Synthesis and in vitro cytotoxicity evaluation of novel naphthindolizinedione derivatives

Article abstract of DOI:10.1002/ardp.200600160

Novel 6,11-dioxo-6,11-dihydro-benzo[f]pyrido[1,2-a]indole-12-carboxamide derivatives and the corresponding 7,10-dihydroxy analogues were designed in accordance with Moore's and Pindur's theory and synthesized based on the structural similarity with known antitumour agents such as ellipticine, daunorubicin, mitoxantrone and 9-aminoacridine-4 carboxamide derivatives. These compounds, including structural variations of the amide side chain, were evaluated in the NCI panel of human tumour cell lines, from which 6,11-dioxo-6,11-dihydro-benzo[f]pyrido[1,2-a]indole-(2-dimethylamino-ethyl) -12-carboxamide 11a was found to be the most potent agent within the series. It showed good selectivity towards leukaemia, colon and renal cancer cell lines, with significant GI₅₀ values, from lower than 10 nM to 0.2 μM. Moreover, its cytotoxicity against the adriamicine-resistant breast tumour cell line at a concentration lower than 1 μM turned out to be higher than the values using the clinical anticancer agents, daunorubicin and mitoxantrone.

Full text of DOI:10.1002/ardp.200600160

Arch. Pharm. Chem. Life Sci. 2007, 340, 147–153
A. Defant et al.
147
Full Paper
Synthesis and In Vitro Cytotoxicity Evaluation of Novel
Naphthindolizinedione Derivatives
Andrea Defant, Graziano Guella and Ines Mancini
Laboratorio di Chimica Bioorganica, Dipartimento di Fisica, Universitꢀ degli studi di Trento, Trento, Italy
Novel 6,11-dioxo-6,11-dihydro-benzo[f]pyrido[1,2-a]indole-12-carboxamide derivatives and the
corresponding 7,10-dihydroxy analogues were designed in accordance with Moore's and Pindur's
theory and synthesized based on the structural similarity with known antitumour agents such
as ellipticine, daunorubicin, mitoxantrone and 9-aminoacridine-4 carboxamide derivatives.
These compounds, including structural variations of the amide side chain, were evaluated in
the NCI panel of human tumour cell lines, from which 6,11-dioxo-6,11-dihydro-benzo[f]py-
rido[1,2-a]indole-(2-dimethylamino-ethyl)-12-carboxamide 11a was found to be the most potent
agent within the series. It showed good selectivity towards leukaemia, colon and renal cancer
cell lines, with significant GI50 values, from lower than 10 nM to 0.2 lM. Moreover, its cytotoxi-
city against the adriamicine-resistant breast tumour cell line at a concentration lower than
1
lM turned out to be higher than the values using the clinical anticancer agents, daunorubicin
and mitoxantrone.
Keywords: Antitumor activity / Cytotoxic activity / DNA-topoisomerases / Polycyclic amines / Synthesis /
Received: September 29, 2006; accepted: December 19, 2006
DOI 10.1002/ardp.200600160
Introduction
vivo, is still partly unknown. In accordance with Moore's
and Pindur's theory [5, 6], the structural properties
Current cancer drug research ideally seeks to define can- required to maximise the activity of DNA intercalators
didate compounds that interact with molecular targets include a planar tricyclic or tetracyclic ring system with
that are unique to tumour cells, forming the basis for a length of 3–4 ꢀ, a width of 6–8 ꢀ and a surface area of
2
current cancer drugs with fewer side effects. Much of the 28 ꢀ , and a p-conjugated quinone moiety. The redox
success in cancer chemotherapy has involved the use of activity of quinones has also been found to play a role in
drugs that damage the DNA of tumour cells, simulta- the radical cleavage of DNA, as shown in significant
neously showing the highest non-toxicity for the normal examples including doxorubicin, daunorubicin 2 and
cells. In this context, molecules of natural origin such as mitoxantrone 3 [6, 7]. The presence of two hydroxyl
ellipticine 1 [1] and daunorubicin 2 [2] or of synthetic groups in para positions on the aromatic nucleus (as seen
derivation, such as mitoxantrone 3 [3] and N-[2-(dimethy- in mitoxantrone) substantially increases the activity
lamino)ethyl]acridine-4-carboxamide (termed DACA) ser- against both leukaemia and some solid tumours in mice
ies, including the 9-amino-5-methyl derivative 4 [4] [8]. In addition, the presence of a cationic side chain at a
(
Fig. 1) have been shown to interact with DNA intimately physiological pH, positioned at a fixed distance (about
by intercalation. However, their potent antitumour 8 ꢀ) on a DACA structure (N-[2-(dimethylamino)ethyl]acri-
effect, which is due to interaction with the drug-DNA in dine-4-carboxamide) (as in compound 4), has been
reported to be essential for bioactivity, playing an addi-
tional binding contact with guanosine-cytosine base
Correspondence: Ines Mancini, Laboratorio di Chimica Bioorganica, Di-
partimento di Fisica, Universitꢀ degli studi di Trento, via Sommarive 14, I-
8050 Povo Trento, Italy
E-mail: mancini@science.unitn.it
Fax: +39 0461 882-009
pairs in the DNA [9].
To the best of our knowledge, only one example of a
naphthindolizinedione derivative has been studied for
3
i
2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

148
A. Defant et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 147–153
Reagents and conditions: (i) ethyl acetoacetate, pyridine, 2-propanol reflux, 4 h; 75%
for 7, 54% for 8; (ii) methyl acetoacetate, pyridine, 2-propanol reflux, 4 h; 75% for 9,
5
4% for 10; (iii) amine in excess, 808C, 3 h, 98%.
