The structure-activity relationships of A-ring-substituted aromathecin topoisomerase i inhibitors strongly support a camptothecin-like binding mode

Article abstract of DOI:10.1016/j.bmc.2010.06.040

Aromathecins are inhibitors of human topoisomerase I (Top1). These compounds are composites of several heteroaromatic systems, namely the camptothecins and indenoisoquinolines, and they possess notable Top1 inhibition and cytotoxicity when substituted at position 14. The SAR of these compounds overlaps with indenoisoquinolines, suggesting that they may intercalate into the Top1-DNA complex similarly. Nonetheless, the proposed binding mode for aromathecins is purely hypothetical, as an X-ray structure is unavailable. In the present communication, we have synthesized eight novel series of A-ring-substituted (positions 1-3) aromathecins, through a simple, modular route, as part of a comprehensive SAR study. Certain groups (such as 2,3-ethylenedioxy) moderately improve Top1 inhibition, and, often, antiproliferative activity, whereas other groups (2,3-dimethoxy and 3-substituents) attenuate bioactivity. Strikingly, these trends are very similar to those previously observed for the A-ring of camptothecins, and this considerable SAR overlap lends further support (in the absence of crystallographic data) to the hypothesis that aromathecins bind in the Top1 cleavage complex as interfacial inhibitors in a 'camptothecin-like' pose.

Full text of DOI:10.1016/j.bmc.2010.06.040

Bioorganic & Medicinal Chemistry 18 (2010) 5535–5552
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The structure–activity relationships of A-ring-substituted aromathecin
topoisomerase I inhibitors strongly support a camptothecin-like binding mode
Maris A. Cinelli a, Andrew E. Morrell a, Thomas S. Dexheimer b, Keli Agama b, Surbhi Agrawal b,
Yves Pommier b, Mark Cushman a,
*
a Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, and the Purdue Center for Cancer Research,
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:
Aromathecins are inhibitors of human topoisomerase I (Top1). These compounds are composites of sev-
eral heteroaromatic systems, namely the camptothecins and indenoisoquinolines, and they possess nota-
ble Top1 inhibition and cytotoxicity when substituted at position 14. The SAR of these compounds
overlaps with indenoisoquinolines, suggesting that they may intercalate into the Top1-DNA complex
similarly. Nonetheless, the proposed binding mode for aromathecins is purely hypothetical, as an
X-ray structure is unavailable. In the present communication, we have synthesized eight novel series
of A-ring-substituted (positions 1–3) aromathecins, through a simple, modular route, as part of a compre-
hensive SAR study. Certain groups (such as 2,3-ethylenedioxy) moderately improve Top1 inhibition, and,
often, antiproliferative activity, whereas other groups (2,3-dimethoxy and 3-substituents) attenuate bio-
activity. Strikingly, these trends are very similar to those previously observed for the A-ring of camptot-
hecins, and this considerable SAR overlap lends further support (in the absence of crystallographic data)
to the hypothesis that aromathecins bind in the Top1 cleavage complex as interfacial inhibitors in a ‘cam-
ptothecin-like’ pose.
Received 3 May 2010
Revised 11 June 2010
Accepted 14 June 2010
Available online 20 June 2010
Keywords:
Topoisomerase 1
Aromathecins
Anticancer
Camptothecin-like
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
the interface of Top1-DNA cleavage complexes in an intercalative
mode,10–12 where it stacks between the base pairs of the Top1-
Topoisomerase I (Top1) is an enzyme that is critical for efficient
DNA replication and cell division. As DNA is highly supercoiled, it
must also be relaxed prior to cellular processes such as replication
and transcription. The enzyme acts by binding to and nicking dou-
ble-stranded DNA through the action of a nucleophilic tyrosine res-
idue (Tyr723). Within the covalent Top1-DNA cleavage complex,
the scissile DNA strand undergoes ‘controlled rotation’1–3 around
the nonscissile strand, relieving the supercoils. The hydroxyl group
of the scissile strand’s 50 end then re-ligates the broken strand and
the enzyme is released.1 As it plays a pivotal role in cellular prolif-
eration, Top1 is often overexpressed in human tumors. High levels
of this enzyme have been found in lung, colorectal, and ovarian
cancers4–6 and elevated Top1 levels in breast cancers are also asso-
ciated with poor patient prognosis.7
DNA cleavage complex and, stabilized chiefly by pi–pi stacking,13
sterically prevents the re-ligation reaction. It also forms key hydro-
gen bonds with Top1 amino acid residues.1,10–12 The resulting
covalent Top1-DNA adduct then produces collisions with advanc-
ing replication forks and transcription complexes, which triggers
irreversible DNA damage and apoptosis.14,15
Although the camptothecins are potent and possess high cyto-
toxicity, they also suffer from well-identified drawbacks, including
short duration of action, poor solubility, resistance mutants,16 and
high toxicity.17,18 Additionally, the E-ring lactone of camptothecin
is readily opened to its hydroxycarboxylate form in vivo.19 This
form is less active and binds strongly to human blood proteins.20
One promising class of noncamptothecin Top1 poisons is the
indenoisoquinolines, such as MJ-III-65 (4).21,12 These compounds
possess high anti-Top1 activity, are cytotoxic, and are more stable
because they lack the hydroxylactone. Through comprehensive
SAR studies,21–24 two clinical candidates, indotecan (5) and indim-
itecan (6) were developed and have begun Phase 1 clinical trials at
the National Cancer Institute.25,26
The only FDA-approved Top1 inhibitors are topotecan (2) and
irinotecan (3),8 drugs based on the pentacyclic antitumor alkaloid
camptothecin (1), (Fig. 1) which was originally isolated from the
Chinese tree Camptotheca acuminata.8,9 Camptothecin binds at
We described in two previous communications27,28 the design,
synthesis, and biological evaluation of substituted 12H-5,11a-
* 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 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.040

5536
M. A. Cinelli et al. / Bioorg. Med. Chem. 18 (2010) 5535–5552
12
7
11
9
rationale provided by existing camptothecin and indenoisoquino-
1
OH
N
HO
O
line SARs and the ‘overlapping’ SAR hypothesis, we prepared 8 no-
vel series of aromathecins (series 27 through 34) substituted on
both the A-ring and position 14. The A-ring substituents encom-
pass a variety of steric, electronic, and H-bonding properties,
allowing for maximum exploration of chemical space and elucida-
tion of those elements required for binding and optimal bioactivity.
OH 14
20
10
N
N
O
21
O
N
19O
N
5
17
O
O
Camptothecin
Topotecan
2
1
2. Chemistry
O
N
N
Although there are several routes to rosettacin and aromathecin
derivatives,32,33 our route proceeds through tricyclic synthon 8.34
The previously developed route to 828 (see Scheme 3 for structure)
proceeds in good yield from commercially available starting
materials and can be scaled up readily. This ketone can then be con-
densed with substituted o-aminochloroaceto- or -chlorobutyr-
ophenones to provide versatile aromathecin cores that can be
readily functionalized, making the assembly of substituted aromat-
hecins rapid and modular. Following existing camptothecin SARs, a
variety of groups (halogens, ethers, the methylenedioxy group, and
a methyl group) were chosen for the study. To vary the nature of the
A-ring substituent, substituted precursor amino aryl ketones were
first prepared.
The preparation of some of these ketone coupling partners is
described in Scheme 1. To install the 2,3-ethylenedioxy group,
1,4-benzodiozan-6-amine (9) was chloroacetylated19 using Sugas-
awa’s Friedel–Crafts conditions to yield 10, and chlorobutyrated
likewise to afford 11.35–37 As the methylenedioxy group (for 2,3-
methylenedioxyaromathecins) is not compatible with the strong
Lewis acids utilized in the chloroacetylation, the modified zinc-cat-
alyzed procedure of Luzzio et al.19 was employed, and beginning
with 3,4-methylenedioxyaniline (12) the ketone 15 was eventually
obtained in low yield but high purity. The chloroacetylation of
4-aminoveratrole (16) proceeded in the absence of additional cat-
alyst (due to the electron-donating effects of the methoxy groups)
to afford 17. 3-Fluoro-4-methylaniline (18) and 4-chloroaniline
(19) were also chloroacetylated to afford their respective ketones
20 and 21, albeit in low yield. The chloroacetylation of 3-chloroan-
iline (22), however, afforded an inseparable mixture of 23 and 24,
which were converted into their respective acetanilides 25 and 26
to aid in purification. These compounds were separated and
hydrolyzed to yield 23 and 24, in a ratio consistent with that re-
O
HO
O
N
O
N
O
O
Irinotecan
O
3
O
H3CO
H3CO
O
O
O
O
N
O
N
H3CO
H3CO
Indotecan
H
N
N
5
OH
O
MJ-III-65
4
4
3
1
O
5
O
2
N
6
7
O
8
9
H3CO
H3CO
14
N
N
N
N
12
11
10
O
O
Indimitecan
Rosettacin
7
6
Figure 1. Representative Top1 poisons.
diazadibenzo[b,h]fluoren-11-ones,
called
‘aromathecins’.27–30
These compounds can be thought of as composites of the camptot-
hecins and indenoisoquinolines, in which the E-ring of the former
has been ‘aromatized’ (replaced by a benzene ring). The majority of
these compounds, when substituted at position 14, possess greater
Top1 inhibitory and antiproliferative activity than the unsubstitut-
ed core compound, rosettacin (7).31 Molecular models indicate that
these 14-substituents overlap spatially with the lactam substitu-
ents of indenoisoquinolines, which are both proposed to project
into the major groove of the DNA-Top1 complex and hydrogen
bond to Top1 amino acids and water in the DNA major groove.27,28
Because of the high degree of SAR overlap at these positions (down
to specific substituents), it was proposed that ‘common’ SAR
elements are shared between the indenoisoquinolines and aroma-
thecins. The SAR studies also support the hypothesis that aromat-
hecins intercalate in a fashion similar to indenoisoquinolines.10–12
Due to the overall similarity in shape and structure to camptothec-
ins, it also was suggested that the aromathecins may bind in a dis-
tinctively ‘camptothecin-like’ pose.13,15 These models are purely
hypothetical, however, and because aromathecins are difficult to
crystallize with the enzyme, no X-ray structure of any aromathecin
in ternary complex with DNA and Top1 is currently available.
The present study was undertaken in order to explore the
hypothesis that the biological activity of the aromathecin system
could be modulated and eventually improved through substitution
at positions other than 14. To this date, no A-ring (positions 1–4)-
substituted aromathecins have been evaluated. Using some of the
H2N
R1
R2
H2N
R1
R2
a
O
n
Cl
9, R1, R2 = -O(CH2)2O-
16, R1, R2 = -OCH3
18, R1 = F, R2 = CH3
19, R1 = H, R2 = Cl
10, R1, R2 = -O(CH2)2O-, n = 1
11, R1, R2 = -O(CH2)2O-, n = 3
17, R1, R2 = -OCH3, n = 1
20, R1 = F, R2 = CH3, n = 1
21, R1 = H, R2 = Cl, n = 1
12, R1, R2 = -OCH2O-
15, R1, R2 = -OCH2O-, n = 1
b
d
AcHN
O
O
O
AcHN
c
O
O
14
13
Cl
Scheme 1. Reagents and conditions: (a) (i) BCl3ꢀMe2S, 1,2-dichloroethane, 0 °C; (ii)
chloroacetonitrile (to afford 10), 4-chlorobutyronitrile (to afford 11), AlCl3 (to afford
all except 16), reflux; (iii) 2 M HCl, reflux; (b) AcO2, Et3N, H2O, rt; (c) chloroacetyl
chloride, ZnCl2, CH3NO2; (d) concd HCl, reflux.

M. A. Cinelli et al. / Bioorg. Med. Chem. 18 (2010) 5535–5552
5537
ported by Sugasawa et al.36 (Scheme 2). Compound 23 was used to
prepare the 3-chloroaromathecin series, and 24, the 1-chloro
series.
The condensation of these ketones with 8 was performed under
Friedlander conditions,27 and the chloroalkylated aromathecin
cores 27a–34a were obtained in modest to excellent yield. In the
majority of cases, a stoichiometric amount (or less) of p-TsOH
was sufficient, although several cases did require higher tempera-
tures, longer times, and (in the case of 28a) catalytic amounts of
AcOH (Scheme 3).
Aromathecin analogues 27b–i (2,3-ethylenedioxy), 29b–f (2,3-
methylenedioxy), 30b–e (2,3-dimethoxy), 31b–g (2-methyl-3-flu-
oro), 32b–f (2-chloro) 33b–e (3-chloro) and 34b–d (1-chloro)
(Scheme 4) were prepared by simple SN2 displacement of the
unhindered, benzylic chloride by a nucleophile at room or slightly
elevated temperature in DMSO or DMF. Many nucleophiles were
chosen to explore an assortment of 14-substituents and to search
for possible synergistic or antagonistic effects with the A-ring sub-
stituents. Nucleophiles available as salts [(S)-pyrrolidin-3-ol38 and
N,N-dimethylamine] were used in the presence of excess triethyl-
amine. This displacement reaction proceeded overnight in most
cases to yield the substituted aromathecins in modest to excellent
yield, although displacement by imidazole (to prepare 27c, 30c, 32f
and 33e) was performed at a higher temperature. Some ethylene-
dioxy analogues (27d and 27f) were converted into trifluoroacetate
salts to aid in solubility. Likewise, analogues 34b–d were converted
to their hydrochloride salts.
H2N
Cl
O
H2N
Cl
a
O
23
Cl
22
Cl
+
b
H2N
Cl
AcHN
Cl
24
O
25
Cl
+
Cl
O
c
23 and 24
AcHN
Cl
Finally, to synthesize the ‘extended’ 30-substituted propyl ethyl-
enedioxy series 28b–g (Scheme 5), an in situ Finkelstein reaction27
was performed at high temperatures using both an excess of nucle-
ophile and sodium iodide to compensate for the decreased electro-
philicity of the terminal alkyl chloride.
26
Scheme 2. Reagents and conditions: (a) (i) BCl3ꢀMe2S, 1,2-dichloroethane, 0 °C; (ii)
chloroacetonitrile, AlCl3, reflux; (iii) 2 M HCl, reflux; (b) Ac2O, 80 °C; (c) concd HCl,
EtOH, 0 °C to reflux.
H2N
R1
R2
3. Results and discussion
O
Aromathecin analogues (with the exception of chlorinated com-
pounds 29a, 31a, and 32–34a, and azides 28f and 31f, due to the
historically low bioactivities of these compounds) were assayed
in the National Cancer Institute’s Developmental Therapeutics
Assay,39,40 where they were tested against the eight cancer cell line
subpanels described in Table 1 as well as a leukemia panel
(approximately 60 cell lines total). After an initial one-dose assay
(at 10ꢁ5 M), selected compounds were tested at five concentrations
ranging from 10ꢁ8 to 10ꢁ4 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 (10ꢁ8 to 10ꢁ4 M) are
assigned GI50 values of either 10ꢁ8 or 10ꢁ4 M. For comparative
purposes, Top1 and antiproliferative activity data for camptothecin
(1),17 indenoisoquinoline 4,21,41 clinical leads 5 and 6,25 and rosett-
acin (7) are included.
n
Cl
R3
10, R1, R2 = -O(CH2)2O-, n = 1
11, R1, R2 = -O(CH2)2O-, n = 3
15, R1, R2 = -OCH2O-, n = 1
17, R1, R2 = -OCH3, n = 1
20, R1 = F, R2 = CH3, n = 1
21, R1 = H, R2 = Cl, n = 1
23, R1 = Cl, R2 = H, n = 1
24, R1, R2 = H, R3 = Cl, n = 1
O
N
a
O
8
R1
3
Top1 inhibition was measured by a compound’s ability to in-
duce enzyme-linked DNA cleavage, and is graded by the following
semiquantitative rubric relative to 1 lM camptothecin: 0, no
N
R2
2
1
R3
N
O
n
Cl
inhibitory activity; +, between 20 and 50% activity; ++, between
50 and 75% activity; +++, between 75% and 95% activity; ++++,
equipotent to or more potent. Compounds that are between two
scores are delineated with parentheses [i.e., between ++ and +++
is ++(+)].
As can be observed in Figure 2, substitution on the A-ring very
easily changes a compound’s anti-Top1 activity for a given 14-sub-
stituent (here, the aminopropylimidazole side chain), although no
‘synergistic’ effect was observed with the 14-substituents. Several
important SAR trends can be gleaned from these data. The ethyl-
enedioxy group, in general, is well tolerated on the A-ring, and
for all except 27g, anti-Top1 activity is either unchanged or
27a, R1, R2 = -O(CH2)2O-, R3 = H, n = 1
28a, R1, R2 = -O(CH2)2O-, R3 = H, n = 3
29a, R1, R2 = -OCH2O-, R3 = H, n = 1
30a, R1, R2 = -OCH3, R3 = H, n = 1
31a, R1 = F, R2 = CH3, R3 = H, n = 1
32a, R1, R3 = H, R2 = Cl, n = 1
33a, R1 = Cl, R2, R3 = H, n = 1
34a, R1, R2 = H, R3 = Cl, n = 1
Scheme 3. Reagents and conditions: (a) p-TsOH, benzene or toluene, AcOH (for 11),
reflux overnight.

