Synthesis of cytotoxic indenoisoquinoline topoisomerase I poisons

Article abstract of DOI:10.1021/jm9803323

A number of indenoisoquinolines were prepared and evaluated for cytotoxicity in human cancer cell cultures and for activity vs topoisomerase 1 (top1). The two most cytotoxic indenoisoquinolines proved to be cis-6- ethyl-5,6,12,13-tetrahydro-2,3-dimethoxy-8,9-(methylenedioxy)-5,11-dioxo- 11H-indeno[1,2-c]isoquinoline (21) and cis-6-allyl-5,6,12,13-tetrahydro-2,3- dimethoxy-8,9-(methylenedioxy)-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (22), both of which displayed submicromolar mean graph midpoints when tested in 55 human cancer cell cultures. Two of the most potent top i inhibitors were 6- (3-carboxy-1-propyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (26) and 6-ethyl-2,3-dimethoxy-8,9-(methylenedioxy)-11H-indeno[1,2- c]isoquinolinium Chloride (27), both of which also inhibited top2, unwound DNA, and are assumed to be DNA intercalators. However, two additional potent top1 inhibitors, 6-allyl-5,6-dihydro-2,3-dimethoxy-8,9-(methylenedioxy)- 5,11-dioxo-11H-indeno[1,2-c]isoquinoline (13c) and 5,6-dihydro-6-(4- hydroxybut-1-yl)-2-3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2- c]isoquinoline (19a), did not unwind DNA and did not affect top2. Some of the DNA cleavage sites detected in the presence of the indenoisoquinolines were different from those seen with the camptothecins. The cleavage sites induced by the indenoisoquinolines were reversed by salt treatment, which is consistent with the reversible trapping of top1 clearable complexes by the indenoisoquinolines. In general, the potencies of the indenoisoquinolines as top1 inhibitors did not correlate with their potencies as cytotoxic agents, as some of the most cytotoxic agents had little if any effect on top1. On the other hand, the most potent of the indenoisoquinolines vs top1 were not the most cytotoxic. In several cases, moderate activity was observed for both cytotoxicity and activity vs top1.

Full text of DOI:10.1021/jm9803323

446
J . Med. Chem. 1999, 42, 446-457
Syn th esis of Cytotoxic In d en oisoqu in olin e Top oisom er a se I P oison s
Dirk Strumberg,† Yves Pommier,† Kenneth Paull,‡ Muthusamy J ayaraman,§ Pamela Nagafuji,§ and
Mark Cushman*,§
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy, Purdue University,
West Lafayette, Indiana 47906, Information Technology Branch, Developmental Therapeutics Program, Division of Cancer
Treatment and Diagnosis, National Cancer Institute, Bethesda, Maryland 20892, and Laboratory of Molecular Pharmacology,
Division of Basic Sciences, National Cancer Institute, Bethesda, Maryland 20892-4255
Received May 29, 1998
A number of indenoisoquinolines were prepared and evaluated for cytotoxicity in human cancer
cell cultures and for activity vs topoisomerase 1 (top1). The two most cytotoxic indenoisoquino-
lines proved to be cis-6-ethyl-5,6,12,13-tetrahydro-2,3-dimethoxy-8,9-(methylenedioxy)-5,11-
dioxo-11H-indeno[1,2-c]isoquinoline (21) and cis-6-allyl-5,6,12,13-tetrahydro-2,3-dimethoxy-
8,9-(methylenedioxy)-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (22), both of which displayed
submicromolar mean graph midpoints when tested in 55 human cancer cell cultures. Two of
the most potent top1 inhibitors were 6-(3-carboxy-1-propyl)-5,6-dihydro-5,11-dioxo-11H-indeno-
[1,2-c]isoquinoline (26) and 6-ethyl-2,3-dimethoxy-8,9-(methylenedioxy)-11H-indeno[1,2-c]iso-
quinolinium chloride (27), both of which also inhibited top2, unwound DNA, and are assumed
to be DNA intercalators. However, two additional potent top1 inhibitors, 6-allyl-5,6-dihydro-
2,3-dimethoxy-8,9-(methylenedioxy)-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (13c) and 5,6-
dihydro-6-(4-hydroxybut-1-yl)-2,3-dimethoxy-8,9-methylenedioxy-5,11-dioxo-11H-indeno[1,2-c]-
isoquinoline (19a ), did not unwind DNA and did not affect top2. Some of the DNA cleavage
sites detected in the presence of the indenoisoquinolines were different from those seen with
the camptothecins. The cleavage sites induced by the indenoisoquinolines were reversed by
salt treatment, which is consistent with the reversible trapping of top1 cleavable complexes
by the indenoisoquinolines. In general, the potencies of the indenoisoquinolines as top1
inhibitors did not correlate with their potencies as cytotoxic agents, as some of the most cytotoxic
agents had little if any effect on top1. On the other hand, the most potent of the indenoiso-
quinolines vs top1 were not the most cytotoxic. In several cases, moderate activity was observed
for both cytotoxicity and activity vs top1.
Sch em e 1
Some time ago, we reported the synthesis of an
indenoisoquinoline 1.1 Compound 1 was subsequently
found to be cytotoxic in human cancer cell cultures.
More recently, a COMPARE analysis2-4 indicated that
the cytotoxicity profile of 1 is similar to that of the
topoisomerase 1 (top1) inhibitors camptothecin and
saintopin.5 When tested for activity against top1, com-
pound 1 was in fact found to induce DNA cleavage in
tors, it is not a DNA intercalator. The present study was
undertaken in order to investigate the structural pa-
rameters associated with the biological activity of 1 and
to exploit it as lead compound for the development of
new top1 inhibitors and potential anticancer agents.
the presence of top1.5 However, the cleavage site
specificity differed from that of camptothecin in that
compound 1 did not cleave at all of the sites character-
istic of camptothecin, while some DNA cleavage sites
were unique to compound 1. In addition, compound 1
did not produce detectable DNA unwinding, suggesting
that in contrast to other noncamptothecin top1 inhibi-
Ch em istr y
A number of indenoisoquinolines 3-8 lacking the
methylenedioxy and methoxy substituents of 1 were
synthesized by reacting commercially available benz-
[d]indeno[1,2-b]pyran-5,11-dione (2) with various pri-
mary amines (Scheme 1). The reactions were carried out
at room temperature in chloroform and the yields were
generally high. These compounds were synthesized in
order to determine whether simple and readily available
† Laboratory of Molecular Pharmacology, NCI.
‡ Information Technology Branch, NCI. This paper is dedicated in
memory of Dr. Kenneth Paull.
§ Purdue University.
10.1021/jm9803323 CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/06/1999

Synthesis of Cytotoxic Indenoisoquinolines
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3 447
Sch em e 2
Sch em e 3
afforded the corresponding protected intermediates 15a
and 15b.12 The imines 17a and 17b were synthesized
by treating the O-TBDMS protected aminols 15a and
15b with piperonal (16) in chloroform in the presence
of anhydrous magnesium sulfate. Condensation of the
Schiff bases 17a and 17b with 4,5-dimethoxyhomoph-
thalic anhydride (10b) afforded the cis 3,4-disubstituted
isoquinolones 18a and 18b. The cis stereochemistry of
18a and 18b was confirmed by a 6 Hz coupling constant
observed for the C-3 and C-4 methine signals.13 Treat-
ment of 18a or 18b with thionyl chloride resulted in
deprotection of the terminal alcohol, Friedel-Crafts
reaction to form the five-membered ring, and dehydro-
genation to afford 19a and 19b.
compounds would retain any of the activity of the parent
compound 1. The idea to synthesize these compounds
was also given encouragement by the antineoplastic
activity recently reported for oracin (9), which has been
found to induce G2 cell cycle arrest and apoptosis in
Burkitt’s lymphoma cells.6-11
Several dihydro derivatives 20-23 were also pre-
pared. The syntheses of 20 and 23 were carried out as
described previously.14,15 Compounds 21 and 22 were
prepared by treatment of the acids 12k and 12c with
Eaton’s reagent (10% P2O5 in methanesulfonic acid).
To accommodate additional substituents on the two
aromatic rings of the indenoisoquinoline system, an
alternative synthesis was executed which was based on
the condensation of Schiff bases 11 with homophthalic
anhydrides 10 to afford cis substituted isoquinolones 12,
followed by conversion to the desired products 13 in the
presence of thionyl chloride (Scheme 2).1 Using this
method, a series of 11 additional indenoisoquinolines
13a -k were synthesized. These compounds incorporate
a variety of substituents at C-2, C-3, N-6, C-8, C-9, and
C-10 of the ring system.
A modification of this route was carried out in order
to synthesize compounds containing an alcohol group
at the end of an alkyl chain located at N-6 (Scheme 3).
Treatment of 4-amino-1-butanol (14a ) or 5-amino-1-
pentanol (14b) with tert-butyldimethylsilyl chloride
according to the procedure of Corey and Venkateswarlu
Treatment of 21 with borane-tetrahydrofuran complex
in refluxing THF for 1 h resulted in reduction of the
ketone to afford 24. When 21 was treated with the same
reagent in refluxing THF for 12 h, reduction of both the

448 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
Ta ble 1. Cytotoxicities of Indenoisoquinoline Analogues
Strumberg et al.
cytotoxicity (GI50 in µM)a
lung
HOP-62
colon
HCT-116
CNS
SF-539
melanoma
UACC-62
ovarian
OVCAR-3
renal
SN12C
prostate
DU-145
breast
MDA-MB-435
top1
compd
MGMb
cleavagec
1
2
3
1.3
>100
35
>100
41
>100
4.2
>100
73
>100
68
>100
37
>100
96
>100
20
85
++
0
0
4
0
5
6
7
8
61
>100
>100
>100
4.6
84
>100
4.4
14.3
>100
8.2
>100
>100
74
93
>100
>100
>100
7.4
>100
>100
>100
3.4
>100
>100
>100
41
98
81
16
14
45
30
0
0
13
4.4
22
17
3.4
16
12
20
72
46
3.2
3.9
54
2.9
2.3
+
+
+
+
+
+
(
0
0
0
0
0
(
+
+
(
(
0
(
0
0
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
19a
19b
20
21
22
23
24
25
26
27
>100
37
>100
9.0
2.2
94
6.6
78
38
3.2
16
4.0
>100
5.2
2.6
4.2
45
21
50
>100
>100
>100
>100
>100
>100
42
42
70
82
72
95
78
2.4
3.2
45
>100
>100
>100
80
>100
>100
>100
58
>100
>100
3.0
>100
>100
39
>100
>100
2.0
>100
>100
88
>100
>100
>100
3.6
76
93
>100
>100
>100
>100
2.3
>100
55
>100
>100
24
2.2
0.70
8.7
9.4
0.29
0.36
2.6
1.4
>100
2.1
0.99
>100
2.6
10
>100
3.7
>100
0.36
5.1
4.1
0.42
0.77
2.0
0.29
0.34
3.1
0.42
0.18
0.38
6.7
0.44
1.78
2.1
17
0.45
1.4
5.0
0.81
0.98
>100
30
>100
35
>100
>100
24
>100
>100
26
>100
41
>100
25
100
50
31
48
13
87
22
40
13
73
27
2.7
48
58
>100
++
++
56
1.9
14
a
The cytotoxicity GI50 values are the concentrations corresponding to 50% growth inhibition. b Mean graph midpoint for growth inhibition
of all human cancer cell lines successfully tested. The entries with population standard deviations are the results of two determinations,
and the ones without population standard deviations are the result of single determinations. c ++: greater than 50% of the activity of 1
µM camptothecin; +: between 20 and 50% of the activity of 1 µM camptothecin; (: less than 20% of the activity of 1 µM camptothecin;
0: inactive. Each drug was tested at concentrations ranging between 0.1 and 300 µM (for example, see Figure 2).
ketone and amide carbonyls occurred to yield 25. The
stereochemistry of the hydroxyl group results from the
approach of the reducing reagent to the less sterically
hindered, convex surface of the indenoisoquinoline 21.
Biologica l Resu lts a n d Discu ssion
The indenoisoquinolines were examined for antipro-
liferative activity against the human cancer cell lines
in the National Cancer Institute screen, in which the
activity of each compound was evaluated with ap-
proximately 55 different cancer cell lines of diverse
tumor origins. The GI50 values obtained with selected
cell lines, along with the mean graph midpoint (MGM)
values, are summarized in Table 1. The MGM is based
on a calculation of the average GI50 for all of the cell
lines tested (approximately 55) in which GI50 values
below and above the test range (10-4 to 10-8 M) are
taken as the minimum (10-8 M) and maximum (10-4
M) drug concentrations used in the screening test.4 In
addition, the relative activities of the compounds in the
top1 cleavage assay are listed in Table 1.
In general, most of the new indenoisoquinolines were
even less cytotoxic in human cancer cell cultures than
the moderately active (MGM 20 µM) lead compound 1.
However, a few members of the series proved to be more
cytotoxic than 1, including the N-allyl analogue 13c
(MGM 4.2 µM), the N-ethyl homologue 13k (MGM 2.4
µM), analogue 19a (MGM 3.2 µM) having an N-(4′-
hydroxybutyl) substituent, and the three dihydro de-
rivatives 20 (MGM 5.0 µM), 21 (MGM 0.81 µM), and
22 (MGM 0.98 µM). The N-ethylisoquinolinium species
27 (MGM 13 µM) and the relatively simple indenoiso-
quinolines 7 (MGM 16 µM) and 8 (MGM 14 µM), both
lacking substituent on the aromatic rings, were slightly
more cytotoxic than 1. Whereas the isoquinolinium salt
27 was comparable to 1 regarding top1 cleavage activity,
We were interested in obtaining an indenoisoquino-
line derivative having an acidic group which might be
converted into a more water-soluble salt. The carboxylic
acid 26 was obtained by oxidation of indenoisoquinoline
7 with J ones reagent.
Dehydration, as well as dehydrogenation, of the
alcohol 25 occurred in the presence of palladium in
charcoal in refluxing acetic acid. Treatment of the
product with aqueous NaCl provided the indenoiso-
quinolinium salt 27.

