Synthesis and biological evaluation of 14-(aminoalkyl-aminomethyl)aromathecins as topoisomerase I inhibitors: Investigating the hypothesis of shared structure-activity relationships

Article abstract of DOI:10.1016/j.bmc.2009.08.066

The aromathecin topoisomerase I (top1) inhibitors offer promising scaffolds for the development of novel cancer chemotherapeutics. They are 'composites' of the camptothecin and indenoisoquinoline top1 inhibitors. Interestingly, some structure-activity relationship (SAR) overlap between the aromathecins and the indenoisoquinolines has been observed. For both classes, placement of certain polar groups in similar regions of the heteroaromatic system improves top1 inhibitory and antiproliferative activities. A series of water-soluble aromathecins substituted at position 14 with diaminoalkanes of various lengths has been prepared. These compounds all possess similar antiproliferative potency, but a general trend is observed: aromathecins with longer diaminoalkane substituents (>6 carbons) possess lower anti-top1 activity than their smaller counterparts (2-4 carbons), presumably as a result of unfavorable hydrophobic interactions. This trend is also noted with the indenoisoquinolines, revealing additional SAR overlap that supports the hypothesis that there is a 'universal' top1 inhibitor SAR.

Full text of DOI:10.1016/j.bmc.2009.08.066

Bioorganic & Medicinal Chemistry 17 (2009) 7145–7155
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of 14-(aminoalkyl-aminomethyl)-
aromathecins as topoisomerase I inhibitors: Investigating the hypothesis
of shared structure–activity relationships
Maris A. Cinelli a, Brenda Cordero a, Thomas S. Dexheimer b, Yves Pommier b, Mark Cushman a,
*
a Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, and the Purdue Cancer Center, Purdue University,
West Lafayette, IN 47907, USA
b Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892-4255, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2009
Revised 22 August 2009
Accepted 29 August 2009
Available online 6 September 2009
The aromathecin topoisomerase I (top1) inhibitors offer promising scaffolds for the development of novel
cancer chemotherapeutics. They are ‘composites’ of the camptothecin and indenoisoquinoline top1 inhib-
itors. Interestingly, some structure–activity relationship (SAR) overlap between the aromathecins and the
indenoisoquinolines has been observed. For both classes, placement of certain polar groups in similar
regions of the heteroaromatic system improves top1 inhibitory and antiproliferative activities. A series
of water-soluble aromathecins substituted at position 14 with diaminoalkanes of various lengths has
been prepared. These compounds all possess similar antiproliferative potency, but a general trend is
observed: aromathecins with longer diaminoalkane substituents (>6 carbons) possess lower anti-top1
activity than their smaller counterparts (2–4 carbons), presumably as a result of unfavorable hydrophobic
interactions. This trend is also noted with the indenoisoquinolines, revealing additional SAR overlap that
supports the hypothesis that there is a ‘universal’ top1 inhibitor SAR.
Keywords:
Topoisomerase I
Aromathecins
Anticancer
Polyamine
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Alternative top1 inhibitors have been based on natural products
such as luotonin A (4)19 and the indolocarbazoles.20 Optimization
Topoisomerase I (top1) breaks one strand of double-stranded
DNA. The cleaved strand then rotates around the uncleaved strand,
resulting in relaxation of DNA supercoils necessary for replication
and transcription. The broken DNA strand then re-ligates and
relaxed DNA is released.1,2 Top1 is overexpressed in human can-
cers,3,4 and interest in top1 as a therapeutic target was stimulated
by the isolation of camptothecin (1) and the later development of
topotecan (2) and irinotecan (3),5 which inhibit the DNA religation
reaction by intercalating at the cleavage site, eventually leading to
apoptosis (Fig. 1).2,6–9
Topotecan and irinotecan are potent anticancer drugs, but are
limited by reversibility of their ternary drug–DNA–enzyme com-
plexes, which necessitates long infusion times for maximum ther-
apeutic benefit. Drug efflux pumps10 and resistance mutations11,12
also limit their efficacy. Side effects include diarrhea, immune sup-
pression, and hemorrhagic cystitis.1,13,14 The E-ring hydroxylac-
tone exists in equilibrium with an open hydroxyacid form that
binds to human serum albumin, thus compromising bioavailabil-
ity.1,15–18
of the latter class has provided edotecarin (5).13,21 The indenoiso-
quinolines, such as 6, are synthetic compounds based on the lead
NSC 314622 (7).22–25 Two indenoisoquinolines are slated to begin
clinical trials at the NIH.26
The 12H-5,11a-diazadibenzo[b,h]fluoren-11-one system, called
‘rosettacin’ (8)27 when unsubstituted and ‘aromathecin’ when
substituted, was first discovered in the natural product 22-
hydroxyacuminatine (9).28 This system is an analogue of campto-
thecin, with the E-ring lactone replaced by a benzene ring. As
such, the system is a ‘hybrid’ of the camptothecin and indenoiso-
quinoline systems. Although initial attempts to develop aromat-
hecins were less than fruitful,29 subsequent efforts led to the
synthesis of the more promising 14-substituted aromathecins.30
These compounds, substituted with amino alcohols and nitrogen
heterocycles, possess both greater anti-top1 and antiproliferative
activity than rosettacin and a greater ability to inhibit top1 than
22-hydroxyacuminatine.
Interestingly, indenoisoquinoline top1 inhibitory activities are
increased by the same substituents (ethanolamine, morpholine,
imidazole, etc.)23,24 that increase the activity of the aromathec-
ins.23,24 Molecular modeling and crystallography8 indicate that the
lactam region of the indenoisoquinoline system (where these sub-
stituents are located) overlaps spatially with both the 7-position of
* Corresponding author. Tel.: +1 765 494 1465; fax: +1 765 494 6790.
E-mail address: cushman@pharmacy.purdue.edu (M. Cushman).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.066

7146
M. A. Cinelli et al. / Bioorg. Med. Chem. 17 (2009) 7145–7155
2. Chemistry
12
7
11
9
OH
N
HO
O
1
N
O
OH
10
14
There are several routes known to the aromathecin system,
N
21
O
20
N
including the condensation of pyrroloquinolinewith phthalides,28,29
pyridone benzannulation and Heck coupling,36 and variants of the
Friedlander condensation.30,37 Our synthesis of 14-substituted aro-
mathecins proceeds through pyrroloquinolinedione 16 (Scheme
1), first prepared by Shamma and Novak in 1968.38 Intermediate
11 was prepared by the facile decarboxylation of commercially
19O
N
O
5
17
O
1
Topotecan
2
available trans-4-hydroxy-L
-proline (10).39 Condensation of 11 with
O
O
N
N
3-chlorophthalide 13 (easilyprepared from2-carboxybenzaldehyde
12)40 yielded the hydroxyamide 14. Previously, this compound was
oxidized and cyclized to 16 via oxidation with pyridinium dichro-
mate and subsequent treatment with polyphosphoric acid.30 An im-
proved variant of this oxidation–cyclization has been discovered.
Subjecting 14 to Swern conditions and a brief reflux period affords
16 rapidly (via intermediate 15). These new conditions furnish the
key intermediate in improved yield (50–60%) without recourse to
heavy metal oxidants and extensive purification. 14-Chloromethyl-
aromathecin (19)30 was prepared by Friedlander condensation of
ketone 16 with aminoacetophenone 18 (Scheme 2). Compound 18
was in turn prepared from aniline (17) and chloroacetonitrile using
the Sugasawa modification of the Friedel–Crafts acylation.41,42
Compound 19 undergoes SN2 displacement easily at its benzylic
chloride (Scheme 3). The diamine moieties were installed as their
mono-Boc-protected carbamates to prevent polymerization under
the displacement conditions and to aid in purification. Following
Morrell et al.’s procedure, diamines 20–30 were readily protected
using Boc2O to afford the carbamates 31–41.31 Treatment of 19
with excess protected diamine in DMSO at room temperature
afforded the aromathecin carbamates 42–52. Compounds 42–52
were readily deprotected using methanolic HCl either in chloro-
form, or in the case of diaminoheptane analogue 47, refluxing ben-
zene, to yield the water-soluble aromathecin salts 53–63.
Elemental analysis proved these analogues were trihydrochloride
salts, protonated on both amines and the quinoline nitrogen.
HO
O
N
O
N
Irinotecan
3
O
HO
HO
N
NH
N
N
O
O
N
O
4
OH
N
N
HO
H
H
OH
OH
O
5
O
O
OH OH
O
H3CO
H3CO
H
N
N
OH
O
O
6
O
O
H3CO
H3CO
N
CH3
4
3
1
3. Results and discussion
O
5
2
N
6
7
7
8
9
Aromathecin analogues were assayed for cytotoxic activity in
the National Cancer Institute’s Developmental Therapeutics
14
N
HO
12
11
O
10
N
8
OH
N
O
b
c
O
9
O
Cl
O
O
12
Figure 1. Representative top1 inhibitors.
HO
HO
13
camptothecin and the 14-position of the aromathecin system in
their respective ternary cleavage complexes. These substituents
likely project into the DNA major groove in the cleavage complex.30
This spatial overlap suggests a significant degree of structure–activ-
ity relationship overlap between these two systems.8 The similar sub-
stituent effects suggest that other aspects of the indenoisoquinoline
SAR might be ‘translated’ to the aromathecin system.
