β-Carbolinedione derivatives as topoisomerase I inhibitors

Article abstract of DOI:10.1002/(SICI)1521-4184(19997)332:7<249::AID-ARDP249>3.0.CO;2-F

Pyrrolo[3,4-c]-β-carbolinedione dimers 5-14 were synthesized from furo[3,4-c]-β-carbolinediones and diamines by solvent-free TaCl₅/silica catalyzed reaction under microwave irradiation. The inhibitory property of these target-compounds, the starting materials 2, 31, 32, and the N-alkylated pyrrolo[3,4-c]-β-carbolinediones 16, 17, 20-30 was tested against the relaxation of supercoiled pRB322 DNA by calf thymus topoisomerases I and II. Some of these compounds, especially 7 and 23 proved to be selective inhibitors of topoisomerase I.

Full text of DOI:10.1002/(SICI)1521-4184(19997)332:7<249::AID-ARDP249>3.0.CO;2-F

Topoisomerase I Inhibitors
249
β-Carbolinedione Derivatives as Topoisomerase I Inhibitors✩
a)
a)
a)
a)
b)
Siavosh Mahboobi *, Stella Eluwa , Sunil Kumar KC , Markus Koller , and Karin Störl
a) Institut für Pharmazie, Naturwissenschaftliche Fakultät IV - Chemie und Pharmazie, Universität, D-93040 Regensburg, Germany
b) Institut für Molekularbiologie, Biologisch-pharmazeutische Fakultät, Friedrich-Schiller-Universität, D-07745 Jena, Germany
Key Words: Topoisomerase I inhibition; β-carbolinedione derivatives; microwave irradiation
common to several topoisomerase inhibitors seems to be their
Summary
[6–9]
DNA-intercalating property
.
Pyrrolo[3,4-c]-β-carbolinedione dimers 5–14 were synthesized
from furo[3,4-c]-β-carbolinediones and diamines by solvent-free
TaCl5/silica catalyzed reaction under microwave irradiation. The
inhibitory property of these target compounds, the starting mate-
rials 2, 31, 32, and the N-alkylated pyrrolo[3,4-c]-β-carbolinedio-
nes 16, 17, 20–30 was tested against the relaxation of supercoiled
pRB322 DNA by calf thymus topoisomerases I and II. Some of
these compounds, especially 7 and 23 proved to be selective
inhibitors of topoisomerase I.
Chemistry
The substituted β-carboline dimers 5–14 were synthesized
by refluxing the corresponding β-carbolinedicarboxylic acid
anhydrides 3, 4, prepared from the imides 1, 2 analogously to
[1]
the procedure of Teressa et. al. , and diamines in toluene
(Scheme 1). This method, however, has some disadvantages.
Since the anhydrides are not readily soluble in toluene we
need relatively large amounts of solvent. The reaction is very
slow and was not completed even after refluxing for two
weeks. The yields could be slightly raised by an excess of
anhydride. As an alternative to this method, we have found
microwave irradiation to be useful. Thus, the β-carboline
dimers were synthesized by irradiating the corresponding
diamines and β-carboline anhydrides, adsorbed on activated
silica gel in the presence of TaCl in a microwave oven
according to the method of Chandrasekar elaborated for
N-alkyl imides . This method affords improved yields; the
reaction is finished within 45 min and can easily be followed
due to the yellow colour of the dimers contrasting to the
colourless starting materials.
Introduction
Several types of substances showing polycyclic aza-aro-
matic structures as well as aminoalkyl moieties display anti-
[1–4]
tumor activity
. Referred to their heterocyclic incre-
5
ments, these substances include isoquinolines, carbazoles,
and carbolines (cf. A–D). Another classification could be
made concerning the type of bonding of the aminoalkyl
substituent either at the indole or at the imide N. Finally,
bifunctional aminoalkyl moieties lead to compounds with
dimeric structures (cf. B and D).
[10]
H
N
O
O
O
H
H
N
N
R
R
O
O
i
CH3
CH3
N
HO
N
N
N
N
HN
N
H
N
H
N
N
H
N
H
N
H
A
B
CH3
CH3
1 R = H
3 R = H
2 R = OCH3
4 R = OCH3
N
O
H
O
O
N
O
H
N
R
N
N
O
N
H
ii
R
O
D
C
(X)
O
O
N
N
Figure 1. Antitumoral aminoalkyl heteroaromatics.
R
R
O
O
N
N
N
H
N
H
It was therefore our intention to synthesize derivates of
structures including both an indolic and an imidic function.
We used the pyrrolo[3,4-c]-β-carbolinediones 1, 2 as start-
5 - 14
[5]
Scheme 1. (X) is defined in Table 1 – Reagents: i) KOH in EtOH/H2O (1:1);
ii) method a: toluene, H2NCH2CH2(X)CH2CH2NH2, reflux, 14 days, or
method b: TaCl5-SiO2, microwave, 450 W, 45 min.
ing materials.
Topoisomerase inhibition has been recognized as a specific
target for potential antitumor agents. DNA topoisomerases
represent a class of enzymes capable of changing the topol-
Alkylation of the pyrrolo[3,4-c]-β-carbolinediones 1 and 2
ogy and conformation of nuclear DNA, being able to cleave should provide access to another class of products. At first,
and rejoin one strand (topoisomerase I) of the phosphodiester we tried to make use of the expectedly different reactivity of
backbone or both (topoisomerase II). One important feature the indole and imide positions, caused by their unequal NH
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
0365-6233/99/0707/0249 $17.50 +.50/0

250
Mahboobi, Eluwa, Kumar, Koller, and Störl
Table 1
CH2Ph
O
H
N
O
O
O
N
O
O
O
i
ii
iii
15
N
N
N
N
N
N
(H3C)2N
(H3C)2N
(H3C)2N
18
19
20
Scheme 3. Reagents: i) K2CO3, acetone, (CH3)2NCH2CH2I•HI, reflux; ii)
KOH in EtOH/H2O (5:1); iii) NH4OAc, melt.
indole-N, while having a free imide NH. 18 could be con-
verted, with loss of benzylamine, to the anhydride 19 with
KOH in ethanol/H O, which in turn was melted with
2
NH OAc without any further purification to yield the desired
4
imide 20 (Scheme 3).
