590 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 4
Moreau et al.
543.1641; found, 543.1642. 1H NMR (400 MHz, DMSO-d6):
1.02 (3H, s), 3.71 (3H, s, OCH3), 3.90 (1H, pt, J ) 9.5 Hz),
3.95 to 4.07 (3H, m), 4.16 (1H, d, J ) 10.3 Hz), 4.95 (1H, pt, J
) 9.5 Hz), 5.60 (1H, d, J ) 6.4 Hz, OH), 6.35 (1H, t, J ) 4.0
Hz, OH), 6.67 (1H, d, J ) 9.5 Hz, H1′), 7.43 (1H, t, J ) 7.1
Hz), 7.45 (1H, t, J ) 7.1 Hz), 7.64 (1H, t, J ) 7.1 Hz), 7.69
(1H, t, J ) 7.1 Hz), 7.79 (1H, d, J ) 8.7 Hz), 8.04 (1H, d, J )
8.7 Hz), 9.14 (1H, d, J ) 8.7 Hz), 9.20 (1H, d, J ) 7.1 Hz),
11.22 (1H, s, Nimide-H), 11.75 (1H, s, Nindole-H). 13C NMR (100
MHz, DMSO-d6): 19.1 (CH3), 58.3 (C6′), 60.3 (OCH3), 73.3, 73.6,
77.2, 77.3, 81.8 (C1′, C2′, C3′, C4′, C5′), 111.3, 112.2, 120.6, 121.1,
124.4, 124.7, 127.0, 127.1 (C tert arom), 117.1, 118.5, 119.3,
121.3, 121.4, 121.5, 128.8, 129.7, 140.7, 141.1 (C quat arom),
168.0, 170.9, 171.1 (CdO).
(26 µL, 0.3 mmol) was then added, and the mixture was
refluxed for 48 h prior to adding water. After workup identical
to that for 9 and purification by flash chromatography (eluent,
EtOAc-CH2Cl2, 50:50), compound 10 (31 mg, 0.045 mmol, 15%
yield) was obtained as a yellow solid. Mp: 155 °C. IR (KBr)
νCdO 1710, 1760 cm-1, νNH,OH 3300-3620 cm-1. HRMS (FAB+)
(M + H)+: calcd for C33H33N3O9Br, 694.1400; found, 694.1425.
1H NMR (400 MHz, DMSO-d6): 1.77 (2H, m), 1.93 (2H, m),
3.59 (2H, pt, J ) 6.9 Hz), 3.71 (3H, s, OCH3), 3.74-4.10 (6H,
m), 4.24 (2H, pt, J ) 6.4 Hz), 4.64 (2H, s), 5.05 (1H, d, J ) 5.4
Hz, OH), 5.36 (1H, d, J ) 5.4 Hz, OH), 6.24 (1H, t, J ) 4.0 Hz,
OH), 6.37 (1H, d, J ) 8.9 Hz, H1′), 7.43 (2H, t, J ) 7.9 Hz),
7.65 (2H, t, J ) 8.4 Hz), 7.78 (1H, d, J ) 8.4 Hz), 8.03 (1H, d,
J ) 8.7 Hz), 9.10 (1H, d, J ) 8.4 Hz), 9.17 (1H, d, J ) 7.4 Hz),
11.73 (1H, s, Nindole-H). 13C NMR (100 MHz, DMSO-d6): 26.9,
28.8, 34.5, 38.9 (CH2), 58.5 (C6′), 60.1 (OCH3), 64.5 (CH2), 73.2,
76.3, 77.2, 77.3, 84.2 (C1′, C2′, C3′, C4′, C5′), 111.9, 112.3, 120.6,
120.9, 124.2, 124.3, 127.1, 127.3 (C tert arom), 117.3, 118.0,
118.8, 119.7, 120.9, 121.3, 128.4, 129.8, 140.9, 142.3 (C quat
arom), 168.2, 168.7, 168.8 (CdO).
P r otein Kin a se C In h ibition . Protamine sulfate was from
Merck (Darmstadt, Germany). Unless specified, chemicals
were from Sigma (St. Louis, MO). [γ-33P] ATP (1000-3000 Ci/
mmol) was obtained from Amersham. Recombinant baculo-
viruses from protein kinase C subtypes were supplied by Dr.
Silvia Stabel, Ko¨ln, Germany. Expression and partial purifica-
tion of PKCs together with measurements of activities were
carried out as previously described.13 Data show IC50 values
expressed in micromolar units.