Indenoisoquinolines substituted with di- and polyamines are po-
tent top1 inhibitors.31,32 It is known in general that di- and poly-
amine conjugates are effective ‘DNA-targeting’ moieties for many
classes of intercalating and anti-top1 compounds.31–35 In 2007, Mor-
rell et al. determined that diamines with two- to four-carbon spacers
were optimal substituents for bioactivity when placed on indenoiso-
quinolines.31 The question as to whether this SAR trend could also be
applied to aromathecins served as a rationale for the synthesis of
14-[(aminoalkyl)-aminomethyl)aromathecins 53–63.
OOC
a
.
N
N
HOOC
COOH
H
H
H
10
11
O
O
OH
CHO
CHO
e
d
N
N
O
N
O
O
16
14
15
Scheme 1. Reagents and conditions: (a) (i) cat. 2-cyclohexen-1-one, cyclohexanol,
reflux; (ii) maleic acid, EtOAc, rt; (b) FeCl3, SOCl2, reflux; (c) MeOH, Et3N, rt; (d) (i)
DMSO, (COCl)2, CH2Cl2, ꢀ78 °C; (ii) Et3N, ꢀ78 °C–rt; (e) reflux.

M. A. Cinelli et al. / Bioorg. Med. Chem. 17 (2009) 7145–7155
7147
pounds were tested at five concentrations ranging from 10ꢀ8 to
10ꢀ4 M. Cytotoxicity results are reported as GI50 values for selected
cell lines from each subpanel, and overall antiproliferative potency
is quantified as a mean-graph midpoint (MGM) in Table 1. The
MGM is a measure of the average GI50 against all cell lines tested,
where compounds whose GI50 values fall outside the concentration
range tested (10ꢀ8–10ꢀ4 M) are assigned GI50 values of either 10ꢀ8
M or 10ꢀ4 M. For comparative purposes, the activities of campto-
O
N
H2N
O
H2N
a
O
b
16
Cl
17
18
thecin (1), the indenoisoquinolines
6
(MJ-III-65),1,23,25 and
NSC314622 (7),22 and rosettacin (8) are reported.
N
Top1 inhibition was evaluated by measurement of top1-depen-
dent DNA cleavage at four concentrations, and inhibition data are
expressed semiquantitatively using the following rubric: 0, no
N
Cl
inhibitory activity; +, between 20% and 50% the activity of 1
camptothecin (1); ++, between 50% and 75% the activity of 1
camptothecin; +++, between 75% and 100% the activity of 1
camptothecin; and ++++, equipotent to or more potent than 1
lM
lM
lM
lM
O
19
Scheme 2. Reagents and conditions: (a) (i) BCl3ꢁMe2S, 1,2,-dichloroethane, 0 °C; (ii)
chloroacetonitrile, AlCl3, reflux; (iii) 2 M HCl, reflux; (b) p-TsOH, toluene, reflux.
camptothecin. Top1 inhibitory data for 1 and 6–8 are also included
in Table 1.
Compounds 53–54, 56–57, 59, 61, and 63 were assayed for anti-
proliferative activity. These aromathecins, on average, display high-
er antiproliferative potency than many of the 14-substituted
aromathecins previously investigated.30 These water-soluble salts
are more easily formulated than many of the free bases previously
tested. In addition, it is well known that positively charged substitu-
ents improve DNA targeting, binding, and intercalative potential
throughsimple electrostatic complementarity to negativelycharged
DNA. This effect has been observed for camptothecins, indenoiso-
quinolines, acridines, and other polyaromatic hydrocarbon-based
systems when substituted with di- and polyamines, amidines, and
guanidines.31–35,45–47
screen.43,44 Although some highly potent Boc-polyaminocamptot-
hecins have been reported,33 indenoisoquinoline carbamates are
largely inactive.31,45 Therefore, the intermediate carbamates 42–
52 were not tested.
Compounds accepted for testing were assayed against approxi-
mately 60 cell lines originating from various human tumors.43,44
Following an initial one-dose assay (at 10ꢀ5 M), selected com-
N
Interestingly, with the exception of compound 61, little vari-
ance in antiproliferative activity is observed in the compounds
tested, regardless of side chain length. Anomalously, compound
61 actually induced cell growth by approximately 30% at a concen-
Cl
N
19
O
a
NH2
n
NHBoc
n
H2N
H2N
tration of 10 lM in the initial one-concentration assay, although
b
both the large observed variance between individual cell lines
and the general trend among the rest of this series indicate that
this result may be an artifact of testing. For indenoisoquinolines
and other compounds, it was observed that an increasing C log P
correlated well with decreasing cytotoxicity, possibly as a result
of poor solubility, inefficient DNA-targeting, or other hydrophobic
effects.31 There is no correlation for aromathecins, however; com-
pounds 53 and 63 are equipotent, and compound 57, more hydro-
phobic than 53, is more active.
20: n = 2
21: n = 3
22: n = 4
23: n = 5
24: n = 6
25: n = 7
26: n = 8
27: n = 9
28: n = 10
29: n = 11
30: n = 12
31: n = 2
32: n = 3
33: n = 4
34: n = 5
35: n = 6
36: n = 7
37: n = 8
38: n = 9
39: n = 10
40: n = 11
41: n = 12
For top1 inhibition, the shorter diaminoalkanes confer good
anti-top1 activity upon the aromathecin core, inducing top1-med-
iated DNA breakage as shown in Figure 2. These compounds induce
DNA cleavage patterns resembling those caused by both camptot-
hecins and indenoisoquinolines. Activity generally decreases with
increasing side-chain length, however. With the exception of 56,
up to six atoms between proximal and distal amines are tolerated
readily for aromathecins. Activity decreases beyond this length,
and compounds with ten or more carbons are completely inactive.
An identical trend is noted for indenoisoquinolines, complete with
compounds derived from 1,5-pentanediamine possessing signifi-
cantly less anti-top1 activity than those derived from 1,6-hexane-
diamine.31 It is unknown why the pentanediamino compounds
possess such low anti-top1 activity.
It has been confirmed by X-ray crystallography that the lactam
substituents of indenoisoquinolines project into the major groove
of the ternary complex.8 Hypothetical models predict that the
14-position substituents of aromathecins project into the same re-
gion, where they likely interact favorably with water and amino
acids such as Asn352.48,49 Figure 3 shows a hypothetical model
of compound 53, the most potent top1 inhibitor from this series,
N
N
NH
N
c
NH
NH2
N
O
O
n
NHBoc
n
. 3 HCl
53: n = 2
54: n = 3
55: n = 4
56: n = 5
57: n = 6
58: n = 7
59: n = 8
60: n = 9
61: n = 10
62: n = 11
63: n = 12
42: n = 2
43: n = 3
44: n = 4
45: n = 5
46: n = 6
47: n = 7
48: n = 8
49: n = 9
50: n = 10
51: n = 11
52: n = 12
Scheme 3. Reagents and conditions: (a) Boc2O, CHCl3, rt; (b) DMSO, rt; (c) 3 M HCl,
MeOH, CHCl3, rt or 3 M HCl, MeOH, benzene, reflux (47).

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M. A. Cinelli et al. / Bioorg. Med. Chem. 17 (2009) 7145–7155
Table 1
Cytotoxicities and topoisomerase I inhibitory activity of 14-diamine-substituted aromathecin analogues
Compd
Cytotoxicity (GI50 in
l
M)a
MGMb
Top 1 cleavagec
Lung
HOP-62
Colon
HCT-116
CNS
SF-539
Melanoma
UACC-62
Ovarian
OVCAR-3
Renal
SN12C
Prostate
DU-145
Breast
MDA-MB-435
1
6
7
8
0.01
0.02
1.3
68.2
1.4
9.8
—
2.6
1.2
—
2.8
—
—
—
6.5
0.03
0.10
35
32.7
1.5
1.5
—
3.2
0.63
—
1.8
—
0.01
0.04
41
66.7
8.3
4.9
—
7.2
0.74
—
1.9
—
0.01
0.03
4.2
97.2
12.6
4.8
—
1.9
2.0
—
1.8
—
—
—
1.9
0.22
0.5
73
39.8
1.7
1.4
—
8.1
1.4
—
2.5
—
—
—
1.9
0.02
<0.01
68
>100
1.5
2.1
—
3.2
0.51
—
1.8
—
0.01
<0.01
37
>100
1.4
1.1
—
1.1
0.33
—
1.1
—
0.04
0.79
96
41.8
11.0
6.8
—
12.6
1.5
—
3.1
—
0.040 0.019
0.11
20.0
58.9
2.8 0.11
2.1 0.38
—
2.6 1.65
1.0
—
1.9
—
—
++++
++++
++
++
53
54
55e
56
57
58e
59
60e
61d
62e
63
+++
++(+)
+++
+
++(+)
++
+
+
0
0
—
—
2.0
—
—
1.8
—
—
2.5
—
—
2.0
—
—
1.7
—
2.6
0
a
b
c
The cytotoxicity GI50 values are the concentrations corresponding to 50% growth inhibition.
Mean graph midpoint for growth inhibition of all human cancer cell lines successfully tested, ranging from 10ꢀ8 to 10ꢀ4 M.
Compound-induced DNA cleavage due to top1 inhibition is graded by the following rubric relative to 1 M camptothecin: 0, no inhibitory activity; +, between 20% and
l
50% activity; ++, between 50% and 75% activity; +++, between 75% and 100% of activity; ++++, equipotent.
d
Not selected for further testing; refer to text for details.
