Design, synthesis, and biological evaluation of 14-substituted aromathecins as topoisomerase I inhibitors

Article abstract of DOI:10.1021/jm800259e

The aromathecin or "rosettacin" class of topoisomerase I (top1) inhibitors is effectively a "composite" of the natural products camptothecin and luotonin A and the synthetic indenoisoquinolines. The aromathecins have aroused considerable interest following the isolation and total synthesis of 22-hydroxyacuminatine, a rare cytotoxic natural product containing the 12H-5,11a-diazadibenzo[b,h]fluoren-11-one system. We have developed two novel syntheses of this system and prepared a series of 14-substituted aromathecins as novel antiproliferative topoisomerase I poisons. These inhibitors are proposed to act via an intercalation and "poisoning" mechanism identical to camptothecin and the indenoisoquinolines. Many of these compounds possess greater antiproliferative activity and anti-top 1 activity than the parent unsubstituted compound (rosettacin) and previously synthesized aromathecins, as well as greater top1 inhibitory activity than 22-hydroxyacuminatine. In addition to potentially aiding solubility and localization to the DNA-enzyme complex, nitrogenous substituents located at the 14-position of the aromathecin system have been proposed to project into the major groove of the top1-DNA complex and hydrogen-bond to major-groove amino acids, thereby stabilizing the ternary complex.

Full text of DOI:10.1021/jm800259e

J. Med. Chem. 2008, 51, 4609–4619
4609
Design, Synthesis, and Biological Evaluation of 14-Substituted Aromathecins as
Topoisomerase I Inhibitors
Maris A. Cinelli,† Andrew Morrell,† Thomas S. Dexheimer,‡ Evan S. Scher,‡ Yves Pommier,‡ and Mark Cushman*,†
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, and the Purdue Cancer
Center, Purdue UniVersity, West Lafayette, Indiana 47907, and Laboratory of Molecular Pharmacology, Center for Cancer Research,
National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255
ReceiVed March 10, 2008
The aromathecin or “rosettacin” class of topoisomerase I (top1) inhibitors is effectively a “composite” of
the natural products camptothecin and luotonin A and the synthetic indenoisoquinolines. The aromathecins
have aroused considerable interest following the isolation and total synthesis of 22-hydroxyacuminatine, a
rare cytotoxic natural product containing the 12H-5,11a-diazadibenzo[b,h]fluoren-11-one system. We have
developed two novel syntheses of this system and prepared a series of 14-substituted aromathecins as novel
antiproliferative topoisomerase I poisons. These inhibitors are proposed to act via an intercalation and
“poisoning” mechanism identical to camptothecin and the indenoisoquinolines. Many of these compounds
possess greater antiproliferative activity and anti-top1 activity than the parent unsubstituted compound
(rosettacin) and previously synthesized aromathecins, as well as greater top1 inhibitory activity than 22-
hydroxyacuminatine. In addition to potentially aiding solubility and localization to the DNA-enzyme complex,
nitrogenous substituents located at the 14-position of the aromathecin system have been proposed to project
into the major groove of the top1-DNA complex and hydrogen-bond to major-groove amino acids, thereby
stabilizing the ternary complex.
Introduction
The indenoisoquinolines (including 4 and 5), a class of
noncamptothecin top1 poisons based on the lead compound 6
(NSC 314622),22 were developed as an alternative to the
camptothecins.22–25 Preclinical development of several inde-
noisoquinolines has recently begun.26 The success of the
camptothecins and the indenoisoquinolines has led to consid-
eration of other heterocyclic systems that might combine the
best features of both series.
Topoisomerase I (top1) is an enzyme that is crucial for DNA
replication and transcription. Through these normal cellular
processes, duplex DNA acquires a considerable degree of both
positive and negative supercoiling. Top1 solves the topological
problems supercoiling causes to allow efficient replication and
transcription. Mechanistically, top1 acts through a nucleophilic
tyrosine residue that nicks a single strand of the phosphodiester
backbone of DNA and allows a “controlled rotation” of the DNA
about the nonscissile strand, thus relaxing the double helix.1,2
Because top1 is preferentially expressed in the S-phase of
the cell cycle and has been found in high levels in several solid
human tumors,3,4 it has long been considered an attractive target
for the design of cancer chemotherapeutics.1,5–7 In 1966, Wall
and Wani isolated the cytotoxic alkaloid camptothecin (1) from
the Chinese tree Camptotheca acuminata.8 Camptothecin and
its semisynthetic analogues such as topotecan (2) and irinotecan
(3) inhibit top1 by intercalating into the DNA-enzyme complex.
The steric bulk of the inhibitors prevents top1’s religation of
the nicked DNA, thus “poisoning” the cleavage complex and
triggering apoptosis.1,9,10
The aromathecin27–30 class of top1 poisons, previously
described as stable hybrids of indenoisoquinolines and camp-
tothecins,27 also bear similarity to the natural product luotonin
A (7), a weaker top1 poison isolated from Peganum nigellas-
trum.31 Several series of modified and substituted luotonins have
been published, and some analogues have greater antiprolifera-
tive activity than the parent compound.32–34 22-Hydroxyacumi-
natine (8), a rare natural product isolated from Camptotheca
acuminata,28,35 contains the 12H-5,11a-diazadibenzo[b,h]fluo-
ren-11-one system, of which the unsubstituted core has been
named “rosettacin”30 (9), with substituted compounds being
named “aromathecins.”28,29 Because of the similarity in structure
and proposed mechanism of action of aromathecins to camp-
tothecins, luotonins, indenoisoquinolines, and 22-hydroxyacumi-
natine, the aromathecin system is more accurately described as
a composite of many of these heteroaromatic structures.29
Representative top1 inhibitors, including the relevant indenoiso-
quinolines 4-6, are shown in Figure 1, while the clinically
useful camptothecin derivatives irinotecan and topotecan are
shown in Figure 2.
Effortstoimprovethesolubilityandpotencyofcamptothecin5,11–18
have provided 2 and 3,1,5,11,12 the only FDA-approved top1
inhibitors for the treatment of cancer. Despite the clinical success
of these compounds, camptothecin derivatives still suffer from
poor solubility, reversibility of cleavage-complex formation,
and dose-limiting toxicity.1,3,12,19 Another flaw of the camp-
tothecins is in the E-ring lactone, which exists in equilibrium
with its ring-open, hydroxycarboxylate form in vivo.1,3 While
the hydroxyacid form retains some of its potency, it possesses
a high affinity for human serum albumin.20,21
Initial efforts to develop aromathecins focused on the
synthesis of 9 and 10.27 Unfortunately, these compounds were
weak top1 poisons and displayed poor growth inhibition.27 We
have discovered in the present study that substitution of the 14-
position of aromathecins with amines, amino alcohols, and
nitrogenous heterocycles confers both improved antiproliferative
potency and top1 inhibition over both 9 and aromathecin 10
* To whom correspondence should be addressed. Phone: 765-494-1465.
Fax: 765-494-6790. E-mail: cushman@pharmacy.purdue.edu.
†
Purdue University.
National Cancer Institute, NIH.
‡
10.1021/jm800259e CCC: $40.75
2008 American Chemical Society
Published on Web 07/17/2008

4610 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Cinelli et al.
Figure 3. Ligand overlay of aromathecin 27d (green) and indenoiso-
quinoline 4 (magenta). The positions of the lactam nitrogen and 14-
position are indicated in their respective colors. The carboxylate group
of 4 is perpendicular to the plane of the ring system.
DNA-top1 complex. These studies are consistent with the
solved crystal structures of indenoisoquinolines in ternary
complex with DNA and top1.10 Hypothetically, these substit-
uents hydrogen-bond with water and major-groove amino acids
and increase the stability of the ternary complex. A ligand
overlay of the crystal structure of the indenoisoquinoline
(4)-top1-DNA ternary complex10 and a hypothetical model
of the aromathecin 27d-top1-DNA ternary complex, showing
this substituent overlap, is displayed in Figure 3. On the basis
of this hypothesis, mono- and trimethylene analogues 27a-k
and 28a-g were designed and synthesized.
Figure 1. Representative top1 inhibitors.
Chemistry
Several routes to aromathecin derivatives have been devel-
oped. 22-Hydroxyacuminatine (8), rosettacin (9), and compound
10 were previously synthesized in our laboratory via the
condensation of a pyrroloquinoline with appropriately substituted
phthalide derivatives.27,28 Notable routes to 22-hydroxyacumi-
natine and the 12H-5,11a-diazadibenzo[b,h]fluoren-11-one sys-
tem include pyridone benzannulation and Heck coupling38 and
aza-Diels-Alder reactions.39 Recently, Pin et al. have published
a novel route to rosettacin and 14-methyl- and 14-phenylaro-
mathecin, employing an N-amidoacylation/aldol condensation
with a benzotriazole ester to form the key intermediate.29
14-Substituted aromathecins 27a-k and 28a-g were pre-
pared from oxatricyclic ketone 23 (Scheme 1). Ketone 23 was
previously prepared in Shamma and Novak’s attempted syn-
thesis of camptothecin, which was also the first reported
synthesis of rosettacin.36 This compound was prepared by two
new routes, both beginning with commercially available amino
acids. The first route is outlined in Scheme 1. Beginning with
the ethyl carbamate 12 of methyl glycinate (11), 3-pyrrolidinone
ethylene ketal (15) was prepared via a one-pot Michael
addition-Dieckmann condensation and decarboxylation, fol-
lowed by ketalization of carbamate 13 to yield 14.40–42 Removal
of the carbamate functionality and reaction with chlorophthalide
17, readily prepared from 2-carboxybenzaldehyde (16),43 yielded
ketal 18. Ketal 18 was cyclized directly to 23 using a
combination of polyphosphoric acid and 85% phosphoric acid,
following the final step of Shamma and Novak’s work.
Figure 2. Structures of irinotecan and topotecan.
and confers improved top1 inhibitory activity over 22-hydroxy-
acuminatine (8).
As reported in the present communication, a new series of
14-substituted aromathecins have been synthesized via two novel
routes that proceed through the known tricyclic ketone 23,36
building on structure-activity relationships established for both
camptothecins and indenoisoquinolines. Substitution of the
analogous 7-position of camptothecin with hydrogen bond
donor-acceptor groups capable of increasing solubility im-
proves biological activity over the parent compound.3,14,16,17 For
indenoisoquinolines, groups such as amino, imidazole, mor-
pholine, and N,N-dimethylamine, located on the lactam nitrogen
at a distance of 2-3 methylene units from the aromatic core,
confer superior top1 inhibition and antiproliferative activity.23,37
In addition to likely solubilizing the aromatic core, molecular
modeling studies indicate that substituents at the 14-position
of the aromathecin core and camptothecin’s 7-position occupy
the same region in space as the lactam substituents of inde-
noisoquinolines, projecting out into the major groove of the
Because of variable yields and loss of material associated
with protection-deprotection steps, the more elegant route
depicted in Scheme 2 was developed as an alternative, protecting
group-free pathway to the key intermediate. Preparation of 23
began with catalytic decarboxylation of commercially available
trans-4-hydroxy-L-proline (19) to yield the amino alcohol 20

Substituted Aromathecins as Top1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4611
Scheme 3a
Scheme 1a
a Reagents and conditions: (a) (i) BCl3 ·Me2S, 1,2,-dichloroethane, 0 °C,
(ii) chloroacetonitrile (for synthesis of 25), 4-chlorobutyronitrile (for
synthesis of 26), AlCl3, reflux; (iii) 2 M HCl, reflux; (b) p-TsOH, benzene,
reflux.
a Reagents and conditions: (a) EtOCOCl, CHCl3, reflux; (b) (i) NaH,
benzene, reflux, (ii) methyl acrylate, reflux, (iii) 0 6 M HCl, reflux; (c)
ethylene glycol, cat. p-TsOH, benzene, reflux; (d) KOH, H2O, reflux; (e)
cat. FeCl3, SOCl2, reflux; (f) THF, Et3N, room temp; (g) polyphosphoric
acid, 85% phosphoric acid, CH2Cl2, 100 °C.
substitution of 27a with imidazole to provide 27d required
higher temperatures, displacement by the remaining amines
at room temperature readily afforded analogues 27e-k
(Scheme 4).