Synthesis of Cytotoxic Indenoisoquinolines
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3 449
activity similar to 1 because it is structurally related
to the cytotoxic benzophenanthridine alkaloid fagar-
onine (28), a known inhibitor of both top1 and top2.16
The most potent of the new indenoisoquinolines vs
top1 proved to be 26 and 27. Both of these compounds
were examined for induction of DNA cleavage in the 3′-
end-labeled PvuII/HindIII fragment of pBluescript SK-
(-) phagemid DNA in the presence of top15 (Figure 1).
The results were compared with those for the lead
compound 1 and camptothecin (29). Some of the cleav-
age sites detected in the presence of 26, 27, and 1 were
different from those induced by camptothecin (29). The
indenoisoquinolines 26, 27, and 1 induced several top1
cleavage sites that were not observed with camptothecin
(29). In Figure 1, the numbers on the left of the gel
picture correspond to the camptothecin-induced cleav-
age sites,5 while the letters on the right correspond to
additional cleavage sites induced by the indenoisoquino-
lines. The bands corresponding to cleavage at identical
sites varied in intensity between the indenoisoquino-
lines and camptothecin. The DNA cleavage sites induced
among the indenoisoquinolines also varied in intensity.
For example, the cleavage site labeled “i” was much
more intense for 26 than it was for either 27 or 1.
A wider array of compounds were tested at various
concentrations, and an example of the results is dis-
played in Figure 2. The top1 inhibition data are sum-
marized in Table 1. In general, except for 13k , which
had very weak activity, the indenoisoquinolines induced
F igu r e 1. Comparison of top1-mediated DNA cleavage in-
duced by compounds 29, 26, 27, and 1 (NSC 314622).5 The
DNA fragment used corresponds to the 3′-end-labeled PVU II/
Hind III fragment of pBluescript SK(-) phagemid DNA. DNA
was reacted with top1 in the presence of the indicated drugs
(29, 1.0 µM; 26 100.0 µM; 27 and 1, 10.0 µM). Reactions were
at room temperature for 30 min and were stopped by adding
0.5% SDS. DNA fragments were separated on 16% denaturing
polyacrylamide gels. FA: purine ladder. Left lane: DNA
reacted with top1 in the absence of any drug. The numbers
on the left correspond to the camptothecin (29) induced
cleavage sites, whereas the letters on the right correspond to
additional cleavage sites induced by 26, 27, and 1.5,24
the other more cytotoxic analogues were significantly
less potent than 1 in the top1 cleavage assay. The
isoquinolinium salt 27 might be expected to have
F igu r e 2. Comparison of top1-mediated DNA cleavages at different drug concentrations induced by the indicated drugs in
pBluescript SK(-) phagemid DNA. The indicated drug concentrations are in µM/L. Reactions were at room temperature for 30
min and stopped by adding 0.5% SDS. DNA fragments were separated on 16% denaturing polyacrylamide gels. Top1 was present
in all reactions except in the control lane. Control: DNA with neither top1 nor any drug. Numbers and letters to the right are as
in Figure 1.

450 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
Strumberg et al.
tions but does not induce unwinding. Alternatively,
higher concentrations of 19a may induce a conforma-
tional change in the enzyme through a direct interac-
tion. Similar possible effects have been proposed pre-
viously to explain the bell-shaped curves produced by
saintopin E, which also does not cause DNA unwind-
ing.18
Camptothecin (29) induces DNA strand breaks by
stabilizing the cleavage complexes and inhibiting DNA
religation.21,22 However, increasing salt concentration
can reverse the camptothecin-induced cleavage com-
plexes, and this method has been used to compare the
molecular interactions between camptothecin deriva-
tives and top1 cleavage complexes. The cleavage sites
induced by camptothecin and the indenoisoquinoline
derivatives 1, 13c, 19a , 26, and 27 were reversed by
salt treatment (data not shown). This reversibility is
consistent with the reversible trapping of top1 cleavage
complexes by the indenoisoquinolines.
F igu r e 3. Top1-mediated DNA cleavage at different drug
concentrations induced by the drugs indicated on the top on
pBluescript SK(-) phagemid DNA. The activity of 1.0 µΜ
camptothecin (29) was arbitrarily set to 100% and the activity
of the indicated drugs is given in relation to the camptothecin
standard. Individual points represent the mean of three to four
independent experiments with a 10% variability.
In general, a planar indenoisoquinoline system ap-
pears to be a necessary, although not sufficient, condi-
tion for potent activity in the top1 cleavage assay. The
nonplanar systems 20-25 were all inactive or displayed
weak activity vs top1 (Table 1). A direct comparison can
be made between the planar indenoisoquinoline 1 and
the corresponding nonplanar, cis dihydro compound 20.
Compound 1 displays good activity in the top1 cleavage
assay, whereas the activity of 20 is weak. On the other
hand, indenoisoquinolines 3-6 and 13f-j are all planar
ring systems that are inactive as top1 inhibitors.
It is of interest to compare the results obtained with
the N-(4′-hydroxybutyl) compound 7 with those of the
corresponding acid 26 in the top1 cleavage assay. Both
of these simple indenoisoquinolines lack substituents
in the aromatic rings and differ only in the oxidation
state of the terminal carbon of the N-substituent. There
is a significant increase in top1 inhibitory activity in
going from the alcohol 7 to the corresponding carboxylic
acid 26.
Since a number of top1 poisons also inhibit top2,22
we tested the induction of top2 cleavage complexes by
indenoisoquinolines. Figure 5 shows a representative
experiment with the compounds that were the most
active against top1. Compound 26 induced top2 cleavage
complexes at sites which often did not overlap with the
top2 sites induced by VP-16 (etoposide). Compound 27
had only marginal top2 activity at 100 µM, and com-
pounds 13c, 19a , and 1 had no effect on top2 cleavage
activity (Figure 5). Compounds 7 and 8 also exhibited
weak top2 activity, and compounds 13b, 13k , 20, 21,
similar cleavage patterns. The indenoisoquinolines were
generally less potent than camptothecin. The relative
effective concentrations compared with 0.1 µM camp-
tothecin were 10 for 19a , 50 for 1 and 27, and 500 for
26. With some of the compounds (e.g., 27), the activity
seemed to increase initially as the concentration was
increased, but then it declined at higher concentrations.
This is reflected in Figure 3, which was obtained after
a more extensive investigation of the most potent
indenoisoquinolines. The increase and then decrease in
activity vs concentration indicates that the compound
suppresses top1-mediated DNA cleavage at higher drug
concentrations, a result which is similar to the bell-
shaped curves seen with DNA unwinding or intercalat-
ing poisons.17-19 To investigate the possibility that some
of the most potent indenoisoquinolines could be unwind-
ing DNA and thus causing inhibition of top1 activity at
higher drug concentrations, they were examined for
DNA unwinding activity. The unwinding assay using
supercoiled DNA in the presence of top1 is a simple
procedure to detect DNA intercalation.20 The results in
Figure 4 show that the indenoisoquinoline 27 in fact
does unwind DNA, as does 26 at higher concentrations.
On the other hand, the indenoisoquinoline 19a , like the
lead compound 1, does not appear to unwind DNA.
Thus, the reduced efficiency of 19a for inducing top1-
mediated DNA cleavage at high concentration does not
appear to be related to DNA unwinding. It is possible
that 19a might interact with DNA in a manner that
causes inhibition of top1 activity at higher concentra-
F igu r e 4. Native supercoiled SV40 DNA (lane 1) was incubated with top1 in the absence of any drug (lane 2) or in the presence
of the indicated drugs (concentrations in µΜ/L) for 30 min at 37 °C. Reactions were stopped with 0.5% SDS (final concentration).
The DNA was then separated in a 0.8% agarose gel stained with ethidium bromide.