Declined for one-dose cytotoxicity testing by the NCI.
e
in ternary complex with top1 and DNA. The aromathecin core is
proposed to intercalate between the base pairs flanking the top1-
induced cleavage site, where the quinoline nitrogen faces toward
the minor groove and may interact with Arg364. Although it is
out of hydrogen-bonding distance (4.47 Å) in this model, this con-
tact is closer in other aromathecin models,30 as well as in campto-
thecin and topotecan crystal structures,7,8 and hydrogen bonding
might still be possible in the present case with induced fit. The dia-
mine side chain projects into the major groove, where it is pro-
posed to hydrogen bond with a flanking nucleobase. Water
molecules likely participate in these interactions, and this hypo-
thetical network of water-mediated H-bonds and polar contacts
with the positively charged amines is shown in Figure 4. In this
well-solvated environment, it can be seen how increasingly hydro-
phobic side-chains (such as those over eight carbons as in 60–63)
would yield unfavorable interactions by possibly disturbing this
intricate network of water molecules. Larger side chains may also
be hindered by steric or entropic factors.
The fact that aromathecin diamines behave in a manner that is
identical to analogous indenoisoquinolines with regard to top1
inhibition lends additional support to the proposed binding mode
(where the 14-position faces the major groove). This behavior
can be rationalized through molecular modeling. Figure 5 depicts
the ligands 53 and 64 (Fig. 6) in an overlay of the hypothetical
Figure 2. Top1-mediated DNA cleavage induced by aromathecins 53, 55, 57, 58, 60, and 63. Lane 1: DNA alone; lane 2: Top1 alone; lane 3: 1, 1
lM; lane 4: 6, 1 lM; lane 5: 8,
100 M; lanes 6–29: 53, 55, 57, 58, 60, and 63 at 0.1, 1, 10, and 100 M, respectively, from left to right. Numbers and arrows on right indicate arbitrary cleavage site positions.
l
l

M. A. Cinelli et al. / Bioorg. Med. Chem. 17 (2009) 7145–7155
7149
Figure 3. Hypothetical model of aromathecin 53 in ternary complex with top1 and DNA showing proposed hydrogen bonds. Water molecules are red spheres; distances are
between heavy atoms. The stereoview is programmed for wall-eyed viewing.
O
N
NH3
O
64
Figure 6. Structure of Indenoisoquinoline 64.
quinolines,23,31 anthracenes,46 camptothecins,33 and aromathec-
ins30 alike. Dallavalle et al. propose that beyond Coulombic
attraction of the drugs to DNA, the cellular pharmacology of posi-
Figure 4. Hypothetical model of water-mediated hydrogen bonds and polar
tively-charged ‘polyamine’ conjugates is an exceedingly complex
contacts between diaminoethyl side chain of aromathecin 53, Asn352, and DNA
interplay of intrinsic molecular structure, hydrophobicity, and sub-
cellular localization.33 This is consistent with Morrell et al.’s
hypotheses that differential ADME properties are responsible for
these discrepancies.31
base pairs. Water molecules are indicated in red, other structures are colored by
atom type. Distances are between heavy atoms.
If such a disparity exists between cytotoxicity and top1 inhibi-
tion, then what is the macromolecular target of compounds such
as 56 and 63 that possess little anti-top1 activity? A COMPARE anal-
ysis was performed on data from compound 56, which, with its low
anti-top1 activity, is effectively a ‘targetless’ aromathecin. The NCI’s
COMPARE algorithm attempts to correlate the antiproliferative po-
tency of a compound with a possible target or mechanism of ac-
tion.44,50,51 When the data for 56 were used to seed a COMPARE
search, the program returned compounds such as amsacrine47 and
teniposide with a moderately high (>0.75) correlation. These com-
pounds act predominantly through inhibition of topoisomerase II
(top2). When compounds 56, 57, 59, and 63 were assayed for the
ability to induce top2-mediated DNA cleavage, however, little inhib-
itory activity was found. It is also worth noting that at high concen-
trations, longer diamines like 60 and 63 suppress the formation of
top1-DNA cleavage complexes (as shown in Fig. 3),31 although the
shorter diamines do not. This could indicate that the antiprolifera-
tive activity of compounds such as 63 may involve topoisomerase
binding or non-specific DNA intercalation. Also, the role of cellular
polyamine transporters in the pharmacokinetics of these molecules
cannot be discounted.52
Figure 5. Ligand overlay of hypothetical ternary complex models of aromathecin
53 (green) and indenoisoquinoline 64 (cyan). The positions of the lactam nitrogen
and 14-position are also shown in their respective colors.
The high degree of overlap in the SAR at the indenoisoquinoline
lactam nitrogen and the aromathecin 14-position, down to specific
substituents, is significant, and supports the hypothesis that there
is a ‘common’ or ‘universal’ structure–activity relationship among
different classes of top1 inhibitors. Different substituents
have been ‘translated’ effectively between camptothecins and
indenoisoquinolines in optimization of the latter class’ indenone
ring.53 This SAR overlap is also seen between camptothecins and
models of their ternary complexes.8,31 The aromatic regions sit
approximately in the same position, the side chains occupy identi-
cal regions spatially, and both interact with Asn352 and water mol-
ecules (not shown).
It is worth noting the disparities between cytotoxicity and top1
inhibition across this analogue series. Such a disparity is not
unprecedented and has been previously observed in indenoiso-

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M. A. Cinelli et al. / Bioorg. Med. Chem. 17 (2009) 7145–7155
aromathecins in translation of active substituents from the for-
mer’s 7-position (such as the N-methylpiperazine of lurtotecan
and camptothecin analogues)1,17 to the 14-position of aromathec-
ins.30 There is also overlap between the SARs of indenoisoquino-
lines, isoquinocinnolinones, and dibenzonaphthyridones in the
usage of common lactam and ring substituents.54,55 Indeed, Staker
et al. have described certain structural features common to all
effective top1 inhibitors, including major groove substituents, a
planar ring that faces the nonscissile strand, and a hydrogen-bond
acceptor facing the minor groove.8 These new results could thus
support the translation of other substituents to the aromathecin
class to produce novel potent top1 inhibitors.
compounds 11,39 13,49 14,30 and 1841,42 were prepared as previ-
ously described and as depicted in Schemes 1 and 2.
5.2. Synthesis of precursors
5.2.1. 2,3-Dihydropyrrolo[1,2-b]isoquinoline-1,5-dione (16)38
Anhydrous CH2Cl2 (170 mL) was cooled, under an argon atmo-
sphere, to ꢀ78 °C. DMSO (2.96 g, 37.9 mmol) was added, followed,
dropwise, by oxalyl chloride (2.40 g, 18.9 mmol). Upon cessation of
gas evolution (10 min), compound 1430 (3.78 g, 17.2 mmol), as a
solution in CH2Cl2 (20 mL), was added slowly, and the resultant
opaque off-white solution was stirred for 15 min. Triethylamine
(8.69 g, 86 mmol) was added, and the mixture became orange.
After stirring for 5 min at ꢀ78 °C, the mixture was warmed to room
temperature (TLC indicated the presence of 15), and darkened as
cyclization began. The mixture was stirred for 30 min and heated
at reflux for 2 h. The black solution was cooled and washed with
H2O (2 ꢂ 200 mL). The aqueous layer was extracted with CHCl3
(150 mL). The combined organic layers were washed with H2O
(2 ꢂ 200 mL), satd NaCl (200 mL), dried over anhydrous sodium
sulfate, treated with decolorizing carbon (1.00 g), and filtered.
The filtrate was concentrated to yield a pale-orange microcrystal-
line solid (2.01 g, 59%) after washing with MeOH (40 mL) and ether
(20 mL): mp 180–183 °C (lit.38 mp 191–192 °C). 1H NMR (300 MHz,
CDCl3) d 8.54 (d, J = 8.1 Hz, 1H), 7.80–7.65 (m, 3H), 7.29 (s, 1H),
4.42 (t, J = 7.0 Hz, 2H), 2.99 (t, J = 7.3 Hz, 2H).
4. Conclusions
In conclusion, a new series of water-soluble 14-substituted aro-
mathecins bearing diaminoalkanes of various lengths has been
synthesized by a facile route beginning with commercially avail-
able starting materials. An improved preparation of the key inter-
mediate in aromathecin synthesis is also reported. The analogues
were assayed against top1 and various human cancer cell lines.
This series contains the most cytotoxic aromathecins synthesized
thus far, their improved antiproliferative potency likely due to in-
creased solubility and multiple DNA-targeting positive charges. In
general, top1 inhibition decreases with increasing side chain
length, likely reflecting the effect of unfavorable hydrophobic
clashes in the charged and well-solvated major groove of the ter-
nary complex. Hypothetical models seem to support this assump-
tion, as well as the hypothesis that hydrogen bonding may play a
significant role in ternary complex stabilization. No correlation is
observed between cytotoxicity and top1 inhibition, however, indi-
cating top1 is not the sole determinant of antiproliferative potency.
Interestingly, the top1 inhibition trend is identical to that observed
for indenoisoquinolines. In addition to supporting our proposed
binding mode, these data indicate additional SAR overlap with this
related class of top1 inhibitors, and possibly lend support to the
hypothesis that there may be features common to all effective
top1 poisons.