Scheme 1. Synthesis of naphthindolizinedione derivatives.
Figure 1. Chemical structures of known DNA intercalator mole-
cules: ellipticine 1, daunorubicin 2, mitoxantrone 3 and 9-amino-
5
-methyl DACA derivative 4.
tetracyclic planar system was thought to increase the
potential bioactivity of these molecules.
antitumour activity, the ester 7. It was evaluated both for
topo I and topo II inhibition and for activity as a cytotoxic
agent against A549, col2 and SNU-638 cell lines (IC50
values of 16.3, 15.4 and 0.2 lg/mL, respectively) [10].
Results and discussion
Although selective, the latter data indicated a moderate Chemistry
cytotoxic activity in cultured stomach cancer cells.
Target compounds 11a–c and 12a–c were obtained
This study was aimed at the synthesis and the investi- according to the reactions depicted in Scheme 1. Precur-
gation of the cytotoxicity against a large panel of human sors methyl 6,11-dioxo-6,11-dihydro-benzo[f]pyrido [1,2-
tumour cell lines for a new series of tetracyclic com- a]indole-12-carboxylate 9 and methyl 7,10-dihydroxy-
pounds with structural variations both on the heterocyc- 6,11-dioxo-6,11-dihydro-benzo[f]pyrido[1,2-a]indole 12-
lic moiety and by the presence of an amide chain, or by carboxylate 10 were obtained through a one-pot cyclisa-
hydroxyl groups on the aromatic system. In our prelimin- tion starting from the commercially available 2,3-
ary design based on Moore's and Pindur's theory [5–7], dichloro-1,4-naphtoquinone 5 or 2,3-dichloro-5,8-dihy-
the AM1 semi-empirical calculations carried on the tar- droxy-1,4-naphtoquinone 6, respectively, by refluxing
get compounds 11a–c and 12a–c showed minimised them in ethanol with pyridine and methyl acetoacetate
structures with values of 2.954 and 8.437 ꢀ relating to [11]. Higher yields were obtained by using 2-propanol as
the distances C1–C4 and C3–C8, respectively. In addi- opposed to the better-known procedure starting with the
tion, the presence of an amide side chain in this common corresponding ethyl ester substrate and using the pro-
i
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www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2007, 340, 147–153
Novel Naphthindolizinedione Derivatives
149
Table 1. In vitro cytotoxic activity of the compounds 8, 11a–11c
and 12a–12c in the NCI three cell line / one dose pre-screening.
51 lM [10] for the tested A549 cell line, all the amides
1a, 11b and 12a–12c resulted as being more potent
1
against the same lung tumour cell line, with amide 11a
showing a more than 100-fold better value.
Moreover, a drastic enhancement of cytotoxicity and a
good selectivity within the series of the substituted
amides under investigation were observed by changing
the side chain. Indeed, the GI50 values change from
Compound Growth percentage (at conc. = 100 lM) Activity
Lung NCI-H460 Breast MCF7 CNS SF-268
8
100
0
0
50
–1
31
0
121
0
0
66
–1
40
0
109
0
Inactive
Active
Active
Inactive
Active
Active
Active
1
1
1
1
1
1
1a
1b
1c
2a
2b
2c
44
77
–2
84
82
–4
– 6
F10 M for the amide 11c to F10 M in the case of 11b,
–
8
and become even better for 11a, showing a a10 M value
in the case of leukaemia HL60 (TB) cell line. The differ-
ence in bioactivity of N,N-dimethylaminoethyl as far as
the N,N-diethylaminoethyl chain is concerned, is similar
cess of cyclization in ethanol [11, 12]. The choice of to the result of modified anthracene-9,10-diones, where
methyl esters 9 and 10 as intermediates for the prepara- the structural features of the side chain and the antileu-
tion of compounds 11a–c and 12a–c, respectively, was kaemic activity were correlated. In particular, this study
required by the successive formation of the correspond- exhibited that both the steric variation on the distal
ing amides. In fact, the amide preparation by direct con- nitrogen and the basic characteristics of the amino group
version of methyl ester was demonstrated by us to be a significantly affected the antitumour bioactivity [15]. As
better procedure than the alternative route relying on far as the difference in the substituents on the quinone
amide formation via the acyl chloride prepared from the moiety are concerned, the data for compound 12b are
carboxylic acid, which, in turn, can be obtained by the quite similar to those obtained for 11b, indicating that
hydrolysis of the esters 7 and 8 [13]. Thus, compounds the additional presence of the hydroxyl groups does not
1
1a–c and 12a–c were obtained by a two-step sequence play an important role. Instead, a striking outcome is the
from the commercial naphtoquinones 5 and 6, with an effect of the hydroxyl aromatic groups in 12a in compari-
overall yield of 74% and 53%, respectively. The molecular son with the most active compound 11a where such
composition of the products was supported by HR-EIMS groups are missing. A strong reduction in cytotoxicity
measurements and ESI-MS tandem fragmentation experi- observed for 12a was unpredictable on the base of the
ments, whereas their structures were fully characterised known data for ametantrone and the dihydroxyl analo-
by extensive NMR analysis, including NOESY, 1D- and 2D- gue mitoxantrone, the latter showing higher activity
1
13
one bond and long range H- C hetero-correlated experi- against both leukaemia and some solid tumours [8].
ments (see Experimental, section 4).