Synthesis of Cytotoxic Indenoisoquinolines
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3 451
dard. 1H NMR spectra were determined at 300 MHz. Chemical
ionization mass spectra (CIMS) were determined using isobu-
tane as the reagent gas. Microanalyses were performed at the
Purdue University Microanalysis Laboratory. Analytical thin-
layer chromatography was carried out on Analtech silica gel
GF 1000 micron glass plates. Compounds were visualized with
short wavelength UV light or phosphomolybdic acid indicator.
Silica gel flash chromatography was performed using 230-
400 mesh silica gel.
6-Eth yl-5,6-d ih yd r o-5,11-d ik eto-11H-in d en o[1,2-c]iso-
qu in olin e (3). Ethylamine (0.2 mL, 3 mmol) was added to a
stirred solution of commercially available (Aldrich) benz[d]-
indeno[1,2-b]pyran-5,1l-dione (2) (0.49 g, 2 mmol) in CHCl3
(10 mL). The bright orange mixture was stirred overnight. To
the reaction mixture CHCl3 (100 mL) was added, and the
mixture was washed with H2O (3 × 25 mL) and brine (1 × 25
mL), dried (MgSO4), and concentrated under reduced pressure
to give an orange-red solid (0.43 g, 75%): mp 188-189 °C;
IR (thin film) 2986, 1690, 1656, 1611, 1549, 1503, 1430, 1320,
1
1197, 991 cm-1; H NMR (DMSO-d6) δ 8.66 (d, J ) 8.3 Hz, 1
H), 8.32 (d, J ) 7.9 Hz, 1 H), 7.69 (dt, J ) 8.4 and 1.4 Hz, 1
H), 7.60 (dd, J ) 8.0 and 1.4 Hz, 1 H), 7.52 (d, J ) 6.9 Hz, 1
H), 7.40 (m, 3 H), 4.56 (q, J ) 7.2 Hz, 2 H), 1.53 (t, J ) 7.2 Hz,
3 H); CIMS m/z (relative intensity) 276 (MH+, 100). Anal.
Calcd for C18H13NO2: C, H, N.
5,6-Dih yd r o-5,11-d ik eto-6-p r op yl-11H-in d en o[1,2-c]iso-
qu in olin e (4). Propylamine (0.3 mL, 3 mmol) was added to a
stirred solution of benz[d]indeno[1,2-b]pyran-5,1l-dione (2)
(0.49 g, 2 mmol) in CHCl3 (10 mL). The red solution stirred
overnight before CHCl3 (75 mL) was added, and the mixture
was washed with H2O (3 × 20 mL) and brine (1 × 20 mL),
dried (MgSO4), and concentrated under reduced pressure to
give a yellow-orange solid (0.32 g, 55%): mp 166-167 °C; IR
(neat) 2967, 1660, 1502, 1427, 1317, 1193, 959 cm-1; 1H NMR
(CDCl3) δ 8.69 (d, J ) 8.0 Hz, 1 H), 8.33 (d, J ) 9.0 Hz, 1 H),
7.70 (dt, J ) 9.0 and 3.0 Hz, 1 H), 7.62 (d, J ) 6.2 Hz, 1 H),
7.40 (m, 4 H), 4.46 (t, J ) 8.0 Hz, 2 H), 1.92 (m, 2 H), 1.12 (t,
J ) 7.4 Hz, 3 H); CIMS m/z (relative intensity) 290 (MH+, 100).
Anal. Calcd for C19H15NO2: C, H, N.
6-Cyclop r op yl-5,6-d ih yd r o-5,11-d ik eto-11H-in d en o[1,2-
c]isoqu in olin e (5). Cyclopropylamine (10 mL) was added to
a stirred solution of benz[d]indeno[1,2-b]pyran-5,1l-dione (2)
(0.28 g, 1.1 mmol) in CHCl3 (10 mL). The red solution stirred
overnight before CHCl3 (50 mL) was added, and the mixture
was washed with H2O (3 × 20 mL) and brine (1 × 20 mL),
dried (MgSO4), and concentrated under reduced pressure to
give a red solid (0.3 g, 91%): mp 206-208 °C; IR (thin film)
3751, 1665, 1500, 1420, 1311, 1083, 950 cm-1; 1H NMR (CDCl3)
δ 8.62 (d, J ) 7.7 Hz, 1 H), 8.29 (d, J ) 8.4 Hz, 1 H), 7.88 (d,
J ) 7.0 Hz, 1 H), 7.69 (dt, J ) 6.9 and 1.2 Hz, 1 H), 7.59 (dd,
J ) 6.1 and 1.3 Hz, 1 H), 7.40 (m, 3 H), 3.37 (m, 1 H), 1.45 (q,
J ) 6.8 Hz, 2 H), 0.99 (m, 2 H); CIMS m/z (relative intensity)
288 (MH+, 100). Anal. Calcd for C19H13NO2: C, H, N.
F igu r e 5. Comparison of top2-mediated DNA cleavages by
compounds 1, 19a , 13c, 27, 26, and VP-16 (etoposide). The
DNA fragment used corresponds to a fragment of the human
c-myc proto-oncogene.23 Reactions were performed at 37 °C for
30 min and stopped by adding SDS (0.5% final concentration).
DNA fragments were separated on an 8% denaturing poly-
acrylamide gel. Top2 was present in all reactions except in
lane 1. The indicated drug concentrations above each lane are
in µM/L. The concentration of VP-16 was 100 µM. FA indicates
the purine ladder.
and 22 had no effect on top2 cleavage (data not shown).
These results indicate that the indenoisoquinolines are
prominently top1 inhibitors, except for the two deriva-
tives 26 and 27 that are dual top1 and top2 inhibitors
and also produce DNA unwinding.
In conclusion, the present study was undertaken in
order to maximize the cytotoxicity of the lead compound
1 in human cancer cell cultures as well as its activity
as a top1 poison. The first objective was realized in the
cytotoxic indenoisoquinolines 7, 8, 13c, 13k , 19a , 20,
21, 22, and 27, all of which displayed a lower MGM than
the lead compound 1 (Table 1). With regard to the
second objective, several top1 inhibitors were synthe-
sized which rival the topoisomerase activity of 1, includ-
ing 13c, 19a , 26, and 27. One obvious point of further
interest is that with the possible exception of 19a and
13c, the two activities did not maximize in the same
compounds, suggesting that the activity of some of the
more cytotoxic compounds may not be due to their
activity vs top1. The situation is complicated by such
factors as cellular uptake and possible conversion of
parent compounds to metabolites which may have
increased activity vs top1.
5,6-Dih yd r o-5,11-d ik eto-6-(m eth oxyca r bon ylm eth yl)-
11H-in d en o[1,2-c]isoqu in olin e (6). Triethylamine (2.7 mL,
19.4 mmol) was added to a stirred solution of glycine methyl
ester hydrochloride (1.57 g, 12.5 mmol) in chloroform (30 mL).
After 1 h, benz[d]indeno[1,2-b]pyran-5, 1l-dione (2) (1.24 g, 5.0
mmol) was added to the mixture. The red mixture stirred an
additional 4 h before CHCl3 (100 mL) was added, and the
mixture was washed with H2O (3 × 50 mL) and brine (1 × 50
mL), dried (MgSO4), and concentrated under reduced pressure
to give an orange-red solid (1.48 g, 92%): mp 248-251 °C;
IR (thin film) 2956, 1735, 1667, 1609, 1502, 1426, 1227, 981
1
cm-1; H NMR (CDCl3) δ 8.68 (d, J ) 8.0 Hz, 1 H), 8.32 (d, J
) 8.2 Hz, 1 H), 7.73 (dt, J ) 7.1 and 1.3 Hz, 1 H), 7.61 (m, 1
H), 7.47 (dt, J ) 7.1 and 1.1 Hz, 1 H), 7.37 (m, 2 H), 7.26 (m,
1 H), 5.34 (s, 2 H), 3.79 (s, 3 H); CIMS m/z (relative intensity)
320 (MH+, 100). Anal. Calcd for C19H13NO4: C, H, N.
Exp er im en ta l Section
5,6-Dih yd r o-6-(4-h yd r oxy-1-bu tyl)-5,11-d ik eto-11H-in -
d en o[1,2-c]isoqu in olin e (7). 4-Amino-1-butanol (0.891 g, 10
mmol) was added to a chloroform (30 mL) solution of benz[d]-
indeno[1,2-b]pyran-5,1l-dione (2) (2.48 g, 10 mmol), and the
reaction mixture was stirred at room temperature for 2 days.
Melting points were determined in capillary tubes and are
uncorrected. Infrared spectra were obtained using CHCl3 as
1
the solvent unless otherwise specified. H NMR spectra were
obtained using CDCl3 as solvent and TMS as internal stan-

