Design and synthesis of novel amino-substituted xanthenones and benzo[b]xanthenones: Evaluation of their antiproliferative activity and their ability to overcome multidrug resistance toward MES-SA/D x 5 cells

Article abstract of DOI:10.1016/j.bmc.2005.12.003

A number of new xanthenone and benzo[b]xanthenone amino derivatives and their pyrazole-fused counterparts have been designed and synthesized possessing structural analogy to the potent anticancer agent 9-methoxypyrazoloacridine. The synthesis of the compo

Full text of DOI:10.1016/j.bmc.2005.12.003

Bioorganic & Medicinal Chemistry 14 (2006) 2910–2934
Design and synthesis of novel amino-substituted xanthenones
and benzo[b]xanthenones: Evaluation of their
antiproliferative activity and their ability to overcome
multidrug resistance toward MES-SA/D · 5 cells
Ioannis K. Kostakis,a Nicole Pouli,a Panagiotis Marakos,a,*
Alexios-Leandros Skaltsounis,b Harris Pratsinisc and Dimitris Kletsasc
aDivision of Pharmaceutical Chemistry, Department of Pharmacy, University of Athens,
Panepistimiopolis-Zografou, Athens 15771, Greece
bDivision of Pharmacognosy, Department of Pharmacy, University of Athens, Panepistimiopolis-Zografou, Athens 15771, Greece
cLaboratory of Cell Proliferation and Ageing, Institute of Biology, NCSR ꢀDemokritos,ꢁ 15310 Athens, Greece
Received 9 November 2005; revised 2 December 2005; accepted 2 December 2005
Available online 11 January 2006
Abstract—A number of new xanthenone and benzo[b]xanthenone amino derivatives and their pyrazole-fused counterparts have
been designed and synthesized possessing structural analogy to the potent anticancer agent 9-methoxypyrazoloacridine. The synthe-
sis of the compounds proceeds through nucleophilic substitution of 1-chloro-4-nitroxanthenone or the corresponding benzo[b]xan-
thenone by the appropriately substituted amine or hydrazine, reduction of the nitro group, and conversion into the suitable
dialkylaminoacetamides. This method cannot be applied for synthesis of the pyrazole-fused benzo[b]xanthenones, consequently a
different, simple, and high-yielding synthetic procedure was developed for the preparation of the target molecules. In vitro cytotoxic
potencies of the new derivatives toward the murine leukemia L1210 cell line, human colorectal adenocarcinoma (HT-29), and
human uterine sarcoma (MES-SA and its 100-fold resistant to doxorubicin variant MES-SA/D · 5) cell lines are described and com-
pared to those of reference drugs. The compounds exhibited significant cytotoxic activity against the tested cell lines and in addition
they retain activity against the multidrug resistant MES-SA/D · 5 subline, showing resistant factors close to 1. A number of deriv-
atives were found to posses DNA binding capacity, according to a standard ethidium bromide displacement assay. The majority of
the studied compounds induce a G2/M arrest, although among them some G1 or S blockers have also been identified.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
compounds for the development of structurally related
analogues. These drugs act by initially binding to cellu-
lar DNA sequences, which is usually followed by the
formation of a ternary complex with DNA topoisomer-
ase II.1 This process produces a distortion of DNA
structure, double strand break, perturbations of DNA
synthesis, and ultimately they induce apoptosis in sus-
ceptible cells. Intercalation into DNA is considered to
be an essential, but not the sole, determinant for their
cell growth inhibitory activity.2
Among different classes of chemotherapeutic agents
used against malignant diseases, compounds that inter-
act reversibly with DNA double helix have attracted
particular attention, due to their high therapeutic poten-
tial. A number of them, such as the antitumor antibiotic
actinomycin D, the 9-anilinoacridine amsacrine, and the
anthracycline derivatives doxorubicin and daunomycin,
have been extensively studied and were also used as lead
In recent years, a number of similarly acting aminoalkyl-
substituted anthracenediones were developed exempli-
fied by the drug mitoxantrone (1, Fig. 1) currently used
in the treatment of acute leukemias and of breast can-
cers.3,4 Unfortunately, the prolonged clinical application
of quinone chemotypes has encountered problems, such
Keywords: Aminoxanthenones; Aminobenzo[b]xanthenones; Amino-
benzopyrano[4,3,2-cd]indazoles; Aminobenzo[g]benzopyrano[4,3,2-
cd]indazoles; Cytotoxic activity; DNA binding affinity; Cell-cycle
selectivity; Multidrug resistance.
*
Corresponding author. Tel.: +30 210 7274830; fax: +30 210
7274747; e-mail: marakos@pharm.uoa.gr
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.12.003

I. K. Kostakis et al. / Bioorg. Med. Chem. 14 (2006) 2910–2934
2911
H
N
CH3
OH
OH
O
O
HN
OH
OH
N
N
N
H3CO
CH3
.2 HCl
N
H
HN
NO2
N
H
1 Mitoxantrone dihydrochloride
Figure 1. Structures of mitoxantrone and PZA.
2 PZA
as the development of cardiotoxicity5 and multidrug
resistance (MDR).6 This led to the development of
anthrapyrazoles7 and benzothiopyranoindazoles,8 which
are characterized by the modification of the central qui-
none to a quasi-iminoquinone group, with an apparent
suppression of redox cycling and free radical generation,
thought to be responsible for cardiac damage.9 The
anthrapyrazoles demonstrate high activity,12 but, in
contrast to the anthracyclines, they are weak iron chela-
tors10 and appear considerably less cardiotoxic, in com-
parison to doxorubicin.11 All these molecules share
common structural features, such as a planar, or
semi-planar, polycyclic chromophore and one or two
polymethylenediamine fragments, as side chains bearing
cationic charges, which improve solubility under physi-
ological conditions and enable electrostatic binding to
the phosphate moieties of DNA. Much effort has been
devoted to the modification of the chromophores, in
order to optimize their characteristics and a number
of acridine derivatives, structurally related to the
anthrapyrazoles, have also been developed. The most
representative examples in this area include imidazoacri-
dones,13 pyrazoloacridines,14 triazoloacridones15, and
pyrimidoacridines.16
mophore provided compounds able to overcome
MDR.24 Among them 9-methoxypyrazoloacridine
(PZA, 2, Fig. 1) was selected for clinical evaluation from
a series of compounds that combine the DNA complex-
ing activity of the acridine chromophore with the poten-
tial hypoxic cell selectivity derived from a reducible nitro
group substitution on the ring.25–27 Studies with PZA
showed that bioreduction occurs, since the correspond-
ing 5-amino derivative is the major metabolite of this
drug in mice.28 In addition to the experimentally con-
firmed hypoxia selectivity, PZA demonstrates unusual
solid tumor selectivity and cytotoxicity to non-dividing
cells,29–31 while retaining full activity against resistant
cell lines.32–34 It has been reported that PZA targets
topoisomerase I and II simultaneously, without stabiliz-
ing the topoisomerase–DNA cleavable complexes,32,35
and has been thus suggested that it should act by a novel
mechanism. The therapeutic potential of PZA is current-
ly being evaluated in clinical combination studies, with
the aim of circumventing drug resistance.12,14,36–38
We have previously reported on the synthesis and anti-
proliferative activity of a number of xanthenone and
thioxanthenone amino derivatives, as well as their pyra-
zole-fused counterparts. These derivatives showed inter-
esting cytotoxicity against the murine leukemia L1210
cell line, as well as against some human solid tumor cell
lines.39–42 In a preliminary report, we have described the
synthesis of some amino-substituted xanthenone and
benzopyrano[4,3,2-cd]indazole nitro derivatives, with di-
rect structural similarity to PZA.43 These compounds
exhibited interesting cytotoxic properties against a panel
of cell lines and retain activity against the multidrug
resistant MES-SA/D · 5 subline. Prompted by these
findings we decided to further explore the above-men-
tioned scaffolds and present here the preparation and
biological evaluation of some xanthenone and
benzo[b]xanthenone amino derivatives bearing either a
nitro substitution or a second basic side chain that could
possibly assist in more effective DNA-binding.
However, the anthrapyrazoles cannot circumvent the
problem of the development of acquired or intrinsic
resistance (MDR).17,18 Changes in intracellular drug
metabolism or in the activity of target enzymes have
been implicated in MDR, while physical obstacles to
drug delivery might also play a role, due to deficient
blood flow in growing solid tumors.19 Nevertheless,
the most important mechanism involves some ATP-
binding membrane proteins, such as the product of hu-
man MDR-1 gene P-glycoprotein (Pgp), which are
responsible for the active efflux of cytotoxic drugs out
of resistant cells.20 It has been reported that the anthra-
pyrazole-induced resistance of tumor cells can be over-
come in vitro with the use of verapamil or other
calcium blockers,17 able to modulate the function of
the above-mentioned exporting pumps.21,22 The effec-
tiveness of this drug combination has not yet been eval-
uated in vivo and is obviously far from therapeutic
applications. On the other hand, intense research efforts
have been made to design novel chemotherapeutic
agents able to counteract the mechanisms of the undesir-
able MDR phenomenon.23
2. Results and discussion
2.1. Chemistry
Commercially available ethyl salicylate (3) or ethyl 3-hy-
droxy-2-naphthoate (4) was used as starting material
(Scheme 1). Treatment of each ester with 2,4-dichloroni-
trobenzene (5) resulted in a mixture of isomeric diaryle-
The incorporation of a fused five- or six-membered het-
erocyclic ring into the anthracenedione or acridine chro-

2912
I. K. Kostakis et al. / Bioorg. Med. Chem. 14 (2006) 2910–2934
Scheme 1. Reagents and conditions: (a) K2CO3, Cu2O, DMF, 110 ꢁC, 8 h; (b) EtOH, NaOH 40%, rt, 30 min; (c) PPA 110 ꢁC, 1 h; (d)
H2NCH2CH2R1, pyridine, reflux, 1–3 h; (e) H2, Pd/C 10%, EtOH, 50 psi, rt, 3–4h; (f) ClCOCH2Cl, Et3N, THF, rt, 90 min; (g) secondary amine,
EtOH abs, reflux, 10–12 h.
thers (6, 8 and 7, 9, respectively). The mixture of the es-
ters 6 and 8 was separated by column chromatography
and each ester was isolated in pure form and identified
by means of 1H and 13C NMR spectral data, using both
direct and long-range heteronuclear correlation experi-
ments (HMBC and HMQC sequencies).43 The forma-
tion of both naphthoates 7 and 9 was evident from an
1H NMR spectrum recorded in their mixture, however
we could isolate pure only the desired isomer 9 by tritur-
ation, since all our attempts to separate this mixture by
column chromatography were unsuccessful. Each one of
the isomers 8 and 9 was then saponified under mild con-
ditions and the resulting carboxylic acids, 10 and 11,
respectively, without further purification, were ring
closed upon treatment with PPA providing the 1-chlo-
ro-4-nitroxanthenones 12 and 13. The nitro derivatives
14a–14d and 15a–15d were prepared by the nucleophilic
substitution of the chloro group of 12 or 13, respective-
ly, by appropriately substituted dialkylaminoethylam-
ines. The 4-nitro group was then easily reduced by
hydrogenation over palladium on activated carbon to
provide the amino derivatives 16a–16d and 17a–17d.
Since these derivatives were highly unstable in solution,
probably due to fast air oxidation, they were not isolat-
ed, but converted to the corresponding chloracetamides
18a–18d and 19a–19d, by treatment with chloracetyl
chloride. Reaction of these amides with the suitable sec-
ondary amines resulted in the target compounds 20a–
20d and 21a–21d.
yethylhydrazine to provide the carbinol 22 (Scheme
2).43 Compound 22 was converted to the mesylate 23,
which was treated with the appropriately substituted
secondary amines to result in the amino derivatives
24a–24d. Reduction of the nitro group of compounds
24a–24d provided the intermediate amines 25a–25d,
which were again not isolated or identified due to insta-
bility, but converted into the chloracetamides 26a–26d.
Reaction of the compounds 26a–26d with the suitable
secondary amines gave the benzopyranoindazoles 27a–
27d. Our attempts to apply an analogous methodology
for the preparation of the corresponding benzoxanthe-
none analogues were not successful because treatment
of the chloride 13 with 2-hydroxyethylhydrazine provid-
ed only minor amounts of the desired carbinol 29
(Scheme 2), the major product being 28. This compound
should have resulted from the displacement of the nitro
group by the reagent, presumably upon initial formation
of 29, which renders the nitro group of this heterocyclic
system highly susceptible to nucleophilic substitution.
Consequently, we have developed a new methodology
for the preparation of the desired derivatives. We have
thus used 3-hydroxy-2-naphthoic acid (30, Scheme 3),
which was treated with 1,3-cyclohexanedione to result
in the diketone 31. Oxidation of 31 in the presence of
DDQ in boiling toluene provided the phenol 32 in
78% overall yield. The preparation of the phenol 32
has been previously reported through two different
methodologies. The first involves the reaction of 3-hy-
droxy-2-naphthoic acid with resorcinol in the presence
of zinc chloride and provided the product in 27% yield.44
The second proceeds through the oxidation of
In order to prepare the corresponding pyrazole-fused
amino derivatives, 12 was reacted with 2-hydrox-

I. K. Kostakis et al. / Bioorg. Med. Chem. 14 (2006) 2910–2934
2913
R
R1
N
O
N
N
O
N
c
(from 23)
a
a: R1= N(CH3)2
b: R1= N(C2H5)2
c: R1= N(CH2)4
d: R1= N(CH2)5
12
NO2
R2
22 R= OH
23 R= OMs
24a-d R2= NO2
25a-d R2= NH2
26a-d R2= NHCOCH2Cl
27a-d R2= NHCOCH2R1
d
e
c
b
OH
OH
N
O
N
N
N
O
N
a
+
13
NO2
H2N
28
OH
29
Scheme 2. Reagents and conditions: (a) H2NNHCH2CH2OH, pyridine, rt, 12 h; (b) CH3SO2Cl, CH2Cl2, Et3N, rt, 4 h; (c) secondary amine, EtOH
abs, reflux, 10–12 h; (d) H2, Pd/C 10%, EtOH, 50 psi, rt, 6 h; (e) ClCOCH2Cl, THF, Et3N, rt, 1–2 h.
O
O
O
OR
CO2H
OH
b
a
O
O
32 R= H
33 R= Ts
31
30
c
d
(from 33)
R
R
N
N
N
N
f
(from 35)
a: R1= N(CH3)2
b: R1= N(C2H5)2
c: R1= N(CH2)4
d: R1= N(CH2)5
O
O
R2
36 R= OMs, R2= NO2
38a-d R= R1, R2=NO2
39a-d R= R1, R2= NH2
40a-d R= R1, R2= NHCOCH2Cl
41a-d R= R1, R2= NHCOCH2R1
34 R= OH
35 R= OMs
g
h
e
i
g
Scheme 3. Reagents and conditions: (a) PPA, 1,3-cyclohexanedione, 150 ꢁC, 2 h; (b) DDQ, toluene, reflux, 40 min; (c) 4-toluenesulfonylchloride,
acetone, Na2CO3, reflux, 3.5 h; (d) H2NNHCH2CH2OH, pyridine, reflux, 9 h; (e) CH3SO2Cl, CH2Cl2, Et3N, rt, 4 h; (f) HNO3 fuming, CH3CO2H, rt,
30 min; (g) secondary amine, EtOH abs, reflux, 8–10 h; (h) H2, Pd/C 10%, EtOH, 50 psi, rt, 5 h; (i) ClCOCH2Cl, Et3N, THF, rt, 1–2 h.
OMs
NO2
1-hydroxy -5a,6,11,11a-tetrahydrobenzo [b] xanthene-
12-one, derived from the cycloaddition reaction of the
suitable chromene carboxaldehyde.45 Nevertheless, our
procedure is quite simple, high yielding, and can be used
as a general scope reaction for the preparation of
1-hydroxyxanthenones. The phenol 32 was then easily
converted to the tosylate 33, which was reacted with
2-hydroxyethylhydrazine. The resulting carbinol 34
was treated with mesylchloride to provide the mesylate
35, which was then nitrated to give the 5-nitro derivative
36 in good yield, together with a small amount of the 3-
nitro isomer 37 (Fig. 2). Compound 36 was used for the
preparation of the target nitroamines 38a–38d, as well as
for the synthesis of the diamines 41a–41d via a proce-
dure analogous to the one presented for the correspond-
N
O
N
37
Figure 2. Structure of the nitro-isomer 37.
ing pyrazoloxanthenones 27a–27d. The air-sensitive
amines 39a–39d were not isolated, but converted in
one step into the chloracetamides 40a–40d.
Taking advantage of the intermediates 33 and 35, we
were also able to prepare the non-substituted amino

2914
I. K. Kostakis et al. / Bioorg. Med. Chem. 14 (2006) 2910–2934
R1
The vast majority of the new compounds showed inter-
O
O
HN
esting cytotoxic activity against all the tested cell lines,
with IC50 values varying typically within the range of
0.3–25 lM.
a
33
In general, the xanthenone and benzo[b]xanthenone
nitro-substituted amino derivatives (14a–14d and
15a–15d), as well as the corresponding dialkylaminoace-
tamides (20a–20d and 21a–21d), appear considerably
more active when compared with the corresponding
non-substituted analogues (42a–42d and 44a–44d). In
the case of the corresponding pyrazole-fused analogues,
the substituted (24a–24d, 27a–27d, 38a–38d, and
41a–41d) and non-substituted (43a–43d and 45a–45d)
derivatives possess comparable cytotoxicity. The data
indicate that the replacement of the nitro group with a
second basic side chain affords a similar or a slightly
higher cytotoxicity in the tested cell lines. Furthermore,
the insertion of a fourth benzene ring into the xanthe-
none chromophore gives compounds that retain cyto-
toxicity, although a clear improvement in the activity
of the parent molecules is not observed. Noticeably,
the non-substituted compounds 43a–43d are consider-
ably weaker than 45a–45d against the L1210 cell line.
42a-d
a: R1= N(CH3)2
b: R1= N(C2H5)2
c: R1= N(CH2)4
d: R1= N(CH2)5
R1
N
O
N
b
35
43a-d
Scheme 4. Reagents and conditions: (a) H2NCH2CH2R1, pyridine,
reflux, 16 h; (b) secondary amine, EtOH abs, reflux, 10 h.
R1
R1
O
O
HN
N
N
O
44a-d
a: R1= N(CH3)2
b: R1= N(C2H5)2
c: R1= N(CH2)4
d: R1= N(CH2)5
45a-d
The most promising derivatives against the L1210 cell
line appear to be the dialkylaminoacetamido-substituted
benzo[b]xanthenones 21a–21d and 41a–41d. The pyrrol-
idine analogue 21c emerges as the most active, showing
strong cytotoxicity with an IC50 value of 0.36 lM,
directly comparable with the corresponding value of
PZA, which has been reported to be 0.424 lM.26
Figure 3. Structure of the previously reported43 amino-substituted
xanthenones 44a–44d and benzopyranoindazoles 45a–45d.
derivatives 42a–42d and 43a–43d, respectively (Scheme
4), which would be useful for comparative reasons, con-
cerning the structure–activity relationship studies. We
also cite herein complete experimental data of the
aminoxanthenones 14a–14d and 44a–44d as well as of
their pyrazole-fused analogues 24a–24d and 45a–45d
(Fig. 3), which have been reported previously, in a pre-
liminary communication.43
The compounds possess an interesting antiprolifera-
tive activity against the colorectal adenocarcinoma
HT-29 cell line. In this case, the pyrazole-fused
xanthenones and benzo[b]xanthenones bearing the
dialkylaminoacetamido side chain (27a–27d and
41a–41d, respectively) are the most potent compounds
within the series.
For biological evaluation purposes, the free base forms
of the majority of the target amines were converted into
their water-soluble hydrochloride addition salts by treat-
ment with hydrochloric acid in methanol. However,
compounds 21d and 41a–41d were tested at the free base
form, since their hydrochloride, fumarate or malonate
addition salts were highly hygroscopic.
Concerning the activity against the uterine sarcoma
MES-SA and MES-SA/D · 5 cell lines, on average the
pyrazole-fused derivatives are more active than the cor-
responding amino-substituted analogues. Furthermore,
the majority of the nitro-substituted derivatives and
the dialkylaminoacetamides exhibit an interesting pro-
file of IC50 values, below 3 lM. Among them the pyra-
zole-fused analogues 38b, 38c, and 27c proved to be
the most cytotoxic compounds, possessing antiprolifera-
tive activity in the submicromolar range (IC50 values of
0.55, 0.57, and 0.59 lM, respectively).
2.2. Biological activity
The in vitro cytotoxic activity of the new compounds
was evaluated in the established model of the murine
leukemia cell line L1210, and in three human solid tu-
mor cell lines: colorectal adenocarcinoma HT-29, uter-
ine sarcoma MES-SA as well as its variant MES-SA/
D · 5, reported to be 100-fold resistant to doxorubi-
cin.46 The results, including reference compounds
mitoxantrone and doxorubicin, are presented in Tables
1 and 2. The previously reported cytotoxicity data of
the structurally related analogues 14a–14d, 24a–24d,
44a–44d, and 45a–45d43 are also included in the Tables,
in order to manifest clearly the structure–activity rela-
tionships within this class of compounds.
The ability of all the tested compounds to overcome
the multidrug resistance of the MES-SA/D · 5 cell line
is clearly indicated by the resistant factor (RF) values,
which are all practically equal to 1. From a compari-
son of any group of analogously substituted deriva-
tives, it is evident that they retain full
antiproliferative activity against P-glycoprotein-over-
expressing cells. Worth mentioning, the RF to doxoru-
bicin was found to be 98.25 as expected,46 while the
RF to mitoxantrone was 11.55.

I. K. Kostakis et al. / Bioorg. Med. Chem. 14 (2006) 2910–2934
2915
Table 1. Inhibition of proliferation of the amino-substituted xanthenone and benzo[b]xanthenone derivatives (IC50 values in lMa) and DNA binding
(EC50 values in lM)
Compound
R1
R2
L1210
HT-29
MES-SA
MES-SA/D · 5
RFe
EC50 (lMf)
g
14ab,i
14bb
14cd,i
14db,i
15ab
15bb
15cb
15db
20ac
20bc
20cc
20dc
21ac
21bc
21cc
21dd
42ab
42bb
42cb
42db
44ab,i
44bb
44cb,i
44db,i
Mx
N(CH3)2
N(C2H5)2
N(CH2)4
N(CH2)5
N(CH3)2
N(C2H5)2
N(CH2)4
N(CH2)5
N(CH3)2
N(C2H5)2
N(CH2)4
N(CH2)5
N(CH3)2
N(C2H5)2
N(CH2)4
N(CH2)5
N(CH3)2
N(C2H5)2
N(CH2)4
N(CH2)5
N(CH3)2
N(C2H5)2
N(CH2)4
N(CH2)5
NO2
NO2
3.13 ( 0.31)
4.96 ( 1.16)
1.48 ( 0.67)
5.88 ( 1.47)
1.93 ( 0.88)
3.90 ( 0.74)
3.24 ( 0.75)
3.00 ( 0.60)
1.09 ( 0.11)
3.90 ( 0.86)
3.58 ( 0.23)
4.34 ( 1.12)
2.13 ( 0.69)
1.24 ( 0.23)
0.36 ( 0.08)
5.92 ( 2.91)
26.1 ( 4.69)
9.31 ( 0.18)
9.19 ( 2.10)
9.66 ( 1.38)
6.58 ( 1.65)
5.00 ( 1.55)
9.00 ( 1.88)
4.59 ( 0.97)
0.077 ( 0.010)
0.080 ( 0.005)
4.66 ( 0.84)
3.20 ( 0.45)
2.53 ( 0.39)
6.49 ( 2.29)
1.81 ( 0.71)
9.43 ( 2.05)
3.43 ( 1.08)
2.26 ( 0.64)
7.23 ( 4.50)
5.99 ( 2.34)
5.05 ( 3.14)
7.45 ( 1.97)
4.16 ( 1.15)
1.22 ( 0.41)
1.44 ( 0.40)
9.68 ( 4.12)
23.4 ( 4.91)
10.1 ( 0.45)
11.1 ( 0.88)
13.8 ( 3.17)
21.6 ( 7.51)
10.9 ( 3.88)
26.6 ( 2.55)
26.5 ( 5.11)
0.020 ( 0.004)
0.320 ( 0.180)
1.56 ( 0.62)
1.25 ( 0.09)
0.78 ( 0.11)
1.59 ( 0.18)
2.38 ( 0.66)
6.31 ( 1.56)
3.01 ( 0.84)
7.81 ( 2.27)
1.59 ( 0.35)
2.72 ( 0.47)
1.70 ( 0.07)
1.84 ( 0.61)
2.34 ( 0.75)
1.16 ( 0.15)
2.40 ( 0.11)
6.71 ( 2.00)
23.4 ( 6.21)
6.40 ( 0.98)
7.60 ( 0.90)
6.60 ( 0.75)
9.03 ( 2.61)
8.38 ( 1.71)
14.61 ( 4.16)
8.31 ( 2.19)
0.003 ( 0.000)
0.016 ( 0.008)
0.95 ( 0.20)
2.02 ( 0.29)
0.55 ( 0.07)
1.56 ( 0.12)
1.1 ( 0.39)
0.61
g
1.62
0.70
0.98
0.46
1.84
0.41
0.58
0.98
0.99
1.08
0.78
1.31
0.92
0.52
0.81
0.36
1.11
0.67
0.86
0.73
0.80
0.53
0.73
11.55
98.25
NO2
NO2
7.9 ( 0.31)
g
h
g
h
g
NO2
NO2
11.6 ( 3.19)
1.23 ( 0.51)
4.55 ( 1.75)
1.56 ( 0.41)
2.69 ( 0.39)
1.84 ( 0.29)
1.43 ( 0.37)
3.06 ( 0.44)
1.07 ( 0.11)
1.25 ( 0.09)
5.41 ( 1.76)
8.51 ( 1.38)
7.10 ( 1.32)
5.10 ( 0.54)
5.70 ( 0.97)
6.62 ( 1.96)
6.68 ( 1.27)
7.76 ( 1.80)
6.04 ( 1.75)
0.030 ( 0.020)
1.56 ( 0.10)
NO2
NO2
NHCOCH2R1
NHCOCH2R1
9.0 ( 0.83)
g
NHCOCH2R1
NHCOCH2R1
10.9 ( 0.95)
g
NHCOCH2R1
NHCOCH2R1
1.2 ( 0.05)
g
NHCOCH2R1
NHCOCH2R1
3.3 ( 0.45)
g
h
g
h
g
g
g
H
H
H
H
H
H
H
H
43.2 ( 5.91)
g
0.7 ( 0.75)
Dx
a The results represent means ( standard deviation) of three independent experiments and are expressed as IC50, the concentration that reduced by
50% the optical density of treated cells with respect to untreated controls.
b Hydrochloride.
c Dihydrochloride.
d Free base form.
e IC50-resistant cells/IC50-sensitive cells.
f The results represent means of two individual experiments ( 1–10%) and are expressed as EC50, the concentration of the compound that causes a
50% reduction in the fluorescence of the calf thymus DNA/ethidium bromide complex.
g Not tested.
h >100 lM.
i Results previously reported in Ref. 43.
A competitive fluorescence displacement assay was per-
formed to determine the binding capacity of selected
compounds to calf thymus DNA. However, this assay
does not provide evidence concerning the exact mode
of interaction with the nucleic acids (e.g., intercalation
and /or groove binding mechanism). The results are
expressed in EC50 values and are presented in the last
column of Tables 1 and 2. The data on binding indicate
that the xanthenone and benzo[b]xanthenone amino
derivatives that possess a dialkylaminoacetamido substi-
tution (20a, 20c, 21a, and 21c) are effective DNA ligands
capable of displacing ethidium bromide (Table 1). On
the other hand, Table 2 shows that the pyrazole-fused
analogues are weak DNA ligands, with the exception
of the dialkylaminoacetamido-xanthenones 27a and
27c. Even if there is no quantitative correspondence
between binding with DNA and in vitro cytotoxicity,
it seems that the most cytotoxic derivatives possess high
affinity for DNA.
presented in Table 3. Most of the studied compounds
provoke, as expected, a G2/M arrest. It is known that
pyrazoloacridine and mitoxantrone block the cell cycle
in the G2 phase.33,47 Two compounds (42a and 44c)
with IC50s higher than the concentration used for
FACS analysis had, indeed, no significant effect on
the cell-cycle phase distribution. On the other hand,
the two benzo[b]xanthenone amino derivatives bearing
the nitro group (15a and 15c) tend to cause accumu-
lation of MES-SA cells in the G1 phase, while the
dimethylaminoethylamino-substituted xanthenylaceta-
mide 20a and its rigid, pyrazole-fused analogue 27a,
proved to be even more potent G1 blockers. Further-
more, the pyrrolidinethylamino-substituted benzo[b]-
xanthenylacetamide 21c and the corresponding
pyrazole-fused derivative 41c induced an S-phase ar-
rest of the cells. Especially in the series of the ami-
no-substituted compounds (Table 1),
a
high
percentage of cells accumulated in the G2 or S phases
correlates with high DNA-binding capacity (e.g., 21a,
21c or 14c), while the most potent G1 blockers 20a
and 27a bind also with high affinity to DNA. Interest-
ingly, 21c, as well as, 21a, 15a, 15c, and 43a induced
Cell-cycle perturbations induced after incubation of
exponentially growing MES-SA uterine sarcoma cells
with a number of new compounds for 24 h are