Among the series of tetracyclic compounds which
were investigated, 11a turned out to be the most power-
ful cytotoxic molecule, with remarkable values against
Biological activity
Compounds 8, 11a–c and 12a–c were evaluated for their some specific leukaemia, colon and renal cancer cell
in vitro activity against cancer cell lines by the National lines. Moreover, its sub-micromolar cytotoxicity against
Cancer Institute (NCI), following its anticancer drug the breast cell line resistant to adriamicine (NCI/ADR-
development programme based on automated sulphor- RES) was comparable to ellipticine 1 and DACA derivative
hodamine blue (SRB) cytotoxicity assay [14]. Compounds 4, or also higher than the values of the reference com-
8
and 11c showed low growth inhibition (GI50 values pounds daunorubicin 2 and mitoxantrone 3 (Table 2).
–
4
F10 M) in the pre-screened panel including three
highly sensitive human tumour lines (MCF-7 breast, NCI-
H640 lung and SF-268 glioma), as summarised in Table 1.
Therefore, they were not selected for the advanced test-
Conclusions
ing in the full 60 cell lines screening, whereas 11a–b and This study focused on the design and synthesis of novel
2a–c underwent the latter advanced bioassays (Table 2).
naphthindolizinedione derivatives 11a–c and 12a–c, the
In Table 2 their most significant GI50 values are structures of which summarise some peculiarities pre-
1
reported compared with the corresponding data for the sent in antitumour molecules such as ellipticine, daunor-
antitumour agents 1–4. These data indicate a decisive ubicin, mitoxantrone and DACA derivatives. Contrary to
role of the amide chain when compared to the corre- the case of daunorubicin and mitoxantrone, the presence
sponding ethyl esters. To look at this in more detail, of two hydroxyl groups in para positions on the aromatic
whereas the known ester 7 exhibited value of GI50
=
nucleus does not improve the cytotoxicity, whereas
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150
A. Defant et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 147–153
Table 2. Inhibition of in vitro human cancer cell lines by compounds 11a–b and 12a–c, in comparison with the antitumour agents
–4.
1
a)
Type of cancer
Cell line
Cytotoxicity GI50 (lM)
b)
1
1a
11b
4.07
n.d.
n.d.
10.3
12a
12b
12c
1
2
3
4
Leukemia
CCRF-CEM
HL60(TB)
K562
MOLT-4
RPMI-8226
SR
A549/ATCC
HOP-92
NCI-H226
NCI-H322M
NCI-H460
COLO 205
HCT-116
HCT-15
HT29
KM12
SW-620
SF-295
SNB-19
LOX IMVI
SK-MEL-5
IGROV-1
OVCAR-8
ACHN
1.64 l 0.94
a0.0100
2.55
2.30
0.738
0.771
4.97
0.293
0.806
3.62
1.82
0.825
0.361
1.84
n.d.
1.58
2.44
1.48
0.996
1.75
3.52
1.12
2.86
1.97
1.89
0.693
3.53
2.10
2.41
1.95
6.04
2.75
3.75
n.d.
n.d.
2.41
3.12
2.17
5.92
4.46
8.71
7.61
2.86
37.4
n.d.
16.0
0.702
29.8
0.365
0.398
0.794
0.501
0.251
0.631
n.d.
0.0158
0.0158
0.0631
0.00316
0.0199
0.0158
0.125
0.0117
0.0398
0.316
0.00501
0.251
0.0631
1.58
0.0794
0.316
0.0199
n.d.
0.00631
0.0100
0.126
0.00501
0.200
0.00794
0.0126
0.0158
0.0158
0.251
0.00631
0.100
0.0501
0.251
0.251
0.501
0.0501
0.0200
0.0158
0.0200
0.0501
0.200
0.0398
0.0126
0.0100
0.00794
0.316
0.0100
0.0100
0.0158
0.0100
0.0200
0.0100
0.0158
0.0631
0.0501
0.0398
0.0100
0.0316
0.0316
0.0158
0.0251
0.0501
0.0251
0.0316
0.0501
0.0316
0.126
0.0316
0.0631
0.0158
0.0158
0.0158
0.100
0.398
0.050
0.0477
0.992 l 0.01
0.268 l 0.025
0.853 l 0.48
0.318 l 0.11
0.395 l 0.097
1.28 l 0.70
0.412 l 0.37
6.92 l 6.08
3.91 l 0.5
3.03
2.00
4.25
2.08
4.344
4.42
2.16
Non-Small
Cell Lung
48.6
30.4
48.7
43.9
38.0
36.3
18.0
46.3
48.1
28.3
55.8
36.7
>100
36.6
18.5
33.5
66.7
49.3
20.3
57.7
29.8
38.6
34.5
36.3
0.501
0.501
0.158
0.316
0.158
0.501
0.251
0.398
0.794
0.501
0.398
0.794
0.794
0.501
1.00
0.794
0.398
n.d.
1.26
1.00
1.99
0.630
n.d.
Colon
0.31 l 0.21
0.194 l 0.184
0.277 l 0.23
0.146 l 0.094
6.55 l 6.05
1.68 l 1.27
5.61 l 4.79
0.574 l 0.22
0.275 l 0.25
1.39 l 0.77
2.65 l 2.0
0.444 l 0.084
0.169 l 0.159
5.66 l 5.14
0.278 l 0.25
3.51 l 3.10
0.638 l 0.59
10.0
12.5
3.65
3.49
5.47
4.87
4.82
3.95
15.6
3.64
14.3
7.88
4.72
2.25
3.60
2.27
2.90
4.41
6.03
7.05
3.43
7.00
25.5
3.32
12.4
10.6
5.54
3.60
4.71
5.32
14.8
5.56
10.4
8.51
CNS
0.0398
0.0501
n.d.