452 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
Strumberg et al.
The reaction mixture turned dark red. The reaction mixture
was taken in chloroform (100 mL) washed with water (2 × 50
mL), 0.5 N HCl (50 mL), and brine (100 mL), dried (Na2SO4),
and concentrated to give the crude product. The product was
filtered through a short column of silica gel and the polar
fraction concentrated to afford a reddish brown solid which
was crystallized from 2-propanol to yield the product (2.56 g,
anhydride (10a ) (3.24 g, 20 mmol) was added to a chloroform
(20 mL) solution of the imine 11b (4.1 g, 20 mmol), and the
mixture was stirred at room temperature for 45 min, after
which the TLC showed the complete disappearance of the
starting materials. The reaction mixture was concentrated to
remove chloroform completely. The residue was dissolved in
hot ethyl acetate (100 mL) and left at room temperature for
12 h. The colorless crystals that separated were filtered and
dried to give pure 12b (6.57 g, 89%): mp 178-181 °C; IR (KBr)
80%): mp 160-162 °C; IR (KBr) 3300, 1695, 1645, 1615 cm-1
;
1H NMR (CDCl3) δ 8.63 (d, J ) 8.1 Hz, 1 H), 8.26 (d, J ) 8.1
Hz, 1 H), 7.70-7.15 (m, 6 H), 4.51 (t, J ) 7.8 Hz, 2 H), 3.77 (t,
J ) 6.1 Hz, 2 H), 1.99 (p, J ) 8.0 and 7.5 Hz, 2 H), 1.83 (s, 1
H, D2O exchangeable), 1.75 (t, J ) 6.6 Hz, 2 H). Anal. Calcd
for C20H17NO3: C, H, N.
1
1712, 1634, 1600 cm -1; H NMR (CDCl3) δ 8.08 (dd, J ) 1.0
and 7.5 Hz, 1 H), 7.52 (d, J ) 7.5 Hz, 1 H), 7.40-7.28 (m, 2
H), 6.51-6.45 (m, 2 H), 6.37 (s, 1 H), 5.75 (dd, J ) 1.1 and 6.4
Hz, 2 H), 4.87 (d, J ) 6.2 Hz, 1 H), 4.51 (d, J ) 6.2 Hz, 1 H),
3.93 (dt, J ) 7.2 and 6.6 Hz, 1 H), 2.73 (dt, J ) 7.2 and 6.6
Hz, 1 H), 1.52 (p, J ) 7.2 Hz, 2 H), 1.26 (hextet, J ) 7.3 Hz,
2 H), 0.83 (t, J ) 7.2 Hz, 3 H); CIMS m/z (relative intensity)
368 (MH+, 100); EIMS m/z (relative intensity) 367 (M+, 5), 322
(30), 135 (100). Anal. Calcd for C21H21NO5: C, H, N.
5,6-Dih yd r oxy-6-(5-h yd r oxy-1-p en tyl)-5,11-d ik eto-11H-
in d en o[1,2-c]isoqu in olin e (8). 5-Amino-1-pentanol (1.03 g,
10 mmol) was added to a chloroform (20 mL) solution of benz-
[d]indeno[1,2-b]pyran-5,1l-dione (2) (2.48 g, 10 mmol), and the
reaction mixture was stirred at room temperature overnight.
The reaction mixture turned dark red. The reaction mixture
was taken in chloroform (100 mL), washed with water (2 ×
50 mL), 0.5 N HCl (50 mL), and brine (100 mL), dried (Na2-
SO4), and concentrated to give the crude product. The TLC
showed traces of starting material. The product was filtered
through a short column of silica gel and the polar fraction
concentrated to get a reddish-brown solid which was crystal-
lized from 2-propanol to afford the product (2.53 g, 76%): mp
6-(1-Bu tyl)-5,6-dih ydr o-5,11-diketo-8,9-m eth ylen edioxy-
11H-in d en o[1,2-c]isoqu in olin e (13b). Thionyl chloride (30
mL) was added dropwise to the acid 12b (3.35 g, 0.089 mol)
with stirring. The resulting solution was stirred at room
temperature for 12 h, after which the solution turned dark
pink. Benzene (20 mL) was added to the reaction mixture, and
it was concentrated under reduced pressure. The resulting
residue was purified by column chromatography (acetone:
hexane, 1:4) followed by crystallization (EtOAc/hexane) to
obtain pure indenoisoquinoline 13b (1.37 g, 44%): mp 200-
1
146-148 °C; IR (KBr) 2996, 1698, 1642, 1615 cm-1; H NMR
(CDCl3) δ 8.63 (d, J ) 8.1 Hz, 1 H), 8.27 (d, J ) 8.1 Hz, 1 H),
7.67 (d, J ) 8.4 Hz, 1 H), 7.56 (d, J ) 6.8 Hz, 1 H), 7.45-7.30
(m, 4 H), 4.47 (t, J ) 7.9 Hz, 2 H), 3.71 (t, J ) 5.9 Hz, 2 H),
1.92 (p, J ) 7.9 and 7.4 Hz, 2 H), 1.82 (s, 1 H, D2O
exchangeable), 1.78-1.55 (m, 4 H); CIMS m/z (relative inten-
sity) 334 (MH+, 100). Anal. Calcd for C21H19NO3: C, H, N.
1
201 °C; IR (KBr) 1691, 1665, 1631 cm -1; H NMR (CDCl3) δ
8.50 (d, J ) 8.1 Hz, 1 H), 8.21 (d, J ) 8 Hz, 1 H), 7.61 (t, J )
8 Hz, 1 H), 7.34 (t, J ) 8 Hz, 1 H), 6.98 (s, 1 H), 6.87 (s, 1 H),
6.03 (s, 2 H), 4.34 (t, J ) 8 Hz, 2 H), 1.80 (p, J ) 8 Hz, 2 H),
1.51 (hextet, J ) 8 Hz, 2 H), 1.01 (t, J ) 8 Hz, 3 Hz); 13C NMR
(CDCl3) δ 189.01, 163.1, 154.85, 151.21, 148.97, 133.58, 132.18,
132.05, 130.50, 128.29, 126.39, 122.82, 122.69, 107.48, 105.12,
104.84, 102.57, 44.13, 31.33, 20.1, 13.73; CIMS m/z (relative
intensity) 348 (MH+, 100); EIMS m/z (relative intensity) 347
cis-4-Ca r boxy-3,4-d ih yd r o-N-m eth yl-3-(3′,4′-m eth ylen e-
d ioxyp h en yl)-1(2H)isoqu in olon e (12a ). Homophthalic an-
hydride (10a ) (0.81 g, 5 mmol) was added to a stirred solution
of 3,4-methylenedioxybenzylidenemethylamine (11a ) (0.82 g,
5 mmol) in chloroform (5 mL). After 30 min, the precipitated
product was filtered from the yellow solution and washed with
chloroform to give a pale yellow solid (1.2 g, 74%): mp 165-
(M+, 60), 330 (10), 318 (30), 291 (100). Anal. Calcd for C21H17
NO4: C, H, N.
-
3,4-Meth ylen ed ioxyben zylid en ea llyla m in e (11c). Ally-
lamine (1.71 g, 30 mmol) was added to a solution of piperonal
(4.5 g, 30 mmol) in chloroform (30 mL) in the presence of
anhydrous magnesium sulfate (3 g), and the reaction mixture
was stirred at room temperature overnight. The reaction
mixture was filtered, the residue washed with chloroform (10
mL), and the combined filtrate was concentrated under
reduced pressure to afford a yellow oil (5.55 g, 98%): IR (neat)
1679, 1639, 1600 and 1585 cm-1; 1H NMR (CDCl3) δ 8.05 (s, 1
H), 7.31 (s, 1 H), 7.04 (d, J ) 8 Hz, 1 H), 6.10-6.00 (m, 1 H),
5.88 (s, 2 H), 5.04 (m, 2 H), 4.10 (m, 2 H).
1
167 °C; H NMR (DMSO-d6) δ 7.99 (d, J ) 7.5 Hz, 1 H), 7.48
(m, 3 H), 6.76 (d, J ) 8.0 Hz, 1 H), 6.52 (d, J ) 8.0 Hz, 1 H),
6.43 (s, 1 H), 5.93 (s, 2 H), 5.03 (d, J ) 6.2 Hz, 1 H), 4.64 (d,
J ) 6.1 Hz, 1 H), 2.89 (s, 3 H); CIMS m/z (relative intensity)
326 (MH+, 100).
5,6-Dih yd r o-5,11-d ik eto-6-m eth yl-8,9-m eth ylen ed ioxy-
11H-in d en o[1,2-c]isoqu in olin e (13a ). Thionyl chloride (8.1
mL) was added with stirring to the cis acid 12a (0.7 g, 2.1
mmol). The yellowish-brown mixture became orange within
15 min and after 30 min was red. After 4 h, the reaction
mixture was diluted with benzene (25 mL) and evaporated to
dryness. The brownish-red solid was recrystallized from
methanol and passed through a short column (SiO2) and eluted
with chloroform to give a brown solid (0.14 g, 24%): mp 310-
cis-N-Allyl-4-car boxy-3,4-dih ydr o-6,7-dim eth oxy-3-(3′,4′-
m et h ylen ed ioxyp h en yl)-1(2H )isoq u in olon e (12c). 4,5-
Dimethoxyhomophthalic anhydride (10b) (1.11 g, 5 mmol) was
added to a chloroform (10 mL) solution of the imine 11c (0.945
g, 5 mmol) and the mixture was stirred at room temperature
for 45 min, after which the TLC showed the complete disap-
pearance of the starting materials and a white precipitate
formed in the reaction mixture. The precipitated product was
filtered off, washed with chloroform (5 mL), and dried to give
pure 12c (1.43 g, 70%): mp 235-238 °C; IR (KBr) 3000, 1736,
1
312 °C; IR (thin film) 2358, 1652, 1540, 1506, 1292 cm-1; H
NMR (DMSO-d6) δ 8.43 (d, J ) 8.0 Hz, 1 H), 8.16 (d, J ) 8.0
Hz, 1 H), 7.75 (t, J ) 7.5 Hz, 1 H), 7.56 (s, 1 H), 7.44 (t, J )
7.6 Hz, 1 H), 7.15 (s, 1 H), 6.19 (s, 2 H), 3.92 (s, 3 H); CIMS
m/z (relative intensity) 306 (MH+, 100). Anal. Calcd for C18H11
-
NO4: C, H, N.
1
1686, 1615 cm-1; H NMR (DMSO-d6) δ 13.0 (bs, 1 H), 7.52
3,4-Meth ylen ed ioxybezylid en ebu tyla m in e (11b). Pip-
eronal (7.5 g, 50 mmol) and n-butylamine (6 mL, 75 mmol)
were stirred in chloroform (100 mL) in the presence of
anhydrous MgSO4 (5 g) at room temperature for 4 h. The
mixture was filtered, and the residue was washed with
chloroform (20 mL). The combined filtrate was concentrated
under reduced pressure to afford a yellow oil (9.8 g, 96%): IR
(neat) 1649, 1643, 1604 cm-1; 1H NMR (CDCl3) δ 8.11 (s, 1 H),
7.31 (d, J ) 1.2 Hz, 1 H), 7.06 (dd, J ) 1.2 and 7.9 Hz, 1 H),
6.79 (d, J ) 7.8 Hz, 1 H), 5.95 (s, 2 H), 3.53 (t, J ) 6.6 Hz, 2
H), 1.63 (p, J ) 7.3 Hz, 2 H), 1.37 (hextet, J ) 7.3 Hz, 2 H),
0.91 (t, J ) 7.3 Hz, 3 H).
(s, 1 H), 7.13 (s, 1 H), 6.76 (d, J ) 8.5 Hz, 1 H), 6.52 (d, J )
8.5 Hz, 1 H), 6.44 (s, 1 H), 5.94 (s, 2 H), 5.85-5.70 (m, 1 H),
5.16 (dd, J ) 3.5 and 17.5 Hz, 2 H), 4.92 (d, J ) 6.5 Hz, 1 H),
4.57 (d, J ) 6.5 Hz, 1 H), 3.82 (s, 3 H), 3.75 (s, 3 H), 3.20-3.10
(m, 2 H). CIMS m/z (relative intensity) 412 (MH+, 100). Anal.
Calcd for C22H21NO7: C, H, N.
6-Allyl-2,3-d im eth oxy-5,6-d ih yd r o-5,11-oxo-8,9-(m eth -
ylen ed ioxy)-11H-in d en o[1,2-c]isoqu in olin e (13c). Treat-
ment of 12c (2.05 g, 5 mmol) with Eaton’s reagent (10% P2O5
in methanesulfonic acid, 60 mL) at room temperature with
stirring in an open flask for 24 h resulted in a mixture of 22
and 13c. The two products were separated by column chro-
matography on silica gel (230-400 mesh) using hexane:acetone
cis-N-(1-Bu t yl)-4-ca r b oxy-3,4-d ih yd r o-3-(3′,4′-m et h yl-
en ed ioxyp h en yl)-1(2H)isoqu in olon e (12b). Homophthalic

