd r o-5H-5-oxop yr r olo[2,1-c]-
[1,4]ben zod ia zep in -8-yl]oxy]eth yl]ca r boxa m id o]p yr r ole-
2-ca r b oxa m id o]p yr r ole -2-ca r b oxa m id o]p r op ion a m i-
1
d in e Hyd r och lor id e (23). Yellow solid, mp 265-266 °C. H
NMR (DMSO-d6): δ 1.9 (m, 5H), 2.61 (t, 2H), 2.78 (t, 2H), 3.2
(m, 2H), 3.4 (m, 3H), 3.77 (s, 3H), 3.8 (s, 3H), 3.83 (s, 3H),
4.27 (m, 2H), 6.86 (s, 1H), 6.92 (s, 2H); 7.07 (s, 1H), 7.19 (s,
2H), 8.3 (t, 1H), 8.68 (s, 2H), 9.02 (s, 2H), 9.94 (s, 1H), 10.1 (s,
1H). [R]D25 ) +30.8 (CH3OH, c ) 0.5). MS (MALDI-TOF): 633
[M + 1]+. TR (retention time) ) 10.35 min. Anal. (C31H38Cl-
N9O6): C, H, Cl, N.
[R]D ) +29.8 (MeOH, c ) 1.24). Anal. (C28H35Cl4N7O8): C,
25
H, Cl, N.
3-[1-Meth yl-4-[1-m eth yl-4-[[(2′-[[(11S,11a S)-11-h ydr oxy-
7-m et h oxy-10-N-[[(t r ich lor oet h yl)oxy]ca r b on yl]-1,2,3,-
10,11,11a-h exah ydr o-5H-pyr r olo[2,1-c][1,4]ben zodiazepin -
8-yl]oxy]et h yl]ca r b oxa m id o]p yr r ole-2-ca r b oxa m id o]-
p yr r ole-2-ca r boxa m id o]p r op ion a m id in e Hyd r och lor id e
3-[1-Meth yl-4-[1-m eth yl-4-[1-m eth yl-4-[[2′-[[(11S,11a S)-
11-h yd r oxy-7-m et h oxy-1,2,3,11a -t et r a h yd r o-5H -5-oxo-
p yr r olo[2,1-c][1,4]ben zod ia zep in -8-yl]oxy]eth yl]ca r box-
a m id e]p yr r ole-2-ca r boxa m id o]p yr r ole-2-ca r boxa m id o]-
p yr r ole-2-ca r boxa m id o]p r op ion a m id in e Hyd r och lor id e
1
1
(19). Gray solid, mp 165-167 °C. H NMR (DMSO-d6): δ 1.9
(24). White solid, mp 263-264 °C (dec). H NMR (DMSO-d6):
(m, 5H), 2.61 (t, 2H), 2.78 (t, 2H), 3.2 (m, 2H), 3.4 (m, 3H),
3.77 (s, 3H), 3.8 (s, 3H), 3.83 (s, 3H), 4.27 (m, 2H), 4.61 (d,
J ) 12 Hz, 1H), 5.1 (d, J ) 12 Hz, 1H), 5.5 (m, 1H), 6.86 (s,
1H), 6.92 (s, 2H), 7.07 (s, 1H), 7.19 (s, 2H), 8.3 (t, 1H), 8.68 (s,
2H), 9.02 (s, 2H), 9.94 (s, 1H), 10.1 (s, 1H). FAB-MS (MALDI-
δ 1.9 (m, 4H), 2.6 (t, J ) 6.4 Hz, 2H), 2.78 (m, 2H), 3.4 (m,
4H), 3.73 (s, 3H), 3.77 (s, 3H), 3.80 (s, 3H), 3.84 (s, 3H), 4.3
(m, 2H), 5.7 (s, 1H); 6.9-7.3 (m, 8H), 8.2 (t, J ) 7.3 Hz, 2H),
8.68 (s, 2H), 9.01 (s, 2H),9.94 (s, 1H), 9.95 (s, 1H), 10.1 (s, 1H).