The reactions of 1 and 2 with the appropriate ω-chloroal-
kyl-dimethylamine hydrochlorides and K CO in refluxing
2
3
acetone lead to the bisaminoalkylated products21–30 in good
yields (Scheme 4).
R1
N
H
O
O
N
R
R
O
O
i
N
N
N
R1
N
H
1 R = H
2 R = OCH3
21 - 30
Scheme 4. R and R1 are defined in Table 2. Reagents: i) K2CO3, acetone,
R1-X, reflux.
Table 2
H
N
R1
N
O
O
R
R
O
O
i
N
N
N
H
N
H
R
H
H
R1
1 R = H
2 R = OCH3
15
16
CH2Ph
CH2CH2N(CH3)2
17 OCH3 CH2CH2CH2N(CH3)3
Scheme 2. Reagents: i) NaH, DMF, 0 °C, R1Cl.
acidity. If NaH is used in a molar ratio of 1.1:1 to deprotonate
1 or 2, respectively, in DMF at 0 °C, imide-substituted prod-
ucts 15, 16, and 17 are obtained with benzyl chloride, 2-chlo-
roethyl-dimethylamine, and 3-chloropropyl-dimethylamine
(the latter as their free bases) (Scheme 2). These results show
the higher acidity of the imide NH as compared to that of the
indole NH.
K CO in refluxing acetone in the presence of 2-iodoethyl-
2
3
dimethylamine hydroiodide produced 18. Compounds of this
type open a synthetic route to structures substituted at the
Arch. Pharm. Pharm. Med. Chem. 332, 249–254 (1999)

Topoisomerase I Inhibitors
251
tography was carried out on Al-sheets coated with 60F245 silica. Column
chromatography was carried out using Merck 60 (70–230 mesh ASTM) silica
gel.
Topoisomerase Inhibition
Both the dimeric (5–14) as well as the bisaminoalkylated
(21–30) and the monoaminoalkylated (16, 17, 20) β-carbol-
ines were tested for their topoisomerase inhibition. As further
1,3-Dihydro-5-phenyl-6H-furo[3,4-c]pyrido[3,4-b]indole-1,3-dione (3)
samples, we included the monomeric β-carbolines 2, 31, and
1 (5.00 g, 15.96 mmol) was refluxed in a solution of KOH (150 g, 2.67
mol) in of 50% aqueous ethanol (600 ml) for 1 h. The mixture was cooled in
an ice bath and acidified with concd. HCl. The precipitate occurring on
addition of of H2O (3 l) was filtered, washed with ice water, and dried under
oil pump vacuum to yield 2.44 g (7.76 mmol, 49%) of pale yellow crystals,
mp 312 °C (ethanol).– Anal. (C19H10N2O3).– IR (KBr): ν = 3030–2875 (CH)
cm–1; 1770, 1710 (C=O); 1625, 1600, 1570, 1485 (C=C).– 1H-NMR/250
MHz ([D6]DMSO): δ = 7.35–8.18 (m, 9H, aromatic H), 12.04 (s; 1H, NH
indole, D2O exchange); MS (EI, 70 eV): m/z (%) = 314 (100) [M+•].
[5]
32 bearing free NH groups
.
1,3-Dihydro-9-methoxy-5-phenyl-6H-furo[3,4-c]pyrido[3,4-b]indole-
1,3-dione (4)
Table 3 shows the results of inhibition assays with calf
thymus topoisomerases I and II at supercoiled pBR 322
4 was prepared as described for 3. Yield 2.30 g (6.69 mmol, 42%) pale
brown solid (no purification possible), mp 295 °C.– IR (KBr): ν = 3380 (NH)
cm–1; 3065–2910 (CH); 1775, 1720 (C=O); 1620, 1575, 1495 (C=C).–
1H-NMR/250 MHz ([D6]DMSO): δ = 3.82 (s, 3H, OCH3), 7.26–8.64 (m,
8H, aromatic H), 11.91 (s, 1H, NH indole, D2O exchange).– MS (EI, 70 eV):
m/z (%) = 344 (45) [M+•].
[11]
DNA. Also included are literature data for camptothecin, a
[12]
[13]
topoisomerase I inhibitor and etoposide , a topoisom-
erase II inhibitor.
Table 3
1,3-Bis{N-[2-(5-phenylpyrrolo[3,4-c]pyrido[3,4-b]indole-1,3(2H)dion-
2-yl)ethyl]-1-piperidine-4-yl}propane (5)
——————————————————————————————
Compound
Concentration
(µg/ml)
Inhibition (%) of
topoisomerase
Method a) A mixture of the corresponding amines (0.31 mmol) and
β-carboline anhydrides 3 or 4 (0.62 mmol) in dry toluene (250 ml) was
refluxed under N2 for 14 d. Toluene was removed in vacuo and the residue
was purified by column chromatography on silica gel eluting with ethyl
acetate/dichloromethane/methanol 4:4:1.
Method b) The corresponding amine (0.1 mmol) and anhydride 3 or 4
(0.2 mmol) were dissolved in dry methanol/chloroform (1:1; 5 ml) under N2.
Then activated (dried overnight above 100 °C) silica gel (1 g) was added to
this solution, the suspension was evaporated carefully in vacuo and dried
(0.1 Torr) at room temp. for 30 min. After shaking vigorously for 1 h,
TaCl5–silica gel[10] (75 mg) was admixed thoroughly. This mixture was
irradiated in a microwave oven at 450 W for 45 min, cooled, transferred onto
a silica gel column and eluted by ethyl acetate/dichloromethane/methanol
4:4:1.