Synthesis of Cytotoxic Indenoisoquinolines
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3 453
(4:1) to afford 22 (842 mg, 43%) and 12c as a purple solid
product (588 mg, 30%) after recrystallization from ethyl
acetate-hexane: mp 290-294 °C; IR (KBr) 2370, 1698, 1653,
undissolved solid was filtered off to obtain pure indenoiso-
quinoline 13e (0.716 g, 65%): mp 310-312 °C; IR (KBr) 1695,
1
1652, 1619, 1578 cm-1; H NMR (CDCl3) δ 8.02 (s, 1 H), 7.66
1
1551, 1484 cm-1; H NMR (CDCl3) δ 7.97 (s, 1 H), 7.63 (s, 1
(s, 1 H), 7.4-7.20 (m, 5 H), 7.02 (s, 1 H), 6.74 (s, 1 H), 5.99 (s,
2 H), 5.69 (s, 2 H), 4.04 (s, 3 H), 3.97 (s, 3 H); 13C NMR (CDCl3)
δ 162.54, 155.03, 148.72, 135.44, 132.52, 130.22, 129.19,
127.67, 125.64, 108.32, 105.24, 103.03, 102.47, 56.31, 47.80,
and 56.03. CIMS m/z (relative intensity) 442 (MH+, 100). Anal.
Calcd for C26H19NO6‚0.8H2O: C, H, N.
H), 6.99 (s, 1 H), 6.91 (s, 1 H), 6.20-6.05 (m, 1 H), 6.06 (s, 2
H), 5.31 (d, J ) 10.5 Hz, 1 H), 5.20-5.00 (m, 3 H), 3.33 (s, 3
H), 3.97 (s, 3 H). Anal. Calcd for C22H17NO6: C, H, N.
cis-N-(1-Bu t yl)-4-ca r b oxy-3,4-d ih yd r o-6,7-d im et h oxy-
3-(3′,4′-m eth ylen ed ioxyp h en yl)-1(2H)isoqu in olon e (12d ).
4,5-Dimethoxyhomophthalic anhydride 10b (2.22 g, 10 mmol)
was added to a chloroform (10 mL) solution of the imine (11b)
(2.1 g, 10 mmol) and the mixture was stirred at room
temperature for 45 min, after which the TLC showed the
complete disappearance of the starting materials and a white
precipitate formed in the reaction mixture. The precipitated
product was filtered off, washed with chloroform (5 mL), and
dried to give pure 12d (3.45 g, 81%): mp 242-244 °C; IR (KBr)
3,4-Meth ylen ed ioxyben zylid en e-p-a n isid in e (11f). Pip-
eronal (15 g, 0.1 mol) and p-anisidine (12.3 g, 0.1 mol) were
stirred in methylene chloride (100 mL) in the presence of
anhydrous MgSO4 (5 g) at room temperature for 4 h. The
mixture was filtered, the residue was washed with methylene
chloride (20 mL), and the combined filtrate was concentrated
under reduced pressure to afford a yellow solid. The crude
product was crystallized in 95% ethanol to give a white
crystalline solid (22.38 g, 87%): mp 113-114 °C; IR (KBr) 1636
1
1732, 1640, 1610, 1600 cm-1; H NMR (CDCl3 + DMSO-d6) δ
1
and 1617 cm-1; H NMR (CDCl3) δ 8.33 (s, 1 H), 7.51 (d, J )
7.56 (s, 1 H), 7.08 (s, 1 H), 6.55-6.48 (m, 2 H), 6.40 (s, 1 H),
5.79 (d, J ) 2.5 Hz, 2 H), 4.86 (d, J ) 6.2 Hz, 1 H), 4.45 (d, J
) 6.2 Hz, 1 H), 3.88 (dt, J ) 7.4 and 6.1 Hz, 1 H), 3.84 (s, 3H),
3.76 (s, 3 H), 2.71 (dt, J ) 7.5 and 6.1 Hz, 1 H), 1.49 (p, J )
7.3 Hz, 2 H), 1.26 (hextet, J ) 7.3 Hz, 2 H), 0.83 (t, J ) 7.3
Hz, 3 H). Anal. Calcd for C23H25NO7: C, H, N.
1.2 Hz, 1 H), 7.2-7.10 (m, 3 H), 6.95-6.80 (m, 3 H), 6.01 (s, 2
H), 3.80 (s, 3 H).
cis-N-(p-An isyl)-4-ca r boxy-3,4-d ih yd r o-6,7-d im eth oxy-
3-(3′,4′-m eth ylen ed ioxyp h en yl)-1(2H)isoqu in olon e (12f).
4,5-Dimethoxyhomophthalic anhydride (10b) (1.11 g, 5 mmol)
was added to a chloroform (10 mL) solution of the imine 11f
(1.275 g, 5 mmol) and the mixture was stirred at room
temperature for 12 h, after which the TLC showed the
complete disappearance of the starting materials and a white
precipitate formed in the reaction mixture. The precipitated
product was filtered off, washed with chloroform (5 mL), and
dried to afford pure 12f (1.36 g, 60%): mp >350 °C; IR (KBr)
6-(1-Bu tyl)-5,6-d ih yd r o-5,11-d ik eto-2,3-d im eth oxy-8,9-
m eth ylen edioxy-11H-in den o[1,2-c]isoqu in olin e (13d). Thio-
nyl chloride (30 mL) was added dropwise to the acid 12d (2.135
g, 5 mmol) with stirring. The resulting solution was stirred at
room temperature for 12 h, after which the solution turned
dark pink. Benzene (20 mL) was added to the reaction mixture
and it was concentrated under reduced pressure. Benzene (50
mL) was added to the resulting residue, and the pink solid
was filtered off to obtain pure indenoisoquinoline 13d (1.3 g,
1
1644, 1639, 1599 cm-1; H NMR (DMSO-d6) δ 7.60-7.30 (m,
5 H), 7.20-6.80 (m, 4 H), 6.10 (s, 2 H), 5.30 (d, J ) 6 Hz, 1 H),
4.77 (d, J ) 6 Hz, 1 H), 3.78 (s, 3 H), 3.71 (s, 3 H), 3.01 (s, 3
65%): mp 280-284 °C; IR (KBr) 1699, 1653, 1646, 1578 cm-1
;
H).
1H NMR (CDCl3) δ 7.99 (s, 1 H), 7.62 (s, 1 H), 7.04 (s, 1 H),
6.92 (s, 1 H), 6.07 (s, 2 H), 4.39 (t, J ) 7.6 Hz, 2 H), 4.01 (s, 3
H), 3.96 (s, 3 H), 1.82 (p, J ) 7.3 Hz, 2 H), 1.68-1.55 (m, 2 H),
1.02 (t, J ) 7.3 Hz, 3 Hz). Anal. Calcd for C23H21NO6‚0.1 H2O:
C, H, N.
6-(p-An isyl)-2,3-d im eth oxy-5,6-d ih yd r o-5,11-d ik eto-8,9-
m eth ylen edioxy-11H-in den o[1,2-c]isoqu in olin e (13f). Thio-
nyl chloride (9 mL) was added dropwise to the acid 12f (0.822
g, 2 mmol) with stirring. The resulting solution was stirred at
room temperature for 5 h, after which the solution turned
purple. Benzene (20 mL) was added to the reaction mixture,
and it was concentrated under reduced pressure. The resulting
residue was passed through a short column of silica gel (230-
400 mesh), eluting with chloroform. Concentration of the
eluent resulted in a pink solid which was crystallized from
ethyl acetate to obtain pure indenoisoquinoline 13f (0.436 g,
53%): mp 360-364 °C; IR (KBr) 1692, 1652, 1625, and 1552
cm-1; 1H NMR (CDCl3) δ 7.94 (s, 1 H), 7.60 (s, 1 H), 7.34 (d, J
) 8.1 Hz, 2 H), 7.10 (d, J ) 8 Hz, 2 H), 6.88 (s, 1 H), 5.90 (s,
2 H), 5.05 (s, 1 H), 4.02 (s, 3 H), 3.93 (s, 3 H), 3.91 (s, 3 H);
CIMS m/z (relative intensity) 458 (MH+, 100). Anal. Calcd for
C26H19NO7: C, H, N.
3,4-Meth ylen edioxyben zyliden eben zylam in e (11e). Pip-
eronal (4.5 g, 30 mmol) and benzylamine (3.21 g, 30 mmol)
were stirred in methylene chloride (30 mL) in the presence of
anhydrous MgSO4 (5 g) at room temperature for 4 h. The
mixture was filtered, the residue was washed with methylene
chloride (20 mL), and the combined filtrate was concentrated
under reduced pressure to afford a white solid (7.03 g, 98%):
mp 69-70 °C; IR (KBr) 1638, 1618, 1602 cm-1 1H NMR
;
(CDCl3) δ 8.18 (s, 1 H), 7.33 (d, J ) 1.3 Hz, 1 H), 7.30-7.10
(m, 5 H), 7.06 (dd, J ) 1.3 and 8.0 Hz, 1 H), 6.74 (d, J ) 8 Hz,
1 H), 5.90 (s, 2 H), 4.69 (s, 2 H).
cis-N-Ben zyl-4-ca r b oxy-3,4-d ih yd r o-6,7-d im et h oxy-3-
(3′,4′-m et h ylen ed ioxyp h en yl)-1(2H )isoq u in olon e (12e).
4,5-Dimethoxyhomophthalic anhydride (10b) (1.11 g, 5 mmol)
was added to a chloroform (10 mL) solution of the imine 11e
(1.19 g, 5 mmol), and the mixture was stirred at room
temperature for 2 h, after which the TLC showed the complete
disappearance of the starting materials and a white precipitate
formed in the reaction mixture. The precipitated product was
filtered off, washed with chloroform (5 mL), and dried to give
pure 12e (1.89 g, 82%): mp 262-264 °C; IR (KBr) 1736, 1654,
1647, 1618, 1595, 1575 cm-1; 1H NMR (DMSO-d6) δ 7.56 (s, 1
H), 7.35-7.20 (m, 5 H), 7.13 (s, 1 H), 6.75 (d, J ) 8.3 Hz, 1 H),
6.51 (d, J ) 8.1 Hz, 1 H), 6.43 (s, 1 H), 5.93 (s, 2 H), 5.25 (d,
J ) 15.6 Hz, 1 H), 4.86 (d, J ) 5.6 Hz, 1 H), 4.51 (d, J ) 5.3
Hz, 1 H), 3.83 (s, 3 H), 3.74 (s, 3 H), 3.39 (d, J ) 15.6 Hz, 1 H).
3,4-Diben zyloxyben zylid en em eth yla m in e (11g). 3,4-
Dibenzyloxybenzaldehyde (7.96 g, 25.0 mmol) was added to a
40% aqueous solution of methylamine (10 mL), and the
reaction mixture was stirred at room temperature for 3 h. The
mixture was extracted with ether (4 × 75 mL), the ether layers
were combined, and the solution washed with saturated
aqueous sodium chloride (75 mL), dried (MgSO4), and concen-
trated under reduced pressure to give an off-white solid (7.7
g, 94%): mp 56-57 °C; IR (KBr) 3031, 2936, 2832, 1648, 1600,
1582, 1509, 1454, 1431, 1267, 1171, 1137, 1017, 735, 696 cm-1
;
1H NMR (CDCl3) δ 8.14 (s, 1 H), 7.35 (m, 11 H), 7.11 (dd, J )
8.1 and 1.0 Hz, 1 H), 6.93 (d, J ) 8.1 Hz, 1 H), 5.18 (s, 4 H),
3.46 (s, 3 H); CIMS m/z (relative intensity) 332 (MH+, 100).
Anal. Calcd for C22H21NO2: C, H, N.
6-Ben zyl-5,6-d ih yd r o-5,11-d ik eto2,3-d im eth oxy-8,9-m e-
th ylen ed ioxy-11H-in d en o[1,2-c]isoqu in olin e (13e). Thio-
nyl chloride (10 mL) was added dropwise to the acid 12e (1.15
g, 2.5 mmol) with stirring. The resulting mixture was stirred
at room temperature for 5 h, after which the solution turned
purple. Benzene (20 mL) was added to the reaction mixture,
and it was concentrated under reduced pressure. Carbon
tetrachloride was added to the resulting residue, and the
cis-3-(3′,4′-Diben zyloxyp h en yl)-4-ca r boxy-3,4-d ih yd r o-
N-m eth yl-1-(2H)-isoqu in olon e (12g). Homophthalic anhy-
dride (10a ) (0.81 g, 5 mmol) was added to a stirred solution of
3,4-dibenzyloxybenzylidenemethylamine (11g) (1.66 g, 5 mmol)
in chloroform (5 mL). After 30 min, ether was added, and the
resulting precipitate was filtered and washed with ether to
give a pale yellow solid (0.9 g, 36%): mp 170-172 °C; IR (thin
film) 3030, 1731, 1625, 1514, 1263, 1137, 1014 cm-1; 1H NMR