2916
I. K. Kostakis et al. / Bioorg. Med. Chem. 14 (2006) 2910–2934
Table 2. Inhibition of proliferation of the pyrazole-fused xanthenone and benzo[b]xanthenone derivatives (IC50 values in lMa) and DNA binding
(EC50 values in lM)
Compound
R1
R2
L1210
HT-29
MES-SA
MES-SA/D · 5
RFe
EC50 (lMf)
24ab,i
24bb
24cb,i
24db,i
38ab
38bb
38cb
38db
27ac
27bc
27cc
27dc
41ad
41bd
41cd
41dd
43ab
43bb
43cb
43db
45ab,i
45bb
45cb,i
45db,i
Mx
N(CH3)2
N(C2H5)2
N(CH2)4
N(CH2)5
N(CH3)2
N(C2H5)2
N(CH2)4
N(CH2)5
N(CH3)2
N(C2H5)2
N(CH2)4
N(CH2)5
N(CH3)2
N(C2H5)2
N(CH2)4
N(CH2)5
N(CH3)2
N(C2H5)2
N(CH2)4
N(CH2)5
N(CH3)2
N(C2H5)2
N(CH2)4
N(CH2)5
NO2
NO2
1.81 ( 0.41)
5.43 ( 0.86)
3.52 ( 0.35)
10.7 ( 1.85)
1.91 ( 0.64)
4.63 ( 1.19)
1.18 ( 0.27)
5.60 ( 1.55)
1.75 ( 0.28)
5.58 ( 1.20)
1.41 ( 0.30)
4.87 ( 1.05)
8.87 ( 1.89)
2.01 ( 0.44)
1.77 ( 0.36)
1.65 ( 0.18
2.88 ( 0.51)
8.83 ( 1.87)
8.09 ( 0.99)
38.4 ( 5.33)
1.17 ( 0.20)
6.37 ( 0.75)
1.79 ( 0.21)
2.74 ( 0.70)
0.077 ( 0.010)
0.080 ( 0.005)
7.49 ( 0.31)
54.10 ( 2.65)
15.8 ( 2.38)
41.6 ( 7.99)
3.41 ( 0.28)
1.85 ( 0.21)
1.83 ( 0.46)
5.15 ( 0.82)
3.41 ( 0.50)
5.77 ( 1.46)
2.26 ( 0.08)
7.22 ( 1.79)
3.74 ( 0.61)
1.48 ( 0.15)
0.39 ( 0.11)
2.23 ( 0.47)
7.84 ( 1.49)
9.22 ( 2.53)
10.7 ( 3.25)
12.1 ( 2.87)
20.9 ( 9.45)
11.5 ( 2.67)
11.5 ( 1.89)
9.92 ( 3.41)
0.020 ( 0.004)
0.320 ( 0.180)
1.03 ( 0.58)
1.98 ( 0.61)
1.13 ( 0.07)
4.05 ( 0.75)
1.30 ( 0.40)
0.55 ( 0.08)
0.57 ( 0.12)
3.87 ( 1.51)
1.65 ( 0.23)
1.87 ( 0.65)
0.59 ( 0.25)
1.63 ( 0.18)
3.55 ( 0.40)
1.12 ( 0.26)
1.55 ( 0.25)
1.95 ( 0.27)
1.40 ( 0.65)
4.73 ( 1.09)
3.00 ( 1.15)
7.50 ( 1.45)
2.79 ( 0.69)
6.55 ( 0.73)
1.75 ( 0.16)
4.40 ( 0.90)
0.003 ( 0.000)
0.016 ( 0.008)
0.63 ( 0.25)
1.95 ( 0.44)
1.47 ( 0.21)
5.38 ( 1.26)
0.39 ( 0.05)
0.30 ( 0.02)
0.51 ( 0.17)
1.49 ( 0.10)
1.74 ( 0.19)
1.62 ( 0.33)
0.86 ( 0.24)
4.57 ( 0.51)
1.84 ( 0.21)
1.28 ( 0.33)
0.96 ( 0.06)
1.17 ( 0.19)
1.45 ( 0.57)
4.53 ( 0.76)
6.20 ( 1.68)
6.90 ( 0.95)
2.76 ( 1.08)
5.78 ( 0.80)
1.58 ( 0.12)
3.88 ( 0.35)
0.030 ( 0.020)
1.56 ( 0.10)
0.61
38 ( 2.40)
g
0.98
1.29
1.33
0.30
0.55
0.89
0.39
1.05
0.87
1.46
2.80
0.52
1.14
0.62
0.60
1.04
0.96
2.07
0.92
0.99
0.88
0.90
0.88
11.55
98.25
g
g
h
g
h
g
NO2
NO2
NO2
NO2
NO2
NO2
NHCOCH2R1
NHCOCH2R1
9.6 ( 1.05)
g
NHCOCH2R1
NHCOCH2R1
12.6 ( 1.53)
g
h
g
NHCOCH2R1
NHCOCH2R1
NHCOCH2R1
NHCOCH2R1
24.3 ( 3.03)
g
h
g
h
g
H
H
H
H
H
H
H
H
32.6 ( 1.14)
g
58.6 ( 6.66)
g
0.7 ( 0.75)
Dx
a The results represent means ( standard deviation) of three independent experiments and are expressed as IC50, the concentration that reduced by
50% the optical density of treated cells with respect to untreated controls.
b Hydrochloride.
c Dihydrochloride.
d Free base form.
e IC50-resistant cells/IC50-sensitive cells.
f The results represent means of two individual experiments ( 1–10%) and are expressed as EC50, the concentration of the compound that causes a
50% reduction in the fluorescence of the calf thymus DNA/ethidium bromide complex.
g Not tested.
h >100 lM.
i Results previously reported in Ref. 43.
Table 3. Cell-cycle phase distribution (%)a
significant percentages of apoptosis, as judged by the
appearance of a sub-diploid peak (12.3%, 15.7%,
27.7%, 11.4%, and 16.7%, respectively, compared to
1.3% of the untreated cells; data not shown in
Table 3).
Compound
G0/G1
S
G2/M
14cb
15a
15c
41.70 ( 2.12)
69.02 ( 4.19)
62.38 ( 3.78)
86.91 ( 7.18)
59.86 ( 4.44)
27.01 ( 4.81)
37.72 ( 2.47)
59.41 ( 3.71)
56.92 ( 2.83)
53.35 ( 3.73)
30.00 ( 4.90)
46.07 ( 1.78)
60.37 ( 3.88)
81.97 ( 6.14)
45.67 ( 0.47)
29.54 ( 1.76)
27.53 ( 6.83)
33.06 ( 3.08)
22.63 ( 1.19)
45.28 ( 6.91)
40.10 ( 4.15)
54.70 ( 4.80)
20.79 ( 3.67)
19.83 ( 3.35)
27.23 ( 5.11)
9.91 ( 2.35)
12.70 ( 1.71)
6.97 ( 3.16)
53.52 ( 0.47)
32.66 ( 4.12)
29.54 ( 3.54)
25.51 ( 1.96)
43.40 ( 8.12)
35.28 ( 3.37)
20.93 ( 4.51)
7.71 ( 2.67)
0.10 ( 0.10)
1.37 ( 0.82)
59.74 ( 0.28)
20.87 ( 4.21)
14.81 ( 0.91)
18.81 ( 3.13)
12.40 ( 1.25)
35.57 ( 2.84)
37.41 ( 5.51)
11.15 ( 1.35)
10.39 ( 3.22)
3.18 ( 1.12)
27.44 ( 3.50)
66.02 ( 5.19)
8.77 ( 2.94)
7.93 ( 1.49)
13.54 ( 2.88)
21.14 ( 3.25)
26.60 ( 5.75)
18.65 ( 0.64)
18.70 ( 2.34)
10.32 ( 3.16)
54.23 ( 0.54)
69.09 ( 3.49)
12.73 ( 4.37)
46.08 ( 3.60)
62.57 ( 3.88)
35.91 ( 4.77)
47.50 ( 6.66)
9.74 ( 1.96)
20a
20c
21a
21c
44cb
42a
42c
24ab
38a
38c
3. Conclusions
In summary, the present study deals with the synthesis
of a number of new xanthenone and benzo[b]xanthe-
none amino derivatives bearing structural analogy to
PZA. The compounds inhibit the proliferation of a pan-
el of cancer cell lines, although probably not via a com-
mon mechanism, since they exhibit significant variation
in their DNA binding capacity and they arrest the cells
in different phases of the cell-cycle. In general, it seems
that the insertion of a second basic side chain in the
chromophore results in the improvement of cytotoxicity
and DNA binding affinity of the derivatives. An inter-
esting feature of this class of compounds is the lack of
cross-resistance with doxorubicin against the MES-SA/
D · 5 cell line, a finding that would require further stud-
ies in the future.
27a
27c
41a
41c
45ab
45cb
43a
43c
Control
a Mean ( SD) of three independent experiments.
b Results previously reported in Ref. 43.

I. K. Kostakis et al. / Bioorg. Med. Chem. 14 (2006) 2910–2934
2917
4. Experimental
J = 8 Hz, 1 Hz, 1H, H-5), 7.96 (d, J = 9 Hz, 1H, H-30),
8.03 (dd, J = 8 Hz, 1H, H-6); 13C NMR (CDCl3,
50 MHz) d 13.81 (CH2CH3), 61.42 (CH2CH3), 117.65
(C-60), 122.21 (C-40), 122.95 (C-3), 124.16 (C-1), 126.25
(C-5), 126.92 (C-2), 132.72 (C-6), 134.34 (C-4), 138.13
(C-20), 140.20 (C-50), 152.76 (C-10, C-2), 164.45 (CO).
Anal. Calcd for C15H12ClNO5: C, 56.00; H, 3.76; N,
4.35. Found: C, 55.94; H, 3.51; N, 4.41.
4.1. Chemistry
All chemicals were purchased from Aldrich Chemical
Co. Melting points were determined with a Buchi appa-
¨
1
ratus and are uncorrected. H NMR spectra and 2D
spectra were recorded on a Bruker Avanche 400 instru-
ment, whereas 13C NMR spectra were recorded on a
Bruker AC 200 spectrometer in deuterated solvents
and were referenced to TMS (d scale). The signals of
1H and 13C spectra were unambiguously assigned by
using 2D NMR techniques: 1H–1H COSY, NOESY
4.1.2. Ethyl 3-(5-chloro-2-nitrophenyloxy)-2-naphthoate
(9). This compound was prepared by a procedure anal-
ogous to that of 8 starting from 4. From the resulting
mixture of the naphthoates 7 and 9 we have only isolat-
ed the desired isomer 9 (33%) by trituration in boiling
ethanol. Mp 112–114 ꢁC (EtOH); 1H NMR (CDCl3,
400 MHz) d 1.18 (t, J = 7 Hz, 3H, CH2CH3), 4.21 (q,
J = 7 Hz, 2H, CH2CH3), 6.73 (d, J = 2 Hz, 1H, H-60),
7.02 (dd, J = 9 Hz, 2 Hz, 1H, H-40), 7.62 (t, J = 8 Hz,
1H, H-6), 7.64 (s, 1H, H-4), 7.68 (t, J = 8 Hz, 1H, H-
7), 7.86 (d, J = 8 Hz, 1H, H-5), 8.02 (d, J = 9 Hz, 1H,
H-30), 8.03 (d, J = 8 Hz, 1H, H-8), 8.67 (s, 1H, H-1);
13C NMR (CDCl3, 50 MHz) d 13.89 (CH2CH3), 61.53
(CH2CH3), 117.73 (C-60), 120.45 (C-4), 122.10 (C-40),
122.76 (C-3), 126.95 (C-30), 127.05 (C-7), 127.25 (C-5),
129.12 (C-6), 129.23 (C-8), 130.63 (C-8a), 134.82 (C-1),
135.71 (C-4a), 138.15 (C-20), 140.22 (C-50), 149.01 (C-
3), 153.58 (C-10), 168.53 (CO). Anal. Calcd for
C19H14ClNO5: C, 61.38; H, 3.80; N, 3.77. Found: C,
61.09; H, 3.64, N, 3.89.
1
HMQC, and HMBC. For the H–15N GHMQC spec-
trum, data were acquired as 3072 · 400 data points with
a total of 290 transients accumulated per t1 increment.
The F1 spectral window employed was set from 100 to
400 ppm and the F2 from 0 to 10 ppm correspondingly.
Flash chromatography was performed on Merck silica
gel 60 (0.040–0.063 mm). Analytical thin-layer chroma-
tography (TLC) was carried out on precoated
(0.25 mm) Merck silica gel F-254 plates. Elemental anal-
yses were performed at the Microanalytical Sections of
the National Hellenic Research Foundation on a Per-
kin-Elmer PE 240C Elemental Analyzer (Norwalk,
CT) and are within 0.4% of the theoretical values.
4.1.1. Ethyl 2-(3-chloro-4-nitrophenyloxy)benzoate (6)
and ethyl 2-(5-chloro-2-nitrophenyloxy)benzoate (8). To
a solution of ethyl salicylate (3, 33.37 g, 201 mmol) in
dry DMF (50 mL), 2,4-dichloronitrobenzene (5, 38.4 g,
200 mmol), powdered K2CO3 (27.74 g, 201 mmol), and
Cu2O (2.85 g, 20.1 mmol) were added under argon and
the mixture was heated at 110 ꢁC for 8 h. The reaction
mixture was then filtered hot, the filter cake was washed
with CH2Cl2, the filtrate was concentrated in vacuo, and
the residue was dissolved in CH2Cl2, washed with water,
dried (Na2SO4), and evaporated to dryness. Flash chro-
matography on silica gel using a mixture of cyclohexane/
EtOAc 100:5 as the eluent provided the title compounds
6 and 8 (23% and 64%, respectively).
4.1.3. 1-Chloro-4-nitro-9H-xanthen-9-one (12).48 To a
suspension of 8 (1.29 g, 4 mmol) in ethanol at room tem-
perature was added dropwise a cold 40% NaOH solu-
tion (2 ml). The mixture was stirred for 30 min at
room temperature and then poured into water and acid-
ified with 18% HCl solution. The resulting 2-(5-chloro-2-
nitrophenyloxy)benzoic acid (10) was filtered, air-dried,
and dissolved in hot polyphosphoric acid. The mixture
was heated at 110 ꢁC for 1 h and upon cooling it was
poured into ice water. The precipitate was filtered and
air-dried to give crude 12, which was purified by column
chromatography (silica gel) using a mixture of CH2Cl2/
cyclohexane 1:4–3:1 as the eluent. Yield: 81%; mp
>270 ꢁC (EtOH); 1H NMR (DMSO-d6, 400 MHz) d
7.53 (td, J = 8 Hz, 1 Hz, 1H, H-7), 7.62 (d, J = 8 Hz,
1H, H-5), 7.66 (d, J = 8 Hz, 1H, H-2), 7.90 (dt,
J = 8 Hz, 1 Hz, 1H, H-6), 8.14 (d, J = 8 Hz, 1 Hz, 1H,
H-8), 8.43 (d, J = 8 Hz, 1H, H-3); 13C NMR (DMSO-
d6, 50 MHz) d 118. 35 (C-5), 120.44 (C-9a), 122.14 (C-
8a), 125.76 (C-7), 126.67 (C-8), 126.74 (C-2), 130.42
(C-3), 136.51 (C-4), 136.61 (C-6), 138.87 (C-1), 150.07
(C-4a), 154.27 (C-10a), 174.29 (C-9).
Data for ethyl 2-(3-chloro-4-nitrophenyloxy)benzoate
(6): mp 76–78 ꢁC (Et2O/n-hexane); H NMR (CDCl3,
1
400 MHz) d 1.17 (t, J = 7 Hz, 3H, CH2CH3), 4.25 (q,
J = 7 Hz, 2H, CH2CH3), 6.96 (d, J = 2 Hz, 1H, H-20),
6.84 (dd, J = 9 Hz, 2 Hz, 1H, H-60), 7.16 (d, J = 8 Hz,
1H, H-3), 7.41 (dt, J = 8 Hz, 1 Hz, 1H, H-4), 7.63 (dt,
J = 8 Hz, 1 Hz, 1H, H-5), 7.98 (d, J = 9 Hz, 1H, H-50),
8.06 (dd, J = 8 Hz, 1H, H-6); 13C NMR (CDCl3,
50 MHz) d 14.15 (CH2CH3), 61.45 (CH2CH3), 114.85
(C-60), 118.85 (C-20), 123.32 (C-3), 124.46 (C-1), 126.45
(C-4), 128.01 (C-50), 129.74 (C-40), 132.62 (C-6), 134.43
(C-5), 141.63 (C-30), 152.86 (C-2), 162.16 (C-10), 164.54
(CO). Anal. Calcd for C15H12ClNO5: C, 56.00; H,
3.76; N, 4.35. Found: C, 55.87; H, 3.40; N, 4.28.
4.1.4. 1-Chloro-4-nitro-12H-benzo[b]xanthen-12-one (13).
This compound was prepared by a procedure analogous
to that of 12 starting from 9. The crude intermediate 3-
(5-chloro-2-nitrophenyloxy)-2-naphthoic acid (11) was
ring closed as described in the foregoing procedure.
Data for ethyl 2-(5-chloro-2-nitrophenyloxy)benzoate
(8): mp 87–89 ꢁC (Et2O/n-hexane); H NMR (CDCl3,
1
1
Yield: 65%; mp >270 ꢁC (DMF); H NMR (DMSO-
400 MHz) d 1.16 (t, J = 7 Hz, 3H, CH2CH3), 4.23 (q,
J = 7 Hz, 2H, CH2CH3), 6.71 (d, J = 2 Hz, 1H, H-60),
7.11 (dd, J = 9 Hz, 2 Hz, 1H, H-40), 7.18 (d, J = 8 Hz,
1H, H-3), 7.41 (dt, J = 8 Hz, 1 Hz, 1H, H-4), 7.63 (dt,
d6, 400 MHz) d 7.14 (d, J = 8 Hz, 1H, H-2), 7.51 (t,
J = 8 Hz, 1H, H-9), 7.62 (t, J = 8 Hz, 1H, H-8), 7.90
(d, J = 8 Hz, 1H, H-7), 7.92 (s, 1H, H-6), 8.01 (d,
J = 8 Hz, 1H, H-10), 8.12 (d, J = 8 Hz, 1H, H-3), 8.44