Melanoma
Ovarian
Renal
0.100
0.0794
0.0251
0.0501
0.0794
0.251
1.25
0.316
0.0443
CAKI-1
SN12C
UO-31
NCI/ADR-RES
11.6
Breast
5.44
3.37
6.446
3.98
0.199
0.0591
MDA-MB-231/ATCC 0.573 l 0.49
MGM # 0.944
0.749
a)
GI50 values, defined as the concentration that inhibits growth by 50% from NCI screening. About the 60 cell lines panel investi-
gated for the compounds 11a–b and 12a–c, only the data for cell lines giving values =10 M are reported. n.d. = not determined,
for compounds 1-4, values accessible by NCI database.
Average values from re-testing of the compound showing a good MGM value (Mean graph medium).
MGM as average GI50 (lM) over all cell lines investigated.
–
6
b)
#
changes in the amide side chain play a decisive role. In pounds and we are grateful to Mr. A. Sterni and Mr. M. Rossi
particular, the N,N-dimethylethylamino chain has been for their technical support with mass and UV spectra, respec-
found to be the most relevant structural modification for tively.
obtaining the highest activity and selectivity towards leu-
kaemia, colon and renal cancer cell lines, with GI50 values
from lower than 10 nM to 0.2 lM. Moreover, the cytotoxi-
city of compound 11a against the sub-panel breast cancer
Experimental
cell line, resistant to adriamicine (NCI/ADR-RES), resulted
in comparable or higher values than those of all the refer-
ence compounds 1–4.
Following these results, further efforts will be
Chemistry
General
All evaporations were carried out at room temperature at
reduced pressure. Yields are given for reacted compounds. All
reagents and solvents were purchased from Sigma Aldrich Eur-
addressed to the study of this most active compound as a
potential DNA intercalator, as well as to investigate the
effect of structural modifications on the naphthindolizi-
nedione skeleton.
ope (Milan, Italy) and used without purification. TLC was carried
on Kieselgel 60 PF254 and RP-18 F254 (Merck, Darmstadt, Germany)
and flash-chromatography (FC) was carried out on Merck silica
gel 60, 20-50 lm; HPLC analysis for compounds 11a–c and 12 a–
c was realized on 2561 cm columns packed with Merck-LiChro-
spher RP-18 (7 lm) under UV monitoring at k254 nm and sol-
vent flux 1 mL/min. M.p.: Reichert Thermovapor microscope (C.
We would like to thank the National Cancer Institute (NCI)
at Bethesda, USA for the antitumour screening tests of our com-
i
2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2007, 340, 147–153
Novel Naphthindolizinedione Derivatives
151
Reichert, Vienna, Austria). UV spectra (kmax in nm) were taken
with a Perkin-Elmer Lambda-3 spectrophotometer, (Perkin
Elmer, Norwalk, CT, USA). NMR spectra were taken with an
Avance 400 Bruker spectrometer (Bruker BioSpin GmbH, Rhein-
2 3 2 3
–OCH CH ), 4.50 (q, 2H, J = 7.20 Hz, OCH CH ), 7.16 (m, 1H, H-3)
7.17 (s, 2H, H-8 and H-9), 7.43 (m, 1H, H-2), 8.26 (br d, 1H, J = 9.1
Hz, H-1), 9.75 (br d, 1H, J = 6.90 Hz, H-4), 12.88 (s, 1H, OH), 13.05
1
3
(s, 1H, OH); C-NMR (100 MHz, CDCl
3 3
) d: 14.25 (q, CH ), 61.60 (t,
1
13
stetten, Germany); H at 400 MHz and C at 100 MHz in CDCl
3
, d
OCH ), 107.24 (s, C-12), 117.40 (d, C-3), 129.28 (two d, C-8 and C-9),
2
values in ppm rel. to SiMe (0 ppm), J values in Hz. Structural
4
121.13 (d, C-1), 128.01 (d, C-2), 128.53 (d, C-4), 128.10 (s, C-5a),
139.98 (s, C-12a), 157.37, 158.46 (two s, C-7 and C-10), 163.29 (s,
1
1
assignments are from H, H-COSY, heteronuclear single quan-
tum correlation (HSQC), Heteronuclear Multiple Bond Correla-
tion (HMBC) and 2D NOESY experiments; multiplicities from
+
COOEt), 178.37 (s, C-6), 184.72 (s, C-11); EI-MS m/z (%): 351 [M 9]
+
(51), 305 (100), 277 (39); HR(EI)MS: 351.0728 €0.003 (C19
calc. 351,0743).