Scheme 2a
Amine 28c was prepared from azide 28b using the Staudinger
methodology as described above (Scheme 5). Because of the
decreased electrophilicity of the terminal chloride of 28a,
increased reaction temperatures were required for substitution.
Additionally, substitution with the desired amines required
adding sodium iodide along with excess amine. The in situ
Finkelstein reaction, followed by displacement of the resulting
iodides with the required amines, yielded analogues 28d-g,
which were isolated as their trifluoroacetate salts.
Biological Results and Discussion
All aromathecin analogues were assayed for antiproliferative
activity in the National Cancer Institute’s Developmental
Therapeutics screen. Each compound was evaluated against
approximately 60 cell lines originating from various human
tumors.47,48 After an initial one-dose assay, selected compounds
were tested at five concentrations encompassing the range from
10-8 to 10-4 M. Results are reported as GI50 values for selected
cell lines from each subpanel, and overall antiproliferative effects
are quantified as a mid-graph midpoint (MGM) in Table 1. The
MGM is a measure of the average GI50 against all cell lines
tested. For completeness and comparison, the activities of
camptothecin (1), indenoisoquinolines 51,23,49 and 6,22 rosettacin
(9), and dimethoxyaromathecin (10),27 in addition to the top1
inhibitory activity of 22-hydroxyacuminatine (8), are reported.
Top1 inhibition was assayed by measurement of top1-
mediated DNA cleavage, and inhibition data are expressed
semiquantitatively as follows: 0, no inhibitory activity; +,
between 20% and 50% the activity of 1 µM camptothecin (1);
++, between 50% and 75% the activity of 1 µM camptothecin;
+++, between 75-100% the activity of 1 µM camptothecin;
and ++++, equipotent to or more potent than 1 µM camp-
tothecin. Top1 inhibitory data for aromathecins and comparative
compounds are also included in Table 1.
a Reagents and conditions: (a) (i) cat. 2-cyclohexen-1-one, cyclohexanol,
reflux, (ii) maleic acid, EtOAc, room temp; (b) MeOH, Et3N, room temp;
(c) PDC, CH2Cl2, reflux; (d) polyphosphoric acid, CHCl3, reflux.
as its hydrogen maleate salt.44 Condensation of 20 with 17
provided hydroxyamide 21, which gave a mixture of the
oxidation products 22 and 23 upon reaction with pyridinium
dichromate. This mixture was completely cyclized to 23 by
treatment with polyphosphoric acid. Although this step is
modest-yielding (30-45%), it provided 23 in high purity.
14-Chloromethylaromathecin 27a and 14-chloropropylaro-
mathecin 28a were prepared by Friedlander condensation of 23
with aminoacetophenone 25 and aminobutyrophenone 26,
respectively (Scheme 3). These ketones were prepared from
aniline (24) and chloroacetonitrile or 4-chlorobutyronitrile via
the aminohaloborane modification of the Friedel-Crafts acy-
lation, as reported by Sugasawa et al.45,46 It is anticipated that
the synthesis of future aromathecin derivatives can become, in
essence, modular, enabling access to numerous substituted
aromathecins through 23 and various substituted acetophenones.
The benzylic chloride of intermediate 27a is easily substituted
by a variety of nucleophiles in DMSO. Displacement of the
chloride by sodium azide yielded 27b, which was readily
converted to amine 27c by Staudinger reduction. Although
Compounds 27b, 27k, and 28a,b are inactive against top1.
Nonetheless, compounds 27b, 27k, and 28a were tested in the
National Cancer Institute’s 60-cell line screen at an initial high
dose (10 µM) and were not selected for further testing because
of their low activities. Compounds 27f, 27k, and 28g, although
not selected for five-dose testing, induced 14%, 25.5%, and

4612 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Cinelli et al.
Scheme 4a
donor–acceptor interactions improves top1 inhibitory activity
relative to 9 and the 14-unsubstituted dimethoxyaromathecin
10. In addition, most aromathecins are better top1 inhibitors
than 8 (a one “+” inhibitor). Figure 4 indicates the presence of
top1-mediated DNA breaks induced by aromathecins 27a,d,g,i
and 28d,f. Interestingly, the cleavage patterns resemble both
those induced by camptothecins and by indenoisoquinolines.
The most active aromathecin (28f) displays a predominant top1
cleavage site at position 62 (Figure 4, lanes 27-30), also
observed for the identically substituted indenoisoquinoline 5.23,49
MGM values were improved over 9 and 10 for the majority
of 14-substituents tested. These groups vary considerably in size
and conformational flexibility, indicating a moderate tolerance
by the ternary complex at this position. Especially effective at
improving anti-top1 activity in the aminomethylene series is
the imidazolyl moiety of 27d. The chiral hydroxypyrrolidinyl
group of 27i, a group not previously investigated in the
development of camptothecins or indenoisoquinolines, also
conferred increased anti-top1 activity relative to rosettacin.
It is difficult to compare activity of aromathecins to com-
pounds other than 9 and 10, however. Luotonin A (7) was not
tested in the National Cancer Institute assay but has displayed
top1 inhibitory activity31,32,34 and antiproliferative activity
against the human lung carcinoma line H460.31 22-Hydroxy-
acuminatine (8) was also not tested in the National Cancer
Institute assay, although previous studies report activity against
murine leukemia KB and P388 cell lines.35,39 However, it was
determined in 2006 that 22-hydroxyacuminatine’s cytotoxicity
did not appear to be top1 dependent.28
a Reagents and conditions: (a) amine or amine salt + Et3N, DMSO, room
temp, 62-100 °C (27d); (b) NaN3, DMSO; (c) (i) (EtO3)P, benzene, reflux,
(ii) 3 M HCl, MeOH, reflux.
Scheme 5a
The improved top1 inhibitory activity and antiproliferative
potency of 14-substituted aromathecins over the parent com-
pound may be due in part to improved solubility as the
substituents at the 7-position of camptothecin and the substit-
uents of irinotecan and topotecan greatly enhance activity
through solubilizing the aromatic core.1,3,14,16,17 For the aro-
mathecins, this hypothesis may be corroborated in part by the
inactivity of compounds 27b and 28a,b. No definite correlation
between growth inhibition, top1 inhibition, or cLogP has been
observed for the aromathecin class, although it has been
observed for certain indenoisoquinolines.37 Compound 27e,
which has a higher cLogP (3.67) than 9 (3.37), inhibits cell
growth nearly 10 times as well and is also a more potent top1
inhibitor. Conversely, compound 27k has a lower cLogP (3.32)
but is inactive against top1 compared to rosettacin 9.
It has also been hypothesized1,5,10,23 that side chains of top1
inhibitors that project toward the major groove of the
DNA-enzyme ternary complex (as seen in Figure 3) may aid
in stabilization through hydrogen bonding interactions with
water or top1 amino acids found in the major groove. We have
previously published a hypothetical model of 9 merged into a
DNA-camptothecin crystal structure, hypothesizing that 9
intercalated in a manner similar to camptothecin.27 Figure 5
shows a hypothetical model of 27d merged into the DNA-top1
crystal structure. The construction of this model was aided by
the availability of the crystal structures of camptothecin (1) and
indenoisoquinolines in ternary complex with top1 and DNA10
(see Experimental Section for modeling details). The aromatic
core of compound 27d is calculated to intercalate between the
base pairs without obvious steric hindrance and is likely
stabilized by π-stacking interactions. The imidazolyl group
projects on the outer range of H bonding distance with Asn352
(heavy-atom distance of 3.77 Å). It appears the imidazolyl group
may be able to rotate somewhat to make this contact. Other
models indicate the monomethylene analogues make hydrogen
a Reagents and conditions: (a) amine, NaI, DMSO, 100 °C; (b) NaN3,
DMSO, 100 °C; (c) (i) (EtO3)P, benzene, reflux, (ii) 3 M HCl, MeOH,
reflux.
21.4% average growth inhibition, respectively, in the presence
of the inhibitor at a concentration of 10 µM across all cell lines
tested.
In general, among the monomethylated aromathecin series
27a-j, substitution at the 14-position of the aromathecin “core”
with solubilizing groups capable of forming hydrogen bond

Substituted Aromathecins as Top1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4613
Table 1. Antiproliferative and Topoisomerase I Inhibitory Activities of 14-Substituted Aromathecin Analogues
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
top 1
compd
MGMb
cleavagec
1
5
0.01
0.02
1.3
0.03
0.10
35
0.01
0.04
41
0.01
0.03
4.2
0.22
0.5
73
0.02
>0.01
68
0.01
>0.01
37
0.04
0.79
96
0.0405
0.11
20.0
++++
++++
++
6
8d
+
9
10
68.2
>100
26.5
9.8
10.0
>100
32.7
57.3
43.4
1.5
13.2
1.9
66.7
>100
7.4
97.2
>100
>100
11.7
3.4
>100
39.8
>100
>100
2.9
>100
>100
>100
2.9
21.5
>100
>100
>100
>100
2.8
41.8
>100
>100
9.4
58.9
91.2
40.7
3.9
12.6
6.2
++
+
+
27a
27c
27d
27e
27fe
27g
27h
27i
27j
28cf
28d
28e
28f
28ge
4.9
4.7
1.8
++
17.0
11.5
4.9
>100
+++
++(+)
++
2.1
3.5
>100
28.6
3.6
3.9
>100
5.4
2.6
>100
2.7
6.3
>100
5.5
2.7
>100
16.9
2.8
>100
7.0
>100
>100
10.3
7.0
5.2
46.8
7.6
++(+)
++(+)
++(+)
++
+
+++
++
9.0
3.2
4.7
3.8
6.4
4.4
3.5
3.2
3.5
2.0
5.0
1.2
1.6
3.9
1.6
0.74
3.5
12.9
1.5
1.4
1.8
5.0
10.6
6.3
4.5
5.9
4.1
3.3
20.3
13.2
4.2
>100
1.7
1.6
5.9
2.8
++++
+++
a 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, ranging from 10-8 to 10-4 M. c Compound-induced DNA cleavage due to top1 inhibition is graded by the following rubric relative to
1 µM camptothecin: 0, no inhibitory activity; +, between 20% and 50% activity; ++, between 50% and 75% activity; +++, between 75% and 95% of
activity; ++++, equipotent. d 22-Hydroxyacuminatine was not tested in the National Cancer Institute assay. e These compounds were not selected for
further testing; refer to text for details. f Currently undergoing five-dose testing.