DNA Meltin g Tem p er a tu r e Stu d ies. Melting curves were
measured using an Uvikon 943 spectrophotometer coupled to
a Neslab RTE111 cryostat. For each series of measurements,
12 samples were placed in a thermostatically controlled cell
holder, and the quartz cells (10 mm path length) were heated
by circulating water. The measurements were performed in
BPE buffer pH 7.1 (6mM Na2HPO4, 2 mM NaH2PO4, 1 mM
EDTA). The temperature inside the cell was measured with a
platinum probe; it was increased over the range 20-100 °C
with a heating rate of 1 °C/min. The “melting” temperature
Tm was taken as the midpoint of the hyperchromic transition
(from the first-derivative plot).
12-(3-O-Br om om et h ylca r b on yl-4-O-m et h yl-â-D-glu co-
pyr an osyl)-6,7,12,13-tetr ah ydr oin dolo[2,3-a ]pyr r olo[3,4-c]-
ca r ba zole-5,7-d ion e (8). To a solution of dechlorinated rebec-
camycin (350 mg, 0.70 mmol) in THF (12 mL) was added
tBuOK (79 mg). The mixture was stirred at room temperature
for 0.5 h before bromoacetyl bromide (61 µL, 0.70 mmol) was
added. The mixture was stirred at room temperature for 24 h
prior to adding water. After extraction with EtOAc, the organic
phase was dried over MgSO4. The solvent was removed and
the residue purified by flash chromatography (eluent, EtOAc-
CH2Cl2, 30:70) to give 8 (30 mg, 0.05 mmol, 7% yield) as a
yellow solid and 5 (58 mg, 0.09 mmol, 13% yield). Mp: 225
°C. IR (KBr): νCdO 1705, 1750 cm-1, νNH,OH 3200-3600 cm-1
.
HRMS (FAB+) (M+): calcd for C29H24N3O8Br, 621.0746; found,
621.0746. 1H NMR (400 MHz, DMSO-d6): 3.63 (3H, s, OCH3),
3.80 (1H, m, H2′), 3.99 (3H, m, 2H6′, H4′), 4.09 (1H, d, J ) 13.2
Hz), 4.21 (1H, d, J ) 12.8 Hz), 4.28 (1H, d, J ) 9.9 Hz, H5′),
5.38 (1H, d, J ) 5.9 Hz, OH2′), 5.42 (1H, pt, J ) 9.8 Hz, H3′),
6.37 (1H, t, J ) 4.0 Hz, OH6′), 6.63 (1H, d, J ) 8.9 Hz, H1′),
7.43 (2H, t, J ) 7.4 Hz), 7.64 (1H, dt, J 1 ) 7.9 Hz, J 2 ) 1.0
Hz), 7.66 (1H, dt, J 1 ) 7.4 Hz, J 2 ) 1.5 Hz), 7.77 (1H, d, J )
8.4 Hz), 8.03 (1H, d, J ) 8.8 Hz), 9.15 (1H, d, J ) 8.4 Hz),
9.20 (1H, d, J ) 7.4 Hz), 11.22 (1H, s, Nimide-H), 11.58 (1H, s,
Nindole-H). 13C NMR (100 MHz, DMSO-d6): 27.1 (CH2Br), 58.3
(C6′), 60.0 (OCH3), 70.9, 75.3, 76.7, 77.0, 78.6, 83.5 (C1′, C2′,
C3′, C4′, C5′), 111.7, 112.2, 120.6, 120.9, 124.6 (2C), 127.0, 127.2
(C tert arom), 117.2, 118.7, 119.6, 121.1, 121.3, 121.5, 128.1,
129.7, 140.8, 142.1 (C quat arom), 166.6, 170.9, 171.0 (CdO).
F lu or escen ce m ea su r em en ts were carried out on
a
Perkin-Elmer LS50B spectrofluorimeter. All measurements
were made using a 10 mm light path cell in a 0.01 M ionic
strength buffer (9.3 mM NaCl, 2 mM Na acetate, 0.1 mM
EDTA) using 20 µM DNA and 2 µM ethidium bromide.28 The
DNA-ethidium complex was excited at 546 nm and the
fluorescence measured at 595 nm.
Top oisom er a se I In h ibition . (1) DNA Rela xa tion Ex-
p er im en ts. Supercoiled pAT DNA (0.5 µg) was incubated with
6 units of topoisomerase I (TopoGen, Colombus, OH) at 37 °C
for 1 h in the relaxation buffer (50 mM Tris pH 7.8, 50 mM
KCl, 10 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA) in the
presence of the test drug at 30 µM. Reactions were terminated
by adding SDS to 0.25% and proteinase K to 250 µg/mL. DNA
samples were then added to the electrophoresis dye mixture
(3 µL) and electrophoresed in a 1% agarose gel containing
ethidium bromide (1 µg/mL). After 2 h of electrophoresis at
room temperature (about 180 V), the gel was washed with
water and then photographed under UV light.