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M. A. Cinelli et al. / Bioorg. Med. Chem. 18 (2010) 5535–5552
R1
R1, R2 = -O(CH2)2O-, R3 = H, 27c
R1, R2 = -OCH3, R3 = H, 30c
R1, R3 = H, R2 = Cl, 32f
N
R2
R3
R1 = Cl, R2, R3 = H, 33e
N
O
N
b
N
R1
3
R1
3
N
R2
2
N
R2
2
a
1
1
R3
R3
N
14
N
14
R4
Cl
O
O
R1, R2 = -O(CH2)2O-, R3 = H, 27d . CF3COOH
R1, R2 = -OCH2O-, R3 = H, 29b
R1, R2 = -OCH3, R3 = H, 30d
R1 = F, R2 = CH3, R3 = H, 31b
R1, R3 = H, R2 = Cl, 32c
27a, R1, R2 = -O(CH2)2O-, R3 = H
29a, R1, R1 = -OCH2O-, R3 = H
30a, R1, R2 = -OCH3, R3 = H
31a, R1 = F, R2 = CH3, R3 = H
32a, R1, R3 = H, R2 = Cl
R4 = -
R4 = -
N
N
N
O
R1 = Cl, R2, R3 = H, 33c
R1, R2 = H, R3 = Cl, 34b . 3 HCl
33a, R1 = Cl, R2, R3 = H
34a, R1, R2 = H, R3 = Cl
R1, R2 = -O(CH2)2O-, R3 = H, 27e
R1, R2 = -O(CH2)2O-, R3 = H, 27f . CF3COOH
R1, R2 = -OCH2O-, R3 = H, 29c
R1 = F, R2 = CH3, R3 = H, 31e
R1, R3 = H, R2 = Cl, 32e
c
F
R4 = NH(CH2)2OH
R4 = -N(CH3)2
N
R1, R2 = -O(CH2)2O-, R3 = H, 27b
R1, R2 = -OCH2O-, R3 = H, 29d
N
N3
R1, R2 = -O(CH2)2O-, R3 = H, 27g
R1, R2 = -OCH2O-, R3 = H, 29e
R1, R2 = -OCH3, R3 = H, 30b
O
31f
R4 = -
N
OH
R1 = F, R2 = CH3, R3 = H, 31c
R1, R3 = H, R2 = Cl, 32d
R1 = Cl, R2, R3 = H, 33d
d
.
R1, R2 = H, R3 = Cl, 34c HCl
F
R1, R2 = -O(CH2)2O-, R3 = H, 27h
R1, R2 = -OCH2O-, R3 = H, 29f
N
N
N
R1, R2 = -OCH3, R3 = H, 30e
R4 = -
NH
N
N
R1 = F, R2 = CH3, R3 = H, 31d
R1, R3 = H, R2 = Cl, 32b
NH2
O
R1 = Cl, R2, R3 = H, 33b
. 2 HCl
R1, R2 = H, R3 = Cl, 34d . 3 HCl
31g
R4 = -
NH
R1, R2 = -O(CH2)2O-, R3 = H, 27i
O
Scheme 4. Reagents and conditions: (a) (i) Amine or amine salt + Et3N, DMSO or DMF; (ii) HCl/MeOH (series 34) or CF3COOH (27d and f); (b) imidazole, DMSO or DMF, 60–
100 °C; (c) for 31a, NaN3, DMSO, rt; (d) (i) (EtO)3P, benzene, reflux; (ii) HCl/MeOH, reflux.
improved over the analogous unsubstituted compounds. For the
monomethylene compounds 27b, 27e, and 27i, Top1 inhibitory
activity is improved, and for 27a, 27c, 27d, 27f and 27h, the anti-
Top1 activity is unchanged. Although they are potent Top1 poisons,
more variability (with the same comparison to the unsubstituted
series) is seen in the extended (28) series. General variability was
also observed for extended unsubstituted compounds alone.27
As antiproliferative activity involves factors more complicated
than Top1 inhibition alone (as evidenced by low correlation be-
tween Top1 inhibition and cytotoxicity27,28,42) the GI50 values are
variable in both the monomethylene and extended series, although
improvements in potency on a per-analogue basis are observed in
several cases, and many are more potent than clinical candidate 5.
Examining the antiproliferative activity of this entire series of eth-
ylenedioxy compounds (series 27 and 28), although some were not
selected for five-dose testing, all compounds (except for 28a, which
was not tested for cytotoxicity) possessed at least some activity. At
a concentration of 10 lM, compound 27a inhibited cell growth at a
mean 10.3%, 27d at 23%, 27i at 10.8%, 28e at 19%, and 27h at 14.3%.
It is unknown exactly how the ethylenedioxy group may en-
hance potency, although it is reported that this group improves
activity for camptothecin derivatives, such as in the clinical candi-
date lurtotecan (Fig. 3 and 35)1,19,43–45 For camptothecins, place-
ment of ethylenedioxy (or methylenedioxy) groups at the

M. A. Cinelli et al. / Bioorg. Med. Chem. 18 (2010) 5535–5552
5539
effect was not observed for 2,3-methylenedioxyaromathecins.
These compounds still retain notable antiproliferative activity
O
O
a
(compound 29d possessed 22.3% inhibition at 10 lM as well) but
N
there is no trend toward improved Top1 inhibition. In our experi-
ence, these compounds were very insoluble, and unfavorable phys-
ical properties could hinder obtaining significant SAR data.
28b, R = -NH(CH2)2OH
N
Other A-ring substituents lend additional support to a proposed
camptothecin-like binding mode. In the elegant studies by Monroe
Wall and colleagues, it is reported that methoxy substituents,
including 10,11-dimethoxy substitution, attenuate bioactivity
when placed on the A-ring of camptothecin.51,52 Remarkably, this
trend is also observed for the aromathecins, indicating more SAR
overlap. Compounds 30a–30e all possess minimal (all 0 or 0/+)
anti-Top1 activity. Aromathecin 30b has only 14.2% mean growth
O
28c, R = - N
N
28a, R = Cl
R
28d, R = -
N
O
b
28e, R = -N(CH3)2
inhibition at 10 lM, and the remainder of the series was declined
O
for testing by the NCI. The bulkier, free-rotating methoxy groups
could simply pose a steric liability toward the nonscissile strand
or decrease the number of planar conformations available due to
repulsions between the groups. Even before crystallography, it
was proposed that the inactivity of dimethoxycamptothecins was
caused by a steric ‘blocking’ effect.52,53
O
N
N
O
The 2-methyl-3-fluoro compounds 31b–g were originally syn-
thesized to mimic fluorinated camptothecins such as exatecan
(36) and diflomotecan (37)1,17 (Fig. 3). Disappointingly, no in-
creases in Top1 inhibitory activity were seen in this series, and
antiproliferative activity generally decreased (neither compound
28f
N3
31c nor 31e induced >25% growth inhibition at 10 lM). This is
c
not entirely surprising, as other 10,11-disubstitution patterns are
reported to have deactivating effects for camptothecins as well. A
comparison between compounds in this series and the clinical can-
didates in Figure 3 is perhaps unjustified due to the presence of
other structural motifs (e.g., chiral amines, homolactones) that aro-
mathecins lack. The increased antitumor activity observed for
these fluorinated camptothecins could also be due to their lipophil-
icity, which increases partitioning to lipids and slows the rate of
lactone hydrolysis in vitro and in vivo.54
O
O
N
N
O
. 2 HCl
H2N
28g
In light of these deactivating disubstitution patterns, it is worth
mentioning that substitution at position 11 tends to diminish anti-
Top1 activity for camptothecins, regardless of the substituent.55
This effect is identical for aromathecins, observed in the placement
of both a methoxy (series 30) and fluorine (series 31) at the anal-
ogous 3-position. Placement of chlorine at this position (series
33) also abolishes most of the Top1 inhibitory activity, indicating
the same steric constraints that hinder camptothecins at this posi-
tion exist for the aromathecin system as well. Modeling compound
33b (Fig. 6) in ternary complex with Top1 and DNA indicates that
when this compound is minimized in our proposed ‘camptothecin-
like’ binding mode (Fig. 4), the bulky, electronegative chlorine
could pose steric and electronic liabilities when projected toward
the phosphodiester backbone of the nonscissile strand. Upon min-
imization, the planar aromatic system of this model distorts to pos-
sibly avoid these clashes (Fig. 6). Indeed, Staker et al.’s work
indicates that this region is also sterically constrained in crystal
structures of indenoisoquinolines and indolocarbazoles.10,11 Addi-
tionally, prior to these studies, Wang et al. proposed that modifica-
tions at this region of camptothecin (positions 9–11) could greatly
affect its biochemical activity due to close proximity to flanking
nucleotides.56 Nonetheless, cyclic groups (e.g., ethylenedioxy) are
more restrained and could theoretically be better accommodated,
as cyclic groups are also proposed to face the nonscissile strand
for indenoisoquinolines.57 Even if substituted camptothecins
bound in an alternative mode to fit these groups, it is likely that
aromathecins behave similarly due to the trends in bioactivity.
Antiproliferative activity for the 3-chloro series is also variable;
(cf. compound 33b with 33d and 33e, the latter two of which did
Scheme 5. Reagents and conditions: (a) Amine, NaI, DMSO or DMF, 100 °C; (b)
NaN3, DMSO, 100 °C (c) (i) (EtO)3P, benzene, reflux; (ii) 3 M HCl, MeOH, reflux.
analogous 10,11 position may improve solubility over the parent
compound, although it is clear that improved solubility is not solely
responsible for improved potency, as racemic 9,10-methylenedi-
oxycamptothecin is only 20% as active as the 10,11-isomer.45 For
hexacyclic camptothecin analogues, hypotheses ranging from ‘ex-
tended coplanarity’42,46 to increased positive charge density47 have
been posited. In our earlier proposed binding mode (Fig. 4) the aro-
matic system (here, of 27h, which was chosen due to its initial high
potency against Top1) intercalates between the base pairs and the
A-ring of the aromathecins abuts the nonscissile DNA strand as the
ligand in the crystal structures of topotecan and camptothecin
does.27,28,48 The E-ring faces the cleaved strand, and the quinoline
nitrogen of the B-ring faces the general direction of Arg364, which
it may hydrogen bond to (although it is out of distance in Fig. 4).
Overlaying hypothetical models of aromathecin 27h with the
ligand in a camptothecin crystal structure (Fig. 5) roughly superim-
poses the A-rings of the two systems. These compounds’ ethylene-
dioxy groups could be positioned similarly and thus exert their
effect via similar mechanisms, which could lend support to the
proposed binding mode shown in Figure 4.
10,11-Methylenedioxycamptothecins are potent cytotoxic Top1
inhibitors,45,46,49,50 and are usually more so than the corresponding
ethylenedioxycamptothecins, possibly due to the dioxolane ring’s
ability to better hold coplanarity.46 Disappointingly, the same
not inhibit cell growth beyond 15% at 10 lM).

5540
M. A. Cinelli et al. / Bioorg. Med. Chem. 18 (2010) 5535–5552
Table 1
Antiproliferative potencies and topoisomerase I inhibitory activities of A-ring- and 14-substituted aromathecin analogue series 27–32
Compd
Cytotoxicitya (GI50 in
lM)
MGMb
Top1 cleavagec
Lung
HOP-62
Colon
HCT-116
CNS
SF-539
Melanoma
UACC-62
Ovarian
OVCAR-3
Renal
SN12C
Prostate
DU-145
Breast
MCF-7
117
0.01
0.02
1.78
<0.01
>100
—
3.31
19.5
—
3.80
5.69
1.01
—
0.03
0.10
1.15
<0.01
57.3
—
1.17
>100
—
1.33
2.63
0.82
—
0.01
0.04
0.04
0.037
>100
—
2.69
4.37
—
0.81
4.26
0.49
—
0.01
0.03
0.03
<0.01
>100
—
1.66
21.4
—
0.99
1.16
0.46
—
0.22
0.5
74.1
0.085
>100
—
2.19
18.6
—
1.70
1.95
1.84
—
0.02
<0.01
0.813
<0.01
>100
—
2.51
81.3
—
2.57
5.75
0.94
—
0.01
<0.01
0.155
<0.01
>100
—
2.19
>100
—
0.93
2.04
1.02
—
0.01
<0.01
0.37
0.01
60.3
—
0.30
0.30
—
0.30
0.44
0.27
—
0.0405 0.0187f
0.21 0.19
4.64 1.25
0.079 0.023
91.2
—
2.95
19.05
++++
++++
++++
++++
++
417
520
620
7
27a
27b
27c
27dd
27e
27f
27g
27hd
27i
+
+++
+++
++(+)
++(+)
++(+)
++
—
1.78 0.165
2.33 0.24
1.24 0.21
—
++
++
—
—
—
—
—
—
—
—
—
28a
28b
28c
28d
28e
28g
29b
29c
29d
29e
29f
30a
30b–ee
31b
31c
31d
31e
31g
32b
32c
32d
32e
32f
—
—
—
—
—
—
—
—
—
0
++
1.01
0.57
0.54
—
2.14
17.0
0.66
—
0.48
5.01
—
—
20.1
—
0.82
1.95
1.42
—
1.95
1.45
0.95
—
0.85
1.32
—
—
1.80
—
0.49
1.95
1.15
—
1.86
11.7
1.35
—
0.60
2.14
—
—
6.46
—
0.46
0.93
1.15
—
1.62
1.62
0.47
—
0.35
3.39
—
—
16.2
—
1.84
7.08
1.84
—
1.95
16.2
1.55
—
>100
4.26
—
—
14.6
—
0.94
12.6
1.13
—
1.86
2.34
1.91
—
0.79
5.89
—
—
16.0
—
1.80
20.2
—
—
1.78
—
10.2
1.54
2.09
19.0
—
1.02
2.88
0.76
—
1.82
51.3
1.82
—
—
2.57
—
—
2.63
—
0.27
0.19
0.06
—
1.05
0.38
0.28
—
0.07
0.48
—
—
1.80
—
2.35 0.215
3.24
0.82 0.03
—
2.14
8.91
2.00
—
6.91
5.50
—
—
6.31 0.145
—
11.3 0.95
14.4 1.81
—
++(+)
++(+)
+++
+++
++
++(+)
++
++
++
0
0 or 0/+
++(+)
++
36.7
11.2
—
3.05
18.4
—
1.10
9.23
—
>100
14.2
—
4.68
16.8
—
6.24
12.2
—
2.21
1.62
—
0
++(+)
+(+)
++
+(+)
+(+)
++
+
+
+
+
—
—
—
—
—
—
—
—
2.29
1.05
7.24
0.46
2.00
18.1
—
0.15
1.10
2.14
0.40
1.82
13.5
—
1.58
1.26
4.36
0.39
1.86
15.1
—
1.41
0.74
2.04
0.31
1.91
17.0
—
1.51
1.86
6.46
0.69
2.04
15.1
—
2.75
1.66
—
0.48
—
23.4
—
—
3.31
0.37
0.23
0.19
1.66
10.9
—
1.85 0.34
1.44
5.02
1.02
2.51
14.5
—
—
4.37
33b
33c
33d
33e
34b
34c
34d
—
—
—
—
—
—
—
0
0
+
+++
1.86
1.66
2.51
1.41
1.55
1.58
1.51
1.95
1.90
15.5
3.09
7.24
4.17
2.63
4.90
13.8
3.71
6.03
9.77
3.98
1.02
1.00
0.37
0.55
2.40
3.09
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 10ꢁ8 to 10ꢁ4 molar.
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 95% activity; ++++, equipotent to or more potent. Compounds that are ranked between two scores are
delineated with parentheses [i.e., between ++ and +++ is ++(+)].
d
Some compounds such as this were not selected for further testing; refer to text for details.
These compounds were declined for testing by the NCI.
For MGM GI50 values in which a standard error appears, the GI50 values for individual cell lines are the average of two determinations; values without standard error are
e
f
from one determination. The values for 1, 4, 5, and 6 are from many determinations.
Halogens improve anti-Top1 potency for camptothecins when
placed at positions 9 and 10.45 The placement of chlorine at posi-
tion 2 of the aromathecin system is tolerated in some cases but
does not confer any advantages. Interestingly, these compounds
possess relatively high antiproliferative activity (even 32b, which
was not tested in the 5-dose assay, inhibited cell growth at nearly
logues synthesized in one study,45 the equivalent 1-chloroaro-
mathecin series cannot be considered an outlier to the
overlapping SAR and binding mode hypotheses. Many groups
report that, in addition to halogens, a variety of 9-substituents
including hydroxyl groups and amines,45,58 oxyimino moieties,59
alkyl chains60 and nitro groups61 can aid in improving Top1 inhibi-
tion, antitumor activity, bioavailability, and pharmacokinetics. Per-
haps the effects of the 9-substituent could be more general than
previously thought.59 Due to synthetic difficulties, no additional
1-substituted aromathecins have been prepared, and the full role
1-substitution plays has yet to be determined.
30% at 10 lM). Nonetheless, the exact nature of this effect is un-
known and several of these compounds may undergo additional
testing. Ultimately, action at a second target besides Top1 may
be responsible, although preliminary studies with aromathecins
indicated that they do not inhibit Top2.28 Unfortunately, the iso-
meric 1-chloro series (34b–d) possessed less anti-Top1 activity in
two out of three cases, but molecular models (not shown) did
not indicate any obvious steric or electronic encumbrances.
Although 9-chlorocamptothecin was among the most potent ana-
The idea that aromathecins may bind in a manner similar to
camptothecins is not entirely novel. The former’s 14 substituents,
capable of hydrogen bonding, are calculated to project into the
major groove of the ternary complex.27,28 Many 7-substituted