Figure 4. Top1-mediated DNA cleavage induced by aromathecins 27a,d,g,i, 28d, and 28f: (lane 1) AG; (lane 2) DNA alone; (lane 3) top1 alone;
(lane 4) camptothecin (1), 1 µM; (lane 5) 5, 1 µM; (lane 6) rosettacin (9), 100 µM; (lanes 7-30) (for compounds 27a,d,g,i, 28d, and 28f) top1 +
indicated compound at 0.1, 1, 10, and 100 µM, respectively.
bonding contacts with Asn352, Thr747, and the carbonyl of
Ile427 (structures not shown). The pyridine nitrogen of the
aromathecin core faces the minor groove, where it appears to
interact with Arg364, identical to the camptothecin class of top1
inhibitors. The presence of a lone pair of electrons at this
position has also proven to be critical for many classes of top1
inhibitors, such as indenoisoquinolines and indolocarbazoles.10
To further probe the characteristics of the 14-position and
investigate interactions deeper within the major groove, ana-
logues 28a-g were prepared. It was demonstrated in several
studies of indenoisoquinolines that approximately three meth-
ylene units between the aromatic core and a “distal” functionality
were ideal for biological activity.23,37 Unfortunately, the results
of this modification proved rather variable for the aromathecins
with regard to top1 inhibition and antiproliferative activity. A
notable exception is compound 28f, which is the most potent
aromathecin top1 inhibitor synthesized to this date. It is not
known why this substituent confers such potent activity,
although certain amino alcohol substitutions conferred similar
activity upon indenoisoquinolines.50 It is also possible that 27f

4614 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Cinelli et al.
Figure 5. Hypothetical model for the binding of aromathecin 27d in the ternary complex of DNA, top1, and the inhibitor. The diagram is programmed
for wall-eyed (relaxed) viewing.
Infrared spectra were obtained as films on salt plates using CHCl3
as the solvent except where otherwise specified, using a Perkin-
Elmer Spectrum One FT-IR spectrometer, and are baseline-
corrected. 1H NMR spectra were obtained at 300 or 500 MHz, using
a Bruker ARX300 and Bruker Avance 500 (TXI 5 mm probe),
respectively. Mass spectral analyses were performed at the Purdue
University Campus-Wide Mass Spectrometry Center. ESIMS was
performed using a FinniganMAT LCQ Classic mass spectrometer
system. EI/CIMS was performed using a Hewlett-Packard Engine
or GCQ FinniganMAT mass spectrometer system. Combustion
microanalyses were performed at the Purdue University Mi-
croanalysis Laboratory using a Perkin-Elmer Series II CHNS/O
model 2400 analyzer; reported values are within 0.4% of calculated
values. Analytical thin-layer chromatography was performed on
Baker-flex silica gel IB2-F plastic-backed TLC plates. Preparative
thin-layer chromatography was performed on Analtech silica gel
1500 µm glass plates. Compounds were visualized with both short-
and long-wavelength UV light. Silica gel flash chromatography was
performed using 40-63 µm flash silica gel.
acts in a manner similar to indenoisoquinoline 5, which bears
an identical side chain.23,49
It is worth noting the inconsistencies observed between
antiproliferative activity and top1 inhibition with certain aro-
mathecin analogues. Compound 27a, despite its poor anti-top1
activity, has greater antiproliferative potency than rosettacin (9).
In addition, preliminary assays indicate intense cytotoxicity for
compound 28c (-47.5% cell growth in the presence of 10 µM
inhibitor, indicating a net cell kill). Clearly, the antiproliferative
activity of these two compounds is not due to inhibition of top1.
It is unknown how these compounds exert their cytotoxic effect.
Perhaps the mechanism is similar to that originally observed
with 8. Also, differences in compound metabolism and uptake
by cells have previously been proposed for certain indenoiso-
quinolines showing similar disparities.23
A
formal
COMPARE48,51,52 analysis, as was performed for 6,22 will be
performed on these “targetless” aromathecins pending further
data.
Methyl N-Ethoxycarbonylglycinate (12).40 The hydrochloride salt
of methyl glycinate (11) (12.6 g, 0.1 mol) was diluted with CHCl3
(200 mL). Triethylamine (25.26 g, 0.250 mol) was added, and the
reaction mixture was cooled to 0 °C. Ethyl chloroformate (22.21
g, 0.205 mol) was then added, and the mixture was heated at reflux
for 24 h. The solution was washed sequentially with H2O (2 ×
150 mL), 10% aqueous HCl (100 mL), and saturated NaCl (100
mL), dried over anhydrous sodium sulfate, and concentrated to
provide an orange oil (14.8 g, 92%). 1H NMR (300 MHz, CDCl3)
δ 5.15 (bs, 1 H), 4.17 (q, J ) 7.1 Hz, 2 H), 3.98 (d, J ) 5.6 Hz,
2 H), 3.76 (s, 3 H), 1.30 (t, J ) 7.1 Hz, 3 H).
For many classes of DNA-binding drugs, the addition of
amine substituents, especially polyamine substituents, facilitates
localization to DNA in addition to aiding solubility. These
nitrogenous substituents are protonated at physiological pH and
increase the drugs’ Coulombic attraction to the negatively
charged DNA.50,53,54
Conclusion
In conclusion, two novel synthetic routes to aromathecin
analogues have been developed from inexpensive, commercially
available precursors. Two series of 14-substituted aromathecins
have been prepared via these routes, bearing substituents
separated from the aromatic core by short “linker” regions.
These novel “composite” structures of camptothecin, luotonins,
and indenoisoquinolines have been evaluated against human
top1 and numerous human tumor cell lines with promising
results. These results establish that 14-substitution, on a whole,
serves to improve top1 inhibitory and antiproliferative potency.
This effect is likely a combination of increased solubility (as
seen with 7-substituted camptothecins), charge complementarity
with DNA, and hydrogen bonding (as proposed for indenoiso-
quinolines). Although increasing side chain length to that
optimal for indenoisoquinolines did result in the discovery of a
top1 inhibitor equipotent to camptothecin, the results of side
chain elongation were largely variable with respect to both top1
inhibition and antiproliferative activity. Nonetheless, these data
have served to reinvigorate interest in a class of compounds
previously thought to be inactive and have since become the
impetus for developing further favorable 14-substitutions.
N-Ethoxycarbonyl-3-pyrrolidinone (13).41,42 Compound 12 (8.350
g, 29.59 mmol) was diluted with benzene (50 mL). Sodium hydride
(1.420 g, 59.18 mmol) was added, and the mixture was heated at
reflux for 30 min. Methyl acrylate (3.057 g, 35.51 mmol) was added,
and the reaction mixture was heated at reflux for 8 h and then
allowed to stir at room temperature for 16 h. Then 6 M HCl (30
mL) was added, and the reaction mixture was heated at reflux for
16 h. The reaction mixture was allowed to cool to room temperature,
and the aqueous and organic phases were separated. The aqueous
phase was extracted with EtOAc (3 × 30 mL), and the combined
organic layer was washed with saturated NaHCO3 (3 × 30 mL)
and saturated NaCl (30 mL). The organic layer was dried over
sodium sulfate and concentrated to provide a yellow oil (3.089 g,
73%). 1H NMR (300 MHz, CDCl3) δ 4.39-4.01 (m, 2 H),
3.82-3.78 (m, 4 H), 2.60 (t, J ) 7.8 Hz, 2 H), 1.27 (q, J ) 7.2
Hz, 3 H); CIMS m/z (rel intensity) 158 (MH+, 100).
N-Ethoxycarbonyl-3-pyrrolidinone Ethylene Ketal (14).41 Com-
pound 13 (2.858 g, 18.18 mmol) was diluted with benzene (50 mL).
Ethylene glycol (2.257 g, 36.37 mmol) was added, followed by
p-TsOH (0.346 g, 1.82 mmol). A Dean-Stark trap was affixed,
and the reaction mixture was heated at reflux for 20 h, allowed to
cool to room temperature, and washed with water (3 × 25 mL)
and saturated NaCl (25 mL). The organic layer was dried over
sodium sulfate and concentrated to provide a yellow oil (3.108 g,
Experimental Section
General Procedures. Melting points were determined in capil-
lary tubes using a Mel-Temp apparatus and are not corrected.
1
81%). H NMR (CDCl3) δ 4.14 (q, J ) 7.1 Hz, 2 H), 3.99-3.94

Substituted Aromathecins as Top1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4615
(m, 4 H), 3.56 (q, J ) 7.5 Hz, 2 H), 3.43 (d, J ) 7.6 Hz, 2 H),
2.05-2.00 (m, 2 H), 1.28 (t, J ) 7.1 Hz, 3 H).
2,3-Dihydropyrrolo[1,2-b]isoquinoline-1,5-dione (23, Method
2).36 Pyridinium dichromate (5.147 g, 13.68 mmol) was diluted with
anhydrous CH2Cl2 (30 mL) under an argon atmosphere. A solution
of 21 (2.00 g, 9.12 mmol) in anhydrous CH2Cl2 (15 mL) was added,
and the mixture was heated at reflux for 19.5 h. The mixture was
cooled and filtered, and the dark-brown filter cake was washed with
CHCl3 (4 × 30 mL). The filtrate was filtered through a pad of Celite,
and the pad was washed with CHCl3 (3 × 30 mL). The filtrate was
concentrated to yield a dark-brown oil that was then diluted with
CHCl3 (40 mL). Polyphosphoric acid (6.15 g) was added, and the
mixture was heated at reflux for 2 h and 10 min. The mixture was
cooled and poured into ice-water (100 mL). The residue in the
flask was stirred with ice-water (3 × 40 mL). The aqueous mixture
was extracted with CHCl3 (5 × 100, 1 × 50 mL). The organic
layers were washed with saturated NaCl (250 mL), dried over
anhydrous sodium sulfate, adsorbed onto SiO2 (7.273 g), and
purified by flash column chromatography (SiO2), eluting with 1%
MeOH in CHCl3. The solvent was evaporated and the resulting
solid was recrystallized from boiling EtOH (20 mL) to yield an
iridescent orange solid (0.590 g, 33%): mp 180-184 °C (lit.36 mp
191-192 °C). The 1H NMR spectrum was identical to compound
23 prepared by method 1 above.
3-Pyrrolidinone Ethylene Ketal (15).36 Compound 14 (3.108 g,
14.71 mmol) was diluted with water (25 mL). Potassium hydroxide
(2.800 g, 49.91 mmol) was added, and the solution was heated at
reflux for 16 h. The reaction mixture was allowed to cool to room
temperature and extracted with CH2Cl2 (4 × 30 mL), and the
combined organic layer was washed with saturated NaCl (30 mL).
The organic layer was dried over sodium sulfate and concentrated
to provide a light-yellow oil (0.887 g, 44%). 1H NMR (300 MHz,
CDCl3) δ 3.97-3.86 (m, 4 H), 3.07 (t, J ) 7.2 Hz, 2 H), 2.89 (s,
2 H), 2.15 (s, 1 H), 1.98 (t, J ) 7.4 Hz, 2 H).
3-Chloro-1(3H)-isobenzofuranone (17).43 2-Carboxybenzalde-
hyde (16, 10.00 g, 66.61 mmol) and ferric chloride (0.030 g, 0.185
mmol) were diluted with thionyl chloride (25 mL) and the mixture
was heated at reflux for 1 h. The reaction mixture was allowed to
cool to room temperature and concentrated to provide a brown oil.