454 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
Strumberg et al.
(CDCl3) δ 8.19 (dd, J ) 6.5 and 1.9 Hz, 1 H), 7.36 (m, 10 H),
7.09 (d, J ) 4.9 Hz, 1 H), 6.74 (d, J ) 8.9 Hz, 1 H), 6.68 (d, J
) 8.9 Hz, 1 H), 6.51 (m, 2 H), 5.03 (d, J ) 7.2 Hz, 2 H), 4.92
(d, J ) 6.1 Hz, 2 H), 4.8 (d, J ) 6.3 Hz, 2 H), 4.5 (d, J ) 6.2
Hz, 2 H), 2.98 (s, 3 H); CIMS m/z (relative intensity) 494 (MH+,
100). Anal. Calcd for C31H27NO5: C, H, N.
H), 3.59 (s, 9 H); CIMS m/z (relative intensity) 432 (MH+, 100).
Anal. Calcd for C22H25NO8: C, H, N.
5,6-Dih yd r o-5,11-d ik et o-6-m et h yl-2,3,8,9,10-p en t a -
m eth oxy-11H-in den o[1,2-c]isoqu in olin e (13i). Thionyl chlo-
ride (15 mL) was added with stirring to the cis acid 12i (1.2 g,
2.8 mmol). The result was a yellow mixture that became dark
red within 15 min. After 4 h, the reaction mixture was diluted
with benzene (25 mL) and evaporated to dryness. The purple
solid was dissolved in chloroform, and ether was added to give
a precipitate that was collected and washed with ether to give
a purple solid (0.75 g, 7.1%): IR (neat) 2944, 1653, 1471, 1255,
8,9-Diben zyloxy-5,6-d ih yd r o-5,11-d ik eto-6-m eth yl-11H-
in d en o[1,2-c]isoqu in olin e (13g). Thionyl chloride (8.1 mL)
was added with stirring to the cis acid 12g (0.7 g, 2.1 mmol).
The result was a yellowish-brown mixture that became orange
within 15 min and after 30 min was red. After 4 h, the reaction
mixture was diluted with benzene (25 mL) and evaporated to
dryness. The brownish-red solid was recrystallized from
methanol and passed through a short column (silica gel),
eluting with chloroform, to give a brown solid (0.14 g, 24%):
mp 198-200 °C; 1H NMR (DMSO-d6) δ 8.43 (d, J ) 8.0 Hz, 1
H), 8.16 (d, J ) 8.0 Hz, 1 H), 7.75 (t, J ) 7.5 Hz, 1 H), 7.39 (m,
13 H), 5.34 (s, 1 H), 5.29 (s, 1 H), 3.93 (s, 1 H); CIMS m/z
1
1116, 1019 cm-1; H NMR (CDCl3) δ 8.15 (s, 1 H), 7.69 (s, 1
H), 7.02 (s, 1 H), 4.11 (s, 3 H), 4.05 (s, 3 H), 4.02 (s, 3 H), 3.99
(s, 6 H), 3.91 (s, 3 H); CIMS m/z (relative intensity) 412 (MH+,
100).
cis-4-Car boxy-3,4-dih ydr o-N-m eth yl-3-(3′,4′,5′-tr im eth ox-
yp h en yl)-1(2H)isoqu in olon e (12j). Homophthalic anhydride
(10a ) (0.32 g, 2 mmol) was added to a stirred solution of 3,4,5-
trimethoxybenzylidenemethylamine (11i) (0.46 g, 2 mmol) in
chloroform (5 mL). After 45 min, ether was added dropwise to
the homogeneous mixture, and the resulting precipitate was
filtered from the yellow solution and washed with ether to give
a pale yellow solid (0.43 g, 60%): mp 194-195 °C; IR (neat)
(relative intensity) 474 (MH+, 100). Anal. Calcd for C31H23
NO4: C, H, N.
-
cis-3-(3′,4′-Diben zyloxyp h en yl)-4-ca r boxy-3,4-d ih yd r o-
N-m eth yl-6,7-d im eth oxy-1-(2H)-isoqu in olon e (12h ). 4,5-
Dimethoxyhomophthalic anhydride (10b) (0.56 g, 2.5 mmol)
was added to a stirred solution of 3,4-dibenzyloxybenzyliden-
emethylamine (11g) (0.83 g, 2.5 mmol) in chloroform (3 mL).
After 30 min, the yellow mixture became heterogeneous, and
ether was added to further precipitate the product. The light
yellow precipitate was collected and washed with chloroform
to give a solid (0.59 g, 44%): mp 194-196 °C; 1H NMR (CDCl3)
δ 7.49 (s, 1 H), 7.34 (m, 11 H), 7.18 (s, 1 H), 6.91 (d, J ) 8.3
Hz, 1 H), 6.79 (s, 1 H), 6.57 (d, J ) 8.3 Hz, 1 H), 5.02 (s, 2 H),
4.98 (d, J ) 6.1 Hz, 1 H), 4.92 (s, 2 H), 4.50 (d, J ) 5.8 Hz, 1
H), 3.78 (s, 3 H), 3.74 (s, 3 H), 2.81 (s, 3 H); FABMS (m-NBA)
m/z (relative intensity) 554 (MH+, 100).
1
2830, 1620, 1549, 1459, 1185, 1123 cm-1; H NMR (CDCl3) δ
8.13 (s, 1 H), 7.99 (d, J ) 7.2 Hz, 1 H), 7.52 (m, 4 H), 6.32 (s,
2 H), 5.04 (d, J ) 5.9 Hz, 1 H), 4.63 (d, J ) 6.0 Hz, 1 H), 3.58
(s, 3 H), 3.55 (s, 6 H), 2.94 (s, 3 H); CIMS m/z (relative
intensity) 372 (MH+, 100). Anal. Calcd for C20H21NO6: C, H,
N.
5,6-Dih yd r o-5,11-d ik et o-6-m et h yl-8,9,10-t r im et h oxy-
11H-in d en o[1,2-c]isoqu in olin e (13j). Thionyl chloride (10
mL) was added with stirring to 12j (200 mg, 0.5 mmol). After
4 h, the reaction mixture was diluted with benzene (50 mL)
and evaporated to dryness. The dark orange solid was dis-
solved in chloroform, and ether was added to give a dark
orange solid (16 mg, 10%): mp 194-195 °C; IR (neat) 2938,
1665, 1463, 1400, 1292, 1125, 1007, 976 cm-1; 1H NMR (CDCl3)
δ 8.67 (d, J ) 7.8 Hz, 1 H), 8.32 (d, J ) 8.0 Hz, 1 H), 7.68 (t,
J ) 8.0 Hz, 1 H), 7.45 (t, J ) 7.8 Hz, 1 H), 7.04 (s, 1 H), 4.09
(s, 3 H), 4.06 (s, 3 H), 3.97 (s, 3 H), 3.89 (s, 3 H); CIMS m/z
8,9-Diben zyloxy-5,6-d ih yd r o-5,11-d ik eto-6-m eth yl-2,3-
d im eth oxy-11H-in d en o[1,2-c]isoqu in olin e (13h ). Thionyl
chloride (15 mL) was added with stirring to the cis acid 12h
(1.2 g, 2.2 mmol). The result was an orange mixture that
became dark red within 15 min. After 6 h, the reaction mixture
was diluted with benzene (25 mL) and evaporated to dryness.
Chloroform (7 mL) was added to the purple solid, and the solid
was collected and washed with ether to give a light purple solid
(0.75 g, 64%): mp 238-240 °C; IR (thin film) 3027, 2963, 1685,
(relative intensity) 352 (MH+, 100). Anal. Calcd for C20H17
NO5: C, H, N.
-
3,4-Meth ylen ed ioxyben zylid en eeth yla m in e (11k ). Pip-
eronal (20.1 g, 0.14 mol) and a 70% aqueous solution of
ethylamine (20 mL) were stirred at room temperature for 3 h.
The mixture was extracted with ether (4 × 50 mL). The ether
layers were combined, washed with aqueous sodium chloride
(50 mL), dried (MgSO4), and concentrated under reduced
pressure to give a white crystalline powder (24.56 g, 93%): mp
47-48 °C; IR (KBr) 2963, 2836, 1645, 1603, 1498, 1480, 1441,
1649, 1493, 1458, 1252, 1203, 1088, 1014 cm-1 1H NMR
;
(CDCl3) δ 7.96 (s, 1 H), 7.62 (s, 1 H), 7.38 (m, 10 H), 7.21 (s, 1
H), 7.11 (s, 1 H), 5.23 (d, J ) 5.2 Hz, 4 H), 4.02 (s, 3 H) 3.95
(s, 3 H), 3.81 (s, 3 H); CIMS m/z (relative intensity) 534 (MH+,
22). Anal. Calcd for C33H27NO6: C, H, N.
3,4,5-Tr im eth oxyben zylid en em eth yla m in e (11i). 3,4,5-
Trimethoxybenzaldehyde (7.81 g, 40.0 mmol) and a 40%
aqueous solution of methylamine (20 mL) were stirred at room
temperature for 2.5 h. The mixture was extracted with ether
(4 × 75 mL), the ether layers were combined, and the solution
was washed with saturated aqueous sodium chloride (75 mL),
dried (MgSO4), and concentrated under reduced pressure to
give a colorless oil (7.94 g, 95%): IR (neat) 2940, 2840, 1646,
1
1252, 1092, 1031, 959, 926 cm-1; H NMR (CDCl3) δ 8.15 (s,
1 H), 7.32 (d, J ) 1.3 Hz, 1 H), 7.09 (dd, J ) 1.4 and 6.0 Hz,
1 H), 6.80 (d, J ) 8.0 Hz, 1 H), 5.98 (s, 2 H), 3.59 (qd, J ) 6.0
and 1.2 Hz, 2 H), 1.26 (t, J ) 7.3 Hz, 3 H); CIMS m/z (relative
intensity) 178 (MH+, 100). Anal. Calcd for C10H11NO2: C, H,
N.
cis-4-Ca r boxy-N-eth yl-3-(3′,4′-m eth ylen ed ioxyp h en yl)-
6,7-d im eth oxy-3,4-d ih yd r o-1(2H)isoqu in olon e (12k ). 3,4-
Methylenedioxybenzylideneethylamine (11k) (0.89 g, 5.0 mmol)
was stirred in chloroform (5.0 mL), and 4,5-dimethoxyhomoph-
thalic anhydride (10b) (1.11 g, 5.0 mmol) was added. After 30
min, the yellow precipitate was filtered and washed with
chloroform to give a pale yellow solid (0.58 g, 29%): mp 231-
233 °C (dec); IR (KBr) 2937, 1732, 1615, 1594, 1573, 1254,
1223, 1174, 1089, 1034, 986, 898 cm-1; 1H NMR (DMSO-d6) δ
7.50 (s, 1 H), 7.15 (s, 1 H), 6.76 (d, J ) 7.8 Hz, 1 H), 6.57 (d,
J ) 8.1 Hz, 1 H), 6.48 (s, 1 H), 5.94 (s, 2 H), 5.03 (d, J ) 6.2
Hz, 1 H), 4.51 (d, J ) 6.2 Hz, 1 H), 3.79 (dq, J ) 6.9 Hz, 1 H),
3.80 (s, 3 H), 3.73 (s, 3 H), 2.96 (dq, J ) 6.9 Hz, 1 H), 1.01 (t,
J ) 6.9 Hz, 3 H); FABMS (m-NBA) m/z (relative intensity)
400 (MH+, 100).
1576, 1500, 1453, 1407, 1369, 1323, 1230, 1115, 1013 cm-1
;
1H NMR (CDCl3) δ 8.18 (d, J ) 1.3 Hz, 1 H), 6.95 (s, 2 H),
3.89 (s, 6 H), 3.87 (s, 3 H), 3.50 (d, J ) 1.3 Hz, 3 H); CIMS m/z
(relative intensity) 210 (MH+, 100). Anal. Calcd for C11H15
NO3: C, H, N.
-
cis-4-Ca r boxy-3,4-d ih yd r o-N-m eth yl-6,7-d im eth oxy-3-
(3′,4′,5′-tr im eth oxyp h en yl)-1(2H)isoqu in olon e (12i). 4,5-
Dimethoxyhomophthalic anhydride (10b) (0.22 g, 1 mmol) was
added to a stirred solution of 3,4,5-trimethoxybenzylidenem-
ethylamine (11i) (0.23 g, 1 mmol) in chloroform (5 mL). After
30 min, the bright yellow homogeneous solution was tan, and
no solid was observed. Ether was added dropwise and the
resulting precipitate was filtered and washed with ether to
give a fine white solid (0.1 g, 20%): mp 229-231 °C; IR (neat)
2928, 1743, 1593, 1418, 1329, 1241, 1167, 1119 cm-1; 1H NMR
(CDCl3) δ 7.50 (s, 1 H), 7.15 (s, 1 H), 6.38 (s, 2 H), 5.0 (d, J )
5.9 Hz, 1 H), 4.48 (d, J ) 5.9 Hz, 1 H), 3.79 (s, 3 H), 3.72 (s, 3
6-Eth yl-5,6-d ih yd r o-5,11-d ik eto-2,3-d im eth oxy-8,9-m e-
th ylen ed ioxy-11H-in d en o[1,2-c]isoqu in olin e (13k ). Thio-
nyl chloride (6.0 mL) was added with stirring to the cis acid