M. A. Cinelli et al. / Bioorg. Med. Chem. 18 (2010) 5535–5552
5541
Figure 2. Top1-mediated DNA cleavage induced by aromathecins 33b, 34d, 30e, 32b, 27h, 29f, and 31d. Lane 1: DNA alone; lane 2: Top1 alone; lane 3: 1, 1
l
M; lane 4: 4,
1 lM; lanes 5–32: lanes 6–29: 33b, 34d, 30e, 32b, 27h, 29f, and 31d at 0.1, 1, 10, and 100 lM, respectively, from left to right. Numbers and arrows on left indicate arbitrary
cleavage site positions.
O
N
were assayed for Top1 inhibition and antiproliferative activity. De-
spite the lack of a strong correlation between anti-Top1 activity
and cytotoxicity, it has been shown that varying the steric bulk, elec-
tronegativity, and hydrogen bonding properties of these A-ring sub-
stituents can drastically change the bioactivity of the aromathecin
system. The nature of these SAR trends mirror those previously ob-
served for camptothecins (with respect to the ethylenedioxy group,
disubstitution, and positions 11 and 3), and these data, coupled with
prior knowledge of positions 7 and 14, strongly support the hypoth-
esis that these two systems share many essential SAR elements,
which could provide a viable avenue for further optimization of this
system. As there is no crystal structure available for aromathecins,
these results also provide the only experimental evidence support-
ing our proposed camptothecin-like binding mode.
O
OH
N
O
CH3
N
O
N
O
Lurtotecan
35
F
CH3
OH
N
O
O
N
H2N
5. Experimental
O
Exatecan
5.1. General procedures
36
F
Reagents and solvents were purchased from commercial ven-
dors and were used without further purification. Melting points
were determined in capillary tubes using a Mel-Temp apparatus
and are not corrected. Infrared spectra were obtained as films on
salt plates using CHCl3 or CDCl3 as the solvent unless 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 or Bruker Avance 500 (QNP
probe or TXI 5 mm/BBO probe), respectively. Mass spectral analy-
ses 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 spec-
trometer system. APCI-MS was performed using an Agilent 6320
Trap mass spectrometer. Purity of all biologically relevant com-
pounds is ꢂ95% by combustion microanalysis. Combustion micro-
analyses were performed by Galbraith Laboratories (Knoxville, TN),
Midwest Microlab LLC (Indianapolis, IN), or at the Purdue Univer-
OH
F
N
O
N
O
O
Diflomotecan
37
Figure 3. A-ring substituted camptothecins.
camptothecins (Fig. 3) also possess similar substituents that would
also sit in the major groove region if they bound in the same pose
as camptothecin and topotecan.10,15
4. Conclusions
In conclusion, eight novel series of A-ring-substituted aromathe-
cin analogues were prepared by a practical, modular route beginning
with commercially available starting materials. These analogues
sity Microanalysis Laboratory using
a Perkin-Elmer Series II
CHNS/O model 2400 analyzer. All reported values are within 0.4%

5542
M. A. Cinelli et al. / Bioorg. Med. Chem. 18 (2010) 5535–5552
Figure 4. Hypothetical model for binding of 2,3-ethylenedioxyaromathecin 27h in a Top1-DNA complex. The ligand is colored in magenta and all other relevant structures
are labeled and colored by atom type. Distances are from heavy atom to heavy atom; the diagram is programmed for wall-eyed (relaxed) viewing.
diluted with 1,2-dichloroethane (30–110 mL) and the mixture was
cooled to 0 °C under an argon atmosphere. The aniline (1–4 g,
ꢃ10–26 mmol) was added dropwise, or, in the case of solid ani-
lines, as a solution in a minimal amount of dichloroethane. The
resulting chunky precipitate was stirred for 15 min, and chloro-
acetonitrile (for compounds 10, 17, 20–21, and 23–24) or chlo-
robutyronitrile (for 11) was added. For all but 17, aluminum
chloride (approximately 1.2 equiv based on the aniline) was then
added, and the mixture was warmed to room temperature, stirred
for approximately 15 min and heated at reflux for between 1.5–4 h
(typical time: 3 h). The mixture was cooled, and 2 M HCl (an
amount equivalent to the solvent volume of the reaction) was
added. The mixture was heated to reflux for 0.5 h, and then cooled
and poured into ice water (>100 mL) and the aqueous and organic
layers were separated. The aqueous layer was exhaustively ex-
tracted with CH2Cl2, and the organic phases were washed with
Figure 5. Ligand overlay of aromathecin 27h (magenta) and camptothecin (1,
water and satd NaCl and dried over anhydrous sodium sulfate.
yellow). Relevant positions are indicated in their respective colors and the A-ring is
Concentration afforded crude products that were purified by pre-
labeled.
cipitation, crystallization, or column chromatography.
a
-chloro-4,5-ethylenedioxyacetophenone (10)19
of calculated values. Analytical thin-layer chromatography was
5.2.1. 2-Amino-
Boron trichloride-methyl sulfide complex (1.78 g, 9.92 mmol),
compound (1.00 g, 6.61 mmol), chloroacetonitrile (0.624 g,
8.27 mmol) and aluminum chloride (1.32 g, 9.92 mmol), in dichlo-
roethane, provided a yellow solid that was isolated by precipitating
with EtOAc–hexanes (0.251 g, 17%): mp 110 °C (lit.19 mp 130 °C).
1H NMR (300 MHz, CDCl3) d 7.13 (s, 1H), 6.15 (s, 1H), 4.56 (s,
2H), 4.31–4.28 (m, 2H), 4.22–4.19 (m, 2H).
performed on Baker-flex silica gel IB2-F plastic-backed TLC plates.
Compounds were visualized with both short and long wavelength
UV light. Silica gel flash chromatography was performed using 40–
9
63 l
m flash silica gel. Precursor compounds 827,28 and 13, 14, and
1519,62 were prepared according to literature procedures and as de-
picted in Schemes 1 and 2.
5.2. General procedure for chloroacylation of substituted
anilines (10–11, 17, 20–21, and 23–24)35–37
5.2.2. 1-(7-Amino-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4-
chloro-1-butanone (11)
Boron trichloride-methyl sulfide complex (approximately 1.80–
5.20 g, 10.0–29.0 mmol, or 1.1–1.5 equiv, based on the aniline) was
Boron trichloride-methyl sulfide complex (5.23 g, 29.1 mmol),
compound 9 (4.00 g, 26.5 mmol), 4-chlorobutyronitrile (3.42 g,
Figure 6. Hypothetical model for binding of 3-chloroaromathecin 33b in a Top1-DNA complex. The ligand is colored in green and all other relevant structures are labeled and
colored by atom type. Distances are from heavy atom to heavy atom. The C, D, and E rings of the aromathecin are coplanar; note the substantial out-of-plane bending to
relieve possible steric and/or electronic clashes on the A-ring side. Distances are heavy atom to heavy atom. The diagram is programmed for wall-eyed (relaxed) viewing.

M. A. Cinelli et al. / Bioorg. Med. Chem. 18 (2010) 5535–5552
5543
33.1 mmol) and aluminum chloride (3.89 g, 29.1 mmol), in dichlo-
roethane, afforded a residue that was adsorbed onto SiO2 (7.64 g),
and purified by flash column chromatography (SiO2, 59.6 g), elut-
ing with CH2Cl2, to yield a clear-yellow oil. Residual 4-chlorobuty-
ronitrile was removed in vacuo by gentle heating in a water bath
(50 °C). The crude product was obtained as a sticky red solid,
(2.00 g, 29%) contaminated with a small amount of nitrile. This
material was used in the next step without further purification
(to prepare 26a). 1H NMR (300 MHz, CDCl3) d 7.34 (s, 1H), 6.49
(s, 1H), 4.31–4.23 (m, 4H), 3.68 (t, J = 6.3 Hz, 2H), 3.06 (t,
J = 7.0 Hz, 2H), 2.25–2.10 (m, 2H).
(8.52 g). The residue was purified by flash column chromatography
(SiO2, 54.9 g), eluting with a gradient of 10% hexanes in CH2Cl2 to
1% MeOH in CH2Cl2 to yield, first, 25 (0.780 g, 13%), as white nee-
dles after recrystallization from 50% EtOH in CH2Cl2 and washing
with hexanes: mp 131–132 °C (lit36 mp 139–140 °C). 1H NMR
(300 MHz, CDCl3) d 11.4 (br s, 1H), 8.91 (d, J = 2.0 Hz, 1H), 7.77
(d, J = 8.5 Hz, 1H), 7.14 (dd, J = 6.9, 1.9 Hz, 1H), 4.73 (s, 2H), 2.26
(s, 3H) and then 26 (0.270 g, 5%), as an orange solid: mp 115–
117 °C (lit36 mp 130–132 °C). 1H NMR (300 MHz, CDCl3) d 8.19
(br s, 1H), 8.02 (d, J = 8.2 Hz, 1H), 7.44 (t, J = 8.2 Hz, 1H), 7.27 (t,
J = 9.5 Hz, 1H), 4.71 (s, 2H), 2.19 (s, 3H).
5.2.3. 2-Amino-
a
-chloro-4,5-(dimethoxy)acetophenone (17)36
5.2.6.2. Hydrolysis of the acetanilides: 4-chloro-2-amino-a-
Boron trichloride-methyl sulfide complex (3.69 g, 20.6 mmol),
compound 16 (3.00 g, 19.6 mmol), and chloroacetonitrile (1.78 g,
23.5 mmol), in dichloroethane, afforded a brown semisolid that
was dissolved in CH2Cl2 and filtered through a pad of SiO2 to re-
move black impurities. The pad was rinsed with CH2Cl2 (120 mL)
until the filtrate ran clear and colorless. The filtrate was concen-
trated to yield a brown solid (2.33 g, 52%) after washing with hex-
anes (40 mL): mp 113–115 °C (lit36 mp 123–124 °C). 1H NMR
(300 MHz, CDCl3) d 7.00 (s, 1H), 6.50–6.20 (br s, 2H), 6.12 (s, 1H),
4.58 (s, 2H), 3.89 (s, 3H), 3.84 (s, 3H).
chloroacetophenone (23)36. Compound 25 (0.780 g, 3.17 mmol)
was dissolved in EtOH (40 mL), and concd HCl (5 mL) was added.
The mixture was heated to reflux for 1 h, and the resultant dark or-
ange solution was cooled and poured into ice water (150 mL). 2 M
NaOH was added until the pH was approximately 9. The mixture
was extracted with CH2Cl2 (3 ꢄ 150 mL), and the organic phase
was washed with H2O (200 mL), dried over anhydrous sodium sul-
fate, and concentrated to yield 23 as an orange iridescent solid
(0.600 g, 93% from 25, 12% from 22): mp 126–128 °C (lit36 mp
134–135 °C). 1H NMR (300 MHz, CDCl3) d 7.58 (d, J = 8.7 Hz, 1H),
6.71 (d, J = 1.9 Hz, 1H), 6.65 (dd, J = 7.5, 2.0 Hz, 1H), 6.39 (br s,
2H), 4.63 (s, 2H).
5.2.4. 2-Amino-4-fluoro-5-methyl-a-chloroacetophenone (20)
Boron trichloride-methyl sulfide complex (5.16 g, 28.8 mmol),
aniline 18 (3.00 g, 23.0 mmol), chloroacetonitrile (2.26 g, 30.0
mmol), and aluminum chloride (4.79 g, 36.0 mmol), in dichloroeth-
ane, afforded a residue that was adsorbed onto SiO2 (8.84 g) and
purified by flash column chromatography (SiO2), eluting with a gra-
dient of 50% hexanes in CH2Cl2 to CH2Cl2 to yield the title compound.
Additional column fractions yielded residue that, after recrystalliza-
tion from boiling hexanes (100 mL), afforded the product as fine
yellow-green needles. A total of 0.781 g (16%) was obtained: mp
111–113 °C. IR (film) 3449, 3341, 2946, 1656, 1639, 1592, 1553,
1229, 1139, 867, 849, 781, 619; 1H NMR (300 MHz, CDCl3) d 7.47
(d, J = 8.3 Hz, 1H), 6.35 (d, J = 11.5 Hz, 1H), 6.30 (br s, 2H), 4.62 (s,
2H), 2.18 (s, 3H); CIMS m/z (rel intensity) 202 (MH+, 100).
5.2.7. 6-Chloro-2-amino-a
-chloroacetophenone (24)36
Compound 26 (0.270 g, 1.10 mmol) was diluted in EtOH
(12 mL), and concd HCl (3 mL) was added. The mixture was heated
at reflux for 1 h 15 min, cooled, and poured into ice water (50 mL).
2 M NaOH was added until the pH was approximately 9, and the
mixture was extracted with CH2Cl2 (2 ꢄ 40 mL). The organic layers
were washed with H2O (1 ꢄ 100 mL), dried over anhydrous sodium
sulfate, and concentrated. The residue was adsorbed onto SiO2
(5.0 g), and was purified by flash column chromatography (SiO2,
28.5 g), eluting with CH2Cl2 to yield
a yellow-green solid
(0.158 g, 71% from 26, 3.3% from 22): mp 50.5–52 °C (lit36 mp
60–61 °C). 1H NMR (300 MHz, CDCl3) d 7.16 (t, J = 8.1 Hz, 1H),
6.76 (d, J = 7.9 Hz, 1H), 6.63 (d, J = 8.3 Hz, 1H), 4.97 (br s, 1H),
4.74 (s, 2H).
5.2.5. 5-Chloro-2-amino-a
-chloroacetophenone (21)36
Boron trichloride-methyl sulfide complex (3.08 g, 17.2 mmol),
4-chloroaniline (19, 2.00 g, 15.6 mmol), chloroacetonitrile (1.47 g,
19.5 mmol), and aluminum chloride (2.50 g, 18.7 mmol), in dichlo-
roethane, afforded a black gum. This residue was adsorbed onto
SiO2 (4.17 g) and purified by flash column chromatography (SiO2,
56.8 g), eluting with 50% hexanes in CH2Cl2, to yield an iridescent
green solid (0.230, 7.2%): mp 130–133 °C (lit36 mp 140–141 °C.)
1H NMR (300 MHz, CDCl3) d 7.59 (d, J = 2.3 Hz, 1H), 7.27–7.23 (m,
1H), 6.67 (d, J = 8.9 Hz, 1H), 6.30 (br s, 1H), 4.64 (s, 2H).
5.3. Synthesis of aromathecin cores (27a–34a)
5.3.1. 14-Chloromethyl-2,3-ethylenedioxy-12H-5,11a-dia-
zadibenzo[b,h]fluoren-11-one (27a)
Compound 8 (0.100 g, 0.502 mmol) and compound 10 (0.114 g,
0.502 mmol) were diluted with benzene (30 mL). p-TsOH monohy-
drate (0.005 g, 0.026 mmol) was added and the solution was
heated at reflux for 16 h using a Dean-Stark trap to collect azeotr-
oped water. The solution was concentrated, diluted with CHCl3
(175 mL) and washed with satd NaHCO3 (3 ꢄ 50 mL) and satd NaCl
(50 mL). The organic layer was dried over sodium sulfate, concen-
trated, and purified by flash column chromatography (SiO2), elut-
ing with a gradient of CHCl3 to 3% MeOH in CHCl3 to provide a
yellow solid (0.107 g, 55%): mp 275 °C (dec). IR (KBr) 1655, 1628,
1605, 1288, 1243, 1226, and 1068 cmꢁ1; 1H NMR (300 MHz, CDCl3)
d 8.55 (d, J = 8.1 Hz, 1H), 7.79–7.57 (m, 5H), 7.55 (s, 1H), 5.40 (s,
2H), 4.95 (s, 2H), 4.45 (s, 4H); ESIMS m/z (rel intensity) 391/393
(MH+, 96/33). Anal. Calcd for C22H15ClN2O3ꢀ0.5H2O: C, 66.09; H,
4.03; N, 7.01. Found: C, 66.02; H, 3.97; N, 7.14.
5.2.6. 4-Chloro-2-amino-a-chloroacetophenone (23), 6-chloro-
2-amino-a-chloroacetophenone (24), and their respective N-
acetyl derivates 25 and 2636
Boron trichloride-methyl sulfide complex (4.63 g, 25.9 mmol),
compound 22 (3.00 g, 23.5 mmol), chloroacetonitrile (2.21 g,
29.5 mmol), and aluminum chloride (3.76 g, 28.2 mmol), in dichlo-
roethane, yielded a green solid. Both 23 and 24 were present by
TLC. The residue was adsorbed onto SiO2 (11.8 g), and was purified
by flash column chromatography (SiO2, 91.4 g), eluting with 25%
hexanes in CH2Cl2, to remove unwanted by-products.
5.2.6.1. Synthesis and separation of 25 and 26. The obtained res-
idue containing 23 and 24 was diluted with acetic anhydride
(10 mL), and the mixture was heated to 80 °C for 30 min, upon
which it became a dark red. The mixture was concentrated to re-
move excess acetic anhydride, and was adsorbed onto SiO2
5.3.2. 14-(30-Chloropropyl)-2,3-ethylenedioxy-12H-5,11a-dia-
zadibenzo[b,h]fluoren-11-one (28a)
Compound 11 (0.481 g, 1.88 mmol) was suspended in CHCl3
(5 mL). Compound 8 (0.300 g, 1.50 mmol) was added, followed
by p-TsOH (0.285 g, 1.50 mmol), glacial acetic acid (2 mL), and