The oil was diluted with hexanes (20 mL) and concentrated to
provide a brown solid. The solid was extracted with boiling hexanes
(5 × 50 mL), and concentration of the extract provided a white
solid (10.96 g, 98%): mp 52-56 °C (lit.43 mp 57-59 °C). 1H NMR
(300 MHz, CDCl3) δ 7.95-7.93 (m, 1 H), 7.82 (t, J ) 7.4 Hz, 1
H), 7.68-7.60 (m, 2 H), 7.10 (s, 1 H).
2,3-Dihydropyrrolo[1,2-b]isoquinoline-1,5-dione (23, Method
1).36 Compound 15 (2.167 g, 15.69 mmol) was diluted with THF
(30 mL) and triethylamine (10 mL). Compound 17 was then added,
and the solution was allowed to stir at room temperature for 30
min. The reaction mixture was concentrated, diluted with water
(50 mL), and extracted with CHCl3 (4 × 40 mL). The combined
organic layers were washed with saturated NaCl (40 mL) and dried
over sodium sulfate. The solution was filtered and concentrated to
provide compound 18, which was used without further purification
in the next step. Compound 18 was diluted with polyphosphoric
acid (10.00 g), dichloromethane (5 mL), and phosphoric acid (85%,
5 mL). The reaction mixture was heated at 100 °C for 3 h and then
allowed to cool to room temperature. The reaction mixture was
diluted with ice-water (100 mL) and extracted with CHCl3 (7 ×
100 mL). The combined organic layer was washed with saturated
NaCl (100 mL), dried over sodium sulfate, and concentrated to
provide the product 23 as an orange-brown solid (2.117 g, 88%).
1H NMR (300 MHz, CDCl3) δ 8.53 (d, J ) 7.9 Hz, 1 H), 7.79-7.64
(m, 3 H), 7.27 (s, 1 H), 4.42 (t, J ) 6.9 Hz, 2 H), 7.98 (t, J ) 7.3
Hz, 2 H).
2-Amino-r-chloroacetophenone (25).45 Boron trichloride-methyl
sulfide complex (1.059 g, 5.906 mmol) was diluted with dichlo-
roethane (15 mL) and cooled to 0 °C. Aniline (24, 0.500 g, 5.369
mmol) was added dropwise, and the solution was allowed to stir at
0 °C for 10 min. Chloroacetonitrile (0.507 g, 6.711 mmol) was
added, followed by aluminum chloride (0.787 g, 5.906 mmol), and
the solution was allowed to gradually warm to room temperature.
After 10 min, the reaction mixture was heated at reflux for 3 h.
The solution was allowed to cool to room temperature, 2 M HCl
(15 mL) was added, and the reaction mixture was heated at reflux
for 30 min. The reaction mixture was diluted with water (20 mL)
and extracted with CH2Cl2 (3 × 20 mL). The combined organic
layers were washed with saturated NaCl (25 mL) and dried over
sodium sulfate. Concentration provided a yellow solid (0.194 g,
21%) that was isolated by washing with hexanes: mp 106-109 °C
1
(lit.45 mp 112-113 °C). H NMR (300 MHz, CDCl3) δ 7.65 (dd,
J ) 8.2 and 1.4 Hz, 1 H), 7.34-7.28 (m, 1 H), 6.72-6.64 (m, 2
H), 4.70 (s, 2 H).
1-(o-Aminophenyl)-4-chloro-1-butanone (26).46 Boron tri-
chloride-methyl sulfide complex (6.353 g, 35.43 mmol) was diluted
with dichloroethane (70 mL) and cooled to 0 °C. Aniline (24, 3.000
g, 32.21 mmol) was added dropwise, and the solution was allowed
to stir at 0 °C for 10 min. 4-Chlorobutyronitrile (4.170 g, 40.27
mmol) was added, followed by aluminum chloride (4.724 g, 35.43
mmol), and the solution was allowed to gradually warm to room
temperature. After 10 min, the reaction mixture was heated at reflux
for 2.5 h. The solution was allowed to cool to room temperature,
10% aqueous HCl (70 mL) was added, and the reaction mixture
was heated at reflux for 30 min. The reaction mixture was allowed
to stir at room temperature for 24 h, and the organic layer was
separated. The aqueous layer was extracted with CH2Cl2 (3 × 50
mL). The combined organic layers were washed with saturated NaCl
(50 mL) and dried over sodium sulfate. Concentration provided a
crude yellow-brown oil that was purified by flash column chroma-
tography (SiO2), eluting with a gradient of hexanes to 50%
EtOAc-hexanes. The solvent was evaporated, and the resulting
product was diluted with Et2O (50 mL) and treated with 3 M HCl
in MeOH (10 mL) and allowed to stir at room temperature for 20
min. The salt was filtered and washed with Et2O (50 mL) to provide
a white solid. The solid was dissolved in saturated NaHCO3 (150
mL) and extracted with CHCl3 (3 × 50 mL). The combined organic
layers were washed with saturated NaCl (50 mL), dried over sodium
sulfate, filtered, and concentrated to provide a yellow oil (2.022 g,
(S)-Pyrrolidin-3-ol Hydrogen Maleate (20).44 trans-4-Hydroxy-
L-proline (19) (10.00 g, 76.26 mmol) was diluted with cyclohexanol
(60 mL). 2-Cyclohexen-1-one (1.0 mL) was added, and the mixture
was heated at reflux for 3 h. After the dark red solution was cooled
to room temperature, maleic acid (8.950 g, 77.09 mmol) was added
portionwise over a 30 min period, keeping the internal temperature
below 35 °C. Ethyl acetate (140 mL) was then added dropwise
over 1 h to precipitate a pale-orange amorphous solid (12.46 g,
80%) after filtration: mp 83-86 °C (lit.44 mp 90-91 °C). 1H NMR
(300 MHz, CD3OD) δ 6.25 (s, 2 H), 4.60-4.50 (m, 1 H), 3.30-3.29
(bm, 2 H), 3.22-3.10 (bm, 2 H), 2.10-2.00 (m, 2H).
N-(o-Formylbenzoyl)-(S)-pyrrolidin-3-ol (21). Compound 20
(3.000 g, 14.62 mmol) was diluted with MeOH (30 mL) and Et3N
(13 mL). Compound 17 (2.096 g, 12.43 mmol) was added, and the
mixture was stirred overnight at room temperature for 22 h. The
solution was concentrated, and the brown residue dissolved in H2O
(20 mL) and extracted with CHCl3 (4 × 50 mL). The organic layers
were dried over anhydrous sodium sulfate and concentrated, and
the resulting brown oil was flash chromatographed (SiO2), eluting
with 20:1 CH2Cl2-MeOH, to afford an orange-yellow amorphous
solid (2.160 g, 79%): mp 71-73 °C. IR (film) 3379, 2947, 2886,
1
1698, 1613, 1597, 1439 cm-1; H NMR (300 MHz, CD3OD) δ
1
10.0 (s, 1 H), 8.01 (d, J ) 7.7 Hz, 1 H), 7.75 (dt, J ) 12.0 and 6.7
Hz, 2 H), 7.49 (dt, J ) 6.4 and 1.3 Hz), 4.50 (m, 0.5 H), 4.35 (m,
0.5 H), 3.79-3.65 (m, 2 H), 3.31-3.00 (m, 2 H), 2.20-1.80 (m,
2 H); CIMS m/z (rel intensity) 220 (MH+, 100).
32%) that solidified upon standing: mp 51-55 °C. H NMR (300
MHz, CDCl3) δ 7.79 (dd, J ) 8.5 and 1.6 Hz, 1 H), 7.31-7.25
(m, 1 H), 6.70-6.65 (m, 2 H), 3.70 (t, J ) 6.3 Hz, 2 H), 3.18 (t,
J ) 7.1 Hz, 2 H), 2.25 (pent, J ) 6.9 Hz, 2 H).

4616 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Cinelli et al.
14-Chloromethyl-12H-5,11a-diazadibenzo[b,h]fluoren-11-one
(27a). Compound 23(0.176 g, 0.884 mmol) and compound 25 (0.150
g, 0.884 mmol) were diluted with benzene (100 mL). p-Toluene-
sulfonic acid monohydrate (0.168 g, 0.884 mmol) was added, and
the solution was heated at reflux for 24 h using a Dean-Stark trap
to collect the azeotroped water. The solution was concentrated,
diluted with NaHCO3 (150 mL), and extracted with CHCl3 (6 ×
100 mL) and saturated NaCl (100 mL). The organic layer was dried
over sodium sulfate, concentrated, and purified by flash column
chromatography (SiO2), eluting with a gradient of CHCl3 to 5%
MeOH in CHCl3, to provide a yellow solid (0.174 g, 59%) after
washing with MeOH: mp 270 °C (dec). IR (KBr) 1661, 1638, 761,
temperature for 19 h. The solution was diluted with CHCl3 (50
mL) and washed with H2O (4 × 40 mL) and saturated NaCl (50
mL). The organic layer was dried over anhydrous sodium sulfate
and concentrated, and the residue was purified by flash column
chromatography (SiO2), eluting with 7% MeOH in CHCl3 to yield
a pale-yellow amorphous solid (0.058 g, 65%) after washing with
hexanes: mp 204-207 °C. IR (film) 2932, 2789, 1661, 1633, 1604,
1
753, 687 cm-1; H NMR (300 MHz, CDCl3) δ 8.57 (d, J ) 8.4
Hz, 1 H), 8.36 (d, J ) 8.6 Hz, 1 H), 8.23 (d, J ) 7.9 Hz, 1 H),
7.78-7.57 (m, 5 H), 7.62 (s, 1 H), 5.47 (s, 2 H), 4.06 (s, 2 H),
2.61 (bs, 4 H), 2.47 (bs, 4 H), 2.29 (s, 3 H); ESIMS m/z (rel
intensity) 397 (MH+, 80), 297 (MH+ - C5H11N2, 100). Anal.
(C25H24N4O·0.6H2O) C, H, N.
1
754, and 688 cm-1; H NMR (300 MHz, CDCl3) δ 8.56 (d, J )
8.1 Hz, 1 H), 8.27 (d, J ) 7.6 Hz, 1 H), 8.19 (d, J ) 8.4 Hz, 1 H),
7.86-7.69 (m, 4 H), 7.65 (s, 1 H), 7.62-7.56 (m, 1 H), 5.44 (s, 2
H), 5.05 (s, 2 H); ESIMS m/z (rel intensity) 333/335 (MH+, 100/
35). Anal. (C20H13ClN2O) C, H, N.
14-(1-Morpholinomethyl)-2H-5,11a-diazadibenzo[b,h]fluoren-11-
one (27f). Compound 27a (0.064 g, 0.192 mmol) was diluted with
DMSO (25 mL), and morpholine (0.050 g, 0.576 mmol) was added.
The solution was stirred at room temperature for 20 h. The solution
was diluted with CHCl3 (40 mL) and then washed with H2O (4 ×
40 mL) and saturated NaCl (40 mL). The organic layer was dried
over anhydrous sodium sulfate and concentrated, and the resulting
residue was purified by flash column chromatography (SiO2), eluting
with EtOAc, which afforded a pale-yellow amorphous solid (0.056
g, 77%) after washing with hexanes: mp 242-244 °C (dec). IR
14-Azidomethyl-12H-5,11a-diazadibenzo[b,h]fluoren-11-one (27b).