Yield a) 26 mg (0.029 mmol, 9%), b) 24 mg (0.027 mmol, 27%) yellow
crystals, mp 257 °C (ethanol).– Anal. (C55H52N8O4).– IR (KBr): ν = 3300
(NH) cm–1; 3050–2925 (CH); 1755, 1715 (C=O); 1640, 1595, 1480, 1450
(C=C).– 1H-NMR/250 MHz ([D6]DMSO): δ = 1.28–1.67 (m, 32H), 7.39–
8.80 (m, 18H, aromatic H), 12.27 (s, 2H, 2 × NH indole, D2O exchange).–
MS (FD): m/z (%) = 889 [M+•].
I
II
——————————————————————————————
7
100
50
90
60
90
10
60
70
95
40
90
100
50
0
0
0
0
0
10
0
95
30
50
0
0
100
50
22
23
27
17
2
31
32
>50
100
100
100
100
100
100
50
Camptothecin
25
30
6
Etoposide
0
——————————————————————————————
Other tested samples did not show activity in both tests.
These data indicate that aminoalkylation is able to enhance
the specificity for inhibition of topoisomerase I (cpds. 7, 22,
Compounds 6–14 were prepared analogously.
N,N′-Bis[2-(5-phenylpyrrolo[3,4-c]pyrido[3,4-b]indole-1,3(2H)dion-2-yl)-
27, and 17) in contrast to N-unsubstituted analogues (cpds. 2, ethyl]-N,N′-dimethyl-1,2-diaminoethane (6)
31, 32), which inhibit both topoisomerases.
Yield a) 22 mg ( 0.028 mmol, 9%), b) 18 mg (0.023 mmol, 23%) yellow
crystals, mp 168 °C (ethanol).– Anal. (C46H38N8O4).– IR (KBr): ν = 3450
(NH), 3050–2925 (CH) cm–1; 1765, 1710 (C=O); 1630, 1580, 1495, 1460
(C=C).– 1H-NMR/250 MHz ([D6]DMSO): δ = 2.20 (s, 6H, 2 × CH3), 2.55
(t, J = 6.3 Hz, 4H, 2 × CH2), 3.35–3.51 (m, 4H, 2 × CH2), 3.77 (t, J = 6.3 Hz,
4H, 2 × CH2), 7.21–8.64 (m, 18H, aromatic H), 12.11 (s, 2H, 2 × NH indole,
D2O exchange).– MS (FD): m/z (%) = 766 [M+• ].
Experimental
Chemistry
Melting points were determined on a Reichert Thermovar 300419 hot-
stage microscope and are uncorrected. Fourier-transform IR spectra (KBr)
were recorded on a Nicolet 510 FTIR spectrometer. 1H-NMR spectra were
recorded on a Bruker AC250 (250 MHz) or Bruker ARX400 (400 MHz)
spectrometer, all δ values (internal standard: TMS) are in the ppm scale. Mass
spectra were obtained with Varian MAT 311A (EI) and Varian MAT 95 (FD)
spectrometers. Elemental analyses were carried out at the Microanalytical
Laboratory of the University of Regensburg. All reactions were performed
in flame- or oven-dried vessels under nitrogen which had been dried over
self-indicating silica gel, concentrated H2SO4 and KOH. The microwave
oven was from Phillips, NN 5556/5506 and had 900 W. Thin layer chroma-
1,4-Bis[2-(5-phenylpyrrolo[3,4-c]pyrido[3,4-b]indole-1,3(2H)dion-2-yl)-e
thyl]piperazine (7)
Yield a) 29 mg (0.037 mmol, 11%), b) 20 mg (0.026, 26%) yellow crystals,
mp 160 °C (ethanol).– Anal. (C46H36N8O4).– IR (KBr): ν = 3270 (NH) cm–1
;
3050–2925 (CH); 1765, 1710 (C=O); 1630, 1580, 1495, 1460 (C=C).–
1H-NMR/250 MHz ([D6]DMSO): δ = 1.31–1.64 (m, 16H), 7.23–8.81 (m,
18H, aromatic H), 11.68 (s, 2H, 2 × NH indole, D2O exchange).– MS (FD):
m/z (%) = 764 [M+•].
Arch. Pharm. Pharm. Med. Chem. 332, 249–254 (1999)

252
Mahboobi, Eluwa, Kumar, Koller, and Störl
1,1-Bis[2-(5-phenylpyrrolo[3,4-c]pyrido[3,4-b]indole-1,3(2H)dion-2-yl)-
ethyl]-4,4′-bipiperidine (8)
3330 (NH) cm–1; 2930 (CH); 1765, 1715 (C=O).– 1H-NMR NMR/400 MHz
([D5]pyridine): δ = 1.97 (t, J = 6.7 Hz, 4H, 2 × CH2), 2.35–2.38 (m, 12H, 6 ×
CH2), 4.02 (s, 6H, 2 × OCH3), 4.06 (t, J = 7.0 Hz, 4H, 2 × CH2), 7.36–8.87
(m, 16H, aromatic H), 13.27 (s, 2H, 2 × NH indole, CD3OD exchange).– MS
(FD): m/z (%) = 862 [M+• ].
Yield a) 21 mg (0.024 mmol, 7%), b) 26 mg (0.031, 31%) yellow crystals,
mp 288 °C (ethanol).– Anal. (C52H46N8O4).– IR (KBr): ν = 3460 (NH) cm–1
;
3050–2925 (CH); 1765, 1710 (C=O); 1630, 1580, 1495, 1460 (C=C).–
1H-NMR/250 MHz ([D6]DMSO): δ = 1.33–1.62 (m, 26H), 7.23–8.81 (m,
18H, aromatic H), 12.35 (s, 2H, 2 × NH indole, D2O exchange).– MS (FD):
m/z (%) = 846 [M+• ].