5544
M. A. Cinelli et al. / Bioorg. Med. Chem. 18 (2010) 5535–5552
toluene (60 mL). The mixture was heated at reflux for 17.5 h, using
a Dean-Stark trap. The mixture was cooled to room temperature,
concentrated, and the residue was suspended in a mixture of
MeOH (30 mL) and CHCl3 (100 mL). The suspension was washed
with satd NaHCO3 (200 mL) and H2O (200 mL). The aqueous layer
was extracted with CHCl3 (50 mL). The organic layers were washed
with H2O (200 mL), satd NaCl (125 mL), dried over anhydrous so-
dium sulfate, and treated with decolorizing carbon (0.100 g). The
mixture was filtered, concentrated, adsorbed onto SiO2 (3.80 g),
and purified by flash column chromatography (SiO2, 62.8 g), elut-
ing with CHCl3, to yield a yellow amorphous solid (0.275 g, 44%)
after washing with MeOH (10 mL): mp 255–258 °C. IR (film)
3401, 2930, 1659, 1627, 1603, 1506, 1440, 1287, 1244, 1068,
was cooled, concentrated, and the residue was suspended in CHCl3
(100 mL) and washed with satd NaHCO3 (100 mL). The aqueous
layers were extracted with CHCl3 (100 mL). The combined organic
layers were washed with H2O (200 mL), following which the aque-
ous layer was again extracted with CHCl3 (50 mL). The combined
organic layers were washed with satd NaCl (200 mL). The aqueous
phase was again extracted with CHCl3 (50 mL). The combined or-
ganic layers were dried over anhydrous sodium sulfate, concen-
trated, and adsorbed onto SiO2 (8.30 g). Purification by flash
column chromatography (SiO2), eluting with a gradient of CHCl3
to 4% MeOH in CHCl3, resulted in precipitation of the compound
on the column. Elution was continued with CHCl3. The obtained or-
ange powder was boiled in tetrahydrofuran (80 mL), filtered, and
washed with CHCl3 and ether to yield the title compound as a pale
orange powder (0.464 g, 63%): mp 300–303 °C (dec). IR (KBr pellet)
3028, 3923, 2363, 2342, 1657, 1631, 1602, 1480, 1435, 1341, 1232,
915, 870, 687 cmꢁ1
; d 8.55 (d,
1H NMR (300 MHz, CDCl3)
J = 8.2 Hz, 1H), 7.77–7.52 (m, 5H), 7.48 (s, 1H), 5.31 (s, 2H), 4.43
(s, 4H), 3.69 (t, J = 6.0 Hz, 2H), 3.27 (t, J = 7.5 Hz, 2H), 2.27–2.17
(m, 2H). Anal. Calcd for C24H19ClN2O3ꢀ0.75H2O: C, 66.67; H, 4.78;
N, 6.48. Found: C, 66.28; H, 4.46; N, 6.40.
1222, 1133, 899, 768, 726, 690 cmꢁ1 1H NMR (300 MHz, CDCl3) d
;
8.57 (d, J = 7.8 Hz, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.86–7.57 (m, 5H),
5.44 (s, 2H), 5.03 (s, 2H), 2.58 (s, 3H); ESIMS m/z (rel intensity)
365 (MH+, 100).
5.3.3. 14-Chloromethyl-2,3-methylenedioxy-12H-5,11a-diaza-
dibenzo[b,h]fluoren-11-one (29a)
Compound 8 (0.200 g, 1.00 mmol) and compound 15 (0.236 g,
1.10 mmol) were diluted with toluene (30 mL) and p-TsOH
(0.190 g, 1.00 mmol) was added. The mixture was heated at reflux,
using a Dean-Stark trap, for 23.5 h. The bright orange suspension
was cooled and concentrated, and the residue was suspended in
CHCl3 (100 mL) and washed with satd NaHCO3 (100 mL). The aque-
ous layer was extracted with CHCl3 (5 ꢄ 50 mL). The combined or-
ganic layers were dried over anhydrous sodium sulfate and
concentrated to yield a yellow amorphous solid (0.310 g, 82%) after
washing with THF (40 mL) and drying: mp 325–327 °C (dec). IR
(film) 2916, 1657, 1619, 1597, 1499, 1462, 1339, 1252, 1034,
5.3.6. 2-Chloro-14-chloromethyl-12H-5,11a-diazadibenzo
[b,h]fluoren-11-one (32a)
Compound 8 (0.200 g, 1.00 mmol), compound 21 (0.230 g,
1.12 mmol) and p-TsOH (0.190 g, 1.00 mmol) were diluted with
toluene (30 mL) and the mixture was heated at reflux for 13.5 h
using a Dean-Stark trap. The mixture was cooled, concentrated,
and the residue was diluted with CHCl3 (100 mL). The solution
was washed with satd NaHCO3 (100 mL). H2O (100 mL) was added,
and the aqueous phase was exhaustively extracted with CHCl3
(1 ꢄ 50, 6 ꢄ 25 mL). The resultant cloudy suspension was dried
over anhydrous sodium sulfate and concentrated to yield a yel-
low-green solid (0.339 g, 92%) after washing with MeOH (50 mL)
and ether (10 mL): mp 276–277 °C (dec). IR (film) 2917, 1665,
866, 688 cmꢁ1 1H NMR (300 MHz, CDCl3) d 8.56 (d, J = 8.1 Hz,
;
1H), 7.79–7.70 (m, 2H) 7.60–7.53 (m, 3H), 7.43 (s, 1H), 6.21 (s,
2H), 5.40 (s, 2H), 4.95 (s, 2H); ESIMS m/z (rel intensity) 377/379
(MH+, 100/35).
1537, 1442, 1343, 1138, 822, 751, 686 cmꢁ1 1H NMR (300 MHz,
;
CDCl3) d 8.57 (d, J = 8.0 Hz, 1H), 8.21 (d, J = 9.0 Hz, 1H), 8.15 (d,
J = 2.1 Hz, 1H), 7.81–7.59 (m, 5H), 5.46 (s, 2H), 5.01 (s, 2H); ESIMS
m/z (rel intensity) 367 (MH+, 100).
5.3.4. 14-Chloromethyl-2,3-dimethoxy-12H-5,11a-diaza-
dibenzo[b,h]fluoren-11-one (30a)
Compound 8 (0.600 g, 3.01 mmol), compound 17 (0.760 g,
3.31 mmol) and p-TsOH (0.572 g, 3.01 mmol) were diluted with
benzene (80 mL). The mixture was heated at reflux for 17 h using
a Dean-Stark trap to collect azeotroped water. The mixture was
cooled and the precipitate was filtered. The tan solid collected
was washed with MeOH (10 mL), diluted with CHCl3, and the or-
ganic phase was washed with satd NaHCO3 (200 mL), H2O
(2 ꢄ 200 mL), and dried over anhydrous sodium sulfate. The solu-
tion was concentrated and adsorbed onto SiO2 (7.12 g) and the res-
idue was purified by flash column chromatography (SiO2), eluting
with CHCl3 to 0.5% MeOH in CHCl3, to yield a yellow amorphous so-
lid (0.267 g, 23%) after washing with cold CHCl3 (5 mL), MeOH
(10 mL), and ether (20 mL): mp 308–311 °C (dec). IR (KBr) 3427,
1654, 1626, 1606, 1590, 1576, 1507, 1482, 1434, 1358, 1255,
5.3.7. 3-Chloro-14-chloromethyl-12H-5,11a-diazadibenzo
[b,h]fluoren-11-one (33a)
Compound 8 (0.200 g, 1.00 mmol) and compound 23 (0.230 g,
1.12 mmol) were diluted with toluene (40 mL) and p-TsOH
(0.190 g, 1.00 mmol) was added. The mixture was heated at reflux
for 17 h using a Dean-Stark trap. The mixture was cooled and con-
centrated, and the residue was diluted with CHCl3 (100 mL), and
the suspension was washed with satd NaHCO3 (100 mL). The aque-
ous layer was extracted with CHCl3 (4 ꢄ 50 mL), and the organic
layers were dried over anhydrous sodium sulfate and concentrated
to yield a yellow solid (0.314 g, 86%) after washing with MeOH
(25 mL): mp 301.5–303 °C (dec). IR (film) 3584, 3369, 2922,
1656, 1622, 1604, 1449, 1342, 1183, 1075, 923, 751, 688 cmꢁ1
;
1H NMR (300 MHz, CDCl3) d 8.58 (d, J = 7.8 Hz, 1H), 8.28 (d,
J = 2.0 Hz, 1H), 8.15 (d, J = 9.0 Hz, 1H), 7.80–7.61 (m, 5H), 5.46 (s,
2H), 5.04 (s, 2H); ESIMS m/z (rel intensity) 368 (MH+, 65), 331
(MH–HCl, 100).
1222, 1012, 844, 687 cmꢁ1 1H NMR (300 MHz, CDCl3) d 8.55 (d,
;
J = 7.9 Hz, 1H), 7.75–7.72 (m, 2H), 7.59–7.54 (m, 3H), 7.32 (s, 1H),
5.42 (s, 2H), 5.01 (s, 2H), 4.10 (s, 6H); ESIMS m/z (rel intensity)
393/335 (MH+, 100/33). Anal. Calcd for C22H17ClN2O3ꢀ1.5H2O: C,
62.94; H, 4.80; N, 6.67. Found: C, 63.04; H, 4.57; N, 6.67.
5.3.8. 1-Chloro-14-chloromethyl-12H-5,11a-diazadibenzo
[b,h]fluoren-11-one (34a)
5.3.5. 14-Chloromethyl-3-fluoro-2-methyl-12H-5,11a-diaza-
dibenzo[b,h]fluoren-11-one (31a)
Compound 8 (0.175 g, 0.88 mmol) and compound 24 (0.200 g,
0.98 mmol) were diluted with toluene (40 mL) and p-TsOH
(0.167 g, 0.88 mmol) was added. The mixture was heated to reflux
for 4.5 h using a Dean-Stark trap. The mixture was stirred at room
temperature for 13 h. The mixture was concentrated, and the res-
idue was diluted with CH2Cl2 (150 mL), and the suspension was
washed with satd NaHCO3 (150 mL). The aqueous layer was
Compound 8 (0.400 g, 2.01 mmol), compound 20 (0.506 g,
2.51 mmol) and p-TsOH (0.382 g, 2.01 mmol) were diluted with
benzene (70 mL) and the mixture was heated at reflux using a
Dean-Stark trap. After 16.5 h, another 0.070 g (0.35 mmol) of 20
was added, and reflux was continued for 1 h 20 min. The mixture