H13NO
6
9,
1
3
Attached Proton Test (APT) experiment. C-NMR data were
deduced by HMBC correlations for compounds 8, 11a, 12b and
1
2c. Electron-impact (EI) mass spectra (m/z; rel.%) and HR-EI data
Methyl-6,11-dioxo-6,11-dihydro-benzo[f]pyrido[1,2-
a]indole 12-carboxylate 9
Orange solid; m. p. 1918C (acetic acid) [lit. [11]: 190–1918C]; H-
were taken with a Kratos-MS80 mass spectrometer (Kratos, Ltd.,
Manchester, UK) with home-built computerised acquisition soft-
ware. ESI-MS data, and tandem fragmentation spectra (MS ),
1
n
TM
3 3
NMR (400 MHz, CDCl ) d: 4.07 (s, 3H, OCH ), 7.16 (t, 1H, J = 7.00
were taken with a Bruker Esquire-LC spectrometer (Bruker Dal-
Hz, H-3), 7.43 (m, 1H, H-2), 7.71 (m, 2H, H-8 and H-9), 8.22 (br d,
H, J = 7.70 Hz, H-7 and H-10), 8.31(d, 1H, J = 9.20 Hz , H-1), 9.85
tonik GmbH, Bremen, Germany), equipped with an electrospray
ionization ion source used in positive ion mode by injection of
the sample into the source from a methanol solution. Semi-
empirical calculations were achieved using the free MOPAC 6.0
software.
2
+
(
br d, 1H, J = 6.80 Hz, H-4); EI-MS m/z (%): 305 [M 9] (62), 274 (100),
47 (23); HR(EI)MS: 305.0693 l 0.005 (C18 , calc. 305.0688).
2
H11NO
4
Methyl-7,10-dihydroxy-6,11-dioxo-6,11-dihydro-
Preparation of esters 7–10
To a mixture of commercially available 2,3-dichloro-1,4-naphto-
quinone 5 (0.284 g, 1.25 mmol) or 2,3-dichloro-5,7-dihydroxy-
benzo[f]pyrido[1,2-a]indole 12-carboxylate 10
1
Orange-brown powder, m. p. 2148C (hexane/ethyl acetate); H-
NMR (400 MHz, CDCl ) d: 4.01 (s, 3H, OCH ), 7.19 (t, 1H, J = 6.90
3
3
1
,4-naphtoquinone 6 (0.324 g, 1.25 mmol) and ethyl acetoace-
Hz, H-3), 7.20 (s, 2H, H-8 and H-9), 7.46 (m, 1H, H-2), 8.30 (br d, 1H,
J = 9.1 Hz, H-1), 9.80 (br d, 1H, J = 6.90 Hz, H-4), 12.90 (s, 1H, OH),
13.10 (s, 1H, OH); EIMS m/z (%): 337 [M 9] (40), 305 (100), 277 (28).
tate (0.24 mL, 1.88 mmol) or methyl acetoacetate (0.20 mL,
+
1
7
4
.88 mmol), dissolved in 2-propanol (13 mL), pyridine (0.60 mL,
.5 mmol) was added and the resultant solution was refluxed for
h. After cooling, the solvent was removed in vacuo and the resi-
General procedure for the conversion to amides
The methyl ester (0.020 g, 0.066 mmol of 9 or 0.060 mmol of 10)
was stirred in the presence of a molar excess of amine (0.5 mL) at
due was partitioned between water and dichloromethane, the
organic phases were separated (63), combined and washed in
sequence with 0.1 M aq. HCl, and water, dried on anhydrous
Na SO and concentrated in vacuo. The residue was subjected to
2 4
flash-chromatography on silica gel with hexane / EtOAc gradient
8
08C for 3 h. The crude mixture was evaporated in vacuo, water
5 mL) was added to the residue or treated with a phosphate buf-
fer solution at pH 6 (5 mL) for the preparation of the amides
2a–c, and extracted with dichloromethane (63), to give a com-
bined organic phase, which was washed with brine. After drying
over anhydrous Na SO and evaporation, the residue was sub-
(
elution, to give the respective esters 7, 9 (each 75%) and 8, 10
1
(
each 54%). The purity of ester 8, later subjected to bioassays, was
tested to be higher than 99.9% by RP-18 HPLC analysis with
9.5:0.5 MeOH/TFA (t 4.9 min).
2
4
9
R
jected to preparative TLC eluted with CH
give pure amides in 98% yields.
2 2
Cl / MeOH (99.5:0.5) to
Ethyl 6,11-dioxo-6,11-dihydro-benzo[f]pyrido[1,2-a]indole
2-carboxylate 7
The purity of each amide was tested to be higher than 99.8%
by RP-18 HPLC analysis with MeOH/TFA (99.5:0.5): 11a (t
15.1 min), 11b (t 13.3 min), 11c (t 3.7 min), 12a (t 3.4 min), 12b
4.0 min).