2,3-Dihydro-2-benzyl-5-phenyl-1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]indole-
1,3-dione (15)
A solution of 100 mg (0.32 mmol) 1 in 0.5 ml of dry DMF was cooled to
0 °C, 10.5 mg (0.35 mmol) NaH (80% in paraffin) were added portionwise,
and the mixture was stirred for 1 h at 0 °C. Benzyl chloride (0.037 ml;
0.35 mmol) was added, and the reaction mixture was stirred at 0 °C over-
night, poured onto 5 ml of satd. NaHCO3 solution and extracted with
dichloromethane (2 × 10 ml). The organic phase was washed with water (2 ×
5 ml) and dried over Na2SO4. Evaporation of the solvent in vacuo and
purification of the residue by column chromatography on silica gel eluting
with dichloromethane/ethyl acetate 10:1 yielded 75 mg (0.19 mmol, 58%)
15 as yellow crystals, mp 269 °C (ethanol).– Anal. (C26H17N3O2).– IR
(KBr): ν = 3405 (NH) cm–1; 3050–2980 (CH); 1765, 1705 (C=O); 1630,
1605, 1585, 1570 (C=C).– 1H-NMR/250 MHz ([D6]DMSO): δ = 4.90 (s, 2H,
CH2), 7.30–7.55 (m, 6H, 5H benzyl + 1 aromatic H), 7.63–7.85 (m, 5H
phenyl), 8.05 (m, 2H, aromatic H), 8.74 (d, J = 8.0 Hz, 1H, aromatic H),
12.25 (s, 1H, NH indole, D2O exchange).– MS (EI, 70 eV): m/z (%) = 403
(82) [M+• ], 375 (6) [M–CO]+• , 244 (100) [M–159]+, 91 (21) [C7H7]+.
16 and 17 were prepared analogously.
1,4-Bis[3-(5-phenylpyrrolo[3,4-c]pyrido[3,4-b]indole-1,3(2H)dion-2-yl)-
propyl]piperazine (9)
Yield a) 29 mg (0.036 mmol, 11%), b) 17 mg (0.022, 22%) yellow crystals,
mp 327 °C (ethanol). Anal. (C48H40N8O4).– IR (KBr): ν = 3350 (NH) cm–1
;
3130–2925 (CH); 1775, 1710 (C=O); 1625, 1590, 1500, 1460 (C=C).–
1H-NMR/250 MHz ([D6]DMSO): δ = 1.28–1.67 (m, 20 H), 7.39–8.80 (m,
18H, aromatic H), 12.26 (s, 2H, 2 × NH indole, D2O exchange).– MS (FD):
m/z (%) = 792 [M+• ].
1,3-Bis{N-[2-(9-methoxy-5-phenylpyrrolo[3,4-c]pyrido[3,4-b]indole-
1,3(2H)dion-2-yl)ethyl]piperidine-4-yl}propane (10)
Yield a) 21 mg (0.021 mmol, 7%), b) 23 mg (0.024 mmol, 24%) yellow
powder, mp > 300 °C (ethanol).– Anal. (C57H56N8O6).– IR (KBr): ν = 3395
(NH) cm–1; 2925 (CH); 1765, 1720 (C=O).– 1H-NMR/400 MHz ([D5]pyri-
dine): δ = 1.02–1.32 (m, 12H), 1.56 (d, J = 12 Hz, 4H, 2 × CH2), 1.99 (t, J =
11 Hz, 4H, 2 × CH2), 2.85 (t, J = 6.5 Hz, 4H, 2 × CH2), 3.14 (d, J = 10 Hz,
4H, 2 × CH2), 4.00 (s, 6H, 2 × OCH3), 4.14 (t, J = 6.5 Hz, 4H, 2 × CH2),
7.30–8.90 (m, 16H, aromatic H), 13.22 (s; 2H, 2 × NH indole, CD3OD
exchange).– MS (FD): m/z (%) = 948 [M+•].
2,3-Dihydro-2-[2-(N,N-dimethylamino)ethyl]-5-phenyl-1H,6H-pyrrolo-
[3,4-c]pyrido[3,4-b]indole-1,3-dione (16)
Yield 37 mg (0.10 mmol, 30%) yellow crystals, mp 233 °C (ethanol).–
Anal. (C23H20N4O2).– IR (KBr): ν = 3450 (NH) cm–1; 3050–2970 (CH);
1
1770, 1710 (C=O); 1585, 1500 (C=C).– H-NMR/250 MHz (CDCl3): δ =
N,N′-Bis[2-(9-methoxy-5-phenylpyrrolo[3,4-c]pyrido[3,4-b]indole-
1,3(2H)dion-2-yl)ethyl]-N,N′-dimethyl-1,2-diaminoethane (11)
2.32 (s, 6H, N(CH3)2), 2.70 (t, J = 6.4 Hz, 2H, CH2), 4.25 (t, J = 6.4 Hz, 2H,
CH2), 7.36–8.85 (m, 9H, aromatic H), 9.36 (s, 1H, NH indole, CD3OD
exchange).– MS (EI, 70 eV): m/z (%) = 384 (5) [M+•], 58 (100)
[(CH3)2N=CH2]+.
Yield a) 28 mg (0.034 mmol, 11%), b) 51 mg (0.062 mmol, 62%) yellow
powder, mp > 300 °C (ethanol).– Anal. (C48H44N8O6).– IR (KBr): ν = 3320
(NH) cm–1; 3060–2945 (CH); 1765, 1715 (C=O).– 1H-NMR/400 MHz
([D5]pyridine): δ = 2.35 (s, 6H, 2 × CH3), 2.64 (s, 4H, 2 × CH2), 2.84 (t, J =
6.7 Hz, 4H, 2 × CH2), 3.95 (s, 6H, 2 × OCH3), 4.03 (t, J = 6.7 Hz, 4H, 2 ×
CH2), 7.33–8.78 (m, 16H, aromatic H) 13.17 (s, 2H, 2 × NH indole, CD3OD
exchange).– MS (FD): m/z (%) = 828 [M+•].