2918
I. K. Kostakis et al. / Bioorg. Med. Chem. 14 (2006) 2910–2934
(s, 1H, H-11); 13C NMR (DMSO-d6, 50 MHz) d 114.97
(C-6), 119.97 (C-12a), 121.53 (C-11a), 126.94 (C-9),
127.46 (C-2), 127.79 (C-7), 128.33 (C-11), 130.43 (C-3,
C-8), 130.61 (C-10, C-10a), 135.62 (C-6a), 136.27 (C-
4), 140.16 (C-1), 150.12 (C-4a), 151.05 (C-5a), 179.35
(C-12). Anal. Calcd for C17H8ClNO4: C, 62.69; H,
2.48; N, 4.30. Found: C, 62.38; H, 2.41; N, 4.05.
J = 8 Hz, 1H, H-7), 7.48 (d, J = 8 Hz, 1H, H-5), 7.65
(t, J = 8 Hz, 1H, H-6), 8.11 (dd, J = 8 Hz, 1 Hz, 1H,
H-8), 8.21 (d, J = 9 Hz, 1H, H-3), 10.60 (t, J = 5 Hz,
1H, D2O exch, NH); 13C NMR (CDCl3, 50 MHz) d
23.52 (N(CH2CH2)2), 42.30 (NHCH2CH2), 54.06
(N(CH2CH2)2), 54.07 (NHCH2CH2), 103.10 (C-2),
104.93 (C-9a), 117.74 (C-5), 121.40 (C-8a), 124.79
(C-7), 125.35 (C-4), 125.56 (C-8), 133.31 (C-3), 134.78
(C-6), 153.05 (C-1), 154.23 (C-10a), 155.37 (C-4a),
178.63 (C-9). Anal. Calcd for C19H19N3O4ÆHClÆ
1/4H2O: C, 57.91; H, 5.18; N, 10.66. Found: C, 58.03;
H, 4.97; N, 10.41.
4.1.5.
yl)ethane-1,2-diamine (14a). A solution of 12 (271 mg,
0.983 mmol) and 2-(dimethylamino)ethylamine
N,N-Dimethyl-N0-(4-nitro-9-oxo-9H-xanthen-1-
(793 mg, 9 mmol) in dry pyridine (6 mL) was refluxed
for 90 min. After cooling, the mixture was vacuum-
evaporated, extracted with ethyl acetate–water, the
organic layer was dried (Na2SO4) and evaporated to
dryness. The residue was purified by column chromatog-
raphy (silica gel, CH2Cl2/MeOH 93:7) to provide 14a
(300 mg, 93%); mp (hydrochloride) >270 ꢁC (EtOH);
1H NMR (CDCl3, 400 MHz) d 2.37 (s, 6H, 2 x CH3),
2.69 (t, J = 7 Hz, 2H, NHCH2CH2), 3.44 (q, J = 7 Hz,
5 Hz, 2H, NHCH2CH2), 6.43 (d, J = 9 Hz, 1H, H-2),
7.41 (td, J = 8 Hz, 1 Hz, 1H, H-7), 7.60 (dd, J = 8 Hz,
1 Hz, 1H, H-5), 7.74 (dt, J = 8 Hz, 1 Hz, 1H, H-6),
8.25 (dd, J = 8 Hz, 1 Hz, 1H, H-8), 8.34 (d, J = 9 Hz,
1H, H-3), 10.74 (t, J = 5 Hz, 1H, D2O exch, NH); 13C
NMR (CDCl3, 50 MHz) d 41.17 (NHCH2CH2), 45.77
(2· CH3), 57.37 (NHCH2CH2), 103.24 (C-2), 105.33
(C-9a), 118.09 (C-5), 121.69 (C-8a), 125.00 (C-7),
125.84 (C-8), 126.82 (C-4), 133.60 (C-3), 135.00 (C-6),
153.32 (C-1), 154.51 (C-10a), 155.54, (C-4a), 179.10
(C-9). Anal. Calcd for C17H17N3O4ÆHClÆ3/2H2O: C,
52.24; H, 5.42; N, 10.75. Found: C, 52.43; H, 5.37; N,
10.57.
4.1.8. 4-Nitro-1-[2-(piperidin-1-yl)ethylamino]-9H-xan-
thene-9-one (14d). This compound was prepared by an
analogous procedure as described for the preparation
of 14a. Yield: 93%; mp (hydrochloride) >270 ꢁC
1
(EtOH); H NMR (CDCl3, 400 MHz) d 1.54 (m, 2H,
4-piperidine-H), 1.74 (m, 4H, 3,5-piperidine-H), 2.62
(m, 4H, 2,6-piperidine-H), 2.72 (t, J = 7 Hz, 2H,
NHCH2CH2), 3.41 (q, J = 7 Hz, 5 Hz, 2H,
NHCH2CH2), 6.41 (d, J = 9 Hz, 1H, H-2), 7.36 (t,
J = 8 Hz, 1H, H-7), 7.50 (d, J = 8 Hz, 1H, H-5), 7.71
(t, J = 8 Hz, 1H, H-6), 8.18 (dd, J = 8 Hz, 1 Hz, 1H,
H-8), 8.30 (d, J = 9 Hz, 1H, H-3), 10.69 (t, J = 5 Hz,
1H, D2O exch, NH); 13C NMR (CDCl3, 50 MHz) d
24.33 (4-piperidine-C), 25.94 (3,5-piperidine-C), 40.65
(NHCH2CH2),
54.54
(2,6-piperidine-C),
56.67
(NHCH2CH2), 103.32 (C-2), 105.23 (C-9a), 118.02 (C-
5), 121.62 (C-8a), 124.90 (C-7), 125.47 (C-4), 125.78
(C-8), 133.46 (C-3), 134.89 (C-6), 153.31 (C-1), 154.41
(C-10a), 155.51 (C-4a), 178.89 (C-9). Anal. Calcd for
C20H21N3O4ÆHClÆH2O: C, 56.94; H, 5.73; N, 9.96.
Found: C, 56.86; H, 5.69; N, 10.08.
4.1.6.
N,N-Diethyl-N0-(4-nitro-9-oxo-9H-xanthen-1-
yl)ethane-1,2-diamine (14b). This compound was pre-
pared by an analogous procedure as described for the
preparation of 14a. Yield: 95%; mp (hydrochloride)
241–243 ꢁC (EtOH); 1H NMR (CDCl3, 400 MHz) d
1.10 (t, J = 7 Hz, 6H, 2· CH2CH3), 2.63 (q, J = 7 Hz,
4H, 2· CH2CH3), 2.80 (t, J = 7 Hz, 2H, NHCH2CH2),
3.39 (q, J = 7 Hz, 5 Hz, 2H, NHCH2CH2), 6.40 (d,
J = 9 Hz, 1H, H-2), 7.38 (t, J = 8 Hz, 1H, H-7), 7.55
(d, J = 8 Hz, 1H, H-5), 7.70 (t, J = 8 Hz, 1H, H-6),
8.20 (dd, J = 8 Hz, 1 Hz, 1H, H-8), 8.29 (d, J = 8 Hz,
1H, H-3), 10.70 (t, J = 5 Hz, 1H, D2O exch, NH); 13C
NMR (CDCl3, 50 MHz) d 11.35 (2· CH2CH3), 40.87
4.1.9. N,N-Dimethyl-N0-(4-nitro-12-oxo-12H-benzo[b]-
xanthen-1-yl)ethane-1,2-diamine (15a). This compound
was prepared by an analogous procedure as described
for the preparation of 14a, starting from 13. Yield:
88%; mp (hydrochloride) >270 ꢁC (EtOH); 1H NMR
(CDCl3, 400 MHz) d 2.41 (s, 6H, 2· CH3), 2.73 (t,
J = 6 Hz, 2H, NHCH2CH2), 3.48 (q, J = 6 Hz, 5 Hz,
2H, NHCH2CH2), 6.47 (d, J = 8 Hz, 1H, H-2), 7.52 (t,
J = 8 Hz, 1H, H-9), 7.66 (t, J = 8 Hz, 1H, H-8), 7.96
(d, J = 8 Hz, 1H, H-7), 8.04 (s, 1H, H-6), 8.06 (d,
J = 8 Hz, 1H, H-10), 8.36 (d, J = 8 Hz, 1H, H-3), 8.85
(s, 1H, H-11), 10.8 (t, J = 5 Hz, 1H, D2O exch, NH);
13C NMR (CDCl3, 50 MHz) d 41.12 (NHCH2CH2),
45.44 (2· CH3), 57.33 (NHCH2CH2), 103.37 (C-2),
104.51 (C-12a), 114.04 (C-6), 120.83 (C-11a), 125.13
(C-4), 126.02 (C-9), 127.33 (C-7), 127.55 (C-11), 129.25
(C-8), 129.61 (C-10), 130.15 (C-10a), 133.81 (C-3),
136.60 (C-6a), 150.55 (C-5a), 155.58 (C-1), 155.62,
(C-4a), 179.66 (C-12). Anal. Calcd for C21H19N3O4Æ
HClÆ1/2H2O: C, 59.65; H, 5.01; N, 9.94. Found: C,
59.71; H, 4.81; N, 9.92.
(NHCH2CH2),
47.08
(2·
CH2CH3),
51.67
(NHCH2CH2), 103.21 (C-2), 105.06 (C-9a), 117.93 (C-
5), 121.54 (C-8a), 124.91 (C-7), 125.70 (C-4), 125.74
(C-8), 133.37 (C-3), 134.91 (C-6), 153.44 (C-1), 154.37
(C-10a), 155.46, (C-4a), 179.01 (C-9). Anal. Calcd for
C19H21N3O4ÆHClÆH2O: C, 55.68; H, 5.90; N, 10.25.
Found: C, 55.51; H, 5.98; N, 10.33.
4.1.7. 4-Nitro-1-[2-(pyrrolidin-1-yl)ethylamino]-9H-xan-
thene-9-one (14c). This compound was prepared by an
analogous procedure as described for the preparation
of 14a. Yield: 95%; mp (hydrochloride) >270 ꢁC
4.1.10.
N,N-Diethyl-N0-(4-nitro-12-oxo-12H-benzo[b]-
xanthen-1-yl)ethane-1,2-diamine (15b). This compound
was prepared by an analogous procedure as described
for the preparation of 14a, starting from 13. Yield:
89%; mp (hydrochloride) >270 ꢁC (EtOH); 1H NMR
(CDCl3, 400 MHz) d 1.12 (t, J = 7 Hz, 6H, 2· CH2CH3),
1
(EtOH); H NMR (CDCl3, 400 MHz) d 1.80 (m, 4H,
N(CH2CH2)2), 2.59 (m, 4H, N(CH2CH2)2), 2.80 (t,
J = 7 Hz, 2H, NHCH2CH2), 3.43 (q, J = 7 Hz, 5 Hz,
2H, NHCH2CH2), 6.33 (d, J = 9 Hz, 1H, H-2), 7.33 (t,

I. K. Kostakis et al. / Bioorg. Med. Chem. 14 (2006) 2910–2934
2919
2.68 (q, J = 7 Hz, 4H, 2· CH2CH3), 2.86 (t, J = 6 Hz,
2H, NHCH2CH2), 3.45 (q, J = 6 Hz, 5 Hz, 2H,
NHCH2CH2), 6.43 (d, J = 8 Hz, 1H, H-2), 7.53 (t,
J = 8 Hz, 1H, H-9), 7.64 (t, J = 8 Hz, 1H, H-8), 7.93
(d, J = 8 Hz, 1H, H-7), 8.02 (s, 1H, H-6), 8.04 (d,
J = 8 Hz, 1H, H-10), 8.37 (d, J = 8 Hz, 1H, H-3), 8.86
(s, 1H, H-11), 10.81 (t, J = 5 Hz, 1H, D2O exch, NH);
13C NMR (CDCl3, 50 MHz) d 11.87 (2· CH2CH3),
41.56 (NHCH2CH2), 47.02 (2· CH2CH3), 50.88
(NHCH2CH2), 103.54 (C-2), 104.63 (C-12a), 114.04
(C-6), 120.92 (C-11a), 125.22 (C-4), 126.02 (C-9),
127.36 (C-7), 127.53 (C-11), 129.20 (C-8), 129.64 (C-
10), 129.75 (C-10a), 133.76 (C-3), 136.44 (C-6a),
150.55 (C-5a), 155.60 (C-1), 155.67, (C-4a), 179.39 (C-
12). Anal. Calcd for C23H23N3O4ÆHClÆH2O: C, 60.06;
H, 5.70; N, 9.14. Found: C, 59.88; H, 5.74; N, 9.11.
4.1.13.
2-Chloro-N-[1-(2-dimethylaminoethylamino)-9-
oxo-9H-xanthen-4-yl]acetamide (18a). A solution of
14a (1.31 g, 4 mmol) in absolute EtOH (40 mL) was
hydrogenated in the presence of 10% Pd/C (100 mg), un-
der a pressure of 50 psi at room temperature for 3 h. The
resulting mixture was filtered through a Celite pad and
the filtrate was evaporated to dryness to result in oil cor-
responding to the 4-aminoderivative 16a. Without fur-
ther purification, the residue was dissolved under
argon in dry THF (10 mL) and to this solution were
added dropwise at 0 ꢁC triethylamine (1.67 mL,
12 mmol) and
a
solution of chloracetylchloride
(0.32 mL, 4 mmol) in dry THF (5 mL). The resulting
solution was stirred for 10 min at 0 ꢁC and at room tem-
perature for another 90 min. The solvent was then evap-
orated to dryness and the residue was purified by
column chromatography (silica gel) using a mixture of
CH2Cl2/MeOH 8:1 as the eluent to provide compounds
4.1.11.
4-Nitro-1-[2-(pyrrolidin-1-yl)ethylamino]-12H-
1
benzo[b]xanthene-12-one (15c). This compound was pre-
pared by an analogous procedure as described for the
preparation of 14a, starting from 13. Yield: 87%; mp
(hydrochloride) >270 ꢁC (EtOH); 1H NMR (CDCl3,
400 MHz) d 1.92 (m, 4H, N(CH2CH2)2), 2.62 (m, 4H,
N(CH2CH2)2), 2.88 (t, J = 6 Hz, 2H, NHCH2CH2),
3.49 (q, J = 6 Hz, 5 Hz, 2H, NHCH2CH2), 6.42 (d,
J = 8 Hz, 1H, H-2), 7.46 (t, J = 8 Hz, 1H, H-9), 7.58
(t, J = 8 Hz, 1H, H-8), 7.90 (d, J = 8 Hz, 1H, H-7),
7.94 (s, 1H, H-6), 7.97 (d, J = 8 Hz, 1H, H-10), 8.29
(d, J = 8 Hz, 1H, H-3), 8.73 (s, 1H, H-11), 10.80 (t,
J = 5 Hz, 1H, D2O exch, NH); 13C NMR (CDCl3,
50 MHz) d 23.67 (N(CH2CH2)2), 45.53 (NHCH2CH2),
54.26 (N(CH2CH2)2), 54.27 (NHCH2CH2), 103.37 (C-
2), 104.40 (C-12a), 113.99 (C-6), 120.74 (C-11a),
125.02 (C-4), 126.01 (C-9), 127.33 (C-7), 127.41 (C-11),
129.21 (C-8), 129.59 (C-10), 130.09 (C-10a), 133.73 (C-
3), 136.56 (C-6a), 150.41 (C-5a), 155.65 (C-1), 155.69,
(C-4a), 179.49 (C-12). Anal. Calcd for C23H21N3O4ÆH-
ClÆ1/2H2O: C, 61.54; H, 5.16; N, 9.36. Found: C,
61.69; H, 4.98; N, 9.28.
18a (1.24 g, 83%); mp >270 ꢁC (EtOAc/n-hexane); H
NMR (CDCl3, 400 MHz) d 2.67 (s, 6H, 2· CH3), 2.97
(t, J = 7 Hz, 2H, NHCH2CH2), 3.64 (q, J = 7 Hz,
5 Hz, 2H, NHCH2CH2), 4.29 (s, 2H, COCH2), 6.47 (d,
J = 9 Hz, 1H, H-2), 7.35 (t, J = 8 Hz, 1 Hz, 1H, H-7),
7.40 (d, J = 8 Hz, 1H, H-5), 7.67 (t, J = 8 Hz, 1H, H-
6), 8.18 (m, 2H, H-3, H-8), 8.73 (s, 1H, D2O exch,
NHCO), 9.43 (t, J = 5 Hz, 1H, D2O exch, NH); 13C
NMR (CDCl3, 50 MHz) d 43.04 (COCH2), 43.89
(NHCH2CH2), 45.31 (2· CH3), 56.16 (NHCH2CH2),
102.91 (C-2), 106.55 (C-9a), 112.80 (C-4), 117.10 (C-5),
121.66 (C-8a), 124.26 (C-7), 126.18 (C-8), 129.74 (C-3),
134.56 (C-6), 148.04 (C-1), 148.42 (C-4a), 154.40
(C-10a), 163.81 (NHCO), 179.39 (C-10). Anal. Calcd
for C19H20ClN3O3: C, 61.04; H, 5.39; N, 11.24. Found:
C, 61.33; H, 5.09; N, 11.39.
Spectral data for the intermediate N,N-dimethyl-N0-
(4-amino-9-oxo-9H-xanthen-1-yl)ethane-1,2-diamine
(16a): H NMR (DMSO-d6, 400 MHz) d 2.48 (s, 6H,
2· CH3), 2.82 (t, J = 7 Hz, 2H, NHCH2CH2), 3.40 (q,
J = 7 Hz, 5 Hz, 2H, NHCH2CH2), 6.27 (d, J = 8 Hz,
1H, H-2), 6.98 (d, J = 8 Hz, 1 Hz, 1H, H-3), 7.21 (t,
J = 8 Hz, 1 Hz, 1H, H-7), 7.29 (d, J = 8 Hz, 1 Hz,
1H, H-5), 7.53 (t, J = 8 Hz, 1 Hz, 1H, H-6), 8.11 (d,
J = 8 Hz, 1H, H-8), 8.81 (t, J = 5 Hz, 1H, D2O exch,
NH).
1
4.1.12.
4-Nitro-1-[2-(piperidin-1-yl)ethylamino]-12H-
benzo[b]xanthene-12-one (15d). This compound was pre-
pared by an analogous procedure as described for the
preparation of 14a, starting from 13. Yield: 87%; mp
(hydrochloride) >270 ꢁC (EtOH); 1H NMR (CDCl3,
400 MHz) d 1.53 (m, 2H, 4-piperidine-H), 1.71 (m, 4H,
3,5-piperidine-H), 2.55 (m, 4H, 2,6-piperidine-H), 2.76
(t, J = 6 Hz, 2H, NHCH2CH2), 3.51 (q, J = 6 Hz,
5 Hz, 2H, NHCH2CH2), 6.43 (d, J = 8 Hz, 1H, H-2),
7.59 (t, J = 8 Hz, 1H, H-9), 7.61 (t, J = 8 Hz, 1H, H-
8), 7.91 (d, J = 8 Hz, 1H, H-7), 7.98 (s, 1H, H-6), 8.04
(d, J = 8 Hz, 1H, H-10), 8.32 (d, J = 8 Hz, 1H, H-3),
8.78 (s, 1H, H-11), 10.81 (t, J = 5 Hz, 1H, D2O exch,
NH); 13C NMR (CDCl3, 50 MHz) d 24.99 (4-piperi-
dine-C), 25.89 (3,5-piperidine-C), 40.59 (NHCH2CH2),
54.51 (2,6-piperidine-C), 56.62 (NHCH2CH2), 103.44
(C-2), 104.44 (C-12a), 113.97 (C-6), 120.78 (C-11a),
124.99 (C-4), 125.95 (C-9), 127.26 (C-7), 127.41 (C-11),
129.15 (C-8), 129.57 (C-10), 130.07 (C-10a), 133.67 (C-
3), 136.53 (C-6a), 150.43 (C-5a), 155.62 (C-1), 155.68,
(C-4a), 179.39 (C-12). Anal. Calcd for C24H23N3O4Æ
HClÆ1/2H2O: C, 62.27; H, 5.44; N, 9.08. Found: C,
62.42; H, 5.17; N, 8.82.
4.1.14. 2-Chloro-N-[1-(2-diethylaminoethylamino)-9-oxo-
9H-xanthen-4-yl]acetamide (18b). This compound was
prepared by an analogous procedure as described for
the preparation of 18a. Yield: 87%; mp 194–196 ꢁC
1
(EtOAc/n-hexane); H NMR (CDCl3, 400 MHz) d 1.10
(t, J = 7 Hz, 6H, 2· CH2CH3), 2.65 (q, J = 7 Hz, 4H,
2· CH2CH3), 2.81 (t, J = 7 Hz, 2H, NHCH2CH2), 3.34
(q, J = 7 Hz, 5 Hz, 2H, NHCH2CH2), 4.28 (s, 2H,
COCH2), 6.42 (d, J = 9 Hz, 1H, H-2), 7.29–7.44 (m,
2H, H-5, H-7), 7.65 (t, J = 8 Hz, 1H, H-6), 8.23 (m,
2H, H-3, H-8), 8.67 (s, 1H, D2O exch, NHCO), 9.40
(t, J = 5 Hz, 1H, D2O exch, NH); 13C NMR (CDCl3,
50 MHz) d 11.68 (2· CH2CH3), 41.16 (NHCH2CH2),
43.11 (COCH2), 47.15 (2· CH2CH3), 51.27
(NHCH2CH2), 103.10 (C-2), 106.26 (C-9a), 111.70 (C-
4), 116.96 (C-5), 121.96 (C-8a), 124.09 (C-7), 126.33
(C-8), 129.49 (C-3), 134.23 (C-6), 148.02 (C-1), 149.01

2920
I. K. Kostakis et al. / Bioorg. Med. Chem. 14 (2006) 2910–2934
(C-4a), 154.41, (C-10a), 163.60 (NHCO), 179.15 (C-9).
Anal. Calcd for C21H24ClN3O3: C, 62.76; H, 6.02; N,
10.46. Found: C, 62.60; H, 5.88; N, 10.60.
54.62 (2,6-piperidine-C), 57.25 (NHCH2CH2), 102.98
(C-2), 106.43 (C-9a), 112.88 (C-4), 117.11 (C-5), 121.62
(C-8a), 124.00 (C-7), 126.44 (C-8), 129.02 (C-3), 134.22
(C-6), 147.33 (C-1), 147.62 (C-4a), 154.11 (C-10a),
163.72 (NHCO), 179.52 (C-9). Anal. Calcd for
C22H24ClN3O3: C, 63.84; H, 5.84; N, 10.15. Found: C,
63.71; H, 5.70; N, 9.87.
Spectral data for the intermediate N,N-diethyl-N0-(4-
amino-9-oxo-9H-xanthen-1-yl)ethane-1,2-diamine
(16b): 1H NMR (CD3OD, 400 MHz) d 1.11 (t,
J = 7 Hz, 6H, 2· CH2CH3), 2.49 (q, J = 7 Hz, 4H,
2· CH2CH3), 2.73 (t, J = 7 Hz, 2H, NHCH2CH2),
3.21 (q, J = 7 Hz, 5 Hz, 2H, NHCH2CH2), 6.29
(d, J = 8 Hz, 1H, H-2), 7.04 (d, J = 8 Hz, 1H, H-
3), 7.33 (t, J = 8 Hz, 1H, H-7), 7.40 (d, J = 8 Hz,
1H, H-5), 7.64 (t, J = 8 Hz, 1H, H-6), 8.16 (d,
J = 8 Hz, 1H, H-8), 9.36 (t, J = 5 Hz, 1H, D2O
exch, NH).
Spectral data for the intermediate 4-amino-1-[2-(piperi-
din-1-yl)ethylamino]-9H-xanthene-9-one
(16d):
1H
NMR (CD3OD, 400 MHz) d 1.51 (m, 2H, 4-piperi-
dine-H), 1.68 (m, 4H, 3,5-piperidine-H), 2.53 (m, 4H,
2,6-piperidine-H), 2.86 (t, J = 7 Hz, 2H, NHCH2CH2),
3.47 (q, J = 7 Hz, 5 Hz, 2H, NHCH2CH2), 6.38 (d,
J = 9 Hz, 1H, H-2), 6.99 (d, J = 8 Hz, 1H, H-3), 7.38
(t, J = 8 Hz, 1H, H-7), 7.45 (d, J = 8 Hz, 1H, H-5),
7.67 (t, J = 8 Hz, 1H, H-6), 8.14 (d, J = 9 Hz, 1H, H-
8), 9.01 (t, J = 5 Hz, 1H, D2O exch, NH).
4.1.15. 2-Chloro-N-[1-[2-(pyrrolidin-1-yl)ethylamino]-9-
oxo-9H-xanthen-4-yl]acetamide (18c). This compound
was prepared by an analogous procedure as described
for the preparation of 18a. Yield: 83%; mp 259–260 ꢁC
4.1.17. 2-Chloro-N-[1-(2-dimethylaminoethylamino)-12-
oxo-12H-benzo[b]xanthen-4-yl]acetamide (19a). This
compound was prepared by an analogous procedure as
described for the preparation of 18a, starting from 15a.
1
(EtOAc/n-hexane); H NMR (CDCl3, 400 MHz) d 1.91
(m, 4H, N(CH2CH2)2), 2.83 (m, 4H, N(CH2CH2)2),
2.98 (t, J = 7 Hz, 2H, NHCH2CH2), 3.55 (q, J = 7 Hz,
5 Hz, 2H, NHCH2CH2), 4.29 (s, 2H, COCH2), 6.48 (d,
J = 9 Hz, 1H, H-2), 7.36 (t, J = 8 Hz, 1H, H-7), 7.40
(d, J = 8 Hz, 1H, H-5), 7.67 (t, J = 8 Hz, 1H, H-6),
8.23 (m, 2H, H-3, H-8), 8.68 (s, 1H, D2O exch, NHCO),
9.44 (t, J = 5 Hz, 1H, D2O exch, NH); 13C NMR
1
Yield: 81%; mp 244–246 ꢁC (EtOH); H NMR (CDCl3,
400 MHz) d 2.39 (s, 6H, 2· CH3), 2.71 (t, J = 7 Hz, 2H,
NHCH2CH2), 3.40 (q, J = 7 Hz, 2H, NHCH2CH2), 4.41
(s, 2H, NHCOCH2), 6.45 (d, J = 8 Hz, 1H, H-2), 7.48 (t,
J = 8 Hz, 1H, H-9), 7.62 (t, J = 8 Hz, 1H, H-8), 7.78 (s,
1H, H-6), 7.88 (d, J = 8 Hz, 1H, H-7), 8.06 (d, J = 8 Hz,
1H, H-10), 8.31 (d, J = 8 Hz, 1H, H-3), 8.82 (s, 1H, D2O
exch, NHCO), 8.84 (s, 1H, H-11), 9.58 (t, J = 5 Hz, 1H,
D2O exch, NH); 13C NMR (CDCl3, 50 MHz) d 40.96
(NHCH2CH2), 45.55 (CH2CH2N(CH3)2), 57.85
(NHCH2CH2), 43.16 (COCH2), 103.17 (C-2), 104.12
(C-12a), 112.45 (C-4), 112.53 (C-6), 121.51 (C-11a),
125.56 (C-9), 126.92 (C-7), 127.98 (C-11), 128.97 (C-8),
129.71 (C-10), 129.82 (C-10a), 130.00 (C-3), 136.35 (C-
6a), 149.30 (C-4a), 149.38 (C-1), 150.86 (C-5a), 163.64
(CDCl3, 50 MHz)
d 23.48 (N(CH2CH2)2), 41.38
(NHCH2CH2), 43.15 (COCH2), 54.26 (NHCH2CH2),
54.28 (N(CH2CH2)2), 103.13 (C-2), 106.44 (C-9a),
112.14 (C-4), 117.10 (C-5), 121.88 (C-8a), 124.23 (C-7),
126.29 (C-8), 129.71 (C-3), 134.42 (C-6), 148.09 (C-1),
148.71 (C-4a), 154.49 (C-10a), 163.71 (NHCO), 179.37
(C-9). Anal. Calcd for C21H22ClN3O3: C, 63.08; H,
5.55; N, 10.51. Found: C, 63.25; H, 5.69; N, 10.32.
Spectral data for the intermediate 4-amino-1-[2-(pyrroli-
din-1-yl)ethylamino]-9H-xanthene-9-one
(16c):
1H
NMR (CD3OD, 400 MHz) 1.93 (m, 4H,
(NHCO),
179.77
(C-12).
Anal.
Calcd
for
d
C23H22ClN3O3: C, 65.17; H, 5.23; N, 9.91. Found: C,
65.34; H, 5.35; N, 9.64.
N(CH2CH2)2), 2.90 (m, 4H, N(CH2CH2)2), 3.01 (t,
J = 7 Hz, 2H, NHCH2CH2), 3.57 (q, J = 7 Hz, 5 Hz,
2H, NHCH2CH2), 6.43 (d, J = 8 Hz, 1H, H-2), 7.09
(d, J = 8 Hz, 1H, H-3), 7.31 (t, J = 8 Hz, 1H, H-7),
7.41 (d, J = 8 Hz, 1H, H-5), 7.65 (t, J = 8 Hz, 1H, H-
6), 8.21 (d, J = 8 Hz, 1H, H-8), 9.95 (t, J = 5 Hz, 1H,
D2O exch, NH).
Spectral data for the intermediate N,N-dimethyl-N0-(4-
amino-12-oxo-12H-benzo[b]xanthen-1-yl)ethane-1,2-di-
amine (17a): 1H NMR (CDCl3, 400 MHz) d 2.43 (s, 6H,
2· CH3), 2.71 (t, J = 7 Hz, 2H, NHCH2CH2), 3.43 (q,
J = 7 Hz, 5 Hz, 2H, NHCH2CH2), 6.34 (d, J = 8 Hz,
1H, H-2), 7.11 (d, J = 8 Hz, 1H, H-3), 7.46 (t,
J = 8 Hz, 1H, H-9), 7.57 (t, J = 8 Hz, 1H, H-8), 7.78
(s, 1H, H-6), 7.85 (d, J = 8 Hz, 1H, H-7), 8.01 (d,
J = 8 Hz, 1H, H-10), 8.80 (s, 1H, H-11), 9.11 (t,
J = 5 Hz, 1H, D2O exch, NH).
4.1.16.
2-Chloro-N-[1-[2-(piperidin-1-yl)ethylamino]-9-
oxo-9H-xanthen-4-yl]acetamide (18d). This compound
was prepared by an analogous procedure as described
for the preparation of 18a. Yield: 82%; mp 264–266 ꢁC
1
(EtOAc/n-hexane); H NMR (CDCl3, 400 MHz) d 1.60
(m, 2H, 4-piperidine-H), 1.88 (m, 4H, 3,5-piperidine-
H), 3.06 (m, 4H, 2,6-piperidine-H), 3.10 (t, J = 7 Hz,
2H, NHCH2CH2), 3.62 (q, J = 7 Hz, 5 Hz, 2H,
NHCH2CH2), 4.27 (s, 2H, COCH2), 6.31 (d, J = 9 Hz,
1H, H-2), 7.16 (d, J = 8 Hz, 1H, H-5), 7.23 (t,
J = 8 Hz, 1H, H-7), 7.53 (t, J = 8 Hz, 1H, H-6), 7.97
(m, 2H, H-3, H-8), 8.71 (s, 1H, D2O exch, NHCO),
9.17 (t, J = 5 Hz, 1H, D2O exch, NH); 13C NMR
(CDCl3, 50 MHz) d 23.85 (4-piperidine-C), 25.68 (3,5-
piperidine-C), 39.85 (NHCH2CH2), 42.98 (COCH2),
4.1.18.
2-Chloro-N-[1-(2-diethylaminoethylamino)-12-
oxo-12H-benzo[b]xanthen-4-yl]acetamide (19b). This
compound was prepared by an analogous procedure as
described for the preparation of 18a, starting from 15b.
Yield: 84%; mp >270 ꢁC (EtOH); 1H NMR (CDCl3,
400 MHz) d 1.11 (t, J = 7 Hz, 6H, N(CH2CH3)2), 2.70
(q, J = 7 Hz, 4H, N(CH2CH3)2), 2.84 (t, J = 7 Hz, 2H,
NHCH2CH2), 3.41 (q, J = 7 Hz, 2H, NHCH2CH2),
4.36 (s, 2H, NHCOCH2), 6.47 (d, J = 8 Hz, 1H, H-2),
7.47 (t, J = 8 Hz, 1H, H-9), 7.62 (t, J = 8 Hz, 1H,