5.2.2. 14-Chloromethyl-12H-5,11a-diazadibenzo[b,h]fluoren-
11-one (19)30
Compound 16 (0.284 g, 1.43 mmol) and compound 1849 (0.291 g,
1.72 mmol) were diluted with toluene (35 mL) and p-TsOH (0.272 g,
1.43 mmol) was added. The mixture was heated at reflux (using a
Dean–Stark trap to collect azeotroped water) for 17 h. The bright-or-
ange suspension was cooled, concentrated, and diluted with CHCl3
(100 mL). The organic phase was washed with satd NaHCO3 (200 mL)
and the aqueous layer was extracted with CHCl3 (100 mL), and the
combined organic layers were washed with 5% NaHCO3 and satd
NaCl (100 mL each). The solution was dried over anhydrous sodium
sulfate and concentrated to yield a yellow amorphous solid (0.425 g,
89%) after washing with MeOH (50 mL): mp 274–280 °C [(dec), lit.30
mp 270 °C (dec)]. 1H NMR (300 MHz, CDCl3) d 8.56 (d, J = 8.1 Hz, 1H),
8.27 (d, J = 7.9 Hz, 1H), 8.19 (d, J = 8.2, 1H), 7.84–7.59 (m, 6H), 5.44(s,
2H), 5.04 (s, 2H).
5. Experimental
5.1. General procedures
Reagents and solvents were purchased from commercial
vendors and were used without further purification. Analytical
thin-layer chromatography was performed on Baker-flex silica
gel IB2-F plastic-backed TLC plates. Preparative thin-layer chroma-
tography was performed on Analtech silica gel G1200
lM glass
plates. Compounds were visualized with both short and long-
5.3. General procedure for synthesis of mono-Boc
diaminoalkanes 31–4131
Boc2O (0.5 g, 2.29 mmol) was dissolved in CHCl3 (10 mL) and
added dropwise to the diamine (11.4 mmol), dissolved in CHCl3
(50 mL). The reaction mixture was allowed to stir overnight at
room temperature. The solution was concentrated, adsorbed onto
SiO2, and purified by flash column chromatography (SiO2), eluting
with 10% MeOH–1% Et3N in CHCl3 to afford the mono-Boc-pro-
tected diamines in acceptable purity after drying in vacuo.
wavelength UV light. Silica gel flash chromatography was
performed using 40–63 lm flash silica gel. Melting points were
determined in capillary tubes using a Mel-Temp apparatus and
are uncorrected. Infrared spectra were obtained as films on salt
plates using CHCl3, CDCl3, or CHCl3/MeOH as the solvent unless
otherwise specified, using a Perkin–Elmer Spectrum One FT-IR
spectrometer. All spectra are baseline-corrected. 1H NMR spectra
were obtained at 300 or 500 MHz, using a Bruker ARX300 and
Bruker Avance 500 (QNP and TXI 5 mm probe), respectively. Mass
spectral analyses were performed at the Purdue University
Campus-Wide Mass Spectrometry Center. ESIMS (electrospray ion-
ization mass spectrometry) was performed using a FinniganMAT
LCQ Classic mass spectrometer system. Combustion microanalyses
were performed at the Purdue University Microanalysis Laboratory
using a Perkin–Elmer Series II CHNS/O model 2400 analyzer and all
reported values are within 0.4% of calculated values. Precursor
5.3.1. Mono-Boc-1,2-diaminoethane (31)
From 20, the general procedure afforded the desired product as a
clear, pale-yellow viscous oil (0.382 g, 100% with minor impurities):
1H NMR (300 MHz, CDCl3) d 4.90 (br s, 1H), 3.19 (q, J = 5.8 Hz, 2H),
2.80 (t, J = 6.0 Hz, 2H), 1.43 (s, 11H).
5.3.2. Mono-Boc-1,3-diaminopropane (32)
From 21, the general procedure afforded the desired product as
a clear, pale-yellow viscous oil (0.330 g, 83%): 1H NMR (300 MHz,

M. A. Cinelli et al. / Bioorg. Med. Chem. 17 (2009) 7145–7155
7151
CDCl3) d 4.97 (br s, 1H), 3.21 (q, J = 6.2 Hz, 2H), 2.75 (t, J = 6.6 Hz,
2H), 1.62–1.53 (m, 2H), 1.41 (s, 11H).
perature for 19 h, poured into H2O (100 mL), and extracted with
CHCl3 (1 ꢂ 100 mL, 1 ꢂ 60 mL). The organic layers were washed
with H2O (2 ꢂ 140 mL, 2 ꢂ 280 mL), dried over anhydrous sodium
sulfate, and concentrated. The residue was adsorbed onto SiO2, and
purified by flash column chromatography (SiO2), eluting with 0.4%
Et3N in CHCl3. The obtained solid was further purified by prepara-
tive TLC (SiO2, 1.5% MeOH, and a few drops of Et3N in CHCl3) to
yield a yellow amorphous solid (0.059 g, 62%) after washing with
ether: mp 170–173 °C. IR (film) 3436, 2975, 1712, 1655, 1617,
5.3.3. Mono-Boc-1,4-diaminobutane (33)
From 22, the general procedure afforded the desired product as
a clear yellow viscous oil (0.443 g, 100% with minor impurities): 1H
NMR (300 MHz, CDCl3) d 4.74 (br s, 1H), 3.10 (q, J = 6.1 Hz, 2H),
2.70 (t, J = 6.7 Hz, 2H), 1.50–1.43 (m, 4H), 1.41 (s, 11H).
5.3.4. Mono-Boc-1,5-diaminopentane (34)
1598, 1502, 1456, 1364, 1171, 753, 687 cmꢀ1 1H NMR (300 MHz,
;
From 23, the general procedure afforded the desired product as
a viscous yellow oil (0.418 g, 90%): 1H NMR (300 MHz, CDCl3) d
4.56 (br s, 1H), 3.12 (q, J = 6.3 Hz, 2H), 2.71 (t, J = 6.7 Hz, 2H),
1.67–1.28 (m, 17H).
CDCl3) d 8.54 (d, J = 8.1 Hz, 1H), 8.25 (dd, J = 12.2 Hz, 8.3 Hz, 2H),
7.79–7.56 (m, 6H), 5.43 (s, 2H), 4.92 (br s, 1H), 4.37 (s, 2H), 3.31
(q, J = 5.6 Hz, 2H), 2.91 (t, J = 5.8 Hz, 2H), 1.68 (s, 1H, under residual
solvent peak), 1.42 (s, 9H); ESIMS m/z (rel intensity) 457 (MH+, 65),
357, (MH+ꢀBoc, 100). Anal. Calcd for C27H28N4O3ꢁ0.75H2O: C,
68.99; H, 6.33; N, 11.92. Found: C, 68.76; H, 6.51; N, 11.89.
5.3.5. Mono-Boc-1,5-diaminohexane (35)
From 24, the general procedure afforded the desired product as
a yellow semisolid (0.428 g, 86%): 1H NMR (300 MHz, CDCl3) d 4.54
(br s, 1H), 3.13 (q, J = 6.5 Hz, 2H), 2.70 (t, J = 6.6 Hz, 2H), 1.43 (s,
11H), 1.34–1.30 (m, 8H).
5.4.2. 14-(30-tert-Boc-aminopropyl-10-aminomethyl)-12H-
5,11a-diazadibenzo[b,h]fluoren-11-one (43)
Compound 19 (0.070 g, 0.210 mmol) was diluted in DMSO
(25 mL) and compound 32 (0.110 g, 0.631 mmol) was dissolved
in DMSO (1 mL) and added, and the mixture was sonicated briefly
to break up the starting material. The mixture was stirred at room
temperature for 16 h, poured into H2O (100 mL), and extracted
with CHCl3 (2 ꢂ 70 mL, 2 ꢂ 40 mL). The organic layers were
washed with H2O (4 ꢂ 140 mL), dried over anhydrous sodium sul-
fate, and concentrated. The residue was adsorbed onto SiO2, and
purified by flash column chromatography (SiO2), eluting with
0.6% Et3N in CHCl3. The obtained solid was further purified by pre-
parative TLC (SiO2, 1.5% MeOH, few drops Et3N in CHCl3) to yield a
yellow amorphous solid (0.061 g, 62%) after washing with ether:
mp 145–158 °C. IR (film) 3307, 2975, 1696, 1659, 1622, 1602,
1512, 1365, 1345, 1170, 754, 687 cmꢀ1; 1H NMR (300 MHz, CDCl3)
d 8.56 (d, J = 7.9 Hz, 1H), 8.30 (d, J = 8.5 Hz, 1H), 8.24 (d, J = 8.2 Hz,
1H), 7.79–7.57 (m, 6H), 5.47 (s, 2H), 4.87 (br s, 1H), 4.36 (s, 2H),
3.22 (q, J = 6.4 Hz, 2H), 2.85 (t, J = 6.5 Hz, 2H), 1.75–1.70 (m, 3H),
1.39 (s, 9H); ESIMS m/z (rel intensity) 471 (MH+, 35), 371,
(MH+ꢀBoc, 100). Anal. Calcd for C28H30N4O3: C, 71.47; H, 6.43; N,
11.91. Found: C, 71.12; H, 6.64; N, 11.81.
5.3.6. Mono-Boc-1,7-diaminoheptane (36)
From 25, the general procedure afforded the desired product as
a clear, pale-yellow viscous oil (0.387 g, 73%): 1H NMR (500 MHz,
CDCl3) d 4.54 (br s, 1H), 3.20–3.00 (m, 2H), 2.68 (t, J = 7.0 Hz, 2H),
1.62–1.55 (m, 4H), 1.42 (s, 9H), 1.30–1.10 (m, 8H).