Compound 27a (0.070 g, 0.210 mmol) and sodium azide (0.022 g,
0.346 mmol) were diluted with DMSO (20 mL), and the mixture
was stirred at room temperature for 19 h. The solution was diluted
with CHCl3 (30 mL), and more CHCl3 was then added until the
organic phase was clear. The organic layer was washed with H2O
(4 × 25 mL) and saturated NaCl (25 mL). The organic layer was
dried over anhydrous sodium sulfate and concentrated, and the
residue was washed and filtered with MeOH, redissolved in CHCl3,
and purified by flash column chromatography (SiO2), eluting with
CHCl3 to provide a pale-yellow amorphous solid (0.051 g, 71%)
after washing with MeOH: mp 210-212 °C (dec). IR (film) 2918,
(film) 2929, 1661, 1602, 756, 689 cm-1 1H NMR (300 MHZ,
;
CDCl3) δ 8.58 (d, J ) 8.0 Hz, 1 H), 8.39 (d, J ) 8.3 Hz, 1 H),
8.24 (d, J ) 8.5 Hz, 1 H), 7.82-7.58 (m, 5 H), 7.66 (s, 1 H), 5.27
(s, 2 H), 4.06 (s, 2 H), 3.73 (t, J ) 4.2 Hz, 4 H), 2.59 (t, J ) 4.3
Hz, 4 H); ESIMS m/z (rel intensity) 384 (MH+, 100). Anal.
(C24H21N3O2) C, H, N.
2102, 1665, 1639, 1605, 745, 685 cm-1 1H NMR (300 MHz,
;
14-(N-Ethanolaminomethyl)-2H-5,11a-diazadibenzo[b,h]fluoren-
11one (27g). Compound 27a (0.065 g, 0.195 mmol) was diluted
with DMSO (25 mL), and ethanolamine (0.048 g, 0.781 mmol)
was added. The mixture was stirred at room temperature for 20 h,
poured into CHCl3 (40 mL), and washed with H2O (4 × 40 mL).
A small amount of methanol was added to aid solubility. The
organic layers were dried over anhydrous sodium sulfate and
concentrated, and the residue was purified by flash column
chromatography (SiO2), eluting with a gradient of 6% MeOH in
CHCl3 to 9% MeOH in CHCl3, to provide a fine yellow powder
(0.029 g, 42%) after washing with hexanes: mp 198.5-204 °C
(dec). IR (film) 2925, 1656, 1618, 1599, 753, 689 cm-1; 1H NMR
(500 MHz, DMSO-d6) δ 8.38-8.31 (m, 2 H), 8.14 (d, J ) 8.1 Hz,
1 H), 7.97 (d, J ) 7.9 Hz, 1 H), 7.81 (q, J ) 7.2 Hz, 2 H),
7.69-7.56 (m, 3 H), 5.44 (s, 2 H), 4.57 (t, J ) 5.3 Hz, 1 H), 4.32
(s, 2 H), 3.54 (q, J ) 5.6 Hz, 2 H), 2.73 (t, J ) 5.7 Hz, 2 H);
ESIMS m/z (rel intensity) 358 (MH+, 100). Anal.
(C22H19N3O2 ·1.25H2O) C, H, N.
CDCl3) δ 8.58 (d, J ) 7.6 Hz, 1 H), 8.28 (d, J ) 8.2 Hz, 1 H),
8.11 (d, J ) 8.3 Hz, 1 H), 7.84-7.60 (m, 5 H), 7.67 (s, 1 H), 5.50
(s, 2 H), 5.01 (s, 2 H); ESIMS m/z (rel intensity) 340 (MH+, 100).
Anal. (C20H13N5O·0.6H2O) C, H, N.
14-Aminomethyl-12H-5,11a-diazadibenzo[b,h]fluoren-11-one Di-
hydrochloride (27c). Compound 27b (0.056 g, 0.165 mmol) was
diluted with benzene (20 mL), triethyl phosphite (0.069 g, 0.413
mmol) was added, and the solution was heated at reflux for 19 h.
The solution was cooled to room temperature, 3 M methanolic HCl
(10 mL) was added, and the dark-orange solution was heated at
reflux for 3 h. The precipitate was vacuum filtered to yield a bright
red flaky solid (0.046 g, 73%) after washing with MeOH: mp
278-284 °C (dec, salt), 265-269 °C (dec, free base). IR (film)
2920, 1656, 1633, 1597, 751, 690 cm-1 1H NMR (300 MHz,
;
CDCl3, CD3OD, Et3N) δ 8.43 (d, J ) 7.4 Hz, 1 H), 8.17 (t, J )
8.0 Hz, 2 H), 7.76-7.54 (m, 5 H), 7.70 (s, 1 H), 5.42 (s, 2 H),
4.37 (2 H); ESIMS m/z (rel intensity) 314 (MH+, 100). Anal.
(C20H17Cl2N3O), C, H, N.
14-(N,N-Dimethylaminomethyl)-2H-5,11a-diazadibenzo[b,h]fluo-
ren-11-one (27h). Compound 27a (0.050 g, 0.150 mmol) was diluted
with DMSO (25 mL), and N,N-diethylamine hydrochloride (0.035
g, 0.429 mmol) and Et3N (0.045 g, 0.445 mmol) were added. The
solution was stirred at room temperature for 20 h and then diluted
with CHCl3 (40 mL) and then washed with H2O (4 × 40 mL) and
saturated NaCl (40 mL). The organic layer was dried over
anhydrous sodium sulfate and concentrated. Flash column chro-
matography of the residue (SiO2), eluting with EtOAc, yielded a
very pale yellow amorphous solid (0.033 g, 64%) after washing
with hexanes: mp 201-202.5 °C. IR (film) 2942, 2819, 2768, 1661,
14-(1-Imidazolylmethyl)-2H-5,11a-diazadibenzo[b,h]fluoren-11-
one (27d). Compound 27a (0.085 g, 0.225 mmol) and imidazole
(0.052 g, 0.766 mmol) were diluted with DMSO (25 mL) and heated
to 62 °C for 3 h, and the resultant clear pale-orange solution was
subsequently stirred at room temperature for 15 h. TLC showed
incomplete reaction, so additional imidazole (0.255 mmol) was
added, and the solution was heated at 100 °C for 1 h. The solution
was diluted with CHCl3 (50 mL) and washed with H2O (4 × 40
mL). The organic layer was dried over anhydrous sodium sulfate
and concentrated, and the residue was purified by flash column
chromatography (SiO2), eluting with a gradient of CHCl3 to
CHCl3-4% MeOH, to yield a yellow amorphous solid (0.041 g,
44%) after washing with diethyl ether: mp 294-297 °C (dec). IR
1
1634, 1604, 753, 688 cm-1; H NMR (300 MHz, CDCl3,) δ 8.58
(d, J ) 8.1, 1 H), 8.37 (d, J ) 8.3 Hz, 1 H), 8.23 (d, J ) 8.9 Hz,
1 H), 7.80-7.58 (m, 5 H), 7.63 (s, 1 H), 5.45 (s, 2 H), 3.97 (s, 2
H), 2.35 (s, 6 H); ESIMS m/z (rel intensity) 345 (MH+, 100). Anal.
(C22H19N3O·0.3H2O) C, H, N.
(film) 1660, 1631, 1600, 762, 687 cm-1 1H NMR (300 MHz,
;
CDCl3) δ 8.52 (d, J ) 8.0 Hz, 1 H), 8.28 (d, J ) 8.4 Hz, 1 H),
8.02 (d, J ) 8.5 Hz, 1 H), 7.82-7.57 (m, 5 H), 7.65 (s, 1 H),
7.60-7.47 (m, 1 H), 7.09 (bs, 1 H), 6.90 (bs, 1 H), 5.67 (s, 2 H),
5.26 (s, 2 H); ESIMS m/z (rel intensity) 365 (MH+, 100). Anal.
(C23H16N4O·0.75H2O) C, H, N.
14-[1-(N-Methylpiperazinylmethyl)]-2H-5,11a-diazadibenzo[b,
h]fluoren-11-one (27e). Compound 27a (0.075 g 0.225 mmol) was
diluted with DMSO (25 mL), and N-methylpiperazine (0.068 g,
0.675 mmol) was added. The solution was stirred at room
14-[N-(S)-3-Hydroxypyrrolidinomethyl]-12H-5,11a diazadiben-
zo[b,h]fluoren-11-one (27i). Compound 27a (0.060 g, 0.180 mmol)
and compound 20 (0.111 g, 0.541 mmol) were diluted with DMSO
(25 mL), and Et3N (0.182 g, 1.80 mmol) was added. The solution
was stirred at room temperature for 19 h, diluted with CHCl3 (40
mL), and washed with H2O (4 × 30 mL). The organic layer was
dried over anhydrous sodium sulfate and concentrated, and the
residue was purified by flash column chromatography (SiO2), eluting

Substituted Aromathecins as Top1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4617
with a gradient of CHCl3 to 4% MeOH in CHCl3, to yield a
flocculent yellow solid (0.048 g, 69%) after washing with hexanes:
mp 220 °C (dec). IR (film) 2918, 2849, 1658, 1601, 1619, 1479,
with H2O (3 × 200 mL), dried over anhydrous sodium sulfate, and
concentrated to afford an off-white amorphous solid, which was
isolated by washing with ether and MeOH and purified by
preparative TLC (SiO2, CHCl3) to yield a pale-yellow amorphous
solid (0.062 g, 40%) after washing with ether: mp 185-188 °C (dec).
IR (film) 2918, 2108, 1659, 1626, 1604, 1506 1441, 1341, 1287, 1244,
1067, 687 cm-1; 1H NMR (300 MHz, CDCl3) δ 8.56 (d, J ) 7.9 Hz,
1 H), 8.24 (d, J ) 8.2 Hz, 1 H), 8.11 (d, J ) 8.3 Hz, 1 H), 7.79-7.58
(m, 6 H), 5.35 (s, 2 H), 3.50 (t, J ) 6.3 Hz, 2 H), 3.29 (t, J ) 7.7 Hz,
2 H), 2.20-2.00 (m, 2 H); ESIMS m/z (rel intensity), 368, (MH+,
100). Anal. (C22H17N5O·0.5H2O) C, H, N.
14-(3-Aminopropyl)-2H-5,11a-diazadibenzo[b,h]fluoren-11-
one Dihydrochloride (28c). Compound 28b (0.040 g, 0.109 mmol)
was diluted with benzene (25 mL), and triethyl phosphite (0.045
g, 0.272 mmol) was added. The solution was heated at reflux for
19 h and cooled, and methanolic HCl (3 M, 15 mL) was added,
followed by heating at reflux for another 3 h. The mixture was
cooled and concentrated to afford a bright-yellow amorphous solid
(0.040 g, 88%) after washing with CHCl3 and ether and drying in
vacuo: mp 290-295 °C (dec). IR (KBr) 3434, 2923, 2874, 1658,
1620, 1601, 1478, 1343, 755, 689 cm-1; 1H NMR (300 MHz, D2O)
δ 7.14-7.11 (m, 3 H), 7.00-6.80 (m, 4 H), 6.60-6.40 (m, 1 H),
6.25 (s, 1 H), 4.26 (s, 2 H), 3.10 (t, J ) 7.3 Hz, 2 H), 2.60-2.40
(m, 2 H), 1.80-1.60 (bm, 2 H); ESIMS m/z (rel intensity), 342
(MH+, 100). Anal. (C22H21Cl2N3O·H2O) C, H, N.