I. K. Kostakis et al. / Bioorg. Med. Chem. 14 (2006) 2910–2934
2921
H-8), 7.79 (s, 1H, H-6), 7.89 (d, J = 8 Hz, 1H, H-7), 8.06 (d,
J = 8 Hz, 1H, H-10), 8.31 (d, J = 8 Hz, 1H, H-3), 8.78 (s,
1H, D2O exch, NHCO), 8.83 (s, 1H, H-11), 9.56 (t,
J = 5 Hz, 1H, D2O exch, NH); 13C NMR (CDCl3,
4.1.20. 2-Chloro-N-[1-[2-(piperidin-1-yl)ethylamino]-12-
oxo-12H-benzo[b]xanthen-4-yl]acetamide (19d). This
compound was prepared by an analogous procedure as
described for the preparation of 18a, starting from 15d.
1
50 MHz)
d
11.34 (CH2CH2N(CH2CH3)2), 41.27
Yield: 80%; mp 256–258 ꢁC (EtOH); H NMR (CDCl3,
(CH2CH2N(CH2CH3)2), 43.25 (NHCOCH2), 47.16
(CH2CH2N(CH2CH3)2), 51.43 (CH2CH2N(CH2CH3)2),
102.91 (C-2), 105.12 (C-12a), 112.47 (C-6), 112.53 (C-
4), 121.40 (C-11a), 125.64 (C-9), 127.05 (C-7), 127.80
(C-11), 129.04 (C-8), 129.63 (C-10), 129.74 (C-3),
130.02 (C-10a), 136.52 (C-6a), 148.75 (C-4a), 149.10
(C-1), 150.94 (C-5a), 163.72 (NHCO), 179.66 (C-12).
Anal. Calcd for C25H26ClN3O3: C, 66.44; H, 5.80; N,
9.30. Found: C, 66.26; H, 5.62; N, 9.24.
400 MHz) d 1.46 (m, 2H, 4-piperidine-H), 1.62 (m, 4H,
3,5-piperidine-H), 2.56 (m, 4H, 2,6-piperidine-H), 2.74
(t, J = 7 Hz, 2H, NHCH2CH2), 3.47 (q, J = 7 Hz, 5Hz,
2H, NHCH2CH2), 4.38 (s, 2H, COCH2), 6.49 (d,
J = 8 Hz, 1H, H-2), 7.49 (t, J = 8 Hz, 1H, H-9), 7.63
(t, J = 8 Hz, 1H, H-8), 7.80 (s, 1H, H-6), 7.90 (d,
J = 8 Hz, 1H, H-7), 8.06 (d, J = 8 Hz, 1H, H-10), 8.30
(d, J = 8 Hz, 1H, H-3), 8.79 (s, 1H, NHCO), 8.84 (s,
1H, H-11), 9.59 (t, J = 5 Hz, 1H, D2O exch, NH); 13C
NMR (CDCl3, 50 MHz) d 24.11 (4-piperidine-C),
25.27 (3,5-piperidine-C), 40.26 (NHCH2CH2), 42.97
Spectral data for the intermediate N,N-diethyl-N0-(4-
amino-12-oxo-12H-benzo[b]xanthen-1-yl)ethane-1,2-
diamine (17b): 1H NMR (CDCl3, 400 MHz) d 1.07 (t,
J = 7 Hz, 6H, N(CH2CH3)2), 2.54 (q, J = 7 Hz, 4H,
N(CH2CH3)2), 2.71 (t, J = 6 Hz, 2H, NHCH2CH2),
3.20 (q, J = 6 Hz, 5 Hz, 2H, NHCH2CH2), 6.19 (d,
J = 8 Hz, 1H, H-2), 6.97 (d, J = 8 Hz, 1H, H-3),
7.33 (t, J = 8 Hz, 1H, H-9), 7.44 (t, J = 8 Hz, 1H,
H-8), 7.54 (s, 1H, H-6), 7.69 (d, J = 8 Hz, 1H, H-7),
7.90 (d, J = 8 Hz, 1H, H-10), 8.64 (s, 1H, H-11),
8.94 (t, J = 5 Hz, 1H, D2O exch, NH).
(NHCOCH2),
54.46
(2,6-piperidine-C),
56.97
(NHCH2CH2), 103.07 (C-2), 104.98 (C-12a), 112.36
(C-4), 112.46 (C-6), 121.36 (C-11a), 125.70 (C-9),
127.01 (C-7), 127.77 (C-11), 129.03 (C-8), 129.98 (C-
10a), 129.67 (C-10), 130.04 (C-3), 136.47 (C-6a),
148.34 (C-4a), 148.67 (C-1), 150.80 (C-5a), 164.02
(NHCO),
179.52
(C-12).
Anal.
Calcd
for
C26H26ClN3O3: C, 67.31; H, 5.65; N, 9.06. Found: C,
67.00; H, 5.78; N, 9.15.
Spectral data for the intermediate 4-amino-1-[2-(piperi-
din-1-yl)ethylamino]-12H-benzo[b]xanthen-12-one
(17d): H NMR (CDCl3, 400 MHz) d 1.52 (m, 2H,
4.1.19. 2-Chloro-N-[1-[2-(pyrrolidin-1-yl)ethylamino]-12-
oxo-12H-benzo[b]xanthen-4-yl]acetamide (19c). This
compound was prepared by an analogous procedure as
described for the preparation of 18a, starting from 15c.
Yield: 80%; mp 251–253 ꢁC (dec) (EtOH); 1H NMR
(CDCl3, 400 MHz) d 1.91 (m, 4H, N(CH2CH2)2), 2.66
(m, 4H, N(CH2CH2)2), 2.91 (t, J = 7 Hz, 2H,
NHCH2CH2), 3.50 (q, J = 7 Hz, 2H, NHCH2CH2),
4.31 (s, 2H, NHCOCH2), 6.50 (d, J = 8 Hz, 1H, H-2),
7.52 (t, J = 8 Hz, 1H, H-9), 7.64 (t, J = 8 Hz, 1H, H-
8), 7.81 (s, 1H, H-6), 7.92 (d, J = 8 Hz, 1H, H-7), 8.06
(d, J = 8 Hz, 1H, H-10), 8.31 (d, J = 8 Hz, 1H, H-3),
8.81 (s, 1H, D2O exch, NHCO), 8.85 (s, 1H, H-11),
9.59 (t, J = 5 Hz, 1H, D2O exch, NH); 13C NMR
1
4-piperidine-H), 1.68 (m, 4H, 3,5-piperidine-H),
2.55 (m, 4H, 2,6-piperidine-H), 2.91 (t, J = 7 Hz,
2H, NHCH2CH2), 3.48 (q, J = 7 Hz, 5 Hz, 2H,
NHCH2CH2), 6.26 (d, J = 8 Hz, 1H, H-2), 7.02
(d, J = 8 Hz, 1H, H-3), 7.39 (t, J = 8 Hz, 1H, H-
9), 7.48 (t, J = 8 Hz, 1H, H-8), 7.59 (s, 1H, H-6),
7.77 (d, J = 8 Hz, 1H, H-7), 7.97 (d, J = 8 Hz, 1H,
H-10), 8.68 (s, 1H, H-11), 8.98 (t, J = 5 Hz, 1H,
D2O exch, NH).
4.1.21.
2-Dimethylamino-N-[1-(2-dimethylaminoethyl-
amino)-9-oxo-9H-xanthen-4-yl]acetamide (20a). To a
solution of the chloride 18a (375 mg, 1 mmol) in abso-
lute ethanol (10 mL) was added dropwise a 5.6 M etha-
nolic solution of dimethylamine (720 lL, 4 mmol) and
the resulting solution was heated at reflux for 10 h.
Upon cooling, the solvent was vacuum-evaporated and
the residue was purified by column chromatography (sil-
ica gel) using a mixture of CH2Cl2/MeOH 8:1 as the elu-
ent, to provide 20a (363 mg, 95%); mp (dihydrochloride)
195–197 ꢁC (EtOH); 1H NMR (CDCl3, 400 MHz) d 2.41
(s, 6H, CH2CH2N(CH3)2), 2.49 (s, 6H, COCH2N
(CH3)2), 2.74 (t, J = 7 Hz, 2H, NHCH2CH2), 3.16 [s,
2H, COCH2N(CH3)2], 3.41 (q, J = 7 Hz, 5 Hz, 2H,
NHCH2CH2), 6.42 (d, J = 8 Hz, 1H, H-2), 7.29–7.35
(m, 2H, H-5, H-7), 7.65 (td, J = 8 Hz, 1 Hz, 1H, H-6),
8.19–8.26 (m, 2H, H-3, H-8), 9.35 (t, J = 5 Hz, 1H,
D2O exch, NH), 9.41 (s, 1H, D2O exch, NHCO); 13C
(CDCl3, 50 MHz)
d 23.55 (N(CH2CH2)2), 42.23
(NHCH2CH2), 43.16 (COCH2), 54.36 (N(CH2CH2)2),
54.70 (NHCH2CH2), 103.19 (C-2), 105.51 (C-12a),
111.83 (C-4), 112.53 (C-6), 121.48 (C-11a), 125.56 (C-
9), 126.92 (C-7), 127.89 (C-11), 128.98 (C-8), 129.62
(C-10), 129.82 (C-10a), 130.02 (C-3), 136.33 (C-6a),
148.11 (C-4a), 149.30 (C-1), 150.82 (C-5a), 163.64
(NHCO),
179.66
(C-12).
Anal.
Calcd
for
C25H24ClN3O3: C, 66.74; H, 5.38; N, 9.34. Found: C,
66.51; H, 5.04; N, 9.61.
Spectral data for the intermediate 4-amino-1-[2-(pyrr-
olidin-1-yl)ethylamino]-12H-benzo[b]xanthen-12-one
(17c): 1H NMR (CDCl3, 400 MHz) d 1.83 (m, 4H,
N(CH2CH2)2), 2.64 (m, 4H, N(CH2CH2)2), 2.84 (t,
J = 7 Hz, 2H, NHCH2CH2), 3.49 (q, J = 7 Hz,
5 Hz, 2H, NHCH2CH2), 6.32 (d, J = 8 Hz, 1H, H-
2), 7.06 (d, J = 8 Hz, 1H, H-3), 7.42 (t, J = 8 Hz,
1H, H-9), 7.54 (t, J = 8 Hz, 1H, H-8), 7.69 (s, 1H,
H-6), 7.83 (d, J = 8 Hz, 1H, H-7), 7.98 (d,
J = 8 Hz, 1H, H-10), 8.68 (s, 1H, H-11), 9.08 (t,
J = 5 Hz, 1H, D2O exch, NH).
NMR (CDCl3, 50 MHz)
d 40.43 (NHCH2CH2N
(CH3)2), 45.17 (CH2CH2N(CH3)2), 46.01 (COCH2N
(CH3)2), 57.48 (NHCH2CH2N(CH3)2), 63.48 (NHCO
CH2), 102.95 (C-2), 106.41 (C-9a), 112.77 (C-4), 116.84
(C-5), 121.88 (C-8a), 123.87 (C-7), 126.22 (C-8), 130.00
(C-3), 134.08 (C-6), 148.02 (C-1), 148.20 (C-4a), 154.52

2922
I. K. Kostakis et al. / Bioorg. Med. Chem. 14 (2006) 2910–2934
(C-10a), 168.64 (NHCO), 179.33 (C-9). Anal. Calcd for
C21H26N4O3Æ2HClÆ1/2H2O: C, 58.80; H, 6.81; N, 13.06.
Found: C, 58.52; H, 6.54; N, 13.31.
400 MHz) d 1.45 [m, 2H, CH2CH2(4-piperidine-H)],
1.55 [m, 2H, COCH2(4-piperidine-H)], 1.64 [m, 4H,
CH2CH2(3,5-piperidine-H)], 1.74 [m, 4H, COCH2(3,5-
piperidine-H)], 2.54 [m, 4H, CH2CH2(2,6-piperidine-
H)], 2.62 [m, 4H, COCH2(2,6-piperidine-H)], 2.72 (t,
J = 7 Hz, 2H, NHCH2CH2), 3.13 (s, 2H, NHCOCH2),
3.41 (q, J = 7 Hz, 5 Hz, 2H, NHCH2CH2), 6.42 (d,
J = 9 Hz, 1H, H-2), 7.29–7.34 (m, 2H, H-5, H-7), 7.64
(td, J = 8 Hz, 1.5 Hz, 1H, H-6), 8.22 (dd, J = 8 Hz,
1.5 Hz, 1H, H-8), 8.39 (d, J = 9 Hz, 1H, H-3), 9.29 (t,
J = 5 Hz, 1H, D2O exch, NH), 9.71 (s, 1H, D2O exch,
4.1.22. 2-Diethylamino-N-[1-(2-diethylaminoethylamino)-
9-oxo-9H-xanthen-4-yl]acetamide (20b). This compound
was prepared by an analogous procedure as described
for the preparation of 20a. Yield: 93%; mp (dihydro-
chloride) >270 ꢁC (EtOH); 1H NMR (CDCl3,
400 MHz) d 1.05 (t, J = 7 Hz, 6H, CH2CH2N(CH2-
CH3)2), 1.17 (t, J = 7 Hz, 6H, COCH2N(CH2CH3)2),
2.59 (q, J = 7 Hz, 4H, CH2CH2N(CH2CH3)2), 2.71 (q,
J = 7 Hz, 4H, COCH2N(CH2CH3)2), 2.75 (t, J = 7 Hz,
2H, NHCH2CH2), 3.21 (s, 2H, COCH2N(CH2CH3)2),
3.28 (q, J = 7 Hz, 5 Hz, 2H, NHCH2CH2), 6.39 (d,
J = 9 Hz, 1H, H-2), 7.25–7.32 (m, 2H, H-5, H-7), 7.60
(td, J = 8 Hz, 2 Hz, 1H, H-6), 8.21 (dd, J = 8 Hz, 2 Hz,
1H, H-8), 8.37 (d, J = 9 Hz, 1H, H-3), 9.27 (t,
J = 5 Hz, 1H, D2O exch, NH), 9.77 (s, 1H, D2O exch,
NHCO); 13C NMR (CDCl3, 50 MHz)
[CH2CH2(4-piperidine-C)], 24.04 [COCH2(4-piperidine-
C)], 25.55 [CH2CH2(3,5-piperidine-C)], 26.68
d
23.63
[COCH2(3,5-piperidine-C)], 40.14 (NHCH2CH2), 54.51
[CH2CH2(2,6-piperidine-C)], 54.91 [COCH2(2,6-piperi-
dine-C)], 57.12 (NHCH2CH2), 62.78 (NHCOCH2),
103.13 (C-2), 106.37 (C-9a), 113.35 (C-4), 116.62 (C-5),
121.95 (C-8a), 123.86 (C-7), 126.32 (C-8), 128.93 (C-3),
134.15 (C-6), 147.27 (C-1), 147.93 (C-4a), 154.62 (C-
10a), 168.63 (NHCO), 179.21 (C-9). Anal. Calcd for
C27H34N4O3Æ2HClÆ 1/2H2O: C, 59.55; H, 6.85; N,
10.29. Found: C, 59.71; H, 6.50; N, 10.03.
NHCO); 13C NMR (CDCl3, 50 MHz)
d
11.68
(CH2CH2N(CH2CH3)2), 12.67 (COCH2N(CH2CH3)2),
41.13 (CH2CH2N(CH2CH3)2), 47.01 (CH2CH2N-
(CH2CH3)2), 48.66 (COCH2N(CH2CH3)2), 51.20
(CH2CH2N(CH2CH3)2), 58.33 (NHCOCH2), 102.91
(C-2), 106.22 (C-9a), 112.99 (C-4), 116.52 (C-5), 121.88
(C-8a), 123.18 (C-7), 126.18 (C-8), 128.90 (C-3), 133.90
(C-6), 147.28 (C-1), 148.05 (C-4a), 154.49 (C-10a),
169.67 (NHCO), 179.04 (C-9). Anal. Calcd for
C25H34N4O3Æ2HClÆH2O: C, 56.71; H, 7.23; N, 10.58.
Found: C, 56.33; H, 7.01; N, 10.20.
4.1.25.
2-Dimethylamino-N-[1-(2-dimethylaminoethyl-
amino)-12-oxo-12H-benzo[b]xanthen-4-yl]acetamide
(21a). This compound was prepared by an analo-
gous procedure as described for the preparation
of 20a, starting from 19a. Yield: 95%; mp (dihydro-
chloride) 231–233 ꢁC (EtOH); 1H NMR (CDCl3,
400 MHz) d 2.42 (s, 6H, CH2CH2N(CH3)2), 2.57
(s, 6H, COCH2N(CH3)2), 2.74 (t, J = 7 Hz, 2H,
NHCH2CH2), 3.21 [s, 2H, COCH2N(CH3)2] 3.42
(q, J = 7 Hz, 2H, NHCH2CH2), 6.43 (d, J = 8 Hz,
1H, H-2), 7.46 (t, J = 8 Hz, 1H, H-9), 7.57 (t,
J = 8 Hz, 1H, H-8), 7.62 (s, 1H, H-6), 7.84 (d,
J = 8 Hz, 1H, H-7), 7.99 (d, J = 8 Hz, 1H, H-10),
8.27 (d, J = 8 Hz, 1H, H-3), 8.79 (s, 1H, H-11),
9.44 (s, 1H, D2O exch, NHCO), 9.48 (t, J = 5 Hz,
1H, D2O exch, NH); 13C NMR (CDCl3, 50 MHz)
d 40.55 (NHCH2CH2N(CH3)2), 45.22 (CH2CH2-
N(CH3)2), 46.09 (COCH2N(CH3)2), 57.55 (NHCH2CH2-
N(CH3)2), 63.58 (NHCOCH2), 102.99 (C-2), 105.56
(C-12a), 112.18 (C-6), 112.66 (C-4), 121.44 (C-11a),
125.28 (C-9), 126.70 (C-7), 127.74 (C-11), 128.74 (C-8),
129.58 (C-10), 129.59 (C-10a), 130.57 (C-3), 136.14
(C-6a), 148.07 (C-4a), 148.59 (C-1), 150.92 (C-5a),
168.65 (NHCO), 179.61 (C-12). Anal. Calcd for C25H28
N4O3Æ2HClÆ2H2O: C, 55.45; H, 6.33; N, 10.35. Found:
C, 55.19; H, 6.46; N, 10.52.
4.1.23. N-[1-[2-(Pyrrolidin-1-yl)ethylamino]-9-oxo-9H-
xanthen-4-yl]-2-pyrrolidin-1-yl-acetamide (20c). This
compound was prepared by an analogous procedure as
described for the preparation of 20a. Yield: 96%; mp
1
(dihydrochloride) >270 ꢁC (EtOH); H NMR (CDCl3,
400 MHz) d 1.78 (m, 4H, CH2CH2N(CH2CH2)2), 1.91
(m, 4H, COCH2N(CH2CH2)2), 2.60 (m, 4H, CH2CH2N-
(CH2CH2)2), 2.79 (m, 4H, COCH2N(CH2CH2)2), 2.80
(t, J = 7 Hz, 2H, NHCH2CH2), 3.31 (s, 2H,
COCH2N(CH2CH2)2), 3.37 (q, J = 7 Hz, 5 Hz, 2H,
NHCH2CH2), 6.36 (d, J = 9 Hz, 1H, H-2), 7.17 (dd,
J = 8 Hz, 2 Hz, 1H, H-5), 7.26 (td, J = 8 Hz, 2 Hz, 1H,
H-7), 7.57 (td, J = 8 Hz, 2 Hz, 1H, H-6), 8.15 (dd,
J = 8 Hz, 2 Hz, 1H, H-8), 8.25 (d, J = 9 Hz, 1H, H-3),
9.26 (t, J = 5 Hz, 1H, D2O exch, NH), 9.46 (s, 1H,
D2O exch, NHCO); 13C NMR (CDCl3, 50 MHz) d
23.30 (CH2CH2N(CH2CH2)2), 24.08 (COCH2N(CH2-
CH2)2), 41.82 (NHCH2CH2N(CH2CH2)2), 54.10 (CH2-
CH2N(CH2CH2)2), 54.28 (COCH2N(CH2CH2)2), 54.43
(NHCH2CH2 N(CH2CH2)2), 59.17 (NHCOCH2),
102.84 (C-2), 106.15 (C-9a), 112.84 (C-4), 116.52 (C-5),
121.73 (C-8a), 123.68 (C-7), 126.03 (C-8), 129.31 (C-3),
133.97 (C-6), 147.47 (C-1), 147.98 (C-4a), 154.34
(C-10a), 168.71 (NHCO), 179.08 (C-9). Anal. Calcd
for C25H30N4O3Æ2HClÆ 3/4H2O: C, 57.64; H, 6.48; N,
10.75. Found: C, 57.50; H, 6.57; N, 10.83.
4.1.26. 2-Diethylamino-N-[1-(2-diethylaminoethylamino)-
12-oxo-12H-benzo[b]xanthen-4-yl]acetamide (21b). This
compound was prepared by an analogous procedure as
described for the preparation of 20a, starting from 19b.
Yield: 96%; mp (dihydrochloride) 239–241 ꢁC (dec)
(EtOH). 1H NMR (CDCl3, 400 MHz) d 1.14 (t, J =
7 Hz, 6H, CH2CH2N(CH2CH3)2), 1.27 (t, J = 7 Hz,
6H, COCH2N(CH2CH3)2), 2.71 (q, J = 7 Hz, 4H, CH2
CH2N(CH2CH3)2), 2.80 (q, J = 7 Hz, 4H, COCH2
N(CH2CH3)2), 2.86 (t, J = 7 Hz, 2H, NHCH2CH2),
3.29 (s, 2H, COCH2N(CH2CH3)2), 3.43 (q, J = 7 Hz,
2H, NHCH2CH2), 6.48 (d, J = 8 Hz, 1H, H-2), 7.49
4.1.24.
N-[1-[2-(Piperidin-1-yl)ethylamino]-9-oxo-9H-
xanthen-4-yl]-2-piperidin-1-yl-acetamide (20d). This com-
pound was prepared by an analogous procedure as
described for the preparation of 20a. Yield: 96%; mp
1
(dihydrochloride) >270 ꢁC (EtOH); H NMR (CDCl3,