5.3.7. Mono-Boc-1,8-diaminooctane (37)
From 26, the general procedure afforded the product as a color-
less semisolid (0.493 g, 91%): 1H NMR (300 MHz, CDCl3) d 4.50 (br
s, 1H), 3.11 (q, J = 6.8 Hz, 2H), 2.70 (t, J = 6.8 Hz, 2H), 1.60–1.50 (m,
4H), 1.44 (s, 11H), 1.40–1.30 (m, 8H).
5.3.8. Mono-Boc-1,9-diaminononane (38)
From 27, the general procedure afforded the product as a vis-
cous, colorless oil which solidified upon standing (0.437 g, 74%):
1H NMR (300 MHz, CDCl3) d 4.50 (br s, 1H), 3.13 (q, J = 6.5 Hz,
2H), 2.69 (t, J = 6.8 Hz, 2H), 1.40–1.30 (m, 13H), 1.30–1.20 (m,
12H).
5.3.9. Mono-Boc 1,10-diaminodecane (39)
5.4.3. 14-(40-tert-Boc-aminobutyl-10-aminomethyl)-12H-5,11a-
diazadibenzo[b,h]fluoren-11-one (44)
From 28, the general procedure afforded the product as a color-
less semisolid (0.598 g, 96%): 1H NMR (300 MHz, CDCl3) d 4.51 (br
s, 1H), 3.13 (q, J = 6.5 Hz, 2H), 2.69 (t, J = 6.8 Hz, 2H), 1.50–1.30 (m,
15H), 1.30–1.20 (m, 12H).
Compound 19 (0.070 g, 0.210 mmol) was diluted in DMSO
(20 mL) and compound 33 (0.119 g, 0.630 mmol) was dissolved
in DMSO (2 mL) and added. The mixture was stirred at room tem-
perature for 17 h, poured into H2O (100 mL), and extracted with
CHCl3 (60 mL); additionally, MeOH (70 mL) was added to the or-
ganic phase to aid solubility. The organic layer was washed with
H2O (4 ꢂ 150 mL), dried over anhydrous sodium sulfate, and con-
centrated. The obtained residue was adsorbed onto SiO2 and puri-
fied by flash column chromatography (SiO2), eluting with 0.6% Et3N
in CHCl3, to yield a yellow amorphous solid (0.069 g, 68%) after
washing with ether: mp 168–175 °C (dec). IR (film) 3351, 2930,
5.3.10. Mono-Boc-1,11-diaminoundecane (40)
From 29, the general procedure afforded the product as a color-
less semisolid (0.588 g, 90%): 1H NMR (300 MHz, CDCl3) d 4.49 (br
s, 1H), 3.13 (q, J = 6.7 Hz, 2H), 2.70 (t, J = 6.8 Hz, 2H), 1.60–1.40 (m,
17H), 1.30–1.20 (m, 12H).
5.3.11. Mono-Boc-1,12-diaminododecane (41)
From 30, the general procedure afforded the product as a color-
less semisolid (0.547 g, 79%): 1H NMR (300 MHz, CDCl3) d 4.50 (br
s, 1H), 3.11 (q, J = 5.7 Hz, 2H), 2.70 (t, J = 6.9 Hz, 2H), 1.60–1.20 (m,
31H).
1693, 1657, 1621, 1601, 1509, 1365, 1250, 1173, 753, 687 cmꢀ1
;
1H NMR (300 MHz, CDCl3) d 8.56 (d, J = 8.0 Hz, 1H), 8.26 (t,
J = 8.4 Hz, 1H), 7.79–7.57 (m, 6H), 5.47 (s, 2H), 4.67 (br s, 1H),
4.37 (s, 2H), 3.14 (q, J = 6.0 Hz, 2H), 2.80 (t, J = 6.2 Hz, 2H),
1.59–1.55 (m, 5H), 1.42 (s, 9H); ESIMS m/z (rel intensity) 485
(MH+, 27), 385, (MH+ꢀBoc, 100).
5.4. Preparation of aromathecins
5.4.1. 14-(20-tert-Boc-aminoethyl-10-aminomethyl)-12H-5,11a-
diazadibenzo[b,h]fluoren-11-one (42)
5.4.4. 14-(50-tert-Boc-aminopentyl-10-aminomethyl)-12H-
5,11a-diazadibenzo[b,h]fluoren-11-one (45)
Compound 19 (0.070 g, 0.210 mmol) was diluted in DMSO
(20 mL) and 31 (0.101 g, 0.631 mmol) was dissolved in DMSO
(1 mL) and added to the suspension. The mixture was sonicated
briefly to break up the starting material and stirred at room tem-
Compound 19 (0.081 g, 0.243 mmol) and compound 34 (0.147 g,
0.729 mmol) were diluted with DMSO (25 mL), and the mixture was
stirred at room temperature for 19 h. The reaction mixture was di-
luted with H2O (100 mL), and extracted with CHCl3, (2 ꢂ 100 mL),

7152
M. A. Cinelli et al. / Bioorg. Med. Chem. 17 (2009) 7145–7155
with additional H2O (100 mL) being added to break up the resultant
emulsion. The organic layers were washed with H2O (3 ꢂ 100 mL),
dried over anhydrous sodium sulfate, concentrated, adsorbed onto
SiO2, and purified by flash column chromatography (SiO2), eluting
with 0.4% Et3N in CHCl3 to yield a pale-yellow amorphous solid
(0.079 g, 66%) after washing with ether: mp 186–189 °C. IR (film)
3368, 2928, 2856, 1693, 1656, 1619, 1600, 1512, 1453, 1365, 1250,
umn chromatography (SiO2), eluting with a gradient of CHCl3–0.5%
MeOH in CHCl3. The resultant residue was purified further by pre-
parative TLC (1% MeOH in CHCl3) to yield a yellow amorphous solid
(0.048 g, 42%) after washing with ether and hexanes: mp 96–101 °C.
IR (film) 3436, 3361, 3287, 2924, 2854, 1684, 1655, 1618, 1600,
1452, 1364, 1250, 1173, 834, 752, 685 cmꢀ1 1H NMR (300 MHz,
;
CDCl3) d 8.56 (d, J = 8.0 Hz, 1H), 8.26 (t, J = 9.1 Hz, 2H), 7.78–7.56
(m, 6H), 5.46 (s, 2H), 4.61 (br s, 1H), 4.36 (s, 2H), 3.12 (q, J = 6.5 Hz,
2H), 2.76 (t, J = 7.1 Hz, 2H), 1.70–1.20 (m, 22H); ESIMS m/z (rel inten-
sity) 541 (MH+, 32), 441 (MH+ꢀBoc, 100), 563 (MNa+, 37).
1172, 835, 752, 686 cmꢀ1 1H NMR (300 MHz, CDCl3) d 8.56 (d,
;
J = 8.0 Hz, 1H), 8.26 (t, J = 8.8 Hz, 2H), 7.78–7.57 (m, 6H), 5.47 (s,
2H), 4.66 (br s, 1H), 4.67 (s, 2H), 3.12 (q, J = 6.2 Hz, 2H), 2.78 (t,
J = 7.0 Hz, 2H), 1.60–1.35 (m, 16H); ESIMS m/z (rel intensity) 499
(MH+, 57), 997, (2MH+ 100).
5.4.8. 14-(90-tert-Boc-aminononyl-10-aminomethyl)-12H-5,11a-
diazadibenzo[b,h]fluoren-11-one (49)
5.4.5. 14-(60-tert-Boc-aminohexyl-10-aminomethyl)-12H-5,11a-
diazadibenzo[b,h]fluoren-11-one (46)
Compound 19 (0.070 g, 0.210 mmol) was diluted with DMSO
(20 mL) and compound 38 (0.163 g, 0.631 mmol) was added as a
suspension in DMSO (5 mL). The mixture was stirred at room tem-
perature for 20 h, diluted with H2O (100 mL) and extracted with
CHCl3 (2 ꢂ 50 mL, 1 ꢂ 30 mL). The organic layers were washed
with H2O (1 ꢂ 120 mL, 4 ꢂ 100 mL), dried over anhydrous sodium
sulfate, concentrated, adsorbed onto SiO2, and purified by flash col-
umn chromatography (SiO2), eluting with 0.2% Et3N in CHCl3. The
obtained residue was further purified by preparative TLC (SiO2,
1% MeOH, and few drops of Et3N in CHCl3), to afford the desired
product as a yellow amorphous solid (0.054 g, 47%) after washing
with ether: mp 90–98 °C. IR (film) 3438, 3360, 2925, 2854, 1693,
1655, 1618, 1600, 1511, 1452, 1365, 1250, 1173, 836, 752,
Compound 19 (0.070 g, 0.210 mmol) was diluted with DMSO
(20 mL) and compound 35 (0.136 g, 0.631 mmol) was dissolved
in DMSO (3 mL) and pipetted into the suspension. The mixture
was stirred at room temperature for 16 h, poured into H2O
(120 mL) and extracted with CHCl3 (2 ꢂ 50 mL, 1 ꢂ 30 mL). The or-
ganic layers were washed with H2O (1 ꢂ 120 mL, 4 ꢂ 100 mL),
dried over anhydrous sodium sulfate, concentrated, adsorbed onto
SiO2, and purified by flash column chromatography (SiO2), eluting
with 0.4% Et3N in CHCl3. The obtained residue was further purified
by preparative TLC (SiO2, 1% MeOH, and a few drops of Et3N in
CHCl3), to yield a pale-yellow amorphous solid (0.055 g, 51%) after
washing with ether: mp 99–104 °C. IR (film) 3437, 3360, 3285,
2928, 1705, 1655, 1619, 1601, 1452, 1365, 1172, 835, 752,
685 cmꢀ1 1H NMR (300 MHz, CDCl3) d 8.55 (J = 7.8 Hz, 1H), 8.27
;
(dd, J = 13.4 Hz, 8.2 Hz), 7.79–7.56 (m, 6H), 5.47 (s, 2H), 4.56 (br
s, 1H), 4.38 (s, 2H), 3.10 (q, J = 6.7 Hz, 2H), 2.78 (t, J = 7.1 Hz, 2H),
1.74–1.26 (m, 24H); ESIMS m/z (rel intensity), 555 (MH+, 100).