M. A. Cinelli et al. / Bioorg. Med. Chem. 18 (2010) 5535–5552
5545
exhaustively extracted with CH2Cl2 (3 ꢄ 75, 3 ꢄ 50 mL). MeOH was
added to the remaining aqueous suspension, which was extracted
with CH2Cl2 (3 ꢄ 50 mL). This suspension was washed with H2O
(2 ꢄ 100 mL). The suspension and combined organic layers were
dried over anhydrous sodium sulfate and concentrated to yield a
yellow amorphous solid (0.305 g, 95%) after washing with MeOH
(50 mL) and ether (30 mL): mp 300–302 °C (dec). IR (film) 1657,
1628, 1444, 1380, 1206, 1124, 925, 883, 850, 814, 766, 754, 725,
5.4.3. 2,3-Ethylenedioxy-14-(N-methylpiperazinylmethyl)-12H-
5,11a-diazadibenzo[b,h]fluoren-11-one trifluoroacetate (27d)
Compound 27a (0.090 g, 0.230 mmol) and N-methylpiperazine
(0.069 g, 0.690 mmol) in DMSO yielded the desired compound as
a brown solid after flash column chromatography (SiO2), eluting
with a gradient of CHCl3 to 7% MeOH in CHCl3. The obtained com-
pound was washed with ether and diluted with CHCl3 (40 mL) and
trifluoroacetic acid (2 mL) was added. The reaction mixture was al-
lowed to stir at room temperature for 30 min, concentrated, and
the residue was triturated with diethyl ether. The obtained precip-
itate was filtered and washed with diethyl ether (50 mL) to provide
a yellow solid (0.113 g, 86%): mp 238–242 °C. IR (KBr) 3430, 1661,
1505, 1287, 1243, 1181, 1067 cmꢁ1; 1H NMR (300 MHz, DMSO-d6)
d 9.39 (br s, 1H) 8.36 (d, J = 7.4 Hz, 1H), 7.98 (d, J = 7.6 Hz, 1H),
7.82–7.78 (m, 2H), 7.62–7.57 (m, 2H), 7.54 (s, 1H), 5.36 (s, 2H),
4.50–4.00 (br m, 2H), 4.44 (s, 4H), 4.07 (s, 2H), 3.37 (m, 2H), 3.03
(m, 4H), 2.80 (d, J = 4.2 Hz, 3H); ESIMS m/z (rel intensity) 455
(MH+, 100). Anal. Calcd for C29H27F3N4O5ꢀ0.25H2O: C, 60.78; H,
4.84; N, 9.78. Found: C, 60.68; H, 4.74; N, 9.56.
; d 8.61 (d,
688 cmꢁ1 1H NMR (300 MHz, CDCl3/CF3COOD)
J = 8.2 Hz, 1H), 8.47 (dd, J = 6.8, 3.0 Hz, 1H), 8.33 (s, 1H), 8.08–
7.90 (m, 5H), 5.78 (s, 2H), 5.57 (s, 2H); APCI-MS m/z (rel intensity)
367 (MH, base-peak).
5.4. General procedure for synthesis of 14-substituted aromat-
hecins (series 27 and 29–34)27,28
The chlorinated aromathecin core (between 0.050 and 0.115 g,
or 0.140–0.300 mmol) was diluted in DMSO (20–30 mL, for room
temperature reactions), or an equivalent amount of dry DMF (for
some reactions performed at higher temperature). A nucleophile
(between a three- and tenfold excess, typically between 5 and
6 equiv) was then added. In the case of amine salts, approximately
10 equiv of Et3N were added. The mixture was stirred overnight
(between 12 and 24 h). The product was extracted by either dilut-
ing the reaction mixture with CHCl3 (between 50 and 100 mL) and
washing with water (at least 90 mL) and satd NaCl, or alternatively,
by pouring the reaction mixture into H2O (ꢃ100 mL) and extract-
ing with CHCl3 (at least 100 mL). The organic layers were washed
with copious water (between 400 and 600 mL) and (when DMF
was used), sat. NH4Cl (ꢃ200 mL), and were dried over anhydrous
sodium sulfate and concentrated, and the residue was purified by
flash column chromatography (loading by either diluting the resi-
due in an appropriate solvent or adsorbing onto between 3 and 5 g
of SiO2) on 20–30 g SiO2. The obtained solids were then washed
with ether or hexane (30–50 mL) and dried. The preparation of
salts is described under the subheadings for individual compounds.
5.4.4. 2,3-Ethylenedioxy-14-(10-morpholinomethyl)-12H-5,11a-
diazadibenzo[b,h]fluoren-11-one (27e)
Compound 27a (0.090 g, 0.230 mmol) and morpholine (0.060 g,
0.690 mmol) in DMSO yielded the desired compound as a brown
solid after flash column chromatography (SiO2), eluting with a gra-
dient of CHCl3 to 2% MeOH in CHCl3. The product was washed with
ether (50 mL) and treated with TFA (as described for 27d), but only
the free base was obtained upon drying, as a yellow-brown solid
(0.088 g, 86%): mp 150–155 °C (dec). IR (KBr) 1661, 1630, 1507,
1290, 1246, 1200, 1176, 1129, 1068 cmꢁ1 1H NMR (300 MHz,
;
DMSO-d6) d 8.34 (d, J = 8.2 Hz, 1H), 7.96 (d, J = 7.9 Hz, 1H), 7.81–
7.76 (m, 2H), 7.61–7.56 (m, 2H), 7.50 (s, 1H), 5.34 (s, 2H), 4.43 (s,
4H), 3.96 (s, 2H), 3.56 (br s, 4H), 2.50 (br s, 4H); ESIMS m/z (rel
intensity) 442 (MH+, 100). Anal. Calcd for C26H23N2O4ꢀ0.5H2O: C,
69.32; H, 5.37; N, 9.33. Found: C, 69.46; H, 5.04; N, 9.08.
5.4.5. 14-(N-Ethanolaminomethyl)-2,3-ethylenedioxy-12H-
5,11a-diazadibenzo[b,h]fluoren-11-one trifluoroacetate (27f)
Compound 27a (0.090 g, 0.230 mmol) and ethanolamine
(0.042 g, 0.691 mmol) in DMSO afforded the desired compound
as a dark yellow solid after flash column chromatography (SiO2,
eluting with a gradient of CHCl3 to 7% MeOH in CHCl3) and washing
with ether (50 mL). The obtained precipitate was treated with TFA
as described for 27d, and was filtered and washed with diethyl
ether (50 mL) to provide a brown-orange solid (0.101 g, 83%): mp
180–183 °C. IR (KBr) 3424, 1658, 1622, 1505, 1290, 1245,
5.4.1. 14-N,N-Dimethylaminomethyl-2,3-ethylenedioxy-12H-
5,11a-diazadibenzo[b,h]fluoren-11-one (27b)
Compound 27a (0.090 g, 0.230 mmol), dimethylamine hydro-
chloride (0.094 g, 1.15 mmol) and Et3N (0.16 mL), 1.15 mmol), in
DMSO yielded the title compound as a brown solid (0.057 mg,
57%) after flash column chromatography (SiO2, eluting with a gra-
dient of CHCl3 to 4% MeOH in CHCl3) and washing with ether
(50 mL): mp 249–251 °C (dec). IR (KBr) 1659, 1632, 1607, 1505,
1287, 1245, 1064 cmꢁ1 1H NMR (300 MHz, DMSO-d6) d 8.34 (d,
;
1200 cmꢁ1 1H NMR (300 MHz, DMSO-d6) d 8.92 (br s, 2H), 8.37
;
J = 7.7 Hz, 1H), 7.95 (d, J = 7.8 Hz, 1H), 7.81–7.75 (m, 2H), 7.60
(m, 1H), 7.58 (s, 1H), 7.49 (s, 1H), 5.29 (s, 2H), 4.42 (s, 4H), 3.86
(s, 2H), 2.24 (s. 6H); ESIMS m/z (rel intensity) 400 (MH+, 100). Anal.
Calcd for C24H21N3O3: C, 72.16; H, 5.30; N, 10.52. Found: C, 72.08;
H, 5.19; N, 10.24.
(d, J = 8.2 Hz, 1H), 8.00 (d, J = 7.9 Hz, 1H), 7.88 (s, 1H), 7.85–7.79
(m, 1H), 7.64–7.59 (m, 3H), 5.53 (s, 2H), 4.78 (br s, 2H), 4.48 (s,
4H), 3.79 (t, J = 5.51 Hz, 2H), 3.37 (m, 2H); the hydroxyl group is
absent due to line-broadening; ESIMS m/z (rel intensity) 416
(MH+, 100). Anal. Calcd for C26H22F3N3O6: C, 58.98; H, 4.19; N,
7.94. Found: C, 59.22; H, 4.21; N, 7.89.
5.4.2. 2,3-Ethylenedioxy-14-(10-imidazolylmethyl)-2H-5,11a-
diazadibenzo[b,h]fluoren-11-one (27c)
5.4.6. 2,3-Ethylenedioxy-14-[N-(S)-30-hydroxypyrrolid
Compound 27a (0.116 g, 0.297 mmol) and imidazole (0.060 g,
0.891 mmol) in DMSO at 100 °C for 2 h, yielded the title compound
as a yellow solid (0.056 mg, 45%) after flash column chromatogra-
phy (SiO2, eluting with a gradient of CHCl3 to 7% MeOH in CHCl3)
and washing with diethyl ether (50 mL): mp 270 °C (dec). IR
inomethyl]-12H-5,11a diazadibenzo[b,h]fluoren-11-one (27g)
Compound 27a (0.065 g, 0.166 mmol), (S)-pyrrolidin-3-ol
hydrogen maleate38 (0.102 g, 0.499 mmol), and Et3N (0.168 g,
1.66 mmol) in DMSO afforded the desired product as a tan solid
(0.048 g, 64%) after flash column chromatography (SiO2, eluting
with a gradient of CHCl3 to 2% MeOH in CHCl3) and washing with
hexanes: mp 235–237 °C (dec). IR (film) 3369, 2930, 1659, 1623,
(KBr) 1656, 1627, 1605, 1505, 1291, 1246 cmꢁ1
;
1H NMR
(300 MHz, DMSO-d6) d 8.33 (d, J = 7.7 Hz, 1H), 7.96–7.92 (m, 2H),
7.82–7.76 (m, 1H), 7.65 (s, 1H), 7.61–7.54 (m, 3H), 7.25 (s, 1H),
6.92 (s, 1H), 5.80 (s, 2H), 5.20 (s, 2H), 4.42 (s, 4H); ESIMS m/z (rel
intensity) 423 (MH+, 100). Anal. Calcd for C25H18N4O3ꢀ0.75H2O: C,
68.88; H, 4.51; N, 12.85. Found: C, 69.15; H, 4.30; N, 12.66.
1603, 1505, 1441, 1287, 1243, 1068, 687 cmꢁ1
;
1H NMR
(300 MHz, CDCl3) d 8.54 (d, J = 7.9 Hz, 1H), 7.77–7.52 (m, 6H),
5.38 (s, 2H), 4.42 (s, 4H), 4.36 (br s, 1H), 4.08 (s, 2H), 2.94–2.90
(m, 1H), 2.70–2.62 (m, 2H), 2.46–2.42 (m, 1H), 2.26–2.20 (m,

5546
M. A. Cinelli et al. / Bioorg. Med. Chem. 18 (2010) 5535–5552
1H), 1.95 (bd, J = 7.0 Hz, 1H), 1.79–1.75 (m, 1H); ESIMS m/z (rel
intensity) 442 (MH+, 100). Anal. Calcd for C26H23N3O4ꢀH2O: C,
67.96; H, 5.48; N, 9.14. Found: C, 68.28; H, 5.22; N, 9.02.
Anal. Calcd for C23H19N3O4ꢀ1.25H2O: C, 65.16; H, 5.11; N, 9.91.
Found: C, 65.35; H, 4.78; N, 9.87.
5.4.11. 14-(N,N-Dimethylaminomethyl)-2,3-methylenedioxy-
12H-5,11a-diazadibenzo[b,h]fluoren-11-one (29d)
5.4.7. 2,3-Ethylenedioxy-14-[(10-imidazolyl)propylami-
nomethyl]-12H-5,11a-diazadibenzo[b,h]fluoren-11-one (27h)
Compound 27a (0.065 g, 0.166 mmol) and 1-(3-aminopro-
pyl)imidazole (0.062 g, 0.499 mmol) in DMSO afforded the desired
compound as a bright yellow amorphous solid (0.044 g, 56%) after
flash column chromatography (SiO2, eluting with a gradient of
CHCl3 to 3% MeOH, with a few drops of Et3N) and washing with
ether: mp 203–207 °C (dec.) IR (film) 3368, 2929, 1658, 1622,
Compound 29a (0.070 g, 0.186 mmol) and N,N-dimethylamine
(0.56 mL, 2 M in THF) in DMSO afforded the title compound as a
yellow powder (0.046 g, 64%) after flash column chromatography
(SiO2, 29.4 g, eluting with 0.25% Et3N in CHCl3) and washing with
ether (40 mL): mp 272–277 °C (dec). IR (film) 3585, 2916, 1661,
1623, 1605, 1463, 1337, 1247, 1034 cmꢁ1 1H NMR (300 MHz,
;
CDCl3) d 8.56 (d, J = 8.13 Hz, 1H), 7.75–7.66 (m, 3H), 7.60–7.50
(m, 2H), 7.48 (s, 1H), 6.16 (s, 2H), 5.38 (s, 2H), 3.84 (s, 2H), 2.33
(s, 6H); ESIMS m/z (rel intensity) 386 (MH+, 100). Anal. Calcd for
1602, 1506, 1287, 1245, 1067 cmꢁ1 1H NMR (300 MHz, CDCl3) d
;
8.54 (d, J = 8.0 Hz, 1H), 7.76–7.56 (m, 6H), 7.47 (s, 1H), 7.26 (s,
1H), 6.87 (s, 1H), 5.37 (s, 2H), 4.44 (s, 4H), 4.23 (s, 2H), 4.08 (t,
J = 6.9 Hz, 2H), 2.76 (t, J = 6.6 Hz, 2H), 2.01–1.90 (m, 2H); the amine
exchanges with residual water; ESIMS m/z (rel intensity) 480
(MH+, 100). Anal. Calcd for C28H25N5O3ꢀ1.5H2O: C, 66.39; H, 5.57;
N, 13.83. Found: C, 66.42; H, 5.57; N, 13.64.
C
23H19N3O3ꢀ0.5H2O: C, 70.04; H, 5.11; N, 10.65. Found: C, 70.17;
H, 5.02; N, 10.27.
5.4.12. 14-[N-(S)-30-Hydroxypyrrolidinomethyl]-2,3-methyl-
enedioxy-12H-5,11a-diazadibenzo[b,h]fluoren-11-one (29e)
Compound 29a (0.065 g, 0.172 mmol), (S)-pyrrolidin-3-ol
hydrogen maleate (0.141 g, 0.690 mmol) and Et3N (0.347 g,
3.44 mmol) in DMSO afforded the title compound as a yellowish-
tan powder (0.051 g, 69%) after flash column chromatography
(SiO2, 27.6 g, eluting with a gradient of 0.6% MeOH in CHCl3 to
1.75% MeOH in CHCl3) and washing with ether (25 mL): mp 260–
263 °C (dec). IR (film) 3436, 2916, 2343, 1656, 1619, 1601, 1465,
5.4.8. 2,3-Ethylenedioxy-14-(30-morpholinopropylaminometh-
yl)-12H-5,11a-diazadibenzo[b,h]fluoren-11-one (27i)
Compound 27a (0.065 g, 0.166 mmol) and 3-morpholinopropyl-
amine (0.072 g, 0.499 mmol) in DMSO afforded the title compound
as a flocculent yellow amorphous solid (0.052 g, 63%) after flash
column chromatography (SiO2, eluting with a gradient of 0.5%
Et3N in CHCl3 to 1.5% MeOH–1% Et3N in CHCl3) and washing with
diethyl ether: mp 202–205 °C. IR (film) 3401, 2921, 1658, 1629,
1338, 1248, 1035, 944, 758, 687 cmꢁ1
;
1H NMR (300 MHz,
DMSO-d6) d 8.33 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 8.0 Hz, 1H). 7.80
(t, J = 7.2 Hz, 1H), 7.76 (s, 1H), 7.59 (t, J = 7.2 Hz, 1H), 7.51 (s, 1H),
7.44 (s, 1H), 6.26 (s, 2H), 5.32 (s, 2H), 4.72 (d, J = 4.3 Hz, 1H),
4.20–4.10 (m, 1H) 4.05 (d, J = 2.9 Hz, 2H), 2.80–2.66 (m, 2H),
2.50–2.38 (m, 2H), 2.02–1.96 (m, 1H), 1.60–1.50 (m, 1H); ESIMS
m/z (rel intensity) 428 (MH+, 100). Anal. Calcd for
1603, 1506, 1446, 1289, 1244, 1115, 1068, 688 cmꢁ1 1H NMR
;
(300 MHz, CDCl3) d 8.54 (d, J = 8.0 Hz, 1H), 7.77–7.53 (m, 6H),
5.39 (s, 2H), 4.42 (s, 4H), 4.23 (s, 2H), 3.66 (t, J = 4.5 Hz, 4H), 2.81
(t, J = 6.5 Hz, 2H), 2.42–2.38 (m, 6H), 1.77–1.70 (m, 2H), 1.60–
1.50 (br m, obscured by residual water, 1H); ESIMS m/z (rel inten-
sity) 499 (MH+, 100). Anal. Calcd for C29H30N4O4ꢀ1.5H2O: C, 66.27;
H, 6.33; N, 10.66. Found: C, 66.29; H, 6.14; N, 10.63.
C
25H21N3O4ꢀ0.8H2O: C, 67.96; H, 5.16; N, 9.51. Found: C, 67.68;
H, 4.78; N, 9.49.
5.4.9. 2,3-Methylenedioxy-14-[10-(N-methylpiperazinylmethyl)]
-12H-5,11a-diazadibenzo[b,h]fluoren-11-one (29b)
5.4.13. 14-[10-(Imidazolyl)propylamino]methyl)-2,3-methyl-
enedioxy-12H-5,11a-diazadibenzo[b,h]fluoren-11-one (29f)
Compound 29a (0.055 g, 0.146 mmol) and 1-(3-aminopro-
pyl)imidazole (0.091 g, 0.730 mmol) in DMSO afforded the title
compound as a yellow amorphous solid (0.052 g, 77%) after flash
column chromatography (SiO2, 28.1 g, eluting with 0.2% MeOH–
0.2% Et3N in CHCl3 to 4% MeOH–0.2% Et3N in CHCl3) and washing
with ether (25 mL): mp 195–199 °C (dec). IR (film) 3436, 2916,
Compound 29a (0.070 g, 0.185 mmol) and N-methylpiperazine
(0.074 g, 0.743 mmol) in DMSO afforded the title compound as a
pale-yellow amorphous solid (0.063 g, 77%) after flash column
chromatography (SiO2, 24.2 g, eluting with 0.5% MeOH in CHCl3
to 5% MeOH in CHCl3) and washing with ether (60 mL): mp 288–
291 °C (dec). IR (film) 3369, 2926, 2807, 1662, 1622, 1587, 1501,
1464, 1247, 1034, 687 cmꢁ1
;
1H NMR (300 MHz, CDCl3) d 8.56 (d,
2310 1655, 1622, 1602, 1488, 1465, 1238, 1037, 687 cmꢁ1 1H
;
J = 7.9 Hz, 1H), 7.77–7.69 (m, 2H), 7.66 (s, 1H), 7.58–7.54 (m, 2H),
7.48 (s, 1H), 6.18 (s, 2H), 5.39 (s, 2H), 3.93 (s, 2H), 2.70–2.30 (br
m, 8H), 2.29 (s, 3H); ESIMS m/z (rel intensity) 441 (MH+, 100). Anal.
Calcd for C26H24N4O3ꢀ0.25H2O: C, 70.18; H, 5.55; N, 12.59. Found:
C, 70.07; H, 5.45; N, 12.59.
NMR (300 MHz, DMSO-d6) d 8.34 (d, J = 7.8 Hz, 1H), 7.95 (d,
J = 7.8 Hz, 1H), 7.80 (t, J = 6.9 Hz, 1H), 7.72 (s, 1H), 7.59–7.53 (m,
3H), 7.46 (s, 1H), 7.13 (s, 1H), 6.86 (s, 1H), 6.27 (s, 2H), 5.40 (s,
2H), 4.21 (s, 2H), 4.04 (t, J = 6.9 Hz, 2H), 2.64 (br m, 2H), 1.92 (m,
2H); the amine proton is not visible due to residual water in the
solvent; ESIMS m/z (rel intensity) 466 (MH+, 100). Anal. Calcd for
5.4.10. 14-(N-Ethanolaminomethyl)-2,3-methylenedioxy-12H-
5,11a diazadibenzo[b,h]fluoren-11-one (29c)
C
27H23N5O3ꢀ0.75H2O: C, 67.60; H, 5.16; N, 14.62. Found: C, 67.90;
H, 4.85; N, 14.47.
Compound 29a (0.065 g, 0.172 mmol) and ethanolamine
(0.042 g, 0.690 mmol) in DMSO afforded the title compound as a
flocculent yellow solid (0.042 g, 61%) after flash column chroma-
tography (SiO2, 24.8 g, eluting with a gradient of 0.5% MeOH in
CHCl3 to 4% MeOH in CHCl3), washing with ether (25 mL) and dry-
ing in vacuo: mp 225–228 °C (dec). IR (film) 3401, 2917, 2342,
5.4.14. 14-[N-(S)-30-Hydroxypyrrolidinomethyl]-2,3-dimethoxy-
12H-5,11a-diazadibenzo[b,h]fluoren-11-one (30b)
Compound 30a (0.060 g, 0.153 mmol), (S)-pyrrolidin-3-ol
hydrogen maleate (0.094 g, 0.458 mmol) and Et3N (0.154 g,
1.50 mmol) in DMSO afforded the title compound as a yellow amor-
phous solid (0.051 g, 75%) after flash column chromatography (SiO2,
eluting with 2% MeOH in CHCl3), and washing with ether (20 mL)
and hexanes (20 mL): mp 223–226 °C. IR (KBr) 3430, 2933, 2342,
1655, 1618, 1493, 1462, 1337, 1250, 1038, 847, 757, 687 cmꢁ1
;
1H NMR (300 MHz, DMSO-d6) d 8.33 (d, J = 7.9 Hz, 1H), 7.94 (d,
J = 7.9 Hz, 1H), 7.80 (t, J = 7.0 Hz, 1H), 7.73 (s, 1H), 7.59 (t,
J = 7.3 Hz, 1H), 7.52 (s, 1H), 7.45 (s, 1H), 6.26 (s, 2H), 5.37 (s, 2H),
4.55 (t, J = 5.2 Hz, 1H), 4.21 (s, 2H), 3.52 (q, J = 5.4 Hz, 1H), 2.71
(t, J = 5.8 Hz, 2H); the amine proton is not visible due to exchange
with residual water; ESIMS m/z (rel intensity) 402 (MH+, 100).
1660, 1627, 1603, 1505, 1478, 1431, 1250, 1167, 846, 687 cmꢁ1
;
1H NMR (300 MHz, CDCl3) d 8.54 (d, J = 8.1 Hz, 1H), 7.76–7.68 (m,
2H), 7.64 (s, 1H), 7.57–7.52 (m, 3H), 5.39 (s, 2H), 4.40 (br m, 1H),
4.20 (q, J = 13.4 Hz, 2H), 4.09 (s, 3H), 4.05 (s, 3H), 2.94, (q,