I. K. Kostakis et al. / Bioorg. Med. Chem. 14 (2006) 2910–2934
2923
(t, J = 8 Hz, 1H, H-9), 7.61 (t, J = 8 Hz, 1H, H-8), 7.71
(s, 1H, H-6), 7.88 (d, J = 8 Hz, 1H, H-7), 8.05 (d,
J = 8 Hz, 1H, H-10), 8.44 (d, J = 8 Hz, 1H, H-3), 8.83
(s, 1H, H-11), 9.53 (t, J = 5 Hz, 1H, D2O exch, NH),
9.98 (s, 1H, D2O exch, NHCO); 13C NMR (CDCl3,
D2O exch, NH), 9.95 (s, 1H, D2O exch, NHCO); 13C
NMR (CDCl3, 50 MHz) d 23.82 [CH2CH2(4-piperi-
dine-C)], 24.07 [COCH2(4-piperidine-C)], 25.53 [CH2
CH2(3,5-piperidine-C)], 26.59 [COCH2(3,5-piperidine-
C)], 40.32 (NHCH2CH2), 54.35 [CH2CH2(2,6-piperi-
dine-C)], 54.93 [COCH2(2,6-piperidine-C)], 56.83
(NHCH2CH2), 62.86 (NHCOCH2), 103.11 (C-2),
105.77 (C-12a), 112.47 (C-4), 112.56 (C-6), 121.35 (C-
11a), 125.31 (C-9), 126.73 (C-7), 127.62 (C-11), 128.67
(C-8), 129.54 (C-10a), 129.55 (C-10), 130.26 (C-3),
136.22 (C-6a), 147.92 (C-4a), 147.92 (C-1), 150.73 (C-
5a), 169.02 (NHCO), 179.53 (C-12). Anal. Calcd for
50 MHz)
d
11.82 (CH2CH2N(CH2CH3)2), 12.54
(COCH2N(CH2 CH3)2), 41.49 (CH2CH2N(CH2CH3)2),
47.21 (CH2CH2 N(CH2CH3)2), 47.58 (COCH2N-
(CH2CH3)2), 51.56 (CH2CH2N(CH2CH3)2), 58.38
(NHCOCH2), 103.37 (C-2), 106.01 (C-12a), 112.38 (C-
6), 112.52 (C-4), 121.67 (C-11a), 125.32 (C-9), 126.82
(C-7), 127.94 (C-11), 128.59 (C-8), 129.60 (C-10a),
129.63 (C-10), 130.15 (C-3), 136.34 (C-6a), 147.12 (C-
4a), 147.64 (C-1), 151.09 (C-5a), 168.66 (NHCO),
179.79 (C-12). Anal. Calcd for C29H36N4O3Æ2HClÆH2O:
C, 60.10; H, 6.96; N, 9.67. Found: C, 60.42; H, 7.08; N,
9.44.
C31H36N4O : C, 72.63; H, 7.08; N, 10.93. Found: C,
3
72.48; H, 7.31; N, 10.72.
4.1.29. 2-[5-Nitro-2H-benzopyrano[4,3,2-cd]indazol-2-yl]-
1-ethanol (22). A solution of 12 (1.1 g, 4 mmol) and 2-
hydroxyethylhydrazine (684 mg, 9 mmol) in dry pyri-
dine (8 mL) was stirred at room temperature for 12 h.
The reaction mixture was then diluted with CH2Cl2
(100 mL), washed successively with 18% HCl solution
and water, dried (Na2SO4), and evaporated to dryness.
The residue was purified by column chromatography
(silica gel) using a mixture of cyclohexane/EtOAc 1:3
as the eluent, to provide compound 22 (1.06 g, 89%);
mp 211–213 ꢁC (EtOH); 1H NMR (DMSO-d6,
400 MHz) d 3.93 (t, J = 7 Hz, 2H, NCH2CH2OH),
4.47 (t, J = 7 Hz, 2H, NCH2CH2OH), 7.13 (d,
J = 8 Hz, 1H, H-3), 7.37 (t, J = 8 Hz, 1H, H-9), 7.46–
7.58 (m, 2H, H-7, H-8), 7.86 (d, J = 8 Hz, 1H, H-10),
7.99 (d, J = 8 Hz, 1H, H-4); 13C NMR (DMSO-d6,
50 MHz) d 49.84 (N–NCH2CH2OH), 62.11 (NCH2
CH2OH), 104.52 (C-3), 116.00 (C-2b), 116.84 (C-10a),
118.84 (C-7), 122.81 (C-10), 124.82 (C-5), 126.07 (C-9),
126.34 (C-4), 131.20 (C-8), 139.06 (C-10b), 142.75
(C-2a), 146.02 (C-5a), 153.69 (C-6a). Anal. Calcd for
C15H11N3O4: C, 60.61; H, 3.73; N, 14.14. Found: C,
60.75; H, 3.60; N, 14.00.
4.1.27. N-[1-[2-(Pyrrolidin-1-yl)ethylamino]-12-oxo-12H-
benzo[b]xanthen-4-yl]-2-pyrrolidin-1-yl-acetamide (21c).
This compound was prepared by an analogous proce-
dure as described for the preparation of 20a, starting
from 19c. Yield: 96%; mp (dihydrochloride) 198–
200 ꢁC (EtOH); 1H NMR (CDCl3, 400 MHz) d 1.95
(m, 4H, CH2CH2N(CH2CH2)2), 2.01 (m, 4H, COCH2N-
(CH2CH2)2), 2.83 (m, 4H, CH2CH2N(CH2CH2)2), 2.96
(m, 4H, COCH2N(CH2CH2)2), 3.10 (t, J = 6 Hz, 2H,
NHCH2CH2), 3.41 (s, 2H, COCH2N(CH2CH2)2), 3.58
(q, J = 5 Hz, 2H, NHCH2CH2), 6.42 (d, J = 8 Hz, 1H,
H-2), 7.35 (s, 1H, H-6), 7.40 (t, J = 8 Hz, 1H, H-9),
7.48 (t, J = 8 Hz, 1H, H-8), 7.66 (d, J = 8 Hz, 1H, H-
7), 7.92 (d, J = 8 Hz, 1H, H-10), 8.21 (d, J = 8 Hz, 1H,
H-3), 8.59 (s, 1H, H-11), 9.32 (t, J = 5 Hz, 1H, D2O
exch, NH), 9.51 (s, 1H, D2O exch, NHCO); 13C NMR
(CDCl3, 50 MHz) d 23.28 (CH2CH2N(CH2CH2)2),
24.23 (COCH2N(CH2CH2)2), 40.57 (NHCH2CH2N-
(CH2CH2)2), 53.84 (NHCH2CH2 N(CH2CH2)2), 54.16
(CH2CH2N(CH2CH2)2), 54.39 (COCH2N(CH2CH2)2),
59.17 (NHCOCH2), 102.91 (C-2), 105.43 (C-12a),
111.99 (C-4), 113.13 (C-6), 121.05 (C-11a), 125.28
(C-9), 126.66 (C-7), 127.49 (C-11), 128.70 (C-8), 129.32
(C-10a), 129.40 (C-10), 130.02 (C-3), 135.98 (C-6a),
147.63 (C-4a), 147.87 (C-1), 150.55 (C-5a), 168.73
(NHCO), 179.39 (C-12). Anal. Calcd for C29H32N4O3Æ
2HClÆH2O: C, 60.52; H, 6.30; N, 9.74. Found: C,
60.37; H, 6.66; N, 9.91.
4.1.30. 2-[5-Nitro-2H-benzopyrano[4,3,2-cd]indazol-2-yl]-
1-ethanol methanesulfonate (23). A solution of metha-
nesulfonyl chloride (70 lL, 0.9 mmol) in dry CH2Cl2
(5 mL) was added dropwise at 0 ꢁC to a suspension of
triethylamine (279 lL, 2 mmol) and 22 (257 mg,
0.865 mmol) in dry CH2Cl2 (20 mL) and the mixture
was stirred at 0 ꢁC for 10 min and then at room temper-
ature for 4 h. The reaction mixture was then washed
with 1 N HCl and the organic layer was dried (Na2SO4)
and concentrated to dryness. The residue was purified
by column chromatography (silica gel), using a mixture
of cyclohexane/EtOAc 3:1 as the eluent, to give com-
pound 23 (304 mg, 94%). Mp 234–236 ꢁC (EtOAc/n-hex-
ane); 1H NMR (CDCl3, 400 MHz) d 2.87 (s, 3H,
SO2CH3), 4.67–4.75 (m, 4H, N–NCH2CH2), 6.93 (d,
J = 9 Hz, 1H, H-3), 7.37 (t, J = 8 Hz, 1H, H-9), 7.52
(t, J = 8 Hz, 1H, H-8), 7.62 (d, J = 8 Hz, 1H, H-7),
7.97 (d, J = 8 Hz, 1H, H-10), 8.21 (d, J = 9 Hz, 1H, H-
4); 13C NMR (CDCl3, 50 MHz) d 37.06 (SO2CH3)
48.15 (N–NCH2CH2), 67.32 (N–NCH2CH2), 104.29
(C-3), 115.17 (C-2b), 116.75 (C-10a), 118.95 (C-7),
122.81 (C-10), 124.25 (C-5), 126.05 (C-9), 126.19 (C-4),
131.16 (C-8), 138.95 (C-10b), 142.74 (C-2a), 145.75 (C-
5a), 153.06 (C-6a). Anal. Calcd for C16H13N3O6S: C,
4.1.28. N-[1-[2-(Piperidin-1-yl)ethylamino]-12-oxo-12H-
benzo[b]xanthen-4-yl]-2-piperidin-1-yl-acetamide (21d).
This compound was prepared by an analogous proce-
dure as described for the preparation of 20a, starting
from 19d. Yield: 96%; mp 171–173 ꢁC (EtOH); 1H
NMR (CDCl3, 400 MHz) d 1.45 [m, 2H, CH2CH2(4-pi-
peridine-H)], 1.51 [m, 2H, COCH2(4-piperidine-H)],
1.62 [m, 4H, CH2CH2(3,5-piperidine-H)], 1.68 [m, 4H,
COCH2(3,5-piperidine-H)], 2.55 [m, 4H, CH2CH2(2,6-
piperidine-H)], 2.61 [m, 4H, COCH2(2,6-piperidine-
H)], 2.88 (t, J = 7 Hz, 2H, NHCH2CH2), 3.15 (s, 2H,
NHCOCH2), 3.44 (q, J = 7 Hz, 5 Hz, 2H, NHCH2CH2),
6.46 (d, J = Hz, 1H, H-2), 7.44 (t, J = Hz, 1H, H-9), 7.52
(t, J = Hz, 1H, H-8), 7.56 (s, 1H, H-6), 7.73 (d, J = Hz,
1H, H-7), 7.97 (d, J = Hz, 1H, H-10), 8.28 (d, J = Hz,
1H, H-3), 8.74 (s, 1H, H-11), 9.45 (t, J = 5 Hz, 1H,

2924
I. K. Kostakis et al. / Bioorg. Med. Chem. 14 (2006) 2910–2934
51.20; H, 3.49; N, 11.19. Found: C, 51.25; H, 3.21; N,
11.35.
C, 53.97; H, 5.48; N, 13.25. Found: C, 54.16; H, 5.30;
N, 13.07.
4.1.31.
Dimethyl-[2-[5-nitro-2H-benzopyrano[4,3,2-
4.1.34. 5-Nitro-2-[2-(piperidin-1-yl)ethyl]-2H-benzopyr-
ano[4,3,2-cd]indazole (24d). This compound was pre-
pared by an analogous procedure as described for the
preparation of 24a. Yield: 90%; mp (hydrochloride)
cd]indazol-2-yl]ethyl]amine (24a). To a solution of the
mesylate 23 (375 mg, 1 mmol) in absolute ethanol
(12 mL) was added dropwise a 5.6 M ethanolic solution
of dimethylamine (540 lL, 3 mmol) and the resulting
solution was refluxed for 10 h. After cooling, the solvent
was removed under reduced pressure, and the residue
was purified by column chromatography (silica gel,
CH2Cl2/MeOH 94:6) to give 24a (300 mg, 92%); mp
(hydrochloride) >270 ꢁC (EtOH); 1H NMR (CDCl3,
400 MHz) d 2.32 (s, 6H, 2· CH3), 2.89 (t, J = 7 Hz,
2H, N–NCH2CH2), 4.44 (t, J = 7 Hz, 2H, N–
NCH2CH2), 6.81 (d, J = 9 Hz, 1H, H-3), 7.29 (t,
J = 8 Hz, 1H, H-9), 7.43 (t, J = 8 Hz, 1H, H-8), 7.50
(dd, J = 8 Hz, 1 Hz, 1H, H-7), 7.90 (dd, J = 8 Hz,
1 Hz, 1H, H-10), 8.06 (d, J = 9 Hz, 1H, H-4); 13C
NMR (CDCl3, 50 MHz) d 45.39 (2· CH3), 48.03 (N–
NCH2CH2), 58.03 (N–NCH2CH2), 102.18 (C-3),
115.92 (C-2b), 116.96 (C-10a), 119.12 (C-7), 122.98 (C-
10), 125.01 (C-5), 125.67 (C-9), 126.84 (C-4), 130.67
(C-8), 140.00 (C-10b), 142.17 (C-2a), 146.40 (C-5a),
153.60 (C-6a). Anal. Calcd for C17H16N4O3ÆHClÆ2H2O:
C, 53.61; H, 5.56; N, 14.71. Found: C, 53.74; H, 5.75;
N, 14.86.
1
>270 ꢁC (EtOH); H NMR (CDCl3, 400 MHz) d 1.39
(m, 2H, 4-piperidine-H), 1.50 (m, 4H, 3,5-piperidine-
H), 2.43 (m, 4H, 2,6-piperidine-H), 2.81 (t, J = 7 Hz,
2H, N–NCH2CH2), 4.40 (t, J = 7 Hz, 2H, N–
NCH2CH2), 6.78 (d, J = 9 Hz, 1H, H-3), 7.27 (td,
J = 8 Hz, 1 Hz, 1H, H-9), 7.41 (td, J = 8 Hz, 1 Hz, 1H,
H-8), 7.48 (dd, J = 8 Hz, 1 Hz, 1H, H-7), 7.86 (dd,
J = 8 Hz, 1 Hz, 1H, H-10), 8.04 (d, J = 9 Hz, 1H, H-
4); 13C NMR (CDCl3, 50 MHz) d 24.04 (4-piperidine-
C), 25.88 (3,5-piperidine-C), 48.04 (N–NCH2CH2),
54.73 (2,6-piperidine-C), 58.22 (N–NCH2CH2), 102.51
(C-3), 115.81 (C-2b), 116.99 (C-10a), 119.12 (C-7),
122.90 (C-10), 124.85 (C-5), 125.62 (C-9), 126.54 (C-4),
130.62 (C-8), 139.85 (C-10b), 142.24 (C-2a), 146.32 (C-
5a), 153.56 (C-6a). Anal. Calcd for C20H20N4O3Æ
HClÆH2O: C, 57.35; H, 5.53; N, 13.38. Found: C,
57.03; H, 5.74; N, 13.56.
4.1.35. 2-Chloro-N-[2-(2-dimethylaminoethyl)-2H-benzo-
pyrano[4,3,2-cd]indazol-5-yl]acetamide (26a). This com-
pound was prepared by an analogous procedure as
described for the preparation of 18a, starting from
24a. Yield: 77%; mp >270 ꢁC (EtOAc/n-hexane); 1H
NMR (CDCl3, 400 MHz) d 2.33 (s, 6H, CH2CH2N-
(CH3)2), 2.81 (t, J = 7 Hz, 2H, N–NCH2CH2), 4.28 (s,
2H, COCH2), 4.30 (t, J = 7 Hz, 2H, N–NCH2CH2),
6.87 (d, J = 9 Hz, 1H, H-3), 7.21 (t, J = 8 Hz, 1H, H-
9), 7.27–7.35 (m, 2H, H-7, H-8), 7.83 (d, J = 8 Hz, 1H,
H-10), 7.87 (d, J = 9 Hz, 1H, H-4), 8.34 (s, 1H, D2O
exch, NHCO); 13C NMR (CDCl3, 50 MHz) d 42.54
(COCH2), 45.54 (CH2CH2N(CH3)2), 48.08 (N–NCH2
CH2), 58.46 (N–NCH2CH2), 101.68 (C-3), 111.99
(C-5), 116.74 (C-2b), 118.12 (C-7), 118.33 (C-10a),
123.51 (C-10), 124.50 (C-9), 126.00 (C-4), 129.54 (C-8),
137.68 (C-10b), 138.42 (C-2a), 139.21 (C-5a), 153.99
(C-6a), 163.46 (NHCO). Anal. Calcd for C19H19ClN4
O2: C, 61.54; H, 5.16; N, 15.11. Found: C, 61.38; H,
5.00; N, 15.02.
4.1.32. Diethyl-[2-[5-nitro-2H-benzopyrano[4,3,2-cd]ind-
azol-2-yl]ethyl]amine (24b).48 This compound was pre-
pared by an analogous procedure as described for the
preparation of 24a. Yield: 91%; mp (hydrochloride)
268–270 ꢁC (EtOH); 1H NMR (CDCl3, 400 MHz) d
0.92 (t, J = 7 Hz, 6H, (CH2CH3)2), 2.53 (q, J = 7 Hz,
4H, (CH2CH3)2), 2.93 (t, J = 7 Hz, 2H, N–NCH2CH2),
4.34 (t, J = 7 Hz, 2H, N–NCH2CH2), 6.75 (d,
J = 9 Hz, 1H, H-3), 7.27 (t, J = 8 Hz, 1H, H-9), 7.41
(t, J = 8 Hz, 1H, H-8), 7.48 (d, J = 8 Hz, 1H, H-7),
7.86 (d, J = 8 Hz, 1H, H-10), 8.01 (d, J = 9 Hz, 1H, H-
4); 13C NMR (CDCl3, 50 MHz) d 11.85 [(CH2CH3)2],
47.36 [(CH2CH3)2], 48.94 (N–NCH2CH2), 52.58 (N–
NCH2CH2), 102.45 (C-3), 115.65 (C-2b), 116.93 (C-
10a), 119.03 (C-7), 122.85 (C-10), 124.76 (C-5), 125.57
(C-9), 126.45 (C-4), 130.53 (C-8), 139.76 (C-10b),
142.26 (C-2a), 146.26 (C-5a), 153.50 (C-6a).
4.1.33. 5-Nitro-2-[2-(pyrrolidin-1-yl)ethyl]-2H-benzopyr-
ano[4,3,2-cd]indazole (24c). This compound was pre-
pared by an analogous procedure as described for the
preparation of 24a. Yield: 94%; mp (hydrochloride)
4.1.36. 2-Chloro-N-[2-(2-diethylaminoethyl)-2H-benzo-
pyrano[4,3,2-cd]indazol-5-yl]acetamide (26b). This com-
pound was prepared by an analogous procedure as
described for the preparation of 18a, starting from
24b. Yield: 79%; mp >270 C (EtOAc/n-hexane); 1H
NMR (CDCl3, 400 MHz) d 1.01 (t, J = 7 Hz, 6H,
CH2CH2N(CH2CH3)2), 2.59 (q, J = 7 Hz, 4H, CH2CH2-
N(CH2CH3)2), 2.97 (t, J = 7 Hz, 2H, N–NCH2CH2),
4.35 (s, 2H, COCH2), 4.40 (t, J = 7 Hz, 2H, N–NCH2-
CH2), 6.86 (d, J = 9 Hz, 1H, H-3), 7.19 (t, J = 8 Hz,
1H, H-9), 7.26 (d, J = 8 Hz, 1H, H-7), 7.35
(t, J = 8 Hz, 1H, H-8), 7.84 (d, J = 8 Hz, 1H, H-10),
7.88 (d, J = 9 Hz, 1H, H-4), 8.37 (s, 1H, D2O exch,
NHCO); 13C NMR (CDCl3, 50 MHz) d 11.69 (CH2CH2-
N(CH2CH3)2), 42.54 (COCH2), 47.35 (CH2CH2N(CH2-
CH3)2), 48.74 (N–NCH2CH2), 52.43 (N–NCH2CH2),
101.92 (C-3), 111.53 (C-5), 116.88 (C-2b), 118.21 (C-7),
1
>270 ꢁC (EtOH); H NMR (CDCl3, 400 MHz) d 1.75
[m, 4H, N(CH2CH2)2], 2.57 [m, 4H, N(CH2CH2)2],
3.03 (t, J = 7 Hz, 2H, N–NCH2CH2), 4.46 (t, J = 7 Hz,
2H, N–NCH2CH2), 6.78 (d, J = 9 Hz, 1H, H-3), 7.28
(t, J = 8 Hz, 1H, H-9), 7.42 (t, J = 8 Hz, 1H, H-8),
7.49 (d, J = 8 Hz, 1H, H-7), 7.88 (d, J = 8 Hz, 1H, H-
10), 8.05 (d, J = 9 Hz, 1H, H-4). 13C NMR (CDCl3,
50 MHz) d 23.44 [N(CH2CH2)2], 49.32 (N–NCH2CH2),
54.21 [N(CH2CH2)2], 55.24 (N–NCH2CH2), 102.14 (C-
3), 115.82 (C-2b), 116.92 (C-10a), 119.05 (C-7), 122.91
(C-10), 124.93 (C-5), 125.59 (C-9), 126.70 (C-4), 130.59
(C-8), 139.89 (C-10b), 142.06 (C-2a), 146.33 (C-5a),
153.53 (C-6a). Anal. Calcd for C19H18N4O3ÆHClÆ2H2O:

I. K. Kostakis et al. / Bioorg. Med. Chem. 14 (2006) 2910–2934
2925
118.24 (C-10a), 123.16 (C-10), 124.41 (C-9), 126.12 (C-
4), 129.67 (C-8), 137.85 (C-10b), 138.44 (C-2a), 139.11
(C-5a), 154.26 (C-6a), 163.88 (NHCO). Anal. Calcd
for C21H23ClN4O2: C, 63.23; H, 5.81; N, 14.05. Found:
C, 63.44; H, 5.65; N, 13.94.
H-4), 9.52 (s, 1H, D2O exch, NHCO); 13C NMR
(CDCl3, 50 MHz) d 45.65 (CH2CH2N(CH3)2), 46.05
(COCH2N(CH3)2), 48.00 (N–NCH2CH2), 58.55 (N–
NCH2CH2), 63.55 (COCH2), 101.48 (C-3), 112.03 (C-
5), 116.80 (C-2b), 118.20 (C-7), 118.57 (C-10a), 123.16
(C-10), 124.41 (C-9), 125.96 (C-4), 129.67 (C-8), 137.79
(C-10b), 138.34 (C-2a), 139.88 (C-5a), 154.37 (C-6a),
169.03 (NHCO). Anal. Calcd for C21H25N5O2Æ2HClÆ1/
2H2O: C, 54.67; H, 6.12; N, 15.18. Found: C, 54.44;
H, 6.13; N, 15.27.
4.1.37. 2-Chloro-N-[2-(2-pyrrolidin-1-yl-ethyl)-2H-benzo-
pyrano[4,3,2-cd]indazol-5-yl]acetamide (26c). This com-
pound was prepared by an analogous procedure as
described for the preparation of 18a, starting from
24c. Yield: 78%; mp >270 ꢁC (EtOAc/n-hexane); 1H
NMR (CDCl3, 400 MHz) d 1.81 [m, 4H, N(CH2CH2)2],
2.65 [m, 4H, N(CH2CH2)2], 3.09 (t, J = 7 Hz, 2H,
N–NCH2CH2), 4.29 (s, 2H, COCH2), 4.53 (t, J = 7 Hz,
2H, N–NCH2CH2), 6.88 (d, J = 8 Hz, 1H, H-3), 7.21
(t, J = 8 Hz, 1H, H-9), 7.28–7.37 (m, 2H, H-7, H-8),
7.84 (d, J = 8 Hz, 1H, H-4), 7.89 (d, J = 8 Hz, 1H, H-
10), 8.31 (s, 1H, D2O exch, NHCO); 13C NMR (CDCl3,
50 MHz) d 23.45 [N(CH2CH2)2], 42.93 (COCH2), 50.89
(N–NCH2CH2), 54.25 (N–NCH2CH2), 54.28 [N(CH2-
CH2)2], 101.99 (C-3), 112.32 (C-5), 117.11 (C-2b), 117.88
(C-10a), 118.35 (C-7), 123.20 (C-10), 124.67 (C-9), 126.25
(C-4), 130.07 (C-8), 138.05 (C-10b), 138.12 (C-2a), 138.66
(C-5a), 153.92 (C-6a), 163.52 (NHCO). Anal. Calcd for
C21H21ClN4O2: C, 63.55; H, 5.33; N, 14.12. Found: C,
63.61; H, 5.23; N, 14.27.
4.1.40. 2-Diethylamino-N-[2-(2-diethylaminoethyl)-2H-
benzopyrano[4,3,2-cd]indazol-5-yl]acetamide (27b). This
compound was prepared by an analogous procedure as
described for the preparation of 20a, starting from 26b.
Yield: 92%; mp (dihydrochloride) 158–160 ꢁC (EtOH);
1H NMR (CDCl3, 400 MHz) d 1.00 (t, J = 7 Hz, 6H,
CH2CH2N(CH2CH3)2), 1.17 (t, J = 7 Hz, 6H, COCH2N-
(CH2CH3)2), 2.60 (q, J = 7 Hz, 4H, CH2CH2N(CH2-
CH3)2), 2.68 (q, J = 7 Hz, 4H, COCH2N(CH2CH3)2),
2.97 (t, J = 7 Hz, 2H, N–NCH2CH2), 3.21 (s, 2H,
COCH2), 4.39 (t, J = 7 Hz, 2H, N–NCH2CH2), 6.82 (d,
J = 9 Hz, 1H, H-3), 7.12–7.22 (m, 2H, H-7, H-9), 7.29
(t, J = 8 Hz, 1H, H-8), 7.85 (d, J = 8 Hz, 1H, H-10),
8.03 (d, J = 9 Hz, 1H, H-4), 9.52 (s, 1H, D2O exch,
NHCO); 13C NMR (CDCl3, 50 MHz)
d
11.62
(CH2CH2N(CH2CH3)2), 12.64 (COCH2N(CH2CH3)2),
47.38 (CH2CH2N(CH2CH3)2), 47.97 (COCH2N(CH2-
CH3)2), 48.70 (N–NCH2CH2), 52.19 (N–NCH2CH2),
58.18 (COCH2), 101.55 (C-3), 112.36 (C-5), 116.55 (C-
2b), 117.94 (C-7), 118.49 (C-10a), 123.02 (C-10), 124.30
(C-9), 125.07 (C-4), 129.59 (C-8), 137.57 (C-10b), 138.08
(C-2a), 139.08 (C-5a), 154.33 (C-6a), 170.10 (NHCO).
Anal. Calcd for C25H33N5O2Æ2HClÆ3/4H2O: C, 57.52; H,
7.05; N, 13.42. Found: C, 57.45; H, 7.31; N, 13.22.
4.1.38. 2-Chloro-N-[2-(2-piperidin-1-yl-ethyl)-2H-benzo-
pyrano[4,3,2-cd]indazol-5-yl]acetamide (26d). This com-
pound was prepared by an analogous procedure as
described for the preparation of 18a, starting from
24d. Yield: 82%; mp 221–223 ꢁC (dec) (EtOAc/n-hex-
1
ane); H NMR (CDCl3, 400 MHz) d 1.45 (m, 2H, 4-pi-
peridine-H), 1.63 (m, 4H, 3,5-piperidine-H), 2.58 (m,
4H, 2,6-piperidine-H), 2.94 (t, J = 7 Hz, 2H, N–
NCH2CH2), 4.28 (s, 2H, COCH2), 4.49 (t, J = 7 Hz,
2H, N–NCH2CH2), 6.85 (d, J = 9 Hz, 1H, H-3), 7.18
(t, J = 8 Hz, 1H, H-9), 7.27 (d, J = 8 Hz, 1H, H-7),
7.33 (t, J = 8 Hz, 1H, H-8), 7.81 (d, J = 8 Hz, 1H, H-
10), 7.87 (d, J = 9 Hz, 1H, H-4), 8.31 (s, 1H, D2O exch,
NHCO); 13C NMR (CDCl3, 50 MHz) d 23.67 (4-piper-
idine-C), 25.25 (3,5-piperidine-C), 42.89 (COCH2),
46.94 (N–NCH2CH2), 54.58 (2,6-piperidine-C), 57.74
(N–NCH2CH2), 101.92 (C-3), 110.89 (C-5), 116.55 (C-
2b), 118.20 (C-10a), 118.24 (C-7), 123.09 (C-10),
124.56 (C-9), 125.92 (C-4), 129.89 (C-8), 137.97 (C-
10b), 138.71 (C-2a), 140.40 (C-5a), 154.22 (C-6a),
164.11 (NHCO). Anal. Calcd for C22H23ClN4O2: C,
64.31; H, 5.64; N, 13.64. Found: C, 64.58; H, 5.71; N,
13.71.
4.1.41.
N-[2-(2-Pyrrolidin-1-yl-ethyl)-2H-benzopyrano
[4,3,2-cd]indazol-5-yl]-2-pyrrolidin-1-yl-acetamide (27c).
This compound was prepared by an analogous proce-
dure as described for the preparation of 20a, starting
from 26c. Yield: 95%; mp (dihydrochloride) 219–
221 ꢁC (EtOH); 1H NMR (CDCl3, 400 MHz) d 1.75
(m, 4H, CH2CH2N(CH2CH2)2), 1.91 (m, 4H, COCH2N-
(CH2CH2)2), 2.55 (m, 6H, CH2CH2N(CH2CH2)2, N–
NCH2CH2), 2.77 (m, 4H, COCH2N(CH2CH2)2), 3.35
(s, 2H, COCH2), 4.42 (t, J = 7 Hz, 2H, N–NCH2CH2),
6.80 (d, J = 9 Hz, 1H, H-3), 7.13–7.20 (m, 2H, H-7, H-
9), 7.29 (t, J = 8 Hz, 1H, H-8), 7.85 (d, J = 8 Hz, 1H,
H-10), 7.97 (d, J = 9 Hz, 1H, H-4), 9.20 (s, 1H, D2O
exch, NHCO); 13C NMR (CDCl3, 50 MHz) d 23.41
(CH2CH2N(CH2CH2)2), 24.03 (COCH2N(CH2CH2)2),
49.06 (N–NCH2CH2), 54.25 (CH2CH2N(CH2CH2)2),
54.35 (COCH2N(CH2CH2)2, N–NCH2CH2), 59.25
(COCH2), 101.41 (C-3), 112.18 (C-5), 116.62 (C-2b),
118.02 (C-7), 118.50 (C-10a), 123.06 (C-10), 124.31 (C-
9), 125.48 (C-4), 129.56 (C-8), 137.54 (C-10b), 138.09
(C-2a), 139.45 (C-5a), 154.27 (C-6a), 169.15 (NHCO).
Anal. Calcd for C25H29N5O2Æ2HClÆH2O: C, 57.47; H,
6.37; N, 13.40. Found: C, 57.39; H, 6.57; N, 13.33.
4.1.39. 2-Dimethylamino-N-[2-(2-dimethylaminoethyl)-
2H-benzopyrano[4,3,2-cd]indazol-5-yl]acetamide (27a).
This compound was prepared by an analogous proce-
dure as described for the preparation of 20a, starting
from 26a. Yield: 97%; mp (dihydrochloride) 227–
1
229 ꢁC (EtOH); H NMR (CDCl3, 400 MHz) d 2.32 (s,
6H, CH2CH2N(CH3)2), 2.47 (s, 6H, COCH2N(CH3)2),
2.84 (t, J = 7 Hz, 2H, N–NCH2CH2), 3.17 (s, 2H,
COCH2), 4.42 (t, J = 7 Hz, 2H, N–NCH2CH2), 6.83
(d, J = 9 Hz, 1H, H-3), 7.19 (t, J = 8 Hz, 1H, H-9),
7.27 (d, J = 8 Hz, 1H, H-7), 7.33 (t, J = 8 Hz, 1H, H-
8), 7.89 (d, J = 8 Hz, 1H, H-10), 7.96 (d, J = 9 Hz, 1H,
4.1.42. N-[2-(2-Piperidin-1-yl-ethyl)-2H-benzopyrano [4,
3,2-cd]indazol-5-yl]-2-piperidin-1-yl-acetamide (27d).
This compound was prepared by an analogous proce-