685 cmꢀ1 1H NMR (300 MHz, CDCl3) d 8.55 (d, J = 8.1 Hz, 1H),
;
8.25 (t, J = 8.1 Hz, 2H), 7.80–7.54 (m, 6H), 5.46 (s, 2H), 4.63 (br s,
1H), 4.35 (s, 2H), 3.10 (q, J = 6.3 Hz, 2H), 2.77 (t, J = 7.0 Hz, 2H),
1.60–1.30 (m, 18H); ESIMS m/z (rel intensity), 513, (MH+, 80),
413 (MH+ꢀBoc, 100).
5.4.9. 14-(100-tert-Boc-aminodecyl-10-aminomethyl)-12H-
5,11a-diazadibenzo[b,h]fluoren-11-one (50)
Compound 19 (0.070 g, 0.210 mmol) was diluted with DMSO
(20 mL) and compound 39 (0.172 g, 0.630 mmol) was added as a
suspension in DMSO (5 mL). The reaction mixture was stirred at
room temperature for 17 h, diluted with H2O (120 mL), and ex-
tracted with CHCl3 (2 ꢂ 50 mL, 2 ꢂ 80 mL, 1 ꢂ 30 mL). The organic
layers were washed with H2O (5 ꢂ 200 mL), dried over anhydrous
sodium sulfate, concentrated, and the resultant residue was ad-
sorbed onto SiO2 and purified by flash column chromatography
(SiO2) eluting with 0.2% Et3N in CHCl3. The obtained material
was further purified by preparative TLC (0.7% MeOH, and a few
drops of Et3N in CHCl3), to yield a bright pale-yellow amorphous
solid (0.062 g, 52%) after washing with ether: mp 88–95 °C. IR
(film) 3437, 3285, 2924, 2853, 1703, 1655, 1618, 1600, 1453,
5.4.6. 14-(70-tert-Boc-aminoheptyl-10-aminomethyl)-12H-
5,11a-diazadibenzo[b,h]fluoren-11-one (47)
Compound 19 (0.055 g, 0.165 mmol) was diluted with DMSO
(25 mL). Compound 36 (0.114 g, 0.495 mmol) was dissolved in
DMSO (2 mL) and added to the suspension. The mixture was stir-
red at room temperature for 22 h, diluted with CHCl3 (40 mL),
and washed with H2O (4 ꢂ 50 mL). The organic phase was dried
over anhydrous sodium sulfate, concentrated, and the residue
was washed with ether and filtered. The filtrate was diluted with
CHCl3 (30 mL) and washed again with H2O (3 ꢂ 20 mL) to remove
residual DMSO, before it was dried over anhydrous sodium sulfate,
combined with the filtered residue, adsorbed onto SiO2, and
purified by flash column chromatography (SiO2), eluting with a
gradient of CHCl3 to 0.5% MeOH–0.5% Et3N to yield a yellow amor-
phous solid that was further purified by preparative TLC (SiO2, 1.2%
MeOH in CHCl3), to yield a pale-yellow amorphous solid (0.039 g,
45%) after washing with ether: mp 97–105 °C. IR (film) 3436,
2926, 2855, 1691, 1655, 1619, 1601, 1512, 1453, 1365, 1252,
1364, 1172, 834, 752, 685 cmꢀ1 1H NMR (300 MHz, CDCl3) d
;
8.56 (d, J = 8.0 Hz, 1H), 8.27 (dd, J = 13.6 Hz, 8.4 Hz, 2H), 7.80–
7.55 (m, 6H), 5.47 (s, 2H), 4.53 (br s, 1H), 4.37 (s, 2H), 3.10 (q,
J = 5.8 Hz, 2H), 2.77 (t, J = 7.0 Hz, 2H), 1.64–1.25 (m, 26H); ESIMS
m/z (rel intensity), 569 (MH+, 46), 1137 (2MH+, 100).
1174, 836, 753, 685 cmꢀ1
;
1H NMR (300 MHz, CDCl3) d 8.58 (d,
5.4.10. 14-(110-tert-Boc-aminoundecyl-10-aminomethyl)-12H-
5,11a diazadibenzo[b,h]fluoren-11-one (51)
J = 7.8 Hz, 1H), 8.27 (t, J = 9.4 Hz, 2H), 7.80–7.58 (m, 6H), 5.48 (s,
2H), 4.69 (br s, 1H), 4.37 (s, 2H), 3.10 (q, J = 6.3 Hz, 2H), 2.77 (t,
J = 7.0 Hz, 2H), 1.60–1.20 (m, 20H); ESIMS m/z (rel intensity),
527, (MH+, 78), 427 (MH+ꢀBoc, 100).
Compound 19 (0.070 g, 0.210 mmol) and compound 40 (0.180 g,
0.630 mmol) were diluted with DMSO (25 mL) and the mixture was
stirred at room temperature for 16 h. The mixture was diluted
with H2O (100 mL), extracted with CHCl3 (2 ꢂ 50 mL, 2 ꢂ 90 mL,
1 ꢂ 50 mL, 1 ꢂ 30 mL), and the organic layers were washed with
H2O (5 ꢂ 200 mL), before they were dried over anhydrous sodium
sulfate, concentrated, adsorbed onto SiO2, and purified by flash col-
umnchromatography(SiO2), elutingwith0.2%Et3NinCHCl3, toyield
a yellow amorphous solid (0.067 g, 55%) after washing with ether
and hexanes: mp 88–100 °C. IR (film) 3436, 3368, 2923, 2853,
5.4.7. 14-(80-tert-Boc-aminooctyl-10-aminomethyl)-12H-5,11a-
diazadibenzo[b,h]fluoren-11-one (48)
Compound 19 (0.071 g, 0.213 mmol) was diluted with DMSO
(25 mL), and compound 37 was dissolved in DMSO (2 mL) and added
to the suspension. The mixture was stirred at room temperature for
16 h, diluted with CHCl3 (40 mL) and washed with H2O (3 ꢂ 50 mL,
1 ꢂ 80 mL). The organic phase was dried over anhydrous sodium
sulfate, concentrated, adsorbed onto SiO2, and purified by flash col-
1704, 1655, 1618, 1600, 1453, 1365, 1169, 835, 752, 685 cmꢀ1
1H NMR (300 MHz, CDCl3) d 8.58 (d, J = 8.2 Hz, 1H), 8.28 (dd,
;

M. A. Cinelli et al. / Bioorg. Med. Chem. 17 (2009) 7145–7155
7153
J = 13.0 Hz, 8.4 Hz, 2H), 7.82–7.58 (m, 6H), 5.49 (s, 2H), 4.52 (br s,
1H), 4.38 (s, 2H), 3.10 (q, J = 6.6 Hz, 2H), 2.77 (t, J = 7.1 Hz, 2H),
1.59–1.24 (m, 28H); ESIMS m/z (rel intensity), 583 (MH+, 100).
7.10–7.00 (m, 1H), 6.86 (s, 1H). 5.05 (s, 2H), 4.55 (s, 2H), 3.33 (t,
J = 6.8 Hz, 2H), 3.05 (t, J = 7.6 Hz, 2H), 1.90–1.70 (m, 4H); ESIMS
m/z (rel intensity) 385 (MH+, 100). Anal. Calcd for
C24H27Cl3N4OꢁH2O: C, 56.31; H, 5.71; N, 10.95. Found: C, 56.07;
H, 5.44; N, 10.58.
5.4.11. 14-(120-tert-Boc-aminododecyl-10-aminomethyl)-12H-
5,11a diazadibenzo[b,h]fluoren-11-one (52)
5.4.15. 14-(50-Aminopentyl-10-aminomethyl)-12H-5,11a-
diazadibenzo[b,h]fluoren-11-one trihydrochloride (56)
Compound 19 (0.070 g, 0.210 mmol) and compound 41 (0.190 g,
0.631 mmol) were diluted with DMSO (25 mL) and the mixture was
stirred at room temperature for 17 h. The mixture was diluted with
H2O (100 mL), extracted with CHCl3 (1 ꢂ 50 mL, 3 ꢂ 80 mL, 1 ꢂ
50 mL) and the organic layers were washed with H2O (5 ꢂ
200 mL), before they were dried over anhydrous sodium sulfate,
concentrated, adsorbed onto SiO2, and purified by flash column
chromatography (SiO2), eluting with 0.2% Et3N in CHCl3, to yield a
pale-yellowamorphoussolid (0.088 g, 70%) after partially dissolving
in ether and precipitating out with hexanes: mp 81–90 °C. IR (film)
3435, 3369, 2922, 2852, 1689, 1655, 1618, 1600, 1453, 1365, 1168,
634, 752, 685 cmꢀ1; 1H NMR (300 MHz, CDCl3) d 8.58 (d, J = 7.7 Hz,
1H), 8.29 (dd, J = 12.6, 7.9 Hz, 2H), 7.81–7.59 (m, 6H), 5.49 (s, 2H),
4.50 (br s, 1H), 4.38 (s, 2H), 3.10 (q, J = 6.7 Hz, 2H), 2.76 (t,
J = 7.1 Hz, 2H), 1.57–1.23 (m, 30H); ESIMS m/z (rel intensity), 597
(MH+, 100). Anal. Calcd for C37H48N4O3ꢁ0.5H2O: C, 73.36; H, 8.15;
N, 9.25. Found: C, 73.09; H, 8.25; N, 9.23.