14-[3-(1-Imidazolylpropyl)]-12H-5,11a-diazadibenzo[b,h]fluoren-
11-one Trifluoroacetate (28d). Compound 28a (0.100 g, 0.277
mmol), sodium iodide (0.249 g, 1.662 mmol), and imidazole (0.113
g, 1.66 mmol) were diluted with DMSO (30 mL), and the reaction
mixture was heated at 100 °C for 24 h and then allowed to cool to
room temperature. The reaction mixture was diluted with CHCl3
(150 mL) and washed with water (3 × 50 mL) and saturated NaCl
(50 mL). The organic layer was dried over sodium sulfate, filtered,
and concentrated. The crude product was purified by flash column
chromatography (SiO2), eluting with a gradient of CHCl3-1% Et3N
to 3% MeOH in CHCl3-1% Et3N, to provide a yellow solid. The
solid was diluted with CHCl3 (2 mL), and trifluoroacetic acid (5
mL) was added. The reaction mixture was allowed to stir at room
temperature for 2 h and concentrated, and the residue was triturated
with diethyl ether. The precipitate was filtered and washed with
diethyl ether (50 mL) to provide a yellow solid (0.081 g, 58%):
mp 215-220 °C. IR (KBr) 1661, 1631, 1201, and 1128 cm-1; 1H
NMR (300 MHz, DMSO-d6) δ 9.10 (s, 1 H), 8.38 (d, J ) 8.1 Hz, 1
H), 8.27 (d, J ) 8.4 Hz, 1 H), 8.18 (d, J ) 7.6 Hz, 1 H), 8.01 (d, J )
7.9 Hz, 1 H), 7.86-7.79 (m, 3 H), 7.75-7.62 (m, 4 H), 5.40 (s, 2 H),
4.45 (t, J ) 7.3 Hz, 2 H), 3.27 (t, J ) 7.4 Hz, 2 H), 2.28 (bs, 2 H);
ESIMS m/z (rel intensity) 393 (MH+, 100). Anal.
(C27H21F3N4O3 ·0.25H2O) C, H, N.
1
1347, 1125, 755, 688 cm-1; H NMR (300 MHz, CDCl3) δ 8.52
(d, J ) 8.0 Hz, 1 H), 8.36 (d, J ) 8.5 Hz, 1 H), 8.20 (d, J ) 8.0
Hz, 1 H), 7.78-7.51 (m, 5 H), 7.63 (s, 1 H), 5.43 (s, 2 H), 4.35
(bs, 1 H), 4.18 (s, 2 H), 2.92-2.90 (m, 1 H), 2.73-2.71 (m, 2 H),
2.50-2.48 (m, 2 H), 2.23-2.13 (m, 1 H), 1.78-1.74 (m, 1 H);
ESIMS m/z (rel intensity) 384 (MH+, 100). Anal.
(C24H21N3O2 ·0.25H2O) C, H, N.
14-[(1-Imidazolyl)propylaminomethyl]-12H-5,11a-diazadiben-
zo[b,h]fluoren-11-one (27j). Compound 27a (0.060 g, 0.180 mmol)
was diluted with DMSO (25 mL), and 1-(3-aminopropyl)imidazole
(0.068 g, 0.505 mmol) was added. The solution was stirred at room
temperature for 17 h, diluted with CHCl3 (40 mL), and washed
with H2O (4 × 30 mL). The organic layers were dried over
anhydrous sodium sulfate and concentrated, and the resultant dark
yellow solid was purified by flash column chromatography (SiO2),
eluting with a gradient of CHCl3 to 6% MeOH-1% Et3N in CHCl3,
to yield a pale-yellow solid (0.045 g, 60%) after washing with
hexanes: mp 138-140 °C (dec). IR (film) 3413, 3292, 1656, 1620,
1600, 1507, 1451, 1343, 755, 688 cm-1 1H NMR (300 MHz,
;
CDCl3) δ 8.56 (d, J ) 8.0 Hz, 1 H), 8.26 (d, J ) 8.7 Hz, 2 H),
7.83-7.56 (m, 5 H), 7.67 (s, 1 H), 7.43 (s, 1 H), 7.03 (s, 1 H),
6.84 (s, 1 H), 5.45 (s, 2 H), 4.36 (s, 2 H), 4.07 (t, J ) 6.9 Hz, 2 H),
2.78 (t, J ) 6.6 Hz, 2 H), 2.04-1.95 (m, 2 H); ESIMS m/z (rel
intensity) 422 (MH+, 100). Anal. (C26H23N5O·0.75H2O) C, H, N.
14-(3-Morpholinopropylaminomethyl)-12H-5,11a-diazadiben-
zo[b,h]fluoren-11-one (27k). Compound 27a (0.055 g, 0.165 mmol)
was diluted with DMSO (25 mL), and 3-morpholinopropylamine
(0.119 g, 0.826 mmol) was added. The solution was stirred at room
temperature for 19 h, diluted with CHCl3 (40 mL), and washed
with H2O (4 × 30 mL). The organic layers were dried over
anhydrous sodium sulfate and concentrated, and the resultant dark
yellow solid was purified by flash column chromatography (SiO2),
eluting with a gradient of 1% Et3N in CHCl3 to 1% MeOH-1%
Et3N in CHCl3 to yield a flaky yellow solid (0.040 g, 55%) after
washing with diethyl ether: mp 172-175 °C. IR (film) 3445, 3302,
2929, 1656, 1619, 1600, 1451, 1344, 1117, 753, 687 cm-1 1H
;
NMR (300 MHz, CDCl3) δ 8.58 (d, J ) 8.3 Hz, 1 H), 8.28 (t, J )
8.5 Hz, 2 H), 7.82-7.58 (m, 5 H), 7.67 (s, 1 H), 5.48 (s, 2 H),
4.37 (s, 2 H), 3.65 (t, J ) 4.4 Hz, 4 H), 2.81 (bm, 2 H), 2.42-2.37
(m, 6 H), 1.78-1.68 (m, 2 H); ESIMS m/z (rel intensity) 441 (MH+,
100). Anal. (C27H28N4O2 ·0.5H2O) C, H, N.
14-(3-Chloropropyl)-12H-5,11a-diazadibenzo[b,h]fluoren-11-
one (28a). Compound 23 (1.000 g, 5.020 mmol) and compound 26
(1.091 g, 5.522 mmol) were diluted with benzene (125 mL).
p-Toluenesulfonic acid monohydrate (0.955 g, 5.020 mmol) was
added, and the solution was heated at reflux for 5 h using a
Dean-Stark trap to collect the azeotroped water. The solution was
concentrated, and the precipitate was washed with Et2O (50 mL).
The precipitate was dissolved in CHCl3 (500 mL) and washed with
saturated NaHCO3 (3 × 200 mL). The combined aqueous layer
was then extracted with CHCl3 (3 × 200 mL). The organic layers
were combined, dried over sodium sulfate, concentrated, and
purified by flash column chromatography (SiO2), eluting with a
gradient of CHCl3 to 4% MeOH in CHCl3, to provide an off-white
solid (1.642 g, 91%) after washing with Et2O (50 mL): mp 235-237
°C. IR (KBr) 3465, 1654, 1619, 1601, 756, and 689 cm-1; 1H NMR
(300 MHz, DMSO-d6) δ 8.37 (d, J ) 8.0 Hz, 1 H), 8.29 (d, J )
7.6 Hz, 1 H), 8.18 (dd, J ) 8.5 and 1.0 Hz, 1 H), 8.00 (d, J ) 7.8
Hz, 1 H), 7.88-7.67 (m, 3 H), 7.64 (s, 1 H), 7.63-7.58 (m, 1 H),
5.40 (s, 2 H), 3.88 (t, J ) 6.3 Hz, 2 H), 3.37-3.31 (m, 2 H),
2.19-2.14 (m, 2 H); ESIMS m/z (rel intensity) 361/363 (MH+,
100/33). Anal. (C22H17ClN2O·0.5H2O) C, H, N.
14-[3(1-Morpholinopropyl)]-12H-5,11a-diazadibenzo[b,h]fluoren-
11-one Trifluoroacetate (28e). Compound 28a (0.100 g, 0.277
mmol), sodium iodide (0.250 g, 1.66 mmol), and morpholine (0.144
g, 1.66 mmol) were diluted with DMSO (30 mL), and the reaction
mixture was heated at 100 °C for 48 h and then allowed to stir at
room temperature for 16 h. The reaction mixture was diluted with
CHCl3 (150 mL) and washed with water (3 × 50 mL) and saturated
NaCl (50 mL). The organic layer was dried over sodium sulfate,
filtered, and concentrated. The crude product was purified by flash
column chromatography (SiO2), eluting with a gradient of
CHCl3-1% Et3N to 5% MeOH in CHCl3-1% Et3N, to provide a
yellow solid. The solid was diluted with CHCl3
(2 mL), and
trifluoroacetic acid (5 mL) was added. The reaction mixture was
allowed to stir at room temperature for 2 h and concentrated, and
the residue was triturated with diethyl ether. The precipitate was
filtered and washed with diethyl ether (50 mL) to provide a yellow
solid (0.140 g, 79%): mp 190-195 °C. IR (KBr) 1662, 1632, 1197,
14-(3-Azidopropyl)-2H-5,11a-diazadibenzo[b,h]fluoren-11-one
(28b). Compound 28a (0.150 g, 0.416 mmol) and sodium azide
(0.081 g, 1.25 mmol) were diluted with DMSO (35 mL), and the
mixture was heated at 100 °C for 16 h. The mixture was diluted
into H2O (100 mL) and extracted with CHCl3 (1 × 100 mL, 1 ×
80 mL, 1 × 50 mL). The combined organic layers were washed
1
1134 cm-1; H NMR (300 MHz, DMSO-d6) δ 8.39 (d, J ) 8.1
Hz, 1 H), 8.33 (d, J ) 8.6 Hz, 1 H), 8.20 (d, J ) 7.5 Hz, 1 H),
8.02 (d, J ) 8.0 Hz, 1 H), 7.90-7.72 (m, 3 H), 7.71 (s, 1 H),
7.65-7.60 (m, 1 H), 5.43 (s, 2 H), 3.98 (d, J ) 12.5 Hz, 2 H),
3.63 (t, J ) 11.9 Hz, 2 H), 3.44-3.25 (m, 6 H), 3.16-3.03 (m, 2

4618 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Cinelli et al.
H), 2.11 (m, 2 H); ESIMS m/z (rel intensity) 412 (MH+, 100).
Anal. (C28H26F3N3O4) C, H, N.
mixed with 30 µL of loading buffer (80% formamide, 10 mM
sodium hydroxide, 1 mM sodium EDTA, 0.1% xylene cyanol, and
0.1% bromophenol blue, pH 8.0). Aliquots were separated in
denaturing gels (16% polyacrylamide, 7 M urea). Gels were dried
and visualized by using a phosphoimager and ImageQuant software
(Molecular Dynamics, Sunnyvale, CA).