[R]D ) -11.2 (MeOH, c ) 0.16). MS (MALDI-TOF): 756
25
TOF): 826 [M + 2]+. [R]D ) +25.7 (MeOH, c ) 0.75). Anal.
[M + 2]+. TR (retention time) ) 10.94 min. Anal. (C37H44ClN11
-
25
(C34H41Cl4N9O9): C, H, Cl, N.
O7): C, H, Cl, N.
3-[1-Meth yl-4-[1-m eth yl-4-[1-m eth yl-4-[[2′-[[(11S,11a S)-
11-h yd r oxy-7-m et h oxy-10-N-[[(t r ich lor oet h yl)oxy]ca r -
b on yl]-1,2,3,10,11,11a -h exa h yd r o-5H -5-oxop yr r olo[2,1-
c][1,4]ben zodiazepin -8-yl]oxy]eth yl]car boxam ido]pyr r ole-
2-ca r b oxa m id o]p yr r ole-2-ca r b oxa m id o]p yr r ole-2-ca r -
boxam ido]pr opion am idin e Hydr och lor ide (20). Gray solid,
mp 230-232 °C (dec). 1H NMR (CDCl3): δ 1.8-2.0 (m, 5H),
2.62 (t, J ) 6 Hz, 2H), 2.78 (m, 2H), 3.49 (m, 5H), 3.77 (s, 3H),
3.80 (s, 3H), 3.83 (s, 6H), 4.26 (m, 2H), 4.61 (d, J ) 12 Hz,
1H); 5.09 (d, J ) 12 Hz, 1H), 5.49 (dd, J ) 5 and 7 Hz, 1H),
6.80-7.24 (m, 8H), 8.25 (t, J ) 4 Hz, 1H), 8.69 (s, 2H), 9.01 (s,
2H), 9.95 (m, 2H), 10.12 (s, 1H). FAB-MS (MALDI-TOF): 949
3-[1-Meth yl-4-[1-m eth yl-4-[1-m eth yl-4-[1-m eth yl-4-[[2′-
[[(11S,11a S)-11-h yd r oxy-7-m eth oxy-1,2,3,11a -tetr a h yd r o-
5H -5-oxop yr r olo[2,1-c][1,4]b en zod ia zep in -8-yl]oxy]et h -
y l]c a r b o x a m id o ]p y r r o le -2-c a r b o x a m id o ]p y r r o le -2-
c a r b o x a m i d o ]p y r r o l e -2 -c a r b o x a m i d o ]p y r r o l e -2 -
ca r boxa m id o]p r op ion a m id in e Hyd r och lor id e (25). White
1
solid, mp 228-230 °C (dec). H NMR (DMSO-d6): δ 1.8-2.0
(m, 7H), 2.63 (t, 2H), 3.0-3.3 (m, 6H), 3.48 (m, 2H), 3.72-
3.85 (m, 15H), 4.24 (m, 2H), 6.85-7.25 (m, 8H), 8.23 (t, J )
7.3 Hz, 1H), 8.46 (br s, 2H), 8.64 (br s, 2H), 8.98 (s, 1H), 9.94
(s, 1H), 10.1 (s, 1H). [R]D ) -79 (MeOH, c ) 1.2). MS
25
(MALDI-TOF): 878 [M + 2]+. TR (retention time) ) 13.97 min.
[M + 2]+. [R]D ) +74.1 (CHCl3, c ) 0.97). Anal. (C40H47Cl4-
Anal. (C43H50ClN13O8): C, H, Cl, N.
25
N
11O10): C, H, Cl, N.