Compound 45 (0.065 g, 0.130 mmol) was diluted in CHCl3
(40 mL) and filtered to remove particulate matter. Methanolic
HCl (3 M, 10 mL) was added dropwise, and the dark red mixture
was stirred for 2 h at room temperature and concentrated to pro-
vide a bright red amorphous solid (0.056 g, 86%) after washing
with ether and drying in vacuo: mp 245–251 °C (dec). IR (KBr)
3399, 2929, 1654, 1474, 1336, 1217, 766, 683 cmꢀ1 1H NMR
;
(300 MHz, D2O) d 7.75–7.45 (m, 7H), 7.20–7.02 (m, 2H), 5.12 (s,
2H), 4.57 (s, 2H), 3.27 (t, J = 6.9 Hz, 2H), 2.98 (t, J = 6.4 Hz, 2H),
1.80–1.60 (m, 4H), 1.50–1.40 (m, 2H); ESIMS m/z (rel intensity).
399 (MH+, 100). Anal. Calcd for C25H29Cl3N4Oꢁ2H2O: C, 55.21; H,
6.12; N, 10.30. Found: C, 55.29; H, 5.97; N, 10.20.
5.4.16. 14-(60-Aminohexyl-10-aminomethyl)-12H-5,11a-
diazadibenzo[b,h]fluoren-11-one trihydrochloride (57)
Compound 46 (0.050 g, 0.097 mmol) was dissolved in CHCl3
(30 mL) and methanolic HCl (3 M, 10 mL) was added dropwise.
The orange cloudy mixture was stirred at room temperature for
2 h and concentrated to afford a dark red amorphous solid
(0.043 g, 85%) after washing with ether and drying in vacuo: mp
230–235 °C. IR (KBr) 3412, 2928, 1657, 1622, 1596, 1478, 1337,
5.4.12. 14-(20-Aminoethyl-10-aminomethyl)-12H-5,11a-
diazadibenzo[b,h]fluoren-11-one trihydrochloride (53)
Compound 42 (0.050 g, 0.110 mmol) was dissolved in CHCl3
(30 mL). Methanolic HCl (3 M, 10 mL) was added dropwise. The
addition was slightly exothermic. The mixture was stirred at room
temperature for 2 h and concentrated to yield a red-orange solid
after washing with ether and drying in vacuo. The powder was
then washed with a solution of CHCl3 (containing several drops
of MeOH) to remove residual Et3NꢁHCl, yielding the desired prod-
uct as a red powder (0.035 g, 69%) after washing with ether: mp
228–235 °C (dec). IR (KBr) 3400, 2991, 2725, 1651, 1595, 1337,
765, 682 cmꢀ1 1H NMR (300 MHz, D2O) d 7.72 (d, J = 8.4 Hz, 1H),
;
7.60 (s, 2H), 7.51–7.42 (m, 4H), 7.06 (t, J = 5.4 Hz, 1H), 6.95 (s,
1H), 5.08 (s, 2H), 4.54 (s, 2H), 3.25 (t, J = 7.7 Hz, 2H), 2.97 (t,
J = 7.4 Hz, 2H), 1.80–1.60 (m, 4H), 1.30–1.40 (m, 4H); ESIMS m/z
(rel intensity) 413 (MH+, 100). Anal. Calcd for C26H31Cl3N4OꢁH2O:
C, 57.84; H, 6.16; N, 10.38. Found: C, 57.53; H, 6.48; N, 10.03.
; d 7.78 (d,
1156, 770, 684 cmꢀ1 1H NMR (300 MHz, D2O)
5.4.17. 14-(70-Aminoheptyl-10-aminomethyl)-12H-5,11a-
diazadibenzo[b,h]fluoren-11-one trihydrochloride (58)
J = 8.6 Hz, 1H), 7.65 (s, 2H), 7.52–7.39 (m, 4H), 7.08 (t, J = 6.6 Hz,
1H), 7.00 (s, 1H), 5.12 (s, 2H), 4.64 (s, 2H), 3.65 (t, J = 6.8 Hz, 2H),
3.41 (t, J = 7.0 Hz, 2H); ESIMS m/z (rel intensity) 357 (MH+, 100).
Anal. Calcd for C22H23Cl3N4O: C, 56.73; H, 4.98; N, 12.03. Found:
C, 56.90; H, 4.75; N, 11.83.
Compound 47 (0.035 g, 0.066 mmol) was diluted with benzene
(25 mL) and methanolic HCl (3 M, 10 mL) was added dropwise,
upon which the solution became dark red. The mixture was heated
at reflux for 3 h, cooled, and concentrated to yield a bright orange
solid (0.030 g, 85%) after washing with ether: mp 208–215 °C (dec).
IR (KBr) 3400, 2929, 1657, 1620, 1600, 1457, 1340, 1132, 764,
5.4.13. 14-(30-Aminopropyl-10-aminomethyl)-12H-5,11a-
diazadibenzo[b,h]fluoren-11-one trihydrochloride (54)
687 cmꢀ1 1H NMR (300 MHz, D2O) d 7.75–7.69 (m, 3H), 7.58–
;
Compound 43 (0.052 g, 0.111 mmol) was dissolved in CHCl3
(30 mL). Methanolic HCl (3 M, 10 mL) was added dropwise. The
mixture was stirred at room temperature for 2 h and concentrated
to yield a red-orange solid (0.043 g, 82%) after washing with ether
and drying in vacuo: mp 232–240 °C (dec). IR (KBr) 3435, 2922,
7.48 (m, 4H), 7.20–7.08 (m, 2H), 5.13 (s, 2H), 4.56 (s, 2H), 3.23 (t,
J = 8.1 Hz, 2H), 2.95 (t, J = 7.7 Hz, 2H), 1.80–1.50 (m, 4H), 1.30–
1.20 (m, 6H); ESIMS m/z (rel intensity) 427 (MH+, 100). Anal. Calcd
for C27H33Cl3N4Oꢁ1.5H2O: C, 57.60; H, 6.45; N, 9.95. Found: C,
57.47; H, 6.49; N, 9.64.
1659, 1623, 1601, 1479, 1334, 762, 686 cmꢀ1 1H NMR (300 MHz,
;
D2O) d 7.75 (d, J = 8.3 Hz, 1H), 7.61 (s, 2H), 7.52–7.41 (m, 4H),
7.04–6.96 (m, 2H), 5.10 (s, 2H), 4.60 (s, 2H), 3.38 (t, J = 7.9 Hz,
2H), 3.10 (t, J = 7.4 Hz, 2H), 2.20–2.00 (m, 2H); ESIMS m/z (rel
intensity) 371(MH+, 100). Anal. Calcd for C23H25Cl3N4O: C, 57.57;
H, 5.25; N, 11.68. Found: C, 57.91; H, 5.24; N, 11.72.
5.4.18. 14-(80-Aminooctyl-10-aminomethyl)-12H-5,11a-
diazadibenzo[b,h]fluoren-11-one trihydrochloride (59)
Compound 48 (0.042 g, 0.078 mmol) was dissolved in CHCl3
(30 mL) and methanolic HCl (3 M, 10 mL) was added dropwise,
upon which the solution became dark orange-red. The mixture
was stirred at room temperature for 2 h and concentrated to yield
a dark red flaky solid (0.036, 85%) after washing with ether and
drying in vacuo: mp 200–203 °C (dec). IR (KBr) 3401, 2929, 2856,
5.4.14. 14-(40-Aminobutyl-10-aminomethyl)-12H-5,11a-
diazadibenzo[b,h]fluoren-11-one trihydrochloride (55)
1758, 1656, 1597, 1619, 1479, 1338, 1135, 764, 688 cmꢀ1 1H
;
Compound 44 (0.055 g, 0.113 mmol) was dissolved in CHCl3
(30 mL). Methanolic HCl (3 M, 10 mL) was added dropwise. The
mixture was stirred at room temperature for 2 h and concentrated
to yield a red-orange solid (0.050 g, 90%) after washing with ether
and drying in vacuo: mp 222–228 °C (dec). IR (KBr) 3401, 2928,
NMR (300 MHz, D2O) 7.70 d (d, J = 8.1 Hz, 1H), 7.60–7.38 (m, 6H),
7.04–7.02 (m, 1H), 6.89 (s, 1H), 5.04 (s, 2H), 4.50 (s, 2H), 3.22 (t,
J = 7.6 Hz, 2H), 2.94 (t, J = 7.3 Hz, 2H), 1.80–1.50 (m, 4H), 1.30–
1.20 (m, 8H); ESIMS m/z (rel intensity) 441 (MH+, 100). Anal. Calcd
for C28H35Cl3N4OꢁH2O: C, 59.21; H, 6.57; N, 9.86. Found: C, 59.03;
H, 6.81; N, 9.78.