2926
I. K. Kostakis et al. / Bioorg. Med. Chem. 14 (2006) 2910–2934
dure as described for the preparation of 20a, starting
from 26d. Yield: 95%; mp (dihydrochloride) 168–
170 ꢁC (EtOH); 1H NMR (CDCl3, 400 MHz) d 1.41
[m, 2H, CH2CH2(4-piperidine-H)], 1.52 [m, 2H,
COCH2(4-piperidine-H)], 1.62 [m, 4H, CH2CH2(3,5-pi-
peridine-H)], 1.73 [m, 4H, COCH2(3,5-piperidine-H)],
2.44 [m, 4H, CH2CH2(2,6-piperidine-H)], 2.61 [m, 4H,
COCH2(2,6-piperidine-H)], 2.80 (t,J = 7 Hz, 2H, N–
NCH2CH2), 3.05 (s, 2H, COCH2), 4.37 (t, J = 7 Hz,
2H, N–NCH2CH2), 6.79 (d, J = 9 Hz, 1H, H-3), 7.11–
7.18 (m, 2H, H-7, H-9), 7.29 (t, J = 8 Hz, 1H, H-8),
7.83 (d, J = 8 Hz, 1H, H-10), 8.04 (d, J = 9 Hz, 1H, H-
4), 9.44 (s, 1H, D2O exch, NHCO); 13C NMR (CDCl3,
H2O); 1H NMR (DMSO-d6, 400 MHz) d 3.22 (t,
J = 7 Hz, 2H, N–NCH2CH2), 4.51 (t, J = 7 Hz, 2H,
N–NCH2CH2), 7.06 (d, J = 9 Hz, 1H, H-3), 7.43–7.54
(m, 2H, H-9, H-10), 7.86 (d, J = 8 Hz, 1H, H-8), 7.90
(d, J = 8 Hz, 1H, H-11), 8.02 (s, 1H, H-7), 8.05 (d,
J = 9 Hz, 1H, H-4), 8.47 (s, 1H, H-12); 13C NMR
(DMSO-d6, 50 MHz) d 53.04 (N–NCH2CH2), 60.63
(N–NCH2CH2), 105.04 (C-3), 114.97 (C-2b), 115.84
(C-12a), 116.71 (C-7), 122.43 (C-12), 125.88 (C-5),
126.84 (C-10), 126.92 (C-4), 127.67 (C-8), 128.15 (C-9),
128.88 (C-11), 130.08 (C-11a), 134.15 (C-7a), 139.42
(C-12b), 143.22 (C-2a), 145.02 (C-5a), 150.83 (C-6a).
Anal. Calcd for C19H13N3O4: C, 65.70; H, 3.77; N,
12.10. Found: C, 65.88; H, 3.59; N, 12.20.
50 MHz)
d 23.71 [CH2CH2(4-piperidine-C)], 24.04
[COCH2(4-piperidine-C)], 25.47 [CH2CH2(3,5-piperi-
dine-C)], 26.06 [COCH2(3,5-piperidine-C)], 51.30 (N–
NCH2CH2), 54.25 [CH2CH2(2,6-piperidine-C)], 54.40
[COCH2(2,6-piperidine-C)], 57.81 (N–NCH2CH2),
62.15 (COCH2), 101.18 (C-3), 112.03 (C-5), 116.11 (C-
2b), 118.13 (C-7), 118.51 (C-10a), 122.61 (C-10),
123.93 (C-9), 124.27 (C-4), 129.19 (C-8), 136.95 (C-
10b), 138.42 (C-2a), 139.02 (C-5a), 153.85 (C-6a),
168.37 (NHCO). Anal. Calcd for C27H33N5O2Æ2HClÆ
1/2H2O: C, 59.88; H, 6.70; N, 12.93. Found: C, 59.97;
H, 6.84; N, 12.81.
4.1.44. 2,3,4,12-Tetrahydro-1H-benzo[b]xanthene-1,12-
dione (31). A suspension of 30 (207 mg, 1.1 mmol) and
1,3 cyclohexanedione (112 mg, 1 mmol) in polyphos-
phoric acid (3 g) was heated at 150 ꢁC for 2 h. After
cooling, the reaction mixture was poured into crushed
ice, the precipitate was filtered, washed with 10%
Na2CO3 and water, and air-dried. Flash chromatogra-
phy on silica gel using a mixture of cyclohexane/EtOAc
2:1 as the eluent provided 31 (169 mg, 64%); mp 259–
261 ꢁC (EtOAc/n-hexane); 1H NMR (CDCl3,
400 MHz) d 2.18 (m, 2H, H-3), 2.61 (t, J = 6.5 Hz, 2H,
H-4), 3.02 (t, J = 6.5 Hz, 2H, H-2), 7.52 (dt,
J = 8.0 Hz, 1.0 Hz, 1H, H-9), 7.60 (dt, J = 8.0 Hz,
1.0 Hz, 1H, H-8), 7.81 (s, 1H, H-6), 7.86 (dd,
J = 8.0 Hz, 1.0 Hz, 1H, H-7), 8.02 (dd, J = 8.0 Hz,
1.0 Hz, 1H, H-10), 8.78 (s, 1H, H-11). 13C NMR
(CDCl3, 50 MHz) d 19.81 (C-3), 29.84 (C-4), 38.52 (C-
2), 114.13 (C-6), 115.81 (C-12a), 123.39 (C-11a),
126.48 (C-9), 127.28 (C-7), 128.24 (C-11), 129.12 (C-8),
129.78 (C-10), 130.63 (C-10a), 135.67 (C-6a), 150.96
(C-5a), 174.52 (C-12), 179.04 (C-4a), 193.96 (C-1). Anal.
Calcd for C17H12O3: C, 77.26; H, 4.58. Found: C, 77.50;
H, 4.31.
4.1.43. N-(2-Hydroxyethyl)-N-[2-(2-hydroxyethyl)-2H-
benzo[g]benzopyrano[4,3,2-cd]indazol-5-yl]hydrazine
(28). A suspension of 13 (623 mg, 2 mmol) and 2-
hydroxyethylhydrazine (304 mg, 4 mmol) in dry DMSO
(8 mL) was stirred at room temperature for 24 h. The
reaction mixture was then poured into crushed ice and
the precipitate was filtered, washed with water, and air-
dried. The residue was purified by column chromatogra-
phy (silica gel) using a mixture of cyclohexane/EtOAc
1:2 as the eluent, to provide compounds 28 (503 mg,
67%) and 29 (67 mg, 9%).
Data for N-(2-hydroxyethyl)-N-[2-(2-hydroxyethyl)-2H-
benzo[g]benzopyrano[4,3,2-cd]indazol-5-yl]hydrazine
(28): mp >270 ꢁC (DMF); 1H NMR (DMSO-d6,
400 MHz) d 3.91 (q, J = 7 Hz, 2H, N–NCH2CH2),
4.06 (q, J = 7 Hz, 2H, NH2–NCH2CH2), 4.70 (t,
J = 7 Hz, 2H, N–NCH2CH2), 4.78 (t, J = 7 Hz, 2H,
NH2–NCH2CH2), 5.04 (t, J = 7 Hz, 1H, D2O exch,
OH), 5.15 (t, J = 7 Hz, 1H, D2O exch, OH), 7.39 (t,
J = 8 Hz, 1H, H-10), 7.41 (s, 1H, H-7), 7.49 (t,
J = 8 Hz, 1H, H-9), 7.81 (d, J = 8 Hz, 1H, H-4),
7.89 (d, J = 8 Hz, 1H, H-3), 7.91 (d, J = 8 Hz, 1H,
H-8), 7.96 (d, J = 8 Hz, 1H, H-11), 9.56 (s, 1H, H-
12); 13C NMR (DMSO-d6, 50 MHz) d 51.41 (N–
NCH2CH2), 52.46 (NH2–NCH2CH2), 58.29 (N–
NCH2CH2), 60.51 (NH2–NCH2CH2), 108.40 (C-5),
110.64 (C-3), 113.38 (C-4), 113.55 (C-7), 119.41 (C-
12a), 122.78 (C-12), 123.81 (C-10), 126.21 (C-2b),
127.32 (C-8), 127.92 (C-9), 128.43 (C-11), 129.91
(C-11a), 134.42 (C-5a), 134.50 (C-7a), 141.52 (C-
2a), 143.78 (C-12b), 153.93 (C-6a). Anal. Calcd for
C21H20N4O3: C, 67.01; H, 5.36; N, 14.88. Found: C,
67.16; H, 5.70; N, 14.59.
4.1.45. 1-Hydroxy-12H-benzo[b]xanthen-12-one (32). A
solution of 31 (100 mg, 0.365 mmol) and DDQ (86 mg,
0.38 mmol) in dry toluene (10 mL) was refluxed for
40 min. After cooling, the solvent was removed under
reduced pressure, and the residue was purified by
column chromatography (silica gel, cyclohexane to
cyclohexane/EtOAc 9:1); Yield: 88%; mp 210–212 ꢁC
(lit.44 212–214 ꢁC).
4.1.46. 1-[(4-Methylphenyl)sulfonyl]oxy-12H-benzo[b]-
xanthen-12-one (33). To a solution of 32 (524 mg,
2 mmol) in dry acetone (20 ml) were added under argon
4-toluenesulfonyl chloride (419 mg, 2.2 mmol) and
anhydrous sodium carbonate (233 mg, 2.2 mmol), and
the mixture was refluxed for 3.5 h. The solvent was then
vacuum-evaporated, the residue was diluted with
CH2Cl2 and extracted with water, the organic layer
was dried (Na2SO4) and vacuum-evaporated. The resi-
due was purified by column chromatography, using a
mixture of cyclohexane/EtOAc 3:1 as the eluent, to give
compound 33 as an oil (782 mg, 94%); 1H NMR
(CDCl3, 400 MHz) d 2.38 (s, 3H, 40-CH3), 7.08 (d,
J = 9.0 Hz, 1H, H-4), 7.32 (d, J = 8 Hz, 2H, H-30, H-
50), 7.41 (d, J = 9 Hz, 1H, H-2) 7.47 (t, J = 8 Hz, 1H,
Data for 2-[5-nitro-2H-benzo[g]benzopyrano[4,3,2-
cd]indazol-2-yl]-1-ethanol (29): mp 225–227 ꢁC (DMF–

I. K. Kostakis et al. / Bioorg. Med. Chem. 14 (2006) 2910–2934
2927
H-9), 7.55–7.64 (m, 2H, H-3, H-8), 7.79 (s, 1H, H-6),
7.86 (d, J = 8 Hz, 1H, H-7), 7.96 (d, J = 8 Hz, 2H, H-
20, H-60), 8.01 (d, J = 8 Hz, 1H, H-10), 8.81 (s, 1H, H-
11). Anal. Calcd for C24H16O5S: C, 69.22; H, 3.87.
Found: C, 69.04; H, 3.79.
Data for 2-[5-nitro-2H-benzo[g]benzopyrano[4,3,2-
cd]indazol-2-yl]-1-ethanol Methanesulfonate (36). Mp
213–215 ꢁC (EtOH); 1H NMR (CDCl3, 400 MHz) d
2.76 (s, 3H, CH3), 4.62 (t, J = 7 Hz, 2H, N–NCH2CH2),
4.72 (t, J = 7 Hz, 2H, N–NCH2CH2), 6.92 (d, J = 9 Hz,
1H, H-3), 7.45–7.54 (m, 2H, H-9, H-10), 7.84 (d,
J = 8 Hz, 1H, H-8), 7.89 (d, J = 8 Hz, 1H, H-11), 8.03
(s, 1H, H-7), 8.17 (d, J = 9 Hz, 1H, H-4), 8.46 (s, 1H,
H-12); 13C NMR (CDCl3, 50 MHz) d 38.16 (CH3),
48.64 (N–NCH2CH2), 67.12 (N–NCH2CH2), 104.64
(C-3), 114.94 (C-2b), 115.92 (C-12a), 116.00 (C-7),
122.12 (C-12), 125.43 (C-5), 126.28 (C-10), 126.96 (C-
4), 127.65 (C-8), 127.69 (C-9), 128.01 (C-11), 130.66
(C-11a), 133.98 (C-7a), 139.74 (C-12b), 142.73 (C-2a),
145.62 (C-5a), 150.83 (C-6a). Anal. Calcd for
C20H15N3O6S: C, 56.47; H, 3.55; N, 9.88. Found: C,
56.76; H, 3.69; N, 9.98.
4.1.47. 2-(2H-Benzo[g]benzopyrano[4,3,2-cd]indazol-2-
yl)-1-ethanol (34). A solution of 33 (170 mg, 0.41 mmol)
and 2-hydroxyethylhydrazine (154 lL, 2.05 mmol) in
dry pyridine (6 mL) was refluxed for 9 h. After cooling,
the reaction mixture was diluted with CH2Cl2 (100 mL),
washed successively with a 18% HCl solution and water,
dried (Na2SO4), and evaporated to dryness. The residue
was purified by column chromatography (silica gel)
using a mixture of cyclohexane/EtOAc 4:1 as the eluent,
to provide compound 34 (109 mg, 88%); mp 222–224 ꢁC
(EtOH); 1H NMR (DMSO-d6, 400 MHz) d 4.06 (t,
J = 7 Hz, 2H, N–NCH2CH2), 4.44 (t, J = 7 Hz, 2H,
N–NCH2CH2), 6.56 (d, J = 8 Hz, 1H, H-5), 6.92 (d,
J = 8 Hz, 1H, H-3), 7.33–7.48 (m, 3H, H-4, H-9, H-
10), 7.66 (s, 1H, H-7), 7.77 (d, J = 8 Hz, 1H, H-8),
7.85 (d, J = 8 Hz, 1H, H-11), 8.33 (s, 1H, H-12); 13C
NMR (DMSO-d6, 50 MHz) d 49.77 (N–NCH2CH2),
62.26 (NHCH2CH2), 100.77 (C-5), 101.84 (C-3),
114.26 (C-7), 115.91 (C-2b), 118.64 (C-12a), 121.89 (C-
12), 125.54 (C-10), 126.94 (C-9), 127.22 (C-8), 128.11
(C-11), 130.21 (C-4), 130.42 (C-11a), 134.27 (C-7a),
137.84 (C-12b), 141.06 (C-2a), 149.56 (C-5a), 152.34
(C-6a). Anal. Calcd for C19H14N2O2: C, 75.48; H,
4.67; N, 9.27. Found: C, 75.30; H, 4.55; N, 9.16.
Data for 2-[3-nitro-2H-benzo[g]benzopyrano[4,3,2-
cd]indazol-2-yl]-1-ethanol Methanesulfonate (37). Mp
1
227–229 ꢁC (EtOH); H NMR (DMSO-d6, 400 MHz) d
3.11 (s, 2H, CH3), 4.65 (t, J = 7 Hz, 2H, N–NCH2CH2),
5.09 (t, J = 7 Hz, 2H, N–NCH2CH2), 6.83 (d, J = 8 Hz,
1H, H-5), 7.43–7.53 (m, 2H, H-9, H-10), 7.84 (d,
J = 8 Hz, 1H, H-8), 7.86 (s, 1H, H-7), 7.99 (d,
J = 8 Hz, 1H, H-11), 8.27 (d, J = 8 Hz, 1H, H-4), 8.41
(s, 1H, H-12); 13C NMR (DMSO-d6, 50 MHz) d 37.08
(CH3), 52.24 (N–NCH2CH2), 69.80 (NHCH2CH2),
103.84 (C-5), 114.97 (C-7), 115.47 (C-12a), 117.65 (C-
2b), 122.89 (C-12), 126.27 (C-10), 127.71 (C-8), 128.14
(C-9), 128.55 (C-3), 128.83 (C-11), 130.64 (C-11a),
131.75 (C-4), 132.97 (C-2a), 134.17 (C-7a), 138.51 (C-
12b), 150.48 (C-6a), 154.30 (C-5a). Anal. Calcd for
C20H15N3O6S: C, 56.47; H, 3.55; N, 9.88. Found: C,
56.29; H, 3.74; N, 9.66.
4.1.48. 2-(2H-Benzo[g]benzopyrano[4,3,2-cd]indazol-2-
yl)-1-ethanol methanesulfonate (35). This compound was
prepared by a procedure analogous to that of 23. Yield:
93%; mp 199–201 ꢁC (EtOH); 1H NMR (CDCl3,
400 MHz) d 2.86 (s, 3H, CH3), 4.64 (t, J = 7 Hz, 2H, N–
NCH2CH2), 4.71 (t, J = 7 Hz, 2H, N–NCH2CH2), 6.61
(d, J = 8 Hz, 1H, H-5), 6.94 (d, J = 8 Hz, 1H, H-3),
7.36–7.50 (m, 3H, H-4, H-9, H-10), 7.69 (s, 1H, H-7),
7.76 (d, J = 8 Hz, 1H, H-8), 7.88 (d, J = 8 Hz, 1H, H-
11), 8.37 (s, 1H, H-12); 13C NMR (CDCl3, 50 MHz) d
38.43 (CH3), 48.52 (N–NCH2CH2), 66.69 (NHCH2CH2),
100.86 (C-5), 101.98 (C-3), 114.22 (C-7), 116.01 (C-2b),
118.54 (C-12a), 121.96 (C-12), 125.53 (C-10), 127.00
(C-9), 127.32 (C-8), 128.01 (C-11), 130.09 (C-4), 130.43
(C-11a), 134.17 (C-7a), 137.84 (C-12b), 141.56 (C-2a),
149.62 (C-5a), 152.43 (C-6a). Anal. Calcd for C20H16N2
O4S: C, 63.15; H, 4.24; N, 7.36. Found: C, 63.33; H, 4.00;
N, 7.04.
4.1.50. Dimethyl-[2-(5-nitro-2H-benzo[g]benzopyrano [4,3,
2-cd]indazol-2-yl)ethyl]amine (38a). This compound was
prepared by an analogous procedure as described for
the preparation of 24a, starting from 36. Yield: 94%; mp
1
(hydrochloride) 200–202 ꢁC (EtOH); H NMR (CDCl3,
400 MHz) d 2.32 (s, 3H, N(CH3)2), 4.43 (t, J = 7 Hz,
2H, N–NCH2CH2), 2.91 (t, J = 7 Hz, 2H, N–NCH2CH2),
6.91 (d, J = 9 Hz, 1H, H-3), 7.43–7.55 (m, 2H, H-9, H-10),
7.85 (d, J = 8 Hz, 1H, H-8), 7.89 (d, J = 8 Hz, 1H, H-11),
8.01 (s, 1H, H-7), 8.15 (d, J = 9 Hz, 1H, H-4), 8.45 (s,
1H, H-12). 13C NMR (CDCl3, 50 MHz) d 45.53
(CH2CH2N(CH3)2), 47.99 (N–NCH2CH2), 58.56 (N–
NCH2CH2), 102.51 (C-3), 114.89 (C-2b), 115.87 (C-7),
116.11 (C-12a), 122.03 (C-12), 125.30 (C-5), 126.29 (C-
10), 126.96 (C-4), 127.61 (C-8), 127.66 (C-9), 127.95 (C-
11), 130.60 (C-11a), 133.95 (C-7a), 139.88 (C-12b),
142.11 (C-2a), 145.82 (C-5a), 150.54 (C-6a). Anal. Calcd
for C21H18N4O3ÆHClÆH2O: C, 58.81; H, 4.94; N, 13.06.
Found: C, 58.62; H, 4.77; N, 13.33.
4.1.49. 2-[5-Nitro-2H-benzo[g]benzopyrano[4,3,2-cd]ind-
azol-2-yl]-1-ethanol methanesulfonate (36). A solution of
fuming nitric acid (126 lL, 2 mmol) in glacial acetic acid
(1 mL) was added dropwise to a solution of 35 (783 mg,
1.9 mmol) in glacial acetic acid (5 mL) at 5 ꢁC and the
mixture was stirred at this temperature for 5 min, fol-
lowed by 30 min of stirring at room temperature. The
reaction mixture was then poured into crushed ice and
the precipitate was filtered, washed with water and 5%
Na2CO3, and air-dried. The residue was purified by col-
umn chromatography (silica gel), using a mixture of
cyclohexane/EtOAc 3:1 as the eluent, to give compounds
36 (492 mg, 61%) and 37 (129 mg, 16%).
4.1.51. Diethyl-[2-(5-nitro-2H-benzo[g]benzopyrano[4,3,
2-cd]indazol-2-yl)ethyl]amine (38b). This compound was
prepared by an analogous procedure as described for
the preparation of 24a, starting from 36. Yield: 88%;
mp (hydrochloride) >270 ꢁC (EtOH); 1H NMR (CDCl3,
400 MHz) d 0.96 (t, J = 7 Hz, 6H, (CH2CH3)2), 2.58
(q, J = 7 Hz, 4H, (CH2CH3)2), 3.01 (t, J = 7 Hz, 2H,