14-(3-N-Ethanolaminopropyl)-12H-5,11a-diazadibenzo[b,h]fluo-
ren-11-one Trifluoroacetate (28f). Compound 28a(0.100 g, 0.277
mmol), sodium iodide (0.249 g, 1.66 mmol), and ethanolamine
(0.100 mL, 1.66 mmol) were diluted with DMSO (30 mL), and
the reaction mixture was heated at 100 °C for 16 h and then allowed
to cool to room temperature. The reaction mixture was diluted with
CHCl3 (150 mL) and washed with water (3 × 50 mL) and saturated
NaCl (50 mL). The organic layer was dried over sodium sulfate,
filtered, and concentrated. The crude product was purified by flash
column chromatography (SiO2), eluting with a gradient of
CHCl3-1% Et3N to 9% MeOH in CHCl3-1% Et3N, to provide a
white solid. The solid was diluted with CHCl3 (3 mL), and
trifluoroacetic acid (5 mL) was added. The reaction mixture was
allowed to stir at room temperature for 2 h and concentrated, and
the residue was triturated with diethyl ether. The precipitate was
filtered and washed with diethyl ether (50 mL) to provide a yellow
solid (0.101 g, 73%): mp 225-227 °C (dec). IR (KBr) 3383, 1661,
1621, 1602, 1201, 1132 cm-1; 1H NMR (300 MHz, DMSO-d6) δ
8.48 (bs, 2 H), 8.39 (d, J ) 8.1 Hz, 1 H), 8.34 (d, J ) 8.1 Hz, 1
H), 8.19 (d, J ) 8.4 Hz, 1 H), 8.02 (d, J ) 8.0 Hz, 1 H), 7.89-7.80
(m, 2 H), 7.76-7.71 (m, 1 H), 7.70 (s, 1 H), 7.65-7.60 (m, 1 H),
5.40 (s, 2 H), 3.66 (t, J ) 5.0 Hz, 2 H), 3.32 (t, J ) 7.1 Hz, 2 H),
3.16 (bs, 2 H), 3.02 (bs, 2 H), 2.06 (bs, 2 H); ESIMS m/z (rel
intensity) 386 (MH+, 100). Anal. (C26H24F3N3O4 ·0.5H2O) C, H,
N.
14-[3-(N,N-Dimethylaminopropyl)]-12H-5,11a-diazadibenzo[b,
h]fluoren-11-one Trifluoroacetate (28g). Compound 28a (0.100 g,
0.277 mmol), sodium iodide (0.249 g, 1.66 mmol), and dimethy-
lamine (2 M in THF, 1.67 mL, 3.32 mmol) were diluted with
DMSO (30 mL), and the reaction mixture was heated at 100 °C
for 16 h and then allowed to cool to room temperature. The reaction
mixture was diluted with CHCl3 (150 mL) and washed with water
(3 × 50 mL) and saturated NaCl (50 mL). The organic layer was
dried over sodium sulfate, filtered, and concentrated. The crude
product was purified by flash column chromatography (SiO2),
eluting with a gradient of CHCl3-1% Et3N to 4% MeOH in
CHCl3-1% Et3N, to provide a yellow oil. The oil was diluted with
CHCl3 (3 mL), and trifluoroacetic acid (5 mL) was added. The
reaction mixture was allowed to stir at room temperature for 2 h
and concentrated, and the residue was triturated with diethyl ether.
The precipitate was filtered and washed with diethyl ether (50 mL)
to provide a light-yellow solid (0.095 g, 71%): mp 210 °C (dec).
IR (KBr) 3434, 1666, 1633, 1199, 1128 cm-1; 1H NMR (300 MHz,
DMSO-d6) δ 8.38 (d, J ) 7.9 Hz, 1 H), 8.33 (d, J ) 8.2 Hz, 1 H),
8.20 (d, J ) 7.6 Hz, 1 H), 8.02 (d, J ) 8.0 Hz, 1 H), 7.90-7.80
(m, 2 H), 7.76-7.71 (m, 1 H), 7.70 (s, 1 H), 7.65-7.60 (m, 1 H),
5.43 (s, 2 H), 3.28-3.23 (m, 4 H), 2.78 (s, 6 H), 2.09-2.07 (m, 3
H); ESIMS m/z (rel intensity) 370 (MH+, 100). Anal.
(C26H24F3N3O3 ·H2O) C, H, N.
Topoisomerase I-Mediated DNA Cleavage Reactions. Human
recombinant Top1 was purified from Baculovirus as described
previously.55 The 161 bp fragment from pBluescript SK(-)
phagemid DNA (Stratagene, La Jolla, CA) was cleaved with the
restriction endonuclease Pvu II and Hind III (New England Biolabs,
Beverly, MA) in supplied NE buffer 2 (50 µL mixtures) for 1 h at
37 °C and separated by electrophoresis in a 1% agarose gel made
in 1× TBE buffer. The 161 bp fragment was eluted from the gel
slice using the QIAEX II kit (QIAGEN Inc., Valencia, CA).
Approximately 200 ng of the fragment was 3′-end labeled at the
Hind III site by fill-in reaction with [R-32P]-dGTP and 0.5 mM
dATP, dCTP, and dTTP, in React 2 buffer (50 mM Tris-HCl, pH
8.0, 100 mM MgCl2, 50 mM NaCl) with 0.5 unit of DNA
polymerase I (Klenow fragment). Unincorporated 32P-dGTP was
removed using mini Quick Spin DNA columns (Roche, Indianapo-
lis, IN), and the eluate containing the 3′-end-labeled 161 bp
fragment was collected. Aliquots (approximately 50 000 dpm/
reaction) were incubated with topoisomerase I at 22 °C for 30 min
in the presence of the tested drug. Reactions were terminated by
adding SDS (0.5% final concentration). The samples (10 µL) were
Modeling Studies. The structure of the ternary complex,
containing topoisomerase I, DNA, and camptothecin, was down-
loaded from the Protein Data Bank (PDB code 1T8I).10,27 Several
of the atoms were then fixed according to the Sybyl atom types.
Hydrogens were added and minimized using the MMFF94s force
field and MMFF94 charges. Modeled analogues were constructed
in Sybyl 7.3, energy minimized with the MMFF94s force field and
MMFF94 charges, and overlapped with the crystal structure ligand
in the ternary complex, and the crystal structure ligand was then
deleted. The new complex was subsequently subjected to energy
minimization using MMFF94s force field with MMFF94 charges.
During the energy minimization, the structure of the aromathecin
and a surrounding 5 Å sphere were allowed to move, while the
structures of the remaining protein and nucleic acids were frozen.
The energy minimization was performed using the Powell method
with a 0.05 kcal/(mol Å) energy gradient convergence criterion and
a distance-dependent dielectric function. Ligand overlays were
constructed using the indenoisoquinoline crystal structure 1SC7.10
Acknowledgment. This work was made possible by the
National Institutes of Health (NIH) through support of this work
with Research Grant UO1 CA89566 and Training Grant ST32
CA09634-12, by an ACS Medicinal Chemistry Predoctoral
Fellowship sponsored by Pfizer Global Research and Develop-
ment (A.M.), and by a Lynn Fellowship (Purdue University,
M.A.C.). This research was supported in part by the Intramural
Research Program of the NIH, National Cancer Institute, Center
for Cancer Research. M.A.C. thanks Drs. Karl Wood and
Huaping Mo for assistance and valuable discussions.
Supporting Information Available: Elemental analysis results
for compounds 27a-k and 28a-g. This material is available free
of charge via the Internet at http://pubs.acs.org.
References
(1) Pommier, Y. Topoisomerase I inhibitors: camptothecins and beyond.
Nat. ReV. Cancer. 2006, 6, 789–802.
(2) Wang, J. C. Cellular roles of DNA topoisomerases: a molecular
perspective. Nat. ReV. Mol. Cell Biol. 2002, 3, 430–440.
(3) Luzzio, M. J.; Besterman, J. M.; Emerson, D. L.; Evans, M. G.; Lackey,
K.; Leitner, P. L.; McIntyre, G.; Morton, B.; Myers, P. L.; Peel, M.;
Sisco, J. M.; Sternbach, D. D.; Tong, W.; Truesdale, A.; Uehling,
D. E.; Vuong, A.; Yates, J. Synthesis and antitumor activity of novel
water soluble derivatives of camptothecin as specific inhibitors of
topoisomerase I. J. Med. Chem. 1995, 38, 395–401.
(4) Husain, I.; Mohler, J. L.; Seigler, H. F.; and Besterman, J. M. Elevation
of topoisomerase I messenger RNA, protein, and catalytic activity in
human tumors: demonstration of tumor-type specficity and implications
for cancer chemotherapy. Cancer. Res. 1994, 54 (2), 539–546.
(5) Thomas, C. J.; Rahier, N. J.; Hecht, S. M. Camptothecin: current
perspectives. Bioorg. Med. Chem. 2004, 12, 1585–1604.
(6) Meng, L.-H.; Liao, Y.-Z.; Pommier, Y. Non-camptothecin DNA
topoisomerase I inhibitors in cancer chemotherapy. Curr. Top. Med.
Chem. 2003, 3, 305–320.
(7) Bailly, C. Topoisomerase I poisons and suppressors as anticancer drugs.
Curr. Med. Chem. 2000, 7, 39–58.
(8) Wall, M. E.; Wani, M. C.; Cook, C. E.; Palmer, K. H.; McPhail, A. T.;
Sim, G. A. The isolation and structure of camptothecin, a novel
alkaloidal leukemia and tumor inhibitor from Camptotheca acuminata.
J. Am. Chem. Soc. 1966, 88, 3888–3890.
(9) Staker, B. L.; Hjerrild, K.; Feese, M. D.; Behnke, C. A.; Burgin, A. B.;
Stewart, L. The mechanism of topoisomerase I poisoning by a camp-
tothecin analog. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15387–15392.
(10) Staker, B. L.; Feese, M. D.; Cushman, M.; Pommier, Y.; Zembower,
D.; Stewart, L.; Burgin, A. Structures of three classes of anticancer
agents bound to the human topoisomerase I-DNA covalent complex.
J. Med. Chem. 2005, 48, 2336–2345.
(11) Kohn, K. W.; Pommier, Y. Molecular and biological determinants of the
cytotoxic actions of camptothecins. Perspective for the development of

Substituted Aromathecins as Top1 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4619
new topoisomerase I inhibitors. Ann. N.Y. Acad. Sci. 2000, 922, 11–
26.
(12) Pommier, Y.; Pourquier, P.; Fan, Y.; Strumberg, D. Mechanism of
action of eukaryotic DNA topoisomerase I and drugs targeted to the
enzyme. Biochim. Biophys. Acta 1998, 1400, 83–106.
(13) Wall, M. E.; Wani, M. C.; Nicholas, A. W.; Manikumar, G.; Tele,
C.; Moore, L.; Truesdale, A.; Leitner, P.; Besterman, J. M. Plant
Antitumor Agents. 30. Synthesis and structure activity of novel
camptothecin analogs. J. Med. Chem. 1993, 36, 2689–2700.
(14) Xie, Z.; Ootsu, K.; and Akimoto, H. Convergent approach to water-
soluble camptothecin derivatives. Bioorg. Med. Chem. Lett. 1995, 5
(19), 2189–2194.
(15) Wani, M. C.; Nicholas, A. W.; Wall, M. E. Plant antitumor agents.