1
R
Orange-brown solid; m. p. 1578C (ethanol) [lit. [11]:157-1588C];
R
R
R
1
H-NMR (400 MHz, CDCl
.51 (q, 2H, J = 7.20 Hz, OCH
m, 1H, H-2), 7.71 (m, 2H, H-8 and H-9), 8.22 (br d, 2H, J = 7.70 Hz,
H-7 and H-10), 8.31(d, 1H, J = 9.20 Hz , H-1), 9.85 (br d, 1H, J =
3
) d : 1.50 (t, 3H, J = 7.00 Hz, OCH
2
CH
3
),
(t
R R
2.4 min), 12c (t
4
(
2
CH ), 7.16 (t, 1H, J = 7.00 Hz, H-3) 7.43
3
6
,11-Dioxo-6,11-dihydro-benzo[f]pyrido[1,2-a]indole-(2-
1
3
dimethylamino-ethyl)-12-carboxamide 11a
Deep red solid; m. p. 142–1438C (chloroform/methanol); UV/Vis
6
.80Hz, H-4); C-NMR (100 MHz, CDCl
OCH ), 106.10 (s, C-12), 117.32 (d, C-3), 123.62 (s, C-10a), 120.94 (d,
C-1), 126.04 (d, C-2), 127.34, 127.78 ( two d, C-7 and C-10), 128.53
d, C-4), 128.70 (s, C-5a), 133.16, 133.34 ( two d, C-8 and C-9),
33.77 (s, C-6a), 134.24 (s, C-10a), 139.59 (s, C-12a), 163.64 (s,
3 3
) d: 14.33 (q, CH ), 61.03 (t,
2
(
EtOH) kmax nm (e) = 488 (5150), 347 (7230), 326 (12380), 254
1
(
38300), 202 (31230); H-NMR (400 MHz, CDCl
), 2.67 (t, 2H, J = 6.7 Hz, CH N(CH ), 3.66 (pseudo q, 2H, J =
6.7 Hz, CONHCH ), 7.18 (td, 1H, J = 6.9 Hz, 1.3 Hz, H-2), 7.42 (br t,
H, J = 6.9 Hz, H-3), 7.70 (td, 1H, J = 7.5, 1.4 Hz, H-8), 7.76 (td, 1H, J
3
) d: 2.35 (s, 6H,
(
1
N(CH
3
)
2
2
3 2
)
+
2
COOEt), 175.24 (s, C-6), 180.31 (s, C-11). EI-MS m/z (%): 319 [M ]
4
85), 274 (87), 247 (100); HR(EI)MS: 319.0840 l 0.003 (C19H13NO ,
1
=
7
(
7.5, 1.4 Hz, H-9), 8.22 (br t, 1H, J = 7.5 Hz, H-7), 8.25 (br t, 1H, J =
.5 Hz, H-10), 9.12 (br d, 1H, J = 9.2 Hz, H-1), 9.90 (br d, 1H, J = 6.9
calc. 319.0844).
1
3
Hz, H-4), 10.48 (br s, 1H, CONH); C-NMR (100 MHz, CDCl
7.28 (t, CONHCH ), 45.30 (q, N(CH ), 58.23 (t, CH N(CH
3
) d:
),
Ethyl-7,10-dihydroxy-6,11-dioxo-6,11-dihydro-
3
2
3
)
2
2
3
)
2
benzo[f]pyrido[1,2-a]indole 12-carboxylate 8
109.98 (s, C-12), 118.30 (d, C-3), 123.41 (d, C-1), 121.69 and 125.34
(s, C-5a, C-11a), 126.21 (d, C-7), 127.69 (d, C-10), 127.62 (s, C-5a),
127.70 (d, C-4), 127.79 (d, C-2), 133.06 (d, C-8), 133.35 (s, C-10a),
133.97 (s, C-6a), 134.42 (d, C-9), 140.58 (s, C-12a), 140.79 (s, C-5a),
Orange-brown solid; m. p. 179–1818C (ethanol); UV/Vis (EtOH)
k
max nm (e) = 528 (4430), 500 (4690), 343(25890), 248 (16320), 202
1
3
(13290); H-NMR (400 MHz, CDCl ) d: 1.48 (t, 3H, J = 7.20 Hz,
i
2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

152
A. Defant et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 147–153
+
1
3
63.35 (s, CONH), 174.55 (s, C-6), 184.22 (s, C-11); EI-MS m/z (%):
CONH), 177.52 (s, C-6), 186.88 (s, C-11); EI-MS m/z (%): 393 [M ] (3),
+
61 [M 9] (1), 291 (2), 274 (6), 58 (100); HR(EI)MS: 361.1417 l 0.003
323 (3), 306 (10), 58 (100); HR(EI)MS: 393.1315 €0.003
+
+
+
(C
21
H
19
N
3
O
3
9, calc. 361.1426). ESI (+)-MS/MS m/z: 362 ([M+H] ) fi
(C21
H
19
N
3
O
5
9, calc. 393.1325).
317.
7
,10-Dihydroxy-6,11-dioxo-6,11-dihydro-
6
,11-Dioxo-6,11-dihydro-benzo[f]pyrido[1,2-a]indole-(2-
benzo[f]pyrido[1,2-a]indole-(2-diethylamino-ethyl)- 12-
diethylamino-ethyl)-12- carboxamide 11b
carboxamide 12b
Deep red solid; m. p. 2008C (chloroform/methanol); UV/Vis
Purple solid; m. p. 2258C (chloroform/methanol); UV/Vis (EtOH)
1
(
(
6
2
EtOH) kmax nm (e) = 490 (5830), 349 (8560), 319 (14200), 250
kmax nm (e) = 550 (4330), 346 (2530), 250 (14080), 205 (19130); H-
1
46300), 205 (147860); H-NMR (400 Mz, CDCl
3
) d: 1.10 (t, 6H, J =
), 2.64 (q, 4H, J = 6.4 Hz, N(CH CH ), 2.78 (t,
H, J = 6.7 Hz, CH NEt ), 3.59 (pseudo q, 2H, J = 6.7 Hz,
),7.22 (td, 1H, J = 6.9, 1.3 Hz, H-2), 7.45 (br t, 1H, J = 6.9
NMR (400 MHz, CDCl
(q, J = 6.7 Hz, 4H, N(CH
3.68 (pseudo q, 2H, J = 6.7 Hz, CONHCH
2