2928
I. K. Kostakis et al. / Bioorg. Med. Chem. 14 (2006) 2910–2934
N–NCH2CH2), 4.45 (t, J = 7 Hz, 2H, N–NCH2CH2),
6.89 (d, J = 9 Hz, 1H, H-3), 7.44–7.55 (m, 2H, H-9, H-
10), 7.83 (d, J = 8 Hz, 1H, H-8), 7.88 (d, J = 8 Hz, 1H,
H-11), 7.98 (s, 1H, H-7), 8.14 (d, J = 9 Hz, 1H, H-4),
8.43 (s, 1H, H-12); 13C NMR (CDCl3, 50 MHz) d
12.04 [(CH2CH3)2], 46.86 [(CH2CH3)2], 48.54 (N–
NCH2CH2), 52.59 (N–NCH2CH2), 102.54 (C-3),
114.90 (C-2b), 115.91 (C-7), 116.06 (C-12a), 121.95 (C-
12), 125.26 (C-5), 126.27 (C-10), 127.14 (C-4), 127.59
(C-8), 127.62 (C-9), 127.96 (C-11), 130.63 (C-11a),
133.91 (C-7a), 139.93 (C-12b), 142.03 (C-2a), 145.77
(C-5a), 150.63 (C-6a). Anal. Calcd for C23H22N4O3Æ
HClÆH2O: C, 60.46; H, 5.51; N, 12.26. Found: C,
60.00; H, 5.54; N, 12.05.
dure as described for the preparation of 18a, starting
from 38a. Yield: 83%; mp 193–195 ꢁC (dec), (EtOAc/n-
1
hexane); H NMR (CDCl3, 400 MHz) d 2.31 (s, 6H,
CH2CH2N(CH3)2), 2.82 (t, J = 7 Hz, 2H, N–
NCH2CH2), 4.30 (s, 2H, COCH2), 4.31 (t, J = 7 Hz,
2H, N–NCH2CH2), 6.83 (d, J = 9 Hz, 1H, H-3), 7.37–
7.49 (m, 2H, H-9, H-10), 7.57 (s, 1H, H-7), 7.69 (d,
J = 8 Hz, 1H, H-8), 7.76 (d, J = 8 Hz, 1H, H-11), 7.84
(d, J = 9 Hz, 1H, H-4), 8.27 (s, 1H, H-12), 8.35 (s, 1H,
D2O exch, NH) 13C NMR (CDCl3, 50 MHz) d 43.05
(COCH2), 45.56 (CH2CH2N(CH3)2), 48.06 (N–
NCH2CH2), 58.46 (N–NCH2CH2), 102.01 (C-3),
112.94 (C-5), 114.03 (C-7), 116.04 (C-2b), 118.49 (C-
12a), 122.03 (C-12), 125.48 (C-10), 125.57 (C-4),
127.12 (C-8), 127.11 (C-9), 128.13 (C-11), 130.66 (C-
11a), 133.99 (C-7a), 137.48 (C-2a), 138.17 (C-5a),
138.99 (C-12b), 152.04 (C-6a), 163.66 (CONH). Anal.
Calcd for C23H21ClN4O2. Calcd: C: 65.63, H: 5.03, N:
13.31. Found: C: 65.39, H: 4.88, N: 12.97.
4.1.52. 5-Nitro-2-[2-(pyrrolidin-1-yl)ethyl]-2H-benzo[g]-
benzopyrano[4,3,2-cd]indazole (38c). This compound
was prepared by an analogous procedure as described
for the preparation of 24a, starting from 36. Yield:
91%; mp (hydrochloride) 180–182 ꢁC (EtOH); 1H
NMR (CDCl3, 400 MHz) d 1.85 [m, 4H, N(CH2CH2)2],
2.68 [m, 4H, N(CH2CH2)2], 3.17 (t, J = 7 Hz, 2H, N–
NCH2CH2), 4.56 (t, J = 7 Hz, 2H, N–NCH2CH2), 6.91
(d, J = 9 Hz, 1H, H-3), 7.41–7.57 (m, 2H, H-9, H-10),
7.84 (d, J = 8 Hz, 1H, H-8), 7.89 (d, J = 8 Hz, 1H, H-
11), 8.01 (s, 1H, H-7), 8.16 (d, J = 9 Hz, 1H, H-4),
8.46 (s, 1H, H-12); 13C NMR (CDCl3, 50 MHz) d
23.48 [N(CH2CH2)2], 48.16 (N–NCH2CH2), 54.23
[N(CH2CH2)2], 54.83 (N–NCH2CH2), 102.58 (C-3),
114.97 (C-2b), 115.56 (C-7), 115.96 (C-12a), 122.12 (C-
12), 125.39 (C-5), 126.27 (C-10), 126.81 (C-4), 127.59
(C-9), 127.60 (C-8), 127.93 (C-11), 130.61 (C-11a),
133.96 (C-7a), 140.06 (C-12b), 142.15 (C-2a), 145.90
(C-5a), 150.57 (C-6a). Anal. Calcd for C23H20N4O3ÆH-
ClÆH2O: C, 60.72; H, 5.10; N, 12.32. Found: C, 60.69;
H, 5.42; N, 12.58.
4.1.55. 2-Chloro-N-[2-(2-diethylaminoethyl)-2H-benzo-
[g]benzopyrano[4,3,2-cd]indazol-5-yl]acetamide
(40b).
This compound was prepared by an analogous proce-
dure as described for the preparation of 18a, starting
from 38b. Yield: 79%; mp 174–176 ꢁC (EtOAc/n-hex-
1
ane); H NMR (CDCl3, 400 MHz) d 0.96 (t, J = 7 Hz,
6H, (CH2CH3)2), 2.59 (q, J = 7 Hz, 4H, (CH2CH3)2),
2.97 (t, J = 7 Hz, 2H, N–NCH2CH2), 4.22 (s, 2H,
COCH2), 4.39 (t, J = 7 Hz, 2H, N–NCH2CH2), 6.79
(d, J = 9 Hz, 1H, H-3), 7.36–7.47 (m, 2H, H-9, H-10),
7.56 (s, 1H, H-7), 7.65 (d, J = 8 Hz, 1H, H-8), 7.74 (d,
J = 8 Hz, 1H, H-11), 7.80 (d, J = 9 Hz, 1H, H-4), 8.24
(s, 1H, H-12), 8.33 (s, 1H, D2O exch, NH); 13C NMR
(CDCl3, 50 MHz) d 11.73 (CH2CH2N(CH2CH3)2),
43.17 (COCH2), 47.36 (CH2CH2N(CH2CH3)2), 48.73
(N–NCH2CH2), 52.28 (N–NCH2CH2), 101.87 (C–3),
113.11 (C-5), 114.05 (C-7), 115.97 (C-2b), 118.61 (C-
12a), 121.14 (C-12), 124.88 (C-10), 125.76 (C-4),
127.04 (C-8, C-9), 128.09 (C-11), 130.62 (C-11a),
134.05 (C-7a), 137.48 (C-2a), 138.11 (C-5a), 139.26 (C-
12b), 151.93 (C-6a), 163.82 (CONH). Anal. Calcd for
C25H25ClN4O2. Calcd: C: 66.88, H: 5.61, N: 12.48.
Found: C: 66.72, H: 5.85, N: 12.10.
4.1.53. 5-Nitro-2-[2-(piperidin-1-yl)ethyl]-2H-benzo[g]-
benzopyrano[4,3,2-cd]indazole (38d). This compound
was prepared by an analogous procedure as described
for the preparation of 24a, starting from 36. Yield:
86%; mp (hydrochloride) >270 ꢁC (EtOH); 1H NMR
(CDCl3, 400 MHz) d 1.43 (m, 2H, 4-piperidine-H),
1.55 (m, 4H, 3,5-piperidine-H), 2.49 (m, 4H, 2,6-piperi-
dine-H), 2.91 (t, J = 7 Hz, 2H, N–NCH2CH2), 4.45 (t,
J = 7 Hz, 2H, N–NCH2CH2, 6.94 (d, J = 9 Hz, 1H, H-
3), 7.48–7.59 (m, 2H, H-9, H-10), 7.88 (d, J = 8 Hz,
1H, H-8), 7.92 (d, J = 8 Hz, 1H, H-11), 8.05 (s, 1H, H-
7), 8.19 (d, J = 9 Hz, 1H, H-4), 8.49 (s, 1H, H-12); 13C
NMR (CDCl3, 50 MHz) d 24.53 (4-piperidine-C),
26.10 (3,5-piperidine-C), 48.39 (N–NCH2CH2), 54.89
(2,6-piperidine-C), 58.15 (N–NCH2CH2), 102.59 (C-3),
114.87 (C-2b), 116.09 (C-7), 116.24 (C-12a), 122.16 (C-
12), 125.28 (C-5), 126.32 (C-10), 126.90 (C-4), 127.64
(C-8), 127.74 (C-9), 127.98 (C-11), 130.58 (C-11a),
134.02 (C-7a), 139.64 (C-12b), 141.93 (C-2a), 145.68
(C-5a), 150.46 (C-6a). Anal. Calcd for C24H22N4O3ÆH-
ClÆH2O: C, 61.47; H, 5.37; N, 11.95. Found: C, 61.54;
H, 5.25; N, 12.01.
4.1.56. 2-Chloro-N-[2-(2-pyrrolidin-1-yl-ethyl)-2H-benzo-
[g]benzopyrano[4,3,2-cd]indazol-5-yl]acetamide
(40c).
This compound was prepared by an analogous procedure
as described for the preparation of 18a, starting from 38c.
Yield: 79%; mp 185–187 ꢁC (dec), (EtOAc/n-hexane); 1H
NMR (CDCl3, 400 MHz) d 1.77 [m, 4H, N(CH2CH2)2],
2.58 [m, 4H, N(CH2CH2)2], 3.03 (t, J = 7 Hz, 2H, N–
NCH2CH2), 4.28 (s, 2H, COCH2), 4.47 (t, J = 7 Hz, 2H,
N–NCH2CH2), 6.85 (d, J = 9 Hz, 1H, H-3), 7.35–7.47
(m, 2H, H-9, H-10), 7.62 (s, 1H, H-7), 7.71 (d, J = 8 Hz,
1H, H-8), 7.80 (d, J = 8 Hz, 1H, H-11), 7.86 (d,
J = 9 Hz, 1H, H-4), 8.31 (s, 1H, H-12), 8.36 (s, 1H, D2O
exch, NH); 13C NMR (CDCl3, 50 MHz) d 22.67
(CH2CH2N(CH2CH2)2), 42.86 (COCH2), 49.25 (N–
NCH2CH2), 54.25 (CH2CH2N(CH2CH2)2), 55.36 (N–
NCH2CH2), 102.03 (C-3), 113.11 (C-5), 113.99 (C-7),
115.98 (C-2b), 118.46 (C-12a), 122.07 (C-12), 125.43 (C-
10), 125.78 (C-4), 127.05 (C-8, C-9), 128.05 (C-11),
130.61 (C-11a), 133.90 (C-7a), 137.43 (C-2a), 138.11
4.1.54. 2-Chloro-N-[2-(2-dimethylaminoethyl)-2H-benzo-
[g]benzopyrano[4,3,2-cd]indazol-5-yl]acetamide (40a).
This compound was prepared by an analogous proce-

I. K. Kostakis et al. / Bioorg. Med. Chem. 14 (2006) 2910–2934
2929
(C-5a), 138.68 (C-12b), 152.02 (C-6a), 164.36 (CONH).
Anal. Calcd for C25H23ClN4O2. Calcd: C: 67.19, H:
5.19, N: 12.54. Found: C: 67.36, H: 5.07, N: 12.63.
7 Hz, 4H, COCH2N(CH2CH3)2), 2.98 (t, J = 7 Hz,
2H, N–NCH2CH2), 3.26 (s, 2H, COCH2), 4.42 (t,
J = 7 Hz, 2H, N–NCH2CH2), 6.89 (d, J = 9 Hz,
1H, H-3), 7.37–7.49 (m, 2H, H-9, H-10), 7.59 (s,
1H, H-7), 7.75 (d, J = 8 Hz, 1H, H-8), 7.83 (d,
J = 8 Hz, 1H, H-11), 8.14 (d, J = 9 Hz, 1H, H-
4),8.37(s,1H, H-12), 9.65(s, 1H, D2O exch, NH);
13C NMR (CDCl3, 50 MHz) d 12.02 (CH2CH2-
N(CH2CH3)2), 12.75 (COCH2N (CH2CH3) 2),47.
59(CH2CH2N(CH2CH3)2), 49.75 (COCH2N(CH2-
CH3)2), 48.88 (N–NCH2CH2), 52.63 (N–NCH2-
CH2), 58.29 (COCH2), 101.99 (C-3), 113.06 (C-5),
113.87 (C-7), 115.89 (C-2b), 118.57 (C-12a), 121.92
(C-12), 124.93 (C-10), 125.56 (C-4), 127.03 (C-8,
C-9), 128.02 (C-11), 130.59 (C-11a), 133.94 (C-7a),
137.25 (C-2a), 138.09 (C-5a), 139.50 (C-12b),
152.02 (C-6a), 170.22 (CONH). Anal. Calcd for
C29H35N5O2: C, 71.73; H, 7.26; N, 14.42. Found:
C, 71.64; H, 7.48; N, 14.49.
4.1.57. 2-Chloro-N-[2-(2-piperidin-1-yl-ethyl)-2H-benzo-
[g]benzopyrano[4,3,2-cd]indazol-5-yl]acetamide
(40d).
This compound was prepared by an analogous proce-
dure as described for the preparation of 18a, starting
from 38d. Yield: 78%; mp 172–174 ꢁC (dec), (EtOAc/
n-hexane); 1H NMR (CDCl3, 400 MHz) d 1.43 (m,
2H, 4-piperidine-H), 1.58 (m, 4H, 3,5-piperidine-H),
2.50 (m, 4H, 2,6-piperidine-H), 2.86 (t, J = 7 Hz,
2H, N–NCH2CH2), 4.29 (s, 2H, COCH2), 4.42 (t,
J = 7 Hz, 2H, N–NCH2CH2, 6.81 (d, J = 9 Hz, 1H,
H-3), 7.38–7.50 (m, 2H, H-9, H-10), 7.56 (s, 1H,
H-7), 7.68 (d, J = 8 Hz, 1H, H-8), 7.78 (d, J = 8 Hz,
1H, H-11), 7.83 (d, J = 9 Hz, 1H, H-4), 8.26 (s, 1H,
H-12), 8.37 (s, 1H, D2O exch, NH); 13C NMR
(CDCl3, 50 MHz) d 24.22 [CH2CH2(4-piperidine-C)],
25.57 [CH2CH2(3,5-piperidine-C)], 43.17 (COCH2),
47.33 (N–NCH2CH2), 54.63 [CH2CH2(3,5-piperidine-
C)], 58.07 (N–NCH2CH2), 102.03 (C-3), 113.05 (C-
5), 114.07 (C-7), 116.09 (C-2b), 118.52 (C-12a),
122.11 (C-12), 125.29 (C-10), 125.64 (C-4), 127.18
(C-9), 127.26 (C-8), 128.13 (C-11), 130.67 (C-11a),
134.00 (C-7a), 137.46 (C-2a), 138.19 (C-5a), 139.29
(C-12b), 152.12 (C-6a), 163.87 (CONH). Anal. Calcd
for C26H25ClN4O2. Calcd: C: 67.75, H: 5.47, N:
12.15. Found: C: 67.51, H: 5.31, N: 12.04.
4.1.60. N-[2-(2-Pyrrolidin-1-yl-ethyl)-2H-benzo[g]benzo-
pyrano[4,3,2-cd]indazol-5-yl]-2-pyrrolidin-1-yl-acetamide
(41c). This compound was prepared by an analogous
procedure as described for the preparation of 20a,
starting from 40c. Yield: 94%; mp 143–145 ꢁC
(EtOAc/n-hexane); 1H NMR (CDCl3, 400 MHz)
d
1.79 (m, 4H, CH2CH2N(CH2CH2)2), 1.98
(m, 4H, COCH2N(CH2CH2)2), 2.60 (m, 4H, CH2CH2-
N(CH2CH2)2), 2.83 (m, 4H, COCH2N(CH2CH2)2),
3.05 (t,J = 7 Hz, 2H, N–NCH2CH2), 3.40 (s, 2H,
COCH2), 4.51 (t, J = 7 Hz, 2H, N–NCH2CH2), 6.89
(d, J = 9 Hz, 1H, H-3), 7.38–7.51 (m, 2H, H-9, H-10),
7.58 (s, 1H, H-7), 7.75 (d, J = 8 Hz, 1H, H-8), 7.84 (d,
J = 8 Hz, 1H, H-11), 8.06 (d, J = 9 Hz, 1H, H-4), 8.36
(s, 1H, H-12), 9.30 (s, 1H, D2O exch, NH). 13C NMR
(CDCl3, 50 MHz) d 23.52 (CH2CH2N(CH2CH2)2),
24.18 (COCH2N(CH2CH2)2), 49.32 (N–NCH2CH2),
54.36 (CH2CH2N(CH2CH2)2), 54.54 (COCH2N-
(CH2CH2)2), 55.53 (N–NCH2CH2), 59.47 (COCH2),
101.85 (C-3), 112.80 (C-5), 113.94 (C-7), 116.08 (C-2b),
118.51 (C-12a), 121.96 (C-12), 125.56 (C-4, C-10),
127.03 (C-8, C-9), 128.02 (C-11), 130.55 (C-11a),
133.94 (C-7a), 137.36 (C-2a), 138.13 (C-5a), 138.97 (C-
12b), 151.95 (C-6a), 169.33 (CONH). Anal. Calcd for
C29H31N5O2. C: 72.33, H: 6.49, N: 14.54. Found: C:
72.59, H: 6.23, N: 14.72.
4.1.58. 2-Dimethylamino-N-[2-(2-dimethylaminoethyl)-
2H-benzo[g]benzopyrano[4,3,2-cd]indazol-5-yl]acetamide
(41a). This compound was prepared by an analogous
procedure as described for the preparation of 20a, start-
ing from 40a. Yield: 97%; mp 166–168 ꢁC (EtOAc/n-
1
hexane); H NMR (CDCl3, 400 MHz) d 2.33 (s, 6H,
CH2CH2N(CH3)2), 2.50 (s, 6H, COCH2N(CH3)2),
2.88 (t, J = 7 Hz, 2H, N–NCH2CH2), 3.18 (s, 2H,
COCH2), 4.47 (t, J = 7 Hz, 2H, N–NCH2CH2), 6.87
(d, J = 9 Hz, 1H, H-3), 7.35–7.51 (m, 2H, H-9, H-10),
7.65 (s, 1H, H-7), 7.74 (d, J = 8 Hz, 1H, H-8), 7.82 (d,
J = 8 Hz, 1H, H-11), 8.01 (d, J = 9 Hz, 1H, H-4), 8.35
(s, 1H, H-12), 9.20 (s, 1H, D2O exch, NH); 13C NMR
(CDCl3, 50 MHz) d 45.61 (CH2CH2N (CH3)2), 46.12
(COCH2N(CH3)2), 48.03 (N–NCH2 CH2), 58.58 (N–
NCH2CH2), 63.58 (COCH2), 101.81 (C-3), 112.69 (C-
5), 114.09 (C-7), 116.00 (C-2b), 118.43 (C-12a), 121.99
(C-12), 125.56 (C-10), 125.81 (C-4), 127.06 (C-8, C-9),
128.06 (C-11), 130.59 (C-11a), 133.97 (C-7a), 137.54
(C-2a), 138.20 (C-5a), 139.19 (C-12b), 151.95 (C-6a),
169.08 (CONH). Anal. Calcd for C25H27N5O2. Calcd:
C: 69.91, H: 6.34, N: 16.30. Found: C: 69.79, H: 6.56,
N: 16.47.
4.1.61. N-[2-(2-Piperidin-1-yl-ethyl)-2H-benzo[g]benzo-
pyrano[4,3,2-cd]indazol-5-yl]-2-piperidin-1-yl-acetamide
(41d). This compound was prepared by an analogous
procedure as described for the preparation of 20a, start-
ing from 40d. Yield: 95%; mp 192–194 ꢁC (EtOAc/n-
1
hexane); H NMR (CDCl3, 400 MHz) d 1.45 [m, 2H,
CH2CH2(4-piperidine-H)], 1.61 [m, 6H, CH2CH2(3,5-pi-
peridine-H), COCH2(4-piperidine-H)], 1.80 [m, 4H,
COCH2(3,5-piperidine-H)], 2.56 [m, 4H, CH2CH2(2,6-
piperidine-H)], 2.66 [m, 4H, COCH2(2,6-piperidine-H),
2.95 (t,J = 7 Hz, 2H, N–NCH2CH2), 3.17 (s, 2H,
COCH2), 4.50 (t, J = 7 Hz, 2H, N–NCH2CH2), 6.89
(d, J = 9 Hz, 1H, H-3), 7.36–7.48 (m, 2H, H-9, H-10),
7.54 (s, 1H, H-7), 7.74 (d, J = 8 Hz, 1H, H-8), 7.80 (d,
J = 8 Hz, 1H, H-11), 8.12 (d, J = 9 Hz, 1H, H-4), 8.32
(s, 1H, H-12), 9.53 (s, 1H, D2O exch, NH); 13C NMR
4.1.59. 2-Diethylamino-N-[2-(2-diethylaminoethyl)-2H-
benzo[g]benzopyrano[4,3,2-cd]indazol-5-yl]acetamide
(41b). This compound was prepared by an analo-
gous procedure as described for the preparation of
20a, starting from 40b. Yield: 95%; mp 129–131 ꢁC
(EtOAc/n-hexane); 1H NMR (CDCl3, 400 MHz) d
1.01 (t, J = 7 Hz, 6H, CH2CH2N(CH2CH3)2), 1.24
(t, J = 7 Hz, 6H, COCH2N(CH2CH3)2), 2.59 (q,
J = 7 Hz, 4H, CH2CH2N(CH2CH3)2), 2.76 (q, J =

2930
I. K. Kostakis et al. / Bioorg. Med. Chem. 14 (2006) 2910–2934
(CDCl3, 50 MHz) d 24.14 [CH2CH2(4-piperidine-C)],
25.65 [CH2CH2(3,5-piperidine-C), COCH2(4-piperi-
dine-C)], 26.86 [COCH2(3,5-piperidine-C)], 47.44 (N–
NCH2CH2), 54.72 [CH2CH2(3,5-piperidine-C)], 55.23
[COCH2(3,5-piperidine-C)], 58.10 (N–NCH2CH2),
62.95 (COCH2), 102.28 (C-3), 113.45 (C-5), 114.18 (C-
7), 116.17 (C-2b), 118.67 (C-12a), 122.27 (C-12),
125.32 (C-10), 125.87 (C-4), 127.34 (C-9), 127.42 (C-8),
128.30 (C-11), 130.83 (C-11a), 134.21 (C-7a), 137.71
(C-2a), 138.26 (C-5a), 138.77 (C-12b), 152.26 (C-6a),
169.31 (CONH). Anal. Calcd for C31H35N5O2. C:
73.06, H: 6.92, N: 13.74. Found: C: 72.88, H: 6.99, N:
13.96.
400 MHz) d 1.89 (m, 4H, N(CH2CH2)2), 2.79 (m, 4H,
N(CH2CH2)2), 2.96 (t, J = 7 Hz, 2H, NHCH2CH2),
3.49 (q, J = 7 Hz, 5 Hz, 2H, NHCH2CH2), 6.41 (d,
J = 8 Hz, 1H, H-4), 6.54 (d, J = 8 Hz, 1H, H-2), 7.39–
7.47 (m, 2H, H-3, H-8), 7.54 (t, J = 8 Hz, 1H, H-9),
7.70 (s, 1H, H-6), 7.81 (d, J = 8 Hz, 1H, H-7), 7.97 (d,
J = 8 Hz, 1H, H-10), 8.74 (s, 1H, H-11), 9.67 (t,
J = 5 Hz, 1H, D2O exch, NH); 13C NMR (CDCl3,
50 MHz) d 29.86 (N(CH2CH2)2), 41.71 (NHCH2CH2),
54.36 (N(CH2CH2)2), 54.54 (NHCH2CH2), 102.40 (C-
2), 103.58 (C-4), 106.22 (C-12a), 112.51 (C-6), 121.74
(C-11a), 125.08 (C-9), 126.88 (C-7), 127.43 (C-11),
128.61 (C-8), 128.81 (C-10a), 129.56 (C-10), 136.55 (C-
3), 136.44 (C-6a), 151.65 (C-1), 151.80 (C-5a), 158.31
(C-4a), 180.18 (C-12). Anal. Calcd for C23H22N2O2ÆH-
ClÆ1/2H2O: C, 68.39; H, 5.99; N, 6.94. Found: C,
52.37; H, 4.97; N, 5.46.
4.1.62. N,N-Dimethyl-N0-(12-oxo-12H-benzo[b]xanthen-
1-yl)ethane-1,2-diamine (42a). This compound was pre-
pared by an analogous procedure as described for the
preparation of 14a, starting from 33. Yield: 88%; mp
(hydrochloride) 259 ꢁC (dec), (EtOH); 1H NMR (CDCl3,
400 MHz) d 2.38 (s, 6H, 2· CH3), 2.71 (t, J = 7 Hz, 2H,
NHCH2CH2), 3.40 (q, J = 7 Hz, 5 Hz, 2H, NHCH2CH2),
6.43 (d, J = 8 Hz, 1H, H-4), 6.58 (d, J = 8 Hz, 1H, H-2),
7.42–7.51 (m, 2H, H-3, H-8), 7.56 (t, J = 8 Hz, 1H, H-
9), 7.76 (s, 1H, H-6), 7.87 (d, J = 8 Hz, 1H, H-7), 8.02
(d, J = 8 Hz, 1H, H-10), 8.84 (s, 1H, H-11), 9.72 (t,
J = 5 Hz, 1H, D2O exch, NH); 13C NMR (CDCl3,
50 MHz) d 40.87 (NHCH2CH2), 45.46 (2· CH3), 57.82
(NHCH2CH2), 102.29 (C-2), 103.61 (C-4), 106.11 (C-
12a), 112.55 (C-6), 121.68 (C-11a), 126.03 (C-9), 127.12
(C-7), 128.33 (C-11), 128.99 (C-8), 129.05 (C-10a),
129.64 (C-10), 136.59 (C-3), 136.85 (C-6a), 151.52 (C-
5a), 151.66 (C-1), 158.31 (C-4a), 180.18 (C-12). Anal.
Calcd for C21H20N2O2ÆHClÆ2H2O: C, 62.29; H, 6.22; N,
6.92. Found: C, 52.23; H, 6.63; N, 8.92.
4.1.65.
1-[2-(Piperidin-1-yl)ethylamino]-12H-benzo[b]-
xanthene-12-one (42d). This compound was prepared
by an analogous procedure as described for the prepara-
tion of 14a, starting from 33. Yield: 91%; mp (hydro-
chloride) 248–250 ꢁC (EtOH); 1H NMR (CDCl3,
400 MHz) d 1.48 (m, 2H, 4-piperidine-H), 1.66 (m, 4H,
3,5-piperidine-H), 2.55 (m, 4H, 2,6-piperidine-H), 2.75
(t, J = 6 Hz, 2H, NHCH2CH2), 3.43 (q, J = 7 Hz,
5 Hz, 2H, NHCH2CH2), 6.40 (d, J = 8 Hz, 1H, H-4),
6.54 (d, J = 8 Hz, 1H, H-2), 7.37–7.48 (m, 2H, H-3, H-
8), 7.55 (t, J = 8 Hz, 1H, H-9), 7.72 (s, 1H, H-6), 7.81
(d, J = 8 Hz, 1H, H-7), 7.99 (d, J = 8 Hz, 1H, H-10),
8.78 (s, 1H, H-11), 9.65 (t, J = 5 Hz, 1H, D2O exch,
NH). 13C NMR (CDCl3, 50 MHz) d 24.22 (4-piperi-
dine-C), 25.77 (3,5-piperidine-C), 40.39 (NHCH2CH2),
54.65 (2,6-piperidine-C), 57.37 (NHCH2CH2), 102.14
(C-2), 103.68 (C-4), 106.31 (C-12a), 112.50 (C-6),
122.03 (C-11a), 125.07 (C-9), 126.87 (C-7), 127.50 (C-
11), 128.56 (C-8), 128.94 (C-10a), 129.60 (C-10),
136.47 (C-3), 136.66 (C-6a), 151.84 (C-1), 151.90 (C-
5a), 158.37 (C-4a), 181.02 (C-12). Anal. Calcd for
C24H24N2O2ÆHClÆH2O: C, 67.52; H, 6.37; N, 6.56.
Found: C, 67.33; H, 6.71; N, 6.51.
4.1.63. N,N-Diethyl-N0-(12-oxo-12H-benzo[b]xanthen-1-
yl)ethane-1,2-diamine (42b). This compound was pre-
pared by an analogous procedure as described for the
preparation of 14a, starting from 33. Yield: 90%; mp
1
(hydrochloride) 251–253 ꢁC (EtOH); H NMR (CDCl3,
400 MHz) d 1.13 (t, J = 7 Hz, 6H, (CH2CH3)2), 2.67 (q,
J = 7 Hz, 4H, (CH2CH3)2), 2.83 (t, J = 7 Hz, 2H,
NHCH2CH2), 3.35 (q, J = 7 Hz, 5 Hz, 2H, NHCH2CH2),
6.39 (d, J = 8 Hz, 1H, H-4), 6.52 (d, J = 8 Hz, 1H, H-2),
7.39–7.45 (m, 2H, H-3, H-8), 7.53 (t, J = 8 Hz, 1H, H-
9), 7.68 (s, 1H, H-6), 7.81 (d, J = 8 Hz, 1H, H-7), 7.98
(d, J = 8 Hz, 1H, H-10), 8.77 (s, 1H, H-11), 9.64 (t,
J = 5 Hz, 1H, D2O exch, NH); 13C NMR (CDCl3,
50 MHz) d 12.05 [(CH2CH3)2], 41.14 (NHCH2CH2),
47.12 [(CH2CH3)2], 51.14 (NHCH2CH2), 102.34 (C-2),
103.55 (C-4), 106.10 (C-12a), 112.68 (C-6), 122.01 (C-
11a), 125.80 (C-9), 127.10 (C-7), 127.90 (C-11), 129.04
(C-8), 129.32 (C-10a), 129.60 (C-10), 136.67 (C-3),
136.91 (C-6a), 151.60 (C-1), 151.88 (C-5a), 157.73 (C-
4a), 180.34 (C-12). Anal. Calcd for C23H24N2O2ÆHClÆ
1/2H2O: C, 68.05; H, 6.46; N, 6.90. Found: C, 68.27; H,
6.32; N, 7.07.
4.1.66. Dimethyl-[2-[2H-benzo[g]benzopyrano[4,3,2-cd]-
indazol-2-yl]ethyl]amine (43a). This compound was pre-
pared by an analogous procedure as described for the
preparation of 24a, starting from 35. Yield: 95%; mp
(hydrochloride) 269–271 ꢁC (EtOH); 1H NMR
(CDCl3, 400 MHz) d 2.23 [s, 6H, N(CH3)2], 2.90 (t,
J = 7 Hz, 2H, N–NCH2CH2), 4.48 (t, J = 7 Hz, 2H,
N–NCH2CH2), 6.57 (d, J = 8 Hz, 1H, H-5), 6.88 (d,
J = 8 Hz, 1H, H-3), 7.30 (t, J = 8 Hz, 1H, H-4), 7.39
(t, J = 8 Hz, 1H, H-9), 7.44 (t, J = 8 Hz, 1H, H-10),
7.63 (s, 1H, H-7), 7.73 (d, J = 8 Hz, 1H, H-8), 7.82
(d, J = 8 Hz, 1H, H-11), 8.37 (s, 1H, H-12); 13C
NMR (CDCl3, 50 MHz) d 45.78 (2· CH3), 48.13
(N–NCH2CH2), 58.75 (N–NCH2CH2), 101.02 (C-5),
102.05 (C-3), 114.14 (C-7), 116.09 (C-2b), 118.62 (C-
12a), 122.00 (C-12), 125.46 (C-10), 127.04 (C-9),
127.30 (C-8), 128.11 (C-11), 130.31 (C-4), 130.49 (C-
11a), 134.24 (C-7a), 137.92 (C-12b), 141.01 (C-2a),
149.72 (C-5a), 152.36 (C-6a). Anal. Calcd for
C21H19N3OÆHClÆ3/4H2O: C, 66.48; H, 5.71; N, 11.08.
Found: C, 66.09; H, 5.48; N, 11.32.
4.1.64. 1-[2-(Pyrrolidin-1-yl)ethylamino]-12H-benzo[b]-
xanthene-12-one (42c). This compound was prepared
by an analogous procedure as described for the prepara-
tion of 14a, starting from 33. Yield: 88%; mp (hydro-
chloride) 242–244 ꢁC (EtOH). 1H NMR (CDCl3,