(2) Exp er im en ts w ith Lin ea r P la sm id DNA on Aga r ose
Gels.29,30 pBR322 DNA (Boehringer Mannheim, Germany) was
linearized with EcoRI and labeled with R-[32P]-dATP in the
presence of the Klenow fragment of DNA polymerase I. The
labeled DNA was then digested to completion with HindIII.
The cleavage reaction mixture contained 20 mM Tris HCl pH
7.4, 60 mM KCl, 0.5 mM EDTA, 0.5 mM dithiothreitol, 2 ×
104 dpm of R-[32P]-pBR322 DNA, and the indicated drug
concentrations. The reaction was initiated by the addition of
calf thymus topoisomerase I (40 units in 20 µL of reaction
volume) and allowed to proceed for 10 min at 37 °C. Reactions
were stopped by adding SDS to a final concentration of 0.25%
and proteinase K to 250 µg/mL, followed by incubation for 30
min at 50 °C. Samples were denatured by the addition of 10
1,11-Dich lor o-12-(4-O-m eth yl-â-D-glu cop yr a n osyl)-6-N-
(3-ch lor o-2-oxo-p r op yl)-6,7,12,13-tetr a h yd r oin d olo[2,3-a ]-
p yr r olo[3,4-c]ca r ba zole-5,7-d ion e (9). A suspension of NaH
(60% w/w dispersion in mineral oil, 27 mg) in DMF (2 mL)
was added to a solution of rebeccamycin (386 mg, 0.68 mmol)
in DMF (4 mL). The mixture was stirred at room temperature
for 0.5 h before the addition of 1,3-dichloroacetone (86 mg, 0.68
mmol) in DMF (1 mL). The mixture was then stirred at room
temperature for 24 h prior to the addition of water. After
extraction with EtOAc, the organic phase was dried over
MgSO4. The solvent was removed and the residue purified by
flash chromatography (eluent, EtOAc-CH2Cl2, 35:65) to give
compound 9 (22 mg, 0.033 mmol, 5% yield) as a yellow solid.
Mp: 185 °C. IR (KBr) νCdO 1700, 1740, 1755 cm-1, νOH 3300-
3600 cm-1. HRMS (FAB+) (M + H)+: calcd for C30H25Cl3N3O8,
660.0707; found, 660.0699. 1H NMR (400 MHz, DMSO-d6): 3.65
(3H, s, OCH3), 3.58-3.75 (3H, m), 3.9 (1H, m), 4.04 (2H, m),
4.89 (4H, s), 5.07 (1H, d, J ) 5.4 Hz, OH), 5.41 (1H, broad s,
OH), 5.48 (1H, broad s, OH), 6.96 (1H, d, J ) 8.9 Hz, H1′),
7.44 (1H, t, J ) 7.8 Hz), 7.50 (1H, t, J ) 7.8 Hz), 7.75 (2H, t,
J ) 7.9 Hz), 8.99 (1H, d, J ) 7.9 Hz), 9.15 (1H, d, J ) 7.9 Hz),
10.73 (1H, s, Nindole-H). 13C NMR (100 MHz, DMSO-d6): 44.7,
47.0 (2CH2), 59.8 (C6′), 60.1 (OCH3), 72.1, 77.3, 79.1, 80.3 (C2′,
C3′, C4′, C5′), 84.5 (C1′), 116.3, 116.4, 117.8, 119.2, 119.6, 121.1,
123.0, 124.9, 129.7, 129.8, 137.2, 137.9 (C quat arom), 122.2,
122.7, 123.2, 123.9, 127.3, 130.2 (C tert arom), 168.0, 168.1,
196.7 (CdO).
12-(4-O-Met h yl-â-D-glu cop yr a n osyl)-6-N-b u t ylb r om o-
a ceta te-6,7,12,13-tetr a h yd r oin d olo[2,3-a ]p yr r olo[3,4-c]-
ca r ba zole-5,7-d ion e (10). A mixture of dechlorinated rebec-
camycin 2 (150 mg, 0.30 mmol), THF (5 mL), and K2CO3 (41
mg, 0.30 mmol) was refluxed for 0.5 h. Bromoacetyl bromide