23. Synthesis and antileukemic activity of camptothecin analogues.
J. Med. Chem. 1986, 29, 2358–2363.
(16) Jew, S.-S.; Kim, M. G.; Kim, H.-J.; Rho, E.-Y.; Park, H.-G.; Kim,
J.-K.; Han, H.-J.; Lee, H. Synthesis and in vitro cytotoxicity of
C(20)(RS)-camptothecin analogues modified at both B (or A) and E
ring. Bioorg. Med. Chem. Lett. 1998, 8, 1797–1800.
(17) Ahn, S. K.; Choi, N. S.; Jeong, B. S.; Kim, K. K.; Journ, D. J.; Kim,
J. K.; Lee, S. J.; Kim, J. W.; Hong, C. I.; Jew, S. Practical synthesis
of (S)-7-(2-isopropylamino)ethylcamptothecin hydrochloride, potent
topoisomerase I inhibitor. J. Heterocycl. Chem 2000, (Sep-Oct), 1141.
(18) Wani, M. C.; Ronman, P. E.; Lindley, J. T.; Wall, M. E. Plant antitumor
agents. 18. Synthesis and biological activity of camptothecin analogues.
J. Med. Chem. 1980, 23, 554–560.
(33) Mason, J. J.; Bergman, J. Total synthesis of luotonin A and
14-substituted analogues. Org. Biomol. Chem. 2007, 5, 2486–2490.
(34) Dallavalle, S.; Merlini, L.; Beretta, G. L.; Tinelli, S.; Zunino, F.
Synthesis and cytotoxic activity of substituted luotonin A derivatives.
Bioorg. Med. Chem. Lett. 2004, 14, 5757–5761.
(35) Lin, L. Z.; Cordell, G. A. Quinoline alkaloids from Camptotheca
acuminata. Phytochemistry 1989, 28, 1295–1297.
(36) Shamma, M.; Novak, L. Synthetic approaches to camptothecin.
Tetrahedron 1969, 25, 2275–2279.
(37) Morrell, A.; Placzek, M. S.; Steffen, J. D.; Antony, S.; Agama, K.;
Pommier, Y.; Cushman, M. Investigation of the lactam side chain
length necessary for optimal indenoisoquinoline topoisomerase I
inhibition and cytotoxicity in human cancer cell cultures. J. Med.
Chem. 2007, 50, 2040–2048.
(38) Babjak, M.; Kanazawa, A.; Anderson, R. J.; Greene, A. E. Concise
synthesis of 22-hydroxyacuminatine, cytotoxic camptothecinoid from
Camptotheca acuminata, by pyridone benzannulation. Org. Biomol.
Chem. 2006, 4, 407–409.
(39) Zhou, H.-B.; Liu, G.-S.; Yao, Z.-J. Short and efficient total synthesis
of luotonin A and 22-hydroxyacuminatine using a common cascade
strategy. J. Org. Chem. 2007, 72, 6270–6272.
(40) Bhaskar Kanth, J. V.; Periasamy, M. Convenient method for the
synthesis of N-(ethyloxycarbonyl) ester derivatives from amino acids.
Synth. Commun. 1995, 25, 1523–1530.
(41) Dyke, S. F.; Sainsbury, M.; Evans, J. R. Lycorine: studies in synthesis.
Tetrahedron 1973, 29, 213–220.
(42) Roglans, A.; Marquet, J.; Moreno-Manas, M. Preparation of 3-pyrrolidone
and 4-perhydroazepinone. Synth. Commun. 1992, 22, 1249–1258.
(43) Sloan, K. B.; Koch, S. A. M. Effect of nucleophilicity and leaving group
ability on the SN2 reactions of amines with (acyloxy)alkyl R-halides: a
product distribution study. J. Org. Chem. 1983, 48, 635–640.
(44) Houghton, P. G.; Humphrey, G. R.; Kennedy, D. J.; Roberts, D. C.;
Wright, S. H. B. Enantiospecific synthesis of the (4R)-1-
azabicyclo[2.2.1]heptane ring system. J. Chem. Soc., Perkin Trans. 1
1993, 1421–1424.
(19) Gottlieb, J. A.; Luce, J. K. Treatment of malignant melanoma with
camptochecin (NSC-100880). Cancer Chemother. Rep. 1972, 56, 103–
105.
(20) Jaxel, C.; Kohn, K. W.; Wani, M. C.; Wall, M. E.; Pommier, Y.
Structure-activity study of the actions of camptothecin derivatives
on mammalian topoisomerase I: evidence for a specific receptor site
and a relation to antitumor activity. Cancer Res. 1989, 49, 1465–1469.
(21) Haas, N. B.; LaCreta, F. P.; Walczak, J.; Hudes, G. R.; Brennan, J. M.;
Ozols, R. F.; O’Dwyer, P. J. Phase-I pharmacokinetic study of
topotecan by 24-h continuous-infusion weekly. Cancer Res. 1994, 54
(5), 1220–1226.
(45) Sugasawa, T.; Toyoda, T.; Adachi, M.; Sasakura, K. Aminohaloborane
in organic synthesis. 1. Specific ortho substitution reaction of anilines.
J. Am. Chem. Soc. 1978, 100, 4842–4852.
(22) Kohlhagen, G.; Paull, K. D.; Cushman, M.; Nagafuji, P.; and Pommier,
Y. Protein-linked DNA strand breaks induced by NSC 314622, a novel
noncamptothecin topoisomerase I poison. Mol. Pharm. 1998, 54, 50–58.
(23) Nagarajan, M.; Morrell, A.; Fort, B. C.; Meckley, M. R.; Antony, S.;
Kohlhagen, G.; Pommier, Y.; Cushman, M. Synthesis and anticancer
activity of simplified indenoisoquinoline topoisomerase I inhibitors
lacking substituents on the aromatic rings. J. Med. Chem. 2004, 47
(23), 5651–5661.
(24) Morrell, A.; Placzek, M.; Parmley, S.; Grella, B.; Antony, S.; Pommier,
Y.; Cushman, M. Optimization of the indenonone ring of indenoiso-
quinoline topoisomerase I inhibitors. J. Med. Chem. 2007, 50, 4388–
4404.
(46) Cevasco, A. A. Process for the Manufacture of Cycloalkyl and
Haloalkyl o-Aminophenyl Ketones. U.S. Patent 5,405,998, 1995.
(47) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J. New
colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl.
Cancer Inst. 1990, 82 (13), 1107–1112.
(48) Boyd, M. R.; Paull, K. D. Some practical considerations and
applications of the national cancer institute in-vitro anticancer drug
discovery screen. Drug DeV. Res. 1995, 34, 91–109.
(49) Antony, S.; Jayaraman, M.; Laco, G.; Kohlhagen, G.; Kohn, K. W.;
Cushman, M.; Pommier, Y. Differential induction of topoisomerase
I-DNA cleavage complexes by the indenoisoquinoline MJ-III-65
(NSC 706744) and camptothecin: base sequence analysis and activity
against camptothecin-resistant topoisomerase I. Cancer Res. 2003, 63,
7428–7435.
(50) Nagarajan, M.; Xiao, X.; Antony, S.; Kohlhagen, G.; Pommier, Y.;
Cushman, M. Design, synthesis, and biological evaluation of inde-
noisoquinoline topoisomerase I Inhibitors featuring polyamine side
chains on the lactam nitrogen. J. Med. Chem. 2003, 46, 5712–5724.
(51) Paull, K. D.; Shoemaker, R. H.; Hodes, L.; Monks, A.; Scudiero, D. A.;
Rubinstein, L.; Plowman, J.; Boyd, M. R. Display and analysis of
patterns of differential activity of drugs against human tumor cell lines:
development of mean graph and COMPARE algorithm. J. Natl.
Cancer. Inst. 1989, 81, 1088–1092.
(52) Paull, K. D.; Hamel, E.; Malspeis, L. Prediction of Biochemical
Mechanism of Action from the in Vitro Antitumor Screen of the National
Cancer Institute. In Cancer Chemotherapeutic Agents; Foye, W. O.,
Ed.; American Chemical Society: Washington, DC, 1995; pp 9-45.
(53) Delcros, J.-G.; Tomasi, S.; Carrington, S.; Martin, B.; Renault, J.;
Blagbrough, I. S.; Uriac, P. Effect of spermine conjugation on the
cytotoxicity and cellular transport of acridine. J. Med. Chem. 2002,
45, 5098–5111.
(54) Garrett, S. W.; Davies, O. R.; Milroy, D. A.; Wood, P. J.; Pouton,
C. W.; Threadgill, M. D. Synthesis and characterisation of polyamine-
poly(ethylene glycol) constructs for DNA binding and gene delivery.
Biooorg. Med. Chem. 2008, 8, 1779–1797.
(55) Pourquier, P.; Ueng, L.-M.; Fertala, J.; Wang, D.; Park, H.-K.;
Essigmann, J. M.; Bjornsti, M.-A.; Pommier, Y. Induction of reversible
complexes between eukaryotic DNA topoisomerase I and DNA-
containing oxidative base damages. 7,8-Dihydro-8-oxoguanine and
5-hydroxycytosine. J. Biol. Chem. 1999, 274, 8516–8523.
(25) Morrell, A.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman, M.
Synthesis of nitrated indenoisoquinolines as topoisomerase I inhibitors.
Bioorg. Med. Chem. Lett. 2004, 14, 3659–3663.
(26) Antony, S.; Agama, K. K.; Miao, Z.-H.; Takagi, K.; Wright, M. H.;
Robles, A. I.; Varticovski, L.; Nagarajan, M.; Morrell, A.; Cushman, M.;
Pommier, Y. Novel indenoisoquinolines NSC 725775 and NSC 724998
produce persistent topoisomerase I cleavage complexes and overcome
multidrug resistance. Cancer Res. 2007, 67 (21), 10397–10405.
(27) Fox, B. M.; Xiao, X.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Staker,
B. L.; Stewart, L.; Cushman, M. Design, synthesis, and biological
evaluation of cytotoxic 11-alkenylindenoisoquinoline topoisomerase
I inhibitors and indenoisoquinoline-camptothecin hybrids. J. Med.
Chem. 2003, 46, 3275–3282.
(28) Xiao, X.; Antony, S.; Pommier, Y.; Cushman, M. Total synthesis and
biological evaluation of 22-hydroxyacuminatine. J. Med. Chem. 2006,
49, 1408–1412.
(29) Pin, F.; Comesse, S.; Sanselme, M.; Daïch, A. A domino N-
amidoacylation/aldol-type condensation approach to the synthesis of
the Topo-I inhibitor rosettacin and derivatives. J. Org. Chem. 2008,
73 (5), 1975–1978.
(30) Cheng, K.; Rahier, N. J.; Eisenhauer, B. M.; Gao, R.; Thomas, S. J.;
Hecht, S. M. 14-Azacamptothecin: a potent water-soluble topoi-
somerase I poison. J. Am. Chem. Soc. 2005, 127 (3), 838–839.
(31) Cagir, A.; Jones, S. H.; Gao, R.; Eisenhauer, B. M.; Hecht, S. M.;
Luotonin, A. A naturally occurring human DNA topoisomerase I
poison. J. Am. Chem. Soc. 2003, 125, 13628–13629.
(32) Cagir, A.; Eisenhauer, B. M.; Gao, R.; Jones, S. H.; Hecht, S. M.
Synthesis and topoisomerase I inhibitory properties of luotonin A
analogues. Bioorg. Med. Chem. 2004, 12, 6287–6299.
JM800259E