M. A. Cinelli et al. / Bioorg. Med. Chem. 18 (2010) 5535–5552
5547
J = 5.5 Hz, 1H), 2.80–2.70 (m, 2H), 2.48 (q, J = 6.2 Hz, 1H), 2.30–2.20
(m, 1H), 1.90–1.70 (m, 2H); ESIMS m/z (rel intensity) 444 (MH+,
100). Anal. Calcd for C26H25N5O4ꢀ1H2O: C, 67.67; H, 5.90; N, 9.10.
Found: C, 67.38; H, 5.76; N, 9.13.
and washing with ether: mp 238–242 °C (dec). IR (KBr pellet)
3435, 2930, 2790, 2764, 2372, 2348, 1670, 1641, 1504, 1441,
1161, 1139, 821, 686 cmꢁ1 1H NMR (300 MHz, CDCl3) d 8.57 (d,
;
J = 8.0 Hz, 1H), 8.17 (d, J = 7.9 Hz, 1H), 7.80–7.56 (m, 5H), 5.44 (s,
2H), 4.03 (s, 2H), 2.70–2.40 (br m, 8H), 2.54 (s, 3H), 2.30 (s, 3H);
ESIMS m/z (rel intensity) 429 (MH+, 100). Anal. Calcd for
5.4.15. 14-(10-Imidazolylmethyl)-2,3-dimethoxy-2H-5,11a-diaz-
adibenzo[b,h]fluoren-11-one (30c)
C
26H25FN4Oꢀ0.5H2O: C, 71.38; H, 5.99; N, 12.81. Found: C, 71.34;
Compound 30a (0.070 g, 0.178 mmol) and imidazole (0.049 g,
0.713 mmol) were diluted with DMSO (25 mL) under an argon
atmosphere. The mixture was heated to 60 °C for 4 h. Additional
imidazole (0.012 g, 0.178 mmol) was added, and the mixture was
stirred at room temperature for 16 h. TLC indicated that the reac-
tion was incomplete, so additional imidazole (0.012 g, 0.178 mmol)
was added, and the mixture was heated to 100 °C for 1 h. Extrac-
tion (following the general procedure) and flash column chroma-
tography (SiO2, eluting with a gradient of CHCl3 to 5% MeOH in
CHCl3) yielded a yellow amorphous solid (0.056 g, 73%) after wash-
ing with ether (20 mL): mp 309–312 °C (dec). IR (KBr) 3468, 3027,
1654, 1627, 1571, 1509, 1482, 1360, 1256, 1170, 1086, 1033, 853,
H, 5.67; N, 12.70.
5.4.19. 3-Fluoro-14-[N-(S)-30-Hydroxypyrrolidinomethyl]-2-
methyl-12H-5,11a-diazadibenzo[b,h]fluoren-11-one (31c)
Compound 31a (0.070 g, 0.192 mmol) was diluted with DMSO
(25 mL) and (S)-pyrrolidin-3-ol hydrogen maleate (0.118 g,
0.576 mmol) was added, followed by Et3N (0.194 g, 1.92 mmol).
The mixture was stirred at room temperature for 16.5 h. TLC indi-
cated incomplete reaction; 0.040 g (0.192 mmol) of additional pyr-
rolidinol and Et3N (0.065 g, 0.642 mmol) were added, and the
mixture was heated at 50 °C for 45 min. Extraction (by general pro-
cedure) and flash column chromatography (SiO2, eluting with 1%
MeOH in CHCl3 to 1.5% MeOH in CHCl3) afforded a cream-colored
powder (0.050 g, 62%) after washing with ether: mp 239–242 °C
(dec). IR (KBr pellet) 3369, 2903, 2806, 1661, 1614, 1597, 1506,
758, 689 cmꢁ1 1H NMR (300 MHz, DMSO-d6) d 8.34 (d, J = 7.4 Hz,
;
1H), 7.96 (d, J = 6.8 Hz, 1H), 7.94 (s, 1H), 7.82 (t, J = 7.9 Hz, 1H),
7.61 (t, J = 7.3 Hz, 1H), 7.53 (s, 2H), 7.48 (s, 1H), 7.29 (s, 1H), 6.92
(s, 1H), 5.98 (s, 2H), 5.20 (s, 2H), 3.98 (s, 3H), 3.93 (s, 3H); ESIMS
m/z (rel intensity) 425 (MH+, 100). Anal. Calcd for
1482, 1440, 1347, 1229, 1135, 1024, 862, 760, 689 cmꢁ1 1H
;
NMR (300 MHz, DMSO-d6) d 8.37 (dd, J = 4.6, 3.9 Hz, 2H), 7.99 (d,
J = 7.9 Hz, 1H), 7.84–7.78 (m, 2H), 7.63–7.58 (m, 2H), 5.39 (s, 2H),
4.75 (d, J = 4.3 Hz, 1H), 4.23–4.18 (m, 3H), 2.85–2.73 (m, 2H),
2.57–2.42 (m, 2H), 2.50 (s, 3H, obscured by solvent peak), 2.02–
2.98 (m, 1H), 1.70–1.50 (m, 1H); ESIMS m/z (rel intensity) 416
(MH+, 100). Anal. Calcd for C25H22FN3O2ꢀ0.25H2O: C, 71.50; H,
5.40; N, 10.01. Found: C, 71.15; H, 5.04; N, 9.90.
C
25H20N4O3ꢀ1.5H2O: C, 66.51; H, 5.13; N, 12.41. Found: C, 66.64;
H, 4.89; N, 12.53.
5.4.16. 2,3-Dimethoxy-14-[10-(N-methylpiperazinylmethyl)]-2H
-5,11a-diazadibenzo[b,h]fluoren-11-one (30d)
Compound 30a (0.055 g 0.140 mmol), and N-methylpiperazine
(0.042 g, 0.420 mmol) in DMSO afforded the title compound as a
yellow-green amorphous solid (0.046 g, 72%) after flash column
chromatography (SiO2, eluting with a gradient of 1% MeOH in CHCl3
to 5% MeOH in CHCl3), and washing with ether (20 mL): mp 256–
259 °C (dec). IR (KBr) 3435, 2926, 2791, 1665, 1636, 1601, 1504,
5.4.20. 3-Fluoro-14-[(10-imidazolyl)propylaminomethyl]-2-
methyl-12H-5,11a-diazadibenzo[b,h]fluoren-11-one (31d)
Compound 31a (0.070 g, 0.19 mmol) was diluted with DMSO
(25 mL), and 1-(3-aminopropyl)imidazole (0.072 g, 0.08 mmol)
was added. The mixture was stirred at room temperature for
16.5 h. TLC indicated the reaction was incomplete, so an additional
0.024 g (0.192 mmol) of 1-(3-aminopropylimidazole) was added,
and the mixture was heated at 50 °C for 1 h. Extraction (by general
procedure) and flash column chromatography (SiO2, eluting with a
gradient of 1% MeOH in CHCl3 to 6% MeOH–0.2% Et3N in CHCl3)
yielded a bright yellow powder (0.077 g, 89%) after washing with
ether: mp 183–187 °C (dec). IR (KBr pellet) 3435, 3248, 3116,
3084, 1947, 2603, 1659, 1633, 1606, 1507, 1439, 1341, 1231, 1137,
1480, 1431, 1249, 1167, 842, 816, 686 cmꢁ1 1H NMR (300 MHz,
;
CDCl3) d 8.57 (d, J = 8.0 Hz, 1H), 7.77–7.89 (m, 3H), 7.58–7.56 (m,
2H), 7.53 (s, 1H), 5.40 (s, 2H), 4.09 (s, 3H), 4.07 (s, 3H), 4.01 (s,
2H), 2.70–2.30 (br m, 8H), 2.30 (s, 3H); ESIMS m/z (rel intensity)
457 (MH+, 100). Anal. Calcd for C27H28N4O3ꢀ0.5H2O: C, 69.66; H,
6.28; N, 12.03. Found: C, 69.59; H, 6.21; N, 12.13.
5.4.17. 14-[(10-Imidazolyl)propylaminomethyl]-2,3-dimethoxy-
12H-5,11a-diazadibenzo[b,h]fluoren-11-one (30e)
Compound 30a (0.050 g, 0.127 mmol) and 1-(3-aminopro-
pyl)imidazole (0.048 g, 0.381 mmol) in DMSO afforded the title
compound as a yellow powder (0.040 mg, 66%) after flash column
chromatography (SiO2, eluting with a gradient of 0.5% MeOH–0.1%
Et3N in CHCl3 to 2.5% MeOH–0.2% Et3N in CHCl3) and washing with
ether (20 mL): mp 213–216 °C (dec). IR (film) 3306, 2918, 1657,
912, 862, 759, 689 cmꢁ1 1H NMR (300 MHz, DMSO-d6) 8.37 (d,
;
J = 8.0 Hz, 1H), 8.31 (d, J = 8.4 Hz, 1H), 7.99 (d, J = 7.8 Hz, 1H), 7.84–
7.77 (m, 2H), 7.63–7.58 (m, 3H), 7.14 (s, 1H), 6.87 (s, 1H), 5.44 (s,
2H), 4.30 (s, 2H), 4.05 (t, J = 7.0 Hz, 2H), 2.66 (t, J = 6.3 Hz, 2H), 2.50
(s, 3H, obscured by solvent peak), 1.94–1.89 (m, 2H); ESIMS m/z
(rel intensity) 454 (MH+, 100). Anal. Calcd for C27H34FN5Oꢀ0.5H2O:
C, 70.11; H, 5.45; N, 15.14. Found: C, 69.73; H, 5.30; N, 14.97.
1626, 1602, 1507, 1478, 1356, 1253, 1111, 687 cmꢁ1 1H NMR
;
(500 MHz, CDCl3) d 8.54 (d, J = 8.0 Hz, 1H), 7.76 (t, J = 7.6 Hz, 1H),
7.73 (t, J = 7.7 Hz, 1H), 7.57–7.53 (m, 3H), 7.49 (s, 1H), 7.44 (s,
1H), 7.03 (s, 1H), 6.85 (s, 1H), 5.39 (s, 2H), 4.30 (s, 2H), 4.09 (s,
3H), 4.07 (s, 3H), 4.05 (t, J = 6.9 Hz, 2H), 2.77 (t, J = 6.7 Hz, 2H),
2.01 (pent, J = 6.7 Hz, 2H); the amine proton is not visible due to
residual water in the solvent; ESIMS m/z (rel intensity) 482 (MH+,
100). Anal. Calcd for C28H27N5O3ꢀ1H2O: C, 67.32; H, 5.85; N,
14.02. Found: C, 66.95; H, 5.73; N, 13.92.
5.4.21. 14-(N-Ethanolaminomethyl)-3-fluoro-2-methyl-12H-
5,11a-diazadibenzo[b,h]fluoren-11-one (31e)
Compound 31a (0.070 g, 0.192 mmol) was diluted with DMSO
(25 mL) and ethanolamine (0.059 g, 0.960 mmol) was added. The
mixture was stirred overnight at room temperature for 17 h. TLC
indicated incomplete reaction, so additional ethanolamine
(0.029 g 0.480 mmol) was added, and the mixture was heated at
50 °C for 1 h. Extraction (by general procedure) and flash column
chromatography (SiO2, eluting with a gradient of 1% MeOH in CHCl3
to 5% MeOH–0.2% Et3N in CHCl3) afforded a residue that was recrys-
tallized from boiling CHCl3 (20 mL) to yield a pale yellow amor-
phous solid (0.036 g, 48%) after washing with ether: mp 212–
216 °C (dec) IR (KBr pellet) 3378, 3066, 2925, 1656, 1617, 1602,
5.4.18. 3-Fluoro-2-methyl-14-[10-(N-methylpiperazinylmethyl)]
-12H-5,11a-diazadibenzo[b,h]fluoren-11-one (31b)
Compound 31a (0.070 g, 0.192 mmol) and N-methylpiperazine
(0.058 g, 0.575 mmol) in DMSO afforded the desired compound
as a pale yellow-green amorphous solid (0.053g, 64%) after flash
column chromatography (SiO2, eluting with 5% MeOH in CHCl3)
1504, 1438, 1348, 1128, 1110, 1056, 878, 689 cmꢁ1 1H NMR
;