I. K. Kostakis et al. / Bioorg. Med. Chem. 14 (2006) 2910–2934
2931
4.1.67. Diethyl-[2-[2H-benzo[g]benzopyrano[4,3,2-cd]ind-
azol-2-yl]ethyl]amine (43b). This compound was pre-
pared by an analogous procedure as described for the
preparation of 24a, starting from 35. Yield: 96%; mp
12), 125.83 (C-10), 126.93 (C-9), 127.19 (C-8), 128.00
(C-11), 130.09 (C-4), 130.38 (C-11a), 134.13 (C-7a),
137.73 (C-12b), 140.93 (C-2a), 149.53 (C-5a), 152.25 (C-
6a). Anal. Calcd for C24H23N3OÆHClÆ4H2O: C, 60.31;
H, 6.75; N, 8.79. Found: C, 60.97; H, 5.57; N, 8.78.
1
(hydrochloride) 258–260 ꢁC (EtOH). H NMR (CDCl3,
400 MHz) d 1.02 (t, J = 7 Hz, 6H, (CH2CH3)2), 2.60 (q,
J = 7 Hz, 4H, (CH2CH3)2), 3.00 (t, J = 7 Hz, 2H, N–
NCH2CH2), 4.44 (t, J = 7 Hz, 2H, N–NCH2CH2), 6.57
(d, J = 8 Hz, 1H, H-5), 6.88 (d, J = 8 Hz, 1H, H-3),
7.29 (t, J = 8 Hz, 1H, H-4), 7.39 (t, J = 8 Hz, 1H, H-
9), 7.44 (t, J = 8 Hz, 1H, H-10), 7.63 (s, 1H, H-7), 7.73
(d, J = 8 Hz, 1H, H-8), 7.82 (d, J = 8 Hz, 1H, H-11),
8.37 (s, 1H, H-12); 13C NMR (CDCl3, 50 MHz) d
11.94 [(CH2CH3)2], 47.56 [(CH2CH3)2], 48.44 (N–
NCH2CH2), 52.59 (N–NCH2CH2), 100.78 (C-5),
102.14 (C-3), 114.01 (C-7), 115.89 (C-2b), 118.57 (C-
12a), 121.81 (C-12), 125.37 (C-10), 126.92 (C-9),
127.17 (C-8), 127.98 (C-11), 130.04 (C-4), 130.37 (C-
11a), 134.12 (C-7a), 137.69 (C-12b), 140.96 (C-2a),
149.56 (C-5a), 152.28 (C-6a). Anal. Calcd for
C23H23N3OÆHClÆ2H2O: C, 64.25; H, 6.56; N, 9.77.
Found: C, 64.75; H, 5.90; N, 9.70.
4.1.70.
N,N-Dimethyl-N0-(9-oxo-9H-xanthen-1-yl)eth-
ane-1,2-diamine (44a). This compound was prepared
by an analogous procedure as described for the prepa-
ration of 14a, starting from the corresponding tosyl-
ate.49–51 Yield: 80%; mp (hydrochloride) >270 C
1
(EtOH); H NMR (CDCl3, 400 MHz) d 2.33 (s, 6H, 2·
CH3), 2.66 (t, J = 7 Hz, 2H, NHCH2CH2), 3.33 (q,
J = 7 Hz, 5 Hz, 2H, NHCH2CH2), 6.38 (d, J = 8 Hz,
1H, H-4), 6.54 (d, J = 8 Hz, 1H, H-2), 7.28 (t, J = 8 Hz,
1H, H-7), 7.33 (d, J = 8 Hz, 1H, H-5), 7.42 (t, J = 8 Hz,
1H, H-3), 7.61 (t, J = 8 Hz, 1H, H-6), 8.22 (d, J = 8 Hz,
1H, H-8), 9.57 (t, J = 5 Hz, 1H, D2O exch, NH); 13C
NMR (CDCl3, 50 MHz) d 40.98 (NHCH2CH2), 45.54
(2· CH3), 57.85 (NHCH2CH2), 102.07 (C-2), 103.47
(C-4), 106.81 (C-9a), 117.10 (C-5), 122.07 (C-8a),
123.31 (C-7), 126.03 (C-8), 133.98 (C-6), 135.92 (C-3),
151.62 (C-1), 155.26 (C-10a), 158.01 (C-4a), 179.74 (C-
9). Anal. Calcd for C17H18N2O2ÆHClÆH2O: C, 60.62; H,
6.28; N, 8.32. Found: C, 60.44; H, 6.52; N, 8.51.
4.1.68. 2-[2-(Pyrrolidin-1-yl)ethyl]-2H-benzo[g]benzopyr-
ano[4,3,2-cd]indazole (43c). This compound was pre-
pared by an analogous procedure as described for the
preparation of 24a, starting from 35. Yield: 95%; mp
(hydrochloride) >270 ꢁC (EtOH). 1H NMR (CDCl3,
400 MHz) d 1.82 [m, 4H, N(CH2CH2)2], 2.66 [m, 4H,
N(CH2CH2)2], 3.12 (t, J = 7 Hz, 2H, N–NCH2CH2),
4.56 (t, J = 7 Hz, 2H, N–NCH2CH2), 6.60 (d,
J = 8 Hz, 1H, H-5), 6.92 (d, J = 8 Hz, 1H, H-3), 7.31
(t, J = 8 Hz, 1H, H-4), 7.41 (t, J = 8 Hz, 1H, H-9),
7.46 (t, J = 8 Hz, 1H, H-10), 7.65 (s, 1H, H-7), 7.75 (d,
J = 8 Hz, 1H, H-8), 7.85 (d, J = 8 Hz, 1H, H-11), 8.39
(s, 1H, H-12); 13C NMR (CDCl3, 50 MHz) d 23.48
4.1.71. N,N-Diethyl-N0-(9-oxo-9H-xanthen-1-yl)ethane-
1,2-diamine (44b). This compound was prepared by an
analogous procedure as described for the preparation
of 14a, starting from the corresponding tosylate. Yield:
81%; mp (hydrochloride) 201–203 ꢁC (EtOH); 1H
NMR (CDCl3, 400 MHz) d 1.10 (t, J = 7 Hz, 6H,
(CH2CH3)2), 2.67 (q, J = 7 Hz, 4H, (CH2CH3)2), 2.82
(t, J = 7 Hz, 2H, NHCH2CH2), 3.36 (q, J = 7 Hz,
5 Hz, 2H, NHCH2CH2), 6.39 (d, J = 8 Hz, 1H, H-4),
6.52 (d, J = 8 Hz, 1H, H-2), 7.27 (t, J = 8 Hz, 1H, H-
7), 7.32 (d, J = 8 Hz, 1H, H-5), 7.40 (t, J = 8 Hz, 1H,
H-3), 7.59 (t, J = 8 Hz, 1H, H-6), 8.20 (d, J = 8 Hz,
1H, H-8), 9.53 (t, J = 5 Hz, 1H, D2O exch, NH); 13C
NMR (CDCl3, 50 MHz) d 11.35 [(CH2CH3)2], 40.87
[N(CH2CH2)2],
48.95
(N–NCH2CH2),
54.36
[N(CH2CH2)2], 55.46 (N–NCH2CH2), 100.93 (C-5),
102.03 (C-3), 114.05 (C-7), 115.93 (C-2b), 118.46 (C-
12a), 121.88 (C-12), 125.37 (C-10), 126.95 (C-9),
127.17 (C-8), 127.98 (C-11), 130.23 (C-4), 130.37 (C-
11a), 134.12 (C-7a), 137.83 (C-12b), 140.89 (C-2a),
149.56 (C-5a), 152.24 (C-6a). Anal. Calcd for
C23H21N3OÆHClÆ2H2O: C, 64.55; H, 6.12; N, 9.82.
Found: C, 64.81; H, 6.34; N, 9.53.
(NHCH2CH2),
47.08
[(CH2CH3)2],
51.09
(NHCH2CH2), 102.11 (C-2), 103.50 (C-4), 106.77 (C-
9a), 117.03 (C-5), 122.06 (C-8a), 123.24 (C-7), 125.92
(C-8), 133.90 (C-6), 135.85 (C-3), 151.51 (C-1), 155.18
(C-10a), 157.94 (C-4a), 179.59 (C-9). Anal. Calcd for
C19H22N2O2ÆHClÆH2O: C, 62.54; H, 6.91; N, 7.68.
Found: C, 62.33; H, 6.68; N, 7.43.
4.1.69. 2-[2-(Piperidin-1-yl)ethyl]-2H-benzo[g]benzopyr-
ano[4,3,2-cd]indazole (43d). This compound was pre-
pared by an analogous procedure as described for the
preparation of 24a, starting from 35. Yield: 97%; mp
(hydrochloride) >270 ꢁC (EtOH). 1H NMR (CDCl3,
400 MHz) d 1.45 (m, 2H, 4-piperidine-H), 1.60 (m, 4H,
3,5-piperidine-H), 2.53 (m, 4H, 2,6-piperidine-H), 2.92
(t, J = 7 Hz, 2H, N–NCH2CH2), 4.51 (t, J = 7 Hz, 2H,
N–NCH2CH2), 6.56 (d, J = 8 Hz, 1H, H-5), 6.89 (d,
J = 8 Hz, 1H, H-3), 7.29 (t, J = 8 Hz, 1H, H-4), 7.39
(t, J = 8 Hz, 1H, H-9), 7.42 (t, J = 8 Hz, 1H, H-10),
7.62 (s, 1H, H-7), 7.72 (d, J = 8 Hz, 1H, H-8), 7.83 (d,
J = 8 Hz, 1H, H-11), 8.35 (s, 1H, H-12); 13C NMR
(CDCl3, 50 MHz) d 24.13 (4-piperidine-C), 25.89 (3,5-pi-
peridine-C), 47.58 (N–NCH2CH2), 54.82 (2,6-piperidine-
C), 58.31 (N–NCH2CH2), 100.87 (C-5), 102.19 (C-3),
114.03 (C-7), 115.94 (C-2b), 118.51 (C-12a), 121.82 (C-
4.1.72. 1-[2-(Pyrrolidin-1-yl)ethylamino]-9H-xanthene-9-
one (44c). This compound was prepared by an analo-
gous procedure as described for the preparation of
14a, starting from the corresponding tosylate. Yield:
80%; mp (hydrochloride) 256–258 ꢁC (EtOH); 1H
NMR (CDCl3, 400 MHz) d 1.88 (m, 4H, N(CH2CH2)2),
2.77 (m, 4H, N(CH2CH2)2), 2.94 (t, J = 7 Hz, 2H,
NHCH2CH2), 3.52 (q, J = 7 Hz, 5 Hz, 2H,
NHCH2CH2), 6.46 (d, J = 8 Hz, 1H, H-4), 6.56 (d,
J = 8 Hz, 1H, H-2), 7.30 (t, J = 8 Hz, 1H, H-7), 7.36
(d, J = 8 Hz, 1H, H-5), 7.44 (t, J = 8 Hz, 1H, H-3),
7.62 (t, J = 8 Hz, 1H, H-6), 8.21 (d, J = 8 Hz, 1H,
H-8), 9.58 (t, J = 5 Hz, 1H, D2O exch, NH);
13C NMR (CDCl3, 50 MHz) d 23.44 (N(CH2CH2)2),
41.64 (NHCH2CH2), 54.32 (N(CH2CH2)2), 54.51

2932
I. K. Kostakis et al. / Bioorg. Med. Chem. 14 (2006) 2910–2934
(NHCH2CH2), 102.47 (C-2), 103.61 (C-4), 106.92 (C-9a),
117.21 (C-5), 121.99 (C-8a), 123.43 (C-7), 126.00 (C-8),
134.12 (C-6), 136.11 (C-3), 151.47 (C-1), 155.33 (C-10a),
158.05 (C-4a), 179.92 (C-9). Anal. Calcd for
C19H20N2O2ÆHClÆ1/2H2O: C, 64.49; H, 6.27; N, 7.92.
Found: C, 64.68; H, 6.45; N, 8.04.
122.98 (C-10), 124.00 (C-9), 129.70 (C-4), 130.11 (C-8),
137.94 (C-10b), 141.01 (C-2a), 149.96 (C-5a), 154.70 (C-
6a). Anal. Calcd for C19H21N3OÆHClÆ2H2O: C, 60.07;
H, 6.90; N, 11.06. Found: C, 60.35; H, 6.78; N, 11.31.
4.1.76.
2-[2-(Pyrrolidin-1-yl)ethyl]-2H-benzopyrano-
[4,3,2-cd]indazole (45c). This compound was prepared
by an analogous procedure as described for the prepara-
tion of 24a, starting from the corresponding mesylate.
Yield: 93%; mp (hydrochloride) 234–236 ꢁC (EtOH);
4.1.73. 1-[2-(Piperidin-1-yl)ethylamino]-9H-xanthene-9-
one (44d). This compound was prepared by an analogous
procedure as described for the preparation of 14a, starting
from the corresponding tosylate. Yield: 83%; mp (hydro-
chloride) 241 ꢁC (dec) C (EtOH); 1H NMR (CDCl3,
400 MHz) d 1.47 (m, 2H, 4-piperidine-H), 1.66 (m, 4H,
3,5-piperidine-H), 2.54 (m, 4H, 2,6-piperidine-H), 2.73
(t, J = 7 Hz, 2H, NHCH2CH2), 3.41 (q, J = 7 Hz, 5 Hz,
2H, NHCH2CH2), 6.41 (d, J = 8 Hz, 1H, H-4), 6.54 (d,
J = 8 Hz, 1H, H-2), 7.28 (t, J = 8 Hz, 1H, H-7), 7.34 (d,
J = 8 Hz, 1H, H-5), 7.41 (t, J = 8 Hz, 1H, H-3), 7.61 (t,
J = 8 Hz, 1H, H-6), 8.21 (d, J = 8 Hz, 1H, H-8), 9.54 (t,
J = 5 Hz, 1H, D2O exch, NH); 13C NMR (CDCl3,
50 MHz) d 24.11 (4-piperidine-C), 25.65 (3,5-piperidine-
C), 40.32 (NHCH2CH2), 54.58 (2,6-piperidine-C), 57.22
(NHCH2CH2), 102.14 (C-2), 103.58 (C-4), 106.85 (C-
9a), 117.10 (C-5), 122.03 (C-8a), 123.32 (C-7), 126.00
(C-8), 133.97 (C-6), 135.92 (C-3), 151.54 (C-1), 155.26
(C-10a), 158.01 (C-4a), 179.70 (C-9). Anal. Calcd for
C20H22N2O2ÆHClÆ3/4H2O: C, 64.51; H, 6.63; N, 7.52.
Found: C, 64.87; H, 6.59; N, 7.86.
1H NMR (CDCl3, 400 MHz)
d
1.78 [m, 4H,
N(CH2CH2)2], 2.60 [m, 4H, N(CH2CH2)2], 3.03 (t,
J = 7 Hz, 2H, N–NCH2CH2), 4.47 (t, J = 7 Hz, 2H,
N–NCH2CH2), 6.49 (d, J = 8 Hz, 1H, H-5), 6.83 (d,
J = 8 Hz, 1H, H-3), 7.17 (t, J = 8 Hz, 1H, H-9), 7.23–
7.35 (m, 3H, H-4, H-7, H-8), 7.89 (d, J = 8 Hz, 1H, H-
10); 13C NMR (CDCl3, 50 MHz) d 23.49 [N(CH2CH2)2],
49.03 (N–NCH2CH2), 54.36 [N(CH2CH2)2], 55.43 (N–
NCH2CH2), 100.23 (C-5), 101.66 (C-3), 116.69 (C-2b),
118.24 (C-7), 118.46 (C-10a), 123.02 (C-10), 124.01 (C-
9), 129.78 (C-4), 130.22 (C-8), 138.05 (C-10b), 140.88
(C-2a), 149.96 (C-5a), 154.71 (C-6a). Anal. Calcd for
C19H19N3OÆHClÆ2H2O: C, 60.39; H, 6.40; N, 11.12.
Found: C, 60.21; H, 6.73; N, 11.26.
4.1.77. 2-[2-(Piperidin-1-yl)ethyl]-2H-benzopyrano[4,3,2-
cd]indazole (45d). This compound was prepared by an
analogous procedure as described for the preparation
of 24a, starting from the corresponding mesylate. Yield:
94%; mp (hydrochloride) >270 ꢁC (EtOH); 1H NMR
(CDCl3, 400 MHz) d 1.43 (m, 2H, 4-piperidine-H),
1.58 (m, 4H, 3,5-piperidine-H), 2.49 (m, 4H, 2,6-piperi-
dine-H), 2.85 (t, J = 7 Hz, 2H, N–NCH2CH2), 4.45 (t,
J = 7 Hz, 2H, N–NCH2CH2), 6.49 (d, J = 8 Hz, 1H,
H-5), 6.83 (d, J = 8 Hz, 1H, H-3), 7.17 (t, J = 8 Hz,
1H, H-9), 7.57–7.35 (m, 3H, H-4, H-7, H-8), 7.89 (d,
J = 8 Hz, 1H, H-10); 13C NMR (CDCl3, 50 MHz) d
24.18 (4-piperidine-C), 25.94 (3,5-piperidine-C), 47.52
(N–NCH2CH2), 54.80 (2,6-piperidine-C), 58.29 (N–
NCH2CH2), 100.16 (C-5), 101.85 (C-3), 116.70 (C-2b),
118.21 (C-7), 118.46 (C-10a), 122.98 (C-10), 124.01 (C-
9), 129.75 (C-4), 130.11 (C-8), 137.94 (C-10b), 140.96
(C-2a), 149.93 (C-5a), 154.67 (C-6a). Anal. Calcd for
C20H21N3OÆHClÆH2O: C, 64.25; H, 6.47; N, 11.24.
Found: C, 64.33; H, 6.32; N, 11.00.
4.1.74. Dimethyl-[2-[2H-benzopyrano[4,3,2-cd]indazol-2-
yl]ethyl]amine (45a). This compound was prepared by an
analogous procedure as described for the preparation of
24a, starting from the corresponding mesylate. Yield:
91%; mp (hydrochloride) 233–235 ꢁC (EtOH); 1H NMR
(CDCl3, 400 MHz) d 2.77 [s, 6H, N(CH3)2], 3.61 (t,
J = 7 Hz, 2H, N–NCH2CH2), 4.81 (t, J = 7 Hz, 2H, N–
NCH2CH2), 6.53 (d, J = 8 Hz, 1H, H-5), 7.03 (d,
J = 8 Hz, 1H, H-3), 7.17 (t, J = 8 Hz, 1H, H-9), 7.25 (d,
J = 8 Hz, 1H, H-7), 7.29–7.37 (m, 2H, H-4, H-8), 7.83
(d, J = 8 Hz, 1H, H-10); 13C NMR (CDCl3, 50 MHz) d
39.15 (2· CH3), 44.84 (N–NCH2CH2), 56.53 (N–
NCH2CH2), 101.22 (C-5), 101.88 (C-3), 116.55 (C-2b),
117.72 (C-10a), 118.38 (C-7), 123.05 (C-10), 124.12 (C-
9), 130.33 (C-4), 131.17 (C-8), 139.30 (C-10b), 140.99
(C-2a), 149.77 (C-5a), 154.70 (C-6a). Anal. Calcd for
C17H17N3OÆHClÆH2O. C: 61.17, H: 6.04, N: 12.59. Found
(%): C: 60.92, H: 6.13, N: 12.74.
4.2. Biology
4.1.75. Diethyl-[2-[2H-benzopyrano[4,3,2-cd]indazol-2-
yl]ethyl]amine (45b). This compound was prepared by
an analogous procedure as described for the prepara-
tion of 24a, starting from the corresponding mesylate.
Yield: 96%; mp (hydrochloride) 257–259 ꢁC (EtOH); 1H
NMR (CDCl3, 400 MHz) d 1.02 (t, J = 7 Hz, 6H,
(CH2CH3)2), 2.60 (q, J = 7 Hz, 4H, (CH2CH3)2), 2.96
(t, J = 7 Hz, 2H, N–NCH2CH2), 4.39 (t, J = 7 Hz, 2H,
N–NCH2CH2), 6.48 (d, J = 8 Hz, 1H, H-5), 6.82 (d,
J = 8 Hz, 1H, H-3), 7.17 (t, J = 8 Hz, 1H, H-9), 7.25 (d,
J = 8 Hz, 1H, H-7), 7.27–7.31 (m, 2H, H-4, H-8), 7.90
(d, J = 8 Hz, 1H, H-10); 13C NMR (CDCl3, 50 MHz) d
12.06 [(CH2CH3)2], 47.56 [(CH2CH3)2], 48.37 (N–
NCH2CH2), 52.52 (N–NCH2CH2), 100.08 (C-5), 101.81
(C-3), 116.69 (C-2b), 118.24 (C-7), 118.53 (C-10a),
4.2.1. Cell culture and assessment of cytotoxicity. The
new compounds were tested for their cytotoxic activity
on the murine leukemia cell line L1210 (American
Type Culture Collection, Rockville, MD), as well as
on the following human solid tumor cell lines: colorec-
tal adenocarcinoma HT-29, uterine sarcoma MES-SA,
and its 100-fold resistant to doxorubicin46 subline
MES-SA/D · 5 (European Collection of Cell Cultures,
Salisbury, U.K.). L1210 cells were cultured in RPMI
1640 medium (Gibco BRL, Paisley, U.K.) supplement-
ed with penicillin (100 U/mL), streptomycin (100 lg/
mL), and 10% fetal bovine serum (media and antibiot-
ics from Biochrom KG, Berlin, Germany) in an envi-
ronment of 5% CO2, 85% humidity, and 37 ꢁC. HT-
29 cells were cultured in Dulbeccoꢁs minimal essential

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above), and routinely subcultured using a trypsin
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4.2.2. DNA binding assay. An ethidium bromide displace-
ment assay was used to determine intercalation poten-
cy.42,52 The test compounds were added to a 5 mM
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The present study was supported in part from the Spe-
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