2,3-Dihydro-2-[3-(N,N-dimethylamino)propyl]-9-methoxy-5-phenyl-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione (17)
Yield 0.53 g (1.23 mmol, 77%) yellow crystals, mp 227 °C (ethanol).–
Anal. (C25H24N4O3).– IR (KBr): ν = 3415 (NH) cm–1, 3055–2865 (CH);
1765, 1705 (C=O); 1630, 1570, 1490 (C=C).– 1H-NMR/400 MHz (CD3OD):
δ = 1.95 (quin, J = 7.4 Hz, 2H, CH2), 2.27 (s, 6H, N(CH3)2), 2.47 (t, J =
7.4 Hz, 2H, CH2), 3.78 (t, J = 7.3 Hz, 2H, CH2), 3.89 (s, 3H, OCH3), 7.22
(dd, Jo = 9.0 Hz, Jm = 2.6 Hz, 1H, 8-H), 7.48 (dd, Jo = 8.9 Hz, J = 0.5 Hz,
1H, 7-H), 7.56–7.98 (m, 5H phenyl), 8.17 (d, Jm = 2.4 Hz, 1H, 10-H).– MS
(EI, 70 eV): m/z (%) = 428 (11) [M+•], 58 (100) [(H3C)2N=CH2]+.
1,4-Bis[2-(9-methoxy-5-phenylpyrrolo[3,4-c]pyrido[3,4-b]indole-
1,3(2H)dion-2-yl)ethyl]piperazine (12)
Yield a) 23 mg (0.028 mmol, 9%), b) 24 mg (0.029 mmol, 29%) yellow
powder, mp > 300 °C (ethanol).– Anal. (C48H30N8O6).– IR (KBr): ν = 3370
(NH) cm–1; 2980 (CH); 1770, 1720 (C=O).– 1H-NMR/400 MHz ([D5]pyri-
dine): δ = 2.78 (t, J = 6.4 Hz, 4H, 2 × CH2), 3.88 (t, J = 6.0 Hz, 4H, 2 × CH2),
4.01 (s, 6H, 2 × OCH3), 4.06–4.37 (m, 8H, 4 × CH2), 7.34–9.05 (m, 16H,
aromatic H), 13.23 (s, 2H, 2 × NH indole, CD3OD exchange).– MS (FD):
m/z (%) = 814 [M+• ].
2,3-Dihydro-2-benzyl-6-[2-(N,N-dimethylamino)ethyl]-5-phenyl-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione (18)
15 (2.00 g, 4.96 mmol), K2CO3 (1.71 g, 12.39 mmol), and 2-iodoethyl-di-
methylamine hydroiodide (1.78 g, 5.45 mmol) were refluxed in dry acetone
(50 ml) for 24 h. Work-up according to 22 yielded 0.54 g (1.14 mmol, 23%)
as yellow crystals, mp 249 °C (ethanol).– Anal. (C30H26N4O2).– IR (KBr):
ν = 3085–2870 (CH) cm–1; 1765, 1705 (C=O); 1625, 1595, 1590, 1490
(C=C).– 1H-NMR/250 MHz ([D6]DMSO): δ = 1.80 (s, 6H, N(CH3)2), 2.10
(t, J = 7.3 Hz, 2H, CH2), 4.25 (t, J = 7.3 Hz, 2H, CH2), 4.91 (s, 2H, CH2),
7.05–8.93 (m, 14H, aromatic H).– MS (EI, 70 eV): m/z (%) = 474 (0.23)
[M+• ], 58 (100) [(CH3)2N=CH2]+.
1,1′-Bis[2-(9-methoxy-5-phenylpyrrolo[3,4-c]pyrido[3,4-b]indole-
1,3(2H)dion-2-yl)ethyl]-4,4′-bipiperidine (13)
Yield a) 25 mg (0.028 mmol, 9%), b) 27 mg (0.030 mmol, 30%) yellow
powder, mp >300 °C (ethanol).– Anal. (C54H50N8O6).– IR (KBr): ν = 3670,
3555 (NH) cm–1; 3055–2945 (CH); 1765, 1715 (C=O).– 1H-NMR/400 MHz
([D5]pyridine): δ = 1.06–1.49 (m, 6H), 1.49 (d, J = 13.0 Hz, 4H, 2 × CH2),
1.90 (t, J = 12 Hz, 4H, 2 × CH2), 2.81 (t, J = 6.5 Hz, 4H, 2 × CH2), 3.12 (d,
J = 11.0 Hz, 4H, 2 × CH2), 4.00 (s, 6H, 2 × OCH3), 4.11 (t, J = 6.5 Hz, 4H,
2 × CH2), 7.37–8.95 (m, 16H, aromatic H), 13.23 (s, 2H, 2 × NH indole,
CD3OD exchange).– MS (FD): m/z (%) = 906 [M+•].
2,3-Dihydro-6-[2-(N,N-dimethylamino)ethyl]-5-phenyl-1H,6H-pyrrolo-
[3,4-c]pyrido[3,4-b]indole-1,3-dione (20)
18 (0.35 mg, 0.74 mmol) and aqueous 5N KOH (3 ml) were stirred in
ethanol (17 ml) for 1 h at room temp. Concd. HCl was added until a white
precipitate occurred. The mixture was diluted with water, the precipitate was
filtered, washed with water and dried at the oil pump. Crude 19 was heated
with NH4OAc (5 g) at 140 °C for 1 h. After cooling to room temp., satd.