5548
M. A. Cinelli et al. / Bioorg. Med. Chem. 18 (2010) 5535–5552
(300 MHz, DMSO-d6) d 8.35 (t, J = 7.3 Hz, 2H), 7.98 (d, J = 8.0 Hz,
1H), 7.83 (t, J = 10.2 Hz, 2H), 7.62–7.58 (m, 2H), 5.44 (s, 2H), 4.59
(br s, 1H), 4.34 (s, 2H), 3.56 (q, J = 5.4 Hz, 2H), 2.80–2.70 (s, 2H),
2.50 (s, 3H, obscured by solvent peak); ESIMS m/z (rel intensity)
390 (MH+, 100). Anal. Calcd for C23H20FN5O2ꢀ0.3H2O: C, 69.97; H,
5.26; N, 10.64. Found: C, 69.90; H, 5.24; N, 10.57.
(s, 2H), 2.70–2.30 (br m, 8H), 2.30 (s, 3H); ESIMS m/z (rel intensity)
431 (MH+, 100). Anal. Calcd for C25H23ClN4Oꢀ0.25H2O: C, 68.96; H,
5.44; N, 12.87. Found: C, 68.95; H, 5.66; N, 12.90.
5.4.26. 2-Chloro-14-[N-(S)-30-hydroxypyrrolidinomethyl]-12H-
5,11a-diazadibenzo[b,h]fluoren-11-one (32d)
Compound 32a (0.060 g, 0.163 mmol), (S)-pyrrolidin-3-ol
(0.133 g, 0.642 mmol) and Et3N (0.197 g, 1.96 mmol) in DMSO
afforded the title compound as a yellow powder (0.040 g, 59%) after
flash column chromatography (SiO2, 25.6 g, eluting with a gradient
of 1% MeOH in CHCl3 to 1.5% MeOH in CHCl3) and washing with
ether (30 mL): mp 235–238 °C (dec). IR (film) 3369, 2917, 2797,
1659, 1617, 1602, 1496, 1480, 1340, 1088, 827, 754, 730,
5.4.22. 14-Azidomethyl-3-fluoro-2-methyl-12H-5,11a-diaza-
dibenzo[b,h]fluoren-11-one (31f)
Compound 31a (0.070 g, 0.19 mmol) and sodium azide (0.037 g,
0.58 mmol) in DMSO afforded the title compound as a pale yellow
amorphous solid (0.057 g, 79%) after flash column chromatography
(SiO2, eluting with CHCl3) and washing with ether: mp 225–226 °C
(dec). IR (KBr pellet) 3437, 2106, 1661, 1636, 1504, 1430, 1342,
687 cmꢁ1 1H NMR (300 MHz, CDCl3) d 8.55 (d, J = 8.1 Hz, 1H),
;
1142, 877, 688 cmꢁ1
;
1H NMR (300 MHz, DMSO-d6) d 8.35 (d,
8.35 (d, J = 2.2 Hz, 1H), 8.15 (d, J = 9.0 Hz, 1H), 7.77–7.58 (m, 5H),
5.45 (d, J = 2.4 Hz, 2H), 4.40 (br s, 1H), 4.15 (s, 2H), 2.95 (q,
J = 5.8 Hz, 1H), 2.78–2.72 (m, 2H), 2.53–2.47 (m, 1H), 2.30–2.23
(m, 1H), 1.84–1.62 (m, 2H); ESIMS m/z (rel intensity) 418 (MH+,
100). Anal. Calcd for C24H20ClN3O2ꢀ0.5H2O: C, 67.52; H, 4.96; N,
9.84. Found: C, 67.80; H, 5.03; N, 9.80.
J = 7.9 Hz, 1H), 8.26 (d, J = 8.2 Hz, 1H), 7.99 (d, J = 7.9 Hz, 1H),
7.88 (d, J = 10.9 Hz, 1H), 7.83 (t, J = 7.5 Hz, 1H), 5.43 (s, 2H), 4.23
(s, 2H), 2.49 (s, 3H, obscured by solvent peak); ESIMS m/z (rel
intensity) 372 (MH+, 100).
5.4.23. 14-Aminomethyl-3-fluoro-2-methyl-12H-5,11a-diaza-
dibenzo[b,h]fluoren-11-one dihydrochloride (31g)
5.4.27. 2-Chloro-14-(N-ethanolaminomethyl)-12H-5,11a-diaza-
dibenzo[b,h]fluoren-11-one (32e)
Compound 31f (0.047 g, 0.127 mmol) was diluted with benzene
(30 mL). Triethyl phosphite (0.063 g, 0.380 mmol) was added, and
the mixture was heated at reflux for 20 h. The mixture was cooled,
methanolic HCl (3 M, 10 mL) was added, and the mixture was
heated at reflux for 3 h. The solution was cooled and concentrated
to yield an orange solid (0.053 g, 100%) after washing with ether
and drying in vacuo: mp 278–282 °C (dec). IR (KBr pellet) 3428,
2047, 2915, 2866, 2632, 2346, 1902, 1656, 1624, 1603, 1528,
Compound 32a (0.070 g, 0.191 mmol) and ethanolamine
(0.047 g, 0.762 mmol) in DMSO afforded a yellow powder after
flash column chromatography (SiO2, 27.9 g, eluting with a gradient
of 1% MeOH–0.2% Et3N in CHCl3 to 1.5% MeOH–0.2% Et3N in CHCl3)
and washing with ether (100 mL). This solid contained residual
Et3NꢀHCl, and was dissolved in CHCl3 (50 mL, with MeOH added
for solubility) and washed with dilute NaOH (2 ꢄ 25 mL). The
aqueous phase was extracted with CHCl3 (5 ꢄ 25 mL), and the or-
ganic phase was dried over anhydrous sodium sulfate and concen-
trated to yield a yellow powder (0.027 g, 36%) after washing with
ether (30 mL): mp 218–220 °C (dec). IR (film) 3306, 2917, 2833,
1346, 1254, 1128, 802, 689 cmꢁ1 1H NMR (300 MHz, D2O) d
;
7.50–7.40 (m, 2H), 7.36 (d, J = 7.3 Hz, 2H), 7.13 (t, J = 6.6 Hz, 1H),
6.87 (d, J = 10.4 Hz, 1H), 6.59 (s, 1H), 5.00 (s, 2H), 4.47 (s, 2H),
2.19 (s, 3H); the amine is not visible due to exchange with the sol-
vent; ESIMS m/z (rel intensity) 346 (MH+, 100). Anal. Calcd for
1654, 1614, 1494, 1482, 1346, 845, 827, 757, 690 cmꢁ1 1H NMR
;
C
21H18Cl2FN3Oꢀ0.5H2O: C, 59.03; H, 4.48; N, 9.83. Found: C, 58.89;
(300 MHz, CDCl3) d 8.54 (d, J = 8.3 Hz, 1H), 8.25 (d, J = 2.2 Hz, 1H),
8.15 (d, J = 9.1 Hz, 1H), 7.76–7.58 (m, 5H), 5.46 (s, 2H), 4.36 (s,
2H), 3.79 (t, J = 4.6 Hz, 2H), 2.96 (t, J = 5.2 Hz, 2H), 2.08 (br s, 1H);
the hydroxyl proton is not visible due to residual water in the sol-
vent; ESIMS m/z (rel intensity) 392 (MH+, 100). Anal. Calcd for
C22H18ClN3O2ꢀ1H2O: C, 64.47; H, 4.92; N, 10.25. Found: C, 64.35;
H, 4.69; N, 9.93.
H, 4.48; N, 9.63.
5.4.24. 2-Chloro-14-[(10-imidazolyl)propylaminomethyl]-12H-
5,11a-diazadibenzo[b,h]fluoren-11-one (32b)
Compound 32a (0.060 g, 0.163 mmol) and 1-(3-aminopro-
pyl)imidazole (0.102 g, 0.817 mmol) in DMSO afforded the desired
compound as a yellow amorphous solid (0.039 g, 53%) after flash
column chromatography (SiO2, 25.8 g, eluting with a gradient of
1% MeOH–0.25% Et3N in CHCl3 to 2% MeOH–0.25% Et3N in CHCl3)
and washing with ether (100 mL): mp 171–175 °C (dec). IR (film)
3293, 2930, 1659, 1617, 1602, 1497, 1481, 1341, 1089, 825, 752,
5.4.28. 2-Chloro-14-(10-imidazolylmethyl)-12H-5,11a-
diazadibenzo[b,h]fluoren-11-one (32f)
Compound 32a (0.070 g, 0.191 mmol) and imidazole (0.065 g,
0.955 mmol) at 60 °C in DMF afforded the title compound as a yel-
low-green chalky solid (0.054 g, 70%) after flash column chromatog-
raphy (SiO2, 26.7 g, eluting with a gradient of 1.5% MeOH in CHCl3 to
4% MeOH in CHCl3), washing with ether (30 mL) and drying: mp
296–298 °C (dec). IR (film) 2918, 1660, 1632, 1606, 1500, 1341,
697 cmꢁ1 1H NMR (300 MHz, CDCl3) d 8.55 (d, J = 7.8 Hz, 1H),
;
8.27 (s, 1H), 8.17 (d, J = 9.1 Hz, 1H), 7.79–7.58 (m, 5H), 7.44 (s,
1H), 7.03 (s, 1H), 6.86 (s, 1H), 5.43 (s, 2H), 4.30 (s, 2H), 4.08 (t,
J = 6.7 Hz, 2H), 2.78 (t, J = 6.7 Hz, 2H), 2.03–1.98 (m, 2H); the amine
proton is not visible due to residual water in the solvent; ESIMS m/z
(rel intensity) 456 (MH+, 100). Anal. Calcd for C26H22ClN5Oꢀ2.2H2O:
C, 63.01; H, 5.37; N, 14.1. Found: C, 63.37; H, 4.99; N, 13.70.
1230, 1083, 828, 756, 689 cmꢁ1 1H NMR (300 MHz, DMSO-d6) d
;
8.34 (d, J = 2.1 Hz, 1H), 8.31 (d, J = 8.0 Hz, 1H), 8.17 (d, J = 9.0 Hz,
1H), 7.98–7.83 (m, 4H), 7.79 (s, 1H), 7.61 (t, J = 7.7 Hz, 1H), 7.28 (s,
1H), 6.94 (s, 1H), 5.92 (s, 2H), 5.18 (s, 2H); ESIMS m/z (rel intensity)
399 (MH+, 100). Anal. Calcd for C23H15ClN4O ꢀ0.25H2O: C, 68.49; H,
3.87; N, 13.89. Found: C, 68.33; H, 3.90; N, 13.92.
5.4.25. 2-Chloro-14-[10-(N-methylpiperazinylmethyl)]-12H-
5,11a-diazadibenzo[b,h]fluoren-11-one (32c)
Compound 32a (0.060 g, 0.163 mmol) and N-methylpiperazine
(0.066 g, 0.652 mmol) in DMSO afforded the title compound as a
pale-yellow amorphous solid (0.055 g, 78%) after flash column
chromatography (SiO2, 22.9 g, eluting with a gradient of 1% MeOH
in CHCl3 to 4% MeOH in CHCl3) and washing with ether (30 mL):
mp 238–241 °C (dec). IR (film) 2931, 2791, 1662, 1632, 1605,
5.4.29. 3-Chloro-14-[(10-imidazolyl)propylaminomethyl]-12H-
5,11a-diazadibenzo[b,h]fluoren-11-one (33b)
Compound 33a (0.060 g, 0.163 mmol) was diluted with DMSO
(20 mL). 1-(3-Aminopropyl)imidazole (0.102 g, 0.817 mmol) was
added, and the mixture was stirred at room temperature for 18 h.
The reaction was incomplete by TLC, so the mixture was heated to
55 °C for 2 h. The product was extracted (following general proce-
dures) and flash column chromatography (SiO2, 27.0 g, eluting with
1496, 1480, 1456, 1338, 1161, 1089, 1011, 826, 731, 687 cmꢁ1 1H
;
NMR (300 MHz, CDCl3) d 8.58 (d, J = 8.0 Hz, 1H), 8.37 (d, J = 2.2 Hz,
1H), 8.16 (d, J = 9.0 Hz, 1H), 7.78–7.59 (m, 5H), 5.46 (s, 2H), 4.02

M. A. Cinelli et al. / Bioorg. Med. Chem. 18 (2010) 5535–5552
5549
1% MeOH–0.2% Et3N in CHCl3) yielded a yellow-orange amorphous
solid (0.046 g, 61%) after washing with ether (20 mL): mp 163–
176 °C (dec). IR (film) 3401, 2922, 1660, 1622, 1604, 1480, 1343,
as a cream-colored solid after flash column chromatography
(SiO2, 27.6 g, eluting with a gradient of 0.5% MeOH in CHCl3 to
3% MeOH in CHCl3). This solid was washed with ether (25 mL), dis-
solved in CHCl3 (35 mL) and methanolic HCl (3 M, 5 mL) was added
slowly. The mixture was stirred at room temperature for 2 h and
concentrated to yield an orange-yellow iridescent solid (0.047 g,
46%) after washing with ether (50 mL) and drying in vacuo: mp
203–206 °C (dec). IR (KBr) 3400, 2599, 2489, 1651, 1618, 1596,
1230, 1081, 923, 687 cmꢁ1 1H NMR (300 MHz, CDCl3) d 8.54 (d,
;
J = 7.8 Hz, 1H), 8.21 (d, J = 1.5 Hz, 1H), 8.20 (d, J = 8.7 Hz, 1H), 7.78–
7.57 (m, 5H), 7.42 (s, 1H), 7.03 (s, 1H), 6.84 (s, 1H), 5.42 (s, 2H),
4.31 (s, 2H), 4.06 (d, J = 6.9 Hz, 2H), 2.76 (t, J = 6.7 Hz, 2H),
2.01–1.97 (m, 2H), 1.60–1.50 (br s, 1H, obscured by solvent peak);
ESIMS m/z (rel intensity) 456 (MH+, 100). Anal. Calcd for
1344, 1127, 821, 759, 692 cmꢁ1 1H NMR (300 MHz, DMSO-d6) d
;
C
26H22ClN5Oꢀ2.25H2O: C, 62.90; H, 5.38; N, 14.11. Found: C, 62.65;
10.13 (br s, 1H), 8.37 (d, J = 7.8 Hz, 1H), 8.18 (dd, J = 7.7, 1.9 Hz,
1H), 8.02 (d, J = 7.9 Hz, 1H), 7.84–7.77 (m, 3H), 7.70 (s, 1H), 7.65
(t, J = 7.2 Hz, 1H), 5.42 (s, 2H), 4.40 (s, 2H), 3.21–3.28 (m, 2H),
3.00–2.80 (m, 4H), 2.70–2.50 (m, 5H); ESIMS m/z (rel intensity)
431 (MH+, 100). Anal. Calcd for C25H26Cl4N4Oꢀ1.3H2O: C, 53.26;
H, 5.11; N, 9.94. Found: C, 53.63; H, 5.51; N, 9.87.
H, 4.99; N, 13.94.
5.4.30. 3-Chloro-14-[10-(N-methylpiperazinylmethyl)]-12H-5,
11a-diazadibenzo[b,h]fluoren-11-one (33c)
Compound 33a (0.060 g, 0.163 mmol) and N-methylpiperazine
(0.066 g, 0.652 mmol) in DMSO afforded the title compound as a
pale-yellow amorphous solid (0.055 g, 78%) after flash column
chromatography (SiO2, 24.4 g, eluting with a gradient of 1% MeOH
in CHCl3 to 5% MeOH in CHCl3) and washing with ether (20 mL):
mp 226–228 °C (dec). IR (film) 2931, 2798, 1662, 1631, 1605,
5.4.34. 1-Chloro-14-[N-(S)-30-hydroxypyrrolidinomethyl]-12H-
5,11a-diazadibenzo[b,h]fluoren-11-one hydrochloride (34c)
Compound 34a (0.070 g, 0.191 mmol) and (S)-pyrrolidin-3-ol
hydrogen maleate (0.157 g, 0.764 mmol) were diluted with DMSO
(25 mL) and Et3N (0.231 g, 0.764 mmol) was added. The mixture
was stirred at room temperature for 16 h, and then heated to
55 °C for 1.5 h. Extraction followed general procedures, and flash
column chromatography (SiO2, 25.7 g, eluting with a gradient of
0.25% MeOH in CHCl3 to 1.25% MeOH in CHCl3) afforded a yellow
solid. This solid was treated (in CHCl3) with methanolic HCl as de-
scribed for 34b to yield a dark-orange iridescent solid (0.031 g,
37%) after washing with ether (50 mL) and drying in vacuo: mp
208–210 °C (dec). IR (KBr) 3400, 2658, 1659, 1627, 1601, 1440,
1460, 1341, 1291, 1162, 1077, 1011, 918, 751, 686 cmꢁ1 1H NMR
;
(300 MHz, CDCl3) d 8.56 (d, J = 8.1 Hz, 1H), 8.33 (d, J = 9.1 Hz, 1H),
8.20 (d, J = 2.1 Hz, 1H), 7.78–7.54 (m, 5H), 5.43 (s, 2H), 4.03 (s,
2H), 2.70–2.30 (br m, 8H), 2.30 (s, 3H); ESIMS m/z (rel intensity)
431 (MH+, 100). Anal. Calcd for C25H23ClN4Oꢀ0.50H2O: C, 68.25; H,
5.50; N, 12.73. Found: C, 68.47; H, 5.34; N, 12.71.
5.4.31. 3-Chloro-14-[N-(S)-30-hydroxypyrrolidinomethyl]-12H-
5,11a-diazadibenzo[b,h]fluoren-11-one (33d)
1377, 1343, 1125, 1102, 926, 815, 758, 688 cmꢁ1
;
1H NMR
Compound 33a (0.060 g, 0.163 mmol) and (S)-pyrrolidin-3-ol
(0.133 g, 0.648 mmol) were diluted with DMSO (25 mL). Et3N
(0.197 g, 1.96 mmol) was added, and the mixture was stirred at
room temperature for 17 h. The reaction was incomplete by TLC,
so the mixture was heated to 50 °C for 1 h and 30 min. Extraction
followed general procedures, and flash column chromatography
(SiO2, 24.9 g, eluting with a gradient of 1% MeOH in CHCl3 to
1.5% MeOH in CHCl3) yielded a pale-yellow amorphous solid
(0.046 g, 68%) after washing with ether (30 mL): mp 247–249 °C
(dec). IR (film) 3401, 2916, 2799, 1659, 1619, 1603, 1480, 1344,
(500 MHz, DMSO-d6) d 10.1–10.0 (br m, 1H), 8.33 (d, J = 8.0 Hz,
1H), 8.20 (dd, J = 7.8, 3.5 Hz, 1H), 7.99 (d, J = 7.9 Hz, 1H), 7.91 (d,
J = 7.5 Hz, 1H), 7.86 (td, J = 8.1, 2.7 Hz, 1H), 7.81 (t, J = 7.1 Hz, 1H),
7.70 (s, 1H), 7.62 (t, J = 7.2 Hz, 1H), 5.70–5.30 (m, 4H), 4.10 (br s,
0.5H), 4.52 (br s, 0.5H), 4.39 (br s, 0.5H), 3.68–2.54 (m, 3H),
3.27–3.25 (br m, 0.5H), 2.50–2.30 (br m, 0.5H), 2.10–2.00 (br m,
0.5H), 2.00–1.90 (br m, 0.5H), 1.80–1.70 (br m, 0.5H); ESIMS m/z
(rel intensity) 418 (MH+, 100). Anal. Calcd for C24H21Cl2N3O2ꢀ2H2O:
C, 58.78; H, 5.14; N, 8.57. Found: C, 59.01; H, 4.84; N, 8.26.
1190, 922, 687 cmꢁ1
; d 8.56 (d,
1H NMR (300 MHz, CDCl3)
5.4.35. 1-Chloro-14-[(10-imidazolyl)propylaminomethyl]-12H-
5,11a-diazadibenzo[b,h]fluoren-11-one trihydrochloride (34d)
Compound 34a (0.090 g, 0.245 mmol) was diluted with DMSO
(25 mL) and 1-(3-aminopropyl)imidazole (0.153 g, 0.1.22 mmol)
was added. The mixture was stirred at room temperature for 17 h,
and then heated to 50 °C for 2 h. Extraction followed general proce-
dures, and flashcolumn chromatography(SiO2, 27.0 g, eluting with a
gradient of 1% MeOH in CHCl3 to 4% MeOH in CHCl3) afforded a yel-
low solid. This solid was treated with methanolic HCl as described
for 34b to yield the title compound as a flaky red iridescent solid
(0.048 g, 35%) after washing with ether (50 mL) and drying in vacuo:
mp 180–185 °C. IR (film) 3400, 2922, 2726, 1658, 1620, 1601, 1463,
1299, 1203, 1103, 818, 758, 683 cmꢁ1; 1H NMR (500 MHz, DMSO-d6)
d 9.9 (br s, 2H), 9.25 (s, 1H), 8.33 (d, J = 7.9 Hz, 1H), 8.22 (dd, J = 7.8,
1.6 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.89–7.77 (m, 4H), 7.73 (s, 1H),
7.71 (s, 1H), 7.63 (t, J = 7.5 Hz, 1H), 5.80 (s, 2H), 4.99 (s, 2H), 4.41
(t, J = 6.6 Hz, 2H), 3.30–3.20 (br m, 2H), 2.40–2.30 (m, 2H); the pro-
tonated secondary amine is not visible due to exchange with resid-
ual water in the solvent; ESIMS m/z (rel intensity) 456 (MH+, 100).
Anal. Calcd for C26H25Cl4N5Oꢀ2.5H2O: C, 51.16; H, 4.95; N, 11.47.
Found: C, 50.90; H, 5.19; N, 11.11.
J = 8.0 Hz, 1H), 8.35 (d, J = 9.1 Hz, 1H), 8.21 (d, J = 2.1 Hz, 1H),
7.79–7.55 (m, 5H), 5.45 (d, J = 1.6 Hz, 2H), 4.39 (br s, 1H), 4.17 (s,
2H), 3.00–2.90 (m, 1H), 2.73 (d, J = 3.7 Hz, 2H), 2.50–2.44 (m,
1H), 2.30–2.20 (m, 1H), 1.84–1.76 (m, 2H); ESIMS m/z (rel inten-
sity) 418 (MH+, 100). Anal. Calcd for C24H20ClN3O2ꢀ0.75H2O: C,
66.82; H, 5.02; N, 9.74. Found: C, 66.67; H, 4.77; N, 9.50.
5.4.32. 3-Chloro-14-(10-imidazolylmethyl)-12H-5,11a-
diazadibenzo[b,h]fluoren-11-one (33e)
Compound 33a (0.070 g, 0.191 mmol) and imidazole (0.065 g,
0.955 mmol) in DMF at 80 °C afforded the title compound as a
chalky yellow solid (0.054 g, 71%) after flash column chromatogra-
phy (SiO2, 27.4 g, eluting with a gradient of 1.5% MeOH in CHCl3 to
3.5% MeOH in CHCl3), and washing with ether (30 mL): mp 279–
281 °C (dec). IR (film) 3401, 2917, 1658, 1621, 1599, 1498, 1448,
1228, 1079, 687 cmꢁ1 1H NMR (500 MHz, DMSO-d6) d 8.30–8.29
;
(m, 2H), 8.18 (s, 1H), 7.97 (d, J = 7.0 Hz, 1H), 7.89 (s, 1H), 7.78–
7.64 (m, 4H), 7.24 (s, 1H), 6.89 (s, 1H), 5.90 (s, 2H), 5.17 (s, 2H);
ESIMS m/z (rel intensity) 399 (MH+, 100). Anal. Calcd for
C23H15ClN4Oꢀ0.25H2O: C, 66.99; H, 4.03; N, 13.59. Found: C,
66.81; H, 3.81; N, 13.39.
5.5. General procedure for synthesis of extended ethylenedi-
oxyaromathecins 28b–g
5.4.33. 1-Chloro-14-[10-(N-methylpiperazinylmethyl)]-12H-
5,11a-diazadibenzo[b,h]fluoren-11-one trihydrochloride (34b)
Compound 34a (0.070 g, 0.191 mmol) and N-methylpiperazine
(0.076 g, 0.762 mmol) in DMSO afforded the desired compound
Compound 28a (0.180–0.215 mmol) was diluted in DMSO or
DMF (25–30 mL), and sodium iodide (6 equiv) and the nucleophile