1657, 1596, 1476, 1334, 762, 681 cmꢀ1 1H NMR (300 MHz, D2O)
;
d 7.72 (d, J = 8.2 Hz, 1H), 7.60–7.45 (m, 3H), 7.36–7.32 (m, 3H),

7154
M. A. Cinelli et al. / Bioorg. Med. Chem. 17 (2009) 7145–7155
5.4.19. 14-(90-Aminononyl-10-aminomethyl)-12H-5,11a-
diazadibenzo[b,h]fluoren-11-one trihydrochloride (60)
Technologies) encompassing the previous identified top1 cleavage
sites identified in the 161-bp fragment from pBluescript SK(ꢀ)
phagemid DNA was employed. This 117-bp oligonucleotide con-
tains a single 50-cytosine overhang, which was 30-end labeled by
Compound 49 (0.046 g, 0.083 mmol) was dissolved in CHCl3
(20 mL) and methanolic HCl (3 M, 10 mL) was added dropwise.
The mixture was stirred for 2 h 20 min at room temperature, con-
centrated, and azeotroped with benzene, to afford a bright red
amorphous solid (0.038 g, 82%) after washing with ether and dry-
ing in vacuo: mp 192–196 °C (dec). IR (KBr) 3414, 2928, 2854,
fill-in reaction with [a-
32P]-dGTP 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, New England BioLabs). Unincor-
porated 32P-dGTP was removed using mini Quick Spin DNA col-
umns (Roche, Indianapolis, IN), and the eluate containing the 30-
end-labeled DNA substrate was collected. Approximately 2 nM of
radiolabeled DNA substrate was incubated with recombinant
1657, 1622, 1599, 1478, 1336, 761, 688 cmꢀ1 1H NMR (300 MHz,
;
D2O) d 7.73–7.65 (m, 3H), 7.52–7.45 (m, 4H), 7.20–7.00 (m, 2H),
5.10 (s, 2H), 4.53 (s, 2H), 3.21 (t, J = 6.9 Hz, 2H), 2.92 (J = 7.2 Hz,
2H), 1.70–1.50 (m, 4H), 1.40–1.20 (m, 10H); ESIMS m/z (rel inten-
sity) 455 (MH+, 100). Anal. Calcd for C29H37Cl3N4O: C, 61.76; H,
6.61; N, 9.93. Found: C, 61.50; H, 7.00; N, 9.71.
top1 in 20
l
L of reaction buffer [10 mM Tris–HCl (pH 7.5),
g/ml BSA] at
50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, and 15
l
25 °C for 20 min in the presence of various concentrations of com-
pounds. The reactions were terminated by adding SDS (0.5% final
concentration) followed by the addition of two volumes of loading
dye (80% formamide, 10 mM sodium hydroxide, 1 mM sodium
EDTA, 0.1% xylene cyanol, and 0.1% bromphenol blue). Aliquots of
each reaction were subjected to 20% denaturing PAGE. Gels were
dried and visualized by using a Phosphoimager and ImageQuant
software (Molecular Dynamics). For simplicity, cleavage sites were
numbered as previously described in the 161-bp fragment.56
5.4.20. 14-(100-Aminodecyl-10-aminomethyl)-12H-5,11a-
diazadibenzo[b,h]fluoren-11-one trihydrochloride (61)
Compound 50 (0.056 g, 0.096 mmol) was dissolved in CHCl3
(30 mL) and methanolic HCl (3 M, 10 mL) was added dropwise.
The addition was slightly exothermic. The mixture was stirred
for 2 h at room temperature and concentrated to yield a red amor-
phous solid (0.049 g, 87%) after washing with ether and drying in
vacuo: mp 185–190 °C (dec). IR (KBr) 3433, 2925, 2854, 1657,
1622, 1599, 1469, 1335, 764, 688 cmꢀ1
;
1H NMR (300 MHz, D2O)
5.6. Modeling studies
d 7.75–7.69 (m, 3H), 7.56–7.49 (m, 4H), 7.15–7.08 (m, 2H), 5.13
(s, 2H), 4.56 (s, 2H), 3.21 (t, J = 7.4 Hz, 2H), 2.91 (t, J = 7.5 Hz, 2H),
1.70–1.50 (m, 4H), 1.40–1.20 (bm, 12H); ESIMS m/z (rel intensity)
469 (MH+, 100). Anal. Calcd for C30H39Cl3N4Oꢁ0.6H2O: C, 61.19; H,
6.88; N, 9.51. Found: C, 60.97; H, 7.16; N, 9.30.
The crystal structure of topotecan in complex with top1 and a
short DNA fragment were downloaded from the Protein Data Bank
(PDB code 1K4T).8 An atom of Hg and one molecule of PEG were
deleted. Hydrogens were added and minimized by the Powell
method, using the MMFF94s force field and MMFF94 charges. Ana-
logues of aromathecins and indenoisoquinolines were constructed
in Sybyl 8.1 (Tripos, Inc.). Fixed positive charges were assigned to
amines using Sybyl atom types, and the structures were energy
minimized using a conjugate gradient method, MMFF94s force
field, and MMFF94 charges. The structure of the ligand was aligned
onto topotecan using the ‘fit atoms’ function. In the case of 64, the
alignment was based on both the overlay of the crystal structures
1K4T and 1SC7, and on earlier overlays of indenoisoquinolines and
aromathecins.8,30 The aligned ligand was merged into the crystal
structure, and the structure of topotecan was deleted. This new
ternary complex was re-subjected to energy minimization using
a standard Powell method, the MMFF94s force field, and MMFF94
charges, converging to termination at 0.05 kcal/mol Å, with a dis-
tance-dependent dielectric function. The structure of the ligand
and a sphere with a radius of 5 Å were allowed to move during
the minimization, and the surrounding structures were frozen in
an aggregate.
5.4.21. 14-(110-Aminoundecyl-10-aminomethyl)-12H-5,11a-
diazadibenzo[b,h]fluoren-11-one trihydrochloride (62)
Compound 51 (0.056 g, 0.097 mmol) was dissolved in CHCl3
(30 mL) and methanolic HCl (3 M, 10 mL) was added dropwise.
The reaction was slightly exothermic. The mixture was stirred for
2 h at room temperature and concentrated to yield a red-orange
amorphous solid (0.052 g, 91%) after washing with ether and dry-
ing in vacuo: mp 183–188 °C (dec). IR (KBr) 3431, 2925, 2853,
1657, 1622, 1601, 1469, 1336, 762, 688 cmꢀ1 1H NMR (300 MHz,
;
D2O) d 7.75–7.52 (m, 7H), 7.19–7.13 (m, 2H), 5.16 (s, 2H), 4.58 (s,
2H), 3.19 (t, J = 6.2 Hz, 2H), 2.91–2.85 (m, 2H), 1.70–1.50 (m, 4H),
1.40–1.10 (m, 14H); ESIMS m/z (rel intensity) 483 (MH+, 100). Anal.
Calcd for C31H41Cl3N4Oꢁ0.5H2O: C, 61.95; H, 7.04; N, 9.32. Found: C,
61.88; H, 7.31; N, 9.10.
5.4.22. 14-(120-Aminododecyl-10-aminomethyl)-12H-5,11a-
diazadibenzo[b,h]fluoren-11-one trihydrochloride (63)
Compound 52 (0.069 g, 0.115 mmol) was dissolved in CHCl3
(30 mL) and methanolic HCl (3 M, 10 mL) was added dropwise.
The reaction mixture was stirred at room temperature for 2 h
and concentrated to yield a bright red amorphous solid (0.064 g,
92%) after washing with ether and drying in vacuo: mp 200–
204 °C (dec). IR (KBr) 3409, 2924, 2851, 1657, 1619, 1598, 1467,
Acknowledgments
This work was made possible by the National Institutes of
Health (NIH) through support with Research Grant UO1
CA89566. This work was also made possible by the Lynn and
McKeehan Fellowships (Purdue University, M.A.C.), and by the
CIC/SROP program (Purdue University, B.C.). This research was sup-
ported in part by the Intramural Research Program of the NIH, Na-
tional Cancer Institute, Center for Cancer Research. M.A.C. wishes
to thank Drs. Andrew Morrell, Karl Wood, and Huaping Mo for their
assistance and insight.
1337, 764, 688 cmꢀ1 1H NMR (300 MHz, D2O, 60 °C) d 7.98–7.81
;
(m, 4H), 7.72 (t, J = 7.8 Hz, 3H), 7.43–7.37 (m, 2H), 5.29 (s, 2H),
4.71 (s, 2H), 3.23 (t, J = 7.6 Hz, 2H), 3.02–2.92 (m, 2H), 1.71–1.56
(m, 4H), 1.30–1.15 (m, 16H); ESIMS m/z (rel intensity), 497,
(MH+, 100). Anal. Calcd for C32H43Cl3N4OꢁH2O: C, 63.42; H, 7.15;
N, 9.24; Cl, 17.55. Found: C, 63.06; H, 7.48; N, 9.12; Cl, 17.17.
5.5. Topoisomerase I-mediated DNA cleavage reactions
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