NaHCO3 solution (100 ml) was added, and the aqueous layer was extracted
1,4-Bis[3-(9-methoxy-5-phenylpyrrolo[3,4-c]pyrido[3,4-b]indole-
1,3(2H)dion-2-yl)propyl]piperazine (14)
Yield a) 21 mg (0.024 mmol, 8%) b) 26 mg (0.03 mmol, 30%) yellow
powder, mp > 300 °C (ethanol).– Anal. (C50H54N8O6).– IR (KBr): ν = 3670,
Arch. Pharm. Pharm. Med. Chem. 332, 249–254 (1999)

Topoisomerase I Inhibitors
253
with dichloromethane (2 × 30 ml). The organic layer was dried over Na2SO4,
and the solvent was evaporated in vacuo. Purification of the residue by
column chromatography on silica gel eluting with dichloromethane/metha-
nol 10:1 yielded 11 mg (0.03 mmol, 4%) 20 as pale yellow crystals, mp
1.32–1.73 (m; 12H, 2 × β- and 2 × α-CH2), 1.93–1.95 (m; 4H), 2.22 (t, J =
7.7 Hz, 2H, CH2), 2.48–2.53 (m, 4H), 2.69 (t, J = 6.8 Hz, 2H, CH2), 3.97 (t,
J = 6.9 Hz, 2H, CH2), 4.24 (t, J = 7.7 Hz, 2H, CH2), 7.42–9.10 (m, 9H,
aromatic H).– MS (EI, 70 eV): m/z (%) = 535 (2) [M+•], 98 (100) [C6H12N]+.
212 °C (ethanol).– Anal. (C23H20N4O2).– IR (KBr): ν = 3405 (NH) cm–1
,
3075–2965 (CH); 1765, 1705 (C=O); 1625, 1600, 1575, 1490 (C=C).–
1H-NMR/250 MHz ([D6]DMSO): δ = 1.80 (s, 6H, N(CH3)2), 2.06 (t, J = 7.5
Hz, 2H, CH2), 4.23 (t, J = 7.5 Hz, 2H, CH2), 7.33–8.31 (m, 10 H, 9 aromatic
H + NH imide, partly D2O exchange).– MS (EI, 70 eV): m/z (%) = 384 (1.26)
[M+• ], 58 (100) [(CH3)2N=CH2]+.
2,3-Dihydro-2,6-bis[2-(N,N-dimethylamino)ethyl]-9-methoxy-5-phenyl-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione (26)
Yield 0.55 g (1.14 mmol, 71%) yellow crystals, mp 197 °C (ethanol).–
Anal. (C28H31N5O3).– IR (KBr): ν = 3055–2945 (CH) cm–1; 1765, 1710
(C=O); 1630, 1565, 1490 (C=C).– 1H-NMR/250 MHz ([D6]DMSO): δ =
1.79 (s, 6H, N(CH3)2), 2.08 (t, J = 7.2 Hz, 2H, CH2), 2.21 (s, 6H, N(CH3)2),
2.56 (t, J = 6.3 Hz, 2H, CH2), 3.80 (t, J = 6.4 Hz, 2H, CH2), 3.93 (s, 3H,
OCH3), 4.20 (t, J = 7.3 Hz, 2H, CH2), 7.44 (dd, Jo = 9.1 Hz, Jm = 2.6 Hz, 1H,
8-H), 7.61–7.71 (m, 5H phenyl), 7.80 (d, Jo = 9.2 Hz, 1H, 7-H), 8.41 (d, Jm
= 2.5 Hz, 1H, 10-H).– MS (EI, 70 eV): m/z (%) = 485 (1) [M+• ], 58 (100)
[(H3C)2N=CH2]+.
2,3-Dihydro-2,6-bis[2-(N,N-dimethylamino)ethyl]-5-phenyl-1H,6H-pyr-
rolo[3,4-c]pyrido[3,4-b]indole-1,3-dione (21)
1 (0.50 g, 1.60 mmol), K2CO3 (1.15 g, 7.98 mmol), and 2-chloroethyl-di-
methylamine hydrochloride (0.57 g, 3.99 mmol)were refluxed in dry acetone
(20 ml) for 18 h. After cooling to room temp., H2O (100 ml) was added, and
the aqueous layer was extracted with dichloromethane (3 × 30 ml). After
drying over Na2SO4, the solvent was removed in vacuo, and the residue was
purified by column chromatography on silica gel eluting with dichlo-
romethane/methanol 9:1, yielding 21 (0.25 g, 0.55 mmol, 34%) as yellow
crystals, mp 179 °C (ethanol).– Anal. (C27H29N5O2).– IR (KBr): ν = 3085–
2865 (CH) cm–1; 1765, 1705 (C=O); 1625, 1600, 1575, 1490 (C=C).–
1H-NMR/250 MHz ([D6]DMSO): δ = 1.81 (s, 6H, N(CH3)2), 2.11 (t, J = 7.3
Hz, 2H, CH2), 2.21 (s, 6H, N(CH3)2), 2.58 (t, J = 6.3 Hz, 2H, CH2), 3.81 (t,
J = 6.3 Hz, 2H, CH2), 4.24 (t, J = 7.3 Hz, 2H, CH2), 7.47–8.94 (m, 9H,
aromatic H).– MS (EI, 70 eV): m/z (%) = 455 (3) [M+•], 58 (100)
[(CH3)2N=CH2]+.
2,3-Dihydro-2,6-bis[3-(N,N-dimethylamino)propyl]-9-methoxy-5-phenyl-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione (27)
Yield 0.64 g (1.26 mmol, 81%) yellow crystals, mp 166 °C (ethanol).–
Anal. (C30H35N5O3).– IR (KBr): ν = 3065–2860 (CH) cm–1; 1770, 1710
(C=O); 1635, 1605, 1570, 1490 (C=C).– 1H-NMR/400 MHz ([D6]acetone):
δ = 1.50 (quint, J = 7.2 Hz, 2H, CH2), 1.70 (t, J = 6.6 Hz, 2H, CH2), 1.91 (s,
6H, N(CH3)2), 2.19 (s, 6H, N(CH3)2), 2.40 (t, J = 6.8 Hz, 2H, CH2), 2.83 (m,
2H, CH2), 3.84 (t, J = 7.4 Hz, 2H, CH2), 4.00 (s, 3H, OCH3), 4.19 (t, J = 7.4
Hz, 2H, CH2), 7.41 (dd, Jo = 9.1 Hz, Jm = 2.6 Hz, 1H, 8-H), 7.61–7.76 (m,
6H, 7-H + 5H phenyl), 8.58 (d, Jm = 2.6 Hz, 1H, 10-H).– MS (EI, 70 eV):
m/z (%) = 513 (26) [M+•], 58 (100) [(H3C)2N=CH2]+.