and H-9),7.49 (br t, 1H, J = 6.9 Hz, H-2), 8.99 (d, 1H, J = 8.9 Hz, H-1),
9.87 (d, 1H, J = 6.8 Hz, H-4), 10.54 (br s, 1H, CONH), 12.70 and
3
) d: 1.17 (t, 6H, J = 6.7 Hz, N(CH
CH ), 2.70 (t, 2H, J = 6.7 Hz, CH
), 7.23 (m, 3H, H-3, H-8
2
CH
3
)
2
), 2.84
.4 Hz, N(CH
2
CH
3
)
2
2
3
)
2
2
3
)
2
2
NEt ),
2
2
2
CONHCH
2
Hz, H-3), 7.73 (td, 1H, J = 7.5, 1.4 Hz , H-8), 7.78 (td, 1H, J = 7.5, 1.4
Hz, H-9), 8.22 (m, 2H, H-7, H-10), 9.10 (br d, 1H, J = 9.2 Hz, H-1),
1
3
13.17 (two br s, 1H each, 2OH); C-NMR (100 MHz, CDCl
(q, N(CH2C ), 32.58 (t, CONCH ), 42.21 (two t, N(CH CH
(t, CONCH2C ), 110.69 (s, C-12), 112.53, 112.81 (two s, C-6a and C-
3
) d: 11.93
1
3
9.94 (br d, 1H, J = 6.9 Hz, H-4), 10.50 (br s, 1H, CONH); C-NMR
H
3
)
2
2
2
3 2
) ), 51.83
(100 MHz, CDCl
3
) d: 11.80 (q, N(CH
2
(CH
3
)
2
), 32.60 (t, CONCH
2
),
H
2
4
1
5
2.24 (two t, N(CH
18.24 (d, C-3), 123.47 (d, C-1), 121.58, 125.24 (two s, C-11a and C-
a), 126.14 (d, C-7), 127.54, 127.63, 127.66 (three d, C-2, C-4, and
2
CH ), 51.95 (t, CONHCH2C
3
)
2
H
2
), 110.08 (s, C-12),
10a), 118.18 (d, C-3), 123.34 (d, C-1), 125.42 (s, C-5a), 127.88 (d, C-
2), 128.02 (d, C-4), 128.71, 130.79 (two d, C-8 and C-9), 139.65 (s, C-
12a), 157.54, 159.04 (two s, C-7 and C-10), 162.78 (s, CONH),
+
C-10), 132.99 (d, C-8), 133.94 and 133.34 (s, C-6a and C-10a),
177.47 (s, C-6), 185.99 (s, C-11); EI-MS m/z (%): 421 [M 9] (4), 306
+
134.30 (d, C-9), 140.75 (s, C-12a), 163.16 (s, CONH), 174.33 (s, C-6),
(10), 86 (100); HR(EI)MS: 421.1631 l 0.003 (C23
H
23
N
3
O
5
9, calc.
+
183.93 (s, C-11); EI-MS m/z (%): 389 [M ] (4), 274 (13), 86 (100);
421.1638).
+
HR(EI)MS: 389.1731 l 0.003 (C23
H
23
N
3
O
3
9, calc. 389.1739).
7
,10-Dihydroxy-12-[4-(2-hydroxy-ethyl)-piperazine-1-
1
2-[4-(2-Hydroxy-ethyl)-piperazine-1-carbonyl]-
carbonyl]-benzo[f]pyrido[1,2-a]indole-6,11-dione 12c
1
Deep red solid; m. p. 235–2368C (chloroform/methanol); H-
NMR (400 MHz, CDCl ) d: 2.29, 2.59,2.83, 3.39,3.46, 3.61, 3.91,
.05 (series of m, 12H), 7.21 (m, 3H, H-3,H-8 and H-9), 7.37 (br t.,
1H, J = 6.9 Hz, H-2), 7.75 (br d., 1H, J = 9.0 Hz, H-1), 9.62 (br d, 1H, J
benzo[f]pyrido[1,2-a]indole-6,11-dione 11c
Orange solid; m. p. 249–2518C (chloroform/methanol); UV/Vis
3
4
(EtOH) kmax nm (e) = 477 (2950), 320 (6670), 280 (30230), 254
1
(
3
4
29150); H-NMR (100 MHz, CDCl
3
) d: 2.29, 2.59,2.83, 3.39,3.46,
.61, 3.91, 4.05 (series of m, 12H), 7.14 (t, 1H, J = 7.10 Hz, H-3),
.46 (br s, 1H, OH), 7.33 (br t, 1H, J = 7.10 Hz , H-2), 7.69 and 7.75
13
=
6.9 Hz, H-4), 12.85 (s, 1H, OH), 13.04 (s, 1H, OH); C-NMR (100
MHz, CDCl ) d: 41.59, 46.33 (two t, (CH NCO), 52.38, 53.11 (two
t, (CH NCH ), 57.24 (t, CH CH OH), 58.93 (t, CH OH), 109.99 (s, C-
2), 112.84, 113.12 (two s, C-6a and C-10a), 117.53, 119.21 (two d,
C-2 and C-3), 125.88 (s, C-5a), 126.12 (d, C-1), 128.21 (d, C-4),
3
2 2
)
2
)
2
2
2
2
2
(two t, 1H each, J = 7.10 Hz, H-8 and H-9), 7.73 (d, J = 9.00 Hz, H-1),
1
8
1
.14 (d, 1H, J = 7.10 Hz, H-10), 8.28 (d, 1H, J = 7.10 Hz, H-7), 9.68 (d,
1
3
H, J = 7.10 Hz, H-4); C-NMR (100 MHz, CDCl
NCO), 52.58, 53.04 (two t, (CH
OH), 59.53 (t, CH OH), 109.92 (s, C-12), 117.74, 119.73 (two
3
) d: 41.61 and 46.43
1
1
1
28.40, 129.12 (two d, C-8 and C-9), 138.35 (s, C-12a), 158.67,
58.71 (two s, C-7 and C-10), 162.99 (s, CONH), 176.50 (s, C-6),
(two t, (CH
2
)
2
2
)
2
NCH ), 57.56 (t,
2
CH
2
CH
2
2
+
+
84.31 (s, C-11); ESI(+)-MS m/z: 458 [M+Na] , 436 [M+H] , MS/MS
d, C-2 and C-3), 120.68 (s, C-11a), 126.43 (d, C-1 and C-7), 126.85 (d,
C-10), 128.04 (d, C-4), 132.97 (d, C-9), 133.98 (d, C-8), 134.80 (s, C-
–
(
458) fi 328; ESI(-)-MS: 434 [M-H] , MS/MS (434) fi 404, 304, 278.