Synthesis of Cytotoxic Indenoisoquinolines
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3 455
12k (0.58 g, 1.5 mmol), and the reaction mixture became dark
reddish-purple and heterogeneous. After 4 h, the reaction
mixture was diluted with benzene (5 mL) and evaporated to
dryness. The brownish-red solid was loaded onto silica gel and
passed through a short column of silica gel, eluting with
chloroform, to give a brownish-red solid (0.34 g, 60%): mp
291-293 °C; IR 2969, 1694, 1643, 1613, 1555, 1486, 1393,
1610 cm-1; 1H NMR (DMSO-d6, 65 °C) δ 7.91 (s, 1 H), 7.53 (s,
1 H), 7.31 (s, 1 H), 7.06 (s, 1 H), 6.17 (s, 1 H), 4.43 (t, J ) 7.7
Hz, 2 H), 3.90 (s, 3 H), 3.86 (s, 3 H), 3.45 (t, J ) 5.8 Hz, 2 H),
1.88-1.70 (m, 2 H), 1.60-1.50 (m, 2 H); CIMS m/z (relative
intensity) 424 (MH+, 100). Anal. Calcd for C23H21NO7‚
0.5H2O: C, H, N.
5,6-Dih yd r o-6-(4-h yd r oxyp e n t -1-yl)-5,11-d ik e t o-2,3-
d im e t h oxy-8,9-m e t h yle n e d ioxy-11H -in d e n oisoq u in o-
lin e (19b). The indenoisoquinoline 18b was isolated in 79%
yield: mp 288-290 °C; IR (KBr) 3411, 2929, 1698, 1653, 1582,
1550 cm-1; 1H NMR (DMSO-d6, 80 °C) δ 7.91 (s, 1 H), 7.53 (s,
1 H), 7.21 (s, 1 H), 7.07 (s, 1 H), 6.18 (s, 1 H), 4.41 (bs, 2 H),
3.90 (s, 3 H), 3.86 (s, 3 H), 3.60 (bs, 1 H), 3.40 (bs, 2 H), 1.88-
1.70 (m, 2 H), 1.60-1.40 (m, 4 H); CIMS m/z (relative intensity)
438 (MH+, 100). Anal. Calcd for C23H21NO7‚0.3H2O: C, H, N.
1
1308, 1252 cm-1; H NMR (CDCl3) δ 8.02 (s, 1 H), 7.65 (s, 1
H), 7.08 (s, 1 H), 7.01 (s, 1 H), 6.08 (s, 2 H), 4.49 (q, J ) 7.2
Hz, 2 H), 4.03 (s, 3 H), 3.97 (s, 3 H), 1.50 (t, J ) 7.2 Hz, 3 H);
CIMS m/z (relative intensity) 366 (MH+, 4.0). Anal. Calcd for
C21 H17NO6: C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Im in es 17. The
O-TBDMS protected aminols 15 were synthesized using a
reported procedure.12 The imines 17 were synthesized by
treating the O-TBDMS protected aminols (9 mmol) with
piperonal (9 mmol) in chloroform (20 mL) in the presence of
anhydrous magnesium sulfate (2 g) at room temperature for
3 h. The imines were used as such for the next reaction without
further purification. The crude yields of the imines 17 were
quantitative.
Gen er a l P r oced u r e for th e Syn th esis of Isoqu in olon es
18. 4,5-Dimethoxyhomophthalic anhydride (10b) (2.22 g, 10
mmol) was added to a chloroform (20 mL) solution of the imine
17a or 17b (10 mmol), and the mixture was stirred at room
temperature for 12 h, after which the TLC showed the
complete disappearance of the starting materials and a white
precipitate formed in the reaction mixture. The precipitated
product was filtered off and washed with chloroform (5 mL)
and dried to give pure 18a or 18b.
cis-5,6,12,13-Tetr a h yd r o-2,3-d im eth oxy-6-m eth yl-5,11-
d ioxo-8,9-(m eth ylen ed ioxy)-(11H)in d en o[1,2-c]isoqu in o-
lin e (20). This compound was prepared as described previ-
ously.14
cis-6-Eth yl-5,6,12,13-tetr a h yd r o-2,3-d im eth oxy-5,11-d i-
oxo-8,9-(m e t h yle n e d ioxy)-11H -in d e n o[1,2-c]isoq u in o-
lin e (21). The acid 12k (3.99 g, 3 mmol) was added slowly
under nitrogen to a solution of degassed Eaton’s reagent (10%
P2O5 in methanesulfonic acid, 120 mL) with stirring over a
period of 20 min. The reaction mixture was stirred at room
temperature for 4 h, after which the mixture was added
dropwise to water (600 mL) with stirring. The precipitated
white solid was filtered off and dissolved in chloroform (150
mL). The chloroform layer was washed with saturated NaH-
CO3 solution (2 × 50 mL), water (50 mL) and brine (60 mL)
and dried (Na2SO4). Concentration of the organic layer gave
the crude product, which was purified by column chromatog-
raphy (4:1, hexane:ethyl acetate) to obtain pure 21 as a white
solid (2.39 g, 63%). Neutralization of the bicarbonate layer with
concentrated HCl gave the unreacted acid (0.821 g) as a white
solid. Thus the yield based on the recovered starting acid is
79.3%. An analytical sample was prepared by recrystallization
from EtOAc-hexane (1:1) to yield white prisms: mp 169-170
cis-N-(ter t-Bu tyld im eth ylsilyloxybu t-1-yl)-4-ca r boxy-
3,4-dih ydr o-6,7-dim eth oxy-3-(3′,4′-m eth ylen edioxyph en yl)-
1(2H)isoqu in olon e (18a ). The isoquinolone 18a was isolated
in 36% yield: mp 239-240 °C; IR (KBr) 3065, 2944, 1737 cm-1
;
1H NMR (DMSO-d6) δ 7.51 (s, 1 H), 7.11 (s, 1 H), 6.75 (d, J )
8.0 Hz, 1 H), 6.54 (dd, J ) 1.3 and 8.1 Hz, 1 H), 6.46 (d, J )
1.2 Hz, 1 H), 5.93 (s, 2 H), 4.98 (d, J ) 6.1 Hz, 1 H), 4.55 (d,
J ) 6.1 Hz, 1 H), 3.81 (s, 3 H), 3.80-8.70 (m, 1 H), 3.74 (s, 3
H), 3.53 (t, J ) 5.78 Hz, 2 H), 2.95-2.80 (m, 1 H), 1.60-1.35
(m, 4 H), 0.88 (s, 9 H), -0.98 (s, 6 H); 13C NMR (DMSO-d6) δ
170.64, 162.47, 151.21, 147.65, 147.00, 146.81, 131.26, 126.84,
121.55, 121.43, 110.85, 109.81, 107.87, 107.71, 101.06, 62.20,
61.01, 55.44, 47.77, 45.25, 29.67, 29.54, 25.81, 24.07, 17.91,
-5.37; CIMS m/z (relative intensity) 558 (MH+, 80). Anal.
Calcd for C29H39NO8Si: C, H, N.
°C; IR (KBr) 3006, 2994, 1706, 1642, 1601 cm-1 1H NMR
;
(CDCl3) δ 7.59 (s, 1 H), 7.16 (s, 1 H), 7.06 (s, 1 H), 7.00 (s, 1
H), 6.09 (s, 1 H), 6.04 (s, 1 H), 5.04 (d, J ) 6.9 Hz, 1 H), 4.70-
4.53 (m, 1 H), 4.21 (d, J ) 7.0 Hz, 1 H), 3.94 (s, 3 H), 3.88 (s,
3 H), 3.40-3.26 (m, 1 H), 1.35 (t, J ) 7.1 Hz, 3 H); 13C NMR
(CDCl3) δ 198.8, 162.0, 154.7, 152.0, 150.6, 149.4, 148.5, 128.8,
126.4, 120.3, 110.2, 108.6, 104.2, 102.6, 56.6, 56.0, 55.8, 50.4,
43.3, 13.2. Anal. Calcd for C21H19NO6: C, H, N.
cis-6-Allyl-5,6,12,13-tetr a h yd r o-2,3-d im eth oxy-5,11-d i-
oxo-8,9-(m et h ylen ed ioxy)-(11H )in d en o[1,2-c]isoq u in o-
lin e (22). Indenoisoquinoline 22 was synthesized in 72% yield
from the acid 12c in a similar procedure for the synthesis of
indenoisoquinoline 21. The treatment of the isoquinolone 12c
(4.11 g, 10 mmol) with Eaton’s reagent (120 mL) provided the
indenoisoquinoline 22 in 72% (2.83 g) yield: mp 178-180 °C;
IR (KBr) 2990, 1708, 1642, 1600 cm-1; 1H NMR (CDCl3) δ 7.60
(s, 1 H), 7.17 (s, 1 H), 7.07 (s, 1 H), 7.03 (s, 1 H), 6.09 (s, 1 H),
6.05 (s, 1 H), 6.05-5.90 (m, 1 H), 5.45-5.20 (m, 3 H), 5.16 (d,
J ) 6.9 Hz, 1 H), 4.19 (d, J ) 6.9 Hz, 1 H), 3.94 (s, 3 H), 3.88
(s, 3 H), 3.90-3.80 (m, 1 H); 13C NMR (CDCl3) δ 198.8, 162.3,
154.8, 152.3, 150.6, 149.5, 148.6, 132.6, 129.0, 126.6, 120.0,
118.0, 110.4, 108.7, 104.4, 102.7, 102.6, 56.3, 56.1, 55.9, 50.3.
Anal. Calcd for C22H19NO6: C, H, N.
cis-N-(ter t-Bu tyld im eth ylsilyloxyp en t-1-yl)-4-ca r boxy-
3,4-dih ydr o-6,7-dim eth oxy-3-(3′,4′-m eth ylen edioxyph en yl)-
1(2H)isoqu in olon e (18b). The isoquinolone 18b was isolated
in 57% yield: mp 240-242 °C; IR (KBr) 3054, 2933, 1737 cm-1
;
1H NMR (DMSO-d6) δ 12.90 (bs, 1 H), 7.51 (s, 1 H), 7.09 (s, 1
H), 6.75 (d, J ) 8.1 Hz, 1 H), 6.55 (dd, J ) 1.6 and 8.1 Hz, 1
H), 6.47 (d, J ) 1.3 Hz, 1 H), 5.93 (s, 2 H), 4.97 (d, J ) 6.2 Hz,
1 H), 4.53 (d, J ) 6.2 Hz, 1 H), 3.81 (s, 3 H), 3.83-8.70 (m, 1
H), 3.74 (s, 3 H), 3.52 (t, J ) 6.2 Hz, 2 H), 2.85-2.73 (m, 1 H),
1.60-1.30 (m, 6 H), 0.82 (s, 9 H), - 0.99 (s, 6 H). CIMS m/z
(relative intensity) 572 (MH+, 100). Anal. Calcd for C30H41
NO8Si: C, H, N.
-
Gen er a l P r oced u r e for th e Syn th esis of In d en oiso-
qu in olin es 19. Thionyl chloride (10 mL) was added dropwise
to the acid 18 (2 mmol) with stirring. The resulting solution
was stirred at room temperature for 5 h after which the
solution turned purple. Benzene (20 mL) was added to the
reaction mixture, and it was concentrated under reduced
pressure. The resulting residue was passed through a short
column of silica gel (230-400 mesh), eluting with chloroform:
methanol (95:5). Concentration of the eluent resulted in a pink
solid which was crystallized from ethyl acetate to obtain pure
indenoisoquinolines 19. Under the reaction conditions, the
deprotection of the O-TBDMS group was observed and only
the hydroxy compounds were isolated.
5,6-Dih yd r o-5,11-d ik et o-2,3,8-t r im et h oxy-6-m et h yl-9-
[(m eth ylsu lfon yl)oxy]-(11H)in den o[1,2-c]isoqu in olin e (23).
This compound was prepared as described previously.15
6-E t h y l-5,6,12R,13R-t e t r a h y d r o -11â-h y d r o x y -2,3-
d im eth oxy-8,9-(m eth ylen ed ioxy)-5-oxo-11H-in d en o[1,2-
c]isoqu in olin e (24). The indenoisoquinoline 21 (0.381 g, 1
mmol) was heated at reflux with a 1 M solution of borane-
tetrahydrofuran complex (4 mL) in dry THF (30 mL) for 1 h.
After cooling, the reaction mixture was concentrated, the
residue was dissolved in EtOAc (60 mL), and glacial acetic acid
was added dropwise until pH 5. The organic layer was washed
with saturated sodium bicarbonate (2 × 50 mL) and brine,
dried (Na2SO4), and concentrated. The residue on chromato-
5,6-Dih yd r o-5,11-d ik e t o-6-(4-h yd r oxyb u t -1-yl)-2,3-
d im et h oxy-8,9-m et h ylen ed ioxy-(11H )in d en o[1,2-c]iso-
qu in olin e (19a ). The indenoisoquinoline 19a was isolated in
84% yield: mp 304-308 °C; IR (KBr) 3432, 2929, 1696, 1645,