Diamines 22–30 were prepared analogously.
2,3-Dihydro-2,6-bis[3-(N,N-dimethylamino)propyl]-5-phenyl-1H,6H-
pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione (22)
2,3-Dihydro-9-methoxy-5-phenyl-2,6-bis[2-(1-pyrrolidinyl)ethyl]-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione (28)
Yield 0.22 g (0.44 mmol, 28%) yellow crystals, mp 188 °C (ethanol).–
Anal. (C29H33N5O2).– IR (KBr): ν = 3250–2940 (CH) cm–1; 1770, 1710
(C=O); 1560, 1520, 1460 (C=C).– 1H-NMR/250 MHz (CDCl3): δ = 1.44–
4.11 (m, 12H), 2.01 (s, 6H, N(CH3)2), 2.11 (s, 6H, N(CH3)2), 7.29–9.10 (m,
9H, aromatic H).– MS (EI, 70 eV): m/z (%) = 483 (21) [M+• ], 425 (3)
[M–58]+, 399 (4) [M–84]+, 84 (43) [H2C=CH-CH=N(CH3)2]+, 58 (100)
[(CH3)2N=CH2]+.
Yield 0.69 g (1.28 mmol, 80%) yellow crystals, mp 183 °C (ethanol).–
Anal. (C32H35N5O3).– IR (KBr): ν = 3060–2875 (CH) cm–1; 1770, 1715
(C=O); 1635, 1605, 1570, 1490 (C=C).– 1H-NMR/400 MHz ([D6]acetone):
δ = 1.50–1.53 (m, 4H, β-CH2), 1.69–1.72 (m, 4H, α-CH2), 2.07–2.10 (m,
4H, β-CH2), 2.39 (t, J = 7.4 Hz, 2H, CH2), 2.57–2.62 (m, 4H, α-CH2), 2.83
(t, J = 6.4 Hz, 2H, CH2), 3.92 (t, J = 6.6 Hz, 2H, CH2), 3.99 (s, 3H, OCH3),
4.32 (t, J = 7.4 Hz, 2H, CH2), 7.42 (dd, Jo = 9.1 Hz, Jm = 2.7 Hz, 1H, 8-H),
7.61–7.78 (m, 6H, 7-H + 5H phenyl), 8.58 (d, Jm = 2.6 Hz, 1H, 10-H).– MS
(EI, 70 eV): m/z (%) = 537 (1) [M+•], 84 (100) [C4H8N=CH2]+.
2,3-Dihydro-5-phenyl-2,6-bis[2-(1-pyrrolidinyl)ethyl]-1H,6H-pyrrolo-
[3,4-c]pyrido[3,4-b]indole-1,3-dione (23)
2,3-Dihydro-9-methoxy-2,6-bis[2-(1-morpholinyl)ethyl]-5-phenyl-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione (29)
Yield 0.17 g (0.33 mmol, 21%) yellow crystals, mp 192 °C (ethanol).–
Anal. (C31H33N5O2).– IR (KBr): ν = 3030–2875 (CH) cm–1; 1770, 1710
(C=O); 1625, 1600, 1570, 1485 (C=C).– 1H-NMR/250 MHz (CDCl3): δ =
1.62 (m, 4H, β-CH2), 1.76 (m, 4H, α-CH2), 2.10 (m, 4H, β-CH2), 2.38 (t, J
= 8.0 Hz, 2H, CH2), 2.64 (m, 4H, α-CH2), 2 .86 (t, J = 6.8 Hz, 2H, CH2),
3.99 (t, J = 6.8 Hz, 2H, CH2), 4.30 (t, J = 8.0 Hz, 2H, CH2), 7.53–9.10 (m,
9H, aromatic H).– MS (EI, 70 eV): m/z (%) = 507 (2) [M+• ], 422 (0.86)
[M–C5H10N]+, 84 (100) [C5H10N]+.
Yield 0.83 g (1.46 mmol, 91%) yellow crystals, mp 185 °C (ethanol).–
Anal. (C32H35N5O5).– IR (KBr): ν = 3055–2860 (CH) cm–1; 1760, 1705
(C=O); 1630, 1605, 1565, 1490 (C=C).– 1H-NMR/400 MHz ([D6]acetone):
δ = 2.01 (t, J = 4.7 Hz, 4H, CH2 ring), 2.28 (t, J = 7.2 Hz, 2H, CH2 chain),
2.51–2.54 (m, 4H, CH2 ring), 2.70 (t, J = 6.4 Hz, 2H, CH2 chain), 3.29 (t, J
= 4.5 Hz, 4H, CH2 ring), 3.56 (t, J = 4.6 Hz, 4H, CH2 ring), 3.93 (t, J = 6.0 Hz,
2H, CH2 chain), 4.00 (s, 3H, OCH3), 4.33 (t, J = 7.0 Hz, 2H, CH2 chain),
7.43 (dd, Jo = 9.1 Hz, Jm = 2.7 Hz, 1H, 8-H), 7.61–7.80 (m, 5H phenyl), 7.79
(d, Jo = 9.6 Hz, 1H, 7-H), 8.59 (d, Jm = 2.5 Hz, 1H, 10-H).– MS (EI, 70 eV):
m/z (%) = 569 (3) [M+•], 469 (0.4) [M–C5H10NO]+, 100 (100)
[OC4H8N=CH2]+.