1
5
0a), 133.98 (s, C-6a), 134.77 (d, C-9), 136.97 (s, C-12a), 125.97 (s, C-
a), 163.34 (s, CONH), 174.25 (s, C-6), 181.59 (s, C-11); EI-MS m/z
+
(%): 403 [M ] (2), 385 (6), 372 (11), 316 (15), 274 (100); HR(EI)MS:
+
References
403.1519 l 0.003 (C23
H
21
N
3
O
4
9, calc. 403.1532). ESI (+)-MS/MS m/z:
+
4
04 ([M+H] ) fi 274.
[1] K. M. Tewey, G. L. Chen, E. M. Nelson, L. F. Liu, J. Biol.
Chem. 1984, 259, 9182–9187.
7
,10-Dihydroxy-6,11-dioxo-6,11-dihydro-
[
2] R. B. Weiss, Semin. Oncol. 1992, 19, 670–686.
benzo[f]pyrido[1,2-a]indole-(2-dimethylamino -ethyl)-12-
carboxamide 12a
Violet solid; m. p. 174–1758C (chloroform/methanol); UV/Vis
[
3] H. Malonne, G. Atassi, Anti-Cancer Drugs 1997, 8, 811–
822.
[
4] M. R. McCrystal, B. D. Evans, V. J. Harvey, P. I. Thompson,
(EtOH) kmax nm (e) = 557 (14370), 520 (13020), 350 (6260), 275
1
et al., Cancer Chemother. Pharmacol. 1999, 44, 39–44.
(
33300), 246 (42750); H-NMR (400 MHz, CDCl
), 2.69 (t, 2H, J = 6.7 Hz , NCH CH ), 3.68 (pseudo q, 2H, J =
.7 Hz, CONHCH ), 7.22 (m, 3H, H-3, H-8 and H-9), 7.49 (br t, 1H, J
6.9 Hz, H-2), 9.15 (d, 1H, J = 8.9 Hz, H-1), 9.88 (d, 1H, J = 6.8 Hz, H-
), 10.28 (br.s, 1H, CONH), 12.85 and 13.20 (two br s, 2H, 2
3
) d: 2.41 (s, 6H,
N(CH
3
)
2
2
2
[5] M. H. Moore, W. H. Hunyer, B. L. d’Estaintot, O. Kennard,
6
=
4
2
J. Mol. Biol. 1989, 206, 693–705.
[
[
6] U. Pindur, M. Harber, K. Sattler, J. Chem. Educ. 1993, 70,
63–272.
2
1
3
OH); C-NMR(100 MHz, CDCl
N(CH ), 58.34 (t, CH NMe ), 110.73 (s, C-12), 112.57, 112.99 (two
s, C-6a and C-10a), 118.33 (d, C-3), 123.59 (d, C-1), 125.38 (s, C-5a),
3 2
) d: 37.35 (t, CONHCH ), 45.52 (q,
7] A. Bolognese, G. Correale, M. Manfra, A. La Vecchia, et al.,
J. Med. Chem. 2004, 47, 849–858.
3
)
2
2
2
1
1
27.96 (d, C-2), 128.14 (d, C-4), 128.63, 130.66 (two d, C-8 and C-9),
39.80 (s, C-12a), 157.90, 159.28 (two s, C-7 and C-10), 162.81 (s,
[8] K. C. Murdock, R. G. Child, P. F. Fabio, R. B. Angier, et al.,
J. Med. Chem. 1979, 22, 1024–1030.
i
2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2007, 340, 147–153
Novel Naphthindolizinedione Derivatives
153
[
9] G. W. Rewcastle, G. J. Atwell, D. Chambers, B. C. Baguley,
[13] J. M. Khurana, A. Sehgal, Org. Prep. Proced. Int. 1994, 26,
W. A. Denny, J. Med. Chem. 1986, 29, 472–477.
580–583.
[
[
[
10] H. J. Park, H-J. Lee, E-J. Lee, H. J. Hwang, et al., Biosci. Bio-
technol. Biochem. 2003, 67, 1944–1949.
[14] http://dtp.nci.nih.gov/branches/btb/ivclsp.html
[15] A. P. Krapcho, M. E. Petry, Z. Getahun, J. J. Landi, et al., J.
Med. Chem. 1994, 37, 828–837.
11] E. F. Pratt, R. W. Luckenbaugh, R. L. Erickson, J. Org.
Chem. 1954, 19, 176–182.
12] M. S. Mathur, B. D. Tilak, J. Scient. & Ind. Res. 1958, 17B,
33–40 [Chem. Abstr. 1958, 52, 72342].
i
2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com