456 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3
Strumberg et al.
graphic purification (2% methanol in chloroform as eluent)
provided the pure product 24 (0.363 g, 95%). An analytical
sample was prepared by recrystallization from EtOAc-hexane
(3:1) to yield white prisms: mp 189-192 °C; IR (KBr) 3468,
6.15 (s, 2 H), 5.03 (q, J ) 7.2 Hz, 2 H), 4.87 (s, 2 H), 4.15 (s, 3
H), 4.05 (s, 3 H), 1.75 (t, J ) 7.2 Hz, 3 H).
13C NMR (MeOH-
d4) δ 189.5, 162.4, 155.7, 155.0, 152.5, 147.3, 133.4, 130.8,
127.9, 123.6, 107.5, 107.3, 106.6, 105.4, 101.5, 57.7, 57.1, 54.8,
15.7. Anal. Calcd for C21H20NO4Cl‚H2O: C, H, N.
1
2919, 1630, 1594 cm-1; H NMR (CDCl3) δ 7.62 (s, 1 H), 6.95
(s, 1 H), 6.94 (s, 1 H), 6.74 (s, 1 H), 5.99 (s, 1 H), 5.98 (s, 1 H),
4.97 (dd, J ) 5.8 and 7.6 Hz, 1 H), 4.89 (d, J ) 6.4 Hz, 1 H),
3.94 (s, 3 H), 3.93 (s, 3 H), 3.90-3.73 (m, 1 H), 3.59 (t, J ) 5.8
Hz, 1 H), 3.45-3.30 (m, 1 H), 2.03 (d, J ) 7.6 Hz, 1 H, D2O
exchangeable), 1.03 (t, J ) 7.1 Hz, 3 H); 13C NMR (CDCl3) δ
162.8, 152.1, 148.5, 148.4, 148.3, 138.7, 135.2, 128.2, 122.8,
110.5, 109.2, 106.6, 106.0, 101.5, 77.4, 60.0, 55.9, 55.8, 48.3,
37.9, 12.0. Anal. Calcd for C21H21NO6: C, H, N.
Top 1-Med ia ted DNA Clea va ge Rea ction s Usin g 3′-
En d -La beled 161 BP P la sm id DNA. The 161 bp fragment
from pBluescript SK(-) phagemid DNA (Stratagene, La J olla,
CA) was cleaved with the restriction endonuclease Pvu II and
Hind III (New England Biolabs, Beverly, MA) in supplied NE
buffer 2 (10 µL reactions) for 1 h at 37 °C and separated by
electrophoresis in a 1% agarose gel made in 1X TBE buffer.
The 161 bp fragment was eluted from the gel slice (centrilutor
by Amicon) and concentrated in a centricon 50 centrifugal
concentrator (Amicon, Beverly, MA). Approximately 200 ng of
the fragment was 3′-end-labeled at the Hind III site by fill-in
reaction with [R-32P]-dCTP and 0.5 mM dATP, dGTP, and
dTTP, in React 2 buffer (50 mM Tris-HCl, pH 8.0, 100 mM
MgCl2, 50 mM NaCl) with 0.5 units of DNA polymerase I
(Klenow fragment). Labeling reactions were followed by phenol-
chloroform extraction and ethanol precipitation. The resulting
161 bp 3′-end-labeled DNA fragment was resuspended in
water. Aliquots (approximately 50 000 dpm/reaction) were
incubated with top1 at 30 °C for 30 min in the presence of the
tested drug. Reactions were terminated by adding SDS (0.5%
final concentration). After ethanol precipitation, the samples
were resuspended in loading buffer (80% formamide, 10 mM
sodium hydroxide, 1 mM sodium EDTA, 0.1% xylene cyanol,
and 0.1% bromophenol blue, pH 8.0) and separated in a
denaturing gel (16% polyacrylamide, 7 M urea) run at 51 °C.
The gel was dried and visualized by using a Phosphoimager
and ImageQuant software (Molecular Dynamics, Sunnyvale,
CA).
6-E t h y l-5,6,12R,13R-t e t r a h y d r o -11â-h y d r o x y -2,3-
d im et h oxy-8,9-(m et h ylen ed ioxy)-11H -in d en o[1,2-c]iso-
qu in olin e (25). The indenoisoquinoline 21 (2.391 g, 6.27
mmol) was heated at reflux with a 1 M solution of borane-
tetrahydrofuran complex (15 mL) in dry THF (100 mL) for 12
h. After cooling, the reaction mixture was concentrated, the
residue was dissolved in EtOAc (100 mL), and glacial acetic
acid was added dropwise until pH 5. The organic layer was
washed with saturated sodium bicarbonate (2 × 100 mL) and
brine, dried (Na2SO4), and concentrated. The residue on
chromatographic purification (5% ethyl acetate in chloroform
as eluent) provided the pure product 25 (2.13 g, 92%). An
analytical sample was prepared by recrystallization from
2-propanol to yield white crystals: mp 180-184 °C; IR (KBr)
3479, 2909, 1605, 1594 cm-1; 1H NMR (CDCl3) δ 7.76 (s, 1 H),
6.90 (s, 1 H), 6.77 (s, 1 H), 6.66 (s, 1 H), 6.040 (s, 1 H), 6.02 (s,
1 H), 5.34 (dd, J ) 3.1 and 6.6 Hz, 1 H), 4.90 (d, J ) 8.4 Hz,
1 H), 4.15 (d, J ) 16.2 Hz, 1 H), 4.06 (d, J ) 16.2 Hz, 1 H),
3.93 (s, 3 H), 3.89 (s, 3 H), 3.71 (t, J ) 7.5 Hz, 1 H), 2.86-2.70
(m, 1 H), 2.20-2.13 (m, 1 H), 2.03 (d, J ) 3.1 Hz, 1 H, D2O
exchangeable), 1.02 (t, J ) 7.1 Hz, 3 H); 13C NMR (CDCl3) δ
149.4, 149.3, 148.4, 137.9, 131.2, 124.3, 123.4, 110.4, 110.1,
109.9, 104.7, 101.6, 74.0, 73.6, 58.0, 56.2, 56.0, 46.8, 45.6, 9.00.
Anal. Calcd for C21H19NO6‚1.5 H2O: C, H, N.
6-(3-Ca r boxy-1-p r op yl)-5,6-d ih yd r o-5,11-d ik eto-11H-in -
den o[1,2-c]isoqu in olin e (26). The indenoisoquinoline 7 (0.319
g, 1 mmol) was dissolved in acetone (50 mL) and cooled in an
ice bath. J ones reagent was added dropwise to the cold solution
of the alcohol until the red color of the reagent persisted. The
excess J ones reagent was quenched by adding few drops of
isopropyl alcohol. The reaction mixture was filtered through
a small pad of Celite, and the residue was washed with acetone
(50 mL). The combined filtrate was concentrated, the residue
was dissolved in saturated bicarbonate (100 mL), and the
aqueous layer was washed with chloroform (2 × 30 mL). The
aqueous layer was neutralized with concentrated HCl and
extracted in CHCl3 (3 × 50 mL). The combined organic layer
was dried (Na2SO4) and concentrated to afford the acid as an
orange solid. The solid was crystallized from isopropyl alcohol
to yield orange crystals (0.320 g, 96%): mp 204-206 °C; IR
(KBr) 3000 (b), 1708, 1698, 1654 cm-1; 1H NMR (CDCl3) δ 8.68
(d, J ) 8 Hz, 1 H), 8.30 (d, J ) 8 Hz, 1 H), 7.86 (d, J ) 7.4 Hz,
1 H), 7.73 (t, J ) 8.0 Hz, 1 H), 7.61 (d, J ) 7.2 Hz, 1 H), 7.50-
7.30 (m, 3 H), 4.60 (t, J ) 7.8 Hz, 2 H), 3.71 (s, 1 H), 2.60 (t,
J ) 7.0 Hz, 2 H), 2.19 (p, J ) 7.0 Hz, 2 H). Anal. Calcd for
C20H15NO4: C, H, N.
Top 2-Med ia ted DNA Clea va ge Assa ys Usin g 5′-En d -
La beled Hu m a n C-m yc DNA. A 403-base-pair DNA frag-
ment of the human c-myc gene from the junction between the
first intron and the first exon was prepared by PCR between
positions 2671 and 3073 using the oligonucleotides 5′-TGC-
CGCATCCACGAAACTTTGC-3′ as sense primer and 5′-
GAACTGTTCAGTGTTTACCCCG-3′ as antisense primer.
Single-end labeling of these DNA fragments was obtained by
5′-end labeling of the adequate primer oligonucleotide. Ap-
proximately 0.1 µg of the human c-myc DNA that had been
restricted by XhoI and XbaI was used as template for PCR.23
The 5′-end-labeled DNA fragments were equilibrated with or
without a drug in 1% dimethyl sulfoxide, 10 mM Tris-HCl,
pH 7.5, 50 mM KCl, 5 mM MgCl2, 2 mM dithiothreitol, 0.1
mM Na2EDTA, 1 mM ATP, and 15 µg/mL bovine serum
albumin for 5 min before addition of purified human top2 (40-
70 ng) in a 10 µL final reaction volume. The reactions were
performed at 37 °C for 30 min and thereafter stopped by
adding 1% sodium dodecyl sulfate (SDS) and 0.4 mg/mL
proteinase K (final concentrations) followed by an additional
incubation at 50 °C for 30 min. Samples were ethanol-
precipitated before separation of the top2-cleaved fragments
on denaturing polyacrylamide gels. The sequencing gels were
made of 7% polyacrylamide in 1X TBE buffer (90 mM Tris
borate, 2 mM EDTA, pH 8.3). Electrophoresis was performed
at 2500 V (60 W) for 2-5 h. The gels were dried and visualized
using a Phosphoimager and ImageQuant software.
6-E t h yl-2,3-d im et h oxy-8,9-(m et h ylen ed ioxy)-11H -in -
d en o[1,2-c]isoqu in olin iu m Ch lor id e (27). The amino al-
cohol 25 (0.738 g, 2 mmol) was heated at reflux with 5%
palladium on charcoal (0.265 g) in glacial acetic acid (100 mL)
for 20 h. After cooling, the mixture was filtered through a small
pad of Celite, and the solvent was evaporated to give a brown
residue. The residue was dissolved in water (50 mL) and
ethanol (6 mL) to give a light brown solution, to which was
added 15% aqueous sodium chloride (10 mL). A yellow product
precipitated immediately and was filtered, washed with ice
cold water (10 mL), and dried over P2O5 under vacuum
overnight to yield a yellow powder (0.552 g, 72%). An analytical
sample was crystallized from methanol: mp 340-343 °C (dec);
IR (KBr) 3382, 1480, 1305 and 1210 cm-1; H NMR (MeOH-
d4) δ 9.27 (s, 1 H), 7.61 (s, 2 H), 7.46 (s, 1 H), 7.30 (s, 1 H),
SV40 DNA Un w in d in g Assa y. Reaction mixtures (10 µL
final volume) contained 0.3 µg of supercoiled SV40 DNA in
reaction buffer (10 mM Tris-HCl, pH 7.5, 50 mM KCl, 5 mM
MgCl2, 0.1 mM EDTA, 15 µg/mL bovine serum albumin) and
10 units of purified calf thymus top1. Reactions were per-
formed at 37 °C for 30 min and terminated by the addition of
SDS (0.5% final concentration), and then 1.1 µL of 10X loading
buffer (20% Ficol 400, 0.1 M Na2EDTA pH 8, 1.0% SDS. 0.25%
Bromophenol Blue) was then added and reaction mixtures
were loaded onto a 1% agarose gel made in 1X TBE buffer.
After electrophoresis, DNA bands were stained in 10 µg/mL
of ethidium bromide and visualized by transillumination with
UV light (300 nm).

Synthesis of Cytotoxic Indenoisoquinolines
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 3 457
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Ack n ow led gm en t. This work was made possible by
the National Institutes of Health (NIH) through support
of this work with Contract NO1-CM-67260. Dirk Strum-
berg was supported by the Deutsche Forschungsge-
meinschaft (Grant Str 527/1-1), Bonn, Germany.
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