2,3-Dihydro-2,6-bis[2-(1-morpholinyl)ethyl]-5-phenyl-1H,6H-pyrrolo-
[3,4-c]pyrido[3,4-b]indole-1,3-dione (24)
Yield 0.80 g (0.15 mmol, 93%) yellow crystals, mp 177 °C (ethanol).–
Anal. (C31H33N5O4).– IR (KBr): ν = 3085–2870 (CH) cm–1; 1765, 1705
(C=O); 1625, 1595, 1580, 1490 (C=C).– 1H-NMR/250 MHz (CDCl3): δ =
2.01 (t, J = 4.6 Hz, 4H, CH2NCH2), 2.27 (t, J = 7.6 Hz, 2H, CH2), 2.57 (t, J
= 4.4 Hz, 4H, CH2), 2.73 (t, J = 6.3 Hz, 2H, CH2), 3.46 (t, J = 4.6 Hz, 4H,
CH2), 3.65 (t, J = 4.6 Hz, 2H, CH2), 3.98 (t, J = 6.4 Hz, 2H, CH2), 2.73 (t, J
= 7.6 Hz, 2H, CH2), 7.44–8.90 (m, 9H, aromatic H).– MS (EI, 70 eV): m/z
(%) = 539 (1) [M+•], 100 (100) [C5H10NO]+.
2,3-Dihydro-9-methoxy-5-phenyl-2,6-bis[2-(1-piperidinyl)ethyl]-
1H,6H-pyrrolo[3,4-c]pyrido[3,4-b]indole-1,3-dione (30)
Yield 0.84 g (1.49 mmol, 93%) yellow crystals, mp 172 °C (ethanol).–
Anal. (C34H39N5O3).– IR (KBr): ν = 3060–2850 (CH) cm–1; 1770, 1715
(C=O); 1630, 1605, 1570, 1490 (C=C).– 1H-NMR /400 MHz ([D6]acetone):
δ = 1.21–1.47 (m, 4H, γ-CH2), 1.48–1.51 (m, 4H, β-CH2), 1.94–1.97 (m, 4H,
α-CH2), 2.22 (t, J = 7.1 Hz, 2H, CH2), 2.47–2.50 (m, 4H, β-CH2), 2.65 (t, J
= 6.6 Hz, 2H, CH2), 2.79–2.83 (m, 4H, α-CH2), 3.90 (t, J = 6.6 Hz, 2H, CH2),
4.00 (s, 3H, OCH3), 4.29 (t, J = 7.0 Hz, 2H, CH2), 7.42 (dd, Jo = 9.1 Hz, Jm
= 2.7 Hz, 1H, 8-H), 7.60–7.78 (m, 5H phenyl), 7.75 (d, Jo = 9.1 Hz, 1H, 7-H),
8.59 (d, Jm = 2.6 Hz, 1H, 10-H).– MS (EI, 70 eV): m/z (%) = 565 (3) [M+• ],
467 (0.4) [M–C6H12N]+, 98 (100) [C5H10N=CH2]+.
2,3-Dihydro-5-phenyl-2,6-bis[2-(1-piperidinyl)ethyl]-1H,6H-pyrrolo-
[3,4-c]pyrido[3,4-b]indole-1,3-dione (25)
Yield 0.45 g (0.84 mmol, 53%) yellow crystals, mp 146 °C (ethanol).–
Anal. (C33H37N5O2).– IR (KBr): ν = 3050–2855 (CH) cm–1; 1765, 1705
(C=O); 1625, 1695, 1580, 1490 (C=C).– 1H-NMR/250 MHz (CDCl3): δ =
Arch. Pharm. Pharm. Med. Chem. 332, 249–254 (1999)

254
Mahboobi, Eluwa, Kumar, Koller, and Störl
Topoisomerase Inhibition Assay
References
✩
Topoisomerases I and II were isolated from calf thymus glands according
to Strausfeld and Richter[11]. Enzymes were detected by their DNA relaxa-
tion activity. In a standard assay, 0.5 µg supercoiled pRB322 DNA was
assayed in a total volume of 20 µl containing 20 mM Tris-HCl, pH 7.8,
150 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol, 50% glycerol, 100 µg/ml
bovine serum albumine and, in case of topoisomerase II, 1 mM ATP. The
amount of enzyme that relaxes 0.5 µg supercoiled DNA in 30 min is defined
as one unit. Substances to be tested as enzyme inhibitors were employed in
a concentration range usually from 1 to 100 µg/ml. The indicated amounts
of inhibitors were preincubated with the DNA for 10 min in the standard
assay. Then one unit of enzyme was added and the incubation was continued
for 10 min at 37 °C. The reaction was terminated by 5 µl of stopper solution
containing 0.5% sodium dodecylsulfate, 1 mg/ml proteinase K and 1 mM
CaCl2 in 50 mM Tris-HCl, pH 7.9 and incubation at 50 °C for 15 min. 5 µl
of 0.005% bromophenolblue in 50% glycerol were then added and the
samples analyzed on 1% agarose gels in TAE buffer (50 mM Tris-acetate,
pH 8.0, 20 mM Na-acetate, 2 mM EDTA, 18 mM NaCl). Electrophoresis
was carried out at 2 Vcm–1 overnight. The gels were stained with ethidium
bromide (0.5 µg/ml) and photographed under UV illumination. The inhibi-
tory effect of the substances on the relaxation activities of topoisomerases
was estimated by comparison with the complete relaxation activity observed
in the absence of any inhibitor.
Dedicated to Prof. Dr. W. Wiegrebe on the occasion of his 67th
birthday.
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Received: March 15, 1999 [FP375]
Arch. Pharm. Pharm. Med. Chem. 332, 249–254 (1999)