5550
M. A. Cinelli et al. / Bioorg. Med. Chem. 18 (2010) 5535–5552
(between 6 and 12 equiv) were added. The mixture was heated to
100 °C overnight (between 12 and 36 h or until TLC indicated com-
pletion of the reaction), and the mixture was poured into H2O (at
least 100 mL) and extracted thoroughly with CHCl3 (at least
150 mL). The organic layers were washed with copious water (be-
tween 400 and 600 mL) and (when DMF was used), satd NH4Cl
(ꢃ200 mL), and dried over anhydrous sodium sulfate. The solution
was concentrated, adsorbed onto SiO2 (between 3 and 5 g), and
purified by flash column chromatography (SiO2, between 20 and
30 g), to afford the aromathecins as solids after washing with ether
or suspending in methanol and precipitating with ether.
5.5.4. 14-[30-(N,N-Dimethylaminopropyl)]-2,3-ethylenedioxy-
12H-5,11a-diazadibenzo[b,h]fluoren-11-one (28e)
Compound 28a (0.075 g, 0.179 mmol), sodium iodide (0.160 g,
1.08 mmol), and N,N-dimethylamine (1.07 mL of a 2 M solution
in THF) in DMF afforded the title compound as a yellow powder
(0.051 g, 67%) after flash column chromatography (SiO2, 30.9 g,
eluting with a gradient of 0.5% MeOH–0.25% Et3N in CHCl3 to 1%
MeOH–0.5% Et3N in CHCl3) and precipitation by the general proce-
dure: mp 221–225 °C. IR (film) 3400, 2924, 1658, 1622, 1603, 1507,
1448, 1289, 1247, 1067, 918, 751, 688 cmꢁ1 1H NMR (300 MHz,
;
CDCl3) d 8.55 (d, J = 8.1 Hz, 1H), 7.78–7.51 (m, 6H), 5.30 (s, 2H),
4.43 (s, 4H), 3.13 (t, J = 7.5 Hz, 2H), 2.54 (t, J = 7.1 Hz, 2H), 2.26 (s,
6H), 1.95–1.90 (m, 2H); ESIMS m/z (rel intensity) 428 (MH+, 100).
Anal. Calcd for C26H25N3O3ꢀ2H2O: C, 67.37; H, 6.31; N, 9.07. Found:
C, 67.30; H, 6.05; N, 9.00.
5.5.1. 14-[30-(N-Ethanolaminopropyl)]-2,3-ethylenedioxy-12H-
5,11a-diazadibenzo[b,h]fluoren-11-one (28b)
Compound 28a (0.090 g, 0.215 mmol), sodium iodide (0.193 g,
1.29 mmol) and ethanolamine (0.079 g, 1.29 mmol) in DMSO affor-
ded the title compound as a yellow solid (0.044 g, 46%) after flash
column chromatography (SiO2, 29.0 g, eluting with a gradient of
1.5% MeOH–0.5% Et3N in CHCl3 to 6% MeOH–0.5% Et3N in CHCl3)
and precipitation by general procedure: mp 200–205 °C. IR (film)
3584, 2930, 1656, 1621, 1602, 1507, 1441, 1400, 1288, 1246,
5.5.5. 14-(30-Azidopropyl)-2,3-ethylenedioxy-12H-5,11a-diaza-
dibenzo[b,h]fluoren-11-one (28f)
Compound 28a (0.075 g, 0.179 mmol) and sodium azide
(0.058 g, 0.895 mmol) were diluted with anhydrous DMF (25 mL)
under an argon atmosphere. The mixture was heated to 100 °C,
upon which it became a bright pink solution. After 17 h, the color
had discharged to pale orange, and the solution was cooled, soni-
cated briefly, and poured into H2O (100 mL). The mixture was ex-
tracted with CHCl3 (3 ꢄ 50 mL) and the organic layers were
washed with H2O (3 ꢄ 200 mL), satd NH4Cl (200 mL), and dried
over anhydrous sodium sulfate. The residue was adsorbed onto
SiO2 (3.07 g) and purified by flash column chromatography (SiO2,
28.6 g), eluting with CHCl3 to yield a yellow-green microcrystalline
solid (0.059 g, 78%) after washing with MeOH (10 mL) and ether
(50 mL) and drying in vacuo for 72 h: mp 228–230 °C (dec). IR (film)
3584, 2930, 2097, 1660, 1628, 1604, 1507, 1448, 1289, 1248, 1068,
688 cmꢁ1; 1H NMR (300 MHz, CDCl3) d 8.56 (d, J = 8.2 Hz, 1H), 7.78–
7.54 (s, 5H), 7.47 (s, 1H), 5.31 (s, 2H), 4.44 (s, 4H), 3.48 (t, J = 6.4 Hz,
2H), 3.19 (t, J = 7.6 Hz, 2H), 2.08 (m, 2H); ESIMS m/z (rel intensity)
426 (MH+, 100). Anal. Calcd for C24H19N5O3: C, 67.76; H, 4.50; N,
16.46. Found: C, 67.47; H, 4.47; N, 16.29.
1068, 914 cmꢁ1 1H NMR (300 MHz, CDCl3) d 8.54 (d, J = 8.0 Hz,
;
1H), 7.78–7.51 (m, 6H), 5.31 (s, 2H), 4.42 (s, 4H), 3.74 (t,
J = 4.9 Hz, 2H), 3.19 (t, J = 7.5 Hz, 2H), 2.85–2.78 (m, 4H), 2.00–
1.95 (m, 2H); the hydroxyl group and amine are not visible due
to residual water in the solvent; ESIMS m/z (rel intensity) 444
(MH+, 100). Anal. Calcd for C26H25ClN3O4ꢀ1.25H2O: C, 67.01; H,
5.95; N, 9.02. Found: C, 66.76; H, 5.67; N, 8.88.
5.5.2. 2,3-Ethylenedioxy-14-[30-(10-imidazolylpropyl)]-12H-
5,11a-diazadibenzo[b,h]fluoren-11-one (28c)
Compound 28a (0.075 g, 0.179 mmol), sodium iodide (0.160 g,
1.08 mmol), and imidazole (0.073 g, 1.08 mmol) were diluted with
anhydrous DMF (30 mL) under an argon atmosphere. The mixture
was heated to 100 °C for 21 h. As the reaction was incomplete by
TLC, additional imidazole (0.073 g, 1.08 mmol) was added, and
the mixture was kept at 100 °C for an additional 3 h, upon which
a white precipitate formed (presumably NaCl). The mixture was
held at 40 °C for 16.5 h, cooled, and diluted with H2O (100 mL).
The mixture was extracted following general procedures, and the
residue was purified by flash column chromatography (SiO2,
30.0 g), eluting with 1% MeOH–0.5% Et3N in CHCl3, to yield a yellow
amorphous solid (0.040 g, 50%) after washing with ether (25 mL):
mp 275–278 °C. IR (film) 3584, 2930, 1657, 1621, 1602, 1506,
5.5.6. 14-(30-Aminopropyl)-2,3-ethylenedioxy-12H-5,11a-
diazadibenzo[b,h]fluoren-11-one dihydrochloride (28g)
Compound 28f (0.050 g, 0.118 mmol) was diluted with benzene
(25 mL) and triethyl phosphite (0.059 g, 0.354 mmol) was added.
The mixture was heated at reflux for 19 h. TLC indicated complete
consumption of the azide and the mixture was cooled to room
temperature. Methanolic HCl (10 mL, 5 M) was added, and the yel-
low-green mixture was heated at reflux for 3 h, cooled, and con-
centrated to afford a bright yellow amorphous solid (0.055 g,
99%) after washing with ether (30 mL) and drying in vacuo: mp
290–300 °C (dec). IR (KBr) 3445, 3040, 2708, 1665, 1622, 1603,
1447, 1288, 1246, 1067, 917 cmꢁ1 1H NMR (300 MHz, CDCl3) d
;
8.53 (d, J = 8.0 Hz, 1H), 7.77–7.53 (m, 6H), 7.20 (s, 1H), 7.17 (s,
1H), 7.01 (s, 1H), 5.23 (s, 2H), 4.42 (s, 4H), 4.17 (t, J = 7.1 Hz, 2H),
3.05 (t, J = 8.0 Hz, 2H), 2.27–2.17 (m, 2H); ESIMS m/z (rel intensity)
451 (MH+, 100). Anal. Calcd for C27H22N4O3ꢀ1H2O: C, 69.22; H, 5.16;
N, 11.96. Found: C, 69.31; H, 5.24; N, 11.77.
1501, 1480, 1401, 1299, 1257, 1156, 1062, 920, 761, 686 cmꢁ1
;
1H NMR (300 MHz, D2O) d 7.20–7.10 (m, 1H), 7.01–6.78 (m, 3H),
6.37 (s, 1H), 6.28 (s, 1H), 6.05 (s, 1H), 4.32 (s, 2H), 4.14–4.10 (m,
4H), 3.10–3.00 (br m, 2H), 2.50–2.40 (br m, 2H), 1.80–1.70 (br m,
2H); the amino group exchanges with the solvent; ESIMS m/z (rel
intensity) 400 (MH+, 100). Anal. Calcd for C24H23Cl2N3O3ꢀ0.5H2O:
C, 59.88; H, 5.03; N, 8.73. Found: C, 60.06; H, 5.29; N, 8.66.
5.5.3. 2,3-Ethylenedioxy-14-[30-(N-morpholinopropyl)]-12H-
5,11a-diazadibenzo[b,h]fluoren-11-one (28d)
Compound 28a (0.075 g, 0.179 mmol), sodium iodide (0.160 g,
1.08 mmol) and morpholine (0.188 g, 2.15 mmol) in DMF afforded
the title compound as a white amorphous solid (0.060 g, 72%) after
flash column chromatography (SiO2, 30.1 g, eluting with 0.5%
MeOH–0.25% Et3N in CHCl3) and precipitation by the general pro-
cedure: mp 259–260 °C. IR (film) 3584, 2929, 2382, 1659, 1630,
5.6. Topoisomerase I-mediated DNA cleavage reactions
Human recombinant Top1 was purified from Baculovirus as
previously described.63 DNA cleavage reactions were prepared as
previously reported27 (for review see64) with the exception of the
DNA substrate. Briefly, a 117-bp DNA oligonucleotide (Integrated
DNA 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
1603, 1506, 1448, 1398, 1287, 1244, 1116, 1068, 914 cmꢁ1 1H
;
NMR (300 MHz, CDCl3) d 8.56 (d, J = 7.9 Hz, 1H), 7.78–7.54 (m,
6H), 5.32 (s, 2H), 4.42 (s, 4H), 3.76–3.73 (m, 4H), 3.16 (t,
J = 7.3 Hz, 2H), 2.45–2.41 (m, 6H), 1.97–1.93 (m, 2H); ESIMS m/z
(rel intensity) 470 (MH+, 100). Anal. Calcd for C28H27N3O4: C,
71.62; H, 5.80; N, 8.95. Found: C, 71.35; H, 5.75; N, 8.92.

M. A. Cinelli et al. / Bioorg. Med. Chem. 18 (2010) 5535–5552
5551
contains a single 50-cytosine overhang, which was 30-end labeled
by fill-in reaction with [
32P]-dGTP in reaction 2 buffer (50 mM
4. Husain, I.; Mohler, J. L.; Seigler, H. F.; Besterman, J. M. Cancer Res. 1994, 54, 539.
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a-
Tris–HCl, pH 8.0, 100 mM MgCl2, 50 mM NaCl) with 0.5 units of
DNA polymerase I (Klenow fragment, New England BioLabs). Unin-
corporated 32P-dGTP was removed using mini Quick Spin DNA
columns (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
Top1 in 20
lL of reaction buffer [10 mM Tris–HCl (pH 7.5),
50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, and 15
l
g/mL BSA] at
10. Staker, B. L.; Feese, M. D.; Cushman, M.; Pommier, Y.; Zembower, D.; Stewart,
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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% bromophenol blue). Aliquots
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The crystal structure of camptothecin or topotecan in complex
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method, the MMFF94s force field, and MMFF94 charges, converg-
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dielectric function. The structure of the ligand and a sphere with
a radius of 5–8 Å were allowed to move during the minimization,
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overlays were performed by aligning the protein backbones in
Sybyl.
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Acknowledgments
This work was facilitated by the National Institutes of Health
(NIH) through support with Research Grant UO1 CA89566, Train-
ing Grant ST32 CA09634-12, and by the Lynn and McKeehan Fel-
lowships (Purdue University, M.A.C.), and a Purdue Research
Foundation Grant (#203202, M.C. and M.A.C.). This work was also
supported in part by an ACS Medicinal Chemistry Pre-doctoral
Fellowship sponsored by Pfizer Global Research and Development
(A.M.), and by the Intramural Research Program of the NIH, Na-
tional Cancer Institute, Center for Cancer Research. M.A.C. thanks
Drs. Karl Wood, Huaping Mo, and H. Daniel Lee for consultation
and valuable discussion and Joe Fornefeld of Midwest Microlab
for additional assistance.
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