In h ibition of in Vitr o Tu m or Cell Gr ow th . The human
chronic myeloid leukemia K562 and the human lymphoblas-
toid J urkat cell lines were grown in vitro as a stationary
suspension culture in RPMI 1640 medium (Gibco-BRL)
supplemented with 10% fetal calf serum (FCS) (Gibco-BRL),
2 mM L-glutamine (Gibco-BRL), 100 units/mL penicillin, and
100 mg/mL streptomycin. To determine survival after drug
exposure, exponentially growing K562 and J urkat cells were
cultured in the presence of various concentrations of the drugs
and the antiproliferative activity of the drugs was evaluated
after 4 days by counting surviving cells in a model ZBI Coulter
counter (Coulter Electronics, Hialeah, FL). Results were
expressed as IC50 (dose causing 50% inhibition of cell growth
in treated cultures relative to untreated controls). All experi-
ments were repeated at least twice. For each drug concentra-
tion, duplicate cell cultures were used. Vehicle or solvent
controls were run with each experiment. All the compounds
tested were dissolved in DMSO at 1 mg/mL immediately before
the use and diluted just before addition to the cell culture.
3-[1-Meth yl-4-[1-m eth yl-4-[1-m eth yl-4-[1-m eth yl-4-[[2′-
[[(11S,11a S)-11-h yd r oxy-7-m eth oxy-10-N-[[(tr ich lor oeth -
yl)oxy]ca r bon yl]-1,2,3,10,11,11a -h exa h yd r o-5H-5-oxop yr -
r olo[2,1-c][1,4]b e n zod ia ze p in -8-yl]oxy]e t h yl]ca r b ox-
a m id o]p yr r ole-2-ca r boxa m id o]p yr r ole-2-ca r boxa m id o]-
p yr r ole-2-ca r boxa m id o]p yr r ole-2-ca r boxa m id o]p r op ion -
1
a m id in e Hyd r och lor id e (21). Brown solid, mp >300 °C. H
NMR (DMSO-d6): δ 1.8-2.0 (m, 9H), 2.62 (t, 2H), 3.0-3.3 (m,
6H), 3.47 (m, 2H), 3.74-3.84 (m, 15H), 4.23 (m, 2H), 4.61 (d,
J ) 12 Hz, 1H), 5.1 (d, J ) 12 Hz, 1H), 5.49 (dd, J ) 5 and 7
Hz, 1H), 6.84-7.25 (m, 10H), 8.63 (br s, 1H), 8.99 (s, 1H), 9.96
(s, 1H), 10.11 (s, 1H). [R]D ) +16.4 (MeOH, c ) 0.67). Anal.
25
(C46H53Cl4N13O11): C, H, Cl, N.
Gen er a l P r oced u r e for th e Dep r otection of Tr oc fr om
th e Hybr id s (18-21): P r ep a r a tion of Activa ted Hybr id s
(22-24). Troc-protected hybrids (18-21) (0.081 mmol) were
dissolved into a mixture of THF (1 mL) and 1 M NH4Ac (1
mL). Ten percent Cd/Pb couple (55 mg, 0.45 mmol of Cd) was
added portionwise, and the mixture was vigorously stirred
until no starting material was detected by TLC (CHCl3/
CH3OH/HCl, 7/3/0.1). The reaction mixture was filtered over
Celite, and the residue was washed with ethyl acetate aliquots.
The organic solution was evaporated and the crude product
was purified by semipreparative HPLC to give 22-25, respec-
tively.
Ta r get DNA, Oligon u cleotid e P r im er s, a n d P olym -
er a se Ch a in Rea ction . The sequences of the primers used
for polymerase chain reaction15a were the following: ER for-
ward, 5′-GACGCATGATATACTTCACC-3′; ER reverse, 5′-
GCAGAATCAAATATCCAGATG-3′; c-myc forward, 5′-CGTGG-
GGAAAGAAAAAAGTCC-3′; c-myc reverse, 5′-TGCCTCTCGCT-
GGAATTACTACAG-3′; HIV-1-F (forward), 5′-ATTTCATCA-